Bacterial Evolution

Streptococcus pseudopneumoniae (SPPN) is a recently described species of the viridans group streptococci (VGS). Although the pathogenic potential of S. pseudopneumoniae remains uncertain, it is most commonly isolated from patients with underlying medical conditions, such as chronic obstructive pulmonary disease. S. pseudopneumoniae can be distinguished from the closely related species, S. pneumoniae and S. mitis, by phenotypic characteristics, including optochin resistance in the presence of 5% CO 2 , bile insolubility, and the lack of the pneumococcal capsule. Previously, we reported the draft genome sequence of S. pseudopneumoniae IS7493, a clinical isolate obtained from an immunocompromised patient with documented pneumonia. Here, we use comparative genomics approaches to identify similarities and key differences between S. pseudopneumoniae IS7493, S. pneumoniae and S. mitis. The genome structure of S. pseudopneumoniae IS7493 is most closely related to that of S. pneumoniae R6, but several recombination events are evident. Analysis of gene content reveals numerous unique features that distinguish S. pseudopneumoniae from other streptococci. The presence of loci for competence, iron transport, pneumolysin production and antimicrobial resistance reinforce the phylogenetic position of S. pseudopneumoniae as an intermediate species between S. pneumoniae and S. mitis. Additionally, the presence of several virulence factors and antibiotic resistance mechanisms suggest the potential of this commensal species to become pathogenic or to contribute to increasing antibiotic resistance levels seen among the VGS.

bution of each mechanism to the generation of genetic Franc ¸ois Taddei, 3 Miroslav Radman, 3,7 diversity varies between species. It has been estimated and Ivan Matic 3 for Escherichia coli that, in spite of the clonal structure 1 INSERM U 458 of its natural populations (Desjardins et al., 1995), any Ho ˆpital Robert Debre ´single nucleotide change is about 50 times more likely to 48 boulevard Se ´rurier have occurred by recombination (with a partner carrying 75935 Paris Cedex 19 such nucleotide change) than by a de novo mutation 2 Service de Syste ´matique Mole ´culaire (Guttman and Dykhuizen, 1994). The rate of successful (GDR CNRS 1005) gene transfer between bacterial populations in nature Muse ´um National d'Histoire Naturelle depends on numerous factors such as ecological isola-43 rue Cuvier tion, host range of genetic exchange vectors, activity of 75231 Paris Cedex 05 the recipient cell nucleases, DNA sequence divergence 3 INSERM E9916 between recombining molecules, type and efficiency of Faculte ´de Me ´decine Necker-Enfants Malades the recipient cell recombination machinery and, finally, Universite ´Paris V the hybrid fitness (Matic et al., 1996). Some of those 156 rue Vaugirard factors have been studied in the laboratory, but their 75730 Paris Cedex 15 impact on gene flow in nature is generally unknown. 4 INSERM U 155 Molecular mechanisms involved in the control of ho-80 rue du Ge ´ne ´ral Leclerc mologous recombination in bacteria have been exten-94276 Kremlin-Bice ˆtre sively studied. For example, it was shown that the fre-5 ACTGene Recherche et De ´veloppements quency of integration of donor DNA in the chromosome Alle ´e Christophe Colomb of recipient cells decreases exponentially with increas-Ge ´nopole ing sequence divergence between the two recombining 91035 Evry Cedex DNA molecules (Zawadzki et al., 1995; Vulic et al., 1997; France Majewski et al., 2000). The degree and pattern of DNA 6 Department of Genetics and Development divergence determine actions of the enzymes that con-Columbia University trol the outcome of recombination (Radman and Wag-College of Physicians and Surgeons ner, 1993). The initiation of recombination in bacteria New York, New York 10032 depends on the activity of the RecA protein, which is selective for sequence identity only at the initial stage of the strand exchange process (Shen and Huang, 1986). Summary Once initiated, RecA-mediated strand exchange can occur in spite of large numbers of mismatches and even Mutation and subsequent recombination events crelarge heterologies (Bianchi and Radding, 1983; Lichten ate genetic diversity, which is subjected to natural and Fox, 1984). During the strand exchange process, selection. Bacterial mismatch repair (MMR) deficient fidelity of recombination is controlled by the methylmutants, exhibiting high mutation and homeologous directed mismatch repair (MMR) system. MMR proteins recombination rates, are frequently found in natural recognize mispaired and unpaired bases in the joint populations. Therefore, we have explored the possibilheteroduplex regions and block RecA-catalyzed strand ity that MMR deficiency emerging in nature has left transfer (Worth et al., 1994). Thus, MMR is a potent some "imprint" in the sequence of bacterial genomes. inhibitor of recombination between nonidentical DNA Comparative molecular phylogeny of MMR genes from sequences (Rayssiguier et al., 1989; Shen and Huang, natural Escherichia coli isolates shows that, compared 1989; Matic et al., 1995; Vulic et al., 1997). Even low to housekeeping genes, individual functional MMR divergence is sufficient to impede recombination. For genes exhibit high sequence mosaicism derived from example, transductional recombination between two diverse phylogenetic lineages. This apparent horiserovars (Typhimurium and Typhi) of Salmonella enterzontal gene transfer correlates with hyperrecombinaica, whose genomes differ only 1%-2% at DNA setion phenotype of MMR-deficient mutators. The sequence level, is increased 10 2 -to 10 3 -fold in MMR-defiquence mosaicism of MMR genes may be a hallmark cient genetic backgrounds (Zahrt and Maloy, 1997). of a mechanism of adaptive evolution that involves MMR system is also involved in the control of replicamodulation of mutation and recombination rates by tion fidelity. Inactivation of MMR genes increases mutarecurrent losses and reacquisitions of MMR gene tion rates 10 2 -to 10 3 -fold (Glickman and Radman, 1980; functions. Schaaper and Dunn, 1987). Studies of mutation rates in natural populations of E. coli, S. enterica, and Pseudomonas aeruginosa show that MMR-deficient strains of-

The bacterial communities of sponges have been studied using molecular techniques as well as culture-based techniques, but the communities described by these two methods are remarkably distinct. Culture-based methods describe communities dominated by Proteobacteria, and Actinomycetes while molecular methods describe communities dominated by predominantly uncultivated groups such as the Chloroflexi, Acidobacteria, and Acidimicrobidae. In this study, we used a wide range of culture media to increase the diversity of cultivable bacteria from the closely related giant barrel sponges, Xestospongia muta collected from the Florida Keys, Atlantic Ocean and Xestospongia testudinaria, collected from Indonesia, Pacific Ocean. Over 400 pure cultures were isolated and identified from X. muta and X. testudinaria and over 90 bacterial species were represented. Over 16,000 pyrosequences were analyzed and assigned to 976 OTUs. We employed both cultured-based methods and pyrosequencing to look for patterns of overlap between the culturable and molecular communities. Only one OTU was found in both the molecular and culturable communities, revealing limitations inherent in both approaches.

Community-associated methicillin-resistant Staphylococcus aureus (MRSA) sequence type 72 (ST72) has emerged as a nosocomial infection that causes pneumonia, endocarditis, and skin and soft tissue infections (SSTIs) in South Korea (1, 2). Due to the multidrug resistance of community-associated ST72, we evaluated the efficacy of lysostaphin as an effective antistaphylococcal bacteriocin that specifically eradicates S. aureus by cleaving the pentaglycine bridge in the cell wall (3, 4).
Human ST72 isolates (K07-561 and K07-204) used in the study were originally isolated by the Asia Pacific Foundation for Infectious Diseases, in South Korea (2). The different levels of susceptibility/resistance of K07-561 and K07-204 to lysostaphin were evaluated (4). The isolate K07-561 showed lysostaphin susceptibility, while K07-204 displayed an ∼1,000 times higher level of lysostaphin resistance upon treatment with 2 U of lysostaphin for 5 min (Fig. 1). These human ST72 isolates, K07-204 (lysostaphin resistant) and K07-561 (lysostaphin susceptible), represent an attractive model for elucidating the unknown mechanism of antibiotic and enzybiotic resistance. Therefore, we selected the isolates K07-204 and K07-561 as representative strains for genome sequencing to understand the genetic differences between the resistant and sensitive phenotypes.

Cyclophilins are prolyl isomerases with multitude of functions in different cellular processes and pathological conditions. Cyclophilin A (PpiA) of Mycobacterium tuberculosis is secreted during infection in intraphagosomal niche. However, our understanding about the evolutionary origin, secretory mechanism or the interactome of M. tuberculosis PpiA is limited. This study demonstrates through phylogenetic and structural analyses that PpiA has more proximity to human cyclophilins than the prokaryotic counterparts. We report a unique N-terminal sequence (MADCDSVTNSP) present in pathogenic mycobacterial PpiA and absent in non-pathogenic strains. This sequence stretch was shown to be essential for PpiA secretion. The overexpression of full-length PpiA from M. tuberculosis in non-pathogenic Mycobacterium smegmatis resulted in PpiA secretion while truncation of the N-terminal stretch obstructed the secretion. In addition, presence of an ESX pathway substrate motif in M. tuberculosis PpiA suggested possible involvement of Type VII secretion system. Site-directed mutagenesis of key residues in this motif in full-length PpiA also hindered the secretion in M. smegmatis. Bacterial twohybrid screens with human lung cDNA library as target were utilized to identify interaction partners of PpiA from host repertoire, and a number of substrates with functional representation in iron storage, signal transduction and immune responses were detected. The extensive host interactome coupled with the sequence and structural similarity to human cyclophilins is strongly suggestive of PpiA being deployed by M. tuberculosis as an effector mimic against the host cyclophilins.

How pathogenic bacteria adapt and evolve in the complex and variable environment of the host remains a largely unresolved question. Here we have used whole genome sequencing of Salmonella enterica serovar Typhimurium LT2 populations serially passaged in mice to identify mutations that adapt bacteria to systemic growth in mice. We found unique pathoadaptive mutations in two global regulators, phoQ and stpA, which increase the competitive indexes of the bacteria 3-to 5-fold. Also, all mouse-adapted lineages had changed the orientation of the hin invertable element, resulting in production of a FliC type of flagellum. Competition experiments in mice with locked flagellum mutants showed that strains expressing the FliC type of flagellum had a 5-fold increase in competitive index as compared to those expressing FljB type flagellum. Combination of the flagellum cassette inversion with the stpA mutation increased competitive indexes up to 20-fold. These experiments show that Salmonella can rapidly adapt to a mouse environment by acquiring a few mutations of moderate individual effect that when combined confer substantial increases in growth.

Environments can change in incremental fashions, where a shift from one state to another occurs over multiple organismal generations. The rate of the environmental change is expected to influence how and how well populations adapt to the final environmental state. We used a model system, the lytic RNA bacteriophage Φ6, to investigate this question empirically. We evolved viruses for thermostability by exposing them to heat shocks that increased to a maximum temperature at different rates. We observed increases in the ability of many heat-shocked populations to survive high temperature heat shocks. On their first exposure to the highest temperature, populations that experienced a gradual increase in temperature had higher average survival than populations that experienced a rapid temperature increase. However, at the end of the experiment, neither the survival of populations at the highest temperature nor the number of mutations per population varied significantly according to the rate of thermal change. We also evaluated mutations from the endpoint populations for their effects on viral thermostability and growth. As expected, some mutations did increase viral thermostability. However, other mutations decreased thermostability but increased growth rate, suggesting that benefits of an increased replication rate may have sometimes outweighed the benefits of enhanced thermostability. Our study highlights the importance of considering the effects of multiple selective pressures, even in environments where a single factor changes.

The root-nodule bacteria of legumes endemic to the Cape Floristic Region are largely understudied, even though recent reports suggest the occurrence of nodulating Burkholderia species unique to the region. In this study, we considered the diversity and evolution of nodulating Burkholderia associated with the endemic papilionoid tribes Hypocalypteae and Podalyrieae. We identified distinct groups from verified rhizobial isolates by phylogenetic analyses of the 16S rRNA and recA housekeeping gene regions. In order to gain insight into the evolution of the nodulation and diazotrophy of these rhizobia we analysed the genes encoding NifH and NodA. The majority of these 69 isolates appeared to be unique, potentially representing novel species. Evidence of horizontal gene transfer determining the symbiotic ability of these Cape Floristic Region isolates indicate evolutionary origins distinct from those of nodulating Burkholderia from elsewhere in the world. Overall, our findings suggest that Burkholderia species associated with fynbos legumes are highly diverse and their symbiotic abilities have unique ancestries. It is therefore possible that the evolution of these bacteria is closely linked to the diversification and establishment of legumes characteristic of the Cape Floristic Region.

Analysis of pathogen genome data sequenced from clinical and historical samples has made it possible to perform phylogenetic analyses of sexually transmitted infections on a global scale, and to estimate the diversity, distribution, and coevolutionary host relationships of these pathogens, providing insights into pathogen emergence and disease prevention. Deep-sequenced pathogen genomes from clinical studies and ancient samples yield estimates of within-host and between-host evolutionary rates and provide data on changes in pathogen genomic stability and evolutionary responses. Here we examine three groups of pathogens transmitted mainly through sexual contact between modern humans to provide insight into ancient human behavior and history with their pathogens. Exploring ancient pathogen genomic divergence and the ancient viral-host parallel evolutionary histories will help us to reconstruct the origin of present-day geographical distribution and diversity of clinical pathogen infec...

Enteroinvasive E. coli (EIEC), like Shigella , is the etiological agent of bacillary dysentery, a particularly severe syndrome in children in developing countries. All EIEC strains share with Shigella the inability to synthesize lysine decarboxylase (the LDC phenotype). The lack of this function is considered a pathoadaptive mutation whose emergence was necessary to obtain the full expression of invasiveness. Cadaverine, the product of lysine decarboxylation, is a small polyamine which interferes mainly with the inflammatory process induced by dysenteric bacteria. Genes coding for lysine decarboxylase and its transporter constitute a single operon ( cadBA ) and are expressed at low pH under the positive control of CadC. This regulator is an inner membrane protein that is able to sense pH variation and to respond by transcriptionally activating the cadBA genes. In this study we show that, unlike in Shigella , mutations affecting the cad locus in the EIEC strains we have analyzed are ...

Glutaraldehyde is a widely used biocide on the market for about 50 years. Despite its broad application, several reports on the emergence of bacterial resistance, and occasional outbreaks caused by poorly disinfection, there is a gap of knowledge on the bacterial adaptation, tolerance, and resistance mechanisms to glutaraldehyde. Here, we analyze the effects of the independent selection of mutations in the transcriptional regulator yqhC for biological replicates of Escherichia coli cells subjected to adaptive laboratory evolution (ALE) in the presence of glutaraldehyde. The evolved strains showed improved survival in the biocide (11-26% increase in fitness) as a result of mutations in the activator yqhC, which led to the overexpression of the yqhD aldehyde reductase gene by 8 to over 30-fold (3.1-5.2 log2FC range). The protective effect was exclusive to yqhD as other aldehyde reductase genes of E. coli, such as yahK, ybbO, yghA, and ahr did not offer protection against the biocide. We describe a novel mechanism of tolerance to glutaraldehyde based on the activation of the aldehyde reductase YqhD by YqhC and bring attention to the potential for the selection of such tolerance mechanism outside the laboratory, given the existence of YqhD homologs in various pathogenic and opportunistic bacterial species.

Bacterial species of the genus Anaplasma are tick transmitted pathogens that negatively impact on animal productions and generate veterinary and public health concerns. This paper reports the identification, molecular characterization and phylogeny of novel unclassified A. platys-like strains in cats. Interestingly, these novel strains are closely related to conspecific strains recently identified in ruminants, and significantly differ from A. platys. A. platys-like strains in cats, unlike ruminants strains, show tropism for platelets. Results have implications in the diagnostic scenario of animal anaplasmosis and provide background for reconstructing the evolutionary history of species genetically related to A. platys.

Correlations between FimH mutations and virulence were established by studying a collection of human commensal and extraintestinal pathogenic Escherichia coli natural isolates. Pathoadaptive (A27V and, to a lesser extent, A119V) and “commensal-adaptive” (A202V) mutations were evidenced in B2 phylogenetic group strains. fimH phylogenetic analysis indicates that these pathoadaptive mutations occurred several times.

Pantothenate is the metabolic precursor of Coenzyme A, an indispensable cofactor for many fundamental cellular processes. In this study, we show that many bacterial species have acquired multiple copies of pantothenate biosynthesis pathway genes via horizontal and vertical gene transfer events. Some bacterial species were also found to lack panE and panD genes, and depended on alternative enzymes/metabolic sources for pantothenate production. To shed light on the factors responsible for such dynamic evolutionary selections, the structural and functional characteristics of P. aeruginosa ketopantoate reductase (KPR), an enzyme that catalyzes the ratelimiting step and also the most duplicated, was investigated. A comparative analysis of apo and NADP+ bound crystal structures of P. aeruginosa KPR with orthologs, revealed that the residues involved in the interaction with specific phosphate moiety of NADP+ are relatively less conserved, suggesting dynamic evolutionary trajectories in KPRs for redox cofactor selection. Our structural and biochemical data also show that the specific conformational changes mediated by NADPH binding facilitate the cooperative binding of ketopantoate. From drastically reduced catalytic activity for NADH catalyzed the reaction with significantly higher K M of ketopantoate, it appears that the binding of ketopantoate is allosterically regulated to confer redox cofactor specificity. Altogether, our results, in compliance with earlier studies, not only depict the role of lateral gene transfer events in many bacterial species for enhancing pantothenate production but also highlight the possible role of redox cofactor balance in the regulation of pantothenate biosynthesis pathways.

Non-typeable Haemophilus influenzae (NTHi), an opportunistic pathogen of the upper airways of healthy children, can infect the lower airways, driving chronic lung disease. However, the molecular basis underpinning NTHi transition from a commensal to a pathogen is not clearly understood. Here, we performed comparative genomic and transcriptomic analyses of 12 paired, isogenic NTHi strains, isolated from the nasopharynx (NP) and bronchoalveolar lavage (BAL) of 11 children with chronic lung disease, to identify convergent molecular signatures associated with lung adaptation. Comparative genomic analyses of the 12 NP-BAL pairs demonstrated that five were genetically identical, with the remaining seven differing by only 1 to 3 mutations. Within-patient transcriptomic analyses identified between 2 and 58 differentially expressed genes in 8 of the 12 NP-BAL pairs, including pairs with no observable genomic changes. Whilst no convergence was observed at the gene level, functional enrichment...

The evolutionary role of transposable elements (TEs) is still highly controversial. Two key parameters, the transposition rate ( u and w , for replicative and non-replicative transposition) and the excision rate ( e ) are fundamental to understanding their evolution and maintenance in populations. We have estimated u , w and e for six families of TEs (including eight members: IS1, IS2, IS3, IS4, IS5, IS30, IS150 and IS186) in Escherichia coli , using a mutation accumulation (MA) experiment. In this experiment, mutations accumulate essentially at the rate at which they appear, during a period of 80 500 (1610 generations × 50 lines) generations, and spontaneous transposition events can be detected. This differs from other experiments in which insertions accumulated under strong selective pressure or over a limited genomic target. We therefore provide new estimates for the spontaneous rates of transposition and excision in E. coli . We observed 25 transposition and three excision event...

Escherichia coli strain BL21 is one of the widely used bacterial hosts for high-level recombinant protein production and for other applications. Here, we present the complete genome sequence of a commercial version of the Escherichia coli BL21 strain.

Bacterial community composition and functional potential change subtly across gradients in the surface ocean. In contrast, while there are significant phylogenetic divergences between communities from freshwater and marine habitats, the underlying mechanisms to this phylogenetic structuring yet remain unknown. We hypothesized that the functional potential of natural bacterial communities is linked to this striking divide between microbiomes. To test this hypothesis, metagenomic sequencing of microbial communities along a 1,800 km transect in the Baltic Sea area, encompassing a continuous natural salinity gradient from limnic to fully marine conditions, was explored. Multivariate statistical analyses showed that salinity is the main determinant of dramatic changes in microbial community composition, but also of large scale changes in core metabolic functions of bacteria. Strikingly, genetically and metabolically different pathways for key metabolic processes, such as respiration, biosynthesis of quinones and isoprenoids, glycolysis and osmolyte transport, were differentially abundant at high and low salinities. These shifts in functional capacities were observed at multiple taxonomic levels and within dominant bacterial phyla, while bacteria, such as SAR11, were able to adapt to the entire salinity gradient. We propose that the large differences in central metabolism required at high and low salinities dictate the striking divide between freshwater and marine microbiomes, and that the ability to inhabit different salinity regimes evolved early during bacterial phylogenetic differentiation. These findings significantly advance our understanding of microbial distributions and stress the need to incorporate salinity in future climate change models that predict increased levels of precipitation and a reduction in salinity.

Several invasive serogroup B meningococcal strains phylogenetically related to the lineage III (ET-24) exhibited a mutator phenotype as shown by mutagenicity assay using rifampicinresistance as a selection marker. Hypermutation was associated to the presence of defective mutL alleles that were genetically characterized. Interestingly, the mutator phenotype was suppressed when a non-functional recB ET-37 allele, derived from ET-37 meningococcal strains, replaced the functional recB allele in a lineage III strain. In contrast, the same gene replacement did not affect mutation frequencies in a mismatch repair-proficient strain. These results suggested that in MutL-deficient strains spontaneous mutations mostly arise from post-replicative DNA synthesis associated to the activity of the RecBCD recombination pathway.

The sodium-pumping NADH: ubiquinone oxidoreductase (Na +-NQR) is the main ion pump and the primary entry site for electrons into the respiratory chain of many different types of pathogenic bacteria. This enzymatic complex creates a transmembrane gradient of sodium that is used by the cell to sustain ionic homeostasis, nutrient transport, ATP synthesis, flagellum rotation and other essential processes. Comparative genomics data demonstrate that the nqr operon, which encodes all Na +-NQR subunits, is found in a large variety of bacterial lineages with different habitats and metabolic strategies. Here we studied the distribution, origin and evolution of this enzymatic complex. The molecular phylogenetic analyses and the organizations of the nqr operon indicate that Na +-NQR evolved within the Chlorobi/Bacteroidetes group, after the duplication and subsequent neofunctionalization of the operon that encodes the homolog RNF complex. Subsequently, the nqr operon dispersed through multiple horizontal transfer events to other bacterial lineages such as Chlamydiae, Planctomyces and a, b, c and d-proteobacteria. Considering the biochemical properties of the Na +-NQR complex and its physiological role in different bacteria, we propose a detailed scenario to explain the molecular mechanisms that gave rise to its novel redox-dependent sodium-pumping activity. Our model postulates that the evolution of the Na +-NQR complex involved a functional divergence from its RNF homolog, following the duplication of the rnf operon, the loss of the rnfB gene and the recruitment of the reductase subunit of an aromatic monooxygenase.

The definition of bacterial species is based on genomic similarities, giving rise to the operational concept of genomic species, but the reasons of the occurrence of differentiated genomic species remain largely unknown. We used the Agrobacterium tumefaciens species complex and particularly the genomic species presently called genomovar G8, which includes the sequenced strain C58, to test the hypothesis of genomic species having specific ecological adaptations possibly involved in the speciation process. We analyzed the gene repertoire specific to G8 to identify potential adaptive genes. By hybridizing 25 strains of A. tumefaciens on DNA microarrays spanning the C58 genome, we highlighted the presence and absence of genes homologous to C58 in the taxon. We found 196 genes specific to genomovar G8 that were mostly clustered into seven genomic islands on the C58 genome-one on the circular chromosome and six on the linear chromosome-suggesting higher plasticity and a major adaptive role of the latter. Clusters encoded putative functional units, four of which had been verified experimentally. The combination of G8-specific functions defines a hypothetical species primary niche for G8 related to commensal interaction with a host plant. This supports that the G8 ancestor was able to exploit a new ecological niche, maybe initiating ecological isolation and thus speciation. Searching genomic data for synapomorphic traits is a powerful way to describe bacterial species. This procedure allowed us to find such phenotypic traits specific to genomovar G8 and thus propose a Latin binomial, Agrobacterium fabrum, for this bona fide genomic species.

The metabolic adaptation of strong mutator strains was studied to better understand the link between the strong mutator phenotype and virulence. Analysis of the growth curves of isogenic strains of Salmonella, which were previously grown in M63 glucose media, revealed that the exponential phase of growth was reached earlier in an M63 acetate medium with strong mutator strains (mutated in mutS or in mutL) than with normomutator strains (P,0.05). Complemented strains confirmed the direct role of the strong mutator phenotype in this faster metabolic adaptation to the assimilation of acetate. In a mixed cell population, proliferation of strong mutators over normomutators was observed when the carbon source was switched from glucose to acetate. These results add to the sparse body of knowledge about strong mutators and highlight the selective advantage conferred by the strong mutator phenotype to adapt to a switch of carbon source in the environment. This work may provide clinically useful information given that there is a high prevalence of strong mutators among pathogenic strains of Salmonella and that acetate is the principal short chain fatty acid of the human terminal ileum and colon where Salmonella infection is localized.

The genomic stability and integrity of host strains are critical for the production of recombinant proteins in biotechnology. Bacterial genomes contain numerous jumping genetic elements, the insertion sequences (ISs) that cause a variety of genetic rearrangements, resulting in adverse effects such as genome and recombinant plasmid instability. To minimize the harmful effects of ISs on the expression of recombinant proteins in Escherichia coli, we developed an IS-free, minimized E. coli strain (MS56) in which about 23 % of the genome, including all ISs and many unnecessary genes, was removed. Here, we compared the expression profiles of recombinant proteins such as tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) and bone morphogenetic protein-2 (BMP2) in MG1655 and MS56. Hopping of ISs (IS1, IS3, or IS5) into the TRAIL and BMP2 genes occurred at the rate of~10 −8 /gene/h in MG1655 whereas such events were not observed in MS56. Even though IS hopping occurred very rarely (10 −8 /gene/h), cells containing the IS-inserted TRAIL and BMP2 plasmids became dominant (~52 % of the total population) 28 h after fermentation began due to their growth advantage over cells containing intact plasmids, significantly reducing recombinant protein production in batch fermentation. Our findings clearly indicate that IS hopping is detrimental to the industrial production of recombinant proteins, emphasizing the importance of the development of IS-free host strains.

The ~4-Mbp basic genome shared by 32 independent isolates of E. coli representing considerable population diversity has been approximated by whole-genome multiple-alignment and computational filtering designed to remove mobile elements and highly variable regions. Single nucleotide polymorphisms (SNPs) in the 496 basic-genome pairs are identified and clonally inherited stretches are distinguished from those acquired by horizontal transfer (HT) by sharp discontinuities in SNP density. The six least diverged genome-pairs each have only one or two HT stretches, each occupying 42-115-kbp of basic genome and containing at least one gene cluster known to confer selective advantage. At higher divergences, the typical mosaic pattern of interspersed clonal and HT stretches across the entire basic genome are observed, including likely fragmented integrations across a restriction barrier. A simple model suggests that individual HT events are of the order of 10-kbp and are the chief contributor to genome divergence, bringing in almost 12 times more SNPs than point mutations. As a result of continuing horizontal transfer of such large segments, 400 out of the 496 strain-pairs beyond genomic divergence of Δ c~1 .25% share virtually no genomic material with their common ancestor. We conclude that the active and continuing horizontal transfer of moderately large genomic fragments is likely to be mediated primarily by a co-evolving population of phages that distribute random genome fragments throughout the population by generalized transduction, allowing efficient adaptation to environmental changes.

Pseudomonas aeruginosa strain PA14 is an opportunistic human pathogen capable of infecting a wide range of organisms including the nematode Caenorhabditis elegans. We used a non-redundant transposon mutant library consisting of 5,850 clones corresponding to 75% of the total and approximately 80% of the non-essential PA14 ORFs to carry out a genomewide screen for attenuation of PA14 virulence in C. elegans. We defined a functionally diverse 180 mutant set (representing 170 unique genes) necessary for normal levels of virulence that included both known and novel virulence factors. Seven previously uncharacterized virulence genes (ABC transporters PchH and PchI, aminopeptidase PepP, ATPase/molecular chaperone ClpA, cold shock domain protein PA0456, putative enoyl-CoA hydratase/isomerase PA0745, and putative transcriptional regulator PA14_27700) were characterized with respect to pigment production and motility and all but one of these mutants exhibited pleiotropic defects in addition to their avirulent phenotype. We examined the collection of genes required for normal levels of PA14 virulence with respect to occurrence in P. aeruginosa strain-specific genomic regions, location on putative and known genomic islands, and phylogenetic distribution across prokaryotes. Genes predominantly contributing to virulence in C. elegans showed neither a bias for strain-specific regions of the P. aeruginosa genome nor for putatively horizontally transferred genomic islands. Instead, within the collection of virulence-related PA14 genes, there was an overrepresentation of genes with a broad phylogenetic distribution that also occur with high frequency in many prokaryotic clades, suggesting that in aggregate the genes required for PA14 virulence in C. elegans are biased towards evolutionarily conserved genes.

The increase in genetic variability of a population can be selected during adaptation, as demonstrated by the selection of mutator alleles. The dynamics of this phenomenon, named second-order selection, can result in an improved adaptability of bacteria through regulation of all facets of mutation and recombination processes.  2001 Éditions scientifiques et médicales Elsevier SAS bacterial adaptability / mutagenesis / adaptation / evolution

Escherichia coli strain BL21 is one of the widely used bacterial hosts for high-level recombinant protein production and for other applications. Here, we present the complete genome sequence of a commercial version of the Escherichia coli BL21 strain.

The evolution of CRISPR-cas loci, which encode adaptive immune systems in archaea and bacteria, involves rapid changes, in particular numerous rearrangements of the locus architecture and horizontal transfer of complete loci or individual modules. These dynamics complicate straightforward phylogenetic classification, but here we present an approach combining the analysis of signature protein families and features of the architecture of cas loci that unambiguously partitions most CRISPR-cas loci into distinct classes, types and subtypes. The new classification retains the overall structure of the previous version but is expanded to now encompass two classes, five types and 16 subtypes. The relative stability of the classification suggests that the most prevalent variants of CRISPR-Cas systems are already known. However, the existence of rare, currently unclassifiable variants implies that additional types and subtypes remain to be characterized.

Environments can change in incremental fashions, where a shift from one state to another occurs over multiple organismal generations. Therateat which the environment changes is expected to influence how and how well populations adapt to the ultimate environment. We used a model system, the lytic RNA bacteriophage Φ6, to investigate this question empirically. We evolved viruses for thermostability by exposing them to heat shocks that increased to a maximum temperature at different rates. We observed increases in the ability of many heat-shocked populations to survive high temperature heat shocks, and on their first exposure to the highest temperature, populations that experienced a gradual increase in temperature had higher average survival than populations that experienced a rapid temperature increase. However, at the end of the experiment, neither the survival of populations at the highest temperature nor the number of mutations per population varied significantly according to the ...

There have been extensive genome sequencing studies for Escherichia coli strains, particularly for pathogenic isolates, because fast determination of pathogenic potential and/or drug resistance and their propagation routes is crucial. For laboratory E. coli strains, however, genome sequence information is limited except for several well-known strains. We determined the complete genome sequence of laboratory E. coli strain RR1 (HB101 RecA(+)), which has long been used as a general cloning host. A hybrid genome sequence of K-12 MG1655 and B BL21(DE3) was constructed based on the initial mapping of Illumina HiSeq reads to each reference, and iterative rounds of read mapping, variant detection, and consensus extraction were carried out. Finally, PCR and Sanger sequencing-based finishing were applied to resolve non-single nucleotide variant regions with aberrant read depths and breakpoints, most of them resulting from prophages and insertion sequence transpositions that are not present i...

The population structure and diversity of Lactococcus garvieae, an emerging pathogen of increasing clinical significance, was determined at both gene and genome level. Selected lactococcal isolates of various origins were analyzed by a multi locus sequence typing (MLST). This gene-based analysis was compared to genomic characteristics, estimated through the complete genome sequences available in database. The MLST identified two branches containing the majority of the strains and two branches bearing one strain each. One strain was particularly differentiated from the other L. garvieae strains, showing a significant genetic distance. The genomic characteristics, correlated to the MLST-based phylogeny, indicated that this "separated strain" appeared first and could be considered the evolutionary intermediate between Lactococcus lactis and L. garvieae main clusters. A preliminary genome analysis of L. garvieae indicated a pan-genome constituted of about 4100 genes, which inc...

The sodium-pumping NADH: ubiquinone oxidoreductase (Na +-NQR) is the main ion pump and the primary entry site for electrons into the respiratory chain of many different types of pathogenic bacteria. This enzymatic complex creates a transmembrane gradient of sodium that is used by the cell to sustain ionic homeostasis, nutrient transport, ATP synthesis, flagellum rotation and other essential processes. Comparative genomics data demonstrate that the nqr operon, which encodes all Na +-NQR subunits, is found in a large variety of bacterial lineages with different habitats and metabolic strategies. Here we studied the distribution, origin and evolution of this enzymatic complex. The molecular phylogenetic analyses and the organizations of the nqr operon indicate that Na +-NQR evolved within the Chlorobi/Bacteroidetes group, after the duplication and subsequent neofunctionalization of the operon that encodes the homolog RNF complex. Subsequently, the nqr operon dispersed through multiple horizontal transfer events to other bacterial lineages such as Chlamydiae, Planctomyces and a, b, c and d-proteobacteria. Considering the biochemical properties of the Na +-NQR complex and its physiological role in different bacteria, we propose a detailed scenario to explain the molecular mechanisms that gave rise to its novel redox-dependent sodium-pumping activity. Our model postulates that the evolution of the Na +-NQR complex involved a functional divergence from its RNF homolog, following the duplication of the rnf operon, the loss of the rnfB gene and the recruitment of the reductase subunit of an aromatic monooxygenase.

PSSRdb (Polymorphic Simple Sequence Repeats database) (http://www.cdfd.org.in/PSSRdb/) is a relational database of polymorphic simple sequence repeats (PSSRs) extracted from 85 different species of prokaryotes. Simple sequence repeats (SSRs) are the tandem repeats of nucleotide motifs of the sizes 1-6 bp and are highly polymorphic. SSR mutations in and around coding regions affect transcription and translation of genes. Such changes underpin phase variations and antigenic variations seen in some bacteria. Although SSR-mediated phase variation and antigenic variations have been well-studied in some bacteria there seems a lot of other species of prokaryotes yet to be investigated for SSR mediated adaptive and other evolutionary advantages. As a part of our ongoing studies on SSR polymorphism in prokaryotes we compared the genome sequences of various strains and isolates available for 85 different species of prokaryotes and extracted a number of SSRs showing length variations and created a relational database called PSSRdb. This database gives useful information such as location of PSSRs in genomes, length variation across genomes, the regions harboring PSSRs, etc. The information provided in this database is very useful for further research and analysis of SSRs in prokaryotes.

Hemotrophic mycoplasmas (hemoplasmas) are a group of animal pathogens of the Mollicutes class. Recently, the genomes of 8 hemoplasmas have been completely sequenced. The aim of this study was to gain a better understanding of their genomic features and relationship to other Mycoplasma species. The genome structure and dynamics of hemoplasmas were analyzed by evaluating gene synteny, adaptive evolution of paralogous gene families (PGF) and horizontal gene transfer (HGT). The Mollicutes class was then phylogenetically analyzed by constructing a distance matrix of the 16S rRNA genes and a phylogenetic tree with 32 conserved, concatenated proteins. Our results suggest that the hemoplasmas have dynamic genomes. The genome size variation (from 547 to 1,545 genes) indicates substantial gene gain/loss throughout evolution. Poorly conserved gene syntenies among hemoplasmas, positional shuffling of paralogous genes between strains, HGT, and codons under positive selection in PGFs were also observed. When compared to other Mollicutes species, the hemoplasmas experienced further metabolic reduction, and the 16S rRNA gene distance matrix of the available mollicutes suggests that these organisms presently constitute the most divergent clade within its class. Our phylogenetic tree of concatenated proteins showed some differences when compared to the 16S rRNA gene tree, but non-mycoplasma organisms, such as Ureaplasma spp. and Mesoplasma spp., continue to branch within Mycoplasma clades. In conclusion, while the hemoplasmas experienced further metabolic shrinkage through gene loss, PGFs with positively selected codons are likely beneficial to these species. Phylogeny of the mollicutes based on 16S rRNA genes or concatenated proteins do not obey the current taxonomy. The metabolism and genetic diversity of the mollicutes, the presence of HGT, and lack of standard for genus circumscription are likely to hinder attempts to classify these organisms based on phylogenetic analyses.

Non-typeable Haemophilus influenzae (NTHi), an opportunistic pathogen of the upper airways of healthy children, can infect the lower airways, driving chronic lung disease. However, the molecular basis underpinning NTHi transition from a commensal to a pathogen is not clearly understood. Here, we performed comparative genomic and transcriptomic analyses of 12 paired, isogenic NTHi strains, isolated from the nasopharynx (NP) and bronchoalveolar lavage (BAL) of 11 children with chronic lung disease, to identify convergent molecular signatures associated with lung adaptation. Comparative genomic analyses of the 12 NP-BAL pairs demonstrated that five were genetically identical, with the remaining seven differing by only 1 to 3 mutations. Within-patient transcriptomic analyses identified between 2 and 58 differentially expressed genes in 8 of the 12 NP-BAL pairs, including pairs with no observable genomic changes. Whilst no convergence was observed at the gene level, functional enrichment...

In enterobacteria, acid stress induces expression of the cad system which is involved in maintaining intracellular pH at levels compatible with cell survival. Despite its crucial role, the cad operon is silenced in Shigella and in other pathogenic Escherichia coli. In the present review, we will address the question of why and how the cad locus has been sacrificed for the sake of optimal expression of virulence traits.

The sodium-pumping NADH: ubiquinone oxidoreductase (Na +-NQR) is the main ion pump and the primary entry site for electrons into the respiratory chain of many different types of pathogenic bacteria. This enzymatic complex creates a transmembrane gradient of sodium that is used by the cell to sustain ionic homeostasis, nutrient transport, ATP synthesis, flagellum rotation and other essential processes. Comparative genomics data demonstrate that the nqr operon, which encodes all Na +-NQR subunits, is found in a large variety of bacterial lineages with different habitats and metabolic strategies. Here we studied the distribution, origin and evolution of this enzymatic complex. The molecular phylogenetic analyses and the organizations of the nqr operon indicate that Na +-NQR evolved within the Chlorobi/Bacteroidetes group, after the duplication and subsequent neofunctionalization of the operon that encodes the homolog RNF complex. Subsequently, the nqr operon dispersed through multiple horizontal transfer events to other bacterial lineages such as Chlamydiae, Planctomyces and a, b, c and d-proteobacteria. Considering the biochemical properties of the Na +-NQR complex and its physiological role in different bacteria, we propose a detailed scenario to explain the molecular mechanisms that gave rise to its novel redox-dependent sodium-pumping activity. Our model postulates that the evolution of the Na +-NQR complex involved a functional divergence from its RNF homolog, following the duplication of the rnf operon, the loss of the rnfB gene and the recruitment of the reductase subunit of an aromatic monooxygenase.

In enterobacteria, acid stress induces expression of the cad system which is involved in maintaining intracellular pH at levels compatible with cell survival. Despite its crucial role, the cad operon is silenced in Shigella and in other pathogenic Escherichia coli. In the present review, we will address the question of why and how the cad locus has been sacrificed for the sake of optimal expression of virulence traits.

The efficiencies of the stop codons TAA, TAG, and TGA in protein synthesis termination are not the same. These variations could allow many genes to be regulated. There are many similar nucleotide trimers found on the second and third reading-frames of a gene. They are called premature stop codons (PSC). Like stop codons, the PSC in bacterial genomes are also highly bias in terms of their quantities and qualities on the genes. Phylogenetically related species often share a similar PSC profile. We want to know whether the selective forces that influence the stop codons and the PSC usage biases in a genome are related. We also wish to know how strong these trimers in a genome are related to the natural history of the bacterium. Knowing these relations may provide better knowledge in the phylogeny of bacteria A 16SrRNA-alignment tree of 19 well-studied α-, β- and γ-Proteobacteria Type species is used as standard reference for bacterial phylogeny. The genomes of sixty-one bacteria, belon...

Annexins are Ca 2+-binding, membrane-interacting proteins, widespread among eukaryotes, consisting usually of four structurally similar repeated domains. It is accepted that vertebrate annexins derive from a double genome duplication event. It has been postulated that a single domain annexin, if found, might represent a molecule related to the hypothetical ancestral annexin. The recent discovery of a single-domain annexin in a bacterium, Cytophaga hutchinsonii, apparently confirmed this hypothesis. Here, we present a more complex picture. Using remote sequence similarity detection tools, a survey of bacterial genomes was performed in search of annexin-like proteins. In total, we identified about thirty annexin homologues, including single-domain and multi-domain annexins, in seventeen bacterial species. The thorough search yielded, besides the known annexin homologue from C. hutchinsonii, homologues from the Bacteroidetes/Chlorobi phylum, from Gemmatimonadetes, from beta-and delta-Proteobacteria, and from Actinobacteria. The sequences of bacterial annexins exhibited remote but statistically significant similarity to sequence profiles built of the eukaryotic ones. Some bacterial annexins are equipped with additional, different domains, for example those characteristic for toxins. The variation in bacterial annexin sequences, much wider than that observed in eukaryotes, and different domain architectures suggest that annexins found in bacteria may actually descend from an ancestral bacterial annexin, from which eukaryotic annexins also originate. The hypothesis of an ancient origin of bacterial annexins has to be reconciled with the fact that remarkably few bacterial strains possess annexin genes compared to the thousands of known bacterial genomes and with the patchy, anomalous phylogenetic distribution of bacterial annexins. Thus, a massive annexin gene loss in several bacterial lineages or very divergent evolution would appear a likely explanation. Alternative evolutionary scenarios, involving horizontal gene transfer between bacteria and protozoan eukaryotes, in either direction, appear much less likely. Altogether, current evidence does not allow unequivocal judgement as to the origin of bacterial annexins.

Copy number polymorphisms of nucleotide tandem repeat (TR) regions, such as microsatellites and minisatellites, are mutationally reversible and highly abundant in eukaryotic genomes. Studies linking TR polymorphism to phenotypic variation have led some to suggest that TR variation modulates and majorly contributes to phenotypic variation; however, studies in which the authors assess the genome-wide impact of TR variation on phenotype are lacking. To address this question, we quantified relationships between polymorphism levels in 143 genome-wide promoter region TRs across 16 isolates of the filamentous fungus Aspergillus flavus and its ecotype Aspergillus oryzae with expression levels of their downstream genes. We found that only 4.3% of relationships tested were significant; these findings were consistent with models in which TRs act as "tuning," "volume," or "optimality" "knobs" of phenotype but not with "switch" models. Furthermore, the promoter regions of differentially expressed genes between A. oryzae and A. flavus did not show TR enrichment, suggesting that genome-wide differences in molecular phenotype between the two species are not significantly associated with TRs. Although in some cases TR polymorphisms do contribute to transcript abundance variation, these results argue that at least in this case, TRs might not be major modulators of variation in phenotype.

bution of each mechanism to the generation of genetic Franç ois Taddei, 3 Miroslav Radman, 3,7 diversity varies between species. It has been estimated and Ivan Matic 3 for Escherichia coli that, in spite of the clonal structure 1 INSERM U 458 of its natural populations (Desjardins et al., 1995), any Hô pital Robert Debré single nucleotide change is about 50 times more likely to 48 boulevard Sé rurier have occurred by recombination (with a partner carrying 75935 Paris Cedex 19 such nucleotide change) than by a de novo mutation 2 Service de Systé matique Molé culaire (Guttman and Dykhuizen, 1994). The rate of successful (GDR CNRS 1005) gene transfer between bacterial populations in nature Musé um National d'Histoire Naturelle depends on numerous factors such as ecological isola-43 rue Cuvier tion, host range of genetic exchange vectors, activity of 75231 Paris Cedex 05 the recipient cell nucleases, DNA sequence divergence 3 INSERM E9916 between recombining molecules, type and efficiency of Faculté de Mé decine Necker-Enfants Malades the recipient cell recombination machinery and, finally, Université Paris V the hybrid fitness (Matic et al., 1996). Some of those 156 rue Vaugirard factors have been studied in the laboratory, but their 75730 Paris Cedex 15 impact on gene flow in nature is generally unknown. 4 INSERM U 155 Molecular mechanisms involved in the control of ho-80 rue du Gé né ral Leclerc mologous recombination in bacteria have been exten-94276 Kremlin-Bicê tre sively studied. For example, it was shown that the fre-5 ACTGene Recherche et Dé veloppements quency of integration of donor DNA in the chromosome Allé e Christophe Colomb of recipient cells decreases exponentially with increas-Gé nopole ing sequence divergence between the two recombining 91035 Evry Cedex DNA molecules (Zawadzki et al., 1995; Vulic et al., 1997; France Majewski et al., 2000). The degree and pattern of DNA 6 Department of Genetics and Development divergence determine actions of the enzymes that con-Columbia University trol the outcome of recombination (Radman and Wag-College of Physicians and Surgeons ner, 1993). The initiation of recombination in bacteria New York, New York 10032 depends on the activity of the RecA protein, which is selective for sequence identity only at the initial stage of the strand exchange process (Shen and Huang, 1986). Summary Once initiated, RecA-mediated strand exchange can occur in spite of large numbers of mismatches and even Mutation and subsequent recombination events crelarge heterologies (Bianchi and Radding, 1983; Lichten ate genetic diversity, which is subjected to natural and Fox, 1984). During the strand exchange process, selection. Bacterial mismatch repair (MMR) deficient fidelity of recombination is controlled by the methylmutants, exhibiting high mutation and homeologous directed mismatch repair (MMR) system. MMR proteins recombination rates, are frequently found in natural recognize mispaired and unpaired bases in the joint populations. Therefore, we have explored the possibilheteroduplex regions and block RecA-catalyzed strand ity that MMR deficiency emerging in nature has left transfer (Worth et al., 1994). Thus, MMR is a potent some "imprint" in the sequence of bacterial genomes. inhibitor of recombination between nonidentical DNA Comparative molecular phylogeny of MMR genes from sequences (Rayssiguier et al., 1989; Shen and Huang, natural Escherichia coli isolates shows that, compared 1989; Matic et al., 1995; Vulic et al., 1997). Even low to housekeeping genes, individual functional MMR divergence is sufficient to impede recombination. For genes exhibit high sequence mosaicism derived from example, transductional recombination between two diverse phylogenetic lineages. This apparent horiserovars (Typhimurium and Typhi) of Salmonella enterzontal gene transfer correlates with hyperrecombinaica, whose genomes differ only 1%-2% at DNA setion phenotype of MMR-deficient mutators. The sequence level, is increased 10 2-to 10 3-fold in MMR-defiquence mosaicism of MMR genes may be a hallmark cient genetic backgrounds (Zahrt and Maloy, 1997). of a mechanism of adaptive evolution that involves MMR system is also involved in the control of replicamodulation of mutation and recombination rates by tion fidelity. Inactivation of MMR genes increases mutarecurrent losses and reacquisitions of MMR gene tion rates 10 2-to 10 3-fold (Glickman and Radman, 1980; functions. Schaaper and Dunn, 1987). Studies of mutation rates in natural populations of E. coli, S. enterica, and Pseudomonas aeruginosa show that MMR-deficient strains of-7 To whom correspondence should be addressed (e-mail: radman@ necker.fr). ten exceed a frequency of 1% (LeClerc et al., 1996; Matic

Cyclophilins are prolyl isomerases with multitude of functions in different cellular processes and pathological conditions. Cyclophilin A (PpiA) of Mycobacterium tuberculosis is secreted during infection in intraphagosomal niche. However, our understanding about the evolutionary origin, secretory mechanism or the interactome of M. tuberculosis PpiA is limited. This study demonstrates through phylogenetic and structural analyses that PpiA has more proximity to human cyclophilins than the prokaryotic counterparts. We report a unique N-terminal sequence (MADCDSVTNSP) present in pathogenic mycobacterial PpiA and absent in non-pathogenic strains. This sequence stretch was shown to be essential for PpiA secretion. The overexpression of full-length PpiA from M. tuberculosis in non-pathogenic Mycobacterium smegmatis resulted in PpiA secretion while truncation of the N-terminal stretch obstructed the secretion. In addition, presence of an ESX pathway substrate motif in M. tuberculosis PpiA suggested possible involvement of Type VII secretion system. Site-directed mutagenesis of key residues in this motif in full-length PpiA also hindered the secretion in M. smegmatis. Bacterial twohybrid screens with human lung cDNA library as target were utilized to identify interaction partners of PpiA from host repertoire, and a number of substrates with functional representation in iron storage, signal transduction and immune responses were detected. The extensive host interactome coupled with the sequence and structural similarity to human cyclophilins is strongly suggestive of PpiA being deployed by M. tuberculosis as an effector mimic against the host cyclophilins.

Even a single mutation can cause a marked change in a protein's properties. When the mutant protein functions within a network, complex phenotypes may emerge that are not intrinsic properties of the protein itself. Network architectures that enable such dramatic changes in function from a few mutations remain relatively uncharacterized. We describe a remarkable example of this versatility in the well-studied PhoQ/PhoP bacterial signaling network, which has an architecture found in many two-component systems. We found that a single point mutation that abolishes the phosphatase activity of the sensor kinase PhoQ results in a striking change in phenotype. The mutant responds to stimulus in a bistable manner, as opposed to the wild-type, which has a graded response. Mutant cells in on and off states have different morphologies, and their state is inherited over many generations. Interestingly, external conditions that repress signaling in the wild-type drive the mutant to the on state. Mathematical modeling and experiments suggest that the bistability depends on positive autoregulation of the two key proteins in the circuit, PhoP and PhoQ. The qualitatively different characteristics of the mutant come at a substantial fitness cost. Relative to the off state, the on state has a lower fitness in stationary phase cultures in rich medium (LB). However, due to the high inheritance of the on state, a population of on cells can be epigenetically trapped in a low-fitness state. Our results demonstrate the remarkable versatility of the prototypical twocomponent signaling architecture and highlight the tradeoffs in the particular case of the PhoQ/PhoP system.