New tRNA-targeting transposons that hijack phage and vesicles.

Genomic islands are hotspots for horizontal gene transfer (HGT) in bacteria, but, for Prochlorococcus, an abundant marine cyanobacterium, how these islands form has puzzled scientists. With the discovery of tycheposons, a new family of transposons, Hackl et al. provide evidence for elegant new mechanisms of gene rearrangement and transfer among Prochlorococcus and bacteria more broadly.

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.

Characterization of a ST137 multidrug-resistant Campylobacter jejuni strain with a tet(O)-positive genomic island from a bloodstream infection patient.

Campylobacter jejuni (C. jejuni) is a major cause of gastroenteritis and rarely cause bloodstream infection. Herein, we characterized a multidrug-resistant C. jejuni strain LZCJ isolated from a tumor patient with bloodstream infection. LZCJ was resistant to norfloxacin, ampicillin, ceftriaxone, ciprofloxacin and tetracycline. It showed high survival rate in serum and acidic environment. Whole genome sequencing (WGS) analysis revealed that strain LZCJ had a single chromosome of 1,629,078 bp (30.6 % G + C content) and belonged to the ST137 lineage. LZCJ shared the highest identity of 99.66 % with the chicken-derived C. jejuni MTVDSCj20. Four antimicrobial resistance genes (ARGs) were detected, blaOXA-61, tet(O), gyrA (T86I), and cmeR (G144D and S207G). In addition, a 12,746 bp genomic island GI_LZCJ carrying 15 open reading frames (ORFs) including the resistance gene tet(O) was identified. Sequence analysis found that the GI_LZCJ was highly similar to the duck-derived C. jejuni ZS004, but with an additional ISChh1-like sequence. 137 non-synonymous mutations in motility related genes (flgF, fapR, flgS), capsular polysaccharide (CPS) coding genes (kpsE, kpsF, kpsM, kpsT), metabolism associated genes (nuoF, nuoG, epsJ, holB), and transporter related genes (comEA, gene0911) were confirmed in LZCJ compared with the best closed chicken-derived strain MTVDSCj20. Our study showed that C. jejuni strain LZCJ was highly similar to the chicken-derived strain MTVDSCj20 but with a lot of SNPs involved in motility, CPS and metabolism coding genes. This strain possessed a tet(O)-positive genomic island GI_LZCJ, which was closed to duck-derived C. jejuni ZS004, but with an additional ISChh1-like sequence. The above data indicated that the LZCJ strain may originate from foodborne bacteria on animals and the importance of continuous surveillance for the spread of foodborne bacteria.

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.

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.

xenoGI 3: using the DTLOR model to reconstruct the evolution of gene families in clades of microbes.

To understand genome evolution in a group of microbes, we need to know the timing of events such as duplications, deletions and horizontal transfers. A common approach is to perform a gene-tree / species-tree reconciliation. While a number of software packages perform this type of analysis, none are geared toward a complete reconstruction for all families in an entire clade. Here we describe an update to the xenoGI software package which allows users to perform such an analysis using the newly developed DTLOR (duplication-transfer-loss-origin-rearrangement) reconciliation model starting from genome sequences as input.

Comparative genomics analysis and characterization of Shiga toxin-producing Escherichia coli O157:H7 strains reveal virulence genes, resistance genes, prophages and plasmids.

Escherichia coli O157:H7 is a foodborne pathogen that has been linked to global disease outbreaks. These diseases include hemorrhagic colitis and hemolytic uremic syndrome. It is vital to know the features that make this strain pathogenic to understand the development of disease outbreaks. In the current study, a comparative genomic analysis was carried out to determine the presence of structural and functional features of O157:H7 strains obtained from 115 National Center for Biotechnology Information database. These strains of interest were analysed in the following programs: BLAST Ring Image Generator, PlasmidFinder, ResFinder, VirulenceFinder, IslandViewer 4 and PHASTER. Five strains (ECP19-198, ECP19-798, F7508, F8952, H2495) demonstrated a great homology with Sakai because of a few regions missing. Five resistant genes were identified, however, Macrolide-associated resistance gene mdf(A) was commonly found in all genomes. Majority of the strains (97%) were positive for 15 of the virulent genes (espA, espB, espF, espJ, gad, chuA, eae, iss, nleA, nleB, nleC, ompT, tccP, terC and tir). The plasmid analysis demonstrated that the IncF group was the most prevalent in the strains analysed. The prophage and genomic island analysis showed a distribution of bacteriophages and genomic islands respectively. The results indicated that structural and functional features of the many O157:H7 strains differ and may be a result of obtaining mobile genetic elements via horizontal gene transfer. Understanding the evolution of O157:H7 strains pathogenicity in terms of their structural and functional features will enable the development of detection and control of transmission strategies.

Effect of the S008-sgaCD operon on IncC plasmid stability in the presence of SGI1-K or absence of an SGI1 variant.

An IncC or IncA plasmid is needed to enable transfer of SGI1 type integrative mobilisable elements but an IncC plasmid does not stably co-exist with SGI1. However, the plasmid is stably maintained with SGI1-K, a natural SGI1 deletion variant that lacks the sgaDC genes (S007 and S006) and the upstream open reading frame (S008) found in the SGI1 backbone. Here, the effect of the sgaDC genes and S008 on the stability of an IncC plasmid in an Escherichia coli strain with or without SGI1-K was examined. Co-transcription of the S008 open reading frame with the downstream sgaDC genes was established. When a strain containing SGI1-K complemented with a pK18 plasmid that included S008-sgaDC or sgaDC expressed from the constitutive pUC promoter was grown without antibiotic selection, the resident IncC plasmid was rapidly lost but loss was slower when S008 was present. In contrast, SGI1-K and the S008-sgaDC or sgaDC plasmid were quite stably maintained for >100 generations. However, the high copy number plasmids carrying the SGI1-derived S008-sgaDC or sgaDC genes constitutively expressed could not be introduced into an E. coli strain carrying the IncC plasmid but without SGI1-K. Using equivalent plasmids with S008-sgaDC or sgaDC genes controlled by an arabinose-inducible promoter, under inducing conditions the IncC plasmid was stable but the plasmid containing the SGI1-derived genes was rapidly lost. This unexpected observation indicates that there are multiple interactions between the IncC plasmid and SGI1 in which the transcriptional activator genes sgaDC play a role. These interactions will require further investigation.

A Brief Evaluation of a Copper Resistance Mobile Genetic Island in the Bacterial Leaf Spot Pathogen Xanthomonas euvesicatoria pv. perforans.

Due to the continuous use of copper containing bactericides without effective alternative bactericides, copper resistance has become more prevalent in plant pathogens, including Xanthomonas euvesicatoria pv. perforans (formerly Xanthomonas perforans), a predominant cause of bacterial leaf spot disease of tomato and pepper in the Southeastern United States. Previously, reports of copper resistance have been associated with a large conjugative plasmid. However, we have characterized a copper resistance genomic island located within the chromosome of multiple X. euvesicatoria pv. perforans strains. The island is distinct from a previously described chromosomally encoded copper resistance island in X. vesicatoria strain XVP26. Computational analysis revealed the genomic island to contain multiple genes associated with genetic mobility, including both phage-related genes and transposase. Among copper-tolerant strains of X. euvesicatoria pv. perforans isolated from Florida, the majority of strains were found to have the copper resistance chromosomally encoded rather than plasmid borne. Our results suggest that this copper resistance island may have two modes of horizontal gene transfer and that chromosomally encoded copper resistance genes may provide a fitness advantage over plasmid-borne resistance.

Mobile Oxazolidinone Resistance Genes in Gram-Positive and Gram-Negative Bacteria.

Seven mobile oxazolidinone resistance genes, including cfr, cfr(B), cfr(C), cfr(D), cfr(E), optrA, and poxtA, have been identified to date. The cfr genes code for 23S rRNA methylases, which confer a multiresistance phenotype that includes resistance to phenicols, lincosamides, oxazolidinones, pleuromutilins, and streptogramin A compounds. The optrA and poxtA genes code for ABC-F proteins that protect the bacterial ribosomes from the inhibitory effects of oxazolidinones. The optrA gene confers resistance to oxazolidinones and phenicols, while the poxtA gene confers elevated MICs or resistance to oxazolidinones, phenicols, and tetracycline. These oxazolidinone resistance genes are most frequently found on plasmids, but they are also located on transposons, integrative and conjugative elements (ICEs), genomic islands, and prophages. In these mobile genetic elements (MGEs), insertion sequences (IS) most often flanked the cfr, optrA, and poxtA genes and were able to generate translocatable units (TUs) that comprise the oxazolidinone resistance genes and occasionally also other genes. MGEs and TUs play an important role in the dissemination of oxazolidinone resistance genes across strain, species, and genus boundaries. Most frequently, these MGEs also harbor genes that mediate resistance not only to antimicrobial agents of other classes, but also to metals and biocides. Direct selection pressure by the use of antimicrobial agents to which the oxazolidinone resistance genes confer resistance, but also indirect selection pressure by the use of antimicrobial agents, metals, or biocides (the respective resistance genes against which are colocated on cfr-, optrA-, or poxtA-carrying MGEs) may play a role in the coselection and persistence of oxazolidinone resistance genes.

Relationship and distribution of Salmonella enterica serovar I 4,[5],12:i:- strain sequences in the NCBI Pathogen Detection database.

BACKGROUND: Of the > 2600 Salmonella serovars, Salmonella enterica serovar I 4,[5],12:i:- (serovar I 4,[5],12:i:-) has emerged as one of the most common causes of human salmonellosis and the most frequent multidrug-resistant (MDR; resistance to ≥3 antimicrobial classes) nontyphoidal Salmonella serovar in the U.S. Serovar I 4,[5],12:i:- isolates have been described globally with resistance to ampicillin, streptomycin, sulfisoxazole, and tetracycline (R-type ASSuT) and an integrative and conjugative element with multi-metal tolerance named Salmonella Genomic Island 4 (SGI-4). RESULTS: We analyzed 13,612 serovar I 4,[5],12:i:- strain sequences available in the NCBI Pathogen Detection database to determine global distribution, animal sources, presence of SGI-4, occurrence of R-type ASSuT, frequency of antimicrobial resistance (AMR), and potential transmission clusters. Genome sequences for serovar I 4,[5],12:i:- strains represented 30 countries from 5 continents (North America, Europe, Asia, Oceania, and South America), but sequences from the United States (59%) and the United Kingdom (28%) were dominant. The metal tolerance island SGI-4 and the R-type ASSuT were present in 71 and 55% of serovar I 4,[5],12:i:- strain sequences, respectively. Sixty-five percent of strain sequences were MDR which correlates to serovar I 4,[5],12:i:- being the most frequent MDR serovar. The distribution of serovar I 4,[5],12:i:- strain sequences in the NCBI Pathogen Detection database suggests that swine-associated strain sequences were the most frequent food-animal source and were significantly more likely to contain the metal tolerance island SGI-4 and genes for MDR compared to all other animal-associated isolate sequences. CONCLUSIONS: Our study illustrates how analysis of genomic sequences from the NCBI Pathogen Detection database can be utilized to identify the prevalence of genetic features such as antimicrobial resistance, metal tolerance, and virulence genes that may be responsible for the successful emergence of bacterial foodborne pathogens.

A horizontally acquired Legionella genomic island encoding a LuxR type regulator and effector proteins displays variation in gene content and regulation.

The intracellular pathogen Legionella pneumophila translocates >300 effector proteins into host cells, many of which are regulated at the transcriptional level. Here, we describe a novel L. pneumophila genomic island, which undergoes horizontal gene transfer within the Legionella genus. This island encodes two Icm/Dot effectors: LegK3 and a previously uncharacterized effector which we named CegK3, as well as a LuxR type regulator, which we named RegK3. Analysis of this island in different Legionella species revealed a conserved regulatory element located upstream to the effector-encoding genes in the island. Further analyses, including gene expression analysis, mutagenesis of the RegK3 regulatory element, controlled expression studies, and gel-mobility shift assays, all demonstrate that RegK3 directly activates the expression levels of legK3 and cegK3 effector-encoding genes. Additionally, the expression of all the components of the island is silenced by the Fis repressors. Comparison of expression profiles of these three genes among different Legionella species revealed variability in the activation levels mediated by RegK3, which were positively correlated with the Fis-mediated repression. Furthermore, LegK3 and CegK3 effectors moderately inhibit yeast growth, and importantly, they have a strong synergistic inhibitory effect on yeast growth, suggesting these two effectors are not only co-regulated but also might function together.

Prevalence of an Insect-Associated Genomic Region in Environmentally Acquired Burkholderiaceae Symbionts.

Microbial symbionts are critical for the development and survival of many eukaryotes. Recent research suggests that the genes enabling these relationships can be localized in horizontally transferred regions of microbial genomes termed "symbiotic islands." Recently, a putative symbiotic island was found that may facilitate symbioses between true bugs and numerous Burkholderia species, based on analysis of five Burkholderia symbionts. We expanded on this work by exploring the putative island's prevalence, origin, and association with colonization across the bacterial family Burkholderiaceae. We performed a broad comparative analysis of 229 Burkholderiaceae genomes, including 8 new genomes of insect- or soil-associated Burkholderia sequenced for this study. We detected the region in 23% of the genomes; these were located solely within two Burkholderia clades. Our analyses suggested that the contiguous region arose at the common ancestor of plant- and insect-associated Burkholderia clades, but the genes themselves are ancestral. Although the region was initially discovered on plasmids and we did detect two likely instances of horizontal transfer within Burkholderia, we found that the region is almost always localized to a chromosome and does not possess any of the mobility elements that typify genomic islands. Finally, to attempt to deduce the region's function, we combined our data with information on several strains' abilities to colonize the insect's symbiotic organ. Although the region was associated with improved colonization of the host, this relationship was confounded with, and likely driven by, Burkholderia clade membership. These findings advance our understanding of the genomic underpinnings of a widespread insect-microbe symbiosis. IMPORTANCE Many plants and animals form intricate associations with bacteria. These pairings can be mediated by genomic islands, contiguous regions containing numerous genes with cohesive functionality. Pathogen-associated islands are well described, but recent evidence suggests that mutualistic islands, which benefit both host and symbiont, may also be common. Recently, a putative symbiosis island was found in Burkholderia symbionts of insects. We determined that this genomic region is located in only two clades of Burkholderia (the plant- and insect-associated species) and that although it has undergone horizontal transfer, it is most likely a symbiosis-associated region rather than a true island. This region is associated with improved host colonization, although this is may be due to specific Burkholderia clades' abilities to colonize rather than presence of the region. By studying the genomic basis of the insect-Burkholderia symbiosis, we can better understand how mutualisms evolve in animals.

Genomic Island-Mediated Horizontal Transfer of the Erythromycin Resistance Gene erm(X) among Bifidobacteria.

Antibiotic resistance is a serious medical issue driven by antibiotic misuse. Bifidobacteria may serve as a reservoir for antibiotic resistance genes (ARGs) that have the potential risk of transfer to pathogens. The erythromycin resistance gene erm(X) is an ARG with high abundance in bifidobacteria, especially in Bifidobacterium longum species. However, the characteristics of the spread and integration of the gene erm(X) into the bifidobacteria genome are poorly understood. In this study, 10 tetW-positive bifidobacterial strains and 1 erm(X)-positive bifidobacterial strain were used to investigate the transfer of ARGs. Conjugation assays found that the erm(X) gene could transfer to five other bifidobacterial strains. Dimethyl sulfoxide (DMSO) and vorinostat significantly promoted the transfer of the erm(X) from strain Bifidobacterium catenulatum subsp. kashiwanohense DSM 21854 to Bifidobacterium longum subsp. suis DSM 20211. Whole-genome sequencing and comparative genomic analysis revealed that the erm(X) gene was located on the genomic island BKGI1 and that BKGI1 was conjugally mobile and transferable. To our knowledge, this is the first report that a genomic island-mediated gene erm(X) transfer in bifidobacteria. Additionally, BKGI1 is very unstable in B. catenulatum subsp. kashiwanohense DSM 21854 and transconjugant D2TC and is highly excisable and has an intermediate circular formation. In silico analysis showed that the BKGI1 homologs were also present in other bifidobacterial strains and were especially abundant in B. longum strains. Thus, our results confirmed that genomic island BKGI1 was one of the vehicles for erm(X) spread. These findings suggest that genomic islands play an important role in the dissemination of the gene erm(X) among Bifidobacterium species. IMPORTANCE Bifidobacteria are a very important group of gut microbiota, and the presence of these bacteria has many beneficial effects for the host. Thus, bifidobacteria have attracted growing interest owing to their potential probiotic properties. Bifidobacteria have been widely exploited by the food industry as probiotic microorganisms, and some species have a long history of safe use in food and feed production. However, the presence of antibiotic resistance raises the risk of its application. In this study, we analyzed the transfer of the erythromycin resistance gene erm(X) and revealed that the molecular mechanism behind the spread of the gene erm(X) was mediated by genomic island BKGI1. To the best of our knowledge this is the first report to describe the transfer of the gene erm(X) via genomic islands among bifidobacteria. This may be an important way to disseminate the gene erm(X) among bifidobacteria.

Can SGI1 family integrative mobilizable elements overcome entry exclusion exerted by IncA and IncC plasmids on IncC plasmids?

Though IncC and IncA plasmids are compatible, they exert high level exclusion on one another. Here, the question of whether the presence of an SGI1 family element in the donor can overcome the exclusion of an IncC plasmid exerted by an IncC or IncA plasmid in the recipient was investigated. The transfer of the integrative mobilizable element SGI1 and its many variant forms into a new host is dependent on transfer machinery supplied by IncC or IncA plasmids. SGI1 elements include the determinants of a mobilization system and three genes that encode homologues of transfer proteins including TraG. Exclusion of a complete IncC plasmid by a complete IncA or IncC plasmid in the recipient was not ameliorated by an SGI1 element in the donor. However, transfer of the SGI was unaffected indicating that a functional mating apparatus was formed. The presence of only the plasmid-derived eexC or eexA gene in the recipient exerted high level exclusion on an incoming IncC plasmid and this was overcome by an SGI1 variant in the donor. Hence, the SGI affects only entry exclusion and additional plasmid features must influence other routes to plasmid exclusion.

Investigation of class 1 integrons and virulence genes in the emergent Salmonella serovar Infantis in Turkey.

The emerging situation of Salmonella enterica subsp. enterica serovar Infantis (S. Infantis) in Turkey was investigated in terms of virulence genes and mobile genetic elements such as Salmonella genomic island 1 (SGI1) and class 1 (C1) integron to see whether increased multidrug resistance (MDR) and ability to cause human cases is a consequence of their possession. Screening of SGI1 (and its variants) and C1 integrons was done with conventional PCR, while screening of gene cassettes and virulence genes was conducted with real-time PCR for 70 S. Infantis isolates from poultry products. SGI1 or its variants were not detected in any of the isolates. Sixty-eight of 70 isolates were detected to carry one C1 integron of size 1.0 kb. These integrons were detected to carry ant(3″)-Ia gene cassette explaining the streptomycin/spectinomycin resistance. Sequence analysis of gene cassettes belongs to four representing isolates which showed that, although their difference in isolation date and place, genetically, they are 99.9% similar. Virulence gene screening was introduced as genotypic virulence profiles. The most dominant profile for S. Infantis isolates, among twelve genes, was gatC-tcfA, which are known to be related to colonization at specific hosts. This study revealed the high percentage of C1 integron possession in S. Infantis isolates from poultry products in Turkey. It also showed the potential of S. Infantis strains to be resistant to more antimicrobial drugs. Moreover, a dominant profile of virulence genes that are uncommon for non-typhoidal Salmonella (NTS) serovars was detected, which might explain the enhanced growth at specified hosts.

Activation of the Type VI Secretion System in the Squid Symbiont Vibrio fischeri Requires the Transcriptional Regulator TasR and the Structural Proteins TssM and TssA.

Bacteria have evolved diverse strategies to compete for a niche, including the type VI secretion system (T6SS), a contact-dependent killing mechanism. T6SSs are common in bacterial pathogens, commensals, and beneficial symbionts, where they affect the diversity and spatial structure of host-associated microbial communities. Although T6SS gene clusters are often located on genomic islands (GIs), which may be transferred as a unit, the regulatory strategies that promote gene expression once the T6SS genes are transferred into a new cell are not known. We used the squid symbiont Vibrio fischeri to identify essential regulatory factors that control expression of a strain-specific T6SS encoded on a GI. We found that a transcriptional reporter for this T6SS is active only in strains that contain the T6SS-encoding GI, suggesting the GI encodes at least one essential regulator. A transposon screen identified seven mutants that could not activate the reporter. These mutations mapped exclusively to three genes on the T6SS-containing GI that encode two essential structural proteins (a TssA-like protein and TssM) and a transcriptional regulator (TasR). Using T6SS reporters, reverse transcription-PCR (RT-PCR), competition assays, and differential proteomics, we found that all three genes are required for expression of many T6SS components, except for the TssA-like protein and TssM, which are constitutively expressed. Based on these findings, we propose a model whereby T6SS expression requires conserved structural proteins, in addition to the essential regulator TasR, and this ability to self-regulate may be a strategy to activate T6SS expression upon transfer of T6SS-encoding elements into a new bacterial host. IMPORTANCE Interbacterial weapons like the T6SS are often located on mobile genetic elements, and their expression is highly regulated. We found that two conserved structural proteins are required for T6SS expression in Vibrio fischeri. These structural proteins also contain predicted GTPase and GTP binding domains, suggesting their role in promoting T6SS expression may involve sensing the energetic state of the cell. Such a mechanism would provide a direct link between T6SS activation and cellular energy levels, providing a "checkpoint" to ensure the cell has sufficient energy to build such a costly weapon. Because these regulatory factors are encoded within the T6SS gene cluster, they are predicted to move with the genetic element to activate T6SS expression in a new host cell.

Identifying genomic islands with deep neural networks.

BACKGROUND: Horizontal gene transfer is the main source of adaptability for bacteria, through which genes are obtained from different sources including bacteria, archaea, viruses, and eukaryotes. This process promotes the rapid spread of genetic information across lineages, typically in the form of clusters of genes referred to as genomic islands (GIs). Different types of GIs exist, and are often classified by the content of their cargo genes or their means of integration and mobility. While various computational methods have been devised to detect different types of GIs, no single method is capable of detecting all types. RESULTS: We propose a method, which we call Shutter Island, that uses a deep learning model (Inception V3, widely used in computer vision) to detect genomic islands. The intrinsic value of deep learning methods lies in their ability to generalize. Via a technique called transfer learning, the model is pre-trained on a large generic dataset and then re-trained on images that we generate to represent genomic fragments. We demonstrate that this image-based approach generalizes better than the existing tools. CONCLUSIONS: We used a deep neural network and an image-based approach to detect the most out of the correct GI predictions made by other tools, in addition to making novel GI predictions. The fact that the deep neural network was re-trained on only a limited number of GI datasets and then successfully generalized indicates that this approach could be applied to other problems in the field where data is still lacking or hard to curate.

Carbapenem Resistance Determinants Acquired through Novel Chromosomal Integrations in Extensively Drug-Resistant Pseudomonas aeruginosa.

Two novel blaDIM-1- or blaIMP-1-containing genomic islands (GIs) were discovered by whole-genome sequence analyses in four extensively drug-resistant (XDR) Pseudomonas aeruginosa isolates from inpatients at a tertiary hospital in Ghana. The strains were of sequence type 234 (ST234) and formed a phylogenetic clade together with ST111, which is recognized as a global high-risk clone. Their carbapenem resistance was encoded by two Tn402-type integrons, In1592 (blaDIM-1) and In1595 (blaIMP-1), both carrying complete tni mobilization modules. In1595 was bound by conserved 25-bp inverted repeats (IRs) flanked by 5-bp direct repeats (DRs) associated with target site duplication. The integrons were embedded in two GIs that contained cognate integrases and were distinguished by a lower GC content than the chromosomal average. PAGI-97A (52.659 bp; In1592), which encoded a P4-type site-specific integrase of the tyrosine recombinase family in its 3' border, was integrated into tRNA-Pro(ggg) and bracketed by a 49-bp perfect DR created by 3'-end target duplication. GIs with the same structural features, but diverse genetic content, were identified in 41/226 completed P. aeruginosa genomes. PAGI-97B (22,636 bp; In1595), which encoded an XerC/D superfamily integrase in its 5' border, was inserted into the small RNA (sRNA) PrrF1/PrrF2 locus. Specific insertions into this highly conserved locus involved in iron-dependent regulation, all leaving PrrF1 intact, were identified in an additional six phylogenetically unrelated P. aeruginosa genomes. Our molecular analyses unveiled a hospital-associated clonal dissemination of carbapenem-resistant ST234 P. aeruginosa in which the XDR phenotype resulted from novel insertions of two GIs into specific chromosomal sites.

Microbial genomic island discovery, visualization and analysis.

Horizontal gene transfer (also called lateral gene transfer) is a major mechanism for microbial genome evolution, enabling rapid adaptation and survival in specific niches. Genomic islands (GIs), commonly defined as clusters of bacterial or archaeal genes of probable horizontal origin, are of particular medical, environmental and/or industrial interest, as they disproportionately encode virulence factors and some antimicrobial resistance genes and may harbor entire metabolic pathways that confer a specific adaptation (solvent resistance, symbiosis properties, etc). As large-scale analyses of microbial genomes increases, such as for genomic epidemiology investigations of infectious disease outbreaks in public health, there is increased appreciation of the need to accurately predict and track GIs. Over the past decade, numerous computational tools have been developed to tackle the challenges inherent in accurate GI prediction. We review here the main types of GI prediction methods and discuss their advantages and limitations for a routine analysis of microbial genomes in this era of rapid whole-genome sequencing. An assessment is provided of 20 GI prediction software methods that use sequence-composition bias to identify the GIs, using a reference GI data set from 104 genomes obtained using an independent comparative genomics approach. Finally, we present guidelines to assist researchers in effectively identifying these key genomic regions.