Wind River Conference on Prokaryotic Biology—2002
Department of Microbiology, Molecular Biology and Biochemistry, University of Idaho, Moscow, Idaho 83844,1 Department of Biochemistry, Molecular Biology, and Cell Biology, Northwestern University, Evanston, Illinois 60208,2 Infectious Diseases Research, Lilly Research Labs, Eli Lilly and Company, Indianapolis, Indiana 46285,3 Department of Microbiology and Immunology, University of Louisville, School of Medicine, Louisville, Kentucky 400924/, http://www.100md.com
Received 8 August 2002/ Accepted 8 October 2002/, http://www.100md.com
INTRODUCTION/, http://www.100md.com
The Wind River Conference, which was begun as the Transformation Meeting in 1957, was renamed the Wind River Conference on Genetic Exchange and ultimately became the Wind River Conference on Prokaryotic Biology to expand the range of areas of discussion and presentation (3). Initially, the discussion centered on bacterial transformation, and many of the seminal findings in that field were reported at this meeting, but recently the conferences have covered a wide range of subject areas from genetics to physiology of many diverse genera of bacteria. In that aspect this meeting is unique, because students and experts working on many different microbial systems can find a common ground for discussion and expansion of ideas. The Wind River Conferences also encourage students to present and even lead sessions. This has provided an excellent introduction to scientific presentation and discussion for a number of scientists across the country, many of whom regularly attend the Wind River Conferences and bring their students here. Since the meeting is held at a relatively remote site, there is ample opportunity to relax together with speakers and informally discuss anything that is of further interest and to develop contacts and friendships. This year's meeting was the 46th in the series. The site for the meeting has varied over the years but mostly has been in the Estes Park area of Colorado and was fixed in this location several years ago. The 46th Wind River Conference took place from 3 to 8 June 2002 at Aspen Lodge in Estes Park, with 140 participants .
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Attendees of the 46th Wind River Conference on Prokaryotic Biology. In the background is Long's Peak.7j][(s, 百拇医药
The work of Sandy Lacks was highlighted at the meeting. Sandy has been a leader in defining genetic transformation in Streptococcus pneumoniae for the last 40 or more years. His talk at the conference is presented elsewhere in this issue (4). Also, the continual good advice of Phil Harriman (National Science Foundation) was noted and recognized. In his comments, Phil reminisced about his 25 years at the National Science Foundation and the major program incentives that were started, mostly to broaden the base of microbiology. He stressed that the peer review system continues to be rigorous and keeps good science going and predicted that individual projects will continue even as money is targeted toward bigger and multidisciplinary efforts.7j][(s, 百拇医药
The scientific sessions included talks and posters. The subject matter ranged from the genetics to the physiology of a diverse series of microorganisms. Among this diversity of presentations, there were several areas of emphasis which reflect the scientific sense of the Wind River Conferences.
QUORUM SENSING AND TWO-COMPONENT SYSTEMS{z'$*, http://www.100md.com
Barry Wanner (Purdue University) provided the keynote address on two-component regulatory systems. His laboratory is characterizing the intermolecular interactions between histidine kinases and response regulators. He described PhoBAR mutants that have altered reactivity with histidine kinases such as VanS. VanS-mediated activation of some of these mutant response regulators is increased as much as 400-fold compared to the activation of wild-type PhoB by VanS. Intramolecular events controlling response regulators were described by Don Walthers and coworkers (Oregon Health Sciences University), who used an innovative chimera approach to understand the roles played by the interdomain linkers of OmpR and PhoB on their respective effector domains. Despite their overall molecular similarities, the N terminus of PhoB inhibits its C-terminal effector domain, while the N terminus of OmpR activates its effector domain. In both cases, the chimeras demonstrated that this mechanism of action requires the interdomain linker region between the N- and C-terminal domains (7). Tracy Nixon and coworkers (Pennsylvania State University) used X-ray crystallographic data to demonstrate the subtle molecular changes involved in the response regulator output level in Sinorhizobium meliloti. Specifically, the DctD receiver domain exhibits very different dimeric structures, depending on whether it is in the "on" or "off" state. This structural alteration favors sigma factor interaction. C. M. Vahling and K. S. McIver (University of Texas, Southwest Medical Center) used mutational analysis to study the role of Mga, a multiple virulence gene regulator of group A streptococcus in two-component signal transduction. Preliminary results show that one of the two putative receiver domains in this important protein acts as the receiver.
More global studies of two-component regulatory systems were presented by Malcolm Winkler's group (Lilly Research Labs). They used microarray and two-dimensional gel electrophoresis technology to identify the targets of the known two-component regulatory systems in Streptococcus pneumoniae and Haemophilus influenzae. Their laboratory has also studied specific inhibitors of histidine kinase activity. These inhibitors function by either blocking ATP binding or by causing protein aggregation. Finally, Shelly Haydel and coworkers (Washington University) have employed the selective capture of transcribed sequences technique to categorize the expression profiles of the 11 paired two-component regulatory systems, as well as orphan histidine kinase and response regulator genes predicted by the genomic sequence in Mycobacterium tuberculosis. They found that some of these systems operated under all conditions, others operated during growth in broth, some were differentially expressed when inside macrophages, and one response regulator was expressed under any conditions tested.
The discussion of quorum sensing was initiated by Stephen Farrand (University of Illinois), who gave a keynote address on his work on quorum-dependent regulation of Ti plasmid conjugal transfer in Agrobacterium tumefaciens. His studies demonstrate that TraR activated by the acyl-homoserine lactone quorum-sensing signal is targeted for degradation by TraM utilizing a Lon protease-dependent mechanism. This mechanism then serves to deactivate the conjugation system when the environmental conditions no longer favor Ti plasmid transfer. M. Marketon and J. Gonzalez (University of Texas, Dallas) demonstrated that quorum sensing by S. meliloti during root colonization plays a significant regulatory role in production of a symbiotically important exopolysaccharide, exopolysaccharide II. They also found that sinRI regulates the synthesis of exopolysaccharide II and abolishes the invasion of alfalfa nodules by S. meliloti (5). M. Antiporta and G. Dunny (University of Minnesota) described a unique strategy used by enterococci whereby the quorum-sensing peptide is encoded within the signal sequence of a prolipoprotein. Subsequent processing was shown to be mediated by the Eep protein, along with another, unknown protease. The exogenous pheromone is detected by the PrgZ protein, which is homologous to OppA. Everett Pesci's laboratory (East Carolina University) examined a novel aspect of quorum sensing which involved the production of a Pseudomonas quinolone signal molecule to affect the activity of the Pseudomonas aeruginosa LasR regulatory protein. The production of this molecule appears to be regulated by the well-described las and rhl quorum-sensing systems. Also, O. Johnsborg and coworkers (Agricultural University of Norway) explored the molecular interactions of the autoinducing peptide, PlnA, with the N-terminal membrane domain of its receptor, the PlnB sensor protein in Lactobacillus plantarum.
ENVIRONMENTAL STRESS AND RESPONSE SYSTEMSo, 百拇医药
Bacteria respond to adverse conditions by activating genes and synthesizing proteins, which help to survive the challenge. E. Mee and N. Welker (Northwestern University) described the initial isolation of specific proteins which are accelerated in synthesis following a shift from 50 to 65°C in Geobacillus stearothermophilus. Their approach was to isolate proteins from two-dimensional gels and then sequence them. Their initial screen yielded an ortholog to an oxidative stress protein in Bacillus subtilis, a LuxR homolog, and an ABC transporter analog. Their goal is to determine if any of these induced proteins relate to thermophily. S. Katzif and coworkers (Emory University) isolated mutations in cspA, the major cold shock gene of Staphylococcus aureus. Their work suggests a new mechanism in S. aureus whereby CspA may control the pigmentation of this organism and also the expression of RsbU, which is a phosphatase and activator of sigma-B, a transcription factor regulating many of the virulence functions in this organism. C. Spence and C. J. Smith (East Carolina University) described the upregulation of an outer membrane protein in Bacteroides fragilis, following exposure of this anaerobe to molecular oxygen and consequently elaboration of the oxidative stress response. This response was OxyR independent. C. J. Sund, from the same laboratory, reported that thiol peroxidase and its putative transporter protein are both induced by oxidative stress and that this upregulation also occurs in the oxyR mutant. The oxidative response in B. fragilis includes 30 or more proteins in multiple regulons. L. Clements and coworkers (University of Louisville and Genencor International) reported on an aspect of anaerobic growth of B. subtilis. Although anaerobic growth of B. subtilis has been described, these investigators looked at sporulation by this organism under anaerobic conditions, where growth is severely slowed. Sporulation is a very ordered growth-dependent process; nevertheless, these organisms sporulated quite well and made spores that resembled aerobically generated spores. However, the anaerobically generated spores were not quite as heat resistant and germinated more poorly, suggesting that the sporulation process, though successful, is aberrant.
N. Kaldalu and coworkers (Northeastern University) used genome arrays to detail which genes are involved in the control of bacterial death by antibiotics. They have isolated a highly persistent (hip) mutant of Escherichia coli which can survive high doses of several antibiotics. If they inactivate the phoB operon, this tolerance disappears. A putative membrane protein (YebE) appears to be necessary for killing by antibiotics. The gene is induced by antibiotics, and deletion of this gene results in a Hip phenotype.3efd, http://www.100md.com
GENE FUNCTION AND REGULATION3efd, http://www.100md.com
Many advances were reported toward understanding the function and regulation of important genes in both gram-positive and gram-negative bacteria. Several reports described genome-wide methods to elucidate function. D. A. Ribardo and K. S. McIver (University of Texas, Southwestern Medical Center) used traditional transposon mutagenesis and a fusion reporter to identify factors that modulate the function of the multiple virulence gene regulator (mga) of group A streptococci. Insertions in genes encoding a putative membrane protein (rmeB) or in a homolog to the comEC genes reduced or eliminated mga-mediated regulation, respectively. Mutations in any of the 13 two-component systems identified in Streptococcus pyogenes failed to affect mga-mediated activation in the genetic backgrounds and conditions tested to date. S. Hollingshead and coworkers (University of Alabama, Birmingham) used different methods to assemble phylogenetic comparisons of numerous clinical and laboratory isolates of S. pneumoniae, showing that the isolates displayed extreme chromosome plasticity. W.-L. Ng and coworkers (Lilly Research Labs) described microarray analyses of transcripts for S. pneumoniae R6 treated with sublethal concentrations of various translation inhibitors. There were expected and unexpected patterns of regulation, including increased transcription of genes encoding the translation apparatus, decreased transcription of genes encoding amino acid biosynthetic enzymes and aminoacyl-tRNA synthetases, and induction of genes that mediate de novo purine biosynthesis and salvage. Surprisingly, cluster analysis revealed that the global transcription patterns were strongly correlated with the mechanisms of translation inhibition and could be used as signatures for testing putative new translation inhibitors (6).
Specific approaches were used to analyze essentiality and function of genes in pathogenic bacteria. C. Trepod and J. Mott (Pharmacia) amplified relatively small regions (~ 3 kb) around possible essential genes in H. influenzae Rd. They mutagenized these fragments in vitro with a minitransposon that imparts kanamycin resistance and then transformed the fragments back into H. influenzae. The absence of transposon insertions at the 5' end of the genes was used as an initial criterion for possible essentiality. Using a different approach, B. Desai and D. A. Morrison (University of Illinois, Chicago) used an integration vector to create merodiploids containing wild-type and lacZ fusion copies of genes that likely play roles in choline metabolism in S. pneumoniae. The putative SP1860 choline transport operon, the licD2 gene, required for choline insertion into teichoic acid, and the licC gene, which may mediate choline metabolism, were examined by this technique. Only the licD2-lacZ fusion showed significant (30-fold) induction upon choline deprivation (1).
Several papers reported on the in vitro and in vivo function of genes in the important livestock pathogen Brucella abortus. Proteomic studies from the laboratory of R. M. Roop (East Carolina University) revealed that the levels of about 40 polypeptides were different in an hfq mutant compared to the isogenic parental strain. Hfq function had previously been shown to be important in stationary-phase survival and virulence of B. abortus. Some notable polypeptides which were different included the products of an hdeA homolog in the gadBC-gls-hdeA cluster, which in other bacteria mediates acid resistance, and thi-5, which is involved in thiamine biosynthesis. T. D. Brown and R. M Roop constructed gadBC mutants, which showed a 4-log decrease in survival compared to the parental strain upon exposure to pH 3.5 in the stationary phase of growth. This mutant failed to show any defect in an in vivo mouse spleen colonization model. J. E. Baumgartner and R. M. Roop created a thi-5 ortholog mutant, which did not survive as well as the parent in late stationary phase in minimal medium, consistent with deprivation of nutrients. This mutation also did not affect ability of the bacteria to colonize in a mouse spleen model. R. D. Read and coworkers constructed a mutation in the relA ortholog of B. abortus. Consistent with a defect in stringent response, the relA mutant showed lags in growth compared to the parent strain. This mutant showed no significant difference in survival in macrophages but was cleared more rapidly from the spleen than its parent in a mouse model. Issues about polarity of this mutation on downstream genes remain to be resolved.
In addition to the linker region studies reported by Walthers and coworkers (see above), S. Palacios and J. C. Escalante-Semerena (University of Wisconsin) characterized the Salmonella enterica PrpR regulator. This protein is a sigma-54-dependent transcriptional activator and is required for the expression of the genes that catabolize propionate. They showed that deletion of the N-terminal domain locked PrpR into a constitutively active conformation that was no longer sensitive to the coactivator 2-methylcitrate. Furthermore, point mutations that caused various degrees of activation were all confined to the N-terminal region of PrpR and not found in the linker to the other domains of the protein. These results support a model in which the N-terminal domain acts as the negative regulator of PrpR activator function.i, http://www.100md.com
Several papers revealed complicated patterns of transcriptional regulation in a variety of operons and bacteria. I. Nes and coworkers (Agricultural University of Norway) described a complicated three component system that regulates bacteriocin production in L. plantarum C11. This system consists of a peptide pheromone (PlnA), a histidine protein kinase (PlnB), and two similar response regulators (PlnC and PlnD). PlnC acts as an activator and PlnD acts as a repressor of bacteriocin production. Five promoters for bacteriocin synthesis with different strengths provide a strong autoregulatory loop and an ordered decline in gene expression. K. Rice and coworkers (University of Idaho) reported the transcript analyses of the S. aureus cidABC locus, which positively regulates murein hydrolase activity and may encode holin-like proteins. Both reverse transcription-PCR and Northern blot analyses indicated that cidABC is transcribed maximally during early exponential growth and that the cidC gene is contained in transcripts of several sizes. These results suggest that overlapping transcripts from the locus might arise from multiple promoters or transcript processing sites.
G.-R. Wang and coworkers (University of Illinois, Urbana) reported on the complex regulation of the tetQ-rteABC central control region of the conjugative transposon CtnDOT from Bacteroides spp. Using a translation fusion system, they found that none of the genes in the central control region are required directly for induction of expression by tetracycline. Instead, tetracycline induction appears to be mediated by at least three mechanisms: possible activation (~ 14-fold) of the Pq promoter region located upstream of tetQ, translational attenuation (~ 4-fold), and an unknown mechanism possibly involving mRNA stabilization (~ 8-fold). N. Shoemaker and coworkers (University of Illinois, Urbana) dissected the genes needed by the CtnDOT control region for excision of the mobilizable transposon NBU1. Their results showed that only the two-component regulatory system RteAB is necessary. Furthermore, RteAB-mediated induction did not increase the expression of the IntN1 integrase, the PrmN1 putative primase, or the MobN1 mobilization nickase but did require cis-acting regions in the mobN1 and prmN1 genes.
Finally, researchers from the laboratory of F. M. Hulett (University of Illinois, Chicago) presented several reports that further the understanding of the mechanisms of phosphate regulation in B. subtilis. S. Paul and F. M. Hulett discovered that the phoPR operon, encoding the PhoP-PhoR two-component system, which regulates the phosphate starvation response, is transcribed from four separate promoters. The operon is autoregulated by binding phosphorylated Pho (Pho~ P) to three sites in the promoter regions and one site in the phoP coding region. Three different sigma factors and Pho~ P are involved in this complex transcription system, and a good model suggests that contributions of the different promoters depend on growth conditions and stage. A cre element, which could bind the CcpA carbon catabolite protein A, is located upstream of this multipromoter region. By changing medium ingredients, A. P. Taneja and F. M. Hulett were able to obtain comparable growth conditions for ccpA+ and ccpA mutant strains. Using these strains, the authors showed that CcpA acts as a repressor of phoPR transcription under phosphate-replete conditions but does not control the signal for operon activation during phosphate limitation.
Activation of the Pho regulon also depends on other factors, notably the ResDE two-component regulatory system, which is involved in both aerobic and anaerobic respiration and is needed for 80% of Pho regulon activation. W. R. Abdel-Fattah and F. M. Hulett found that transcription from the resABCDE operon also initiates from multiple promoters and is growth and medium dependent. Both Pho~ P and ResD~ P are required for activation of transcription from this operon during phosphate limitation, whereas ResD is sufficient at the same start site under sporulation conditions. To understand the mechanism of ResD action, M. Schau and F. M. Hulett isolated bypass compensatory (cmp) mutations in a deleted resD mutant. Some of these mutations fully restored Pho regulon activation. Mapping by transposons revealed that this restoration of Pho regulon activity required cytochrome bd function and that the cmp mutant contained higher cytochrome bd levels than did the original deletion strain.@x-5n+, 百拇医药
GENETIC TRANSFER
Since this meeting has its roots firmly in bacterial transformation and genetic exchange in general, there were many presentations reporting the use of genetic exchange as a tool for studies on subjects other than the molecular aspects of the exchange reaction itself. Nevertheless, there were also papers that concentrated on the mechanism of genetic exchange.5$, 百拇医药
In the last few years, Don Morrison's laboratory (University of Illinois, Chicago) has been involved in much of the molecular description of S. pneumoniae transformation. P. Luo and D. A. Morrison purified the ComX protein, which is postulated to be a transcription activator for many of the late competence genes in this organism. When the purified protein was added to an RNA polymerase from a comX mutant strain, the holoenzyme could transcribe several competence-specific genes. ComX is short-lived and can be purified only from competent S. pneumoniae cells. K. C. Sung and coworkers, from the same laboratory, used DNA microarrays to identify competence-stimulating peptide-responsive genes in the genome of S. pneumoniae. They identified several genes that were indispensable for transformation and others which had markedly decreased competence. This approach should net most of the genes necessary for development of competence and DNA uptake in this organism.
H. Hirt and coworkers (University of Minnesota School of Medicine) presented data on plasmid transfer in Enterococcus faecalis. This system operates through sex pheromone production and plasmids that respond to this pheromone. The recipients secrete the small peptide, and within 30 min the plasmids in the donor respond to the peptide and are transferred to the recipient. The plasmid codes for a surface protein that fosters cell-to-cell contact. The gene for this protein is highly conserved among plasmids and the surface protein is also a virulence factor for this organism. Conditions in vivo favor plasmid transfer, and the aggregating protein is expressed in human plasma. G. Whittle and coworkers (University of Illinois, Urbana) described the characterization of genes that are involved in conjugal transfer of the Bacteroides transposon CtnDOT. They previously found that the 18-kb transfer region of this transposon could be cloned as a transfer-proficient unit. In this work, they constructed a plasmid-based minimum transfer system, allowing them to uncouple excision and transfer. Using this system, they found a 770-bp fragment that represses transfer protein production. They also reported that in the presence of tetracycline, the RteC regulatory protein activates expression of the putative exc topoisomerase gene, which then increases transfer protein expression and frequency of DNA transfer. This process is negatively regulated in the absence of tetracycline at a region in the 3' end of the exc gene.
BACTERIAL PHYSIOLOGY4cl'a, http://www.100md.com
Details of S. aureus lytSR, a complex regulatory locus which controls the activity of murein hydrolases produced by these cells, were described by T. Patton and K. Rice from K. Bayles' laboratory (University of Idaho). An important component of this regulation is the lrgAB operon, whose expression has been shown to inhibit murein hydrolase activity and results in tolerance to penicillin. The LrgA protein has structural similarities to bacteriophage-encoded holins and may function in a manner analogous to that of an inhibitor holin. Analysis of the S. aureus genome revealed the presence of lrgAB homologs (designated cidABC). The cid operon encodes the holin component of this system, which enhances murein hydrolase activity and increases sensitivity not only to penicillin but also to other agents, such as rifampin and mitomycin C. It is interesting that fresh isolates of S. aureus show a much higher level of cidABC expression and are much more susceptible to killing by rifampin than are laboratory strains.
J. A. Opdyke and coworkers (Emory University) reported on a secondary sigma factor (SigX) in the human pathogen S. pyogenes, group A. There are in fact two identical copies of this gene in these streptococci. The sigX gene is homologous to the comX gene from S. pneumoniae, which, as already described (above), transcribes late competence genes in this organism. Although these bacteria do not develop natural competence, many of the genes containing a possible SigX-dependent promoter are homologues to known competence genes in S. pneumoniae. However, many genes which also contain putative SigX-dependent promoters have no analogy to known competence genes. Nevertheless, these findings suggest that the two copies of sigX gene may play an important role in the biology of this organism and suggest that a transformation system could potentially be engineered in this organism.zq.d, http://www.100md.com
Evidence for directed evolution in B. subtilis was presented by W. Samarri and coworkers (New York City Technical College and Hunter College). Using a wild-type relA strain, a relA null mutant, and a relA-overproducing strain, these workers found a positive correlation during leucine and threonine starvation between reversion rates in leuB and thrA genes, (p)ppGpp levels, and the increased levels of leuB and thrA transcripts produced during nutritional stress. In the RelA overproducer, mRNA levels increased about twofold relative to levels in parental strains, while in the null mutant there was a 65 to 85% decrease in mRNA. Also, in the null mutant the mRNA was far more stable than in the parental strain or the overproducer. The number of revertants was substantially increased in the overproducer after starvation compared to the other strains.
E.A. Ling and E. C. Pesci (East Carolina University) described the construction of a plasmid for P. aeruginosa that can be used for a broad-specificity bioassay to detect the presence of a wide range of different autoinducers of quorum-sensing systems. This bioassay could be a useful tool for the detection of cell-to-cell signals from many bacterial species.r:o, 百拇医药
R. Alcantara and R. M. Roop (East Carolina University) reported that transposon disruptions within B. abortus genes required for both amino acid and purine biosynthesis dramatically reduced the intracellular survival of these bacteria in cultured macrophages. This effect was most pronounced in genes for purine biosynthesis.r:o, 百拇医药
Mycobacterial species contain genes encoding two terminal oxidase complexes, aa3-type cytochrome c oxidase and a bd-type quinol oxidase. Expression of bd oxidase has been linked to virulence in the intracellular pathogens Shigella flexneri and B. abortus. J. A. Meghee and coworkers (University of Mississippi) reported that levels of bd oxidase of Mycobacterium smegmatis are increased in macrophages under conditions of reduced oxygen tension. Since M. smegmatis is a useful model for M. tuberculosis pathogenesis, the bd oxidase could serve as a novel drug target for antimicrobials against this human pathogen.
In prokaryotes, the biosynthesis of coenzyme B12 requires enzymes encoded by at least 24 genes. Although much is known about how this important cofactor is synthesized, some functions still are not well understood. There were a number of papers from the laboratory of J. C. Escalante-Semerena (University of Wisconsin) probing some of the unknown functions. J. VanEssendelft used in vitro conditions to show that CobC, an enzyme that catalyzes one of the late steps of coenzyme B12 biosynthesis in S. enterica, has phosphatase activity against two very different substrate intermediates, one of which is attached to the corrin ring through an aminopropanol moiety. Genetic evidence suggests that CobC is involved in the attachment of aminopropanol-P to the corrin ring. However, this phenotype is carbon source specific and may suggest the existence of redundant functions. J. D. Woodson reported that the cobY gene from the extremely halophilic archaeon Halobacterium salinarum is the nonorthologous replacement for the S. enterica cobU gene, encoding bifunctional CobU, which is required for coenzyme B12 synthesis and for the salvage of cobinamide from the environment. CobY was shown to have nucleotidyltransferase activity in vitro but lacked kinase activity. Phenotypic analysis of a cobY mutant revealed that CobY is required for the synthesis of cobamides and that H. salinarum possesses a highly specific salvage transport system (8). This is interesting in view of the fact that archaeal genome databases showed no orthologs to the function used by bacteria to salvage the precursor cobinamide from the environment.
V. J. Starai and J. C. Escalante-Semerena reported that mutants of S. enterica, which are deficient in sirtuins (NAD+-dependent protein deacetylases), are unable to utilize short-chain fatty acids such as propionate and acetate as carbon and energy sources. Sirtuin-deficient strains lack acetyl coenzyme A (acetyl-CoA) synthetase activity. Purified acetyl-CoA synthetase from these sirtuin-deficient strains was inactive but was converted to the active form upon incubation with homogeneous sirtuin and substrate levels of NAD+. The presenters proposed that sirtuins control the level of acetyl-CoA synthetase in the cell by controlling the acetylation state of the enzyme.}x0m:, 百拇医药
Aminoimidazole ribotide (AIR) is the last common intermediate between the thiamine and purine biosynthetic pathways. PurF, one of five enzymes involved in AIR synthesis, catalyzes the first common step in the biosynthesis of thiamine and purines in which phosphoribosylpyrophosphate and glutamine are converted to phosphoribosylamine (PRA) and glutamate. I. Ramos-Solis and D. M. Downs (University of Wisconsin) showed that mutations in purF result in purine auxotrophy but thiamine auxotrophy is conditional. Genetic and biochemical data show that purF-independent thiamine biosynthesis is carried out by the four subsequent enzymes, synthesizing AIR from PRA. This alternative route requires a functional oxidative pentose phosphate pathway. A point mutation which could bypass this requirement and restore thiamine biosynthesis was mapped to a region of the tryptophan operon. The presenters postulated that an altered enzyme can then catalyze PRA synthesis from ribose-5-phosphate and either glutamine or asparagine. Further conversion of AIR to hydroxymethyl pyrimidine phosphate was described by Dougherty and Downs (University of Wisconsin) (2).
CONCLUSIONS/u'@, 百拇医药
The Wind River Conference on Prokaryotic Biology is unique in that it is a small but very diverse meeting. Scientists working in a plethora of biological systems and using varied techniques can come together and demonstrate that common goals can be achieved. This is shown by the variety of organisms and approaches detailed under each of the meeting summary headings. The conference provides students and scientists in training with the same broad perspective. Students can learn that presentations, where there is no similarity to their work or organism of choice, can still provide ideas and approaches that can apply directly to their own research./u'@, 百拇医药
The 47th Wind River Conference will be 4 to 8 June 2003 at Aspen Lodge in Estes Park, Colo./u'@, 百拇医药
ACKNOWLEDGMENTS/u'@, 百拇医药
We thank Aspen Lodge and acknowledge the generous support for this meeting from Diversa, Eli Lilly and Company, Genencor International, Laboratory for Molecular Biology, University of Illinois, Chicago, National Science Foundation, Oxford University Press, Pharmacia Corporation, and Wiley-Liss Publishers.
REFERENCES5p&\;3v, 百拇医药
Desai, B. V., H. Reiter, and D. A. Morrison. 2003. Choline starvation induces the gene licD2 in Streptococcus pneumoniae. J. Bacteriol. 185:710-025p&\;3v, 百拇医药
Dougherty, M., and D. M. Downs. 2003. The stm4066 gene product of Salmonella enterica serovar Typhimurium has aminoimidazole riboside (AIRs) kinase activity and allows AIRs to satisfy the thiamine requirement of pur mutant strains. J. Bacteriol. 185:714-025p&\;3v, 百拇医药
Goodgal, S. 1982. The origin of the transformation meetings, p. 19-23. In U. N. Streips, S. H. Goodgal, W. R. Guild, and G. A. Wilson (ed.), Genetic exchange: a celebration and a new generation. Marcel Dekker, New York, N.Y.5p&\;3v, 百拇医药
Lacks, S. A. 2003. Rambling and scrambling in bacterial transformation: a historical and personal memoir. J. Bacteriol. 185:1037-025p&\;3v, 百拇医药
Marketon, M. M., S. A. Glenn, A. Eberhard, and J. E. Gonzalez. 2003. Quorum sensing controls exopolysaccharide production in Sinorhizobium meliloti. J. Bacteriol. 185:926-025p&\;3v, 百拇医药
Ng, W.-L., K. M. Kazmierczak, G. T. Robertson, R. Gilmour, and M. E. Winkler. 2003. Transcriptional regulation and signature patterns revealed by microarray analyses of Streptococcus pneumoniae R6 challenged with sublethal concentrations of translation inhibitors. J. Bacteriol. 185:772-025p&\;3v, 百拇医药
Walthers, D., V. K. Tran, and L. J. Kenney. 2003. Interdomain linkers of homologous response regulators determine their mechanism of action. J. Bacteriol. 185:715-025p&\;3v, 百拇医药
Woodson, J. D., R. F. Peck, M. P. Krebs, and J. C. Escalante-Semerena. 2003. The cobY gene of the archaeon Halobacterium sp. strain NRC-1 is required for de novo cobamide synthesis. J. Bacteriol. 185:712-02(Kenneth W. Bayles Neil E. Welker Malcolm E. Winkler and Uldis N. Streips)
Received 8 August 2002/ Accepted 8 October 2002/, http://www.100md.com
INTRODUCTION/, http://www.100md.com
The Wind River Conference, which was begun as the Transformation Meeting in 1957, was renamed the Wind River Conference on Genetic Exchange and ultimately became the Wind River Conference on Prokaryotic Biology to expand the range of areas of discussion and presentation (3). Initially, the discussion centered on bacterial transformation, and many of the seminal findings in that field were reported at this meeting, but recently the conferences have covered a wide range of subject areas from genetics to physiology of many diverse genera of bacteria. In that aspect this meeting is unique, because students and experts working on many different microbial systems can find a common ground for discussion and expansion of ideas. The Wind River Conferences also encourage students to present and even lead sessions. This has provided an excellent introduction to scientific presentation and discussion for a number of scientists across the country, many of whom regularly attend the Wind River Conferences and bring their students here. Since the meeting is held at a relatively remote site, there is ample opportunity to relax together with speakers and informally discuss anything that is of further interest and to develop contacts and friendships. This year's meeting was the 46th in the series. The site for the meeting has varied over the years but mostly has been in the Estes Park area of Colorado and was fixed in this location several years ago. The 46th Wind River Conference took place from 3 to 8 June 2002 at Aspen Lodge in Estes Park, with 140 participants .
fig.ommitted7j][(s, 百拇医药
Attendees of the 46th Wind River Conference on Prokaryotic Biology. In the background is Long's Peak.7j][(s, 百拇医药
The work of Sandy Lacks was highlighted at the meeting. Sandy has been a leader in defining genetic transformation in Streptococcus pneumoniae for the last 40 or more years. His talk at the conference is presented elsewhere in this issue (4). Also, the continual good advice of Phil Harriman (National Science Foundation) was noted and recognized. In his comments, Phil reminisced about his 25 years at the National Science Foundation and the major program incentives that were started, mostly to broaden the base of microbiology. He stressed that the peer review system continues to be rigorous and keeps good science going and predicted that individual projects will continue even as money is targeted toward bigger and multidisciplinary efforts.7j][(s, 百拇医药
The scientific sessions included talks and posters. The subject matter ranged from the genetics to the physiology of a diverse series of microorganisms. Among this diversity of presentations, there were several areas of emphasis which reflect the scientific sense of the Wind River Conferences.
QUORUM SENSING AND TWO-COMPONENT SYSTEMS{z'$*, http://www.100md.com
Barry Wanner (Purdue University) provided the keynote address on two-component regulatory systems. His laboratory is characterizing the intermolecular interactions between histidine kinases and response regulators. He described PhoBAR mutants that have altered reactivity with histidine kinases such as VanS. VanS-mediated activation of some of these mutant response regulators is increased as much as 400-fold compared to the activation of wild-type PhoB by VanS. Intramolecular events controlling response regulators were described by Don Walthers and coworkers (Oregon Health Sciences University), who used an innovative chimera approach to understand the roles played by the interdomain linkers of OmpR and PhoB on their respective effector domains. Despite their overall molecular similarities, the N terminus of PhoB inhibits its C-terminal effector domain, while the N terminus of OmpR activates its effector domain. In both cases, the chimeras demonstrated that this mechanism of action requires the interdomain linker region between the N- and C-terminal domains (7). Tracy Nixon and coworkers (Pennsylvania State University) used X-ray crystallographic data to demonstrate the subtle molecular changes involved in the response regulator output level in Sinorhizobium meliloti. Specifically, the DctD receiver domain exhibits very different dimeric structures, depending on whether it is in the "on" or "off" state. This structural alteration favors sigma factor interaction. C. M. Vahling and K. S. McIver (University of Texas, Southwest Medical Center) used mutational analysis to study the role of Mga, a multiple virulence gene regulator of group A streptococcus in two-component signal transduction. Preliminary results show that one of the two putative receiver domains in this important protein acts as the receiver.
More global studies of two-component regulatory systems were presented by Malcolm Winkler's group (Lilly Research Labs). They used microarray and two-dimensional gel electrophoresis technology to identify the targets of the known two-component regulatory systems in Streptococcus pneumoniae and Haemophilus influenzae. Their laboratory has also studied specific inhibitors of histidine kinase activity. These inhibitors function by either blocking ATP binding or by causing protein aggregation. Finally, Shelly Haydel and coworkers (Washington University) have employed the selective capture of transcribed sequences technique to categorize the expression profiles of the 11 paired two-component regulatory systems, as well as orphan histidine kinase and response regulator genes predicted by the genomic sequence in Mycobacterium tuberculosis. They found that some of these systems operated under all conditions, others operated during growth in broth, some were differentially expressed when inside macrophages, and one response regulator was expressed under any conditions tested.
The discussion of quorum sensing was initiated by Stephen Farrand (University of Illinois), who gave a keynote address on his work on quorum-dependent regulation of Ti plasmid conjugal transfer in Agrobacterium tumefaciens. His studies demonstrate that TraR activated by the acyl-homoserine lactone quorum-sensing signal is targeted for degradation by TraM utilizing a Lon protease-dependent mechanism. This mechanism then serves to deactivate the conjugation system when the environmental conditions no longer favor Ti plasmid transfer. M. Marketon and J. Gonzalez (University of Texas, Dallas) demonstrated that quorum sensing by S. meliloti during root colonization plays a significant regulatory role in production of a symbiotically important exopolysaccharide, exopolysaccharide II. They also found that sinRI regulates the synthesis of exopolysaccharide II and abolishes the invasion of alfalfa nodules by S. meliloti (5). M. Antiporta and G. Dunny (University of Minnesota) described a unique strategy used by enterococci whereby the quorum-sensing peptide is encoded within the signal sequence of a prolipoprotein. Subsequent processing was shown to be mediated by the Eep protein, along with another, unknown protease. The exogenous pheromone is detected by the PrgZ protein, which is homologous to OppA. Everett Pesci's laboratory (East Carolina University) examined a novel aspect of quorum sensing which involved the production of a Pseudomonas quinolone signal molecule to affect the activity of the Pseudomonas aeruginosa LasR regulatory protein. The production of this molecule appears to be regulated by the well-described las and rhl quorum-sensing systems. Also, O. Johnsborg and coworkers (Agricultural University of Norway) explored the molecular interactions of the autoinducing peptide, PlnA, with the N-terminal membrane domain of its receptor, the PlnB sensor protein in Lactobacillus plantarum.
ENVIRONMENTAL STRESS AND RESPONSE SYSTEMSo, 百拇医药
Bacteria respond to adverse conditions by activating genes and synthesizing proteins, which help to survive the challenge. E. Mee and N. Welker (Northwestern University) described the initial isolation of specific proteins which are accelerated in synthesis following a shift from 50 to 65°C in Geobacillus stearothermophilus. Their approach was to isolate proteins from two-dimensional gels and then sequence them. Their initial screen yielded an ortholog to an oxidative stress protein in Bacillus subtilis, a LuxR homolog, and an ABC transporter analog. Their goal is to determine if any of these induced proteins relate to thermophily. S. Katzif and coworkers (Emory University) isolated mutations in cspA, the major cold shock gene of Staphylococcus aureus. Their work suggests a new mechanism in S. aureus whereby CspA may control the pigmentation of this organism and also the expression of RsbU, which is a phosphatase and activator of sigma-B, a transcription factor regulating many of the virulence functions in this organism. C. Spence and C. J. Smith (East Carolina University) described the upregulation of an outer membrane protein in Bacteroides fragilis, following exposure of this anaerobe to molecular oxygen and consequently elaboration of the oxidative stress response. This response was OxyR independent. C. J. Sund, from the same laboratory, reported that thiol peroxidase and its putative transporter protein are both induced by oxidative stress and that this upregulation also occurs in the oxyR mutant. The oxidative response in B. fragilis includes 30 or more proteins in multiple regulons. L. Clements and coworkers (University of Louisville and Genencor International) reported on an aspect of anaerobic growth of B. subtilis. Although anaerobic growth of B. subtilis has been described, these investigators looked at sporulation by this organism under anaerobic conditions, where growth is severely slowed. Sporulation is a very ordered growth-dependent process; nevertheless, these organisms sporulated quite well and made spores that resembled aerobically generated spores. However, the anaerobically generated spores were not quite as heat resistant and germinated more poorly, suggesting that the sporulation process, though successful, is aberrant.
N. Kaldalu and coworkers (Northeastern University) used genome arrays to detail which genes are involved in the control of bacterial death by antibiotics. They have isolated a highly persistent (hip) mutant of Escherichia coli which can survive high doses of several antibiotics. If they inactivate the phoB operon, this tolerance disappears. A putative membrane protein (YebE) appears to be necessary for killing by antibiotics. The gene is induced by antibiotics, and deletion of this gene results in a Hip phenotype.3efd, http://www.100md.com
GENE FUNCTION AND REGULATION3efd, http://www.100md.com
Many advances were reported toward understanding the function and regulation of important genes in both gram-positive and gram-negative bacteria. Several reports described genome-wide methods to elucidate function. D. A. Ribardo and K. S. McIver (University of Texas, Southwestern Medical Center) used traditional transposon mutagenesis and a fusion reporter to identify factors that modulate the function of the multiple virulence gene regulator (mga) of group A streptococci. Insertions in genes encoding a putative membrane protein (rmeB) or in a homolog to the comEC genes reduced or eliminated mga-mediated regulation, respectively. Mutations in any of the 13 two-component systems identified in Streptococcus pyogenes failed to affect mga-mediated activation in the genetic backgrounds and conditions tested to date. S. Hollingshead and coworkers (University of Alabama, Birmingham) used different methods to assemble phylogenetic comparisons of numerous clinical and laboratory isolates of S. pneumoniae, showing that the isolates displayed extreme chromosome plasticity. W.-L. Ng and coworkers (Lilly Research Labs) described microarray analyses of transcripts for S. pneumoniae R6 treated with sublethal concentrations of various translation inhibitors. There were expected and unexpected patterns of regulation, including increased transcription of genes encoding the translation apparatus, decreased transcription of genes encoding amino acid biosynthetic enzymes and aminoacyl-tRNA synthetases, and induction of genes that mediate de novo purine biosynthesis and salvage. Surprisingly, cluster analysis revealed that the global transcription patterns were strongly correlated with the mechanisms of translation inhibition and could be used as signatures for testing putative new translation inhibitors (6).
Specific approaches were used to analyze essentiality and function of genes in pathogenic bacteria. C. Trepod and J. Mott (Pharmacia) amplified relatively small regions (~ 3 kb) around possible essential genes in H. influenzae Rd. They mutagenized these fragments in vitro with a minitransposon that imparts kanamycin resistance and then transformed the fragments back into H. influenzae. The absence of transposon insertions at the 5' end of the genes was used as an initial criterion for possible essentiality. Using a different approach, B. Desai and D. A. Morrison (University of Illinois, Chicago) used an integration vector to create merodiploids containing wild-type and lacZ fusion copies of genes that likely play roles in choline metabolism in S. pneumoniae. The putative SP1860 choline transport operon, the licD2 gene, required for choline insertion into teichoic acid, and the licC gene, which may mediate choline metabolism, were examined by this technique. Only the licD2-lacZ fusion showed significant (30-fold) induction upon choline deprivation (1).
Several papers reported on the in vitro and in vivo function of genes in the important livestock pathogen Brucella abortus. Proteomic studies from the laboratory of R. M. Roop (East Carolina University) revealed that the levels of about 40 polypeptides were different in an hfq mutant compared to the isogenic parental strain. Hfq function had previously been shown to be important in stationary-phase survival and virulence of B. abortus. Some notable polypeptides which were different included the products of an hdeA homolog in the gadBC-gls-hdeA cluster, which in other bacteria mediates acid resistance, and thi-5, which is involved in thiamine biosynthesis. T. D. Brown and R. M Roop constructed gadBC mutants, which showed a 4-log decrease in survival compared to the parental strain upon exposure to pH 3.5 in the stationary phase of growth. This mutant failed to show any defect in an in vivo mouse spleen colonization model. J. E. Baumgartner and R. M. Roop created a thi-5 ortholog mutant, which did not survive as well as the parent in late stationary phase in minimal medium, consistent with deprivation of nutrients. This mutation also did not affect ability of the bacteria to colonize in a mouse spleen model. R. D. Read and coworkers constructed a mutation in the relA ortholog of B. abortus. Consistent with a defect in stringent response, the relA mutant showed lags in growth compared to the parent strain. This mutant showed no significant difference in survival in macrophages but was cleared more rapidly from the spleen than its parent in a mouse model. Issues about polarity of this mutation on downstream genes remain to be resolved.
In addition to the linker region studies reported by Walthers and coworkers (see above), S. Palacios and J. C. Escalante-Semerena (University of Wisconsin) characterized the Salmonella enterica PrpR regulator. This protein is a sigma-54-dependent transcriptional activator and is required for the expression of the genes that catabolize propionate. They showed that deletion of the N-terminal domain locked PrpR into a constitutively active conformation that was no longer sensitive to the coactivator 2-methylcitrate. Furthermore, point mutations that caused various degrees of activation were all confined to the N-terminal region of PrpR and not found in the linker to the other domains of the protein. These results support a model in which the N-terminal domain acts as the negative regulator of PrpR activator function.i, http://www.100md.com
Several papers revealed complicated patterns of transcriptional regulation in a variety of operons and bacteria. I. Nes and coworkers (Agricultural University of Norway) described a complicated three component system that regulates bacteriocin production in L. plantarum C11. This system consists of a peptide pheromone (PlnA), a histidine protein kinase (PlnB), and two similar response regulators (PlnC and PlnD). PlnC acts as an activator and PlnD acts as a repressor of bacteriocin production. Five promoters for bacteriocin synthesis with different strengths provide a strong autoregulatory loop and an ordered decline in gene expression. K. Rice and coworkers (University of Idaho) reported the transcript analyses of the S. aureus cidABC locus, which positively regulates murein hydrolase activity and may encode holin-like proteins. Both reverse transcription-PCR and Northern blot analyses indicated that cidABC is transcribed maximally during early exponential growth and that the cidC gene is contained in transcripts of several sizes. These results suggest that overlapping transcripts from the locus might arise from multiple promoters or transcript processing sites.
G.-R. Wang and coworkers (University of Illinois, Urbana) reported on the complex regulation of the tetQ-rteABC central control region of the conjugative transposon CtnDOT from Bacteroides spp. Using a translation fusion system, they found that none of the genes in the central control region are required directly for induction of expression by tetracycline. Instead, tetracycline induction appears to be mediated by at least three mechanisms: possible activation (~ 14-fold) of the Pq promoter region located upstream of tetQ, translational attenuation (~ 4-fold), and an unknown mechanism possibly involving mRNA stabilization (~ 8-fold). N. Shoemaker and coworkers (University of Illinois, Urbana) dissected the genes needed by the CtnDOT control region for excision of the mobilizable transposon NBU1. Their results showed that only the two-component regulatory system RteAB is necessary. Furthermore, RteAB-mediated induction did not increase the expression of the IntN1 integrase, the PrmN1 putative primase, or the MobN1 mobilization nickase but did require cis-acting regions in the mobN1 and prmN1 genes.
Finally, researchers from the laboratory of F. M. Hulett (University of Illinois, Chicago) presented several reports that further the understanding of the mechanisms of phosphate regulation in B. subtilis. S. Paul and F. M. Hulett discovered that the phoPR operon, encoding the PhoP-PhoR two-component system, which regulates the phosphate starvation response, is transcribed from four separate promoters. The operon is autoregulated by binding phosphorylated Pho (Pho~ P) to three sites in the promoter regions and one site in the phoP coding region. Three different sigma factors and Pho~ P are involved in this complex transcription system, and a good model suggests that contributions of the different promoters depend on growth conditions and stage. A cre element, which could bind the CcpA carbon catabolite protein A, is located upstream of this multipromoter region. By changing medium ingredients, A. P. Taneja and F. M. Hulett were able to obtain comparable growth conditions for ccpA+ and ccpA mutant strains. Using these strains, the authors showed that CcpA acts as a repressor of phoPR transcription under phosphate-replete conditions but does not control the signal for operon activation during phosphate limitation.
Activation of the Pho regulon also depends on other factors, notably the ResDE two-component regulatory system, which is involved in both aerobic and anaerobic respiration and is needed for 80% of Pho regulon activation. W. R. Abdel-Fattah and F. M. Hulett found that transcription from the resABCDE operon also initiates from multiple promoters and is growth and medium dependent. Both Pho~ P and ResD~ P are required for activation of transcription from this operon during phosphate limitation, whereas ResD is sufficient at the same start site under sporulation conditions. To understand the mechanism of ResD action, M. Schau and F. M. Hulett isolated bypass compensatory (cmp) mutations in a deleted resD mutant. Some of these mutations fully restored Pho regulon activation. Mapping by transposons revealed that this restoration of Pho regulon activity required cytochrome bd function and that the cmp mutant contained higher cytochrome bd levels than did the original deletion strain.@x-5n+, 百拇医药
GENETIC TRANSFER
Since this meeting has its roots firmly in bacterial transformation and genetic exchange in general, there were many presentations reporting the use of genetic exchange as a tool for studies on subjects other than the molecular aspects of the exchange reaction itself. Nevertheless, there were also papers that concentrated on the mechanism of genetic exchange.5$, 百拇医药
In the last few years, Don Morrison's laboratory (University of Illinois, Chicago) has been involved in much of the molecular description of S. pneumoniae transformation. P. Luo and D. A. Morrison purified the ComX protein, which is postulated to be a transcription activator for many of the late competence genes in this organism. When the purified protein was added to an RNA polymerase from a comX mutant strain, the holoenzyme could transcribe several competence-specific genes. ComX is short-lived and can be purified only from competent S. pneumoniae cells. K. C. Sung and coworkers, from the same laboratory, used DNA microarrays to identify competence-stimulating peptide-responsive genes in the genome of S. pneumoniae. They identified several genes that were indispensable for transformation and others which had markedly decreased competence. This approach should net most of the genes necessary for development of competence and DNA uptake in this organism.
H. Hirt and coworkers (University of Minnesota School of Medicine) presented data on plasmid transfer in Enterococcus faecalis. This system operates through sex pheromone production and plasmids that respond to this pheromone. The recipients secrete the small peptide, and within 30 min the plasmids in the donor respond to the peptide and are transferred to the recipient. The plasmid codes for a surface protein that fosters cell-to-cell contact. The gene for this protein is highly conserved among plasmids and the surface protein is also a virulence factor for this organism. Conditions in vivo favor plasmid transfer, and the aggregating protein is expressed in human plasma. G. Whittle and coworkers (University of Illinois, Urbana) described the characterization of genes that are involved in conjugal transfer of the Bacteroides transposon CtnDOT. They previously found that the 18-kb transfer region of this transposon could be cloned as a transfer-proficient unit. In this work, they constructed a plasmid-based minimum transfer system, allowing them to uncouple excision and transfer. Using this system, they found a 770-bp fragment that represses transfer protein production. They also reported that in the presence of tetracycline, the RteC regulatory protein activates expression of the putative exc topoisomerase gene, which then increases transfer protein expression and frequency of DNA transfer. This process is negatively regulated in the absence of tetracycline at a region in the 3' end of the exc gene.
BACTERIAL PHYSIOLOGY4cl'a, http://www.100md.com
Details of S. aureus lytSR, a complex regulatory locus which controls the activity of murein hydrolases produced by these cells, were described by T. Patton and K. Rice from K. Bayles' laboratory (University of Idaho). An important component of this regulation is the lrgAB operon, whose expression has been shown to inhibit murein hydrolase activity and results in tolerance to penicillin. The LrgA protein has structural similarities to bacteriophage-encoded holins and may function in a manner analogous to that of an inhibitor holin. Analysis of the S. aureus genome revealed the presence of lrgAB homologs (designated cidABC). The cid operon encodes the holin component of this system, which enhances murein hydrolase activity and increases sensitivity not only to penicillin but also to other agents, such as rifampin and mitomycin C. It is interesting that fresh isolates of S. aureus show a much higher level of cidABC expression and are much more susceptible to killing by rifampin than are laboratory strains.
J. A. Opdyke and coworkers (Emory University) reported on a secondary sigma factor (SigX) in the human pathogen S. pyogenes, group A. There are in fact two identical copies of this gene in these streptococci. The sigX gene is homologous to the comX gene from S. pneumoniae, which, as already described (above), transcribes late competence genes in this organism. Although these bacteria do not develop natural competence, many of the genes containing a possible SigX-dependent promoter are homologues to known competence genes in S. pneumoniae. However, many genes which also contain putative SigX-dependent promoters have no analogy to known competence genes. Nevertheless, these findings suggest that the two copies of sigX gene may play an important role in the biology of this organism and suggest that a transformation system could potentially be engineered in this organism.zq.d, http://www.100md.com
Evidence for directed evolution in B. subtilis was presented by W. Samarri and coworkers (New York City Technical College and Hunter College). Using a wild-type relA strain, a relA null mutant, and a relA-overproducing strain, these workers found a positive correlation during leucine and threonine starvation between reversion rates in leuB and thrA genes, (p)ppGpp levels, and the increased levels of leuB and thrA transcripts produced during nutritional stress. In the RelA overproducer, mRNA levels increased about twofold relative to levels in parental strains, while in the null mutant there was a 65 to 85% decrease in mRNA. Also, in the null mutant the mRNA was far more stable than in the parental strain or the overproducer. The number of revertants was substantially increased in the overproducer after starvation compared to the other strains.
E.A. Ling and E. C. Pesci (East Carolina University) described the construction of a plasmid for P. aeruginosa that can be used for a broad-specificity bioassay to detect the presence of a wide range of different autoinducers of quorum-sensing systems. This bioassay could be a useful tool for the detection of cell-to-cell signals from many bacterial species.r:o, 百拇医药
R. Alcantara and R. M. Roop (East Carolina University) reported that transposon disruptions within B. abortus genes required for both amino acid and purine biosynthesis dramatically reduced the intracellular survival of these bacteria in cultured macrophages. This effect was most pronounced in genes for purine biosynthesis.r:o, 百拇医药
Mycobacterial species contain genes encoding two terminal oxidase complexes, aa3-type cytochrome c oxidase and a bd-type quinol oxidase. Expression of bd oxidase has been linked to virulence in the intracellular pathogens Shigella flexneri and B. abortus. J. A. Meghee and coworkers (University of Mississippi) reported that levels of bd oxidase of Mycobacterium smegmatis are increased in macrophages under conditions of reduced oxygen tension. Since M. smegmatis is a useful model for M. tuberculosis pathogenesis, the bd oxidase could serve as a novel drug target for antimicrobials against this human pathogen.
In prokaryotes, the biosynthesis of coenzyme B12 requires enzymes encoded by at least 24 genes. Although much is known about how this important cofactor is synthesized, some functions still are not well understood. There were a number of papers from the laboratory of J. C. Escalante-Semerena (University of Wisconsin) probing some of the unknown functions. J. VanEssendelft used in vitro conditions to show that CobC, an enzyme that catalyzes one of the late steps of coenzyme B12 biosynthesis in S. enterica, has phosphatase activity against two very different substrate intermediates, one of which is attached to the corrin ring through an aminopropanol moiety. Genetic evidence suggests that CobC is involved in the attachment of aminopropanol-P to the corrin ring. However, this phenotype is carbon source specific and may suggest the existence of redundant functions. J. D. Woodson reported that the cobY gene from the extremely halophilic archaeon Halobacterium salinarum is the nonorthologous replacement for the S. enterica cobU gene, encoding bifunctional CobU, which is required for coenzyme B12 synthesis and for the salvage of cobinamide from the environment. CobY was shown to have nucleotidyltransferase activity in vitro but lacked kinase activity. Phenotypic analysis of a cobY mutant revealed that CobY is required for the synthesis of cobamides and that H. salinarum possesses a highly specific salvage transport system (8). This is interesting in view of the fact that archaeal genome databases showed no orthologs to the function used by bacteria to salvage the precursor cobinamide from the environment.
V. J. Starai and J. C. Escalante-Semerena reported that mutants of S. enterica, which are deficient in sirtuins (NAD+-dependent protein deacetylases), are unable to utilize short-chain fatty acids such as propionate and acetate as carbon and energy sources. Sirtuin-deficient strains lack acetyl coenzyme A (acetyl-CoA) synthetase activity. Purified acetyl-CoA synthetase from these sirtuin-deficient strains was inactive but was converted to the active form upon incubation with homogeneous sirtuin and substrate levels of NAD+. The presenters proposed that sirtuins control the level of acetyl-CoA synthetase in the cell by controlling the acetylation state of the enzyme.}x0m:, 百拇医药
Aminoimidazole ribotide (AIR) is the last common intermediate between the thiamine and purine biosynthetic pathways. PurF, one of five enzymes involved in AIR synthesis, catalyzes the first common step in the biosynthesis of thiamine and purines in which phosphoribosylpyrophosphate and glutamine are converted to phosphoribosylamine (PRA) and glutamate. I. Ramos-Solis and D. M. Downs (University of Wisconsin) showed that mutations in purF result in purine auxotrophy but thiamine auxotrophy is conditional. Genetic and biochemical data show that purF-independent thiamine biosynthesis is carried out by the four subsequent enzymes, synthesizing AIR from PRA. This alternative route requires a functional oxidative pentose phosphate pathway. A point mutation which could bypass this requirement and restore thiamine biosynthesis was mapped to a region of the tryptophan operon. The presenters postulated that an altered enzyme can then catalyze PRA synthesis from ribose-5-phosphate and either glutamine or asparagine. Further conversion of AIR to hydroxymethyl pyrimidine phosphate was described by Dougherty and Downs (University of Wisconsin) (2).
CONCLUSIONS/u'@, 百拇医药
The Wind River Conference on Prokaryotic Biology is unique in that it is a small but very diverse meeting. Scientists working in a plethora of biological systems and using varied techniques can come together and demonstrate that common goals can be achieved. This is shown by the variety of organisms and approaches detailed under each of the meeting summary headings. The conference provides students and scientists in training with the same broad perspective. Students can learn that presentations, where there is no similarity to their work or organism of choice, can still provide ideas and approaches that can apply directly to their own research./u'@, 百拇医药
The 47th Wind River Conference will be 4 to 8 June 2003 at Aspen Lodge in Estes Park, Colo./u'@, 百拇医药
ACKNOWLEDGMENTS/u'@, 百拇医药
We thank Aspen Lodge and acknowledge the generous support for this meeting from Diversa, Eli Lilly and Company, Genencor International, Laboratory for Molecular Biology, University of Illinois, Chicago, National Science Foundation, Oxford University Press, Pharmacia Corporation, and Wiley-Liss Publishers.
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Dougherty, M., and D. M. Downs. 2003. The stm4066 gene product of Salmonella enterica serovar Typhimurium has aminoimidazole riboside (AIRs) kinase activity and allows AIRs to satisfy the thiamine requirement of pur mutant strains. J. Bacteriol. 185:714-025p&\;3v, 百拇医药
Goodgal, S. 1982. The origin of the transformation meetings, p. 19-23. In U. N. Streips, S. H. Goodgal, W. R. Guild, and G. A. Wilson (ed.), Genetic exchange: a celebration and a new generation. Marcel Dekker, New York, N.Y.5p&\;3v, 百拇医药
Lacks, S. A. 2003. Rambling and scrambling in bacterial transformation: a historical and personal memoir. J. Bacteriol. 185:1037-025p&\;3v, 百拇医药
Marketon, M. M., S. A. Glenn, A. Eberhard, and J. E. Gonzalez. 2003. Quorum sensing controls exopolysaccharide production in Sinorhizobium meliloti. J. Bacteriol. 185:926-025p&\;3v, 百拇医药
Ng, W.-L., K. M. Kazmierczak, G. T. Robertson, R. Gilmour, and M. E. Winkler. 2003. Transcriptional regulation and signature patterns revealed by microarray analyses of Streptococcus pneumoniae R6 challenged with sublethal concentrations of translation inhibitors. J. Bacteriol. 185:772-025p&\;3v, 百拇医药
Walthers, D., V. K. Tran, and L. J. Kenney. 2003. Interdomain linkers of homologous response regulators determine their mechanism of action. J. Bacteriol. 185:715-025p&\;3v, 百拇医药
Woodson, J. D., R. F. Peck, M. P. Krebs, and J. C. Escalante-Semerena. 2003. The cobY gene of the archaeon Halobacterium sp. strain NRC-1 is required for de novo cobamide synthesis. J. Bacteriol. 185:712-02(Kenneth W. Bayles Neil E. Welker Malcolm E. Winkler and Uldis N. Streips)