Genome-Wide Transposon Mutagenesis Screens Identify Group A Streptococcus Genes Affecting Susceptibility to ß-Lactam Antibiotics.

Group A streptococcus (GAS) is a Gram-positive human bacterial pathogen responsible for more than 700 million infections annually worldwide. Beta-lactam antibiotics are the primary agents used to treat GAS infections. Naturally occurring GAS clinical isolates with decreased susceptibility to beta-lactam antibiotics attributed to mutations in PBP2X have recently been documented. This prompted us to perform a genome-wide screen to identify GAS genes that alter beta-lactam susceptibility in vitro. Using saturated transposon mutagenesis, we screened for GAS gene mutations conferring altered in vitro susceptibility to penicillin G and/or ceftriaxone, two beta-lactam antibiotics commonly used to treat GAS infections. In the aggregate, we found that inactivating mutations in 150 GAS genes are associated with altered susceptibility to penicillin G and/or ceftriaxone. Many of the genes identified were previously not known to alter beta-lactam susceptibility or affect cell wall biosynthesis. Using isogenic mutant strains, we confirmed that inactivation of clpX (Clp protease ATP-binding subunit) or cppA (CppA proteinase) resulted in decreased in vitro susceptibility to penicillin G and ceftriaxone. Deletion of murA1 (UDP-N-acetylglucosamine 1-carboxyvinyltransferase) conferred increased susceptibility to ceftriaxone. Our results provide new information about the GAS genes affecting susceptibility to beta-lactam antibiotics. IMPORTANCE Beta-lactam antibiotics are the primary drugs prescribed to treat infections caused by group A streptococcus (GAS), an important human pathogen. However, the molecular mechanisms of GAS interactions with beta-lactam antibiotics are not fully understood. In this study, we performed a genome-wide mutagenesis screen to identify GAS mutations conferring altered susceptibility to beta-lactam antibiotics. In the aggregate, we discovered that mutations in 150 GAS genes were associated with altered beta-lactam susceptibility. Many identified genes were previously not known to alter beta-lactam susceptibility or affect cell wall biosynthesis. Our results provide new information about the molecular mechanisms of GAS interaction with beta-lactam antibiotics.

Polymorphisms in Regulator of Cov Contribute to the Molecular Pathogenesis of Serotype M28 Group A Streptococcus.

Two-component systems (TCSs) are signal transduction proteins that enable bacteria to respond to external stimuli by altering the global transcriptome. Accessory proteins interact with TCSs to fine-tune their activity. In group A Streptococcus (GAS), regulator of Cov (RocA) is an accessory protein that functions with the control of virulence regulator/sensor TCS, which regulates approximately 15% of the GAS transcriptome. Whole-genome sequencing analysis of serotype M28 GAS strains collected from invasive infections in humans identified a higher number of missense (amino acid-altering) and nonsense (protein-truncating) polymorphisms in rocA than expected. We hypothesized that polymorphisms in RocA alter the global transcriptome and virulence of serotype M28 GAS. We used naturally occurring clinical isolates with rocA polymorphisms (n = 48), an isogenic rocA deletion mutant strain, and five isogenic rocA polymorphism mutant strains to perform genome-wide transcript analysis (RNA sequencing), in vitro virulence factor assays, and mouse and nonhuman primate pathogenesis studies to test this hypothesis. Results demonstrated that polymorphisms in rocA result in either a subtle transcriptome change, causing a wild-type-like virulence phenotype, or a substantial transcriptome change, leading to a significantly increased virulence phenotype. Each polymorphism had a unique effect on the global GAS transcriptome. Taken together, our data show that naturally occurring polymorphisms in one gene encoding an accessory protein can significantly alter the global transcriptome and virulence phenotype of GAS, an important human pathogen.

RocA Has Serotype-Specific Gene Regulatory and Pathogenesis Activities in Serotype M28 Group A Streptococcus.

Serotype M28 group A streptococcus (GAS) is a common cause of infections such as pharyngitis ("strep throat") and necrotizing fasciitis ("flesh-eating" disease). Relatively little is known about the molecular mechanisms underpinning M28 GAS pathogenesis. Whole-genome sequencing studies of M28 GAS strains recovered from patients with invasive infections found an unexpectedly high number of missense (amino acid-changing) and nonsense (protein-truncating) polymorphisms in rocA (regulator of Cov), leading us to hypothesize that altered RocA activity contributes to M28 GAS molecular pathogenesis. To test this hypothesis, an isogenic rocA deletion mutant strain was created. Transcriptome sequencing (RNA-seq) analysis revealed that RocA inactivation significantly alters the level of transcripts for 427 and 323 genes at mid-exponential and early stationary growth phases, respectively, including genes for 41 transcription regulators and 21 virulence factors. In contrast, RocA transcriptomes from other GAS M protein serotypes are much smaller and include fewer transcription regulators. The rocA mutant strain had significantly increased secreted activity of multiple virulence factors and grew to significantly higher colony counts under acid stress in vitro RocA inactivation also significantly increased GAS virulence in a mouse model of necrotizing myositis. Our results demonstrate that RocA is an important regulator of transcription regulators and virulence factors in M28 GAS and raise the possibility that naturally occurring polymorphisms in rocA in some fashion contribute to human invasive infections caused by M28 GAS strains.

Genomic Landscape of Intrahost Variation in Group A Streptococcus: Repeated and Abundant Mutational Inactivation of the fabT Gene Encoding a Regulator of Fatty Acid Synthesis.

To obtain new information about Streptococcus pyogenes intrahost genetic variation during invasive infection, we sequenced the genomes of 2,954 serotype M1 strains recovered from a nonhuman primate experimental model of necrotizing fasciitis. A total of 644 strains (21.8%) acquired polymorphisms relative to the input parental strain. The fabT gene, encoding a transcriptional regulator of fatty acid biosynthesis genes, contained 54.5% of these changes. The great majority of polymorphisms were predicted to deleteriously alter FabT function. Transcriptome-sequencing (RNA-seq) analysis of a wild-type strain and an isogenic fabT deletion mutant strain found that between 3.7 and 28.5% of the S. pyogenes transcripts were differentially expressed, depending on the growth temperature (35°C or 40°C) and growth phase (mid-exponential or stationary phase). Genes implicated in fatty acid synthesis and lipid metabolism were significantly upregulated in the fabT deletion mutant strain. FabT also directly or indirectly regulated central carbon metabolism genes, including pyruvate hub enzymes and fermentation pathways and virulence genes. Deletion of fabT decreased virulence in a nonhuman primate model of necrotizing fasciitis. In addition, the fabT deletion strain had significantly decreased survival in human whole blood and during phagocytic interaction with polymorphonuclear leukocytes ex vivo We conclude that FabT mutant progeny arise during infection, constitute a metabolically distinct subpopulation, and are less virulent in the experimental models used here.

The highly conserved MraZ protein is a transcriptional regulator in Escherichia coli.

The mraZ and mraW genes are highly conserved in bacteria, both in sequence and in their position at the head of the division and cell wall (dcw) gene cluster. Located directly upstream of the mraZ gene, the Pmra promoter drives the transcription of mraZ and mraW, as well as many essential cell division and cell wall genes, but no regulator of Pmra has been found to date. Although MraZ has structural similarity to the AbrB transition state regulator and the MazE antitoxin and MraW is known to methylate the 16S rRNA, mraZ and mraW null mutants have no detectable phenotypes. Here we show that overproduction of Escherichia coli MraZ inhibited cell division and was lethal in rich medium at high induction levels and in minimal medium at low induction levels. Co-overproduction of MraW suppressed MraZ toxicity, and loss of MraW enhanced MraZ toxicity, suggesting that MraZ and MraW have antagonistic functions. MraZ-green fluorescent protein localized to the nucleoid, suggesting that it binds DNA. Consistent with this idea, purified MraZ directly bound a region of DNA containing three direct repeats between Pmra and the mraZ gene. Excess MraZ reduced the expression of an mraZ-lacZ reporter, suggesting that MraZ acts as a repressor of Pmra, whereas a DNA-binding mutant form of MraZ failed to repress expression. Transcriptome sequencing (RNA-seq) analysis suggested that MraZ also regulates the expression of genes outside the dcw cluster. In support of this, purified MraZ could directly bind to a putative operator site upstream of mioC, one of the repressed genes identified by RNA-seq.

Transcriptome response to nitrosative stress in Rhodobacter sphaeroides 2.4.1.

The facultative photosynthetic bacterium Rhodobacter sphaeroides 2.4.1 has a nitric oxide-response transcriptional regulator, NnrR, and nitric oxide reductase (NOR), although it is incapable of denitrification. To investigate at the genomic level the physiological response to nitrosative stress of R. sphaeroides, the transcriptome profiles of strain 2.4.1 and its NnrR mutant were analyzed before and after exposure to nitrosating agents, S-nitrosoglutathione (GSNO) and sodium nitroprusside (SNP), under microaerobic conditions. GSNO and SNP affected the expression of different but overlapping sets of genes. Only a limited number of these genes, including the genes for NOR, were under the control of NnrR, and those genes were significantly upregulated by GSNO and by SNP. The oxygen-responsive regulator FnrL and a predicted iron-sensing regulator were perhaps also involved in the transcriptome response to reactive nitrogen species. Some genes, including hemN for heme biosynthesis, were subject to dual regulation by NnrR and FnrL.

Analysis of the Genomics and Mouse Virulence of an Emergent Clone of Streptococcus dysgalactiae Subspecies equisimilis.

Streptococcus dysgalactiae subsp. equisimilis is a bacterial pathogen that is increasingly recognized as a cause of severe human infections. Much less is known about the genomics and infection pathogenesis of S. dysgalactiae subsp. equisimilis strains compared to the closely related bacterium Streptococcus pyogenes. To address these knowledge deficits, we sequenced to closure the genomes of seven S. dysgalactiae subsp. equisimilis human isolates, including six that were emm type stG62647. Recently, for unknown reasons, strains of this emm type have emerged and caused an increasing number of severe human infections in several countries. The genomes of these seven strains vary between 2.15 and 2.21 Mbp. The core chromosomes of these six S. dysgalactiae subsp. equisimilis stG62647 strains are closely related, differing on average by only 495 single-nucleotide polymorphisms, consistent with a recent descent from a common progenitor. The largest source of genetic diversity among these seven isolates is differences in putative mobile genetic elements, both chromosomal and extrachromosomal. Consistent with the epidemiological observations of increased frequency and severity of infections, both stG62647 strains studied were significantly more virulent than a strain of emm type stC74a in a mouse model of necrotizing myositis, as assessed by bacterial CFU burden, lesion size, and survival curves. Taken together, our genomic and pathogenesis data show the strains of emm type stG62647 we studied are closely genetically related and have enhanced virulence in a mouse model of severe invasive disease. Our findings underscore the need for expanded study of the genomics and molecular pathogenesis of S. dysgalactiae subsp. equisimilis strains causing human infections. IMPORTANCE Our studies addressed a critical knowledge gap in understanding the genomics and virulence of the bacterial pathogen Streptococcus dysgalactiae subsp. equisimilis. S. dysgalactiae subsp. equisimilis strains are responsible for a recent increase in severe human infections in some countries. We determined that certain S. dysgalactiae subsp. equisimilis strains are genetically descended from a common ancestor and that these strains can cause severe infections in a mouse model of necrotizing myositis. Our findings highlight the need for expanded studies on the genomics and pathogenic mechanisms of this understudied subspecies of the Streptococcus family.

The early divisome protein FtsA interacts directly through its 1c subdomain with the cytoplasmic domain of the late divisome protein FtsN.

In Escherichia coli, FtsN localizes late to the cell division machinery, only after a number of additional essential proteins are recruited to the early FtsZ-FtsA-ZipA complex. FtsN has a short, positively charged cytoplasmic domain (FtsN(Cyto)), a single transmembrane domain (FtsN(TM)), and a periplasmic domain that is essential for FtsN function. Here we show that FtsA and FtsN interact directly in vitro. FtsN(Cyto) is sufficient to bind to FtsA, but only when it is tethered to FtsN(TM) or to a leucine zipper. Mutation of a conserved patch of positive charges in FtsN(Cyto) to negative charges abolishes the interaction with FtsA. We also show that subdomain 1c of FtsA is sufficient to mediate this interaction with FtsN. Finally, although FtsN(Cyto-TM) is not essential for FtsN function, its overproduction causes a modest dominant-negative effect on cell division. These results suggest that basic residues within a dimerized FtsN(Cyto) protein interact directly with residues in subdomain 1c of FtsA. Since FtsA binds directly to FtsZ and FtsN interacts with enzymes involved in septum synthesis and splitting, this interaction between early and late divisome proteins may be one of several feedback controls for Z ring constriction.

New Pathogenesis Mechanisms and Translational Leads Identified by Multidimensional Analysis of Necrotizing Myositis in Primates.

A fundamental goal of contemporary biomedical research is to understand the molecular basis of disease pathogenesis and exploit this information to develop targeted and more-effective therapies. Necrotizing myositis caused by the bacterial pathogen Streptococcus pyogenes is a devastating human infection with a high mortality rate and few successful therapeutic options. We used dual transcriptome sequencing (RNA-seq) to analyze the transcriptomes of S. pyogenes and host skeletal muscle recovered contemporaneously from infected nonhuman primates. The in vivo bacterial transcriptome was strikingly remodeled compared to organisms grown in vitro, with significant upregulation of genes contributing to virulence and altered regulation of metabolic genes. The transcriptome of muscle tissue from infected nonhuman primates (NHPs) differed significantly from that of mock-infected animals, due in part to substantial changes in genes contributing to inflammation and host defense processes. We discovered significant positive correlations between group A streptococcus (GAS) virulence factor transcripts and genes involved in the host immune response and inflammation. We also discovered significant correlations between the magnitude of bacterial virulence gene expression in vivo and pathogen fitness, as assessed by previously conducted genome-wide transposon-directed insertion site sequencing (TraDIS). By integrating the bacterial RNA-seq data with the fitness data generated by TraDIS, we discovered five new pathogen genes, namely, S. pyogenes 0281 (Spy0281 [dahA]), ihk-irr, slr, isp, and ciaH, that contribute to necrotizing myositis and confirmed these findings using isogenic deletion-mutant strains. Taken together, our study results provide rich new information about the molecular events occurring in severe invasive infection of primate skeletal muscle that has extensive translational research implications.IMPORTANCE Necrotizing myositis caused by Streptococcus pyogenes has high morbidity and mortality rates and relatively few successful therapeutic options. In addition, there is no licensed human S. pyogenes vaccine. To gain enhanced understanding of the molecular basis of this infection, we employed a multidimensional analysis strategy that included dual RNA-seq and other data derived from experimental infection of nonhuman primates. The data were used to target five streptococcal genes for pathogenesis research, resulting in the unambiguous demonstration that these genes contribute to pathogen-host molecular interactions in necrotizing infections. We exploited fitness data derived from a recently conducted genome-wide transposon mutagenesis study to discover significant correlation between the magnitude of bacterial virulence gene expression in vivo and pathogen fitness. Collectively, our findings have significant implications for translational research, potentially including vaccine efforts.

Genetic heterogeneity of the Spy1336/R28-Spy1337 virulence axis in Streptococcus pyogenes and effect on gene transcript levels and pathogenesis.

Streptococcus pyogenes is a strict human pathogen responsible for more than 700 million infections annually worldwide. Strains of serotype M28 S. pyogenes are typically among the five more abundant types causing invasive infections and pharyngitis in adults and children. Type M28 strains also have an unusual propensity to cause puerperal sepsis and neonatal disease. We recently discovered that a one-nucleotide indel in an intergenic homopolymeric tract located between genes Spy1336/R28 and Spy1337 altered virulence in a mouse model of infection. In the present study, we analyzed size variation in this homopolymeric tract and determined the extent of heterogeneity in the number of tandemly-repeated 79-amino acid domains in the coding region of Spy1336/R28 in large samples of strains recovered from humans with invasive infections. Both repeat sequence elements are highly polymorphic in natural populations of M28 strains. Variation in the homopolymeric tract results in (i) changes in transcript levels of Spy1336/R28 and Spy1337 in vitro, (ii) differences in virulence in a mouse model of necrotizing myositis, and (iii) global transcriptome changes as shown by RNAseq analysis of isogenic mutant strains. Variation in the number of tandem repeats in the coding sequence of Spy1336/R28 is responsible for size variation of R28 protein in natural populations. Isogenic mutant strains in which genes encoding R28 or transcriptional regulator Spy1337 are inactivated are significantly less virulent in a nonhuman primate model of necrotizing myositis. Our findings provide impetus for additional studies addressing the role of R28 and Spy1337 variation in pathogen-host interactions.

Half-Site DNA sequence and spacing length contributions to PrrA binding to PrrA site 2 of RSP3361 in Rhodobacter sphaeroides 2.4.1.

The consensus DNA binding sequence for PrrA, a global regulator in Rhodobacter sphaeroides 2.4.1, is poorly defined. We have performed mutational analysis of PrrA site 2, of the RSP3361 gene, to which PrrA binds in vitro (J. M. Eraso and S. Kaplan, J. Bacteriol. 191:4341-4352, 2009), to further define the consensus sequence for DNA binding. Two half-sites of equal length, containing 6 nucleotides each, were required for PrrA binding to this DNA sequence. Systematic nucleotide substitutions in both inverted half-sites led to a decrease in binding affinity of phosphorylated PrrA in vitro, the level of which was dependent on the substitution. The reduced binding affinities were confirmed by competition experiments and led to proportional decreases in the expression of lacZ transcriptional fusions to the RSP3361 gene in vivo. The 5-nucleotide spacer region between the half-sites was found to be optimal for PrrA binding to the wild-type half-sites, as shown by decreased PrrA DNA binding affinities to synthetic DNA sequences without spacer regions or with spacer regions ranging from 1 to 10 nucleotides. The synthetic spacer region alleles also showed decreased gene expression in vivo when analyzed using lacZ transcriptional fusions. We have studied three additional DNA sequences to which PrrA binds in vitro. They are located in the regulatory regions of genes positively regulated by PrrA and contain spacer regions with 5 or 8 nucleotides. We demonstrate that PrrA can bind in vitro to DNA sequences with different lengths in the spacer regions between the half-sites.

Regulation of gene expression by PrrA in Rhodobacter sphaeroides 2.4.1: role of polyamines and DNA topology.

In the present study, we show in vitro binding of PrrA, a global regulator in Rhodobacter sphaeroides 2.4.1, to the PrrA site 2, within the RSP3361 locus. Specific binding, as shown by competition experiments, requires the phosphorylation of PrrA. The binding affinity of PrrA for site 2 was found to increase 4- to 10-fold when spermidine was added to the binding reaction. The presence of extracellular concentrations of spermidine in growing cultures of R. sphaeroides gave rise to a twofold increase in the expression of the photosynthesis genes pucB and pufB, as well as the RSP3361 gene, under aerobic growth conditions, as shown by the use of lacZ transcriptional fusions, and led to the production of light-harvesting spectral complexes. In addition, we show that negative supercoiling positively regulates the expression of the RSP3361 gene, as well as pucB. We show the importance of supercoiling through an evaluation of the regulation of gene expression in situ by supercoiling, in the case of the former gene, as well as using the DNA gyrase inhibitor novobiocin. We propose that polyamines and DNA supercoiling act synergistically to regulate expression of the RSP3361 gene, partly by affecting the affinity of PrrA binding to the PrrA site 2 within the RSP3361 gene.

Gene fitness landscape of group A streptococcus during necrotizing myositis.

Necrotizing fasciitis and myositis are devastating infections characterized by high mortality. Group A streptococcus (GAS) is a common cause of these infections, but the molecular pathogenesis is poorly understood. We report a genome-wide analysis using serotype M1 and M28 strains that identified GAS genes contributing to necrotizing myositis in nonhuman primates (NHP), a clinically relevant model. Using transposon-directed insertion-site sequencing (TraDIS), we identified 126 and 116 GAS genes required for infection by serotype M1 and M28 organisms, respectively. For both M1 and M28 strains, more than 25% of the GAS genes required for necrotizing myositis encode known or putative transporters. Thirteen GAS transporters contributed to both M1 and M28 strain fitness in NHP myositis, including putative importers for amino acids, carbohydrates, and vitamins and exporters for toxins, quorum-sensing peptides, and uncharacterized molecules. Targeted deletion of genes encoding 5 transporters confirmed that each isogenic mutant strain was significantly (P < 0.05) impaired in causing necrotizing myositis in NHPs. Quantitative reverse-transcriptase PCR (qRT-PCR) analysis showed that these 5 genes are expressed in infected NHP and human skeletal muscle. Certain substrate-binding lipoproteins of these transporters, such as Spy0271 and Spy1728, were previously documented to be surface exposed, suggesting that our findings have translational research implications.

Integrated analysis of population genomics, transcriptomics and virulence provides novel insights into Streptococcus pyogenes pathogenesis.

Streptococcus pyogenes causes 700 million human infections annually worldwide, yet, despite a century of intensive effort, there is no licensed vaccine against this bacterium. Although a number of large-scale genomic studies of bacterial pathogens have been published, the relationships among the genome, transcriptome, and virulence in large bacterial populations remain poorly understood. We sequenced the genomes of 2,101 emm28 S. pyogenes invasive strains, from which we selected 492 phylogenetically diverse strains for transcriptome analysis and 50 strains for virulence assessment. Data integration provided a novel understanding of the virulence mechanisms of this model organism. Genome-wide association study, expression quantitative trait loci analysis, machine learning, and isogenic mutant strains identified and confirmed a one-nucleotide indel in an intergenic region that significantly alters global transcript profiles and ultimately virulence. The integrative strategy that we used is generally applicable to any microbe and may lead to new therapeutics for many human pathogens.

The use of chromatin immunoprecipitation to define PpsR binding activity in Rhodobacter sphaeroides 2.4.1.

The expression of genes involved in photosystem development in Rhodobacter sphaeroides is dependent upon three major regulatory networks: FnrL, the PrrBA (RegBA) two-component system, and the transcriptional repressor/antirepressor PpsR/AppA. Of the three regulators, PpsR appears to have the narrowest range of physiological effects, which are limited to effects on the structural and pigment biosynthetic activities involved in photosynthetic membrane function. Although a PrrA(-) mutant is unable to grow under photosynthetic conditions, when a ppsR mutation was present, photosynthetic growth occurred. An examination of the double mutant under anaerobic-dark-dimethyl sulfoxide conditions using microarray analysis revealed the existence of an "extended" PpsR regulon and new physiological roles. To characterize the PpsR regulon and to better ascertain the significance of degeneracy within the PpsR binding sequence in vivo, we adapted the chromatin immunoprecipitation technique to R. sphaeroides. We demonstrated that in vivo there was direct and significant binding by PpsR to newly identified genes involved in microaerobic respiration and periplasmic stress resistance, as well as to photosynthesis genes. The new members of the PpsR regulon are located outside the photosynthesis gene cluster and have degenerate PpsR binding sequences. The possible interaction under physiologic conditions with degenerate binding sequences in the presence of other biologically relevant molecules is discussed with respect to its importance in physiological processes and to the existence of complex phenotypes associated with regulatory mutants. This study further defines the DNA structure necessary for PpsR binding in situ.

Role of the global transcriptional regulator PrrA in Rhodobacter sphaeroides 2.4.1: combined transcriptome and proteome analysis.

The PrrBA two-component regulatory system is a major global regulator in Rhodobacter sphaeroides 2.4.1. Here we have compared the transcriptome and proteome profiles of the wild-type (WT) and mutant PrrA2 cells grown anaerobically in the dark with dimethyl sulfoxide as an electron acceptor. Approximately 25% of the genes present in the PrrA2 genome are regulated by PrrA at the transcriptional level, either directly or indirectly, by twofold or more relative to the WT. The genes affected are widespread throughout all COG (cluster of orthologous group) functional categories, with previously unsuspected "metabolic" genes affected in PrrA2 cells. PrrA was found to act as both an activator and a repressor of transcription, with more genes being repressed in the presence of PrrA (9:5 ratio). An analysis of the genes encoding the 1,536 peptides detected through our chromatographic study, which corresponds to 36% coverage of the genome, revealed that approximately 20% of the genes encoding these proteins were positively regulated, whereas approximately 32% were negatively regulated by PrrA, which is in excellent agreement with the percentages obtained for the whole-genome transcriptome profile. In addition, comparison of the transcriptome and proteome mean parameter values for WT and PrrA2 cells showed good qualitative agreement, indicating that transcript regulation paralleled the corresponding protein abundance, although not one for one. The microarray analysis was validated by direct mRNA measurement of randomly selected genes that were both positively and negatively regulated. lacZ transcriptional and kan translational fusions enabled us to map putative PrrA binding sites and revealed potential gene targets for indirect regulation by PrrA.

Bacterial cell wall: thinking globally, actin locally.

The bacterial actin-like protein MreB is thought to form a continuous helical polymer at the membrane to confer rod shape. Two new studies now show that MreB forms discrete dynamic patches that travel circumferentially.

Postgenomic adventures with Rhodobacter sphaeroides.

This review describes some of the recent highlights taken from the studies of Rhodobacter sphaeroides 2.4.1. The review is not intended to be comprehensive, but to reflect the bias of the authors as to how the availability of a sequenced and annotated genome, a gene-chip, and proteomic profile as well as comparative genomic analyses can direct the progress of future research in this system.

Combining microarray and genomic data to predict DNA binding motifs.

The ability to detect regulatory elements within genome sequences is important in understanding how gene expression is controlled in biological systems. In this work, microarray data analysis is combined with genome sequence analysis to predict DNA sequences in the photosynthetic bacterium Rhodobacter sphaeroides that bind the regulators PrrA, PpsR and FnrL. These predictions were made by using hierarchical clustering to detect genes that share similar expression patterns. The DNA sequences upstream of these genes were then searched for possible transcription factor recognition motifs that may be involved in their co-regulation. The approach used promises to be widely applicable for the prediction of cis-acting DNA binding elements. Using this method the authors were independently able to detect and extend the previously described consensus sequences that have been suggested to bind FnrL and PpsR. In addition, sequences that may be recognized by the global regulator PrrA were predicted. The results support the earlier suggestions that the DNA binding sequence of PrrA may have a variable-sized gap between its conserved block elements. Using the predicted DNA binding sequences, a whole-genome-scale analysis was performed to determine the relative importance of the interplay between the three regulators PpsR, FnrL and PrrA. Results of this analysis showed that, compared to the regulation by PpsR and FnrL, a much larger number of genes are candidates to be regulated by PrrA. The study demonstrates by example that integration of multiple data types can be a powerful approach for inferring transcriptional regulatory patterns in microbial systems, and it allowed the detection of photosynthesis-related regulatory patterns in R. sphaeroides.