STC Faculty -- F. de Bruijn, T. Schmidt, M. Thomashow
Postdocs -- A. Milcamps
Graduate Students -- M.E. Davey, B. Stevenson, J. Klappenbach, A. Gustafson
The primary goal of the Microbial Adaptation thrust group has been to understand better the mechanisms that bacteria have evolved to adapt to adverse environmental conditions. The specific environmental conditions that we have been studying are two "stressful" factors that soil organisms are routinely exposed to and result in slow growth rates, namely nutrient limitation and low temperature. Taking into account the fact that the CME was into a "ramping down" phase in regard to NSF funding, we chose to focus our efforts this past year on those experiments relating to the adaptation of bacteria to alternating feast and famine conditions. Our previous results suggested that these studies had the greatest chance of providing broad new insights into microbial adaptation and ecology.
One chosen line of research has focused on determining whether ribosomal RNA operon copy number is a predictor of bacterial life history strategies. As noted previously, there is a striking lack of convergence on the number of ribosomal RNA operons in prokaryotes. While natural selection appears to have eliminated redundancy of most prokaryotic genes, there are as many as 15 copies of the ribosomal RNA (rRNA) operon in some bacterial species. Despite this conspicuous deviation from the majority rule of single copy genes, few phenotypic characteristics have been correlated with rRNA operon copy number. The central role of ribosomes in metabolism and the lack of obvious evolutionary constraint on rRNA operon copy number suggests that the control of ribosome synthesis via alterations in copy number may be an adaptation to the environment. Indeed, in field studies we found a strong correlation between rRNA copy number and the rate at which bacteria from soil were able to establish visible colonies on a dilute (but complex) agar medium. Without exception, bacteria that quickly formed colonies contained five or more copies of the small subunit rRNA gene, while the slowly responding organisms contained an average of two copies.
In parallel laboratory studies with E. coli, a tradeoff for rapid-upshift capacity was observed as an inability to adequately regulate expression of additional plasmid-borne rRNA operons at slow growth rates. To further investigate the potential disadvantage to E. coli populations with seven rRNA operons in slow growth environments with a constantly available limiting resource, the promoters and the 16S and 23S genes in the rRNA operons A and B were deleted both individually and concurrently. Unlike earlier deletions of an rRNA operon, only rRNA operon sequence was removed and no rRNA transcripts (functional or not) were made from the deleted operon(s). These "clean" deletions allowed for the direct assessment of rRNA copy number on the hypothesized differential fitness of E. coli populations with seven rRNA operons relative to those with only five or six functional rRNA operons. In direct batch culture competition experiments, strains with more rRNA operons were able to out-compete strains with fewer operons. As predicted by the model and growth characteristics, this was because those strains with fewer rRNA operons responded more slowly to the influx of nutrients (i.e., had longer lag times) relative to the control strain with seven rRNA operons. These results support the hypothesis that organisms with more copies of rRNA operons on their chromosome are at an advantage under conditions of fluctuating nutrient concentrations because they can respond more rapidly to an influx of nutrients. These observations are also consistent with the hypotheses that multiple rRNA operons provide the capacity for rapid response to added nutrients, and provide a direct correlation between the ecological strategies of bacteria and a fundamental characteristic of their genomes.
The second line of research that we chose to focus on is the regulation of genes in response to nutrient-limitation; i.e. to understand better the mechanisms that soil bacteria have evolved to sense and respond to periodic famine conditions. Previously, we described the isolation and characterization of more than 60 Sinorhizobium meliloti mutant strains carrying Tn5-luxAB transposon inserts in loci that are induced in response to either carbon, nitrogen and O2 limitation. Further testing indicated that the loci tagged in two mutants, N4 and C22, were induced in response to multiple stresses. This was of particular interest as it suggested the genes might be controlled by a global regulatory system involved in bacterial adaptation to oligotrophic conditions. The locus tagged in the N4 strain is induced by both nitrogen and carbon limitation. The tagged gene was found to be hmgA which encodes homogentisate dioxygenase, an enzyme involved in tyrosine degradation; this is the first report of an hmgA homolog in bacteria. Significantly, the locus, which is required for growth on tyrosine as sole carbon source, has an important role in bacterial survival in stationary growth phase. The second mutant, C22, carries a gene fusion that is induced by nitrogen, carbon, oxygen and iron limitation as well as salt stress and during stationary-phase growth. The gene affected, designated ndi, encodes a novel polypeptide. Flanking the ndi gene are ORFs deduced to encode polypeptides with amino acid sequence similarity to a transcriptional regulator and a sugar transporter.
A secondary mutagenesis using the Tn3 derivative, Tn1721, was conducted with strains N4 and C22 in an attempt to learn more about the regulation of the hmgA and ndi genes. Out of two individual collections of 3600 double mutants, several strains were found with altered levels of lux expression under nitrogen and carbon starvation. The Tn1721 tagged loci of two N4::Tn1721 mutants were cloned and DNA sequence analysis revealed significant similarity with a group of transcriptional regulators of the ArsR family (arsenicum resistance). DNA binding studies have been initiated to determine site(s) within hmgA promoter that bind the putative regulatory factor. Interestingly, preliminary results do not reveal any binding which raises the intriguing possibility that the regulator activates the hmgA gene indirectly. In regard to strain C22, one of the Tn1721 tagged strains no longer displayed luminescence under conditions of N-, C-, Fe-, or O2 limitation. The Tn1721 insertion of this tagged locus was found to reside in an ORF encoding a protein with high degree of similarity to a mitochondrial benzodiazepine receptor pK18, as well as an outer membrane oxygen sensor protein TspO in Rhodobacter sphaeroides. Expression studies of the ndi locus showed that the nitrogen responsive regulator NtrC is not involved in the regulation. However, the low oxygen responsive regulator FixL was found to be necessary for full induction of the ndi locus under low oxygen tension.
A total of 23 loci whose expression increased in response to microaerobic (1% oxygen) conditions were also identified. The symbiotic phenotype of the Tn5luxAB mutants was determined by examining inoculated alfalfa seedlings for nodule formation (Nod) and nitrogen fixation activity (Fix) after 5 weeks of growth. All of the mutants were Nod+ and Fix+. The Tn5luxAB-tagged loci were also examined for luminescence in response to carbon and nitrogen deprivation. Induction by multiple stresses was observed for 10 of the 23 mutants suggesting that some of the loci may be involved in a general stress response in S. meliloti. DNA sequencing indicated that OX4 matches fixN, a known oxygen-regulated gene (this results confirmed that the assay conditions were adequate to obtain fusions expressed in response to microaerobic conditions). Other interesting matches included OX219 with cyoC, cytochrome oxidase; OX25 with potE, putrescine-ornithine antiporter; OX106 with exoO, succinoglycan synthesis; OX202 with gltX, glutamyl tRNA synthesis; and OX209 with htpG, heat shock protein. Significantly, there was a lack of similarity of 12 of the loci to deposited sequences suggesting that many of the loci may encode novel genes.
In S. meliloti, FixL/FixJ, a two-component regulatory system that senses microaerobic conditions, coordinately controls the genes encoding functions required for nitrogen fixation and for respiration inside root nodules. This is the only oxygen-sensing regulatory system reported to date in S. meliloti. The 24 Tn5luxAB fusions were therefore examined for regulation by FixL/FixJ. A fixL mutation was introduced into the OX strains via phage transduction and luminescence determined. Analysis of the OX fixL double mutants demonstrated that fixL was required for microaerobic induction of only three of the Tn5luxAB fusions (OX mutants 4, 223, and 226) suggesting that regulatory systems in addition to FixL/FixJ are involved in the microaerobic response of S. meliloti.
The results of the ribosomal RNA operon experiments are important as they indicate that operon copy number plays a role in microbial adaptation to a "feast and famine" lifestyle. This exciting line of research will be continued, now under the funding of a new grant from the National Science Foundation, a situation made possible by the initial funding of the project through the CME. Another CME funded project that has also secured funding, in this case by the NSF LExEN program, is the research on recovery of microorganisms from ancient Siberian permafrost soils; a satellite effort of the Microbial Adaptation thrust area. It was the background strain isolation and characterization made possible by the CME that enabled the Siberian research to obtain the LExEN funding. Additional funding opportunities may also exist. The microbial cells that are currently present in the ancient permafrost are likely to be very old in age; the permafrost layers themselves have been continuously frozen for up to 3 million years. Thus, it is possible that these organisms might serve as a unique model to study mechanisms of resistance to cell aging. This line of investigation is now being given serious consideration and may potentially be funded as part of the new Michigan Life Science Corridor, a large health-related research program that has grown out of the recent settlement between Michigan and the tobacco industry. And finally, the mutants that have transposon inserts in genes that are responsive to multiple nutrient deficiencies remain an extremely valuable resource that will continue to be used in coming years. Not only does this resource promise to yield significant new insight into the role of stress-regulated genes, many of which are novel, in microbial adaptation to nutrient limitation, but in addition, should yield fundamental new insights into how microorganisms sense and respond to changes in the environment.
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