Large Mobile Genetic Elements Research Articles

The Neisseria gonorrhoeae Accessory Genome and Its Association with the Core Genome and Antimicrobial Resistance.

ABSTRACTThe bacterial accessory genome provides the genetic flexibility needed to facilitate environment and host adaptation. In Neisseria gonorrhoeae, known accessory elements include plasmids which can transfer and mediate antimicrobial resistance (AMR); however, chromosomal accessory genes could also play a role in AMR. Here, the gonococcal accessory genome was characterized using gene-by-gene approaches and its association with the core genome and AMR were assessed. The gonococcal accessory gene pool consisted of 247 genes, which were mainly genes located on large mobile genetic elements, phage associated genes, or genes encoding putative secretion systems. Accessory elements showed similar synteny across genomes, indicating either a predisposition for particular genomic locations or ancestral inheritance that are conserved during strain expansion. Significant associations were found between the prevalence of accessory elements and core genome multi-locus sequence types (cgMLST), consistent with a structured gonococcal population despite frequent horizontal gene transfer (HGT). Increased prevalence of putative DNA exchange regulators was significantly associated with AMR, which included a putative secretion system, methyltransferases and a toxin-antitoxin system. Although frequent HGT results in high genetic diversity in the gonococcus, we found that this is mediated by a small gene pool. In fact, a highly organized genome composition was identified with a strong association between the accessory and core genome. Increased prevalence of DNA exchange regulators in antimicrobial resistant isolates suggests that genetic material exchange plays a role in the development or maintenance of AMR. These findings enhance our understanding of gonococcal genome architecture and have important implications for gonococcal population biology.IMPORTANCE The emergence of antimicrobial resistance (AMR) against third generation cephalosporins in Neisseria gonorrhoeae is a major public health concern, as these are antibiotics of last resort for the effective treatment of gonorrhea. Although the resistance mechanisms against this class of antibiotics have not been entirely resolved, resistance against other classes of antibiotics, such as tetracyclines, is known to be mediated through plasmids, which are known gonococcal extra-chromosomal accessory elements. A complete assessment of the chromosomal accessory genome content and its role in AMR has not yet been undertaken. Here, we comprehensively characterize the gonococcal accessory genome to better understand genome architecture as well as the evolution and mechanisms of AMR in this species.

Natural transformation allows transfer of SCCmec-mediated methicillin resistance in Staphylococcus aureus biofilms.

SCCmec is a large mobile genetic element that includes the mecA gene and confers resistance to β-lactam antibiotics in methicillin-resistant Staphylococcus aureus (MRSA). There is evidence that SCCmec disseminates among staphylococci, but the transfer mechanisms are unclear. Here, we show that two-component systems mediate the upregulation of natural competence genes in S. aureus under biofilm growth conditions, and this enhances the efficiency of natural transformation. We observe SCCmec transfer via natural transformation from MRSA, and from methicillin-resistant coagulase-negative staphylococci, to methicillin-sensitive S. aureus. The process requires the SCCmec recombinase genes ccrAB, and the stability of the transferred SCCmec varies depending on SCCmec types and recipients. Our results suggest that natural transformation plays a role in the transfer of SCCmec and possibly other mobile genetic elements in S. aureus biofilms.

The Mobile Genetic Element RD2 Affects Colonization Potential of Different GAS Serotypes.

M-type 28 (M28) Streptococcus pyogenes (group A Streptococcus [GAS]) strains are highly associated with life-threatening puerperal infections. Genome sequencing has revealed a large mobile genetic element, RD2, present in most M28 GAS isolates but not found widely in other serotypes. Previous studies have linked RD2 to the ability of M28 GAS to colonize the vaginal tract. A new study by Roshika and colleagues (R. Roshika, I. Jain, J. Medicielo, J. Wächter, J. L. Danger, P. Sumby, Infect Immun 89:e00722-20, 2021, https://doi.org/10.1128/IAI.00722-20) used gain-of-function mutants in three different GAS serotypes to help determine why RD2 appears to have a serotype preference and what that could mean for GAS mucosal colonization and pathogenesis.

Polinton-like viruses are abundant in aquatic ecosystems

BackgroundPolintons are large mobile genetic elements found in the genomes of eukaryotic organisms that are considered the ancient ancestors of most eukaryotic dsDNA viruses. Originally considered as transposons, they have been found to encode virus capsid genes, suggesting they may actually be integrated viruses; however, an extracellular form has yet to be detected. Recently, circa 25 Polinton-like viruses have been discovered in environmental metagenomes and algal genomes, which shared distantly related genes to both Polintons and virophages (Lavidaviridae). These entities could be the first members of a major class of ancient eukaryotic viruses; however, owing to the lack of available genomes for analysis, information on their global diversity, evolutionary relationships, eukaryotic hosts, and status as free virus particles is limited.ResultsHere, we analysed the metaviromes of an alpine lake to show that Polinton-like virus genome sequences are abundant in the water column. We identify major capsid protein genes belonging to 82 new Polinton-like viruses and use these to interrogate publicly available metagenomic datasets, identifying 543 genomes and a further 16 integrated into eukaryotic genomes. Using an analysis of shared gene content and major capsid protein phylogeny, we define large groups of Polinton-like viruses and link them to diverse eukaryotic hosts, including a new group of viruses, which possess all the core genes of virophages and infect oomycetes and Chrysophyceae.ConclusionsOur study increased the number of known Polinton-like viruses by 25-fold, identifying five major new groups of eukaryotic viruses, which until now have been hidden in metagenomic datasets. The large enrichment (> 100-fold) of Polinton-like virus sequences in the virus-sized fraction of this alpine lake and the fact that their viral major capsid proteins are found in eukaryotic host transcriptomes support the hypothesis that Polintons in unicellular eukaryotes are viruses. In summary, our data reveals a diverse assemblage of globally distributed viruses, associated with a wide range of unicellular eukaryotic hosts. We anticipate that the methods we have developed for Polinton-like virus detection and the database of over 20,000 genes we present will allow for continued discovery and analysis of these new viral groups.4nQk6QRKxQCQHashEWMwNeVideo abstract

Fitness costs of methicillin resistance in Staphylococcus aureus

Staphylococcus aureus is a Gram-positive bacteria, and a common human pathogen. It is a leading cause of healthcare-associated infections worldwide, causing potentially fatal bacteraemia. Infections can be treated using antibiotics, but many strains have quickly developed resistance to the most common antibiotics. Methicillin resistance in Staphylococcus is acquired through the uptake of the Staphylococcal Cassette Chromosome mec (SCCmec). SCCmec is a large mobile genetic element, which can integrate into the Staphylococcus genome. It carries the mecA gene, which confers resistance to broad-spectrum β-lactam antibiotics, including methicillin. Several different SCCmec elements have been described, some of which can carry additional antibiotic resistance genes, alongside mecA. These can include resistance to common antibiotics or resistance to heavy metals. Smaller SCCmec types do not encode any additional antibiotic resistance genes. These have been shown to confer no fitness cost to the bacteria. Because of this, smaller SCCmec types are becoming increasingly more common, particularly in community-associated MRSA infections.

Genomic epidemiology and global diversity of the emerging bacterial pathogen Elizabethkingia anophelis.

Elizabethkingia anophelis is an emerging pathogen involved in human infections and outbreaks in distinct world regions. We investigated the phylogenetic relationships and pathogenesis-associated genomic features of two neonatal meningitis isolates isolated 5 years apart from one hospital in Central African Republic and compared them with Elizabethkingia from other regions and sources. Average nucleotide identity firmly confirmed that E. anophelis, E. meningoseptica and E. miricola represent demarcated genomic species. A core genome multilocus sequence typing scheme, broadly applicable to Elizabethkingia species, was developed and made publicly available (http://bigsdb.pasteur.fr/elizabethkingia). Phylogenetic analysis revealed distinct E. anophelis sublineages and demonstrated high genetic relatedness between the African isolates, compatible with persistence of the strain in the hospital environment. CRISPR spacer variation between the African isolates was mirrored by the presence of a large mobile genetic element. The pan-genome of E. anophelis comprised 6,880 gene families, underlining genomic heterogeneity of this species. African isolates carried unique resistance genes acquired by horizontal transfer. We demonstrated the presence of extensive variation of the capsular polysaccharide synthesis gene cluster in E. anophelis. Our results demonstrate the dynamic evolution of this emerging pathogen and the power of genomic approaches for Elizabethkingia identification, population biology and epidemiology.

SigmaB regulates ccrAB expression and SCCmec excision in methicillin-resistant Staphylococcus aureus

Methicillin-resistant Staphylococcus aureus (MRSA) is a worldwide pathogen that is resistant to practically the entire class of β-lactam antibiotics due to the presence of the mecA gene. The mecA gene is located on a large mobile genetic element referred to as staphylococcal cassette chromosome mec (SCCmec), and the excision and integration of SCCmec are mediated by the Ccr recombinase encoded by ccrAB or ccrC, which are also located on SCCmec. Previous studies have shown that the ccrAB genes are only expressed in a minority of cells and that their expression levels can be affected by certain environmental stimuli, but the molecular mechanisms controlling these phenotypes remain elusive. Here, we found that overexpression of SigB can dramatically enhance ccrA transcription and SCCmec excision in MRSA strain N315, revealing an important role for this alternative sigma factor in the lateral transfer of SCCmec. Further primer extension-blot analysis and 5′RACE (Rapid Amplification of cDNA Ends) indicated that an unrecognized SigB-dependent promoter region, which exists in certain SCCmec type II and IV strains, is responsible for the enhancement, and the ccrAB genes are in fact transcribed in a two-promoter pattern with a low activity of the SigB-dependent promoter under normal growth conditions.

Insights on virulence from the complete genome of Staphylococcus capitis.

Staphylococcus capitis is an opportunistic pathogen of the coagulase negative staphylococci (CoNS). Functional genomic studies of S. capitis have thus far been limited by a lack of available complete genome sequences. Here, we determined the closed S. capitis genome and methylome using Single Molecule Real Time (SMRT) sequencing. The strain, AYP1020, harbors a single circular chromosome of 2.44 Mb encoding 2304 predicted proteins, which is the smallest of all complete staphylococcal genomes sequenced to date. AYP1020 harbors two large mobile genetic elements; a plasmid designated pAYP1020 (59.6 Kb) and a prophage, ΦAYP1020 (48.5 Kb). Methylome analysis identified significant adenine methylation across the genome involving two distinct methylation motifs (1972 putative 6-methyladenine (m6A) residues identified). Putative adenine methyltransferases were also identified. Comparative analysis of AYP1020 and the closely related CoNS, S. epidermidis RP62a, revealed a host of virulence factors that likely contribute to S. capitis pathogenicity, most notably genes important for biofilm formation and a suite of phenol soluble modulins (PSMs); the expression/production of these factors were corroborated by functional assays. The complete S. capitis genome will aid future studies on the evolution and pathogenesis of the coagulase negative staphylococci.

The interplay of restriction-modification systems with mobile genetic elements and their prokaryotic hosts.

The roles of restriction-modification (R-M) systems in providing immunity against horizontal gene transfer (HGT) and in stabilizing mobile genetic elements (MGEs) have been much debated. However, few studies have precisely addressed the distribution of these systems in light of HGT, its mechanisms and its vectors. We analyzed the distribution of R-M systems in 2261 prokaryote genomes and found their frequency to be strongly dependent on the presence of MGEs, CRISPR-Cas systems, integrons and natural transformation. Yet R-M systems are rare in plasmids, in prophages and nearly absent from other phages. Their abundance depends on genome size for small genomes where it relates with HGT but saturates at two occurrences per genome. Chromosomal R-M systems might evolve under cycles of purifying and relaxed selection, where sequence conservation depends on the biochemical activity and complexity of the system and total gene loss is frequent. Surprisingly, analysis of 43 pan-genomes suggests that solitary R-M genes rarely arise from the degradation of R-M systems. Solitary genes are transferred by large MGEs, whereas complete systems are more frequently transferred autonomously or in small MGEs. Our results suggest means of testing the roles for R-M systems and their associations with MGEs.

Large mobile genetic elements carrying resistance genes that do not confer a fitness burden in healthcare-associated meticillin-resistant Staphylococcus aureus

Healthcare-associated (HA) meticillin-resistant Staphylococcus aureus (MRSA) clone CC22 SCCmecIV (EMRSA-15) has recently overtaken CC30/ST36 SCCmecII (EMRSA-16) as the dominant clone in UK hospitals. CC22 SCCmecIV shows greater fitness than CC30 SCCmecII, although both are successful global pathogens. The aim of this study was to test whether mobile genetic elements (MGEs), specifically SCCmec and large plasmids encoding resistance genes, are a burden and contribute to this fitness difference. Thirty-nine clinical isolates of MRSA and meticillin-sensitive S. aureus from lineages CC30 and CC22 with a variety of antibiotic resistance genes were grown in the absence of antibiotics. A range of relative fitness measures were used to compare clinical isolates with and without SCCmecII and SCCmecIV. The same fitness measures were used to compare eight isolates with and without naturally occurring large antibiotic resistance plasmids carrying gentamicin resistance (determined by microarray) and an isolate with an introduced plasmid. Growth rate, competitive ability during co-culture and survival after desiccation were then compared. Carriage of SCCmecII contributed to the reduced fitness of CC30 MRSA. However, we found no evidence of a fitness cost due to carriage of SCCmecIV in CC22, or large antibiotic resistance plasmids in CC30 or multiple resistances in both lineages. In conclusion, many large MGEs are not a fitness burden. Surprisingly, lineage background was the most important determinant of fitness. Our results suggest CC22 SCCmecIV will remain a successful healthcare-associated clone, and resistance to meticillin and gentamicin is likely to be maintained even in the absence of antibiotic pressure.

CRISPR-Cas systems target a diverse collection of invasive mobile genetic elements in human microbiomes

BackgroundBacteria and archaea develop immunity against invading genomes by incorporating pieces of the invaders' sequences, called spacers, into a clustered regularly interspaced short palindromic repeats (CRISPR) locus between repeats, forming arrays of repeat-spacer units. When spacers are expressed, they direct CRISPR-associated (Cas) proteins to silence complementary invading DNA. In order to characterize the invaders of human microbiomes, we use spacers from CRISPR arrays that we had previously assembled from shotgun metagenomic datasets, and identify contigs that contain these spacers' targets.ResultsWe discover 95,000 contigs that are putative invasive mobile genetic elements, some targeted by hundreds of CRISPR spacers. We find that oral sites in healthy human populations have a much greater variety of mobile genetic elements than stool samples. Mobile genetic elements carry genes encoding diverse functions: only 7% of the mobile genetic elements are similar to known phages or plasmids, although a much greater proportion contain phage- or plasmid-related genes. A small number of contigs share similarity with known integrative and conjugative elements, providing the first examples of CRISPR defenses against this class of element. We provide detailed analyses of a few large mobile genetic elements of various types, and a relative abundance analysis of mobile genetic elements and putative hosts, exploring the dynamic activities of mobile genetic elements in human microbiomes. A joint analysis of mobile genetic elements and CRISPRs shows that protospacer-adjacent motifs drive their interaction network; however, some CRISPR-Cas systems target mobile genetic elements lacking motifs.ConclusionsWe identify a large collection of invasive mobile genetic elements in human microbiomes, an important resource for further study of the interaction between the CRISPR-Cas immune system and invaders.

Bioinformatic evidence and characterization of novel putative large conjugative transposons residing in genomes of genera Bacteroides and Prevotella

Bioinformatic evidence of the presence of a large conjugative transposon in ruminal bacterium Prevotella bryantii B(1)4(T) is presented. The described transposon appears to be related to another large conjugative transposon CTnBST, described in Bacteroides uniformis WH207 and to the conjugative transposon CTn3-Bf, which was observed in the genome of Bacteroides fragilis strain YCH46. All three transposons share tra gene regions with high amino acid identity and clearly conserved gene order. Additionally, a second conserved region consisting of hypothetical genes was discovered in all three transposons and named the GG region. This region served as a specific sequence signature and made possible the discovery of several other apparently related hypothetical conjugative transposons in bacteria from the genus Bacteroides. A cluster of genes involved in sugar utilization and metabolism was discovered within the hypothetical CTnB(1)4, to a certain extent resembling the polysaccharide utilization loci which were described recently in some Bacteroides strains. This is the first firm report on the presence of a large mobile genetic element in any strain from the genus Prevotella.

Unexpected Diversity of Staphylococcal Cassette Chromosome mec Type IV in Methicillin-Resistant Staphylococcus aureus Strains

Staphylococcal cassette chromosome mec (SCCmec) is a large mobile genetic element which is used frequently for subtyping of methicillin-resistant Staphylococcus aureus (MRSA) strains. MRSA SCCmec type IV not only predominates among community-acquired MRSA (CA-MRSA) strains but also is associated with several genetic lineages of hospital-acquired MRSA (HA-MRSA) and with other species. The objective of this study was to investigate the diversity of MRSA strains classified as SCCmec type IV by using a multiplex PCR-based reverse line blot (mPCR/RLB) hybridization assay as well as spa typing and pulsed-field gel electrophoresis (PFGE). Sixty-two primer pairs and 63 probes were designed to interrogate each open reading frame (ORF) of SCCmec type IV sequences. A set of 131 MRSA SCCmec type IV isolates were classified into 79 subtypes by this method. There was considerable concordance between SCCmec type IV subtyping, spa typing, and PFGE patterns for clinical isolates, and the stability of SCCmec type IV subtyping was comparable to that of the other two methods. Using an in-house computer program, we showed that a subset of 20 genetic markers could achieve the same level of discrimination between isolates as the full set of 62, with a Simpson's index of diversity of 0.975. SCCmec type IV has a much higher level of diversity than previously suggested. The application of the mPCR/RLB hybridization assay to MRSA SCCmec type IV subtyping can improve the discriminatory power and throughput of MRSA typing and has the potential to enhance rapid infection control surveillance and outbreak detection.