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Bittner, A.N., A. Foltz, and V. Oke (2007) Only one of five groEL genes is required for viability and successful symbiosis in Sinorhizobium meliloti. J. Bacteriol. 189:1884-1889 Many bacterial species contain multiple copies of the genes that encode the chaperone GroEL and its co-chaperone GroES, including all of the fully sequenced root nodulating bacteria that interact symbiotically with legumes to generate fixed nitrogen. In particular, in Sinorhizobium meliloti there are four groESL operons and one groEL gene. To uncover functional redundancies of these genes during growth and symbiosis, we attempted to construct strains containing all combinations of groEL mutations. Although a double groEL1 groEL2 mutant cannot be constructed, we demonstrate that the quadruple groEL1 groESL3 groEL4 groESL5 and groEL2 groESL3 groEL4 groESL5 mutants are viable. Therefore, like E. coli and other species, S. meliloti only requires one groEL for viability and either groEL1 or groEL2 will suffice. The groEL1 groESL5 double mutant is more severely affected for growth at both 30 degrees and 40 degrees C than the single mutants, suggesting overlapping functions in stress response. During symbiosis the quadruple groEL2 groESL3 groEL4 groESL5 mutant acts like the wild type, but the quadruple groEL1 groESL3 groEL4 groESL5 mutant acts like the groEL1 single mutant, which cannot fully induce nod gene expression and forms ineffective nodules. Therefore, the only groEL gene required for symbiosis is groEL1. However, we show that the other groE genes are expressed in the nodule at lower levels, suggesting minor roles during symbiosis. Combining our data with other data, we conclude that groESL1 encodes the housekeeping GroEL/GroES chaperone and that groESL5 is specialized for stress response.
Bittner, A.N., and V. Oke (2006) Multiple groESL operons are not key targets of RpoH1 and RpoH2 in Sinorhizobium meliloti. J. Bacteriol. 188:3507-3515 Among the rhizobia that establish nitrogen-fixing nodules on the roots of host plants, many contain multiple copies of genes encoding the sigma factor RpoH and the chaperone GroEL/GroES. In Sinorhizobium meliloti there are two rpoH genes, four groESL operons, and one groEL gene. rpoH1 mutants are defective for growth at high temperature and form ineffective nodules, rpoH1 rpoH2 double mutants are unable to form nodules, and groESL1 mutants form ineffective nodules. To explore the roles of RpoH1 and RpoH2, we identified mutants that suppress both the growth and nodulation defects. These mutants do not suppress the nitrogen fixation defect. This implies that the functions of RpoH1 during growth and RpoH1/RpoH2 during the initiation of symbiosis are similar but that there is a different function of RpoH1 needed later during symbiosis. We showed that, unlike in Escherichia coli, overexpression of groESL is not sufficient to bypass any of the RpoH defects. Under free-living conditions, we determined that RpoH2 does not control expression of the groE genes, and RpoH1 only controls expression of groESL5. Finally, we completed the series of groE mutants by constructing groESL3 and groEL4 mutants and demonstrated that they do not display symbiotic defects. Therefore, the only groESL operon required by itself for symbiosis is groESL1. Taken together, these results suggest that GroEL/GroES production alone cannot explain the requirements for RpoH1 and RpoH2 in S. meliloti and that there must be other crucial targets.
Oke, V., B.G. Rushing, E.J. Fisher, M. Moghadam Tabrizi, and S.R. Long (2001) Identification of the heat shock sigma factor RpoH and a second RpoH-like protein in Sinorhizobium meliloti. Microbiology 147:2399-2408 Hybridization to a PCR product derived from conserved sigma-factor sequences led to the identification of two Sinorhizobium meliloti DNA segments that display significant sequence similarity to the family of rpoH genes encoding the sigma(32) (RpoH) heat-shock transcription factors. The first gene, rpoH1, complements an Escherichia coli rpoH mutation. Cells containing an rpoH1 mutation are impaired in growth at 37 degrees C under free-living conditions and are defective in nitrogen fixation during symbiosis with alfalfa. A plasmid-borne rpoH1-gusA fusion increases in expression upon entry of the culture into the stationary phase of growth. The second gene, designated rpoH2, is 42% identical to the S. meliloti rpoH1 gene. Cells containing an rpoH2 mutation have no apparent phenotype under free-living conditions or during symbiosis with the host plant alfalfa. An rpoH2-gusA fusion increases in expression during the stationary phase of growth. The presence of two rpoH-like sequences in S. meliloti is reminiscent of the situation in Bradyrhizobium japonicum, which has three rpoH genes.
Barnett, M.J., V. Oke, and S.R. Long (2000) New genetic tools for use in the Rhizobiaceae and other bacteria. Biotechniques 29:244-245 Oke, V., and S.R. Long (1999) Bacterial genes induced within the nodule during the Rhizobium-legume symbiosis. Mol. Microbiol. 32:837-849 During the symbiosis between the bacterium Rhizobium meliloti and plants such as alfalfa, the bacteria elicit the formation of nodules on the roots of host plants. The bacteria infect the nodule, enter the cytoplasm of plant cells and differentiate into a distinct cell type called a bacteroid, which is capable of fixing atmospheric nitrogen. To discover bacterial genes involved in the infection and differentiation stages of symbiosis, we obtained genes expressed at the appropriate time and place in the nodule by identifying promoters that are able to direct expression of the bacA gene, which is required for bacteroid differentiation. We identified 230 fusions that are expressed predominantly in the nodule. Analysis of 23 sequences indicated that only three encode proteins known to be involved in the Rhizobium-legume symbiosis, six encode proteins with homology to proteins not previously associated with symbiosis, and 14 have no significant similarity to proteins of known function. Disruption of a locus that encodes a protein with homology to a cell adhesion molecule led to a defect in the formation of nitrogen-fixing nodules, resulting in an increased number of nitrogen-starved plants. Our isolation of a large number of nodule-expressed genes will help to open the intermediate stages of nodulation to molecular analysis.
Oke, V., and S.R. Long (1999) Bacteroid formation in the Rhizobium-legume symbiosis. Curr. Opin. Microbiol. 2:641-646 During the Rhizobium-legume symbiosis, bacteria enter the cells of host plants and differentiate into nitrogen-fixing bacteroids. Recent mutant screens and expression studies have revealed bacterial genes involved in the developmental pathway and demonstrate how the genetic requirements can vary from one host-microbe system to another. Whether bacteroids are terminally differentiated cells is an ongoing debate and new experimental systems are required to address this issue.
Oke, V., M. Shchepetov, and S. Cutting (1997) SpoIVB has two distinct functions during spore formation in Bacillus subtilis. Mol. Microbiol. 23:223-230 The Bacillus subtilis SpoIVB protein is a critical component of the intercompartmental signal-transduction pathway that activates the sigma factor, sigma K, in the mother cell of the sporulating cell. SpoIVB, synthesized in the forespore chamber, must act across two layers of phospholipid membrane to facilitate proteolytic processing of inactive pro-sigma K to active sigma K. We have used a genetic approach to dissect SpoIVB function and found that this protein has two distinct developmental functions. One function is that of intercompartmental signalling of pro-sigma K processing. The other role is essential to spore formation and is illustrated by mutations of SpoIVB which allow cell-cell signalling of pro-sigma K processing but prevent the formation of viable spores. Using localized and site-specific mutagenesis we have identified a functional domain of SpoIVB that is involved in its non-signalling role.
Halberg, R., V. Oke, and L. Kroos (1995) Effects of Bacillus subtilis sporulation regulatory protein SpoIIID on transcription by sigma K RNA polymerase in vivo and in vitro. J. Bacteriol. 177:1888-1891 SpoIIID is a sequence-specific, DNA-binding protein that activates or represses transcription of different genes by sigma K RNA polymerase in vitro. A Bacillus subtilis strain engineered to produce both sigma K and SpoIIID during growth showed effects of SpoIIID on expression of sigma K-dependent genes that were consistent with the effects of a small amount of SpoIIID on transcription of these genes in vitro, indicating that the strain provides a simple, in vivo method to screen for effects of SpoIIID on transcription of sigma K-dependent genes.
van der Keyl, H., H. Kim, R. Espey, C.V. Oke, and M.K. Edwards (1994) Caenorhabditis elegans sqt-3 mutants have mutations in the col-1 collagen gene. Dev. Dynam. 201:86-94 sqt-3 mutants of Caenorhabditis elegans form dumpy larvae and adults and display allele-specific defects in locomotion, fertility, and viability. We have determined that the sqt-3 locus encodes COL-1 collagen. We physically mapped the col-1 gene to a cosmid on chromosome V whose position is consistent with the location of the sqt-3 gene. We also observed morphological defects in sqt-3 mutants at stages that correlate with the mRNA expression patterns of col-1. Sequence analysis of the col-1 gene in the three temperature-sensitive mutants revealed that each allele of sqt-3 has a unique missense mutation causing arginine or glutamic acid to replace glycine in a Gly-X-Y triple helical domain. These glycine substitutions may result in longer non-collagenous domains, which may decrease the thermal stability or impart additional flexibility to mutant trimers. In addition, we describe four corrections to the published sequence of col-1, including one fifteen nucleotide addition that completes a conserved domai Oke, V., and R. Losick (1993) Multi-level regulation of the sporulation transcription factor sigma-K in Bacillus subtilis. J. Bacteriol. 175:7341-7347 Gene expression in the mother-cell compartment of the Bacillus subtilis sporangium is governed in part by the sporulation transcription factor sigma K. The production of sigma K is controlled at three levels: by a chromosomal rearrangement that generates the sigma K-coding sequence (sigK), by compartment-specific transcription of sigK, and by conversion of the inactive pro-protein product of sigK (pro-sigma K) to sigma K. To investigate the function of these multiple levels of regulation, we constructed a set of strains that bypass the chromosomal rearrangement, pro-protein processing, or both levels of control. Here we show that one of the functions of the chromosomal rearrangement and pro-protein processing is to prevent inappropriate production of sigma K under nonsporulation conditions. In the absence of both of these levels of control, a low level of sigma K-directed gene expression is observed during stationary phase after growth in rich medium. The appearance of sigma K under these conditions is probably due to a low level of sigma K-directed transcription from the sigK promoter in a positive feedback loop. We also report the construction of a strain that produces high levels of sigma K during growth. Using this strain, we demonstrate that the production of sigma K during growth is sufficient to induce a cascade of gene expression that closely mimics late events in the mother-cell line of gene expression.
Cutting, S., V. Oke, A. Driks, R. Losick, S. Lu, and L. Kroos (1990) A forespore checkpoint for mother-cell gene expression during development in Bacillus subtilis. Cell 62:239-250 Gene expression in the mother cell compartment of sporulating cells of B. subtilis is partly governed by the mother cell RNA polymerase sigma factor sigma K. Paradoxically, sigma K-directed gene expression also depends on sigma G, the product of the forespore compartment regulatory gene spoIIIG, and on other forespore regulatory proteins. We now identify mutations in the genes bofA and bofB that relieve the dependence of mother cell gene expression on forespore regulatory proteins but not on sigma K. We establish that the dependence of mother cell gene expression on the forespore regulatory proteins is mediated at the level of the conversion of pro-sigma K to its mature, active form. We propose that the BofA and/or BofB proteins govern this conversion in response to a signal generated by the forespore. Activation of pro-sigma K could be a checkpoint for coordinating gene expression between the mother cell and forespore compartments of the developing sporangium.
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