Dual proteomics of infected macrophages reveal bacterial and host players involved in the Francisella intracellular life cycle and cell to cell dissemination by merocytophagy.

Bacterial pathogens adapt and replicate within host cells, while host cells develop mechanisms to eliminate them. Using a dual proteomic approach, we characterized the intra-macrophage proteome of the facultative intracellular pathogen, Francisella novicida. More than 900 Francisella proteins were identified in infected macrophages after a 10-h infection. Biotin biosynthesis-related proteins were upregulated, emphasizing the role of biotin-associated genes in Francisella replication. Conversely, proteins encoded by the Francisella pathogenicity island (FPI) were downregulated, supporting the importance of the F. tularensis Type VI Secretion System for vacuole escape, not cytosolic replication. In the host cell, over 300 proteins showed differential expression among the 6200 identified during infection. The most upregulated host protein was cis-aconitate decarboxylase IRG1, known for itaconate production with antimicrobial properties in Francisella. Surprisingly, disrupting IRG1 expression did not impact Francisella's intracellular life cycle, suggesting redundancy with other immune proteins or inclusion in larger complexes. Over-representation analysis highlighted cell-cell contact and actin polymerization in macrophage deregulated proteins. Using flow cytometry and live cell imaging, we demonstrated that merocytophagy involves diverse cell-to-cell contacts and actin polymerization-dependent processes. These findings lay the groundwork for further exploration of merocytophagy and its molecular mechanisms in future research.Data are available via ProteomeXchange with identifier PXD035145.

Hypervirulent Klebsiella pneumoniae employs genomic island encoded toxins against bacterial competitors in the gut.

The hypervirulent lineages of Klebsiella pneumoniae (HvKp) cause invasive infections such as Klebsiella-liver abscess. Invasive infection often occurs after initial colonization of the host gastrointestinal tract by HvKp. Over 80% of HvKp isolates belong to the clonal group 23 sublineage I that has acquired genomic islands (GIs) GIE492 and ICEKp10. Our analysis of 12 361 K. pneumoniae genomes revealed that GIs GIE492 and ICEKp10 are co-associated with the CG23-I and CG10118 HvKp lineages. GIE492 and ICEKp10 enable HvKp to make a functional bacteriocin microcin E492 (mccE492) and the genotoxin colibactin, respectively. We discovered that GIE492 and ICEKp10 play cooperative roles and enhance gastrointestinal colonization by HvKp. Colibactin is the primary driver of this effect, modifying gut microbiome diversity. Our in vitro assays demonstrate that colibactin and mccE492 kill or inhibit a range of Gram-negative Klebsiella species and Escherichia coli strains, including Gram-positive bacteria, sometimes cooperatively. Moreover, mccE492 and colibactin kill human anaerobic gut commensals that are similar to the taxa found altered by colibactin in the mouse intestines. Our findings suggest that GIs GIE492 and ICEKp10 enable HvKp to kill several commensal bacterial taxa during interspecies interactions in the gut. Thus, acquisition of GIE492 and ICEKp10 could enable better carriage in host populations and explain the dominance of the CG23-I HvKp lineage.

Speeding genomic island discovery through systematic design of reference database composition.

BACKGROUND: Genomic islands (GIs) are mobile genetic elements that integrate site-specifically into bacterial chromosomes, bearing genes that affect phenotypes such as pathogenicity and metabolism. GIs typically occur sporadically among related bacterial strains, enabling comparative genomic approaches to GI identification. For a candidate GI in a query genome, the number of reference genomes with a precise deletion of the GI serves as a support value for the GI. Our comparative software for GI identification was slowed by our original use of large reference genome databases (DBs). Here we explore smaller species-focused DBs. RESULTS: With increasing DB size, recovery of our reliable prophage GI calls reached a plateau, while recovery of less reliable GI calls (FPs) increased rapidly as DB sizes exceeded ~500 genomes; i.e., overlarge DBs can increase FP rates. Paradoxically, relative to prophages, FPs were both more frequently supported only by genomes outside the species and more frequently supported only by genomes inside the species; this may be due to their generally lower support values. Setting a DB size limit for our SMAll Ranked Tailored (SMART) DB design speeded runtime ~65-fold. Strictly intra-species DBs would tend to lower yields of prophages for small species (with few genomes available); simulations with large species showed that this could be partially overcome by reaching outside the species to closely related taxa, without an FP burden. Employing such taxonomic outreach in DB design generated redundancy in the DB set; as few as 2984 DBs were needed to cover all 47894 prokaryotic species. CONCLUSIONS: Runtime decreased dramatically with SMART DB design, with only minor losses of prophages. We also describe potential utility in other comparative genomics projects.

Combining CRISPR/Cas9 and natural excision for the precise and complete removal of mobile genetic elements in bacteria.

Horizontal gene transfer, facilitated by mobile genetic elements (MGEs), is an adaptive evolutionary process that contributes to the evolution of bacterial populations and infectious diseases. A variety of MGEs not only can integrate into the bacterial genome but also can survive or even replicate like plasmids in the cytoplasm, thus requiring precise and complete removal for studying their strategies in benefiting host cells. Existing methods for MGE removal, such as homologous recombination-based deletion and excisionase-based methods, have limitations in effectively eliminating certain MGEs. To overcome these limitations, we developed the Cas9-NE method, which combines the CRISPR/Cas9 system with the natural excision of MGEs. In this approach, a specialized single guide RNA (sgRNA) element is designed with a 20-nucleotide region that pairs with the MGE sequence. This sgRNA is expressed from a plasmid that also carries the Cas9 gene. By utilizing the Cas9-NE method, both the integrative and circular forms of MGEs can be precisely and completely eliminated through Cas9 cleavage, generating MGE-removed cells. We have successfully applied the Cas9-NE method to remove four representative MGEs, including plasmids, prophages, and genomic islands, from Vibrio strains. This new approach not only enables various investigations on MGEs but also has significant implications for the rapid generation of strains for commercial purposes.IMPORTANCEMobile genetic elements (MGEs) are of utmost importance for bacterial adaptation and pathogenicity, existing in various forms and multiple copies within bacterial cells. Integrated MGEs play dual roles in bacterial hosts, enhancing the fitness of the host by delivering cargo genes and potentially modifying the bacterial genome through the integration/excision process. This process can lead to alterations in promoters or coding sequences or even gene disruptions at integration sites, influencing the physiological functions of host bacteria. Here, we developed a new approach called Cas9-NE, allowing them to maintain the natural sequence changes associated with MGE excision. Cas9-NE allows the one-step removal of integrated and circular MGEs, addressing the challenge of eliminating various MGE forms efficiently. This approach simplifies MGE elimination in bacteria, expediting research on MGEs.

The intricate relationship of G-Quadruplexes and bacterial pathogenicity islands.

The dynamic interplay between guanine-quadruplex (G4) structures and pathogenicity islands (PAIs) represents a captivating area of research with implications for understanding the molecular mechanisms underlying pathogenicity. This study conducted a comprehensive analysis of a large-scale dataset from reported 89 pathogenic strains of bacteria to investigate the potential interactions between G4 structures and PAIs. G4 structures exhibited an uneven and non-random distribution within the PAIs and were consistently conserved within the same pathogenic strains. Additionally, this investigation identified positive correlations between the number and frequency of G4 structures and the GC content across different genomic features, including the genome, promoters, genes, tRNA, and rRNA regions, indicating a potential relationship between G4 structures and the GC-associated regions of the genome. The observed differences in GC content between PAIs and the core genome further highlight the unique nature of PAIs and underlying factors, such as DNA topology. High-confidence G4 structures within regulatory regions of Escherichia coli were identified, modulating the efficiency or specificity of DNA integration events within PAIs. Collectively, these findings pave the way for future research to unravel the intricate molecular mechanisms and functional implications of G4-PAI interactions, thereby advancing our understanding of bacterial pathogenicity and the role of G4 structures in pathogenic diseases.

Novel multidrug resistance genomic islands and transposon carrying blaVEB-1 identified in mcr-positive Aeromonas strains from raw meat in China.

OBJECTIVES: To characterize the genetic environments of ESBL gene blaVEB-1 in mcr-positive Aeromonas strains from raw meat in China. METHODS: Whole genomes of Aeromonas strains were sequenced using the Illumina or Nanopore platforms. Genetic environments of blaVEB-1 were analysed using the BLAST program. RESULTS: The blaVEB-1 gene was detected in five Aeromonas strains carrying the mcr-7-like gene. WGS revealed that all blaVEB-1 genes were located on Aeromonas chromosome, and were carried by two novel different genomic islands named Aeromonas veronii genomic islands AveGI1 and AveGI2, as well as one transposon named Tn7690. AveGI1 is a new member of the Salmonella genomic island 1 family, incorporated into the 3'-end of mnmE (trmE). AveGI2 is a novel genomic island that has a size of 23 180 bp and is incorporated into the 3'-end of syd. The MDR regions of AveGI1 and AveGI2 are two different class 1 integrons containing 10 and five resistance genes, respectively. Tn7690 is a Tn1722 derivative containing In4-type integron and Tn5393, which harbours 10 resistance genes and integrates into different positions on the chromosomes of three strains with the capacity for mobility. CONCLUSIONS: We report chromosomally located novel MDR genomic islands and transposon that carry blaVEB-1 in mcr-positive Aeromonas strains. These genetic elements may mediate the spread of blaVEB-1 in Aeromonas, and may also evolve by capturing new antimicrobial resistance genes or other mobile genetic elements.

The Helicobacter pylori cag pathogenicity island as a determinant of gastric cancer risk.

Helicobacter pylori strains can be broadly classified into two groups based on whether they contain or lack a chromosomal region known as the cag pathogenicity island (cag PAI). Colonization of the human stomach with cag PAI-positive strains is associated with an increased risk of gastric cancer and peptic ulcer disease, compared to colonization with cag PAI-negative strains. The cag PAI encodes a secreted effector protein (CagA) and components of a type IV secretion system (Cag T4SS) that delivers CagA and non-protein substrates into host cells. Animal model experiments indicate that CagA and the Cag T4SS stimulate a gastric mucosal inflammatory response and contribute to the development of gastric cancer. In this review, we discuss recent studies defining structural and functional features of CagA and the Cag T4SS and mechanisms by which H. pylori strains containing the cag PAI promote the development of gastric cancer and peptic ulcer disease.

Genomic island-encoded regulatory proteins in enterohemorrhagic Escherichia coli O157:H7.

Enterohemorrhagic Escherichia coli (EHEC) is an important zoonotic pathogen that is a major cause of foodborne diseases in most developed and developing countries and can cause uncomplicated diarrhoea, haemorrhagic colitis, and haemolytic uraemic syndrome. O islands (OIs), which are unique genomic islands in EHEC O157:H7, are composed of 177 isolated genomic features and harbour 26% of the total genes that are absent in the non-pathogenic E. coli K-12 genome. In the last twenty years, many OI-encoded proteins have been characterized, including proteins regulating virulence, motility, and acid resistance. Given the critical role of regulatory proteins in the systematic and hierarchical regulation of bacterial biological processes, this review summarizes the OI-encoded regulatory proteins in EHEC O157:H7 characterized to date, emphasizing OI-encoded regulatory proteins for bacterial virulence, motility, and acid resistance. This summary will be significant for further exploration and understanding of the virulence and pathogenesis of EHEC O157:H7.

Flexible genomic island conservation across freshwater and marine Methylophilaceae.

The evolutionary trajectory of Methylophilaceae includes habitat transitions from freshwater sediments to freshwater and marine pelagial that resulted in genome reduction (genome-streamlining) of the pelagic taxa. However, the extent of genetic similarities in the genomic structure and microdiversity of the two genome-streamlined pelagic lineages (freshwater "Ca. Methylopumilus" and the marine OM43 lineage) has so far never been compared. Here, we analyzed complete genomes of 91 "Ca. Methylopumilus" strains isolated from 14 lakes in Central Europe and 12 coastal marine OM43 strains. The two lineages showed a remarkable niche differentiation with clear species-specific differences in habitat preference and seasonal distribution. On the other hand, we observed a synteny preservation in their genomes by having similar locations and types of flexible genomic islands (fGIs). Three main fGIs were identified: a replacement fGI acting as phage defense, an additive fGI harboring metabolic and resistance-related functions, and a tycheposon containing nitrogen-, thiamine-, and heme-related functions. The fGIs differed in relative abundances in metagenomic datasets suggesting different levels of variability ranging from strain-specific to population-level adaptations. Moreover, variations in one gene seemed to be responsible for different growth at low substrate concentrations and a potential biogeographic separation within one species. Our study provides a first insight into genomic microdiversity of closely related taxa within the family Methylophilaceae and revealed remarkably similar dynamics involving mobile genetic elements and recombination between freshwater and marine family members.

Novel mechanisms of diversity generation in Acinetobacter baumannii resistance islands driven by Tn7-like elements.

Mobile genetic elements play an important role in the acquisition of antibiotic and biocide resistance, especially through the formation of resistance islands in bacterial chromosomes. We analyzed the contribution of Tn7-like transposons to island formation and diversification in the nosocomial pathogen Acinetobacter baumannii and identified four separate families that recognize different integration sites. One integration site is within the comM gene and coincides with the previously described Tn6022 elements suggested to account for the AbaR resistance island. We established Tn6022 in a heterologous E. coli host and confirmed basic features of transposition into the comM attachment site and the use of a novel transposition protein. By analyzing population features within Tn6022 elements we identified two potential novel transposon-encoded diversification mechanisms with this dynamic genetic island. The activities of these diversification features were confirmed in E. coli. One was a novel natural gain-of-activity allele that could function to broaden transposition targeting. The second was a transposon-encoded hybrid dif-like site that parasitizes the host dimer chromosome resolution system to function with its own tyrosine recombinase. This work establishes a highly active Tn7-like transposon that harnesses novel features allowing the spread and diversification of genetic islands in pathogenic bacteria.

Antirepressor specificity is shaped by highly efficient dimerization of the staphylococcal pathogenicity island regulating repressors: Stl repressor dimerization perturbed by dUTPases.

The excision and replication, thus the life cycle of pathogenicity islands in staphylococci are regulated by Stl master repressors that form strong dimers. It has been recently shown that SaPIbov1-Stl dimers are separated during the activation of the Staphylococcus aureus pathogenicity island (SaPI) transcription via helper phage proteins. To understand the mechanism of this regulation, a quantitative analysis of the dimerization characteristics is required. Due to the highly efficient dimerization process, such an analysis has to involve specific solutions that permit relevant experiments to be performed. In the present work, we focused on two staphylococcal Stls associated with high biomedical interest, namely Stl proteins of Staphylococcus aureus bov1 and Staphylococcus hominis ShoCI794_SEPI pathogenicity islands. Exploiting the interactions of these two Stl proteins with their antirepressor-mimicking interaction partners allowed precise determination of the Stl dimerization constant in the subnanomolar range.

Divergent Selection in Low Recombination Regions Shapes the Genomic Islands in Two Incipient Shorebird Species.

Speciation in the face of gene flow is usually associated with a heterogeneous genomic landscape of divergence in nascent species pairs. However, multiple factors, such as divergent selection and local recombination rate variation, can influence the formation of these genomic islands. Examination of the genomic landscapes of species pairs that are still in the early stages of speciation provides an insight into this conundrum. In this study, population genomic analyses were undertaken using a wide range of sampling and whole-genome resequencing data from 96 unrelated individuals of Kentish plover (Charadrius alexandrinus) and white-faced plover (Charadrius dealbatus). We suggest that the two species exhibit varying levels of population admixture along the Chinese coast and on the Taiwan Island. Genome-wide analyses for introgression indicate that ancient introgression had occurred in Taiwan population, and gene flow is still ongoing in mainland coastal populations. Furthermore, we identified a few genomic regions with significant levels of interspecific differentiation and local recombination suppression, which contain several genes potentially associated with disease resistance, coloration, and regulation of plumage molting and thus may be relevant to the phenotypic and ecological divergence of the two nascent species. Overall, our findings suggest that divergent selection in low recombination regions may be a main force in shaping the genomic islands in two incipient shorebird species.

An efflux pump in genomic island GI-M202a mediates the transfer of polymyxin B resistance in Pandoraea pnomenusa M202.

BACKGROUND: Polymyxin B is considered a last-line therapeutic option against multidrug-resistant gram-negative bacteria, especially in COVID-19 coinfections or other serious infections. However, the risk of antimicrobial resistance and its spread to the environment should be brought to the forefront. METHODS: Pandoraea pnomenusa M202 was isolated under selection with 8 mg/L polymyxin B from hospital sewage and then was sequenced by the PacBio RS II and Illumina HiSeq 4000 platforms. Mating experiments were performed to evaluate the transfer of the major facilitator superfamily (MFS) transporter in genomic islands (GIs) to Escherichia coli 25DN. The recombinant E. coli strain Mrc-3 harboring MFS transporter encoding gene FKQ53_RS21695 was also constructed. The influence of efflux pump inhibitors (EPIs) on MICs was determined. The mechanism of polymyxin B excretion mediated by FKQ53_RS21695 was investigated by Discovery Studio 2.0 based on homology modeling. RESULTS: The MIC of polymyxin B for the multidrug-resistant bacterial strain P. pnomenusa M202, isolated from hospital sewage, was 96 mg/L. GI-M202a, harboring an MFS transporter-encoding gene and conjugative transfer protein-encoding genes of the type IV secretion system, was identified in P. pnomenusa M202. The mating experiment between M202 and E. coli 25DN reflected the transferability of polymyxin B resistance via GI-M202a. EPI and heterogeneous expression assays also suggested that the MFS transporter gene FKQ53_RS21695 in GI-M202a was responsible for polymyxin B resistance. Molecular docking revealed that the polymyxin B fatty acyl group inserts into the hydrophobic region of the transmembrane core with Pi-alkyl and unfavorable bump interactions, and then polymyxin B rotates around Tyr43 to externally display the peptide group during the efflux process, accompanied by an inward-to-outward conformational change in the MFS transporter. Additionally, verapamil and CCCP exhibited significant inhibition via competition for binding sites. CONCLUSIONS: These findings demonstrated that GI-M202a along with the MFS transporter FKQ53_RS21695 in P. pnomenusa M202 could mediate the transmission of polymyxin B resistance.

Remodulation of bacterial transcriptome after acquisition of foreign DNA: the case of irp-HPI high-pathogenicity island in Vibrio anguillarum.

The high-pathogenicity island irp-HPI is widespread in Vibrionaceae and encodes the siderophore piscibactin, as well as the regulator PbtA that is essential for its expression. In this work, we aim to study whether PbtA directly interacts with irp-HPI promoters. Furthermore, we hypothesize that PbtA, and thereby the acquisition of irp-HPI island, may also influence the expression of other genes elsewhere in the bacterial genome. To address this question, an RNAseq analysis was conducted to identify differentially expressed genes after pbtA deletion in Vibrio anguillarum RV22 genetic background. The results showed that PbtA not only modulates the irp-HPI genes but also modulates the expression of a plethora of V. anguillarum core genome genes, inducing nitrate, arginine, and sulfate metabolism, T6SS1, and quorum sensing, while repressing lipopolysaccharide (LPS) production, MARTX toxin, and major porins such as OmpV and ChiP. The direct binding of the C-terminal domain of PbtA to piscibactin promoters (PfrpA and PfrpC), quorum sensing (vanT), LPS transporter wza, and T6SS structure- and effector-encoding genes was demonstrated by electrophoretic mobility shift assay (EMSA). The results provide valuable insights into the regulatory mechanisms underlying the expression of irp-HPI island and its impact on Vibrios transcriptome, with implications in pathogenesis.IMPORTANCEHorizontal gene transfer enables bacteria to acquire traits, such as virulence factors, thereby increasing the risk of the emergence of new pathogens. irp-HPI genomic island has a broad dissemination in Vibrionaceae and is present in numerous potentially pathogenic marine bacteria, some of which can infect humans. Previous works showed that certain V. anguillarum strains exhibit an expanded host range plasticity and heightened virulence, a phenomenon linked to the acquisition of the irp-HPI genomic island. The present work shows that this adaptive capability is likely achieved through comprehensive changes in the transcriptome of the bacteria and that these changes are mediated by the master regulator PbtA encoded within the irp-HPI element. Our results shed light on the broad implications of horizontal gene transfer in bacterial evolution, showing that the acquired DNA can directly mediate changes in the expression of the core genome, with profounds implications in pathogenesis.

Effect of Salmonella pathogenicity island 1 and 2 (SPI-1 and SPI-2) deletion on intestinal colonization and systemic dissemination in chickens.

Salmonella's virulence genes are located in two regions known as Salmonella pathogenicity islands 1 and 2 (SPI-1 and SPI-2). SPI-1 allows the bacteria to invade the intestine, while SPI-2 is important for intracellular survival and replication, although it is also necessary for intestinal disease. The aim of this study was to evaluate the effect of the deletion of SPI-1 or SPI-2 genes on the intestinal and systemic salmonellosis using the avian model. Groups of chickens were orally infected with 1010 Colony-Forming Units (CFU) of S. Typhimurium SL1344 WT strain, as well as mutants ∆SPI-1 or ∆SPI-2. At different times post-infection, 5 chickens from each group were euthanized and examined postmortem. Cecum and liver were taken from each chicken for determination of CFU's, histopathological analysis and immunochemistry. Bacterial colonies were recovered from the liver and cecum samples infected with WT strain, while in the cultures from the organs infected with the mutant strains no colonies were recovered or were drastically affected in the ability to survive. In histopathological analysis, the WT strain produced lesions in liver and ceca, and it was detected by immunohistochemistry throughout the course of the infection. On the other hand, organs of chickens infected with ∆SPI-1 or ∆SPI-2 showed attenuated lesions and the immunohistochemistry revealed less bacteria compared to the WT strain. Taken together, our results show the importance of SPI-1 and SPI-2 genes for the complete intestinal and systemic disease in an in vivo avian model.

Structure of the Portal Complex from Staphylococcus aureus Pathogenicity Island 1 Transducing Particles In Situ and In Isolation.

Staphylococcus aureus is an important human pathogen, and the prevalence of antibiotic resistance is a major public health concern. The evolution of pathogenicity and resistance in S. aureus often involves acquisition of mobile genetic elements (MGEs). Bacteriophages play an especially important role, since transduction represents the main mechanism for horizontal gene transfer. S. aureus pathogenicity islands (SaPIs), including SaPI1, are MGEs that carry genes encoding virulence factors, and are mobilized at high frequency through interactions with specific "helper" bacteriophages, such as 80α, leading to packaging of the SaPI genomes into virions made from structural proteins supplied by the helper. Among these structural proteins is the portal protein, which forms a ring-like portal at a fivefold vertex of the capsid, through which the DNA is packaged during virion assembly and ejected upon infection of the host. We have used high-resolution cryo-electron microscopy to determine structures of the S. aureus bacteriophage 80α portal itself, produced by overexpression, and in situ in the empty and full SaPI1 virions, and show how the portal interacts with the capsid. These structures provide a basis for understanding portal and capsid assembly and the conformational changes that occur upon DNA packaging and ejection.

The horizontal gene transfer of perchlorate reduction genomic island in three bacteria from an ecological niche.

Three new strains of dissimilatory perchlorate-reducing bacteria (DPRB), QD19-16, QD1-5, and P3-1, were isolated from an active sludge. Phylogenetic trees based on 16S rRNA genes indicated that QD19-16, QD1-5, and P3-1 belonged to Brucella, Acidovorax, and Citrobacter, respectively, expanding the distribution of DPRB in the Proteobacteria. The three strains were gram-negative and facultative anaerobes with rod-shaped cells without flagella, which were 1.0-1.6 µm long and 0.5-0.6 µm wide. The three DPRB strains utilized similar broad spectrum of electron donors and acceptors and demonstrated a similar capability to reduce perchlorate within 6 days. The enzyme activity of perchlorate reductase in QD19-16 toward chlorate was higher than that toward perchlorate. The high sequence similarity of the perchlorate reductase operon and chlorite dismutase genes in the perchlorate reduction genomic islands (PRI) of the three strains implied that they were monophyletic origin from a common ancestral PRI. Two transposase genes (tnp1 and tnp2) were found in the PRIs of strain QD19-16 and QD1-5, but were absent in the strain P3-1 PRI. The presence of fragments of IR sequences in the P3-1 PRI suggested that P3-1 PRI had previously contained these two tnp genes. Therefore, it is plausible to suggest that a common ancestral PRI transferred across the strains Brucella sp. QD19-16, Acidovorax sp. QD1-5, and Citrobacter sp. P3-1 through horizontal gene transfer, facilitated by transposases. These results provided a direct evidence of horizontal gene transfer of PRI that could jump across phylogenetically unrelated bacteria through transposase. KEY POINTS: • Three new DPRB strains can effectively remove high concentration of perchlorate. • The PRIs of three DPRB strains are acquired from a single ancestral PRI. • PRIs are incorporated into different bacteria genome through HGT by transposase.

Phosphate-related genomic islands as drivers of environmental adaptation in the streamlined marine alphaproteobacterial HIMB59.

IMPORTANCE: These results shed light on the evolutionary strategies of microbes with streamlined genomes to adapt and survive in the oligotrophic conditions that dominate the surface waters of the global ocean. At the individual level, these microbes have been subjected to evolutionary constraints that have led to a more efficient use of nutrients, removing non-essential genes named as "streamlining theory." However, at the population level, they conserve a highly diverse gene pool in flexible genomic islands resulting in polyclonal populations on the same genomic background as an evolutionary response to environmental pressures. Localization of these islands at equivalent positions in the genome facilitates horizontal transfer between clonal lineages. This high level of environmental genomic heterogeneity could explain their cosmopolitan distribution. In the case of the order HIMB59 within the class Alphaproteobacteria, two factors exert evolutionary pressure and determine this intraspecific diversity: phages and the concentration of P in the environment.

Subinhibitory antibiotic concentrations promote the excision of a genomic island carried by the globally spread carbapenem-resistant Klebsiella pneumoniae sequence type 258.

The ICEKp258.2 genomic island (GI) has been proposed as an important factor for the emergence and success of the globally spread carbapenem-resistant Klebsiella pneumoniae sequence type (ST) 258. However, a characterization of this horizontally acquired element is lacking. Using bioinformatic and experimental approaches, we found that ICEKp258.2 is not confined to ST258 and ST512, but also carried by ST3795 strains and emergent invasive multidrug-resistant pathogens from ST1519. We also identified several ICEKp258.2-like GIs spread among different K. pneumoniae STs, other Klebsiella species and even other pathogen genera, uncovering horizontal gene transfer events between different STs and bacterial genera. Also, the comparative and phylogenetic analyses of the ICEKp258.2-like GIs revealed that the most closely related ICEKp258.2-like GIs were harboured by ST11 strains. Importantly, we found that subinhibitory concentrations of antibiotics used in treating K. pneumoniae infections can induce the excision of this GI and modulate its gene expression. Our findings provide the basis for the study of ICEKp258.2 and its role in the success of K. pneumoniae ST258. They also highlight the potential role of antibiotics in the spread of ICEKp258.2-like GIs among bacterial pathogens.