Xenogeneic nucleoid-associated EnrR thwarts H-NS silencing of bacterial virulence with unique DNA binding

Type III and type VI secretion systems (T3/T6SS) are encoded in horizontally acquired genomic islands (GIs) that play crucial roles in evolution and virulence in bacterial pathogens. T3/T6SS expression is subjected to tight control by the host xenogeneic silencer H-NS, but how this mechanism is counteracted remains to be illuminated. Here, we report that xenogeneic nucleoid-associated protein EnrR encoded in a GI is essential for virulence in pathogenic bacteria Edwardsiella and Salmonella. We showed that EnrR plays critical roles in T3/T6SS expression in these bacteria. Various biochemical and genetic analyses demonstrated that EnrR binds and derepresses the promoter of esrB, the critical regulator of T3/T6SS, to promote their expression by competing with H-NS. Additionally, EnrR targets AT-rich regions, globally modulates the expression of ∼363 genes and is involved in various cellular processes. Crystal structures of EnrR in complex with a specific AT-rich palindromic DNA revealed a new DNA-binding mode that involves conserved HTH-mediated interactions with the major groove and contacts of its N-terminal extension to the minor groove in the symmetry-related duplex. Collectively, these data demonstrate that EnrR is a virulence activator that can antagonize H-NS, highlighting a unique mechanism by which bacterial xenogeneic regulators recognize and regulate foreign DNA.     

Silencing of cryptic prophages in Corynebacterium glutamicum

DNA of viral origin represents a ubiquitous element of bacterial genomes. Its integration into host regulatory circuits is a pivotal driver of microbial evolution but requires the stringent regulation of phage gene activity. In this study, we describe the nucleoid-associated protein CgpS, which represents an essential protein functioning as a xenogeneic silencer in the Gram-positive Corynebacterium glutamicum. CgpS is encoded by the cryptic prophage CGP3 of the C. glutamicum strain ATCC 13032 and was first identified by DNA affinity chromatography using an early phage promoter of CGP3. Genome-wide profiling of CgpS binding using chromatin affinity purification and sequencing (ChAP-Seq) revealed its association with AT-rich DNA elements, including the entire CGP3 prophage region (187 kbp), as well as several other elements acquired by horizontal gene transfer. Countersilencing of CgpS resulted in a significantly increased induction frequency of the CGP3 prophage. In contrast, a strain lacking the CGP3 prophage was not affected and displayed stable growth. In a bioinformatics approach, cgpS orthologs were identified primarily in actinobacterial genomes as well as several phage and prophage genomes. Sequence analysis of 618 orthologous proteins revealed a strong conservation of the secondary structure, supporting an ancient function of these xenogeneic silencers in phage-host interaction.     

Xenogeneic silencing relies on temperature-dependent phosphorylation of the host H-NS protein in Shewanella

Lateral gene transfer (LGT) plays a key role in shaping the genome evolution and environmental adaptation of bacteria. Xenogeneic silencing is crucial to ensure the safe acquisition of LGT genes into host pre-existing regulatory networks. We previously found that the host nucleoid structuring protein (H-NS) silences prophage CP4So at warm temperatures yet enables this prophage to excise at cold temperatures in Shewanella oneidensis. However, whether H-NS silences other genes and how bacteria modulate H-NS to regulate the expression of genes have not been fully elucidated. In this study, we discovered that the H-NS silences many LGT genes and the xenogeneic silencing of H-NS relies on a temperature-dependent phosphorylation at warm temperatures in S. oneidensis. Specifically, phosphorylation of H-NS at Ser42 is critical for silencing the cold-inducible genes including the excisionase of CP4So prophage, a cold shock protein, and a stress-related chemosensory system. By contrast, nonphosphorylated H-NS derepresses the promoter activity of these genes/operons to enable their expression at cold temperatures. Taken together, our results reveal that the posttranslational modification of H-NS can function as a regulatory switch to control LGT gene expression in host genomes to enable the host bacterium to react and thrive when environmental temperature changes.     

Bacterial Genomic Islands: Organization,Function, and Evolutionary Role

Data on the structural organization and evolutionary role of specific bacterial DNA regions known as genomic islands are reviewed. Emphasis is placed on the most extensively studied genomic islands, pathogenicity islands (PAIs), which are present in the chromosome of Gram-negative and Gram-positive pathogenic bacteria and absent from related nonpathogenic strains. PAIs are long DNA regions that harbor virulence genes and often differ in GC content from the remainder of the bacterial genome. Many PAI occur in the tRNA gene loci, which provide a convenient target for foreign gene insertion. Some PAI are highly homologous to each other and contain sequences similar to ISs, phage att sites, and plasmid ori sites, along with functional or defective integrase and transposase genes, suggesting horizontal transfer of PAI among bacteria.     

Intracellular common gardens reveal niche differentiation in transposable element community during bacterial adaptive evolution

The distribution and abundance of transposable elements across the tree of life have significantly shaped the evolution of cellular organisms, but the underlying mechanisms shaping these ecological patterns remain elusive. Here we establish a “common garden” approach to study causal ecological interactions between a xenogeneic conditional lethal sacB gene and the community of transposable insertion sequences (ISs) in a multipartite prokaryote genome. Xenogeneic sacB of low, medium, or high GC content was individually inserted into three replicons of a model bacterium Sinorhizobium fredii, and exhibited replicon- and GC-dependent variation in genetic stability. This variation was largely attributable to multidimensional niche differentiation for IS community members. The transposition efficiency of major active ISs depended on the nucleoid-associated xenogeneic silencer MucR. Experimentally eliminating insertion activity of specific ISs by deleting MucR strongly demonstrated a dominant role of niche differentiation among ISs. This intracellular common garden approach in the experimental evolution context allows not only for evaluating genetic stability of natural and synthetic xenogeneic genes of different sequence signatures in host cells but also for tracking and testing causal relationships in unifying ecological principles in genome ecology.Subject terms: Molecular ecology, Molecular evolution, Mutation, Community ecology     

A phage-encoded nucleoid associated protein compacts both host and phage DNA and derepresses H-NS silencing

Nucleoid Associated Proteins (NAPs) organize the bacterial chromosome within the nucleoid. The interaction of the NAP H-NS with DNA also represses specific host and xenogeneic genes. Previously, we showed that the bacteriophage T4 early protein MotB binds to DNA, co-purifies with H-NS/DNA, and improves phage fitness. Here we demonstrate using atomic force microscopy that MotB compacts the DNA with multiple MotB proteins at the center of the complex. These complexes differ from those observed with H-NS and other NAPs, but resemble those formed by the NAP-like proteins CbpA/Dps and yeast condensin. Fluorescent microscopy indicates that expression of motB in vivo, at levels like that during T4 infection, yields a significantly compacted nucleoid containing MotB and H-NS. motB overexpression dysregulates hundreds of host genes; ∼70% are within the hns regulon. In infected cells overexpressing motB, 33 T4 late genes are expressed early, and the T4 early gene repEB, involved in replication initiation, is up ∼5-fold. We postulate that MotB represents a phage-encoded NAP that aids infection in a previously unrecognized way. We speculate that MotB-induced compaction may generate more room for T4 replication/assembly and/or leads to beneficial global changes in host gene expression, including derepression of much of the hns regulon.     

Novel anti-repression mechanism of H-NS proteins by a phage protein

H-NS family proteins, bacterial xenogeneic silencers, play central roles in genome organization and in the regulation of foreign genes. It is thought that gene repression is directly dependent on the DNA binding modes of H-NS family proteins. These proteins form lateral protofilaments along DNA. Under specific environmental conditions they switch to bridging two DNA duplexes. This switching is a direct effect of environmental conditions on electrostatic interactions between the oppositely charged DNA binding and N-terminal domains of H-NS proteins. The Pseudomonas lytic phage LUZ24 encodes the protein gp4, which modulates the DNA binding and function of the H-NS family protein MvaT of Pseudomonas aeruginosa. However, the mechanism by which gp4 affects MvaT activity remains elusive. In this study, we show that gp4 specifically interferes with the formation and stability of the bridged MvaT–DNA complex. Structural investigations suggest that gp4 acts as an ‘electrostatic zipper’ between the oppositely charged domains of MvaT protomers, and stabilizes a structure resembling their ‘half-open’ conformation, resulting in relief of gene silencing and adverse effects on P. aeruginosa growth. The ability to control H-NS conformation and thereby its impact on global gene regulation and growth might open new avenues to fight Pseudomonas multidrug resistance.     

A silencer promotes the assembly of silenced chromatin independently of recruitment

In Saccharomyces cerevisiae, silenced chromatin occurs at telomeres and the silent mating-type loci HMR and HML. At these sites, the Sir proteins are recruited to a silencer and then associate with adjacent chromatin. We used chromatin immunoprecipitation to compare the rates of Sir protein assembly at different genomic locations and discovered that establishment of silenced chromatin was much more rapid at HMR than at the telomere VI-R. Silenced chromatin also assembled more quickly on one side of HMR-E than on the other. Despite differences in spreading, the Sir proteins were recruited to HMR-E and telomeric silencers at equivalent rates. Additionally, insertion of HMR-E adjacent to the telomere VI-R increased the rate of Sir2p association with the telomere. These data suggest that HMR-E functions to both recruit Sir proteins and promote their assembly across several kilobases. Observations that association of Sir2p occurs simultaneously throughout HMR and that silencing at HMR is insensitive to coexpression of catalytically inactive Sir2p suggest that HMR-E acts by enabling assembly to occur in a nonlinear fashion. The ability of silencers to promote assembly of silenced chromatin over several kilobases is likely an important mechanism for maintaining what would otherwise be unstable chromatin at the correct genomic locations.     

Depletion of CG-Specific Methylation in Mycoplasma hyorhinis Genomic DNA after Host Cell Invasion

Adaptation to the environment requires pathogenic bacteria to alter their gene expression in order to increase long-term survival in the host. Here, we present the first experimental evidence that bacterial DNA methylation affects the intracellular survival of pathogenic Mycoplasma hyorhinis. Using bisulfite sequencing, we identified that the M. hyorhinis DNA methylation landscape was distinct in free-living M. hyorhinis relative to the internalized bacteria surviving in the infected human cells. We determined that genomic GATC sites were consistently highly methylated in the bacterial chromosome suggesting that the bacterial GATC-specific 5-methylcytosine DNA methyltransferase was fully functional both pre- and post-infection. In contrast, only the low CG methylation pattern was observed in the mycoplasma genome in the infective bacteria that invaded and then survived in the host cells. In turn, two distinct populations, with either high or low CG methylation, were detected in the M. hyorhinis cultures continually grown in the rich medium independently of host cells. We also identified that M. hyorhinis efficiently evaded endosomal degradation and uses exocytosis to exit infected human cells enabling re-infection of additional cells. The well-orchestrated changes in the chromosome methylation landscape play a major regulatory role in the mycoplasma life cycle.     

New evidence of an old problem: The coupling of genome replication to cell growth in bacteria

The problem of coordinating genome replication with cell growth in bacteria was posed over four decades ago. Unlike for eukaryotes, this problem has not been completely solved even for Escherichia coli, which has been comprehensively studied by molecular biologists, to say nothing of other bacteria. Current models of the bacterial life cycle solve the coupling problem by introducing a phenomenological hypothesis that considers the dynamic coordination of growth and replication but does not unveil the underlying molecular mechanisms. Here we review the mechanisms regulating genome replication initiation with regards to their coupling to growth processes in the three best investigated bacterial species: E. coli, Bacillus subtilis, and Caulobacter crescentus. A putative correlation between the type of cell growth laws and the actual mechanisms regulating the replication of DNA formed during the process of evolution in various classes of bacteria, is discussed, including those intracellular parasites in which degenerative evolution has discarded most of their genomes. We contemplate the concept of a universal growth law for bacterial cells and some features in the formation of a primitive negative replication regulating mechanism in the context of the coupling problem.     

Horizontal gene transfer regulation in bacteria as a "spandrel" of DNA repair mechanisms

Horizontal gene transfer (HGT) is recognized as the major force for bacterial genome evolution. Yet, numerous questions remain about the transferred genes, their function, quantity and frequency. The extent to which genetic transformation by exogenous DNA has occurred over evolutionary time was initially addressed by an in silico approach using the complete genome sequence of the Ralstonia solanacearum GMI1000 strain. Methods based on phylogenetic reconstruction of prokaryote homologous genes families detected 151 genes (13.3%) of foreign origin in the R. solanacearum genome and tentatively identified their bacterial origin. These putative transfers were analyzed in comparison to experimental transformation tests involving 18 different genomic DNA positions in the genome as sites for homologous or homeologous recombination. Significant transformation frequency differences were observed among these positions tested regardless of the overall genomic divergence of the R. solanacearum strains tested as recipients. The genomic positions containing the putative exogenous DNA were not systematically transformed at the highest frequencies. The two genomic "hot spots", which contain recA and mutS genes, exhibited transformation frequencies from 2 to more than 4 orders of magnitude higher than positions associated with other genes depending on the recipient strain. These results support the notion that the bacterial cell is equipped with active mechanisms to modulate acquisition of new DNA in different genomic positions. Bio-informatics study correlated recombination "hot-spots" to the presence of Chi-like signature sequences with which recombination might be preferentially initiated. The fundamental role of HGT is certainly not limited to the critical impact that the very rare foreign genes acquired mainly by chance can have on the bacterial adaptation potential. The frequency to which HGT with homologous and homeologous DNA happens in the environment might have led the bacteria to hijack DNA repair mechanisms in order to generate genetic diversity without losing too much genomic stability.     

Genomic perspectives on the evolution and spread of bacterial pathogens

Since the first complete sequencing of a free-living organism, Haemophilus influenzae, genomics has been used to probe both the biology of bacterial pathogens and their evolution. Single-genome approaches provided information on the repertoire of virulence determinants and host-interaction factors, and, along with comparative analyses, allowed the proposal of hypotheses to explain the evolution of many of these traits. These analyses suggested many bacterial pathogens to be of relatively recent origin and identified genome degradation as a key aspect of host adaptation. The advent of very-high-throughput sequencing has allowed for detailed phylogenetic analysis of many important pathogens, revealing patterns of global and local spread, and recent evolution in response to pressure from therapeutics and the human immune system. Such analyses have shown that bacteria can evolve and transmit very rapidly, with emerging clones showing adaptation and global spread over years or decades. The resolution achieved with whole-genome sequencing has shown considerable benefits in clinical microbiology, enabling accurate outbreak tracking within hospitals and across continents. Continued large-scale sequencing promises many further insights into genetic determinants of drug resistance, virulence and transmission in bacterial pathogens.     

Sialoglycan-binding patterns of bacterial AB5 toxin B subunits correlate with host range and toxicity,indicating evolution independent of A subunits

Many pathogenic bacteria secrete AB₅ toxins that can be virulence factors. Cytotoxic A subunits are delivered to the cytosol following B subunit binding to specific host cell surface glycans. Some B subunits are not associated with A subunits, for example, YpeB of Yersinia pestis, the etiologic agent of plague. Plague cannot be eradicated because of Y. pestis'' adaptability to numerous hosts. We previously showed selective binding of other B₅ pentamers to a sialoglycan microarray, with sialic acid (Sia) preferences corresponding to those prominently expressed by various hosts, for example, N-acetylneuraminic acid (Neu5Ac; prominent in humans) or N-glycolylneuraminic acid (Neu5Gc; prominent in ruminant mammals and rodents). Here, we report that A subunit phylogeny evolved independently of B subunits and suggest a future B subunit nomenclature based on bacterial species names. We also found via phylogenetic analysis of B subunits, which bind Sias, that homologous molecules show poor correlation with species phylogeny. These data indicate ongoing lateral gene transfers between species, including mixing of A and B subunits. Consistent with much broader host range of Y. pestis, we show that YpeB recognizes all mammalian Sia types, except for 4-O-acetylated ones. Notably, YpeB alone causes dose-dependent cytotoxicity, which is abolished by a mutation (Y77F) eliminating Sia recognition, suggesting that cell proliferation and death are promoted via lectin-like crosslinking of cell surface sialoglycoconjugates. These findings help explain the host range of Y. pestis and could be important for pathogenesis. Overall, our data indicate ongoing rapid evolution of both host Sias and pathogen toxin-binding properties.     

Variation in host preferences of malaria mosquitoes is mediated by skin bacterial volatiles

The host preferences of the anthropophilic mosquito species in the Anopheles gambiae complex (Diptera: Culicidae) are mediated by skin bacterial volatiles. However, it is not known whether these mosquitoes respond differentially to skin bacterial volatiles from non‐human host species. In this study, the responses of two malaria mosquito species in the An. gambiae complex, Anopheles gambiae s.s. (hereafter, An. gambiae) and Anopheles arabiensis, with different host preferences, to volatiles released from skin bacteria were tested. Skin bacteria collected from human, cow and chicken skin significantly increased trap catches; traps containing bacteria collected from human skin caught the highest proportions of An. gambiae and An. arabiensis. Traps with bacteria of human origin caught a significantly higher proportion of An. gambiae than of An. arabiensis, whereas bacterial volatiles from the chicken attracted significantly higher numbers of An. arabiensis than of An. gambiae. Additionally, An. gambiae showed a specialized response to volatiles from four specific bacteria, whereas An. arabiensis responded equally to all species of bacteria tested. Skin bacterial volatiles may therefore play important roles in guiding mosquitoes with different host preferences. The identification of these bacterial volatiles can contribute to the development of an odour blend that attracts mosquitoes with different host preferences.     

Evolutionary insights from Erwinia amylovora genomics

Evolutionary genomics is coming into focus with the recent availability of complete sequences for many bacterial species. A hypothesis on the evolution of virulence factors in the plant pathogen Erwinia amylovora, the causative agent of fire blight, was generated using comparative genomics with the genomes E. amylovora, Erwinia pyrifoliae and Erwinia tasmaniensis. Putative virulence factors were mapped to the proposed genealogy of the genus Erwinia that is based on phylogenetic and genomic data. Ancestral origin of several virulence factors was identified, including levan biosynthesis, sorbitol metabolism, three T3SS and two T6SS. Other factors appeared to have been acquired after divergence of pathogenic species, including a second flagellar gene and two glycosyltransferases involved in amylovoran biosynthesis. E. amylovora singletons include 3 unique T3SS effectors that may explain differential virulence/host ranges. E. amylovora also has a unique T1SS export system, and a unique third T6SS gene cluster. Genetic analysis revealed signatures of foreign DNA suggesting that horizontal gene transfer is responsible for some of these differential features between the three species.     

Spontaneous modulation of a dynamic balance between bacterial genomic stability and mutability: roles and molecular mechanisms of the genetic switch

Bacteria need a high degree of genetic stability to maintain their species identities over long evolutionary times while retaining some mutability to adapt to the changing environment.It is a long unanswered question that how bacteria reconcile these seemingly contradictory biological properties.We hypothesized that certain mechanisms must maintain a dynamic balance between genetic stability and mutability for the survival and evolution of bacterial species.To identify such mechanisms,we analyzed bacterial genomes,focusing on the Salmonella mismatch repair(MMR)system.We found that the MMR gene mutL functions as a genetic switch through a slipped-strand mispairing mechanism,modulating and maintaining a dynamic balance between genetic stability and mutability during bacterial evolution.This mechanism allows bacteria to maintain their phylogenetic status,while also adapting to changing environments by acquiring novel traits.In this review,we outline the history of research into this genetic switch,from its discovery to the latest findings,and discuss its potential roles in the genomic evolution of bacteria.     

Diversity of rhizobia and interactions among the host legumes and rhizobial genotypes in an agricultural-forestry ecosystem

To investigate the diversity of rhizobia and interactions among the host legumes and rhizobial genotypes in the same habitat, a total of 97 rhizobial strains isolated from nine legume species grown in an agricultural-forestry ecosystem were identified into seven genomic species and 12 symbiotic genotypes within the genera Bradyrhizobium, Mesorhizobium, Rhizobium and Sinorhizobium based upon analyses of genomic DNA regions and symbiotic genes. The results evidenced that the symbiotic genotypes of rhizobia were consistent with their hosts of origin; revealed that vertical transfer was the main mechanism in rhizobia to maintain the symbiotic genes but lateral transfer of symbiotic genes might have happened between the closely related rhizobial species; suggested the existence of co-distribution and co-evolution among the legume hosts and compatible rhizobia. All of these data demonstrated that the biogeography of rhizobia was a result of interactions among the host legumes, bacterial genomic backgrounds and environments.