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AbaR-type genomic islands (AbaRs) are prevalent and associated with multiple antimicrobial resistance in Acinetobacter baumannii AbaRs feature varied structural configurations involving different but closely related backbones with acquisition of diverse mobile genetic elements (MGEs) and antimicrobial resistance genes. This study aimed to understand the structural modulation patterns of AbaRs. A total of 442 intact AbaRs, including nonresistance but closely related islands, were mapped to backbones Tn 6019 , Tn 6022 , Tn 6172 /Tn 6173 , and AbGRI1-0 followed by alien sequence characterization. Genetic configurations were then examined and compared. The AbaRs fall into 53 genetic configurations, among which 26 were novel, including one Tn 6019 -type, nine Tn 6022 -type, three Tn 6172 /Tn 6173 -type, nine AbGRI1-type, and four new transposons that could not be mapped to the known backbones. The newly identified genetic configurations involved insertions of novel MGEs like IS Acsp2 , IS Aba42 , IS Aba17 , and IS Aba10 , novel structural modulations driven by known MGEs such as IS CR2 , Tn 2006 , and even another AbaR, and different backbone deletions. Recombination events in AbGRI1-type elements were also examined by identifying hybrid sequences from different backbones. Moreover, we found that the content and context features of AbaRs including the profiles of the MGEs driving the plasticity of these elements and the consequently acquired antimicrobial resistance genes, insertion sites, and clonal distribution displayed backbone-specific patterns. This study provides a comprehensive view of the genetic features of AbaRs. IMPORTANCE AbaR-type genomic islands (AbaRs) are well-known elements that can cause antimicrobial resistance in Acinetobacter baumannii These elements contain diverse and complex genetic configurations involving different but related backbones with acquisition of diverse mobile genetic elements and antimicrobial resistance genes. Understanding their structural diversity is far from complete. In this study, we performed a large-scale comparative analysis of AbaRs, including nonresistance but closely related islands. Our findings offered a comprehensive and interesting view of their genetic features, which allowed us to correlate the structural modulation signatures, antimicrobial resistance patterns, insertion loci, as well as host clonal distribution of these elements to backbone types. This study provides insights into the evolution of these elements, explains the association between their antimicrobial resistance gene profiles and clonal distribution, and could facilitate establishment of a more proper nomenclature than the term "AbaR" that has been variously used., (Copyright © 2020 Bi et al.)

AbaR-type genomic islands (AbaRs) are important elements responsible for antimicrobial resistance in Acinetobacter baumannii This study performed a large-scale identification of AbaRs to understand their distribution and compositions of antimicrobial resistance genes. We identified 2.89-kb left-end and 1.87-kb right-end conserved sequences (CSs) and developed a bioinformatics approach to identify AbaRs, using the CSs as signatures, in 3,148 publicly available genomes. AbaRs were prevalent in A. baumannii , being found in 2,091 genomes. They were sparse in other Acinetobacter species and confined only to this genus. Results from 111 complete genomes showed that over 85% of AbaRs resided on chromosomes. The external flanks adjacent to the inverted repeats available in all identified CSs were mapped to an AbaR-free chromosome or searched in the NCBI database for empty loci to define insertion sites. Surprisingly, 84 insertion sites with diverse origins were revealed, including 51 scattered on the chromosome, 20 plasmid borne, 12 located on prophages, transposons, IS Aba1 , complex AbaRs, and genomic islands of other types, and one uncharacterized, and some were strongly associated with clonal lineages. Finally, we found 994 antimicrobial resistance genes covering 28 unique genes from 70.9% (299/422) of intact AbaRs currently available. The resistance gene profiles displayed an apparent clonal lineage-specific pattern, highlighting the distinct features of AbaRs in global clone 1 (GC1) and GC2. The tet (B) gene was highly specific to the AbaRs in GC2. In conclusion, AbaRs have diverse insertion sites on the chromosome and mobile genetic elements (MGEs) and display distinct antimicrobial resistance gene profiles in different clonal lineages., (Copyright © 2019 American Society for Microbiology.)

Background: Strains of Escherichia coli that are non-typeable by pulsed-field gel electrophoresis (PFGE) due to in-gel degradation can influence their molecular epidemiological data. The DNA degradation phenotype (Dnd(+)) is mediated by the dnd operon that encode enzymes catalyzing the phosphorothioation of DNA, rendering the modified DNA susceptible to oxidative cleavage during a PFGE run. In this study, a PCR assay was developed to detect the presence of the dnd operon in Dnd(+) E. coli strains and to improve their typeability. Investigations into the genetic environments of the dnd operon in various E. coli strains led to the discovery that the dnd operon is harboured in various diverse genomic islands., Results: The dndBCDE genes (dnd operon) were detected in all Dnd(+) E. coli strains by PCR. The addition of thiourea improved the typeability of Dnd(+) E. coli strains to 100% using PFGE and the Dnd(+) phenotype can be observed in both clonal and genetically diverse E. coli strains. Genomic analysis of 101 dnd operons from genome sequences of Enterobacteriaceae revealed that the dnd operons of the same bacterial species were generally clustered together in the phylogenetic tree. Further analysis of dnd operons of 52 E. coli genomes together with their respective immediate genetic environments revealed a total of 7 types of genetic organizations, all of which were found to be associated with genomic islands designated dnd-encoding GIs. The dnd-encoding GIs displayed mosaic structure and the genomic context of the 7 islands (with 1 representative genome from each type of genetic organization) were also highly variable, suggesting multiple recombination events. This is also the first report where two dnd operons were found within a strain although the biological implication is unknown. Surprisingly, dnd operons were frequently found in pathogenic E. coli although their link with virulence has not been explored., Conclusion: Genomic islands likely play an important role in facilitating the horizontal gene transfer of the dnd operons in E. coli with 7 different types of islands discovered so far.

Background: Several strain-specific Klebsiella pneumoniae virulence determinants have been described, though these have almost exclusively been linked with hypervirulent liver abscess-associated strains. Through PCR interrogation of integration hotspots, chromosome walking, island-tagging and fosmid-based marker rescue we captured and sequenced KpGI-5, a novel genomic island integrated into the met56 tRNA gene of K. pneumoniae KR116, a bloodstream isolate from a patient with pneumonia and neutropenic sepsis., Results: The 14.0 kb KpGI-5 island exhibited a genome-anomalous G +  content, possessed near-perfect 46 bp direct repeats, encoded a γ1-chaperone/usher fimbrial cluster (fim2) and harboured seven other predicted genes of unknown function. Transcriptional analysis demonstrated expression of three fim2 genes, and suggested that the fim2A-fim2K cluster comprised an operon. As fimbrial systems are frequently implicated in pathogenesis, we examined the role of fim2 by analysing KR2107, a streptomycin-resistant derivative of KR116, and three isogenic mutants (Δfim, Δfim2 and ΔfimΔfim2) using biofilm assays, human cell adhesion assays and pair-wise competition-based murine models of intestinal colonization, lung infection and ascending urinary tract infection. Although no statistically significant role for fim2 was demonstrable, liver and kidney CFU counts for lung and urinary tract infection models, respectively, hinted at an ordered gradation of virulence: KR2107 (most virulent), KR2107∆fim2, KR2107∆fim and KR2107∆fim∆fim2 (least virulent). Thus, despite lack of statistical evidence there was a suggestion that fim and fim2 contribute additively to virulence in these murine infection models. However, further studies would be necessary to substantiate this hypothesis., Conclusion: Although fim2 was present in 13% of Klebsiella spp. strains investigated, no obvious in vitro or in vivo role for the locus was identified, although there were subtle hints of involvement in urovirulence and bacterial dissemination from the respiratory tract. Based on our findings and on parallels with other fimbrial systems, we propose that fim2 has the potential to contribute beneficially to pathogenesis and/or environmental persistence of Klebsiella strains, at least under specific yet-to-be identified conditions.

Klebsiella pneumoniae is an important bacterial pathogen of man that is commonly associated with opportunistic and hospital-associated infections. Increasing levels of multiple-antibiotic resistance associated with this species pose a major emerging clinical problem. This organism also occurs naturally in other diverse environments, including the soil. Consistent with its varied lifestyle and membership of the Enterobacteriaceae family, K. pneumoniae genomes exhibit highly plastic architecture comprising a core genome backbone interspersed with numerous and varied alien genomic islands. In this study the size of the presently known K. pneumoniae pan-genome gene pool was estimated through analysis of complete sequences of three chromosomes and 31 plasmids belonging to K. pneumoniae strains. In addition, using a PCR-based strategy the genomic content of eight tRNA/tmRNA gene sites that serve as DNA insertion hotspots were investigated in 28 diverse environmental and clinical strains of K. pneumoniae. Sequencing and characterization of five newly identified horizontally-acquired tmRNA-associated islands further expanded the archived K. pneumoniae gene pool to a total of 7648 unique gene members. Large-scale investigation of the content of tRNA/tmRNA hotspots will be useful to identify and/or survey accessory sequences dispersed amongst hundreds to thousands of members of many key bacterial species., (Copyright © 2010 Elsevier B.V. All rights reserved.)

Pseudomonas aeruginosa is a leading cause of hospital-acquired pneumonia and severe chronic lung infections in cystic fibrosis patients. The reference strains PA14 and PAO1 have been studied extensively, revealing that PA14 is more virulent than PAO1 in diverse infection models. Among other factors, this may be due to two pathogenicity islands, PAPI-1 and PAPI-2, both present in PA14 but not in PAO1. We compared the global contributions to virulence of PAPI-1 and PAPI-2, rather than that of individual island-borne genes, using murine models of acute pneumonia and bacteremia. Three isogenic island-minus mutants (PAPI-1-minus, PAPI-2-minus, and PAPI-1-minus, PAPI-2-minus mutants) were compared with the wild-type parent strain PA14 and with PAO1. Our results showed that both islands contributed significantly to the virulence of PA14 in acute pneumonia and bacteremia models. However, in contrast to the results for the bacteremia model, where each island was found to contribute individually, loss of the 108-kb PAPI-1 island alone was insufficient to measurably attenuate the mutant in the acute pneumonia model. Nevertheless, the double mutant was substantially more attenuated, and exhibited a lesser degree of virulence, than even PAO1 in the acute pneumonia model. In particular, its ability to disseminate from the lungs to the bloodstream was markedly inhibited. We conclude that both PAPI-1 and PAPI-2 contribute directly and synergistically in a major way to the virulence of PA14, and we suggest that analysis of island-minus strains may be a more appropriate way than individual gene knockouts to assess the contributions to virulence of large, horizontally acquired segments of DNA.

Background: The Dnd DNA degradation phenotype was first observed during electrophoresis of genomic DNA from Streptomyces lividans more than 20 years ago. It was subsequently shown to be governed by the five-gene dnd cluster. Similar gene clusters have now been found to be widespread among many other distantly related bacteria. Recently the dnd cluster was shown to mediate the incorporation of sulphur into the DNA backbone via a sequence-selective, stereo-specific phosphorothioate modification in Escherichia coli B7A. Intriguingly, to date all identified dnd clusters lie within mobile genetic elements, the vast majority in laterally transferred genomic islands., Methodology: We organized available data from experimental and bioinformatics analyses about the DNA phosphorothioation phenomenon and associated documentation as a dndDB database. It contains the following detailed information: (i) Dnd phenotype; (ii) dnd gene clusters; (iii) genomic islands harbouring dnd genes; (iv) Dnd proteins and conserved domains. As of 25 December 2008, dndDB contained data corresponding to 24 bacterial species exhibiting the Dnd phenotype reported in the scientific literature. In addition, via in silico analysis, dndDB identified 26 syntenic dnd clusters from 25 species of Eubacteria and Archaea, 25 dnd-bearing genomic islands and one dnd plasmid containing 114 dnd genes. A further 397 other genes coding for proteins with varying levels of similarity to Dnd proteins were also included in dndDB. A broad range of similarity search, sequence alignment and phylogenetic tools are readily accessible to allow for to individualized directions of research focused on dnd genes., Conclusion: dndDB can facilitate efficient investigation of a wide range of aspects relating to dnd DNA modification and other island-encoded functions in host organisms. dndDB version 1.0 is freely available at http://mml.sjtu.edu.cn/dndDB/.

The complete sequence (92 770 bp) of a genomic island (GI) named SLG from Streptomyces lividans 66, encoding a novel DNA S-modification system (dnd), was determined. Its overall G+C content was 67.8%, lower than those of three sequenced Streptomyces genomes. Among 85 predicted open reading frames (ORFs) in SLG, 22 ORFs showed little homology with previously known proteins. SLG displays a mosaic structure composed of four modules, indicative of multiple recombination events in its formation. Spontaneous excision and circularization of SLG was observed, and the excision rate appeared to be induced at least fivefold by MNNG exposure. Using constructed mini-islands of SLG, we demonstrated that Slg01, a P4-like integrase, was sufficient to promote SLG integration, excision and circularization. Eleven counterpart dnd clusters, which also mapped to GIs in 10 chromosomes and a plasmid, were found in taxonomically unrelated bacterial species from various geographic niches. Additionally, c. 10% of actinomycetes were found to possess a dnd cluster in a survey involving 74 strains. Comparison of dnd clusters in the 12 bacteria strongly suggests that these dnd-bearing elements might have evolved from a common ancestor similar to plasmid-originated chromosome II of Pseudoalteromonas haloplanktis TAC125.

MobilomeFINDER (http://mml.sjtu.edu.cn/MobilomeFINDER) is an interactive online tool that facilitates bacterial genomic island or 'mobile genome' (mobilome) discovery; it integrates the ArrayOme and tRNAcc software packages. ArrayOme utilizes a microarray-derived comparative genomic hybridization input data set to generate 'inferred contigs' produced by merging adjacent genes classified as 'present'. Collectively these 'fragments' represent a hypothetical 'microarray-visualized genome (MVG)'. ArrayOme permits recognition of discordances between physical genome and MVG sizes, thereby enabling identification of strains rich in microarray-elusive novel genes. Individual tRNAcc tools facilitate automated identification of genomic islands by comparative analysis of the contents and contexts of tRNA sites and other integration hotspots in closely related sequenced genomes. Accessory tools facilitate design of hotspot-flanking primers for in silico and/or wet-science-based interrogation of cognate loci in unsequenced strains and analysis of islands for features suggestive of foreign origins; island-specific and genome-contextual features are tabulated and represented in schematic and graphical forms. To date we have used MobilomeFINDER to analyse several Enterobacteriaceae, Pseudomonas aeruginosa and Streptococcus suis genomes. MobilomeFINDER enables high-throughput island identification and characterization through increased exploitation of emerging sequence data and PCR-based profiling of unsequenced test strains; subsequent targeted yeast recombination-based capture permits full-length sequencing and detailed functional studies of novel genomic islands.

We devised software tools to systematically investigate the contents and contexts of bacterial tRNA and tmRNA genes, which are known insertion hotspots for genomic islands (GIs). The strategy, based on MAUVE-facilitated multigenome comparisons, was used to examine 87 Escherichia coli MG1655 tRNA and tmRNA genes and their orthologues in E.coli EDL933, E.coli CFT073 and Shigella flexneri Sf301. Our approach identified 49 GIs occupying approximately 1.7 Mb that mapped to 18 tRNA genes, missing 2 but identifying a further 30 GIs as compared with Islander [Y. Mantri and K. P. Williams (2004), Nucleic Acids Res., 32, D55-D58]. All these GIs had many strain-specific CDS, anomalous GC contents and/or significant dinucleotide biases, consistent with foreign origins. Our analysis demonstrated marked conservation of sequences flanking both empty tRNA sites and tRNA-associated GIs across all four genomes. Remarkably, there were only 2 upstream and 5 downstream deletions adjacent to the 328 loci investigated. In silico PCR analysis based on conserved flanking regions was also used to interrogate hotspots in another eight completely or partially sequenced E.coli and Shigella genomes. The tools developed are ideal for the analysis of other bacterial species and will lead to in silico and experimental discovery of new genomic islands.