Progress 09/01/02 to 09/30/06
Outputs
This project had two specific goals: to characterize RosR function in R. etli and to identify genes regulated by the RosR-like proteins in different alpha-proteobacteria. 1. Characterization of RosR. We were interested in defining the DNA site to which RosR binds, as a first step towards analysis of RosR function. We sequenced the surrounding regions of several RosR-regulated loci, to identify likely promoter regions. These regions were then cloned into the gusA promoter-probe vector pFUS1. The resulting clones were mobilized into the wild-type R. etli and into a rosR mutant derivative, and quantitative GUS assays were performed. These DNA fragments conferred RosR-dependent GUS activity in vivo. These promoter regions are therefore candidates for direct RosR binding assays. We evaluated several two-plasmid systems for analysis of RosR and RosR-dependent promoters in surrogate hosts such as E. coli, but none were satisfactory for further analysis. Repeated attempts to
purify RosR using previously published protocols were not successful, nor were attempts to purify RosR using other protein purification systems. 2. Analysis of RosR homologs in different bacteria. We constructed mutations in Rhodopseudomonas palustris (rpal3127) and Rhodospirillum rubrum (rrub0158) by inserting a cassette containing a gusA reporter gene into the gene cloned on a plasmid. These mutations were introduced into R. palustris or R. rubrum and mutant strains were identified that had exchanged the mutant version of the gene for the wild-type version. The correct genomic structure was confirmed by Southern analysis. rpal3127 was expressed in R. palustris and rrub0158 was expressed in R. rubrum, based on the results of quantitative GUS assays. Total soluble protein from the wild-type and mutant strains of R. palustris and R. rubrum were subjected to two-dimensional protein gel analysis to identify proteins that were differentially expressed in one strain versus the other. No
proteins were identified that varied between the strains. We tested the ability of Rpal3127 or Rrub0158 to regulate expression of its own gene, but introduction of the wild-type version of the genes (on pHRP309, a replicating plasmid in R. palustris, or on pRK310, a replicating plasmid in R. rubrum) had no effect on expression of rpal3127::gusA or rrub0158::gusA, respectively.
Impacts
This research will improve our understanding of regulation of gene expression by RosR and related proteins. Compared to other transcriptional regulatory proteins, the function of these proteins is not well understood, so this analysis is expected to yield information of fundamental importance.
Publications
- No publications reported this period
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Progress 01/01/05 to 12/31/05
Outputs
This projects aims to characterize RosR function in Rhizobium etli. We are interested in defining the DNA site to which RosR binds. We sequenced the surrounding regions of several RosR-regulated loci, to identify likely promoter regions. These regions were then cloned into the gusA promoter-probe vector pFUS1. The resulting clones were mobilized into the wild-type R. etli and into a rosR mutant derivative, and quantitative GUS assays were performed. These DNA fragments conferred RosR-dependent GUS activity in vivo. We developed a system in E. coli to monitor RosR regulation by cloning RosR onto a low copy plasmid. We set up a two-plasmid system with this rosR-containing plasmid and the various pFUS derivatives containing RosR-regulated promoters and have shown that these promoter fragments confer RosR-dependent GUS expression in E. coli strains, suggesting that RosR functions in E. coli. We are currently developing mutagenesis systems to identify the specific residues
that are required for RosR regulation, as well as developing an in vitro system to test RosR binding.
Impacts
This research will improve our understanding of regulation of gene expression by RosR and related proteins. Since this protein family has not been extensively studied in bacteria, these results will contribute to our basic understanding of gene regulation in bacteria.
Publications
- No publications reported this period
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Progress 01/01/04 to 12/31/04
Outputs
This project has two specific goals: to characterize RosR function in R. etli and to identify genes regulated by the RosR-like proteins in different alpha-proteobacteria. 1. Characterization of RosR. We are interested in defining the DNA site to which RosR binds, as a first step towards analysis of RosR function. We sequenced the surrounding regions of several RosR-regulated loci, to identify likely promoter regions. These regions were then cloned into the gusA promoter-probe vector pFUS1. The resulting clones were mobilized into the wild-type R. etli and into a rosR mutant derivative, and quantitative GUS assays were performed. These DNA fragments conferred RosR-dependent GUS activity in vivo. These promoter regions are therefore candidates for direct RosR binding assays, which are in progress. 2. Analysis of RosR homologs in different bacteria. 2.1 Rhodopseudomonas palustris. We constructed mutations in rpal3127 by inserting a cassette containing a gusA reporter
gene into the gene. These mutations were introduced into R. palustris and mutant strains were identified that had exchanged the mutant version of the gene for the wild-type version. The correct genomic structure was confirmed by Southern analysis. There was no observable colony phenotype associated with the mutant strains, and EPS and LPS analysis showed no differences. rpal3127 was expressed in R. palustris, based on the results of quantitative GUS assays. Total soluble protein from the wild-type and mutant strains was subjected to two-dimensional protein gel analysis to identify proteins that were differentially expressed in one strain versus the other. No proteins were identified that varied between the strains. We tested the ability of Rpal3127 to regulate expression of its own gene, but introduction of rpal3127 on pHRP309 (a replicating plasmid in R. palustris) had no effect on expression of rpal3127::gusA. 2.2. Rhodospirillum rubrum. R. rubrum contains two genes homologous to
rosR, rrub0158 and rrub3130. We constructed mutations similar to those described for rpal3127 above in rrub0158. Mutants were confirmed by PCR analysis. Again, there was no observable colony phenotype associated with the mutant strains. The gene was expressed in R. rubrum. Total soluble protein from the wild-type and mutant strains was subjected to two-dimensional protein gel analysis, as described above, to identify proteins that were differentially expressed in one strain versus the other, but no proteins were identified. We tested the ability of Rrub0158 to regulate expression of its own gene, but introduction of rrub0158 on pRK310 (a replicating plasmid in R. rubrum) had no effect on expression of rrub0158::gusA. We noted that rrub0158 is located immediately upstream of a gene encoding a homolog to the global iron regulatory protein Fur. We used reverse transcriptase PCR to show that the genes were co-transcribed, and that the PCR product representing the two-gene transcript was
not produced when RNA from the rrub0158 mutant strain was used as a template. This would be the first evidence for a role for Rrub0158 in the cell.
Impacts
This research will improve our understanding of regulation of gene expression by RosR and related proteins. Compared to other transcriptional regulatory proteins, the function of these proteins is not well understood, so this analysis is expected to yield information of fundamental importance.
Publications
- No publications reported this period
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Progress 01/01/03 to 12/31/03
Outputs
This research focuses on the analysis of a small family of proteins related to Ros from Agrobacterium tumefaciens and RosR from Rhizobium etli. We identified genes encoding similar proteins in the genomes of related bacteria. We have constructed gene-knockout mutant strains in several of these bacteria. Our results show that the genes coding for Ros homologs are expressed in the cell during growth on laboratory media. Current work is aimed at identifying the function of these proteins in the cell by identifying other proteins whose expression is altered in the mutant strains. We are also interested in understanding the mode of action of RosR, which is thought to be a global regulatory protein. We have identified a number of promoters that are controlled by the RosR protein and are now testing in vitro binding of RosR to these DNA sequences. This work will allow us to determine a consensus binding site for the RosR protein.
Impacts
This research will improve our understanding of regulation of gene expression by RosR and related proteins. Compared to other transcriptional regulatory proteins, the function of these proteins is not well understood, so this analysis is expected to yield information of fundamental importance.
Publications
- No publications reported this period
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