Hypermutable simple sequence repeats (SSRs) are major drivers of phase variation in Campylobacter jejuni. The presence of multiple SSR-mediated phase-variable genes encoding enzymes that modify surface structures, including capsular polysaccharide (CPS) and lipooligosaccharide (LOS), generates extreme cell surface diversity within bacterial populations, thereby promoting adaptation to selective pressures in host environments. Therefore, genetically controlling SSR-mediated phase variation can be important for achieving stable and reproducible research on C. jejuni. Here, we show that natural "cotransformation" is an effective method for C. jejuni genome editing. Cotransformation is a trait of naturally competent bacteria that causes uptake/integration of multiple different DNA molecules, which has been recently adapted to multiplex genome editing by natural transformation (MuGENT), a method for introducing multiple mutations into the genomes of these bacteria. We found that cotransformation efficiently occurred in C. jejuni. To examine the feasibility of MuGENT in C. jejuni, we "locked" different polyG SSR tracts in strain NCTC11168 (which are located in the biosynthetic CPS/LOS gene clusters) into either the ON or OFF configurations. This approach, termed "MuGENT-SSR," enabled the generation of all eight edits within 2 weeks and the identification of a phase-locked strain with a highly stable type of Penner serotyping, a CPS-based serotyping scheme. Furthermore, extensive genome editing of this strain by MuGENT-SSR identified a phase-variable gene that determines the Penner serotype of NCTC11168. Thus, MuGENT-SSR provides a platform for genetic and phenotypic engineering of genetically unstable C. jejuni, making it a reliable approach for elucidating the mechanisms underlying phase-variable expression of specific phenotypes. IMPORTANCE Campylobacter jejuni is the leading bacterial cause of foodborne gastroenteritis in developed countries and occasionally progresses to the autoimmune disease Guillain-Barré syndrome. A relatively large number of hypermutable simple sequence repeat (SSR) tracts in the C. jejuni genome markedly decreases its phenotypic stability through reversible changes in the ON or OFF expression states of the genes in which they reside, a phenomenon called phase variation. Thus, controlling SSR-mediated phase variation can be important for achieving stable and reproducible research on C. jejuni. In this study, we developed a feasible and effective approach for genetically manipulate multiple SSR tracts in the C. jejuni genome using natural cotransformation, a trait of naturally transformable bacterial species that causes the uptake and integration of multiple different DNA molecules. This approach will greatly help to improve the genetic and phenotypic stability of C. jejuni to enable diverse applications in research and development.

Protein filaments play important roles in many biological processes. We discovered an actin homolog in halophilic archaea, which we call Salactin. Just like the filaments that segregate DNA in eukaryotes, Salactin grows out of the cell poles towards the middle, and then quickly depolymerizes, a behavior known as dynamic instability. Furthermore, we see that Salactin affects the distribution of DNA in daughter cells when cells are grown in low-phosphate media, suggesting Salactin filaments might be involved in segregating DNA when the cell has only a few copies of the chromosome.

Clinical development of antibiotics with novel mechanisms of action to kill pathogenic bacteria is challenging, in part, due to the inevitable emergence of resistance. A phenomenon of potential clinical importance that is broadly overlooked in preclinical development is heteroresistance, an often-unstable phenotype in which subpopulations of bacterial cells show decreased antibiotic susceptibility relative to the dominant population. Here, we describe a new globomycin analog, G0790, with potent activity against the Escherichia coli type II signal peptidase LspA and uncover two novel resistance mechanisms to G0790 in the clinical uropathogenic E. coli strain CFT073. Building on the previous finding that complete deletion of Lpp, the major Gram-negative outer membrane lipoprotein, leads to globomycin resistance, we also find that an unexpectedly modest decrease in Lpp levels mediated by insertion-based disruption of regulatory elements is sufficient to confer G0790 resistance and increase sensitivity to serum killing. In addition, we describe a heteroresistance phenotype mediated by genomic amplifications of lspA that result in increased LspA levels sufficient to overcome inhibition by G0790 in culture. These genomic amplifications are highly unstable and are lost after as few as two subcultures in the absence of G0790, which places amplification-containing resistant strains at high risk of being misclassified as susceptible by routine antimicrobial susceptibility testing. In summary, our study uncovers two vastly different mechanisms of resistance to LspA inhibitors in E. coli and emphasizes the importance of considering the potential impact of unstable and heterogenous phenotypes when developing antibiotics for clinical use.IMPORTANCE Despite increasing evidence suggesting that antibiotic heteroresistance can lead to treatment failure, the significance of this phenomena in the clinic is not well understood, because many clinical antibiotic susceptibility testing approaches lack the resolution needed to reliably classify heteroresistant strains. Here we present G0790, a new globomycin analog and potent inhibitor of the Escherichia coli type II signal peptidase LspA. We demonstrate that in addition to previously known mechanisms of resistance to LspA inhibitors, unstable genomic amplifications containing lspA can lead to modest yet biologically significant increases in LspA protein levels that confer a heteroresistance phenotype.

Amikacin and colistin are effective against carbapenem-resistant Klebsiella pneumoniae In 2017, we successively isolated three carbapenem-resistant K. pneumoniae isolates (ST967) from a patient with chronic renal failure in Japan. The first (SMKP01, sputum, day 0) and second (SMKP02, blood, day 14) strains were resistant to most antimicrobials tested but still susceptible to amikacin (MICs of 4 and 0.5 mg/liter, respectively) and colistin (MIC of 0.5 mg/liter for both). The third strain (SMKP03, blood, day 51) was not susceptible to amikacin (MIC, 32 mg/liter), and its MIC for colistin varied (0.5 to 8 mg/liter). Whole-genome sequencing of SMKP01 revealed that 17 of 20 antimicrobial resistance genes, including qnrB91 (a novel qnrB2 variant) and aac(6')-Ib-cr, were located on an 86.9-kb IncFII-IncQ plasmid. The qnrB91 conferred greater fluoroquinolone resistance than qnrB2 SMKP03 aac(6')-Ib-cr that possessed a gene mutation that resulted in an R102W substitution, namely, aac(6')-Ib-D179Y, made a greater contribution to amikacin resistance than did aac(6')-Ib-cr SMKP03 harbored a nonsense mutation in mutS, which encodes a DNA repair enzyme. Introduction of this mutation into SMKP01 (SMKP01mutSA307T) resulted in a dramatic increase (>58-fold) in the frequency of spontaneous amikacin-resistant mutants relative to SMKP01, and the substantial mutants possessed aac(6')-Ib-D179Y SMKP01mutSA307T exhibited an unstable MIC for colistin (0.5 to 8 mg/liter). The results demonstrate that a disruptive mutation in MutS, arising during the clinical course of an infection, created a platform for the acquisition of amikacin nonsusceptibility and colistin heteroresistance in multidrug-resistant K. pneumoniae, mediated by the elevated frequency of spontaneous mutations.IMPORTANCE The emergence of multidrug resistance in pathogens such as Klebsiella pneumoniae is of great clinical concern. Antimicrobial resistance sometimes arises during the course of an infection. Although many studies have reported the emergence of antimicrobial resistance and novel antimicrobial resistance genes in the clinical isolates, the identity of the bacterial factor(s) that generate this emergence is still unclear. We report that a disruptive mutation in MutS, arising during the clinical course of an infection, created a context for the acquisition of colistin resistance and the emergence of a novel variant of the amikacin resistance gene in multidrug-resistant K. pneumoniae via an increase in the frequency of spontaneous mutation. This observation is important for understanding how K. pneumoniae develops multidrug resistance during infection and could potentially lead to new antimicrobial treatments for high-risk pathological microbes.

Gene amplification is believed to play an important role in antibiotic resistance but has been rarely documented in clinical settings because of its unstable nature. We report a rise in MICs from 0.5 to 16 μg/ml in successive Acinetobacter baumannii isolated over 4 days from a patient being treated with tobramycin for an infection by multidrug-resistant A. baumannii, resulting in therapeutic failure. Isolates were characterized by whole-genome sequencing, real-time and reverse transcriptase PCR, and growth assays to determine the mechanism of tobramycin resistance and its fitness cost. Tobramycin resistance was associated with two amplification events of different chromosomal fragments containing the aphA1 aminoglycoside resistance gene part of transposon Tn6020. The first amplification event involved low amplification (6 to 10 copies) of a large DNA fragment that was unstable and conferred tobramycin MICs of ≤ 8 μg/ml. The second event involved moderate (10 to 30 copies) or high (40 to 110 copies) amplification of Tn6020. High copy numbers were associated with tobramycin MICs of 16 μg/ml, impaired fitness, and genetic instability, whereas lower copy numbers resulted in tobramycin MICs of ≤8 μg/ml and no fitness cost and were stably maintained in vitro. Exposure in vitro to tobramycin of the initial susceptible isolate and of the A. baumannii AB0057 reference strain led to similar aphA1 amplifications and elevated tobramycin MICs. To the best of our knowledge, this is the first report of in vivo development of antibiotic resistance secondary to gene amplifications resulting in therapy failure. IMPORTANCE A combination of whole-genome sequencing and mapping were used to detect an antibiotic resistance mechanism, gene amplification, which has been presumed for a long time to be of major importance but has rarely been reported in clinical settings because of its unstable nature. Two gene amplification events in a patient with an Acinetobacter baumannii infection treated with tobramycin were identified. One gene amplification event led to high levels of resistance and was rapidly reversible, while the second event led to low and more stable resistance since it incurred low fitness cost on the host. Gene amplification, with an associated rise in tobramycin MICs, could be readily reproduced in vitro from initially susceptible strains exposed to increasing concentrations of tobramycin, suggesting that gene amplification in A. baumannii may be a more common mechanism than currently believed. This report underscores the importance of rapid molecular techniques for surveillance of drug resistance.

Antisense transcription is widespread in bacteria. By base pairing with overlapping sense RNAs, antisense RNAs (asRNA) can form double-stranded RNAs (dsRNA), which are cleaved by RNase III, a dsRNA endoribonuclease. The ectopic expression of plant Tombusvirus p19 in Escherichia coli stabilizes ∼21-nucleotide (nt) dsRNA RNase III decay intermediates, which enabled us to characterize otherwise highly unstable asRNA by deep sequencing of p19-captured dsRNA. RNase III-produced small dsRNA were formed at most bacterial genes in the bacterial genome and in a plasmid. We classified the types of asRNA in genomic clusters producing the most abundant p19-captured dsRNA and confirmed RNase III regulation of asRNA and sense RNA decay at three type I toxin-antitoxin loci and at a coding gene, rsd Furthermore, we provide potential evidence for the RNase III-dependent regulation of CspD protein by asRNA. The analysis of p19-captured dsRNA revealed an RNase III sequence preference for AU-rich sequences 3 nucleotides on either side of the cleavage sites and for GC-rich sequences in the 2-nt overhangs. Unexpectedly, GC-rich sequences were enriched in the middle section of p19-captured dsRNA, suggesting some unexpected sequence bias in p19 protein binding. Nonetheless, the ectopic expression of p19 is a sensitive method for identifying antisense transcripts and RNase III cleavage sites in dsRNA formed by overlapping sense and antisense transcripts in bacteria.

Prolonged virologic failure on 2nd-line protease inhibitor (PI)-based antiretroviral therapy (ART) without emergence of major protease mutations is well recognized and provides an opportunity to study within-host evolution in long-term viremic individuals. Using next-generation sequencing and in silico haplotype reconstruction, we analyzed whole-genome sequences from longitudinal plasma samples of eight chronically infected HIV-1-positive individuals failing 2nd-line regimens from the French National Agency for AIDS and Viral Hepatitis Research (ANRS) 12249 Treatment as Prevention (TasP) trial. On nonsuppressive ART, there were large fluctuations in synonymous and nonsynonymous variant frequencies despite stable viremia. Reconstructed haplotypes provided evidence for selective sweeps during periods of partial adherence, and viral haplotype competition, during periods of low drug exposure. Drug resistance mutations in reverse transcriptase (RT) were used as markers of viral haplotypes in the reservoir, and their distribution over time indicated recombination. We independently observed linkage disequilibrium decay, indicative of recombination. These data highlight dramatic changes in virus population structure that occur during stable viremia under nonsuppressive ART. IMPORTANCE HIV-1 infections are most commonly initiated with a single founder virus and are characterized by extensive inter- and intraparticipant genetic diversity. However, existing literature on HIV-1 intrahost population dynamics is largely limited to untreated infections, predominantly in subtype B-infected individuals. The manuscript characterizes viral population dynamics in long-term viremic treatment-experienced individuals, which has not been previously characterized. These data are particularly relevant for understanding HIV dynamics but can also be applied to other RNA viruses. With this unique data set we propose that the virus is highly unstable, and we have found compelling evidence of HIV-1 within-host viral diversification, recombination, and haplotype competition during nonsuppressive ART.

Marine phytoplankton and heterotrophic bacteria share a very close but usually changeable relationship. However, the ultimate fate of their unstable relationship on a long-term scale is unclear. Here, the relationship between Synechococcus and heterotrophic bacterial communities underwent a dramatic shift from antagonism to commensalism and eventually to mutualism during long-term cocultivation. The relationship change is attributed to the different (even opposite) effects of diverse bacterial members on Synechococcus and the ratio of beneficial to harmful bacteria. Different bacterial members also interact with each other (e.g., quorum-sensing communication, hostility, or mutual promotion) and drive a dynamic succession in the entire community structure that corresponds exactly to the shift in its relationship with Synechococcus. In the final mutualism stage, a self-sufficient nitrogen cycle, including nitrogen fixation, denitrification, and organic nitrogen degradation, contributed to the healthy survival of Synechococcus for 2 years without an exogenous nutrient supply. This natural selective trait of Synechococcus and heterotrophic bacteria toward mutualism under long-term coexistence provides a novel clue for understanding the ubiquity and competitive advantage of Synechococcus in global oceans. IMPORTANCE Phytoplankton and heterotrophic bacteria have a close but usually changeable relationship. Uncovering the dynamic changes and driving factors of their interrelationships is of great significance for an in-depth understanding of the ecological processes and functions of marine microorganisms. Here, we observed that Synechococcus and heterotrophic bacterial communities underwent a dramatic change in their relationship from antagonism to mutualism during a long-term cocultivation process. We revealed that the interactions between different members of the bacterial community and the combined effects of different bacterial individuals on Synechococcus promoted the dynamic changes of the Synechococcus-bacterium relationship. In the end, a self-sufficient nutrient cycle (especially nitrogen) established by Synechococcus and bacterial communities supported their long-term survival without any external nutrition supply. This study provides novel insight into the interaction between Synechococcus and heterotrophic bacteria in the ocean and provides a novel clue for understanding the ubiquity and competitive advantage of Synechococcus in global oceans.

Using whole-genome sequence (WGS) data from the GenomeTrakr network, a globally distributed network of laboratories sequencing foodborne pathogens, we present a new phylogeny of Salmonella enterica comprising 445 isolates from 266 distinct serovars and originating from 52 countries. This phylogeny includes two previously unidentified S. enterica subsp. enterica clades. Serovar Typhi is shown to be nested within clade A. Our findings are supported by both phylogenetic support, based on a core genome alignment, and Bayesian approaches, based on single-nucleotide polymorphisms. Serovar assignments were refined by in silico analysis using SeqSero. More than 10% of serovars were either polyphyletic or paraphyletic. We found variable genetic content in these isolates relating to gene mobilization and virulence factors which have different distributions within clades. Gifsy-1- and Gifsy-2-like phages appear more prevalent in clade A; other viruses are more evenly distributed. Our analyses reveal IncFII is the predominant plasmid replicon in S. enterica Few core or clade-defining virulence genes are observed, and their distributions appear probabilistic in nature. Together, these patterns demonstrate that genetic exchange within S. enterica is more extensive and frequent than previously realized, which significantly alters how we view the genetic structure of the bacterial species.IMPORTANCE Rapid improvements in nucleotide sequencing access and affordability have led to a drastic increase in availability of genetic information. This information will improve the accuracy of molecular descriptions, including serovars, within S. enterica Although the concept of serovars continues to be useful, it may have more significant limitations than previously understood. Furthermore, the discrete absence or presence of specific genes can be an unstable indicator of phylogenetic identity. Whole-genome sequencing provides more rigorous tools for assessing the distributions of these genes. Our phylogenetic and genetic content analyses reveal how active genetic elements are dynamically distributed within a species, allowing us to better understand genetic reservoirs and underlying bacterial evolution.

Plasmids play an important role in bacterial evolution by transferring niche-adaptive functional genes between lineages, thus driving genomic diversification. Bacterial genomes commonly contain multiple, coexisting plasmid replicons, which could fuel adaptation by increasing the range of gene functions available to selection and allowing their recombination. However, plasmid coexistence is difficult to explain because the acquisition of plasmids typically incurs high fitness costs for the host cell. Here, we show that plasmid coexistence was stably maintained without positive selection for plasmid-borne gene functions and was associated with compensatory evolution to reduce fitness costs. In contrast, with positive selection, plasmid coexistence was unstable despite compensatory evolution. Positive selection discriminated between differential fitness benefits of functionally redundant plasmid replicons, retaining only the more beneficial plasmid. These data suggest that while the efficiency of negative selection against plasmid fitness costs declines over time due to compensatory evolution, positive selection to maximize plasmid-derived fitness benefits remains efficient. Our findings help to explain the forces structuring bacterial genomes: coexistence of multiple plasmids in a genome is likely to require either rare positive selection in nature or nonredundancy of accessory gene functions among the coexisting plasmids.IMPORTANCE Bacterial genomes often contain multiple coexisting plasmids that provide important functions like antibiotic resistance. Using lab experiments, we show that such plasmid coexistence within a genome is stable only in environments where the function they encode is useless but is unstable if the function is useful and beneficial for bacterial fitness. Where competing plasmids perform the same useful function, only the most beneficial plasmid is kept by the cell, a process that is similar to competitive exclusion in ecological communities. This process helps explain how bacterial genomes are structured: bacterial genomes expand in size by acquiring multiple plasmids when selection is relaxed but subsequently contract during periods of strong selection for the useful plasmid-encoded function.

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.

Coenzyme F420 is a microbial redox cofactor that mediates diverse physiological functions and is increasingly used for biocatalytic applications. Recently, diversified biosynthetic routes to F420 and the discovery of a derivative, 3PG-F420, were reported. 3PG-F420 is formed via activation of 3-phospho-d-glycerate (3-PG) by CofC, but the structural basis of substrate binding, its evolution, as well as the role of CofD in substrate selection remained elusive. Here, we present a crystal structure of the 3-PG-activating CofC from Mycetohabitans sp. B3 and define amino acids governing substrate specificity. Site-directed mutagenesis enabled bidirectional switching of specificity and thereby revealed the short evolutionary trajectory to 3PG-F420 formation. Furthermore, CofC stabilized its product, thus confirming the structure of the unstable molecule and revealing its binding mode. The CofD enzyme was shown to significantly contribute to the selection of related intermediates to control the specificity of the combined biosynthetic CofC/D step. These results imply the need to change the design of combined CofC/D activity assays. Taken together, this work presents novel mechanistic and structural insights into 3PG-F420 biosynthesis and evolution and opens perspectives for the discovery and enhanced biotechnological production of coenzyme F420 derivatives in the future. IMPORTANCE The microbial cofactor F420 is crucial for processes like methanogenesis, antibiotics biosynthesis, drug resistance, and biocatalysis. Recently, a novel derivative of F420 (3PG-F420) was discovered, enabling the production and use of F420 in heterologous hosts. By analyzing the crystal structure of a CofC homolog whose substrate choice leads to formation of 3PG-F420, we defined amino acid residues governing the special substrate selectivity. A diagnostic residue enabled reprogramming of the substrate specificity, thus mimicking the evolution of the novel cofactor derivative. Furthermore, a labile reaction product of CofC was revealed that has not been directly detected so far. CofD was shown to provide another layer of specificity of the combined CofC/D reaction, thus controlling the initial substrate choice of CofC. The latter finding resolves a current debate in the literature about the starting point of F420 biosynthesis in various organisms.

We have discovered a new cluster of genes that is found exclusively in the Actinobacteria phylum. This locus includes genes for the 2-aminophenol meta-cleavage pathway and the shell proteins of a bacterial microcompartment (BMC) and has been named aromatics (ARO) for its putative role in the breakdown of aromatic compounds. In this study, we provide details about the distribution and composition of the ARO BMC locus and conduct phylogenetic, structural, and functional analyses of the first two enzymes in the catabolic pathway: a unique 2-aminophenol dioxygenase, which is exclusively found alongside BMC shell genes in Actinobacteria, and a semialdehyde dehydrogenase, which works downstream of the dioxygenase. Genomic analysis reveals variations in the complexity of the ARO loci across different orders. Some loci are simple, containing shell proteins and enzymes for the initial steps of the catabolic pathway, while others are extensive, encompassing all the necessary genes for the complete breakdown of 2-aminophenol into pyruvate and acetyl-CoA. Furthermore, our analysis uncovers two subtypes of ARO BMC that likely degrade either 2-aminophenol or catechol, depending on the presence of a pathway-specific gene within the ARO locus. The precise precursor of 2-aminophenol, which serves as the initial substrate and/or inducer for the ARO pathway, remains unknown, as our model organism Micromonospora rosaria cannot utilize 2-aminophenol as its sole energy source. However, using enzymatic assays, we demonstrate the dioxygenase's ability to cleave both 2-aminophenol and catechol in vitro, in collaboration with the aldehyde dehydrogenase, to facilitate the rapid conversion of these unstable and toxic intermediates. IMPORTANCE Bacterial microcompartments (BMCs) are proteinaceous organelles that are widespread among bacteria and provide a competitive advantage in specific environmental niches. Studies have shown that the genetic information necessary to form functional BMCs is encoded in loci that contain genes encoding shell proteins and the enzymatic core. This allows the bioinformatic discovery of BMCs with novel functions and expands our understanding of the metabolic diversity of BMCs. ARO loci, found only in Actinobacteria, contain genes encoding for phylogenetically remote shell proteins and homologs of the meta-cleavage degradation pathway enzymes that were shown to convert central aromatic intermediates into pyruvate and acetyl-CoA in gamma Proteobacteria. By analyzing the gene composition of ARO BMC loci and characterizing two core enzymes phylogenetically, structurally, and functionally, we provide an initial functional characterization of the ARO BMC, the most unusual BMC identified to date, distinctive among the repertoire of studied BMCs.