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Comeau, A.M., G.F. Hatfull, H.M. Krisch, D. Lindell, N.H. Mann, and D. Prangishvili (2008) Exploring the prokaryotic virosphere. Res. Microbiol. 159:306-313 The world of prokaryotic viruses, including the "traditional" bacteriophages and the viruses of Archaea, is currently in a period of renaissance, brought about largely by our new capabilities in (meta)genomics and by the isolation of diverse novel virus-host systems. In this review, we highlight some of the directions where we believe research on the prokaryotic virosphere will lead us in the near future. Hatfull, G.F., S.G. Cresawn, and R.W. Hendrix (2008) Comparative genomics of the mycobacteriophages: insights into bacteriophage evolution. Res. Microbiol. 159:332-339 The recognition of the vast numbers of bacteriophages in the biosphere has prompted a renewal of interest in understanding their morphological and genetic diversity, and elucidating the evolutionary mechanisms that give rise to them. We have approached these questions by isolating and characterizing a collection of mycobacteriophages that infect a common bacterial host, Mycobacterium smegmatis. Comparative genomic analysis of 50 mycobacteriophages shows that they are highly diverse, although not uniformly so, that they are pervasively mosaic with a multitude of single gene modules, and that this mosaicism is generated through illegitimate recombination. Hatfull, G.F. (2008) Bacteriophage genomics. Curr. Opin. Microbiol. 11:447-453 The past three years have seen an escalation in the number of sequenced bacteriophage genomes with more than 500 now in the NCBI phage database, representing a more than threefold increase since 2005. These span at least 70 different bacterial hosts, with two-thirds of the sequenced genomes of phages representing only eight bacterial hosts. Three key features emerge from the comparative analysis of these genomes. First, they span a very high degree of genetic diversity, suggesting early evolutionary origins. Second, the genome architectures are mosaic, reflecting an unusually high degree of horizontal genetic exchange in their evolution. Third, phage genomes contain a very high proportion of novel genetic sequences of unknown function, and probably represent the largest reservoir of unexplored genes. With an estimated 10(31) bacterial and archael viruses in the biosphere, our view of the virosphere will draw into sharper focus as further bacteriophage genomes are characterized. van Kessel, J.C., L.J. Marinelli, and G.F. Hatfull (2008) Recombineering mycobacteria and their phages. Nat. Rev. Microbiol. 6:851-857 Bacteriophages are central components in the development of molecular tools for microbial genetics. Mycobacteriophages have proven to be a rich resource for tuberculosis genetics, and the recent development of a mycobacterial recombineering system based on mycobacteriophage Che9c-encoded proteins offers new approaches to mycobacterial mutagenesis. Expression of the phage exonuclease and recombinase substantially enhances recombination frequencies in both fast- and slow-growing mycobacteria, thereby facilitating construction of both gene knockout and point mutants; it also provides a simple and efficient method for constructing mycobacteriophage mutants. Exploitation of host-specific phages thus provides a general strategy for recombineering and mutagenesis in genetically naive systems. Marinelli, L.J., M. Piuri, Z. Swigonova, A. Balachandran, L.M. Oldfield, K.e.s.s.e. van, and G.F. Hatfull (2008) BRED: a simple and powerful tool for constructing mutant and recombinant bacteriophage genomes. PLoS ONE 3:e3957 Advances in DNA sequencing technology have facilitated the determination of hundreds of complete genome sequences both for bacteria and their bacteriophages. Some of these bacteria have well-developed and facile genetic systems for constructing mutants to determine gene function, and recombineering is a particularly effective tool. However, generally applicable methods for constructing defined mutants of bacteriophages are poorly developed, in part because of the inability to use selectable markers such as drug resistance genes during viral lytic growth. Here we describe a method for simple and effective directed mutagenesis of bacteriophage genomes using Bacteriophage Recombineering of Electroporated DNA (BRED), in which a highly efficient recombineering system is utilized directly on electroporated phage DNA; no selection is required and mutants can be readily detected by PCR. We describe the use of BRED to construct unmarked gene deletions, in-frame internal deletions, base substitutions, precise gene replacements, and the addition of gene tags. Morris, P., L.J. Marinelli, D. Jacobs-Sera, R.W. Hendrix, and G.F. Hatfull (2008) Genomic characterization of mycobacteriophage Giles: evidence for phage acquisition of host DNA by illegitimate recombination. J. Bacteriol. 190:2172-2182 A characteristic feature of bacteriophage genomes is that they are architecturally mosaic, with each individual genome representing a unique assemblage of individual exchangeable modules. Plausible mechanisms for generating mosaicism include homologous recombination at shared boundary sequences of module junctions, illegitimate recombination in a non-sequence-directed process, and site-specific recombination. Analysis of the novel mycobacteriophage Giles genome not only extends our current perspective on bacteriophage genetic diversity, with more than 60% of the genes unrelated to other mycobacteriophages, but offers novel insights into how mosaic genomes are created. In one example, the integration/excision cassette is atypically situated within the structural gene operon and could have moved there either by illegitimate recombination or more plausibly via integrase-mediated site-specific recombination. In a second example, a DNA segment has been recently acquired from the host bacterial chromosome by illegitimate recombination, providing further evidence that phage genomic mosaicism is generated by nontargeted recombination processes.
van Kessel, J.C., and G.F. Hatfull (2008) Efficient point mutagenesis in mycobacteria using single-stranded DNA recombineering: characterization of antimycobacterial drug targets. Mol. Microbiol. 67:1094-1107 Construction of genetically isogenic strains of mycobacteria is complicated by poor recombination rates and the lack of generalized transducing phages for Mycobacterium tuberculosis. We report here a powerful method for introducing single point mutations into mycobacterial genomes using oligonucleotide-derived single-stranded DNA recombineering and mycobacteriophage-encoded proteins. Phage Che9c gp61-mediated recombination is sufficiently efficient that single base changes can be introduced without requirement for direct selection, with isogenic mutant strains identified simply by PCR. Efficient recombination requires only short (50 nucleotide) oligonucleotides, but there is an unusually strong strand bias and an oligonucleotide targeting lagging strand DNA synthesis can recombine more than 10,000-fold efficiently than its complementary oligonucleotide. This ssDNA recombineering provides a simple assay for comparing the activities of related phage recombinases, and we find that both Escherichia coli RecET and phage lambda Red recombination proteins function inefficiently in mycobacteria, illustrating the utility of developing recombineering in new bacterial systems using host-specific bacteriophage recombinases. ssDNA mycobacterial recombineering provides a simple approach to characterizing antimycobacterial drug targets, and we have constructed and characterized single point mutations that confer resistance to isoniazid, rifampicin, ofloxacin and streptomycin. Ghosh, P., L.A. Bibb, and G.F. Hatfull (2008) Two-step site selection for serine-integrase-mediated excision: DNA-directed integrase conformation and central dinucleotide proofreading. Proc. Natl. Acad. Sci., USA 105:3238-3243 Bacteriophage-encoded serine-integrases are members of the large family of serine-recombinases and catalyze site-specific integrative recombination between a phage attP site and a bacterial attB site to form an integrated prophage. Prophage excision involves a second site-specific recombination event, in which the sites generated by integration, attL and attR, are used as substrates to regenerate attP and attB. Excision is catalyzed by integrase but also requires a phage-encoded recombination directionality factor (RDF). The Bxb1 recombination sites, attP and attB, are small (<50 bp), different in sequence, and quasisymmetrical, and they give rise to attL- and attR-recombinant products that are asymmetric but similar to each other, each being composed of B- and P-type half-sites. We show here that the determination of correct excision products is a two-step process, with a presynaptic RDF-dependent step that aligns attL and attR in the correct orientation and a postsynaptic step in which the nonpalindromic central dinucleotide confers identity to attL and attR and prevents each from recombining with itself. van Kessel, J.C., and G.F. Hatfull (2008) Mycobacterial recombineering. Methods. Mol. Biol. 435:203-215 Although substantial advances have been made in mycobacterial genetics over the past 15 yr, manipulation of mycobacterial genomes and Mycobacterium tuberculosis in particular, continues to be hindered by problems of relatively poor DNA uptake, slow growth rate, and high levels of illegitimate recombination. In Escherichia coli an effective approach to stimulating recombination frequencies has been developed called "recombineering," in which phage-encoded recombination functions are transiently expressed to promote efficient homologous recombination. Although homologs of these recombination proteins are rare among mycobacteriophages, we have identified one phage, Che9c, encoding relatives of both RecE and RecT of the E. coli rac prophage. Expression of the Che9c proteins from an inducible expression system in either slow- or fast-growing mycobacteria provides elevated recombination frequencies and facilitates simple allelic exchange using linear DNA substrates. Mycobacterial recombineering, therefore, offers a simple approach for constructing gene replacement mutants in M. smegmatis and M. tuberculosis. Ojha, A.K., A.D. Baughn, D. Sambandan, T. Hsu, X. Trivelli, Y. Guerardel, A. Alahari, L. Kremer, W.R. .J.r. Jacobs, and G.F. Hatfull (2008) Growth of Mycobacterium tuberculosis biofilms containing free mycolic acids and harbouring drug-tolerant bacteria. Mol. Microbiol. 69:164-174 Successful treatment of human tuberculosis requires 6-9 months' therapy with multiple antibiotics. Incomplete clearance of tubercle bacilli frequently results in disease relapse, presumably as a result of reactivation of persistent drug-tolerant Mycobacterium tuberculosis cells, although the nature and location of these persisters are not known. In other pathogens, antibiotic tolerance is often associated with the formation of biofilms--organized communities of surface-attached cells--but physiologically and genetically defined M. tuberculosis biofilms have not been described. Here, we show that M. tuberculosis forms biofilms with specific environmental and genetic requirements distinct from those for planktonic growth, which contain an extracellular matrix rich in free mycolic acids, and harbour an important drug-tolerant population that persist despite exposure to high levels of antibiotics. Pham, T.T., D. Jacobs-Sera, M.L. Pedulla, R.W. Hendrix, and G.F. Hatfull (2007) Comparative genomic analysis of mycobacteriophage Tweety: evolutionary insights and construction of compatible site-specific integration vectors for mycobacteria. Microbiology 153:2711-2723 Mycobacteriophage Tweety is a newly isolated phage of Mycobacterium smegmatis. It has a viral morphology with an isometric head and a long flexible tail, and forms turbid plaques from which stable lysogens can be isolated. The Tweety genome is 58 692 bp in length, contains 109 protein-coding genes, and shows significant but interrupted nucleotide sequence similarity with the previously described mycobacteriophages Llij, PMC and Che8. However, overall the genome possesses mosaic architecture, with gene products being related to other mycobacteriophages such as Che9d, Omega and Corndog. A gene encoding an integrase of the tyrosine-recombinase family is located close to the centre of the genome, and a putative attP site has been identified within a short intergenic region immediately upstream of int. This Tweety attP-int cassette was used to construct a new set of integration-proficient plasmid vectors that efficiently transform both fast- and slow-growing mycobacteria through plasmid integration at a chromosomal locus containing a tRNA(Lys) gene. These vectors are maintained well in the absence of selection and are completely compatible with integration vectors derived from mycobacteriophage L5, enabling the simple construction of complex recombinants with genes integrated simultaneously at different chromosomal positions. Ojha, A., and G.F. Hatfull (2007) The role of iron in Mycobacterium smegmatis biofilm formation: the exochelin siderophore is essential in limiting iron conditions for biofilm formation but not for planktonic growth. Mol. Microbiol. 66:468-483 Many species of mycobacteria form structured biofilm communities at liquid-air interfaces and on solid surfaces. Full development of Mycobacterium smegmatis biofilms requires addition of supplemental iron above 1 microM ferrous sulphate, although addition of iron is not needed for planktonic growth. Microarray analysis of the M. smegmatis transcriptome shows that iron-responsive genes - especially those involved in siderophore synthesis and iron uptake - are strongly induced during biofilm formation reflecting a response to iron deprivation, even when 2 microM iron is present. The acquisition of iron under these conditions is specifically dependent on the exochelin synthesis and uptake pathways, and the strong defect of an iron-exochelin uptake mutant suggests a regulatory role of iron in the transition to biofilm growth. In contrast, although the expression of mycobactin and iron ABC transport operons is highly upregulated during biofilm formation, mutants in these systems form normal biofilms in low-iron (2 microM) conditions. A close correlation between iron availability and matrix-associated fatty acids implies a possible metabolic role in the late stages of biofilm maturation, in addition to the early regulatory role. M. smegmatis surface motility is similarly dependent on iron availability, requiring both supplemental iron and the exochelin pathway to acquire it. Pope, W.H., P.R. Weigele, J. Chang, M.L. Pedulla, M.E. Ford, J.M. Houtz, W. Jiang, W. Chiu, G.F. Hatfull, R.W. Hendrix, and J. King (2007) Genome sequence, structural proteins, and capsid organization of the cyanophage Syn5: a "horned" bacteriophage of marine synechococcus. J. Mol. Biol. 368:966-981 Marine Synechococcus spp and marine Prochlorococcus spp are numerically dominant photoautotrophs in the open oceans and contributors to the global carbon cycle. Syn5 is a short-tailed cyanophage isolated from the Sargasso Sea on Synechococcus strain WH8109. Syn5 has been grown in WH8109 to high titer in the laboratory and purified and concentrated retaining infectivity. Genome sequencing and annotation of Syn5 revealed that the linear genome is 46,214 bp with a 237 bp terminal direct repeat. Sixty-one open reading frames (ORFs) were identified. Based on genomic organization and sequence similarity to known protein sequences within GenBank, Syn5 shares features with T7-like phages. The presence of a putative integrase suggests access to a temperate life cycle. Assignment of 11 ORFs to structural proteins found within the phage virion was confirmed by mass-spectrometry and N-terminal sequencing. Eight of these identified structural proteins exhibited amino acid sequence similarity to enteric phage proteins. The remaining three virion proteins did not resemble any known phage sequences in GenBank as of August 2006. Cryo-electron micrographs of purified Syn5 virions revealed that the capsid has a single "horn", a novel fibrous structure protruding from the opposing end of the capsid from the tail of the virion. The tail appendage displayed an apparent 3-fold rather than 6-fold symmetry. An 18 A resolution icosahedral reconstruction of the capsid revealed a T=7 lattice, but with an unusual pattern of surface knobs. This phage/host system should allow detailed investigation of the physiology and biochemistry of phage propagation in marine photosynthetic bacteria. Weigele, P.R., W.H. Pope, M.L. Pedulla, J.M. Houtz, A.L. Smith, J.F. Conway, J. King, G.F. Hatfull, J.G. Lawrence, and R.W. Hendrix (2007) Genomic and structural analysis of Syn9, a cyanophage infecting marine Prochlorococcus and Synechococcus. Environ. Microbiol. 9:1675-1695 Cyanobacteriophage Syn9 is a large, contractile-tailed bacteriophage infecting the widespread, numerically dominant marine cyanobacteria of the genera Prochlorococcus and Synechococcus. Its 177,300 bp genome sequence encodes 226 putative proteins and six tRNAs. Experimental and computational analyses identified genes likely involved in virion formation, nucleotide synthesis, and DNA replication and repair. Syn9 shows significant mosaicism when compared with related cyanophages S-PM2, P-SSM2 and P-SSM4, although shared genes show strong purifying selection and evidence for large population sizes relative to other phages. Related to coliphage T4 - which shares 19% of Syn9's genes - Syn9 shows evidence for different patterns of DNA replication and uses homologous proteins to assemble capsids with a different overall structure that shares topology with phage SPO1 and herpes virus. Noteworthy bacteria-related sequences in the Syn9 genome potentially encode subunits of the photosynthetic reaction centre, electron transport proteins, three pentose pathway enzymes and two tryptophan halogenases. These genes suggest that Syn9 is well adapted to the physiology of its photosynthetic hosts and may affect the evolution of these sequences within marine cyanobacteria. Pham, T.T., D. Jacobs-Sera, M.L. Pedulla, R.W. Hendrix, and G.F. Hatfull (2007) Comparative genomic analysis of mycobacteriophage Tweety: evolutionary insights and construction of compatible site-specific integration vectors for mycobacteria. Microbiology 153:2711-2723 Mycobacteriophage Tweety is a newly isolated phage of Mycobacterium smegmatis. It has a viral morphology with an isometric head and a long flexible tail, and forms turbid plaques from which stable lysogens can be isolated. The Tweety genome is 58 692 bp in length, contains 109 protein-coding genes, and shows significant but interrupted nucleotide sequence similarity with the previously described mycobacteriophages Llij, PMC and Che8. However, overall the genome possesses mosaic architecture, with gene products being related to other mycobacteriophages such as Che9d, Omega and Corndog. A gene encoding an integrase of the tyrosine-recombinase family is located close to the centre of the genome, and a putative attP site has been identified within a short intergenic region immediately upstream of int. This Tweety attP-int cassette was used to construct a new set of integration-proficient plasmid vectors that efficiently transform both fast- and slow-growing mycobacteria through plasmid integration at a chromosomal locus containing a tRNA(Lys) gene. These vectors are maintained well in the absence of selection and are completely compatible with integration vectors derived from mycobacteriophage L5, enabling the simple construction of complex recombinants with genes integrated simultaneously at different chromosomal positions. Hatfull, G.F. (2007) Host-microbe interactions: matters of life and death. Curr. Opin. Microbiol. 0: Hanauer, D.I., D. Jacobs-Sera, M.L. Pedulla, S.G. Cresawn, R.W. Hendrix, and G.F. Hatfull (2006) Inquiry learning. Teaching scientific inquiry. Science 314:1880-1881 van Kessel, J.C., and G.F. Hatfull (2006) Recombineering in Mycobacterium tuberculosis. Nat. Methods :In Press Genetic dissection of M. tuberculosis is complicated by its slow growth and its high rate of illegitimate recombination relative to homologous DNA exchange. We report here the development of a facile allelic exchange system by identification and expression of mycobacteriophage-encoded recombination proteins, adapting a strategy developed previously for recombineering in Escherichia coli. Identifiable recombination proteins are rare in mycobacteriophages, and only 1 of 30 genomically characterized mycobacteriophages (Che9c) encodes homologs of both RecE and RecT. Expression and biochemical characterization show that Che9c gp60 and gp61 encode exonuclease and DNA-binding activities, respectively, and expression of these proteins substantially elevates recombination facilitating allelic exchange in both M. smegmatis and M. tuberculosis. Mycobacterial recombineering thus provides a simple approach for the construction of gene replacement mutants in both slow- and fast-growing mycobacteria. Hatfull, G.F., M.L. Pedulla, D. Jacobs-Sera, P.M. Cichon, A. Foley, M.E. Ford, R.M. Gonda, J.M. Houtz, A.J. Hryckowian, V.A. Kelchner, S. Namburi, K.V. Pajcini, M.G. Popovich, D.T. Schleicher, B.Z. Simanek, A.L. Smith, G.M. Zdanowicz, V. Kumar, C.L. Peebles, W.R. .J.r. Jacobs, J.G. Lawrence, and R.W. Hendrix (2006) Exploring the mycobacteriophage metaproteome: phage genomics as an educational platform. PLoS Genet. 2:e92 Bacteriophages are the most abundant forms of life in the biosphere and carry genomes characterized by high genetic diversity and mosaic architectures. The complete sequences of 30 mycobacteriophage genomes show them collectively to encode 101 tRNAs, three tmRNAs, and 3,357 proteins belonging to 1,536 "phamilies" of related sequences, and a statistical analysis predicts that these represent approximately 50% of the total number of phamilies in the mycobacteriophage population. These phamilies contain 2.19 proteins on average; more than half (774) of them contain just a single protein sequence. Only six phamilies have representatives in more than half of the 30 genomes, and only three-encoding tape-measure proteins, lysins, and minor tail proteins-are present in all 30 phages, although these phamilies are themselves highly modular, such that no single amino acid sequence element is present in all 30 mycobacteriophage genomes. Of the 1,536 phamilies, only 230 (15%) have amino acid sequence similarity to previously reported proteins, reflecting the enormous genetic diversity of the entire phage population. The abundance and diversity of phages, the simplicity of phage isolation, and the relatively small size of phage genomes support bacteriophage isolation and comparative genomic analysis as a highly suitable platform for discovery-based education.
Pitcher, R.S., L.M. Tonkin, J.M. Daley, P.L. Palmbos, A.J. Green, T.L. Velting, A. Brzostek, M. Korycka-Machala, S. Cresawn, J. Dziadek, G.F. Hatfull, T.E. Wilson, and A.J. Doherty (2006) Mycobacteriophage exploit NHEJ to facilitate genome circularization. Mol. Cell 23:743-748 Ku-dependent nonhomologous end joining (NHEJ) is a double-strand break repair process conserved in all branches of cellular life but has not previously been implicated in the DNA metabolic processes of viruses. We identified Ku homologs in Corndog and Omega, two related mycobacteriophages of Mycobacterium smegmatis. These proteins formed homodimers and bound DNA ends in a manner identical to other Ku's and stimulated joining of ends by the host NHEJ DNA ligase (LigD). Omega and Corndog are unusual in having short 4 base cos ends that would not be expected to self-anneal and would therefore require NHEJ during phage genome circularization. Consistently, M. smegmatis LigD null strains are entirely and selectively unable to support infection by Corndog or Omega, with concomitant failure of genome circularization. These results establish a new paradigm for sequestration of the host cell NHEJ process by bacteriophage and provide a framework for understanding similar transactions in eukaryotic viral infections.
Nkrumah, L.J., R.A. Muhle, P.A. Moura, P. Ghosh, G.F. Hatfull, W.R. .J.r. Jacobs, and D.A. Fidock (2006) Efficient site-specific integration in Plasmodium falciparum chromosomes mediated by mycobacteriophage Bxb1 integrase. Nat. Methods 3:615-621 Here we report an efficient, site-specific system of genetic integration into Plasmodium falciparum malaria parasite chromosomes. This is mediated by mycobacteriophage Bxb1 integrase, which catalyzes recombination between an incoming attP and a chromosomal attB site. We developed P. falciparum lines with the attB site integrated into the glutaredoxin-like cg6 gene. Transfection of these attB(+) lines with a dual-plasmid system, expressing a transgene on an attP-containing plasmid together with a drug resistance gene and the integrase on a separate plasmid, produced recombinant parasites within 2 to 4 weeks that were genetically uniform for single-copy plasmid integration. Integrase-mediated recombination resulted in proper targeting of parasite proteins to intra-erythrocytic compartments, including the apicoplast, a plastid-like organelle. Recombinant attB x attP parasites were genetically stable in the absence of drug and were phenotypically homogeneous. This system can be exploited for rapid genetic integration and complementation analyses at any stage of the P. falciparum life cycle, and it illustrates the utility of Bxb1-based integrative recombination for genetic studies of intracellular eukaryotic organisms.
Ghosh, P., L.R. Wasil, and G.F. Hatfull (2006) Control of phage bxb1 excision by a novel recombination directionality factor. PLoS Biol. 4:e186 Mycobacteriophage Bxb1 integrates its DNA at the attB site of the Mycobacterium smegmatis genome using the viral attP site and a phage-encoded integrase generating the recombinant junctions attL and attR. The Bxb1 integrase is a member of the serine recombinase family of site-specific recombination proteins and utilizes small (<50 base pair) substrates for recombination, promoting strand exchange without the necessity for complex higher order macromolecular architectures. To elucidate the regulatory mechanism for the integration and excision reactions, we have identified a Bxb1-encoded recombination directionality factor (RDF), the product of gene 47. Bxb1 gp47 is an unusual RDF in that it is relatively large ( 28 kDa), unrelated to all other RDFs, and presumably performs dual functions since it is well conserved in mycobacteriophages that utilize unrelated integration systems. Furthermore, unlike other RDFs, Bxb1 gp47 does not bind DNA and functions solely through direct interaction with integrase-DNA complexes. The nature and consequences of this interaction depend on the specific DNA substrate to which integrase is bound, generating electrophoretically stable tertiary complexes with either attB or attP that are unable to undergo integrative recombination, and weakly bound, electrophoretically unstable complexes with either attL or attR that gain full potential for excisive recombination.
Ojha, A., M. Anand, A. Bhatt, L. Kremer, W.R. Jacobs, Jr., and G.F. Hatfull (2005) GroEL1: a dedicated chaperone involved in mycolic acid biosynthesis during biofilm formation in mycobacteria. Cell 123:861-873 Mycobacteria are unusual in encoding two GroEL paralogs, GroEL1 and GroEL2. GroEL2 is essential - presumably providing the housekeeping chaperone functions - while groEL1 is nonessential, contains the attB site for phage Bxb1 integration, and encodes a putative chaperone with unusual structural features. Inactivation of the Mycobacterium smegmatis groEL1 gene by phage Bxb1 integration allows normal planktonic growth but prevents the formation of mature biofilms. GroEL1 modulates synthesis of mycolates - long-chain fatty acid components of the mycobacterial cell wall - specifically during biofilm formation and physically associates with KasA, a key component of the type II Fatty Acid Synthase involved in mycolic acid synthesis. Biofilm formation is associated with elevated synthesis of short-chain (C56-C68) fatty acids, and strains with altered mycolate profiles - including an InhA mutant resistant to the antituberculosis drug isoniazid and a strain overexpressing KasA - are defective in biofilm formation.
Bibb, L.A., M.I. Hancox, and G.F. Hatfull (2005) Integration and excision by the large serine recombinase phiRv1 integrase. Mol. Microbiol. 55:1896-1910 The Mycobacterium tuberculosis prophage-like element phiRv1 encodes a site-specific recombination system utilizing an integrase of the serine recombinase family. Recombination occurs between a putative attP site and the host chromosome, but is unusual in that the attB site lies within a redundant repetitive element (REP13E12) of which there are seven copies in the M. tuberculosis genome; four of these elements contain attB sites suitable for phiRv1 integration in vivo. Although the mechanism of directional control of large serine integrases is poorly understood, a recombination directionality factor (RDF) has been identified that is required for phiRv1 integrase-mediated excisive recombination in vivo. Here we describe defined in vitro recombination reactions for both phiRv1 integrase-mediated integration and excision and show that the phiRv1 RDF is not only required for excision but inhibits integrative recombination; neither reaction requires DNA supercoiling, host factors, or high-energy cofactors. Integration, excision and excise-mediated inhibition of integration require simple substrates sites, indicating that the control of directionality does not involve the manipulation of higher-order protein-DNA architectures as described for the tyrosine integrases.
Ghosh, P., N.R. Pannunzio, and G.F. Hatfull (2005) Synapsis in phage Bxb1 integration: selection mechanism for the correct pair of recombination sites. J. Mol. Biol. 349:331-348 Recombination by site-specific recombinases is a highly concerted process that requires synapsis of the correct pair of DNA substrates. Phage-encoded serine-integrases are unusual among the serine-recombinase family, which includes transposon resolvases and DNA invertases, in that they utilize two simple but different DNA substrates (attB and attP) and do not require accessory sites, additional proteins, or DNA supercoiling. Synapsis must therefore be directed solely by integrase-DNA interactions. We show here that the Bxb1 serine-integrase binds as a dimer to its two DNA substrates (attB, attP) and recombinant products (attL, attR) with similar affinities. However, synapsis occurs only between attP and attB, and not between any of the other nine possible site combinations. The Bxb1 integrase domain structure, the unusual DNA-binding properties of the integrase, and the characterization of a mutant protein with altered site-discrimination, are consistent with synaptic selectivity being derived from DNA sequence-induced changes in the conformations of integrase-DNA complexes.
Casjens, S.R., E.B. Gilcrease, D.A. Winn-Stapley, P. Schicklmaier, H. Schmieger, M.L. Pedulla, M.E. Ford, J.M. Houtz, G.F. Hatfull, and R.W. Hendrix (2005) The generalized transducing Salmonella bacteriophage ES18: complete genome sequence and DNA packaging strategy. J. Bacteriol. 187:1091-1104 The generalized transducing double-stranded DNA bacteriophage ES18 has an icosahedral head and a long noncontractile tail, and it infects both rough and smooth Salmonella enterica strains. We report here the complete 46,900-bp genome nucleotide sequence and provide an analysis of the sequence. Its 79 genes and their organization clearly show that ES18 is a member of the lambda-like (lambdoid) phage group; however, it contains a novel set of genes that program assembly of the virion head. Most of its integration-excision, immunity, Nin region, and lysis genes are nearly identical to those of the short-tailed Salmonella phage P22, while other early genes are nearly identical to Escherichia coli phages lambda and HK97, S. enterica phage ST64T, or a Shigella flexneri prophage. Some of the ES18 late genes are novel, while others are most closely related to phages HK97, lambda, or N15. Thus, the ES18 genome is mosaically related to other lambdoid phages, as is typical for all group members. Analysis of virion DNA showed that it is circularly permuted and about 10% terminally redundant and that initiation of DNA packaging series occurs across an approximately 1-kbp region rather than at a precise location on the genome. This supports a model in which ES18 terminase can move substantial distances along the DNA between recognition and cleavage of DNA destined to be packaged. Bioinformatic analysis of large terminase subunits shows that the different functional classes of phage-encoded terminases can usually be predicted from their amino acid sequence.
Lee, S., J. Kriakov, C. Vilcheze, Z. Dai, G.F. Hatfull, and W.R. .J.r. Jacobs (2004) Bxz1, a new generalized transducing phage for mycobacteria. FEMS Microbiol. Lett. 241:271-276 We have isolated and characterized a new generalized transducing phage, Bxz1, from soil sampling at a neighboring Wildlife Preservation Park. The hosts of the phage, measured by the formation of plaques, include fast growing Mycobacterium smegmatis and Mycobacterium vaccae. Bxz1 is capable of transducing chromosomal markers, point mutations, and plasmids at frequencies ranging from 10-8 to 10-6 per plaque forming unit between strains of M. smegmatis. We also demonstrated cotransduction of a transposon insertion linked to a point mutation of the ndh gene.
Casjens, S.R., E.B. Gilcrease, W.M. Huang, K.L. Bunny, M.L. Pedulla, M.E. Ford, J.M. Houtz, G.F. Hatfull, and R.W. Hendrix (2004) The pKO2 linear plasmid prophage of Klebsiella oxytoca. J. Bacteriol. 186:1818-1832 Temperate bacteriophages with plasmid prophages are uncommon in nature, and of these only phages N15 and PY54 are known to have a linear plasmid prophage with closed hairpin telomeres. We report here the complete nucleotide sequence of the 51,601-bp Klebsiella oxytoca linear plasmid pKO2, and we demonstrate experimentally that it is also a prophage. We call this bacteriophage phiKO2. An analysis of the 64 predicted phiKO2 genes indicate that it is a fairly close relative of phage N15; they share a mosaic relationship that is typical of different members of double-stranded DNA tailed-phage groups. Although the head, tail shaft, and lysis genes are not recognizably homologous between these phages, other genes such as the plasmid partitioning, replicase, prophage repressor, and protelomerase genes (and their putative targets) are so similar that we predict that they must have nearly identical DNA binding specificities. The phiKO2 virion is unusual in that its phage lambda-like tails have an exceptionally long (3,433 amino acids) central tip tail fiber protein. The phiKO2 genome also carries putative homologues of bacterial dinI and umuD genes, both of which are involved in the host SOS response. We show that these divergently transcribed genes are regulated by LexA protein binding to a single target site that overlaps both promoters.
Dobbins, A.T., M. George Jr., D.A. Basham, M.E. Ford, J.M. Houtz, M.L. Pedulla, J.G. Lawrence, G.F. Hatfull, and R.W. Hendrix (2004) Complete genomic sequence of the virulent Salmonella bacteriophage SP6. J. Bacteriol. 186:1933-1944 We report the complete genome sequence of enterobacteriophage SP6, which infects Salmonella enterica serovar Typhimurium. The genome contains 43,769 bp, including a 174-bp direct terminal repeat. The gene content and organization clearly place SP6 in the coliphage T7 group of phages, but there is approximately 5 kb at the right end of the genome that is not present in other members of the group, and the homologues of T7 genes 1.3 through 3 appear to have undergone an unusual reorganization. Sequence analysis identified 10 putative promoters for the SP6-encoded RNA polymerase and seven putative rho-independent terminators. The terminator following the gene encoding the major capsid subunit has a termination efficiency of about 50% with the SP6-encoded RNA polymerase. Phylogenetic analysis of phages related to SP6 provided clear evidence for horizontal exchange of sequences in the ancestry of these phages and clearly demarcated exchange boundaries; one of the recombination joints lies within the coding region for a phage exonuclease. Bioinformatic analysis of the SP6 sequence strongly suggested that DNA replication occurs in large part through a bidirectional mechanism, possibly with circular intermediates.
Kim, A.I., P. Ghosh, M.A. Aaron, L.A. Bibb, S. Jain, and G.F. Hatfull (2003) Mycobacteriophage Bxb1 integrates into the Mycobacterium smegmatis groEL1 gene. Mol. Microbiol. 50:463-473
Ghosh, P., A.I. Kim, and G.F. Hatfull (2003) The orientation of mycobacteriophage Bxb1 integration is solely dependent on the central dinucleotide of attP and attB. Mol. Cell 12:1101-1111 Integration of the mycobacteriophage Bxb1 genome into its host chromosome is catalyzed by a serine-integrase, a member of the transposon-resolvase family of site-specific recombinases. These enzymes use a concerted mechanism of strand exchange involving double-stranded cleavages with two-base extensions, and covalent protein-DNA linkages via phosphoserine bonds. In contrast to the resolvase/invertase recombination systems--where there are strict requirements for a specific synaptic complex within which the catalytic potential of the enzyme is activated--synapsis of attP and attB by Bxb1 integrase is completely promiscuous, aligning the sites with equal proclivity in parallel and antiparallel alignments. Moreover, the catalytic potential of Bxb1 integrase is fully active in either alignment. As a consequence, the nonpalindromic central dinucleotide (5'-GT) at the center of attP and attB is the sole determinant of Bxb1 prophage orientation, and a single base pair substitution in the two sites is sufficient to eliminate orientation control.
Banaiee, N., M. Bobadilla-del-Valle, P.F. Riska, S. .J.r. Bardarov, P.M. Small, A. Ponce-de-Leon, W.R. .J.r. Jacobs, G.F. Hatfull, and J. Sifuentes-Osornio (2003) Rapid identification and susceptibility testing of Mycobacterium tuberculosis from MGIT cultures with luciferase reporter mycobacteriophages. J. Med. Microbiol. 52:557-561 In a prospective study conducted in a diagnostic laboratory in Mexico City, luciferase reporter mycobacteriophages (LRPs) were evaluated for their utility and performance in identification and antibiotic-susceptibility testing of Mycobacterium tuberculosis complex (MTC) isolates from MGIT-960 cultures. Eighty-four consecutive MGIT cultures recovered from 54 patients were included in this study. The LRPs confirmed mycobacterial growth in 79 (94 %) of 84 MGIT cultures. Failure to confirm growth was due to low inoculum (n = 1) or growth with non-tuberculous mycobacteria (n = 4). The median time to confirmation of MGIT cultures was 1 day (range 1-55). Confirmed cultures were identified with p-nitro-alpha-acetylamino-beta-hydroxypropiophenone (NAP), a selective inhibitor of MTC species, and results obtained with LRPs were compared with those obtained by BACTEC-460. The sensitivity and specificity of the LRP NAP test were respectively 97 and 100 %, and the median turnaround time for identification was 3 days with both methods. The accuracy and speed of the LRPs for susceptibility testing with rifampicin, streptomycin, isoniazid and ethambutol were compared with BACTEC-460 and discrepant results were tested by the conventional agar proportion method. In total, 72 MTC cultures were tested. The overall agreement between the LRPs and BACTEC-460 was 98.6 %. Four isolates (5.6 %) were falsely identified as ethambutol-resistant. The median turnaround time for susceptibility testing was 3 days (range 3-57) with the LRPs and 9 days (range 7-29) with BACTEC-460. LRPs offer an accurate and rapid approach for identification and susceptibility testing of M. tuberculosis from MGIT-960 cultures.
Hendrix, R.W., G.F. Hatfull, and M.C. Smith (2003) Bacteriophages with tails: chasing their origins and evolution. Res. Microbiol. 154:253-257 Comparative genomic analysis of the tailed bacteriophages shows that they are genetically mosaic with respect to each other, implying that horizontal exchange of sequences is an important component of their evolution. Horizontal exchange occurs intensively among closely related phages but also at reduced frequency across the entire population of tailed phages. It results in exchange of homologous functions, exchange of analogous but non-homologous functions as with the prophage integrases, and introduction of novel functions into the genome as with the morons. Extrapolation of these processes back in evolutionary time leads to a speculative model for the origins and early evolution of phages.
Lewis, J.A., and G.F. Hatfull (2003) Control of directionality in L5 integrase-mediated site-specific recombination. J. Mol. Biol. 326:805-821 Mycobacteriophage L5 is a temperate phage that forms lysogens in Mycobacterium smegmatis. These lysogens carry an integrated L5 prophage inserted at a specific chromosomal location and undergo subsequent excision during induction of lytic growth. Both the integrative and excisive site-specific recombination events are catalyzed by the phage-encoded tyrosine integrase (Int-L5) and require the host-encoded protein, mIHF. The directionality of these recombination events is determined by a second phage-encoded protein, Excise, the product of gene 36 (Xis-L5); integration occurs efficiently in the absence of Xis-L5 while excision is dependent upon it. We show here that Xis-L5 binds to attR DNA, introduces a DNA bend, and facilitates the formation of an intasome-R complex. This complex, which requires mIHF, Xis-L5 and Int-L5, readily recombines with a second intasome formed by Int-L5, mIHF and attL DNA (intasome-L) to generate the attP and attB products of excision. Xis-L5 also strongly inhibits Int-L5-mediated integrative recombination but does not prevent either the protein-DNA interactions that form the attP intasome (intasome-P) or the capture of attB, but acts later in the reaction presumably by preventing the formation of a recombinagenic synaptic intermediate. The mechanism of action of Xis-L5 appears to be purely architectural, influencing the assembly of protein-DNA structures solely through its DNA-binding and DNA-bending properties.
Pedulla, M.L., M.E. Ford, T. Karthikeyan, J.M. Houtz, R.W. Hendrix, G.F. Hatfull, A.R. Poteete, E.B. Gilcrease, D.A. Winn-Stapley, and S.R. Casjens (2003) Corrected sequence of the bacteriophage P22 genome. J. Bacteriol. 185:1475-1477 We report the first accurate genome sequence for bacteriophage P22, correcting a 0.14% error rate in previously determined sequences. DNA sequencing technology is now good enough that genomes of important model systems like P22 can be sequenced with essentially 100% accuracy with minimal investment of time and resources.
Lewis, J.A., and G.F. Hatfull (2003) Control of directionality in L5 integrase-mediated site-specific recombination. J. Mol. Biol. 326:805-821 Mycobacteriophage L5 is a temperate phage that forms lysogens in Mycobacterium smegmatis. These lysogens carry an integrated L5 prophage inserted at a specific chromosomal location and undergo subsequent excision during induction of lytic growth. Both the integrative and excisive site-specific recombination events are catalyzed by the phage-encoded tyrosine integrase (Int-L5) and require the host-encoded protein, mIHF. The directionality of these recombination events is determined by a second phage-encoded protein, Excise, the product of gene 36 (Xis-L5); integration occurs efficiently in the absence of Xis-L5 while excision is dependent upon it. We show here that Xis-L5 binds to attR DNA, introduces a DNA bend, and facilitates the formation of an intasome-R complex. This complex, which requires mIHF, Xis-L5 and Int-L5, readily recombines with a second intasome formed by Int-L5, mIHF and attL DNA (intasome-L) to generate the attP and attB products of excision. Xis-L5 also strongly inhibits Int-L5-mediated integrative recombination but does not prevent either the protein-DNA interactions that form the attP intasome (intasome-P) or the capture of attB, but acts later in the reaction presumably by preventing the formation of a recombinagenic synaptic intermediate. The mechanism of action of Xis-L5 appears to be purely architectural, influencing the assembly of protein-DNA structures solely through its DNA-binding and DNA-bending properties.
Pedulla, M.L., M.E. Ford, J.M. Houtz, T. Karthikeyan, C. Wadsworth, J.A. Lewis, D. Jacobs-Sera, J. Falbo, J. Gross, N.R. Pannunzio, W. Brucker, V. Kumar, J. Kandasamy, L. Keenan, S. Bardarov, J. Kriakov, J.G. Lawrence, W.R. Jacobs, R.W. Hendrix, and G.F. Hatfull (2003) Origins of highly mosaic mycobacteriophage genomes. Cell 113:171-182 Bacteriophages are the most abundant organisms in the biosphere and play major roles in the ecological balance of microbial life. The genomic sequences of ten newly isolated mycobacteriophages suggest that the bacteriophage population as a whole is amazingly diverse and may represent the largest unexplored reservoir of sequence information in the biosphere. Genomic comparison of these mycobacteriophages contributes to our understanding of the mechanisms of viral evolution and provides compelling evidence for the role of illegitimate recombination in horizontal genetic exchange. The promiscuity of these recombination events results in the inclusion of many unexpected genes including those implicated in mycobacterial latency, the cellular and immune responses to mycobacterial infections, and autoimmune diseases such as human lupus. While the role of phages as vehicles of toxin genes is well established, these observations suggest a much broader involvement of phages in bacterial virulence and the host response to bacterial infections.
Bardarov, S., S. Bardarov, Jr., M.S. Pavelka, Jr., V. Sambandamurthy, M. Larsen, J. Tufariello, J. Chan, G. Hatfull, and W.R. Jacobs, Jr. (2002) Specialized transduction: an efficient method for generating marked and unmarked targeted gene disruptions in Mycobacterium tuberculosis, M. bovis BCG and M. smegmatis. Microbiology 148:3007-3017 The authors have developed a simple and highly efficient system for generating allelic exchanges in both fast- and slow-growing mycobacteria. In this procedure a gene of interest, disrupted by a selectable marker, is cloned into a conditionally replicating (temperature-sensitive) shuttle phasmid to generate a specialized transducing mycobacteriophage. The temperature-sensitive mutations in the mycobacteriophage genome permit replication at the permissive temperature of 30 degrees C but prevent replication at the non-permissive temperature of 37 degrees C. Transduction at a non-permissive temperature results in highly efficient delivery of the recombination substrate to virtually all cells in the recipient population. The deletion mutations in the targeted genes are marked with antibiotic-resistance genes that are flanked by gammadelta-res (resolvase recognition target) sites. The transductants which have undergone a homologous recombination event can be conveniently selected on antibiotic-containing media. To demonstrate the utility of this genetic system seven different targeted gene disruptions were generated in three substrains of Mycobacterium bovis BCG, three strains of Mycobacterium tuberculosis, and Mycobacterium smegmatis. Mutants in the lysA, nadBC, panC, panCD, leuCD, Rv3291c and Rv0867c genes or operons were isolated as antibiotic-resistant (and in some cases auxotrophic) transductants. Using a plasmid encoding the gammadelta-resolvase (tnpR), the resistance genes could be removed, generating unmarked deletion mutations. It is concluded from the high frequency of allelic exchange events observed in this study that specialized transduction is a very efficient technique for genetic manipulation of mycobacteria and is a method of choice for constructing isogenic strains of M. tuberculosis, BCG or M. smegmatis which differ by defined mutations.
Bibb, L.A., and G.F. Hatfull (2002) Integration and excision of the Mycobacterium tuberculosis prophage-like element, phiRv1. Mol. Microbiol. 45:1515-1526 The genomes of Mycobacterium tuberculosis H37Rv and CDC1551 each contain two prophage-like elements, phiRv1 and phiRv2. The phiRv1 element is not only absent from Mycobacterium bovis BCG but is in different locations within the two sequenced M. tuberculosis genomes; in both cases phiRv1 is inserted into a REP13E12 repeated sequence, which presumably contains the bacterial attachment site, attB, for phiRv1. Although phiRv1 is probably too small to encode infectious phage particles, it may nevertheless have an active integration/excision system and be capable of moving from one chromosomal position to another. We show here that the M. tuberculosis H37Rv phiRv1 element does indeed encode an active site-specific recombination system in which an integrase of the serine recombinase family (Rv1586c) catalyses integration and excision and a small, basic phiRv1-encoded protein (Rv1584c) controls the directionality of re-combination. Integration-proficient plasmid vectors derived from phiRv1 efficiently transform BCG, can utilize four of the seven REP13E12 sites present in BCG as attachment sites, and can occupy more than one site simultaneously.
Hatfull, G.F. (2002) Microbiology. A tail of two specifi-cities. Science 295:2031-2032
Morgan, G.J., G.F. Hatfull, S. Casjens, and R.W. Hendrix (2002) Bacteriophage Mu genome sequence: analysis and comparison with Mu-like prophages in Haemophilus, Neisseria and Deinococcus. J. Mol. Biol. 317:337-359 We report the complete 36,717 bp genome sequence of bacteriophage Mu and provide an analysis of the sequence, both with regard to the new genes and other genetic features revealed by the sequence itself and by a comparison to eight complete or nearly complete Mu-like prophage genomes found in the genomes of a diverse group of bacteria. The comparative studies confirm that members of the Mu-related family of phage genomes are genetically mosaic with respect to each other, as seen in other groups of phages such as the phage lambda-related group of phages of enteric hosts and the phage L5-related group of mycobacteriophages. Mu also possesses segments of similarity, typically gene-sized, to genomes of otherwise non-Mu-like phages. The comparisons show that some well-known features of the Mu genome, including the invertible segment encoding tail fiber sequences, are not present in most members of the Mu genome sequence family examined here, suggesting that their presence may be relatively volatile over evolutionary time.The head and tail-encoding structural genes of Mu have only very weak similarity to the corresponding genes of other well-studied phage types. However, these weak similarities, and in some cases biochemical data, can be used to establish tentative functional assignments for 12 of the head and tail genes. These assignments are strongly supported by the fact that the order of gene functions assigned in this way conforms to the strongly conserved order of head and tail genes established in a wide variety of other phages. We show that the Mu head assembly scaffolding protein is encoded by a gene nested in-frame within the C-terminal half of another gene that encodes the putative head maturation protease. This is reminiscent of the arrangement established for phage lambda.
Lawrence, J.G., G.F. Hatfull, and R.W. Hendrix (2002) Imbroglios of viral taxonomy: Genetic exchange and the failings of phenetic approaches. J. Bacteriol. 184:4891-4905 The practice of classifying organisms into hierarchical groups originated with Aristotle and was codified into nearly immutable biological law by Linneaus. The heart of taxonomy is the biological species, which forms the foundation for higher levels of classification. While long established among sexual eukaryotes, achieving a meaningful species concept for prokaryotes has been an onerous task, and has proven exceedingly difficult for describing viruses and bacteriophages. Moreover, the assembly of viral "species" into higher-order taxonomic groupings has been even more tenuous, based initially on limited numbers of morphological features and more recently on overall genomic similarities. The wealth of nucleotide sequence information that catalyzed a revolution in the taxonomy of free-living organisms obligates a reëvaluation of the concept of viral species, genera, families and higher levels of classification. Just as microbiologists discarded dubious morphological traits in favor of more accurate molecular yardsticks of evolutionary change, virologists can gain new insight into viral evolution through the rigorous analyses afforded by the molecular phylogenetics of viral genes. For bacteriophages, such dissections of genomic sequences reveal fundamental flaws in the Linnean paradigm that necessitate a new view of viral evolution, classification and taxonomy.
Lewis, J.A., and G.F. Hatfull (2001) Control of directionality in integrase-mediated recombination: examination of recombination directionality factors (RDFs) including Xis and Cox proteins. Nucleic Acids Res. 29:2205-2216 Similarity between the DNA substrates and products of integrase-mediated site-specific recombination reactions results in a single recombinase enzyme being able to catalyze both the integration and excision reactions. The control of directionality in these reactions is achieved through a class of small accessory factors that favor one reaction while interfering with the other. These proteins, which we will refer to collectively as recombination directionality factors (RDFs), play architectural roles in reactions catalyzed by their cognate recombinases and have been identified in conjunction with both tyrosine and serine integrases. Previously identified RDFs are typically small, basic and have diverse amino acid sequences. A subset of RDFs, the cox genes, also function as transcriptional regulators. We present here a compilation of all the known RDF proteins as well as those identified through database mining that we predict to be involved in conferring recombination directionality. Analysis of this group of proteins shows that they can be grouped into distinct sub-groups based on their sequence similarities and that they are likely to have arisen from several independent evolutionary lineages. This compilation will prove useful in recognizing new proteins that confer directionality upon site-specific recombination reactions encoded by plasmids, transposons, phages and prophages.
Hatfull, G.F. (2001) Microbiology. The great escape. Science 292:2263-2264 Banaiee, N., M. Bobadilla-Del-Valle, S. .J.r. Bardarov, P.F. Riska, P.M. Small, A. Ponce-De-Leon, W.R. .J.r. Jacobs, G.F. Hatfull, and J. Sifuentes-Osornio (2001) Luciferase reporter mycobacteriophages for detection, identification, and antibiotic susceptibility testing of Mycobacterium tuberculosis in Mexico. J. Clin. Microbiol. 39:3883-3888 The utility of luciferase reporter mycobacteriophages (LRPs) for detection, identification, and antibiotic susceptibility testing of Mycobacterium tuberculosis was prospectively evaluated in a clinical microbiology laboratory in Mexico City, Mexico. Five hundred twenty-three consecutive sputum samples submitted to the laboratory during a 5-month period were included in this study. These specimens were cultivated in Middlebrook 7H9 (MADC), MGIT, and Lowenstein-Jensen (LJ) media. Of the 71 mycobacterial isolates recovered with any of the three media, 76% were detected with the LRPs, 97% were detected with the MGIT 960 method, and 90% were detected with LJ medium. When contaminated specimens were excluded from the analysis, the LRPs detected 92% (54 of 59) of the cultures. The median time to detection of bacteria was 7 days with both the LRPs and the MGIT 960 method. LRP detection of growth in the presence of p-nitro-alpha-acetylamino-beta-hydroxypropiophenone (NAP) was used for selective identification of M. tuberculosis complex (MTC) and compared to identification with BACTEC 460. Using the LRP NAP test, 47 (94%) out of 50 isolates were correctly identified as tuberculosis complex. The accuracy and speed of LRP antibiotic susceptibility testing with rifampin, streptomycin, isoniazid, and ethambutol were compared to those of the BACTEC 460 method, and discrepant results were checked by the conventional proportion method. In total, 50 MTC isolates were tested. The overall agreement between the LRP and BACTEC 460 results was 98.5%. The median LRP-based susceptibility turnaround time was 2 days (range, 2 to 4 days) compared to 10.5 days (range, 7 to 16 days) by the BACTEC 460 method. Phage resistance was not detected in any of the 243 MTC isolates tested. Mycobacteriophage-based approaches to tuberculosis diagnostics can be implemented in clinical laboratories with sensitivity, specificity, and rapidity that compare favorably with those of the MGIT 960 and BACTEC 460 methods. The phages currently provide the fastest phenotypic assay for susceptibility testing.
Jain, S., and G.F. Hatfull (2000) Transcriptional regulation and immunity in Mycobacteriophage Bxb1. Mol. Microbiol. 38:971-985 Mycobacteriophage Bxb1 is a temperate phage of Mycobacterium smegmatis that shares a similar genome organization to mycobacteriophage L5, although the two phages are heteroimmune. We have investigated the regulatory circuitry of Bxb1 and found that it encodes a repressor, gp69, which regulates at least two promoters, an early lytic promoter, Pleft, and the divergent promoter, Pright. Bxb1 gp69 is 41% identical to the L5 repressor (gp71) and binds to repressor binding sites that conform to a similar, but distinct, 13 bp asymmetric consensus sequence to that for the L5 gp71 binding sites. The two phage repressors have a strong preference for their cognate binding sites, thus accounting for their immunity phenotypes. The Bxb1 genome contains 34 putative repressor binding sites located throughout the genome, but situated within short intergenic spaces and orientated in only one direction relative to the direction of transcription. Comparison with the locations of repressor binding sites within the L5 genome provides insights into how these unusual regulatory systems evolve.
Mediavilla, J., S. Jain, J. Kriakov, M.E. Ford, R.L. Duda, W.R. Jacobs Jr., R.W. Hendrix, and G.F. Hatfull (2000) Genome organization and characterization of mycobacteriophage Bxb1. Mol. Microbiol. 38:955-970 Mycobacteriophage Bxb1 is a temperate phage of Mycobacterium smegmatis. The morphology of Bxb1 particles is similar to that of mycobacteriophages L5 and D29, although Bxb1 differs from these phages in other respects. First, it is heteroimmune with L5 and efficiently forms plaques on an L5 lysogen. Secondly, it has a different host range and fails to infect slow-growing mycobacteria, using a receptor system that is apparently different from that of L5 and D29. Thirdly, it is the first mycobacteriophage to be described that forms a large prominent halo around plaques on a lawn of M. smegmatis. The sequence of the Bxb1 genome shows that it possesses a similar overall organization to the genomes of L5 and D29 and shares weak but detectable DNA sequence similarity to these phages within the structural genes. However, Bxb1 uses a different system of integration and excision, a repressor with different specificity to that of L5 and encodes a large number of novel gene products including several with enzymatic functions that could degrade or modify the mycobacterial cell wall.
Belanger, A.E., J.C. Porter, and G.F. Hatfull (2000) Genetic analysis of peptidoglycan biosynthesis in mycobacteria: characterization of a ddlA mutant of Mycobacterium smegmatis. J. Bacteriol. 182:6854-6856 A temperature-sensitive mutant of Mycobacterium smegmatis was characterized that contains a mutation in ddlA, the gene encoding D-alanine:D-alanine ligase. Enzymatic assays using recombinant proteins and D-cycloserine susceptibility indicate that the A365V mutation in the SMEG35 DdlA protein causes a reduction in enzymatic activity in vitro and in vivo.
Hendrix, R.W., J.G. Lawrence, G.F. Hatfull, and S. Casjens (2000) The origins and ongoing evolution of viruses. Trends Microbiol. 8:504-508 Genome analyses of dsDNA tailed bacteriophages argue that they evolve by recombinational reassortment of genes, and by the acquisition of novel genes as simple genetic elements termed morons. These processes suggest a model for early virus evolution, wherein viruses can be regarded less as having derived from cells and more as being partners in their mutual co-evolution.
Peña, C.E.A., J.M. Kahlenberg, and G.F. Hatfull (2000) Assembly and activation of site-specific recombination complexes. Proc. Natl. Acad. Sci., USA 97:7760-7776 Site-specific recombination is responsible for a broad range of biological phenomena, including DNA inversion, resolution of transposition intermediates, and the integration and excision of bacteriophage genomes. Integration of mycobacteriophage L5 is catalyzed by a phage-encoded integrase with recombination occurring between specific attachment sites on the phage and mycobacterial chromosomes (attP and attB, respectively). Although some site-specific recombination systems simply involve binding of the recombinase to the sites of strand exchange, synapsis, and recombination, phage systems typically require the assembly of higher-order structures within which the recombinational potential of integrase is activated. The requirement for these structures derives from the necessity to regulate the directionality of recombinationeither integration or excisionwhich must be closely coordinated with other aspects of the phage growth cycles. We show herein that there are multiple pathways available for the assembly of L5 recombination complexes, including the early synapsis of the attP and attB DNAs. This process is in contrast to the model for lambda integration and illustrates the different usage of molecular machineries to accomplish the same biological outcome.
Peteroy, M., A. Severin, F. Zhao, D. Rosner, U. Lopatin, H. Scherman, A. Belanger, B. Harvey, G.F. Hatfull, P.J. Brennan, and N.D. Connell (2000) Characterization of a Mycobacterium smegmatis mutant that is simultaneously resistant to D-cycloserine and vancomycin. Antimicrob. Agents Ch. 44:1701-1704 A mutant of Mycobacterium smegmatis has been isolated that is simultaneously resistant to both D-cycloserine (D-CS) and vancomycin. Genetic complementation with a PBP4 homolog restores sensitivity to both drugs. Resistance to D-CS and vancomycin in this mutant is most likely due to a novel mechanism involving peptidoglycan assembly at the cell surface.
Hatfull, G.F. (2000) Molecular biology of mycobacteriophages. Pp 37-54 in Molecular Genetics of Mycobacteria, Hatfull, G.F., and W.R. Jacobs, Jr., Ed. ASM Press, Washington DC Ravin, V., N. Ravin, S. Casjens, M.E. Ford, G.F. Hatfull, and R.W. Hendrix (2000) Genomic sequence and analysis of the atypical temperate bacteriophage N15. J. Mol. Biol. 299:53-73 N15 is a temperate bacteriophage that forms stable lysogens in Escherichia coli. While its virion is morphologically very similar to phage lambda and its close relatives, it is unusual in that the prophage form replicates autonomously as a linear DNA molecule with closed hairpin telomeres. We describe here the genomic architecture of N15, and its global pattern of gene expression, which reveal that N15 contains several plasmid-derived genes that are expressed in N15 lysogens. The tel site, at which processing occurs to form the prophage ends is close to the center of the genome in a similar location to that occupied by the attachment site, attP, in lambda and its relatives and defines the boundary between the left and right arms. The left arm contains a long cluster of structural genes that are closely-related to those of the lambda-like phages, but also includes homologs of umuD', which encodes a DNA polymerase accessory protein, and the plasmid partition genes, sopA and sopB. The right arm likewise contains a mixture of apparently phage- and plasmid-derived genes including genes encoding plasmid replication functions, a phage repressor, a transcription antitermination system, as well as phage host cell lysis genes and two putative DNA methylases. The unique structure of the N15 genome suggests that the large global population of bacteriophages may exhibit a much greater diversity of genomic architectures than was previously recognized.
Juhala, R.J., M.E. Ford, R.L. Duda, A. Youlton, G.F. Hatfull, and R.W. Hendrix (2000) Genomic sequences of bacteriophages HK97 and HK022: pervasive genetic mosaicism in the lambdoid bacteriophages. J. Mol. Biol. 299:27-52
We report the complete genome DNA sequences of HK97 (39,732 bp) and HK022 (40,751 bp), dsDNA bacteriophages of Escherichia coli and members of the lambdoid or l-like group of phages. We provide a comparative analysis of these sequences with each other and with two previously determined lambdoid family genome sequences, those of E. coli phage lambda and Salmonella typhimurium phage P22. The comparisons confirm that these phages are genetic mosaics, with mosaic segments separated by sharp transitions in the sequence. The mosaicism provides clear evidence that horizontal exchange of genetic material is a major component of evolution for these viruses. The data suggest a model for evolution in which diversity is generated by a combination of illegitimate and homologous recombination and mutational drift, and selection for function produces a population in which most of the surviving mosaic boundaries are located at gene boundaries or in some cases at protein domain boundaries within genes. Comparisons of these genomes highlight a number of differences that allow plausible inferences of specific evolutionary scenarios for some parts of the genome.
Lewis, J.A., and G.F. Hatfull (2000) Identification and characterization of mycobacteriophage L5 excisionase. Mol. Microbiol. 35:350-360 The well-characterized mycobacteriophage L5 forms stable lysogens in Mycobacterium smegmatis. Establishment of lysogeny involves integration of the phage genome into the chromosome of its mycobacterial hosts through an integrase-mediated site-specific recombination event. As L5 lysogens spontaneously generate free phage particles, prophage excision must also occur, although an L5 excisionase gene had not been identified. We show here that L5 gene 36 encodes the phage excisionase and is a small, heat-stable 56-amino-acid protein that strongly stimulates excisive recombination both in vivo and in vitro. The ability to manipulate the highly directional phage integration and excision reactions will provide powerful tools for the introduction, curing and recovery of foreign genes in recombinant mycobacterial strains.
Belanger, A.E., and G.F. Hatfull (1999) Exponential-phase glycogen recycling is essential for growth of Mycobacterium smegmatis. J. Bacteriol. 181:6670-6678 Bacterial glycogen is a polyglucose storage compound that is thought to prolong viability during stationary phase. However, a specific role for glycogen has not been determined. We have characterized SMEG53, a temperature-sensitive mutant of Mycobacterium smegmatis that contains a mutation in glgE, encoding a putative glucanase. This mutation causes exponentially growing SMEG53 cells to stop growing at 42 degrees C in response to high levels of glycogen accumulation. The mutation in glgE is also associated with an altered growth rate and colony morphology at permissive temperatures; the severity of these phenotypes correlates with the amount of glycogen accumulated by the mutant. Suppression of the temperature-sensitive phenotype, via a decrease in glycogen accumulation, is mediated by growth in certain media or multicopy expression of garA. The function of GarA is unknown, but the presence of a forkhead-associated domain suggests that this protein is a member of a serine-threonine kinase signal transduction pathway. Our results suggest that in M. smegmatis glycogen is continuously synthesized and then degraded by GlgE throughout exponential growth. In turn, this constant recycling of glycogen controls the downstream availability of carbon and energy. Thus, in addition to its conventional storage role, glycogen may also serve as a carbon capacitor for glycolysis during the exponential growth of M. smegmatis.
Riska, P.F., Y. Su, S. Bardarov, L. Freundlich, G. Sarkis, G. Hatfull, C. Carriere, V. Kumar, J. Chan, and W.R. Jacobs, Jr. (1999) Rapid film-based determination of Mycobacterium tuberculosis antibiotic susceptibilities using a luciferase reporter phage and the bronx box. J. Clin. Microbiol. 37:1144-1149 Detecting antibiotic resistance in Mycobacterium tuberculosis is becoming increasingly important with the global recognition of drug-resistant strains and their adverse impact on clinical outcomes. Current methods of susceptibility testing are either time-consuming or costly; rapid, reliable, simple, and inexpensive methods would be highly desirable, especially in the developing world where most tuberculosis is found. The luciferase reporter phage is a unique reagent well-suited for this purpose: upon infection with viable mycobacteria, it produces quantifiable light which is not observed in mycobacterial cells treated with active antimicrobials. In this report, we describe a modification of our original assay, which allows detection of the emitted light with a Polaroid film box designated the Bronx Box. The technique has been applied to 25 M. tuberculosis reference and clinical strains, and criteria are presented which allow rapid and simple discrimination among strains susceptible or resistant to isoniazid and rifampin, the major antituberculosis agents.
Hendrix, R.W., M.C.M. Smith, R.N. Burns, M.E. Ford, and G.F. Hatfull (1999) Evolutionary relationships among diverse bacteriophages and prophages: All the World's a Phage. Proc. Natl. Acad. Sci., USA 96:2192-2197 We report DNA and predicted protein sequence similarities, implying homology, among genes of double-stranded DNA (dsDNA) bacteriophages and prophages spanning a broad phylogenetic range of host bacteria. The sequence matches reported here establish genetic connections, not always direct, among the lambdoid phages of Escherichia coli, phage phiC31 of Streptomyces, phages of Mycobacterium, a previously unrecognized cryptic prophage, phiflu, in the Haemophilus influenzae genome, and two small prophage-like elements, phiRv1 and phiRv2, in the genome of Mycobacterium tuberculosis. The results imply that these phage genes, and very possibly all of the dsDNA tailed phages, share common ancestry. We propose a model for the genetic structure and dynamics of the global phage population in which all dsDNA phage genomes are mosaics with access, by horizontal exchange, to a large common genetic pool but in which access to the gene pool is not uniform for all phage.
Peña, C.E., J.M. Kahlenberg, and G.F. Hatfull (1999) Protein-DNA complexes in L5 integrative recombination. J. Bacteriol. 181:454-461 The temperate mycobacteriophage L5 integrates site specifically into the genomes of Mycobacterium smegmatis, Mycobacterium tuberculosis, and Mycobacterium bovis bacillus Calmette-Guerin. This integrative recombination event occurs between the phage L5 attP site and the mycobacterial attB site and requires the phage-encoded integrase and mycobacterial-encoded integration host factor mIHF. Here we show that attP, Int-L5, and mIHF assemble into a recombinationally active complex, the intasome, which is capable of attB capture and formation of products. The arm-type integrase binding sites within attP play specialized roles in the formation of specific protein-DNA architectures; the intasome is constructed by the formation of intramolecular integrase bridges between one pair of sites, P4-P5, and the attP core, while an additional pair of sites, P1-P2, is required for interaction with attB.
Peña, C.E., J.M. Kahlenberg, and G.F. Hatfull (1998) The role of supercoiling in mycobacteriophage L5 integrative recombination. Nucleic Acids Res. 26:4012-4018 The genome of temperate mycobacteriophage L5 integrates into the chromosomes of its hosts, including Mycobacterium smegmatis, Mycobacterium tuberculosis and bacille Calmette-Guerin. This integrase-mediated site-specific recombination reaction occurs between the phage attP site and the mycobacterial attB site and requires the mycobacterial integration host factor. Here we examine the role of supercoiling in this reaction and show that integration is stimulated by DNA supercoiling but that supercoiling of either the attP or the attB substrate enhances recombination. Supercoiling thus facilitates a post-synaptic recombination event. We also show that, while supercoiling is not required for the production of a recombinagenic intasome, a mutant attP DNA deficient in binding of the host factor acquires a dependence on supercoiling for intasome formation and recombination.
Pedulla, M.L., and G.F. Hatfull (1998) Characterization of the mIHF gene of Mycobacterium smegmatis. J. Bacteriol. 180:5473-5477 Integration of mycobacteriophage L5 requires the mycobacterial integration host factor (mIHF) in vitro. mIHF is a 105-residue heat-stable polypeptide that is not obviously related to HU or any other small DNA-binding proteins. mIHF is most abundant just prior to entry into stationary phase and is essential for the viability of Mycobacterium smegmatis.
Peña, C.E., J. Stoner, and G.F. Hatfull (1998) Mycobacteriophage D29 integrase-mediated recombination in vitro. Gene 225:143-151 Mycobacteriophage D29 is a lytic phage that infects both fast- and slow-growing species of the mycobacteria. D29 forms clear plaques on lawns of Mycobacterium smegmatis and Mycobacterium bovis bacille Calmette-Guerin (BCG) in which a very high proportion of infected cells are killed. However, genomic analysis of D29 demonstrates that it is a close relative of the temperate mycobacteriophage L5, and is presumably a non-temperate derivative of a temperate parent. The D29 genome encodes a putative integrase protein with a primary amino acid sequence similar to that of the L5 integrase; the corresponding int genes fall in colinear positions within the D29 and L5 genomes, immediately flanking and transcribed away from their associated attP sites. We show here that the D29 integrase is functional and catalyzes integrative recombination between the D29 attP site and the M. smegmatis attB site in vitro in an mIHF-dependent manner. D29 integrase also mediates recombination between the L5 attP site and attB DNA and, reciprocally, L5 integrase catalyzes recombination with D29 attP DNA. However, in both in-vitro and in-vivo assays, the D29-encoded integrase recombines the D29 attP more efficiently than the L5 attP, and vice versa, suggesting that each integration system has evolved a degree of specificity of attP recognition. We also present the sequences of the putative attP site and integrase protein of the cryptic prophage-like element phiRv2, and compare them to those of mycobacteriophages L5 and D29.
Ford, M.E., C. Stenstrom, R.W. Hendrix, and G.F. Hatfull (1998) Mycobacteriophage TM4: genome structure and gene expression. Tuber. Lung Dis. 79:63-73 Mycobacteriophage TM4 is a dsDNA-tailed phage that infects both fast-growing and slow-growing strains of mycobacteria. While TM4 has been used extensively for the construction of mycobacterial shuttle phasmids and for the delivery of reporter genes and transposons into mycobacterial cells, little is known about its genetics or molecular biology. We describe here the complete 52,797 bp genome sequence of TM4 and a map of its genome organization. While not a close relative of other mycobacteriophages, TM4 encodes several proteins with sequence similarity to those of other bacteriophages--including L5 and D29--indicating that they have common ancestry. In addition, TM4 encodes proteins with similarity to haloperoxidases, glutaredoxins and the WhiB family of transcriptional regulators. Following infection, TM4 genes are expressed in a defined temporal pattern, with the virion structural proteins expressed late in the phage growth cycle. Understanding the genetics of TM4 will greatly facilitate its use as a tool for the genetic manipulation of the mycobacteria. Ford, M., G.J. Sarkis, A.E. Belanger, R.W. Hendrix, and G.F. Hatfull (1998) Genome structure of mycobacteriophage D29: implications for phage evolution. J. Mol. Biol. 279:143-164 Mycobacteriophage D29 is a lytic phage that infects both fast and slow-growing mycobacterial species. The complete genome sequence of D29 reveals that it is a close relative of the temperate mycobacteriophage L5, whose sequence has been described previously. The overall organization of the D29 genome is similar to that of L5, although a 3.6 kb deletion removing the repressor gene accounts for the inability of D29 to form lysogens. Comparison of the two genomes shows that they are punctuated by a large number of insertions, deletions, and substitutions of genes, consistent with the genetic mosaicism of lambdoid phages.
Klann, A.G., A.E. Belanger, A. Abanes-De Mello, J.Y. Lee, and G.F. Hatfull (1998) Characterization of the dnaG locus in Mycobacterium smegmatis reveals linkage of DNA replication and cell division. J. Bacteriol. 180:65-72 We have isolated a UV-induced temperature-sensitive mutant of Mycobacterium smegmatis that fails to grow at 42 degrees C and exhibits a filamentous phenotype following incubation at the nonpermissive temperature, reminiscent of a defect in cell division. Complementation of this mutant with an M. smegmatis genomic library and subsequent subcloning reveal that the defect lies within the M. smegmatis dnaG gene encoding DNA primase. Sequence analysis of the mutant dnaG allele reveals a substitution of proline for alanine at position 496. Thus, dnaG is an essential gene in M. smegmatis, and DNA replication and cell division are coupled processes in this species. Characterization of the sequences flanking the M. smegmatis dnaG gene shows that it is not part of the highly conserved macromolecular synthesis operon present in other eubacterial species but is part of an operon with a dgt gene encoding dGTPase. The organization of this operon is conserved in Mycobacterium tuberculosis and Mycobacterium leprae, suggesting that regulation of DNA replication, transcription, and translation may be coordinated differently in the mycobacteria than in other bacteria.
Barsom, E.K., and G.F. Hatfull (1997) A putative ABC-transport operon of Mycobacterium smegmatis. Gene 185:127-132 We have recently described the mpr gene of Mycobacterium smegmatis whose product confers resistance to mycobacteriophages L5 and D29 when overproduced (Barsom and Hatfull (1996) Mol. Microbiol. 21, 159-170). We have determined the nt sequence of approximately 3.5 kb immediately adjacent to mpr which appears to encode components of an ATP-binding cassette (ABC) transport system. Four closely-spaced open reading frames (ORF) were identified although two of these may cooperate to produce an integral membrane component of the transport system via a programmed translational frameshift. Another putative protein is also predicted to be an integral membrane protein, while the third is an ABC-transporter protein. We propose that these three putative proteins form a mycobacterial membrane-bound complex involved in protein-dependent transport. This is the first ABC-transport system to be described in mycobacteria.
Fullner, K.J., and G.F. Hatfull (1997) Mycobacteriophage L5 infection of Mycobacterium bovis BCG: implications for phage genetics in the slow-growing mycobacteria. Mol. Microbiol. 26:755-766 Mycobacteriophage L5 is a well-characterized temperate phage that forms stable lysogens in Mycobacterium smegmatis. The host range of L5 is, however, unclear because previous reports suggested that it does not infect slow-growing mycobacteria such as Mycobacterium tuberculosis and bacille Calmette-Guerin (BCG). Moreover, luciferase reporter phage derivatives of L5 failed to produce light from BCG, suggesting that infection is blocked at or before the stage of DNA injection. In this study, we demonstrate that L5 infection of slow growing mycobacteria specifically requires a high concentration of Ca2+, conditions that differs from those required for infection of M. smegmatis by L5 and for infection of BCG by the closely related phage D29. In addition, we show that there are specific genetic determinants of L5 that confer the ability to infect slow growing mycobacteria, without altering infection of M. smegmatis. These observations extend the use of phage L5 for the diagnosis and analysis of tuberculosis and other mycobacterial diseases.
Brown, K.L., G.K. Sarkis, C. Wadsworth, and G.F. Hatfull (1997) Transcriptional silencing by the phage L5 repressor. EMBO J. 16:5914-5921 The success of a temperate bacteriophage is dependent upon its ability to completely shut down expression of its lytic genes during lysogenic growth. Mycobacteriophage L5 accomplishes this by an atypical phage repressor, gp71, which binds to multiple asymmetric DNA sites. L5 gp71 regulates transcription initiation at an early lytic promoter, Pleft, but also affects downstream gene expression at 'stoperator' sites in the phage genome. The L5 genome is replete with stoperator sites located within short intergenic spaces in both the early and late lytic operons and oriented specifically with respect to transcription. Binding of gp71 to these sites results in a strong orientation-dependent polar effect on downstream gene expression and global silencing of prophage gene expression.
Bardarov, S., J. Kriakov, C. Carriere, S. Yu, C. Vaamonde, R.A. McAdam, B.R. Bloom, G.F. Hatfull, and W.R. Jacobs, Jr. (1997) Conditionally replicating mycobacteriophages: a system for transposon delivery to Mycobacterium tuberculosis. Proc. Natl. Acad. Sci., USA 94:10961-10966 Transposon mutagenesis provides a direct selection for mutants and is an extremely powerful technique to analyze genetic functions in a variety of prokaryotes. Transposon mutagenesis of Mycobacterium tuberculosis has been limited in part because of the inefficiency of the delivery systems. This report describes the development of conditionally replicating shuttle phasmids from the mycobacteriophages D29 and TM4 that enable efficient delivery of transposons into both fast- and slow-growing mycobacteria. These shuttle phasmids consist of an Escherichia coli cosmid vector containing either a mini-Tn10(kan) or Tn5367 inserted into a nonessential region of the phage genome. Thermosensitive mutations were created in the mycobacteriophage genome that allow replication at 30 degrees C but not at 37 degrees C (TM4) or 38.5 degrees C (D29). Infection of mycobacteria at the nonpermissive temperature results in highly efficient transposon delivery to the entire population of mycobacterial cells. Transposition of mini-Tn10(kan) occurred in a site-specific fashion in M. smegmatis whereas Tn5367 transposed apparently randomly in M. phlei, Bacille Calmette-Guerin (BCG), and M. tuberculosis. Sequence analysis of the M. tuberculosis and BCG chromosomal regions adjacent to Tn5367 insertions, in combination with M. tuberculosis genomic sequence and physical map data, indicates that the transpositions have occurred randomly in diverse genes in every quadrant of the genome. Using this system, it has been readily possible to generate libraries containing thousands of independent mutants of M. phlei, BCG, and M. tuberculosis.
Peña, C., M.H. Lee, and G.F. Hatfull (1997) Characterization of the mycobacteriophage L5 attachment site, attP. J. Mol. Biol. 266:76-92 Lysogenization of mycobacteriophage L5 involves integration of the phage genome into the Mycobacterium smegmatis chromosome. Integration occurs by a site-specific recombination event between a phage attachment site, attP, and a bacterial attachment site, attB, which is catalyzed by the phage-encoded integrase protein. DNase I footprinting reveals that L5 integrase binds to two types of sites within attP which span an unexpectedly large region of 413 bp: seven arm-type sites (P1 to P7) each of which correspond to a consensus sequence 5'-TGCaaCtcYy, and core-type sites at the points of strand exchange. Mutational analyses indicate that not all of the arm-type sites are required for integration, and that the P3 site and the rightmost pair of sites (P6 and P7) are dispensable for integration. We show that a 252 bp segment of attP DNA is sufficient for efficient integrative recombination and that int can be provided in trans for simple and efficient transformation of the mycobacteria.
Hatfull, G.F. (1996) The molecular genetics of Mycobacterium tuberculosis. Curr. Top. Microbiol. Immunol. 215:29-47 Barsom, E.K., and G.F. Hatfull (1996) Characterization of Mycobacterium smegmatis gene that confers resistance to phages L5 and D29 when overexpressed. Mol. Microbiol. 21:159-170 Bacteriophage infection requires a specific interaction with the outer surface of a bacterial host followed by interaction with the cell membrane and phage DNA injection. Phages of the mycobacteria encounter a cell wall that is rich in unusual lipid- and sugar-containing components which form a formidable barrier that must be passed to gain access to the membrane. We describe here a gene of Mycobacterium smegmatis that confers resistance to mycobacteriophages L5 and D29. The phage-resistance phenotype results not from mutation but from elevated expression of a wild-type gene. It appears that the product of this multicopy phage-resistance (mpr) gene may alter the structure of the host cell wall or membrane, thereby inhibiting productive phage DNA injection. Peña, C.E., J.E. Stoner, and G.F. Hatfull (1996) Positions of strand exchange in mycobacteriophage L5 integration and characterization of the attB site. J. Bacteriol. 178:5533-5536 Mycobacteriophage L5 integrates into the genome of Mycobacterium smegmatis via site-specific recombination between the phage attP site and the bacterial attB site. These two sites have a 43-bp common core sequence within which strand exchange occurs and which overlaps a tRNAGly gene at attB. We show here that a 29-bp segment of DNA is necessary and sufficient for attB function and identify the positions of strand exchange.
Pearson, R.E., S. Jurgensen, G.J. Sarkis, G.F. Hatfull, and W.R. Jacobs, Jr. (1996) Construction of D29 shuttle phasmids and luciferase reporter phages for detection of mycobacteria. Gene 183:129-136 Diseases caused by Mycobacterium tuberculosis, M. leprae and M. avium, cause significant morbidity and mortality worldwide. Effective treatments require that the organisms be speciated and that drug susceptibilities for the causative organisms be characterized. Reporter phage technology has been developed as a rapid and convenient method for identifying mycobacterial species and evaluating drug resistance. In this report we describe the construction of luciferase reporter phages from mycobacteriophage D29 DNA. Shuttle phasmids were first constructed with D29 in order to identify non-essential regions of the D29 genomes and to introduce unique cloning sites within that region. Using this approach, we observed that all of the D29 shuttle phasmids had the cosmid vector localized to one area of the phage genome near one cohesive end. These shuttle phasmids had been constructed with a cosmid that could be readily excised from the D29 genome with different sets of restriction enzymes. Luciferase reporter phages were made by substituting the luciferase cassette for the cosmid vector. Recombinant phages with the luciferase cassette fall into two groups. One group produced light and had the expression cassette oriented with the promoter directing transcription away from the cohesive end. In contrast, the other group had the expression cassette in the opposite orientation and failed to produce light during lytic infection, but did produce light in L5 lysogens which are known to repress D29 promoters. These results suggest that a phage promoter of the D29 phage can occlude the expression of a promoter introduced into this region. D29 luciferase reporter phages are capable of detecting low numbers of L5 lysogens like L5 luciferase phages. However, unlike L5 luciferase phages, D29 luciferase phages can readily infect M. tuberculosis and M. bovis BCG, demonstrating that these phages can be used to evaluate drug susceptibilities of many types of mycobacteria.
Pedulla, M.L., M.H. Lee, D.C. Lever, and G.F. Hatfull (1996) A novel host factor for integration of mycobacteriophage L5. Proc. Natl. Acad. Sci., USA 24:15411-15416 Bacterial integration host factors (IHFs) play central roles in the cellular processes of recombination, DNA replication, transcription, and bacterial pathogenesis. We describe here a novel mycobacterial IHF (mIHF) of Mycobacterium smegmatis and Mycobacterium tuberculosis that stimulates integration of mycobacteriophage L5. mIHF is the product of a single gene and is unrelated at the sequence level to other integration host factors. By itself, mIHF does not bind preferentially to attP DNA, although it significantly alters the pattern of integrase (Int) binding, promoting the formation of specific integrase-mIHF-attP intasome complexes.
Pascopella, L., F.M. Collins, J.M. Martin, M.H. Lee, G.F. Hatfull, C.K. Stover, B.R. Bloom, and W.R. Jacobs, Jr. (1995) Use of in vivo complementation in Mycobacterium tuberculosis to identify a genomic fragment associated with virulence. Infect. Immun. 62:1313-1319 Novel molecular tools and genetic methods were developed to isolate genomic fragments of Mycobacterium tuberculosis that may be associated with virulence. We sought to restore virulence, a characteristic of M. tuberculosis that is correlated with growth rate in mouse spleen and lung tissue, to the avirulent strain H37Ra by complementation. A representative library of the virulent M. tuberculosis strain H37Rv was constructed and transformed into H37Ra. Enrichment for individual faster-growing recombinants was achieved by passage of pools of H37Ra transformants harboring the H37Rv library through mice. A molecular strategy was devised to isolate and clone the H37Rv genomic DNA fragment ivg, which conferred a more rapid in vivo growth rate to H37Ra.
Sarkis, G.J., W.R. Jacobs, Jr., and G.F. Hatfull (1995) L5 luciferase reporter mycobacteriophages: a sensitive tool for the detection and assay of live mycobacteria. Mol. Microbiol. 15:1055-1067 Recombinant bacteriophages provide efficient delivery systems for introducing reporter genes into specific bacterial hosts. We have constructed mycobacteriophage L5 recombinants carrying the firefly luciferase gene inserted into the tRNA region of the phage genome. Infection of Mycobacterium smegmatis by these phages results in expression of the luciferase gene and light emission. Fortuitously, the luciferase gene is expressed continuously in lysogens surviving infection. Synthesis of luciferase from a mycobacterial promoter created by cloning enables the detection of extremely small numbers of M. smegmatis cells. These reporter phages can be used to discriminate between drug-sensitive and drug-resistant strains of M. smegmatis, and may provide tools for the rapid identification and classification of antimycobacterial agents. Nesbit, C.E., M.E. Levin, M.K. Donnelly-Wu, and G.F. Hatfull (1995) Transcriptional regulation of repressor synthesis in mycobacteriophage L5. Mol. Microbiol. 17:1045-1056 Mycobacteriophage L5 is a temperate phage of the mycobacteria that forms stable lysogens in Mycobacterium smegmatis. Lysogeny is maintained by the putative repressor, the gene 71 product, which also mediates immunity to superinfection. We show here that there are three promoters located upstream of gene 71 which are active in an L5 lysogen but which do not require any phage-encoded proteins. In early lytic growth, gene 71 is also transcribed from a promoter, Pleft, located at the right end of the genome and which appears to be a target of gp71 regulation. A model is given for the regulation of L5 life cycles. Hatfull, G.F., L. Barsom, L. Chang, M. Donnelly-Wu, M.H. Lee, M. Levin, C. Nesbit, and G.J. Sarkis (1994) Bacteriophages as tools for vaccine development. Dev. Biol. Stand. 82:43-47 The construction of live recombinant bacterial vaccines requires a reasonably sophisticated genetic system for the introduction, stabilization and expression of foreign antigen genes. Bacteriophages offer a rich collection of tools that can be used for vaccine construction, including site-specific integration-proficient vectors, non-antibiotic selectable markers and signals for efficient transcription and translation of foreign genes. We describe the characterization of a temperate phage of the mycobacteria, mycobacteriophage L5, and application of these phage studies for the construction of recombinant BCG vaccines. Hatfull, G.F., and G.J. Sarkis (1993) DNA sequence, structure and gene expression of mycobacteriophage L5: a phage system for mycobacterial genetics. Mol. Microbiol. 7:395-405 Genetic studies of Mycobacterium tuberculosis and other mycobacterial pathogens have suffered from the lack of a sophisticated genetic system. To address this issue we have developed a viral system through a detailed characterization of mycobacteriophage L5, a temperate phage that infects both fast- and slow-growing mycobacteria. We describe here the complete DNA sequence of the L5 genome and initial characterization of L5 virion structure and gene expression. In addition to providing a genetic 'tool-box' for the mycobacteria we find that L5 offers a new paradigm for dsDNA phages, being phenotypically temperate but employing genetic strategies for phage growth usually associated with lytic bacteriophages. Donnelly-Wu, M.K., W.R. Jacobs, Jr., and G.F. Hatfull (1993) Superinfection immunity of mycobacteriophage L5: applications for genetic transformation of mycobacteria. Mol. Microbiol. 7:407-417 Mycobacteriophage L5 is a temperate phage of the mycobacteria that forms stable lysogens in Mycobacterium smegmatis. We show here that the 183-amino-acid product of L5 gene 71 confers immunity to L5 superinfection, is required for maintenance of the lysogenic state and contains a helix-turn-helix DNA-binding motif--properties associated with repressors of temperate phages. We have utilized these observations to demonstrate the use of L5 gene 71 as a selectable marker for genetic transformation of the mycobacteria. Significantly, the use of L5 gene 71 as a selectable gene avoids the requirement for antibiotic-resistance genes providing an important tool for manipulation of the pathogens Mycobacterium tuberculosis and Mycobacterium avium, and for the construction of recombinant BCG vaccines. Levin, M.E., and G.F. Hatfull (1993) Mycobacterium smegmatis RNA polymerase: DNA supercoiling, action of rifampicin and mechanism of rifampicin resistance. Mol. Microbiol. 8:277-285 We have isolated RNA polymerase from Mycobacterium smegmatis and established conditions for specific transcription initiation in vitro. The M. smegmatis enzyme has a strong dependence on supercoiling of the DNA substrate for transcription from mycobacterial promoters. We also show that RNA polymerase is the target for rifampicin, and that this antibiotic specifically inhibits the transition from synthesis of short oligoribonucleotides to full-length transcripts. RNA polymerase isolated from a rifampicin-resistant mutant of M. smegmatis is less sensitive to rifampicin in vitro, confirming that one mechanism of rifampicin resistance in mycobacteria is through alteration of RNA polymerase. This in vitro transcription system provides a simple method for the characterization of gene expression in mycobacteria including the pathogens Mycobacterium tuberculosis, Mycobacterium avium and Mycobacterium leprae. It also provides a system for evaluating potential anti-mycobacterial drugs. Jacobs, Jr., W.R., R.G. Barletta, R. Udani, J. Chan, G. Kalkut, G. Sosne, T. Kieser, G.J. Sarkis, G.F. Hatfull, and B.R. Bloom (1993) Rapid assessment of drug susceptibilities of Mycobacterium tuberculosis by means of luciferase reporter phages. Science 260:819-822 Effective chemotherapy of tuberculosis requires rapid assessment of drug sensitivity because of the emergence of multidrug-resistant Mycobacterium tuberculosis. Drug susceptibility was assessed by a simple method based on the efficient production of photons by viable mycobacteria infected with specific reporter phages expressing the firefly luciferase gene. Light production was dependent on phage infection, expression of the luciferase gene, and the level of cellular adenosine triphosphate. Signals could be detected within minutes after infection of virulent M. tuberculosis with reporter phages. Culture of conventional strains with antituberculosis drugs, including isoniazid or rifampicin, resulted in extinction of light production. In contrast, light signals after luciferase reporter phage infection of drug-resistant strains continued to be produced. Luciferase reporter phages may help to reduce the time required for establishing antibiotic sensitivity of M. tuberculosis strains from weeks to days and to accelerate screening for new antituberculosis drugs.
Hatfull, G.F. (1993) Genetic transformation of mycobacteria. Trends Microbiol. 1:310-314 Recent advances in methods for introducing DNA into both fast- and slow-growing species of mycobacteria have stimulated the construction of a variety of cloning vectors and the development of a versatile genetic system. These tools will facilitate studies of mycobacterial pathogenicity, antibiotic action and drug resistance, and lead to more effective methods for diagnosis, prevention and treatment of mycobacterial diseases. Lee, M.H., and G.F. Hatfull (1993) Mycobacteriophage L5 integrase-mediated site-specific integration in vitro. J. Bacteriol. 175:6836-6841 Mycobacteriophage L5, a temperate phage of the mycobacteria, forms stable lysogens in Mycobacterium smegmatis via site-specific integration of the phage genome. Recombination occurs within specific phage and bacterial attachment sites and is catalyzed by the phage-encoded integrase protein in vivo. We describe here the overexpression and purification of L5 integrase and its ability to mediate integrative recombination in vitro. We find that L5 integrase-mediated recombination is greatly stimulated by extracts of M. smegmatis but not by Escherichia coli extracts, purified E. coli integration host factor, or purified HU, indicating the presence of a novel mycobacterial integration host factor.
Oyashi, M., and G.F. Hatfull (1992) The cohesive ends of mycobacteriophage L5 DNA. Nucleic Acids Res. 20:3251
Stover, C.K., V.F. de la Cruz, T.R. Fuerst, J.E. Burlein, L.A. Benson, L.T. Bennett, G.P. Bansal, J.F. Young, M.H. Lee, and G.F. Hatfull (1991) New use of BCG for recombinant vaccines. Nature 351:456-460 BCG, a live attenuated tubercle bacillus, is the most widely used vaccine in the world and is also a useful vaccine vehicle for delivering protective antigens of multiple pathogens. Extrachromosomal and integrative expression vectors carrying the regulatory sequences for major BCG heat-shock proteins have been developed to allow expression of foreign antigens in BCG. These recombinant BCG strains can elicit long-lasting humoral and cellular immune responses to foreign antigens in mice.
Lee, M.H., L. Pascopella, W.R. Jacobs, Jr., and G.F. Hatfull (1991) Site-specific integration of mycobacteriophage L5: integration-proficient vectors for Mycobacterium smegmatis, Mycobacterium tuberculosis, and bacille Calmette-Guerin. Proc. Natl. Acad. Sci., USA 88:3111-3115 Mycobacteriophage L5, a temperate phage of mycobacteria, integrates site-specifically into the Mycobacterium smegmatis chromosome. We have identified the int gene and attP site of L5, characterized the chromosomal attachment site (attB), and constructed plasmid vectors that efficiently transform M. smegmatis through stable site-specific integration of the plasmid into the bacterial genome. These integration-proficient plasmids also efficiently transform slow-growing mycobacteria such as the pathogen Mycobacterium tuberculosis and the vaccine strain bacille Calmette-Guerin (BCG). The ability to easily generate stable recombinants in these slow-growing mycobacteria without the requirement for continual selection is of particular importance for the construction of recombinant BCG vaccines and for the isolation and characterization of mycobacterial pathogenic determinants in animal model systems. Integration vectors of this type should be of general use in a number of additional bacterial systems where temperate phages have been identified.
Stark, W.M., N.D. Grindley, G.F. Hatfull, and M.R. Boocock (1991) Resolvase-catalysed reactions between res sites differing in the central dinucleotide of subsite I. EMBO J. 10:3541-3548 The resolvase-catalysed reaction between two res sites in a circular DNA substrate normally gives two circular recombination products linked in a two-noded catenane. Homology between the two res sites at the central overlap dinucleotide of subsite I is important for recombination. Reactions between res sites differing at one position in the central dinucleotide (AC X AT) gave a low yield of recombinants containing mismatched base-pairs, but gave large amounts of a non-recombinant four-noded knot. This result was predicted by a 'simple rotation' model for strand exchange. The mismatch is evidently recognized only after commitment to an initial 180 degrees rotation of the resolvase-linked DNA ends, and it induces a second 180 degrees rotation which restores correct base-pairing at the overlap, giving the four-noded product. Correct base-pairing is not essential for religation, but may be important for release of the products. Characteristic patterns of 4, 6, 8 and 10 node knots, or 4, 8, 12 and 16 node knots were obtained, depending on the reaction conditions and the resolvase. Two pathways for multiple rounds of rotation in 360 degrees steps are inferred. The results support a model for strand exchange by supercoil-directed subunit rotation within a resolvase tetramer.
Droge, P., G.F. Hatfull, N.D. Grindley, and N.R. Cozzarelli (1990) The two functional domains of gamma delta resolvase act on the same recombination site: implications for the mechanism of strand exchange. Proc. Natl. Acad. Sci., USA 87:5336-5340 During site-specific recombination by the gamma delta resolvase, four DNA strands are broken, exchanged, and religated. This exchange is carried out within a DNA-protein complex, the synaptosome, in which the recombination sites, res, are aligned. The domain of resolvase that binds to a res site is distinct from the domain that breaks and rejoins the DNA. We tested whether the catalytic domain acts on the res site to which its binding domain is bound (in cis) or on the opposing res site in the synaptic complex (in trans). We constructed a hybrid synaptosome in which one res site is bound to wild-type resolvase and the other is bound to a mutant resolvase that binds normally but is unable to break DNA. From the pattern of strand breakage in the reaction intermediate containing resolvase covalently attached to DNA, we conclude that resolvase attacks predominantly, if not exclusively, in cis. Because cis breakage and reunion per se cannot lead to recombination, our results support a model in which DNA exchange is guided by an exchange of resolvase subunits between the breakage and reunion events.
Sanderson, M.R., P.S. Freemont, P.A. Rice, A. Goldman, G.F. Hatfull, N.D. Grindley, and T.A. Steitz (1990) The crystal structure of the catalytic domain of the site-specific recombination enzyme gamma delta resolvase at 2.7 A resolution. Cell 63:1323-1329 The crystal structure of the catalytic domain of the site-specific recombination enzyme gamma delta resolvase has been determined at 2.7 A resolution. Its first 120 amino acids form a central five-stranded, beta-pleated sheet surrounded by five alpha helices. In one of the four dyad-related dimers, the two active site Ser-10 residues are 19 A apart, perhaps close enough to contact and become covalently linked to the DNA at the recombination site. This dimer also forms the only closely packed tetramer found in the crystal. The subunit interface at a second dyad-related dimer is more extensive and more highly conserved among the homologous recombinases; however, its active site Ser-10 residues are more than 30 A apart. Side chains, identified by mutations that eliminate catalysis but not DNA binding, are located on the subunit surface near the active site serine and at the interface between a third dyad-related pair of subunits of the tetramer.
Hughes, R.E., G.F. Hatfull, P. Rice, T.A. Steitz, and N.D. Grindley (1990) Cooperativity mutants of the gamma delta resolvase identify an essential interdimer interaction. Cell 63:1331-1338 gamma delta resolvase, a transposon-encoded site-specific recombinase, catalyzes the resolution of the cointegrate intermediate of gamma delta transposition. The recombination reaction involves the formation of a catalytic nucleoprotein complex whose structure is determined by specific protein-DNA and protein-protein interactions. We have isolated many resolvase mutants and have identified four that are unable to mediate a subclass of higher order protein-protein interactions necessary for recombination. This mutant phenotype is characterized by an inability to catalyze recombination, a loss of cooperative binding to res DNA, and a failure to induce looping out of the DNA between two resolvase binding sites within res. The amino acid side chains identified by the cooperativity mutants cluster on a surface of the protein that mediates an interaction between resolvase dimers in a crystallographic tetramer. We have therefore identified a region of resolvase that mediates an interdimer protein-protein interaction necessary for the formation of the recombinogenic synaptic intermediate.
Rimphanitchayakit, V., G.F. Hatfull, and N.D. Grindley (1989) The 43 residue DNA binding domain of gamma delta resolvase binds adjacent major and minor grooves of DNA. Nucleic Acids Res. 17:1035-1050 The carboxyl-terminal domain of gamma delta resolvase binds to each half of the three resolvase binding sites that constitute the recombination site, res. Ethylation inhibition experiments show that the phosphate contacts made by the C-terminal DNA binding domain are similar to those made by intact resolvase, with the exception of a single phosphate at the inside end of each contact region which is contacted solely by the intact resolvase. The DNA binding domain makes essentially identical contacts to all 6 half sites, whereas the intact resolvase makes slightly different contacts to each binding site. Despite its small size, only 43 amino acid residues, the resolvase C-terminal domain interacts with an unusually large segment of DNA. Phosphate contacts extend across an adjacent major and minor groove of DNA and about one third of the circumference around the helix. The minimal binding segment, determined experimentally, is a 12 bp sequence that includes the 9 base pair inverted repeat (common to all half sites), the adjacent 3 base pairs (towards the center of the intact resolvase binding site), and phosphates at both ends.
Hatfull, G.F., M.R. Sanderson, P.S. Freemont, P.R. Raccuia, N.D. Grindley, and T.A. Steitz (1989) Preparation of heavy-atom derivatives using site-directed mutagenesis. Introduction of cysteine residues into gamma delta resolvase. J. Mol. Biol. 208:661-667 The ability to determine protein structures by X-ray crystallography is often thwarted by the difficulty of finding isomorphous heavy-atom derivatives. The crystal structure of the site-specific recombinase, resolvase, has been difficult to determine for this reason. We have overcome this problem by introducing 13 single cysteine substitutions into the resolvase catalytic domain using oligonucleotide mutagenesis. The mutant proteins were screened for their ability to crystallize into the orthorhombic form and bind mercury ions isomorphously. Two mutant proteins provided excellent heavy-atom derivatives. This approach should be of general use and particularly helpful in cases where traditional methods have failed to produce a derivative.
Falvey, E., G.F. Hatfull, and N.D. Grindley (1988) Uncoupling of the recombination and topoisomerase activities of the gamma delta resolvase by a mutation at the crossover point. Nature 332:861-863 In several well-characterized site-specific recombination systems it has been shown that, for efficient recombination, the two recombining sites must have identical DNA sequences across the region between the staggered points of exchange. The precise DNA sequence of this overlap region, however, appears to be of little importance (with the exception of one position in the loxP site of bacteriophage P1 (ref. 6]. In this report we characterize a mutant recombination site for the site-specific recombination enzyme gamma delta resolvase (encoded by the gamma delta transposon), in which the dinucleotide at the crossover point is changed from AT to CT. Our results indicate that identity of the two overlap regions is not sufficient for recombination. Although resolvase binds normally to the mutant site and induces the structural deformation characteristic of the wild-type recombination site, catalysis at the crossover point (cutting and rejoining of DNA strands) is effectively limited to just one of the two strands, allowing resolvase to act as a topoisomerase but not as a recombinational enzyme.
Hatfull, G.F., S.M. Noble, and N.D. Grindley (1987) The gamma delta resolvase induces an unusual DNA structure at the recombinational crossover point. Cell 49:103-110 gamma delta resolvase, a transposon-encoded protein active in site-specific recombination, induces a structural change in the DNA at the recombinational crossover point that results in enhanced intercalation of the foot-printing reagent MPE X Fe(II). The structural change correlates with the formation of a bend in the DNA: a mutant resolvase that binds to the crossover site but induces little or no bend does not promote intercalation. The properties of the mutant protein suggest that the induced structural change, which we propose is a localized kink, is required for recombination.
Hatfull, G.F., and C.M. Joyce (1986) Deletion of the spf (spot 42 RNA) gene of Escherichia coli. J. Bacteriol. 166:746-750 To investigate the function of spot 42 RNA, a small RNA of Escherichia coli, we constructed a strain in which spf, the structural gene for this RNA, is deleted. We achieved this by using a delta att phage lambda carrying a DNA fragment spanning the spf region but with a precise deletion of spf. By integration of this phage at the spf locus and by its subsequent excision, we were able to cross the spf deletion onto the bacterial chromosome. The fact that such a deletion could be obtained indicated that spf is not an essential gene. We did not observe any major defect in delta spf cells, although in one strain background the deletion caused a slight growth impairment.
Hatfull, G.F., and N.D. Grindley (1986) Analysis of gamma delta resolvase mutants in vitro: evidence for an interaction between serine-10 of resolvase and site I of res. Proc. Natl. Acad. Sci., USA 83:5429-5433 The resolvase encoded by the transposon gamma delta mediates a site-specific recombination between the two copies of gamma delta in a cointegrate molecule to yield the final products of transposition. Several mutants of resolvase that lack recombinational activity have been isolated previously, and one of these, which has a serine-to-leucine change at position 10, we have characterized in vitro. We also have constructed a serine-to-cysteine change at position 10 by in vitro mutagenesis and have analyzed this mutant protein in vitro. We find that the cysteine-10 mutant is defective in recombinational activity but binds to the recombinational site, res, similarly to wild-type, as assayed by gel electrophoresis of the protein-DNA complexes. In contrast, the leucine-10 mutant has a specific defect in complex formation with site I, which contains the recombinational crossover point, although it can bind and provide the ancillary functions of resolvase at sites II and III. It has been shown that a phosphoserine linkage is made to the DNA during recombination; because serine-10 is absolutely conserved amongst the family of homologous site-specific recombination proteins, it is a good candidate for the active-site serine. The properties of these resolvase mutants with substitutions at position 10 are consistent with this hypothesis and suggest that serine-10 is in close contact with the DNA at site I.
Gill, D.R., G.F. Hatfull, and G.P. Salmond (1986) A new cell division operon in Escherichia coli. Mol. Gen. Genet. 205:134-145 At 76 min on the E. coli genetic map there is a cluster of genes affecting essential cellular functions, including the heat shock response and cell division. A combination of in-vivo and in-vitro genetic analysis of cell division mutants suggests that the cell division gene fts E is the second gene in a 3 gene operon. A cold-sensitive mutant, defective in the third gene, is also unable to divide at the restrictive temperature, and we designate this new cell division gene fts X. Another cell division gene, fts S, is very close to, but distinct from, the 3 genes of the operon. The fts E product is a 24.5 Kd polypeptide which shows strong homology with a small group of proteins involved in transport. Both the fts E product and the protein coded by the first gene (fts Y) in the operon have a sequence motif found in a wide range of heterogeneous proteins, including the Ras proteins of yeast. This common domain is indicative of a nucleotide-binding site. Robinson, A.C., D.J. Kenan, G.F. Hatfull, N.F. Sullivan, R. Spiegelberg, and W.D. Donachie (1984) DNA sequence and transcriptional organization of essential cell division genes ftsQ and ftsA of Escherichia coli: evidence for overlapping transcriptional units. J. Bacteriol. 160:546-555 The DNA sequence of a cloned segment of the Escherichia coli chromosome containing ftsQ, ftsA, and part of the ftsZ gene was determined and interpreted for genetic complementation and promoter fusion data for the region. The contiguous genes ftsQ, ftsA, and ftsZ were transcribed in the same direction (clockwise on the genetic map) and each had at least one associated promoter which allowed it to be transcribed independently of neighboring genes. ftsA and ftsZ possessed promoters within the coding sequences of the juxtaposed upstream structural genes, and a promoter element for ftsA was surrounded by a region of twofold symmetry which corresponded closely to a symmetrical element in the region of a putative ftsZ promoter. The structural gene of ftsQ consisted of 838 nucleotides, encoding a 276-residue amino acid polypeptide of molecular weight 31,400; the structural gene of ftsA consisted of 1,260 nucleotides, encoding a 420-residue amino acid polypeptide of molecular weight 45,400. The observation that the termination codon of ftsQ overlaps with a potential initiation codon for ftsA suggested that these two genes may be translationally coupled when transcription is initiated upstream of the ftsQ coding sequence.
Begg, K.J., G.F. Hatfull, and W.D. Donachie (1980) Identification of new genes in a cell envelope-cell division gene cluster of Escherichia coli: cell division gene ftsQ. J. Bacteriol. 144:435-437 We report the identification, cloning, and mapping of a new cell division gene, ftsQ. This gene formed part of a cluster of three division genes (in the order ftsQ ftsA ftsZ) which itself formed part of a larger cluster of at least 10 genes, all of which were involved in some step in cell division, cell envelope synthesis, or both. The ftsQAZ group was transcribed from at least two independent promoters.
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