Two efficient recombination systems were combined to produce a versatile method for chromosomal engineering that obviates the need to prepare double-stranded DNA (dsDNA) recombination substrates. A synthetic "targeting oligonucleotide" is incorporated into the chromosome via homologous recombination mediated by the phage Che9c RecT annealase. This oligonucleotide contains a site-specific recombination site for the directional Bxb1 integrase (Int), which allows the simultaneous integration of a "payload plasmid" that contains a cognate recombination site and a selectable marker. The targeting oligonucleotide and payload plasmid are cotransformed into a RecT- and Int-expressing strain, and drug-resistant homologous recombinants are selected in a single step. A library of reusable target-independent payload plasmids is available to generate gene knockouts, promoter replacements, or C-terminal tags. This new system is called ORBIT (for "oligonucleotide-mediated recombineering followed by Bxb1 integrase targeting") and is ideally suited for the creation of libraries consisting of large numbers of deletions, insertions, or fusions in a bacterial chromosome. We demonstrate the utility of this "drag and drop" strategy by the construction of insertions or deletions in over 100 genes in Mycobacteriumtuberculosis and M. smegmatisIMPORTANCE We sought to develop a system that could increase the usefulness of oligonucleotide-mediated recombineering of bacterial chromosomes by expanding the types of modifications generated by an oligonucleotide (i.e., insertions and deletions) and by making recombinant formation a selectable event. This paper describes such a system for use in M. smegmatis and M. tuberculosis By incorporating a single-stranded DNA (ssDNA) version of the phage Bxb1 attP site into the oligonucleotide and coelectroporating it with a nonreplicative plasmid that carries an attB site and a drug selection marker, we show both formation of a chromosomal attP site and integration of the plasmid in a single transformation. No target-specific dsDNA substrates are required. This system will allow investigators studying mycobacterial diseases, including tuberculosis, to easily generate multiple mutants for analysis of virulence factors, identification of new drug targets, and development of new vaccines.

Coordinating chromosome duplication and segregation with cell division is clearly critical for bacterial species with one chromosome. The precise choreography required is even more complex in species with more than one chromosome. The alpha subgroup of bacteria contains not only one of the best-studied bacterial species, Caulobacter crescentus, but also several species with more than one chromosome. Rhodobacter sphaeroides is an alphaproteobacterium with two chromosomes, but, unlike C. crescentus, it divides symmetrically rather than buds and lacks the complex CtrA-dependent control mechanism. By examining the Ori and Ter regions of both chromosomes and associated ParA and ParB proteins relative to cell division proteins FtsZ and MipZ, we have identified a different pattern of chromosome segregation and cell division. The pattern of chromosome duplication and segregation resembles that of Vibrio cholerae, not that of Agrobacterium tumefaciens, with duplication of the origin and terminus regions of chromosome 2 controlled by chromosome 1. Key proteins are localized to different sites compared to C. crescentus OriC1 and ParB1 are localized to the old pole, while MipZ and FtsZ localize to the new pole. Movement of ParB1 to the new pole following chromosome duplication releases FtsZ, which forms a ring at midcell, but, unlike reports for other species, MipZ monomers do not form a gradient but oscillate between poles, with the nucleotide-bound monomer and the dimer localizing to midcell. MipZ dimers form a single ring (with a smaller diameter) close to the FtsZ ring at midcell and constrict with the FtsZ ring. Overproduction of the dimer form results in filamentation, suggesting that MipZ dimers are regulating FtsZ activity and thus septation. This is an unexpected role for MipZ and provides a new model for the integration of chromosome segregation and cell division.IMPORTANCE Cell division has to be coordinated with chromosome segregation to ensure the stable inheritance of genetic information. We investigated this coordination in the multichromosome bacterium Rhodobacter sphaeroides By examining the origin and terminus regions of the two chromosomes, the ParA-like ATPase MipZ and FtsZ, we showed that chromosome 1 appears to be the "master" chromosome connecting DNA segregation and cell division, with MipZ being critical for coordination. MipZ shows an unexpected localization pattern, with MipZ monomers interacting with ParB of the chromosome 1 at the cell poles whereas MipZ dimers colocalize with FtsZ at midcell during constriction, both forming dynamic rings. These data suggest that MipZ has roles in R. sphaeroides in both controlling septation and coordinating chromosome segregation with cell division.

As typical bacterial replicons, circular chromosomes replicate bidirectionally and circular plasmids replicate either bidirectionally or unidirectionally. Whereas the finding of chromids (plasmid-derived chromosomes) in multiple bacterial lineages provides circumstantial evidence that chromosomes likely evolved from plasmids, all experimentally assayed chromids were shown to use bidirectional replication. Here, we employed a model system, the marine bacterial genus Pseudoalteromonas, members of which consistently carry a chromosome and a chromid. We provide experimental and bioinformatic evidence that while chromids in a few strains replicate bidirectionally, most replicate unidirectionally. This is the first experimental demonstration of the unidirectional replication mode in bacterial chromids. Phylogenomic and comparative genomic analyses showed that the bidirectional replication evolved only once from a unidirectional ancestor and that this transition was associated with insertions of exogenous DNA and relocation of the replication terminus region (ter2) from near the origin site (ori2) to a position roughly opposite it. This process enables a plasmid-derived chromosome to increase its size and expand the bacterium's metabolic versatility while keeping its replication synchronized with that of the main chromosome. A major implication of our study is that the uni- and bidirectionally replicating chromids may represent two stages on the evolutionary trajectory from unidirectionally replicating plasmids to bidirectionally replicating chromosomes in bacteria. Further bioinformatic analyses predicted unidirectionally replicating chromids in several unrelated bacterial phyla, suggesting that evolution from unidirectionally to bidirectionally replicating replicons occurred multiple times in bacteria.IMPORTANCE Chromosome replication is an essential process for cell division. The mode of chromosome replication has important impacts on the structure of the chromosome and replication speed. Bidirectional replication is the rule for bacterial chromosomes, and unidirectional replication has been found only in plasmids. To date, no bacterial chromosomes have been experimentally demonstrated to replicate unidirectionally. Here, we showed that the chromids (plasmid-derived chromosomes) in Pseudoalteromonas replicate either uni- or bidirectionally and that a single evolutionary transition from uni- to bidirectionality explains this diversity. These uni- and bidirectionally replicating chromids likely represent two stages during the evolution from a small and unidirectionally replicating plasmid to a large and bidirectionally replicating chromosome. This study provides insights into both the physiology of chromosome replication and the early evolutionary history of bacterial chromosomes.

It is unclear how gene order within the chromosome influences genome evolution. Bacteria cluster transcription and translation genes close to the replication origin (oriC). In Vibrio cholerae, relocation of s10-spc-α locus (S10), the major locus of ribosomal protein genes, to ectopic genomic positions shows that its relative distance to the oriC correlates to a reduction in growth rate, fitness, and infectivity. To test the long-term impact of this trait, we evolved 12 populations of V. cholerae strains bearing S10 at an oriC-proximal or an oriC-distal location for 1,000 generations. During the first 250 generations, positive selection was the main force driving mutation. After 1,000 generations, we observed more nonadaptative mutations and hypermutator genotypes. Populations fixed inactivating mutations at many genes linked to virulence: flagellum, chemotaxis, biofilm, and quorum sensing. Throughout the experiment, all populations increased their growth rates. However, those bearing S10 close to oriC remained the fittest, indicating that suppressor mutations cannot compensate for the genomic position of the main ribosomal protein locus. Selection and sequencing of the fastest-growing clones allowed us to characterize mutations inactivating, among other sites, flagellum master regulators. Reintroduction of these mutations into the wild-type context led to a ≈10% growth improvement. In conclusion, the genomic location of ribosomal protein genes conditions the evolutionary trajectory of V. cholerae. While genomic content is highly plastic in prokaryotes, gene order is an underestimated factor that conditions cellular physiology and evolution. A lack of suppression enables artificial gene relocation as a tool for genetic circuit reprogramming. IMPORTANCE The bacterial chromosome harbors several entangled processes such as replication, transcription, DNA repair, and segregation. Replication begins bidirectionally at the replication origin (oriC) until the terminal region (ter) organizing the genome along the ori-ter axis gene order along this axis could link genome structure to cell physiology. Fast-growing bacteria cluster translation genes near oriC. In Vibrio cholerae, moving them away was feasible but at the cost of losing fitness and infectivity. Here, we evolved strains harboring ribosomal genes close or far from oriC. Growth rate differences persisted after 1,000 generations. No mutation was able to compensate for the growth defect, showing that ribosomal gene location conditions their evolutionary trajectory. Despite the high plasticity of bacterial genomes, evolution has sculpted gene order to optimize the ecological strategy of the microorganism. We observed growth rate improvement throughout the evolution experiment that occurred at expense of energetically costly processes such the flagellum biosynthesis and virulence-related functions. From the biotechnological point of view, manipulation of gene order enables altering bacterial growth with no escape events.

Plant roots influence the soil microbiota via physical interaction, secretion, and plant immunity. However, it is unclear whether the root fraction or soil is more important in determining the structure of the prokaryotic or eukaryotic community and whether this varies between plant species. Furthermore, the leaf (phyllosphere) and root microbiotas have a large overlap; however, it is unclear whether this results from colonization of the phyllosphere by the root microbiota. Soil, rhizosphere, rhizoplane, and root endosphere prokaryote-, eukaryote-, and fungus-specific microbiotas of four plant species were analyzed with high-throughput sequencing. The strengths of factors controlling microbiota structure were determined using permutational multivariate analysis of variance (PERMANOVA) statistics. The origin of the phyllosphere microbiota was investigated using a soil swap experiment. Global microbial kingdom analysis conducted simultaneously on multiple plants shows that cereals, legumes, and Brassicaceae establish similar prokaryotic and similar eukaryotic communities inside and on the root surface. While the bacterial microbiota is recruited from the surrounding soil, its profile is influenced by the root itself more so than by soil or plant species. However, in contrast, the fungal microbiota is most strongly influenced by soil. This was observed in two different soils and for all plant species examined. Microbiota structure is established within 2 weeks of plant growth in soil and remains stable thereafter. A reciprocal soil swap experiment shows that the phyllosphere is colonized from the soil in which the plant is grown.IMPORTANCE Global microbial kingdom analysis conducted simultaneously on multiple plants shows that cereals, legumes, and Brassicaceae establish similar prokaryotic and similar eukaryotic communities inside and on the root surface. While the bacterial microbiota is recruited from the surrounding soil, its profile is influenced by the root fraction more so than by soil or plant species. However, in contrast, the fungal microbiota is most strongly influenced by soil. This was observed in two different soils and for all plant species examined, indicating conserved adaptation of microbial communities to plants. Microbiota structure is established within 2 weeks of plant growth in soil and remains stable thereafter. We observed a remarkable similarity in the structure of a plant's phyllosphere and root microbiotas and show by reciprocal soil swap experiments that both fractions are colonized from the soil in which the plant is grown. Thus, the phyllosphere is continuously colonized by the soil microbiota.

Bacterial toxin-antitoxin (TA) systems encode a toxin and an antitoxin that counteracts the toxin. Such TA systems are found abundantly on bacterial chromosomes and on extrachromosomal genetic elements. The toxin is always a protein. Based on the nature of the antitoxin (protein or RNA) and on their mode of regulation, they are classified into six groups (I to VI). In the group II TA systems, both the toxin and the antitoxin are proteins, and the gene specifying the antitoxin precedes the gene specifying for the toxin. Here, we studied the intracellular localization in Escherichia coli cells of the proteins specified by the following type II TA modules: mazEF, chpBIK, mqsRA, and rnlAB. We visualized the localization of these proteins by fusing them with the fluorescent protein mCherry using recombinant DNA technology. We used fluorescence microscopy and image analysis software to obtain and quantify protein distribution data. With the exception of the chpBIK TA module, we found that the localization of each toxin-antitoxin complex was different from the localization of the toxin itself. Our results demonstrate clearly that the presence of the antitoxin shifts the localization of its respective toxin toward the middle of the cell, which could contribute to the reduction of cellular toxicity. IMPORTANCE Bacterial toxin-antitoxin (TA) systems, which were discovered in 1985, have since been studied extensively. These studies have focused particularly on the distribution of these bacterial TA systems on either plasmids or on bacterial chromosomes, their functionality, their targets, their relation to virulence, and their mechanisms of action. Our study, reported here, is the first to clarify the intracellular localization of the proteins specified for some type II TA systems. We have shown that, with the exception of the chpBIK module, each toxin-antitoxin complex was localized in a different part of the cell than the toxin itself. Our results revealed clearly that the presence of the antitoxin changes the localization of the toxin by moving the toxin toward the middle of the cell. Until now, the general view has been that the antagonistic effect of the antitoxins over their cognate toxins is based only on their direct structural interactions. Here, we show that this antagonistic effect is also a function of a specific change in the intracellular localization of the toxin.

The physiological state of natural competence for transformation allows certain bacteria to take up free DNA from the environment and to recombine such newly acquired DNA into their chromosomes. However, even though conserved components that are required to undergo natural transformation have been identified in several naturally competent bacteria, our knowledge of the underlying mechanisms of the DNA uptake process remains very limited. To better understand these mechanisms, we investigated the competence-mediated DNA transport in the naturally transformable pathogen Vibrio cholerae. Previously, we used a cell biology-based approach to experimentally address an existing hypothesis, which suggested the competence protein ComEA plays a role in the DNA uptake process across the outer membrane of Gram-negative bacteria. Here, we extended this knowledge by investigating the dynamics of DNA translocation across both membranes. More precisely, we indirectly visualized the transfer of the external DNA from outside the cell into the periplasm followed by the shuttling of the DNA into the cytoplasm. Based on these data, we conclude that for V. cholerae, the DNA translocation across the outer and inner membranes is spatially but not temporally coupled.

In this study, we describe the isolation and characterization of novel bacteriophage vB_EcoP_Kapi1 (Kapi1) isolated from a strain of commensal Escherichia coli inhabiting the gastrointestinal tract of healthy mice. We show that Kapi1 is a temperate phage integrated into tRNA argW of strain MP1 and describe its genome annotation and structure. Kapi1 shows limited homology to other characterized prophages but is most similar to the seroconverting phages of Shigella flexneri and clusters taxonomically with P22-like phages. The receptor for Kapi1 is the lipopolysaccharide O-antigen, and we further show that Kapi1 alters the structure of its host's O-antigen in multiple ways. Kapi1 displays unstable lysogeny, and we find that the lysogenic state is more stable during growth in simulated intestinal fluid. Furthermore, Kapi1 lysogens have a competitive advantage over their nonlysogenic counterparts both in vitro and in vivo, suggesting a role for Kapi1 during colonization. We thus report the use of MP1 and Kapi1 as a model system to explore the molecular mechanisms of mammalian colonization by E. coli to ask what the role(s) of prophages in this context might be. IMPORTANCE Although research exploring the microbiome has exploded in recent years, our understanding of the viral component of the microbiome is lagging far behind our understanding of the bacterial component. The vast majority of intestinal bacteria carry prophages integrated into their chromosomes, but most of these bacteriophages remain uncharacterized and unexplored. Here, we isolate and characterize a novel temperate bacteriophage infecting a commensal strain of Escherichia coli. We aim to explore the interactions between bacteriophages and their hosts in the context of the gastrointestinal tract, asking what role(s) temperate bacteriophages may play in growth and survival of bacteria in the gut. Understanding the fundamental biology of gut commensal bacteria can inform the development of novel antimicrobial or probiotic strategies for intestinal infections.

Homologous recombination is an important mechanism directly involved in the repair, organization, and evolution of prokaryotic and eukaryotic chromosomes. We developed a system, based on two genetic cassettes, that allows the measurement of recombinational repair rates between different locations on the chromosome. Using this system, we analyzed 81 different positional combinations throughout the chromosome to answer the question of how the position and orientation of sequences affect intrachromosomal homologous recombination. Our results show that recombination was possible between any two locations tested in this study and that recombinational repair rates varied by just above an order of magnitude. The observed differences in rate do not correlate with distance between the recombination cassettes or with distance from the origin of replication but could be explained if each location contributes individually to the recombination event. The relative levels of accessibility for recombination vary 5-fold between the various cassette locations, and we found that the nucleoid structure of the chromosome may be the major factor influencing the recombinational accessibility of each chromosomal site. Furthermore, we found that the orientation of the recombination cassettes had a significant impact on recombination. Recombinational repair rates for the cassettes inserted as direct repeats are, on average, 2.2-fold higher than those for the same sets inserted as inverted repeats. These results suggest that the bacterial chromosome is not homogenous with regard to homologous recombination, with regions that are more or less accessible, and that the orientation of genes affects recombination rates. IMPORTANCE Bacterial chromosomes frequently carry multiple copies of genes at separate chromosomal locations. In Salmonella, these include the 7 rrn operons and the duplicate tuf genes. Genes within these families coevolve by homologous recombination, but it is not obvious whether their rates of recombination reflect general rates of intrachromosomal recombination or are an evolved property particularly associated with these conserved genes and locations. Using a novel experimental system, we show that recombination is possible between all tested pairs of locations at rates that vary by just above 1 order of magnitude. Differences in rate do not correlate with distance between the sites or distance to the origin of replication but may be explained if each location contributes individually to the recombination event. Our results suggest the existence of bacterial chromosomal domains that are differentially available for recombination and that gene orientation affects recombination rates.

It has recently been demonstrated that bacterial chromosomes are highly organized, with specific positioning of the replication initiation region. Moreover, the positioning of the replication machinery (replisome) has been shown to be variable and dependent on species-specific cell cycle features. Here, we analyzed replisome positions in Mycobacterium smegmatis, a slow-growing bacterium that exhibits characteristic asymmetric polar cell extension. Time-lapse fluorescence microscopy analyses revealed that the replisome is slightly off-center in mycobacterial cells, a feature that is likely correlated with the asymmetric growth of Mycobacterium cell poles. Estimates of the timing of chromosome replication in relation to the cell cycle, as well as cell division and chromosome segregation events, revealed that chromosomal origin-of-replication (oriC) regions segregate soon after the start of replication. Moreover, our data demonstrate that organization of the chromosome by ParB determines the replisome choreography.

The arms race between bacteria and their bacteriophages profoundly influences microbial evolution. With an estimated 1023 phage infections occurring per second, there is strong selection for both bacterial survival and phage coevolution for continued propagation. Many phage resistance systems, including restriction-modification systems, clustered regularly interspaced short palindromic repeat-Cas (CRISPR-Cas) systems, a variety of abortive infection systems, and many others that are not yet mechanistically defined, have been described. Temperate bacteriophages are common and form stable lysogens that are immune to superinfection by the same or closely related phages. However, temperate phages collude with their hosts to confer defense against genomically distinct phages, to the mutual benefit of the bacterial host and the prophage. Prophage-mediated viral systems have been described in Mycobacterium phages and Pseudomonas phages but are predicted to be widespread throughout the microbial world. Here we describe a new viral defense system in which the mycobacteriophage Sbash prophage colludes with its Mycobacterium smegmatis host to confer highly specific defense against infection by the unrelated mycobacteriophage Crossroads. Sbash genes 30 and 31 are lysogenically expressed and are necessary and sufficient to confer defense against Crossroads but do not defend against any of the closely related phages grouped in subcluster L2. The mapping of Crossroads defense escape mutants shows that genes 132 and 141 are involved in recognition by the Sbash defense system and are proposed to activate a loss in membrane potential mediated by Sbash gp30 and gp31.IMPORTANCE Viral infection is an ongoing challenge to bacterial survival, and there is strong selection for development or acquisition of defense systems that promote survival when bacteria are attacked by bacteriophages. Temperate phages play central roles in these dynamics through lysogenic expression of genes that defend against phage attack, including those unrelated to the prophage. Few prophage-mediated viral defense systems have been characterized, but they are likely widespread both in phage genomes and in the prophages integrated in bacterial chromosomes.

Highly expressed genes are commonly located close to the origin of replication of bacterial chromosomes (OriC). This location skew is thought to reflect selective advantages associated with gene dosage effects during the replication cycle. The expression of constitutively expressed genes can vary up to fivefold based on chromosomal location, but it is not clear what level of variation would occur in naturally regulated operons. We tested the magnitude of the chromosome location effect using EF-Tu (tufA, tufB), an abundant protein whose cellular level correlates with, and limits, the maximum growth rate. We translocated the Salmonella tufB operon to four locations across the chromosome. The distance from OriC had only a small effect on growth rate, consistent with this operon having the natural ability to upregulate expression and compensate for reduced gene dosage. In contrast, when the total EF-Tu concentration was limiting for the growth rate (tufA deleted), we observed a strong gene dosage effect when tufB was located further from OriC. However, only a short period of experimental evolution was required before the bacteria adapted to this EF-Tu starvation situation by acquiring genetic changes that increased expression levels from the translocated tufB gene, restoring growth rates. Our findings demonstrate that, at least for the tufB operon, gene dosage is probably not the dominant force selecting for a chromosomal location close to OriC. We suggest that the colocation of highly expressed genes close to OriC might instead be selected because it enhances their coregulation during various growth states, with gene dosage being a secondary benefit.IMPORTANCE A feature of bacterial chromosomes is that highly expressed essential genes are usually located close to the origin of replication. Because bacteria have overlapping cycles of replication, genes located close to the origin will often be present in multiple copies, and this is thought to be of selective benefit where high levels of expression support high growth rate. However, the magnitude of this selective effect and whether other forces could be at play are poorly understood. To study this, we translocated a highly expressed essential operon, tufB, to different locations and measured growth fitness. We found that transcriptional regulation buffered the effects of translocation and that even under conditions where growth rate was reduced, genetic changes that increased the expression of tufB were easily and rapidly selected. We conclude, at least for tufB, that forces other than gene dosage may be significant in selecting for chromosomal location.

Vibrio vulnificus, an opportunistic pathogen, is the causative agent of a life-threatening septicemia and a rising problem for aquaculture worldwide. The genetic factors that differentiate its clinical and environmental strains remain enigmatic. Furthermore, clinical strains have emerged from every clade of V. vulnificus In this work, we investigated the underlying genomic properties and population dynamics of the V. vulnificus species from an evolutionary and ecological point of view. Genome comparisons and bioinformatic analyses of 113 V. vulnificus isolates indicate that the population of V. vulnificus is made up of four different clusters. We found evidence that recombination and gene flow between the two largest clusters (cluster 1 [C1] and C2) have drastically decreased to the point where they are diverging independently. Pangenome and phenotypic analyses showed two markedly different lifestyles for these two clusters, indicating commensal (C2) and bloomer (C1) ecotypes, with differences in carbohydrate utilization, defense systems, and chemotaxis, among other characteristics. Nonetheless, we identified frequent intra- and interspecies exchange of mobile genetic elements (e.g., antibiotic resistance plasmids, novel "chromids," or two different and concurrent type VI secretion systems) that provide high levels of genetic diversity in the population. Surprisingly, we identified strains from both clusters in the mucosa of aquaculture species, indicating that manmade niches are bringing strains from the two clusters together. We propose an evolutionary model of V. vulnificus that could be broadly applicable to other pathogenic vibrios and facultative bacterial pathogens to pursue strategies to prevent their infections and emergence.IMPORTANCEVibrio vulnificus is an emergent marine pathogen and is the cause of a deadly septicemia. However, the genetic factors that differentiate its clinical and environmental strains and its several biotypes remain mostly enigmatic. In this work, we investigated the underlying genomic properties and population dynamics of the V. vulnificus species to elucidate the traits that make these strains emerge as a human pathogen. The acquisition of different ecological determinants could have allowed the development of highly divergent clusters with different lifestyles within the same environment. However, we identified strains from both clusters in the mucosa of aquaculture species, indicating that manmade niches are bringing strains from the two clusters together, posing a potential risk of recombination and of emergence of novel variants. We propose a new evolutionary model that provides a perspective that could be broadly applicable to other pathogenic vibrios and facultative bacterial pathogens to pursue strategies to prevent their infections.

Coordination between chromosome replication and segregation is essential for equal partitioning of genetic material between daughter cells. In bacteria, this is achieved through the proximity of the origin of replication, oriC, and the chromosome partitioning site, parS We report here that in Pseudomonas aeruginosa, segregation but not replication is also controlled at the terminus region of the chromosome. Using the fluorescent repressor operator system (FROS), we investigated chromosome segregation in P. aeruginosa strain PAO1-UW, wherein the chromosome dimer resolution site, dif, is asymmetrically positioned relative to oriC In these cells, segregation proceeded sequentially along the two chromosomal arms and terminated at dif In contrast, chromosome replication terminated elsewhere, opposite from oriC We further found two large domains on the longer arm of the chromosome, wherein DNA segregated simultaneously. Notably, GC-skew, which reflects a bias in nucleotide usage between the leading and lagging strands of the chromosome, switches polarity at the dif locus but not necessarily at the terminus of replication. These data demonstrate that termination of chromosome replication and segregation can be physically separated without adverse effects on bacterial fitness. They also reveal the critical role of the dif region in defining the global layout of the chromosome and the progression of chromosome segregation and suggest that chromosome packing adapts to its subcellular layout.IMPORTANCE Segregation of genetic information is a central event in cellular life. In bacteria, chromosome segregation occurs concurrently with replication, sequentially along the two arms from oriC to dif How the two processes are coordinated is unknown. We explored here chromosome segregation in an opportunistic human pathogen, Pseudomonas aeruginosa, using its strain with markedly unequal chromosomal arms. We found that replication and segregation diverge in this strain and terminate at very different locations, whereas the longer chromosomal arm folds into large domains to align itself with the shorter arm. The significance of this research is in establishing that segregation and replication of bacterial chromosomes are largely uncoupled from each other and that the large-scale structure of the chromosome adapts to its subcellular layout.

In this work, by comparing genomes of closely related individuals of Streptomyces isolated at a spatial microscale (millimeters or centimeters), we investigated the extent and impact of horizontal gene transfer in the diversification of a natural Streptomyces population. We show that despite these conspecific strains sharing a recent common ancestor, all harbored significantly different gene contents, implying massive and rapid gene flux. The accessory genome of the strains was distributed across insertion/deletion events (indels) ranging from one to several hundreds of genes. Indels were preferentially located in the arms of the linear chromosomes (ca. 12 Mb) and appeared to form recombination hot spots. Some of them harbored biosynthetic gene clusters (BGCs) whose products confer an inhibitory capacity and may constitute public goods that can favor the cohesiveness of the bacterial population. Moreover, a significant proportion of these variable genes were either plasmid borne or harbored signatures of actinomycete integrative and conjugative elements (AICEs). We propose that conjugation is the main driver for the indel flux and diversity in Streptomyces populations.IMPORTANCE Horizontal gene transfer is a rapid and efficient way to diversify bacterial gene pools. Currently, little is known about this gene flux within natural soil populations. Using comparative genomics of Streptomyces strains belonging to the same species and isolated at microscale, we reveal frequent transfer of a significant fraction of the pangenome. We show that it occurs at a time scale enabling the population to diversify and to cope with its changing environment, notably, through the production of public goods.

mazEF is a toxin-antitoxin module located on chromosomes of most bacteria. MazF toxins are endoribonucleases antagonized by MazE antitoxins. Previously, we characterized several quorum sensing peptides called "extracellular death factors" (EDFs). When secreted from bacterial cultures, EDFs induce interspecies cell death. EDFs also enhance the endoribonucleolytic activity of Escherichia coli MazF. Mycobacterium tuberculosis carries several mazEF modules. Among them, the endoribonucleolytic activities of MazF proteins mt-1, mt-3, and mt-6 were identified. MazF-mt6 and MazF-mt-3 cleave M. tuberculosis rRNAs. Here we report the in vitro effects of EDFs on the endoribonucleolytic activities of M. tuberculosis MazFs. Escherichia coli EDF (EcEDF) and the three Pseudomonas aeruginosa EDFs (PaEDFs) individually enhance the endoribonucleolytic activities of MazF-mt6 and MazF-mt3 and overcome the inhibitory effect of MazE-mt3 or MazE-mt6 on the endoribonucleolytic activities of the respective toxins. We propose that these EDFs can serve as a basis for a novel class of antibiotics against M. tuberculosisIMPORTANCEMycobacterium tuberculosis is one of the leading causes of death from infectious disease. M. tuberculosis is highly drug resistant, and drug delivery to the infected site is very difficult. In previous studies, we showed that extracellular death factors (EDFs) can work as quorum sensing molecules which participate in interspecies bacterial cell death. In this study, we demonstrated the role of different EDFs in the endoribonucleolytic activities of M. tuberculosis MazFs. Escherichia coli EDF (EcEDF) and the three Pseudomonas aeruginosa EDFs (PaEDFs) individually enhance the endoribonucleolytic activities of MazF-mt6 and MazF-mt3. The current report provides a basis for the use of the EDF peptides EcEDF and PaEDF as novel antibiotics against M. tuberculosis.

Recent works suggest that bacterial gene order links chromosome structure to cell homeostasis. Comparative genomics showed that, in fast-growing bacteria, ribosomal protein genes (RP) locate near the replication origin (oriC). We recently showed that Vibrio cholerae employs this positional bias as a growth optimization strategy: under fast-growth conditions, multifork replication increases RP dosage and expression. However, RP location may provide advantages in a dosage-independent manner: for example, the physical proximity of the many ribosomal components, in the context of a crowded cytoplasm, may favor ribosome biogenesis. To uncover putative dosage-independent effects, we studied isogenic V. cholerae derivatives in which the major RP locus, S10-spc-α (S10), was relocated to alternative genomic positions. When bacteria grew fast, bacterial fitness was reduced according to the S10 relative distance to oriC The growth of wild-type V. cholerae could not be improved by additional copies of the locus, suggesting a physiologically optimized genomic location. Slow growth is expected to uncouple RP position from dosage, since multifork replication does not occur. Under these conditions, we detected a fitness impairment when S10 was far from oriC Deep sequencing followed by marker frequency analysis in the absence of multifork replication revealed an up to 30% S10 dosage reduction associated with its relocation that closely correlated with fitness alterations. Hence, the impact of S10 location goes beyond a growth optimization strategy during feast periods. RP location may be important during the whole life cycle of this pathogen.IMPORTANCE The role of gene order within the bacterial chromosome is poorly understood. In fast growers, the location of genes linked with the expression of genetic information (i.e., transcription and translation) is biased toward oriC It was proposed that the location of these genes helps to maximize their expression by recruiting multifork replication during fast growth. Our results show that such genomic positioning impacts cell fitness beyond fast-growth conditions, probably across the whole life cycle of fast growers. Thus, the genomic position of key highly expressed genes, such as RP, was finely tuned during the evolution of fast-growing bacteria and may also be important in slow growers. In the near future, many more genes whose genomic position impacts bacterial phenotype will be described. These studies will contribute to discovery the rules of genome organization and application of them for the design of synthetic chromosomes and the creation of artificial life forms.

Enterococcus faecium is a gut commensal of humans and animals but is also listed on the WHO global priority list of multidrug-resistant pathogens. Many of its antibiotic resistance traits reside on plasmids and have the potential to be disseminated by horizontal gene transfer. Here, we present the first comprehensive population-wide analysis of the pan-plasmidome of a clinically important bacterium, by whole-genome sequence analysis of 1,644 isolates from hospital, commensal, and animal sources of E. faecium Long-read sequencing on a selection of isolates resulted in the completion of 305 plasmids that exhibited high levels of sequence modularity. We further investigated the entirety of all plasmids of each isolate (plasmidome) using a combination of short-read sequencing and machine-learning classifiers. Clustering of the plasmid sequences unraveled different E. faecium populations with a clear association with hospitalized patient isolates, suggesting different optimal configurations of plasmids in the hospital environment. The characterization of these populations allowed us to identify common mechanisms of plasmid stabilization such as toxin-antitoxin systems and genes exclusively present in particular plasmidome populations exemplified by copper resistance, phosphotransferase systems, or bacteriocin genes potentially involved in niche adaptation. Based on the distribution of k-mer distances between isolates, we concluded that plasmidomes rather than chromosomes are most informative for source specificity of E. faeciumIMPORTANCEEnterococcus faecium is one of the most frequent nosocomial pathogens of hospital-acquired infections. E. faecium has gained resistance against most commonly available antibiotics, most notably, against ampicillin, gentamicin, and vancomycin, which renders infections difficult to treat. Many antibiotic resistance traits, in particular, vancomycin resistance, can be encoded in autonomous and extrachromosomal elements called plasmids. These sequences can be disseminated to other isolates by horizontal gene transfer and confer novel mechanisms to source specificity. In our study, we elucidated the total plasmid content, referred to as the plasmidome, of 1,644 E. faecium isolates by using short- and long-read whole-genome technologies with the combination of a machine-learning classifier. This was fundamental to investigate the full collection of plasmid sequences present in our collection (pan-plasmidome) and to observe the potential transfer of plasmid sequences between E. faecium hosts. We observed that E. faecium isolates from hospitalized patients carried a larger number of plasmid sequences compared to that from other sources, and they elucidated different configurations of plasmidome populations in the hospital environment. We assessed the contribution of different genomic components and observed that plasmid sequences have the highest contribution to source specificity. Our study suggests that E. faecium plasmids are regulated by complex ecological constraints rather than physical interaction between hosts.

Bacteria regulate chromosome replication and segregation tightly with cell division to ensure faithful segregation of DNA to daughter generations. The underlying mechanisms have been addressed in several model species. It became apparent that bacteria have evolved quite different strategies to regulate DNA segregation and chromosomal organization. We have investigated here how the actinobacterium Corynebacterium glutamicum organizes chromosome segregation and DNA replication. Unexpectedly, we found that C. glutamicum cells are at least diploid under all of the conditions tested and that these organisms have overlapping C periods during replication, with both origins initiating replication simultaneously. On the basis of experimental data, we propose growth rate-dependent cell cycle models for C. glutamicumIMPORTANCE Bacterial cell cycles are known for few model organisms and can vary significantly between species. Here, we studied the cell cycle of Corynebacterium glutamicum, an emerging cell biological model organism for mycolic acid-containing bacteria, including mycobacteria. Our data suggest that C. glutamicum carries two pole-attached chromosomes that replicate with overlapping C periods, thus initiating a new round of DNA replication before the previous one is terminated. The newly replicated origins segregate to midcell positions, where cell division occurs between the two new origins. Even after long starvation or under extremely slow-growth conditions, C. glutamicum cells are at least diploid, likely as an adaptation to environmental stress that may cause DNA damage. The cell cycle of C. glutamicum combines features of slow-growing organisms, such as polar origin localization, and fast-growing organisms, such as overlapping C periods.

The ability of Bradyrhizobium spp. to nodulate and fix atmospheric nitrogen in soybean root nodules is critical to meeting humanity's nutritional needs. The intricacies of soybean bradyrhizobia-plant interactions have been studied extensively; however, bradyrhizobial ecology as influenced by phages has received somewhat less attention, even though these interactions may significantly impact soybean yield. In batch culture, four soybean bradyrhizobia strains, Bradyrhizobium japonicum S06B (S06B-Bj), B. japonicum S10J (S10J-Bj), Bradyrhizobium diazoefficiens USDA 122 (USDA 122-Bd), and Bradyrhizobium elkanii USDA 76T (USDA 76-Be), spontaneously (without apparent exogenous chemical or physical induction) produced tailed phages throughout the growth cycle; for three strains, phage concentrations exceeded cell numbers by ~3-fold after 48 h of incubation. Phage terminase large-subunit protein phylogeny revealed possible differences in phage packaging and replication mechanisms. Bioinformatic analyses predicted multiple prophage regions within each soybean bradyrhizobia genome, preventing accurate identification of spontaneously produced prophage (SPP) genomes. A DNA sequencing and mapping approach accurately delineated the boundaries of four SPP genomes within three of the soybean bradyrhizobia chromosomes and suggested that the SPPs were capable of transduction. In addition to the phages, S06B-Bj and USDA 76-Be contained three to four times more insertion sequences (IS) and large, conjugable, broad host range plasmids, both of which are known drivers of horizontal gene transfer (HGT) in soybean bradyrhizobia. These factors indicate that SPP along with IS and plasmids participate in HGT, drive bradyrhizobia evolution, and play an outsized role in bradyrhizobia ecology. IMPORTANCE Previous studies have shown that IS and plasmids mediate HGT of symbiotic nodulation (nod) genes in soybean bradyrhizobia; however, these events require close cell-to-cell contact, which could be limited in soil environments. Bacteriophage-assisted gene transduction through spontaneously produced prophages provides a stable means of HGT not limited by the constraints of proximal cell-to-cell contact. These phage-mediated HGT events may shape soybean bradyrhizobia population ecology, with concomitant impacts on soybean agriculture.