Integrative And Conjugative Elements Research Articles

Diversity and Evolution of Integrative and Conjugative Elements Involved in Bacterial Aromatic Compound Degradation and Their Utility in Environmental Remediation.

Integrative and conjugative elements (ICEs) are mobile DNA molecules that can be transferred through excision, conjugation, and integration into chromosomes. They contribute to the horizontal transfer of genomic islands across bacterial species. ICEs carrying genes encoding aromatic compound degradation pathways are of interest because of their contribution to environmental remediation. Recent advances in DNA sequencing technology have increased the number of newly discovered ICEs in bacterial genomes and have enabled comparative analysis of their evolution. The two different families of ICEs carry various aromatic compound degradation pathway genes. ICEclc and its related ICEs contain a number of members with diverse catabolic capabilities. In addition, the Tn4371 family, which includes ICEs that carry the chlorinated biphenyl catabolic pathway, has been identified. It is apparent that they underwent evolution through the acquisition, deletion, or exchange of modules to adapt to an environmental niche. ICEs have the property of both stability and mobility in the chromosome. Perspectives on the use of ICEs in environmental remediation are also discussed.

Interspecies Horizontal Transfer and Specific Integration of the Mosquitocidal Toxin-Encoding Plasmid pTAND672-2 from Bacillus thuringiensis subsp. israelensis to Lysinibacillus sphaericus.

pTAND672-2, a 144-kb resident plasmid of Bacillus thuringiensis serovar israelensis strain TAND672, was sequenced and characterized. This extrachromosomal element carries mosquitocidal toxin-, conjugation-, and recombinase-encoding genes, together with a putative arbitrium system, a genetic module recently discovered in temperate phages controlling lysogeny-lysis transition and in mobile genetic elements (MGEs) where its function remains clarified. Using conjugation experiments, pTAND672-2 is shown to be a novel integrative and conjugative element (ICE), which can horizontally transfer from B. thuringiensis serovar israelensis to Lysinibacillus sphaericus, another mosquitocidal bacterium, where it integrates into the chromosome. Its integration and circularization are reversible and involve a single-cross recombination between 33-bp specific sites, attB in the chromosome of L. sphaericus and attP in pTAND672-2. CDS143, coding for the putative tyrosine integrase Int143 distantly related to site-specific tyrosine Xer recombinases and phage integrases, can mediate the integration of pTAND672-2 to attB. The B. thuringiensis mosquito-killing genes carried by pTAND672-2 are efficiently transcribed and expressed in L. sphaericus, displaying a slight increased toxicity in this bacterium against Aedes albopictus larvae. The occurrence of pTAND672-2-like plasmids within the Bacillus cereus group was also explored and indicated that they all share a similar genetic backbone with diverse plasmid sizes, ranging from 58 to 225 kb. Interestingly, among them, the pEFR-4-4 plasmid of Bacillus paranthracis EFR-4 and p5 of B. thuringiensis BT-59 also display conjugative capability; moreover, like pTAND672-2 displays a chimeric structure between the pCH_133-e- and pBtoxis-like plasmids, pBTHD789-3 also appears to be mosaic of two plasmids. IMPORTANCE Horizontal transfer of mobile genetic elements carrying mosquitocidal toxin genes may play a driving role in the diversity of mosquitocidal bacteria. Here, the 144-kb mosquitocidal toxin-encoding plasmid pTAND672-2 is the first verified integrative and conjugative element (ICE) identified in Bacillus thuringiensis serovar israelensis. The key tyrosine integrase Int143, involved in the specific integration, is distantly related to other tyrosine recombinases. The study also reports the occurrence and potential interspecies transmission of pTAND672-2-like plasmids with varied sizes in B. thuringiensis, Bacillus paranthracis, and Bacillus wiedmannii isolates belonging to the Bacillus cereus group. This study is important for further understanding the evolution and ecology of mosquitocidal bacteria, as well as for providing new direction for the genetic engineering of biopesticides in the control of disease-transmitting mosquitoes.

Horizontal Gene Transfer of Antibiotic Resistance Genes in Biofilms

Most bacteria attach to biotic or abiotic surfaces and are embedded in a complex matrix which is known as biofilm. Biofilm formation is especially worrisome in clinical settings as it hinders the treatment of infections with antibiotics due to the facilitated acquisition of antibiotic resistance genes (ARGs). Environmental settings are now considered as pivotal for driving biofilm formation, biofilm-mediated antibiotic resistance development and dissemination. Several studies have demonstrated that environmental biofilms can be hotspots for the dissemination of ARGs. These genes can be encoded on mobile genetic elements (MGEs) such as conjugative and mobilizable plasmids or integrative and conjugative elements (ICEs). ARGs can be rapidly transferred through horizontal gene transfer (HGT) which has been shown to occur more frequently in biofilms than in planktonic cultures. Biofilm models are promising tools to mimic natural biofilms to study the dissemination of ARGs via HGT. This review summarizes the state-of-the-art of biofilm studies and the techniques that visualize the three main HGT mechanisms in biofilms: transformation, transduction, and conjugation.

Characterization of an atypical but widespread type IV secretion system for transfer of the integrative and conjugative element (ICEclc) in Pseudomonas putida.

Conjugation of DNA relies on multicomponent protein complexes bridging two bacterial cytoplasmic compartments. Whereas plasmid conjugation systems have been well documented, those of integrative and conjugative elements (ICEs) have remained poorly studied. We characterize here the conjugation system of the ICEclc element in Pseudomonas putida UWC1 that is a model for a widely distributed family of ICEs. By in frame deletion and complementation, we show the importance on ICE transfer of 22 genes in a 20-kb conserved ICE region. Protein comparisons recognized seven homologs to plasmid type IV secretion system components, another six homologs to frequent accessory proteins, and the rest without detectable counterparts. Stationary phase imaging of P. putida ICEclc with in-frame fluorescent protein fusions to predicted type IV components showed transfer-competent cell subpopulations with multiple fluorescent foci, largely overlapping in dual-labeled subcomponents, which is suggestive for multiple conjugation complexes per cell. Cross-dependencies between subcomponents in ICE-type IV secretion system assembly were revealed by quantitative foci image analysis in a variety of ICEclc mutant backgrounds. In conclusion, the ICEclc family presents an evolutionary distinct type IV conjugative system with transfer competent cells specialized in efficient transfer.

Examination of the horizontal gene transfer dynamics of an integrative and conjugative element encoding multidrug resistance in Histophilus somni.

Integrative and conjugative elements (ICEs) are self-transferable mobile genetic elements that play a significant role in disseminating antimicrobial resistance between bacteria via horizontal gene transfer. A recently identified ICE in a clinical isolate of Histophilus somni (ICEHs02) is 72 914 base pairs in length and harbours seven predicted antimicrobial resistance genes conferring resistance to tetracycline (tetR-tet(H)), florfenicol (floR), sulfonamide (Sul2), aminoglycosides (APH(3″)-Ib, APH(6)-Id, APH(3')-Ia), and copper (mco). This study investigatedICEHs02 host range, assessed effects of antimicrobial stressors on transfer frequency, and examined effects of ICEHs02 acquisition on hosts. Conjugation assays examined transfer frequency ofICEHs02to H. somni andPasteurella multocida strains. Polymerase chain reaction assays confirmed the presence of a circular intermediate, ICE-associated core genes, and cargo genes in recipient strains. Susceptibility testing examinedICEHs02-associated resistance phenotypes in recipient strains. Tetracycline and ciprofloxacin induction significantly increased the transfer rates ofICEHs02 in vitro. The copy numbers of the circular intermediate of ICEHs02 per chromosome exhibited significant increases of ∼37-fold after tetracycline exposure and ∼4-fold after ciprofloxacin treatment. The acquisition of ICEHs02 reduced the relative fitness of H. somni transconjugants (TG) by 28% (w=0.72±0.04) and the relative fitness of P. multocida TG was decreased by 15% (w=0.85±0.01).

Deciphering the genetic network and programmed regulation of antimicrobial resistance in bacterial pathogens.

Antimicrobial resistance (AMR) in bacteria is an important global health problem affecting humans, animals, and the environment. AMR is considered as one of the major components in the "global one health". Misuse/overuse of antibiotics in any one of the segments can impact the integrity of the others. In the presence of antibiotic selective pressure, bacteria tend to develop several defense mechanisms, which include structural changes of the bacterial outer membrane, enzymatic processes, gene upregulation, mutations, adaptive resistance, and biofilm formation. Several components of mobile genetic elements (MGEs) play an important role in the dissemination of AMR. Each one of these components has a specific function that lasts long, irrespective of any antibiotic pressure. Integrative and conjugative elements (ICEs), insertion sequence elements (ISs), and transposons carry the antimicrobial resistance genes (ARGs) on different genetic backbones. Successful transfer of ARGs depends on the class of plasmids, regulons, ISs proximity, and type of recombination systems. Additionally, phage-bacterial networks play a major role in the transmission of ARGs, especially in bacteria from the environment and foods of animal origin. Several other functional attributes of bacteria also get successfully modified to acquire ARGs. These include efflux pumps, toxin-antitoxin systems, regulatory small RNAs, guanosine pentaphosphate signaling, quorum sensing, two-component system, and clustered regularly interspaced short palindromic repeats (CRISPR) systems. The metabolic and virulence state of bacteria is also associated with a range of genetic and phenotypic resistance mechanisms. In spite of the availability of a considerable information on AMR, the network associations between selection pressures and several of the components mentioned above are poorly understood. Understanding how a pathogen resists and regulates the ARGs in response to antimicrobials can help in controlling the development of resistance. Here, we provide an overview of the importance of genetic network and regulation of AMR in bacterial pathogens.

TnSmu1 is a functional integrative and conjugative element in Streptococcus mutans that when expressed causes growth arrest of host bacteria.

Integrative and conjugative elements (ICEs) are major drivers of horizontal gene transfer in bacteria. They mediate their own transfer from host cells (donors) to recipients and allow bacteria to acquire new phenotypes, including pathogenic and metabolic capabilities and drug resistances. Streptococcus mutans, a major causative agent of dental caries, contains a putative ICE, TnSmu1, integrated at the 3' end of a leucyl tRNA gene. We found that TnSmu1 is a functional ICE, containing all the genes necessary for ICE function. It excised from the chromosome and excision was stimulated by DNA damage. We identified the DNA junctions generated by excision of TnSmu1, defined the ends of the element, and detected the extrachromosomal circle. We found that TnSmu1 can transfer from S. mutans donors to recipients when co-cultured on solid medium. The presence of TnSmu1 in recipients inhibited successful acquisition of another copy and this inhibition was mediated, at least in part, by the likely transcriptional repressor encoded by the element. Using microscopy to track individual cells, we found that activation of TnSmu1 caused an arrest of cell growth. Our results demonstrate that TnSmu1 is a functional ICE that affects the biology of its host cells.

Antimicrobial resistance and associated genetic background of Histophilus somni isolated from clinically affected and healthy cattle.

Histophilus somni, a member of the Pasteurellaceae family, causes various diseases, including thrombotic meningoencephalitis and respiratory diseases. Here, 166 isolates recovered from Japanese cattle with various diseases between the late 1970s and the 2010s were subjected to susceptibility testing against 14 antimicrobials (ampicillin, amoxicillin, cefazolin, ceftiofur, kanamycin, streptomycin, nalidixic acid, enrofloxacin, danofloxacin, florfenicol, erythromycin, tylosin, oxytetracycline, and fosfomycin). The proportions of antimicrobial-resistant/intermediate isolates were low in the total isolates, with resistance rates ranging from 0% for ceftiofur and florfenicol to 13.2% for ampicillin. However, relatively high minimum inhibitory concentrations (MICs) and resistance/intermediate rates were observed in the isolates from cattle with respiratory diseases; i.e., 21/53 isolates (39.6%) showed resistance or intermediate to one or more antimicrobials for treatment of respiratory diseases, and the resistance/intermediate rates to oxytetracycline, kanamycin, ampicillin, amoxicillin, nalidixic acid, and danofloxacin were 28.3, 24.5, 24.5, 13.2, 1.9, and 1.9%, respectively. Isolates with high MICs tended to possess antimicrobial resistance genes, which may confer antimicrobial resistance phenotypes. In particular, all isolates with MICs of ampicillin/amoxicillin, kanamycin, and oxytetracycline ≥2 μg/mL, ≥512 μg/mL, and ≥4 μg/mL possessed bla ROB - 1, aphA-1, and tetH/tetR, respectively, whereas isolates whose MICs were lower than the above-mentioned values did not possess these resistance genes. These results suggest that the resistance genes detected in this study are primarily responsible for the reduced susceptibility of H. somni strains to these antimicrobials. As integrative and conjugative element (ICEs)-associated genes were detected only in genetically related isolates possessing antimicrobial resistance genes, ICEs may play an important role in the spread of resistance genes in some genetic groups of H. somni strains.

Activation of the integrative and conjugative element Tn916 causes growth arrest and death of host bacteria.

Integrative and conjugative elements (ICEs) serve as major drivers of bacterial evolution. These elements often confer some benefit to host cells, including antibiotic resistance, metabolic capabilities, or pathogenic determinants. ICEs can also have negative effects on host cells. Here, we investigated the effects of the ICE (conjugative transposon) Tn916 on host cells. Because Tn916 is active in a relatively small subpopulation of host cells, we developed a fluorescent reporter system for monitoring activation of Tn916 in single cells. Using this reporter, we found that cell division was arrested in cells of Bacillus subtilis and Enterococcus faecalis (a natural host for Tn916) that contained an activated (excised) Tn916. Furthermore, most of the cells with the activated Tn916 subsequently died. We also observed these phenotypes on the population level in B. subtilis utilizing a modified version of Tn916 that can be activated in the majority of cells. We identified two genes (orf17 and orf16) in Tn916 that were sufficient to cause growth defects in B. subtilis and identified a single gene, yqaR, that is in a defective phage (skin) in the B. subtilis chromosome that was required for this phenotype. These three genes were only partially responsible for the growth defect caused by Tn916, indicating that Tn916 possesses multiple mechanisms to affect growth and viability of host cells. These results highlight the complex relationships that conjugative elements have with their host cells and the interplay between mobile genetic elements.

Florfenicol and oxazolidone resistance status in livestock farms revealed by short- and long-read metagenomic sequencing.

Antibiotic resistance genes (ARGs) as a novel type of environmental pollutant pose a health risk to humans. Oxazolidinones are one of the most important antibiotics for the treatment of Gram-positive bacterial infections in humans. Although oxazolidinones are not utilized in the livestock industry, florfenicol is commonly used on farms to treat bacterial infections, which may contribute to the spread of the cfr, optrA, and poxtA genes on farms. Using metagenomics sequencing, we looked into the antibiotic resistome context of florfenicol and oxazolidinone in 10 large-scale commercial farms in China. We identified 490 different resistance genes and 1,515 bacterial genera in the fecal samples obtained from 10 farms. Florfenicol-resistant Kurthia, Escherichia, and Proteus were widely present in these samples. The situation of florfenicol and oxazolidone resistance in pig farms is even more severe. The total number of genes and the abundance of drug resistance genes were higher in pigs than in chickens, including optrA and poxtA. All the samples we collected had a high abundance of fexA and floR. Through nanopore metagenomic analysis of the genetic environment, we found that plasmids, integrative and conjugative element (ICE), and transposons (Tn7-like and Tn558) may play an important role in the spread of floR, cfr, and optrA. Our findings suggest that florfenicol and oxazolidinone resistance genes have diverse genetic environments and are at risk of co-transmission with, for example, tetracycline and aminoglycoside resistance genes. The spread of florfenicol- and oxazolidinone-resistant bacteria on animal farms should be continuously monitored.

ICEscreen: a tool to detect Firmicute ICEs and IMEs, isolated or enclosed in composite structures.

Mobile Genetic Elements (MGEs) are integrated in bacterial genomes and key elements that drive prokaryote genome evolution. Among them are Integrative and Conjugative Elements (ICEs) and Integrative Mobilizable Elements (IMEs) which are important for bacterial fitness since they frequently carry genes participating in important bacterial adaptation phenotypes such as antibiotic resistance, virulence or specialized metabolic pathways. Although ICEs and IMEs are widespread, they are as yet almost never annotated in public bacterial genomes. To address the need of dedicated strategies for the annotation of these elements, we developed ICEscreen, a tool that introduces two new features to detect ICEs and IMEs in Firmicute genomes. First, ICEscreen uses an efficient strategy to detect Signature Proteins of ICEs and IMEs based on a database dedicated to Firmicutes and composed of manually curated proteins and Hidden Markov Models (HMM) profiles. Second, ICEscreen includes a new original algorithm that detects composite structures of ICEs and IMEs that are frequent in genomes of Firmicutes but are currently not resolved by any other tool. We benchmarked ICEscreen on experimentally supported elements and on a public dataset of 246 manually annotated elements including the genomes of 40 Firmicutes and demonstrate its efficiency to detect ICEs and IMEs.

Activation of TnSmu1, an integrative and conjugative element, by an ImmR-like transcriptional regulator in Streptococcus mutans.

Integrative and conjugative elements (ICEs) are chromosomally encoded mobile genetic elements that can transfer DNA between bacterial strains. Recently, as part of efforts to determine hypothetical gene functions, we have discovered an important regulatory module encoded on an ICE known as TnSmu1 on the Streptococcus mutans chromosome. The regulatory module consists of a cI-like repressor with a helix-turn-helix DNA binding domain immR Smu (immunity repressor) and a metalloprotease immA Smu (anti-repressor). It is not possible to create an in-frame deletion mutant of immR Smu and repression of immR Smu with CRISPRi (CRISPR interference) causes substantial cell defects. We used a bypass of essentiality (BoE) screen to discover genes that allow deletion of the regulatory module. This revealed that conjugation genes, located within TnSmu1, can restore the viability of an immR Smu mutant. Deletion of immR Smu also leads to production of a circular intermediate form of TnSmu1, which is also inducible by the genotoxic agent mitomycin C. To gain further insights into potential regulation of TnSmu1 by ImmRSmu and broader effects on S. mutans UA159 physiology, we used CRISPRi and RNA-seq. Strongly induced genes included all the TnSmu1 mobile element, genes involved in amino acid metabolism, transport systems and a type I-C CRISPR-Cas system. Lastly, bioinformatic analysis shows that the TnSmu1 mobile element and its associated genes are well distributed across S. mutans isolates. Taken together, our results show that activation of TnSmu1 is controlled by the immRA Smu module, and that activation is deleterious to S. mutans, highlighting the complex interplay between mobile elements and their host.

Resistance to Fluoroquinolones in Pseudomonas aeruginosa from Human, Animal, Food and Environmental Origin: The Role of CrpP and Mobilizable ICEs.

Fluoroquinolone resistance and the associated genetic mechanisms were assessed by antimicrobial susceptibility and whole genome sequencing in 56 Pseudomonas aeruginosa strains from human, animal, food and environmental origins. P. aeruginosa PAO1, PA7 and PA14 reference strains were also included in the study. Twenty-two strains (37%) were resistant to, at least, one fluoroquinolone agent. Correlation between the number of changes in GyrA and ParC proteins and the level of fluoroquinolone resistance was observed. Mutations or absence of genes, such as mexZ, mvaT and nalD encoding efflux pumps regulators, were also found in resistant strains. The crpP gene was detected in 43 strains (72.9%; 17 of them non-clinical strains), and coded seven different CrpP variants, including a novel one (CrpP-7). The crpP gene was located in 23 different chromosomal mobile integrative and conjugative elements (ICEs), inserted in two tRNAs integration sites. A great variety of structures was detected in the crpP-ICEs elements, e.g., the fimbriae related cup clusters, the mercury resistance mer operon, the pyocin S5 or S8 bacteriocin encoding genes, and mobilization genes. The location of crpP-like genes in mobilizable ICEs and linked to heavy metal resistance and virulence factors is of significant concern in P. aeruginosa. This work provides a genetic explanation of the fluoroquinolone resistance and crpP-associated pathogenesis of P. aeruginosa from a One-Health approach.

Prevalence and mobility of integrative and conjugative elements within a Streptomyces natural population.

Horizontal Gene Transfer (HGT) is a powerful force generating genomic diversity in bacterial populations. HGT in Streptomyces is in large part driven by conjugation thanks to plasmids, Integrative and Conjugative elements (ICEs) and Actinomycete ICEs (AICEs). To investigate the impact of ICE and AICE conjugation on Streptomyces genome evolution, we used in silico and experimental approaches on a set of 11 very closely related strains isolated from a millimeter scale rhizosphere population. Through bioinformatic searches of canonical conjugation proteins, we showed that AICEs are the most frequent integrative conjugative elements, with the central chromosome region being a hotspot for integrative element insertion. Strains exhibited great variation in AICE composition consistent with frequent HGT and/or gene loss. We found that single insertion sites can be home to different elements in different strains (accretion) and conversely, elements belonging to the same family can be found at different insertion sites. A wide variety of cargo genes was present in the AICEs with the potential to mediate strain-specific adaptation (e.g., DNA metabolism and resistance genes to antibiotic and phages). However, a large proportion of AICE cargo genes showed hallmarks of pseudogenization, consistent with deleterious effects of cargo genes on fitness. Pock assays enabled the direct visualization of conjugal AICE transfer and demonstrated the transfer of AICEs between some, but not all, of the isolates. Multiple AICEs were shown to be able to transfer during a single mating event. Although we did not obtain experimental evidence for transfer of the sole chromosomal ICE in this population, genotoxic stress mediated its excision from the chromosome, suggesting its functionality. Our results indicate that AICE-mediated HGT in Streptomyces populations is highly dynamic, with likely impact on strain fitness and the ability to adapt to environmental change.

Oxazolidinones: mechanisms of resistance and mobile genetic elements involved.

The oxazolidinones (linezolid and tedizolid) are last-resort antimicrobial agents used for the treatment of severe infections in humans caused by MDR Gram-positive bacteria. They bind to the peptidyl transferase centre of the bacterial ribosome inhibiting protein synthesis. Even if the majority of Gram-positive bacteria remain susceptible to oxazolidinones, resistant isolates have been reported worldwide. Apart from mutations, affecting mostly the 23S rDNA genes and selected ribosomal proteins, acquisition of resistance genes (cfr and cfr-like, optrA and poxtA), often associated with mobile genetic elements [such as non-conjugative and conjugative plasmids, transposons, integrative and conjugative elements (ICEs), prophages and translocatable units], plays a critical role in oxazolidinone resistance. In this review, we briefly summarize the current knowledge on oxazolidinone resistance mechanisms and provide an overview on the diversity of the mobile genetic elements carrying oxazolidinone resistance genes in Gram-positive and Gram-negative bacteria.

Whole genome sequencing of the multidrug-resistant Chryseobacterium indologenes isolated from a patient in Brazil.

Chryseobacterium indologenes is a non-glucose-fermenting Gram-negative bacillus. This emerging multidrug resistant opportunistic nosocomial pathogen can cause severe infections in neonates and immunocompromised patients. This study aimed to present the first detailed draft genome sequence of a multidrug-resistant C. indologenes strain isolated from the cerebrospinal fluid of an infant hospitalized at the Neonatal Intensive Care Unit of Brazilian Tertiary Hospital. We first analyzed the susceptibility of C. indologenes strain to different antibiotics using the VITEK 2 system. The strain demonstrated an outstanding resistance to all the antibiotic classes tested, including β-lactams, aminoglycosides, glycylcycline, and polymyxin. Next, C. indologenes was whole-genome-sequenced, annotated using Prokka and Rapid Annotation using Subsystems Technology (RAST), and screened for orthologous groups (EggNOG), gene ontology (GO), resistance genes, virulence genes, and mobile genetic elements using different software tools. The draft genome contained one circular chromosome of 4,836,765 bp with 37.32% GC content. The genomic features of the chromosome present numerous genes related to cellular processes that are essential to bacteria. The MDR C. indologenes revealed the presence of genes that corresponded to the resistance phenotypes, including genes to β-lactamases (blaIND–13, blaCIA–3, blaTEM–116, blaOXA–209, blaVEB–15), quinolone (mcbG), tigecycline (tet(X6)), and genes encoding efflux pumps which confer resistance to aminoglycosides (RanA/RanB), and colistin (HlyD/TolC). Amino acid substitutions related to quinolone resistance were observed in GyrA (S83Y) and GyrB (L425I and K473R). A mutation that may play a role in the development of colistin resistance was detected in lpxA (G68D). Chryseobacterium indologenes isolate harbored 19 virulence factors, most of which were involved in infection pathways. We identified 13 Genomic Islands (GIs) and some elements associated with one integrative and conjugative element (ICEs). Other elements linked to mobile genetic elements (MGEs), such as insertion sequence (ISEIsp1), transposon (Tn5393), and integron (In31), were also present in the C. indologenes genome. Although plasmids were not detected, a ColRNAI replicon type and the most resistance genes detected in singletons were identified in unaligned scaffolds. We provided a wide range of information toward the understanding of the genomic diversity of C. indologenes, which can contribute to controlling the evolution and dissemination of this pathogen in healthcare settings.

Characterization and Fitness Cost of Tn7100, a Novel Integrative and Conjugative Element Conferring Multidrug Resistance in Haemophilus influenzae.

A multidrug-resistant (MDR) strain of Haemophilus influenzae, Hi-228, with phenotypic resistance toward ampicillin, cefotaxime, chloramphenicol, gentamicin, and azithromycin, was isolated in Oslo, Norway. The strain was part of a clonal outbreak (2016–2017) comprising five ST143 strains with identical resistotypes. Hi-228 carries a novel integrative and conjugative element (ICE), Tn7100, contributing to this remarkable and previously unreported MDR profile. Tn7100 contains the following resistance genes: blaTEM−1B, catA2, aac(6′)-Im, aph(2″)-Ib, mef (E), and mel. The latter four are previously unreported or rarely reported in H. influenzae. In this study, we investigated the genetic environment, mechanisms of transfer, impact on phenotypic susceptibility, and fitness cost of this ICE. We found that Tn7100 has an overall structure similar to the previously described ICE Tn6686, with blaTEM−1B and catA2 carried by Tn3 and Tn10, respectively. The major difference between Tn7100 and Tn6686 is that Tn7100 lacks tet(B) but carries the resistance gene pairs aac(6′)-Im and aph(2″)-Ib and mef (E) and mel. The gene pairs are located on the novel transposable elements Tn7470 and Tn7471, which have high sequence identities to a plasmid in Enterobacterales and an ICE in streptococcal species, respectively. Tn7100 does circularize and is transferable, however, at a low frequency. Head-to-head competition experiments showed that uptake of Tn7100 reduces bacterial fitness. Our study shows that MDR strains are capable of clonal spread and that the H. influenzae supragenome comprises an increasingly wide range of transferable resistance genes, with evidence of transfer from unrelated genera. The findings offer a glimpse into the genome dynamics of H. influenzae, highlighting the importance of rational antibiotic usage to contain antimicrobial resistance and the emergence of MDR strains in this important pathogen.

Whole genome sequence of Proteus mirabilis ChSC1905 strain harbouring a new SXT/R391-family ICE

The aim of this study was to characterise the whole genome sequence of a multidrug-resistant (MDR) Proteus mirabilis strain ChSC1905 isolated from a swine farm in China. The genome was sequenced by Illumina NovaSeq and Oxford Nanopore platforms, and it was assembled via Canu v.1.5. The acquired antimicrobial resistance genes (ARGs) were identified by ResFinder. A conjugation experiment was carried out to determine the mobilisation of integrative and conjugative element. Strain ChSC1905 exhibited a MDR phenotype. The genome of strain ChSC1905 was 4 038 038 bp in length with a GC content of 39.1%, which contained 3645 coding sequences and 110 RNA genes. A total of 23 acquired ARGs were identified, among which 21 ARGs including the clinically important resistance genes blaCTX-M-65, cfr, fosA3, and aac(6')-Ib-cr were located on a SXT/R391 integrative and conjugative element (ICE). BLAST analysis showed that this new SXT/R391-family ICE (ICEPmiChnChSC1905 of 143 689 bp) was involved in sequence inversion mediated by ISVsa3 and genetic rearrangement mediated by IS26, and it could be transferred to E. coli EC600. In this study, we report the genome sequence of MDR P. mirabilis strain ChSC1905 that harboured a novel SXT/R391-family ICE (ICEPmiChnChSC1905) involved in genetic rearrangement in China, which promotes the diversity of ICE and should receive more attention.

Meet the President: Russell B. Melchert, PhD, RPh

<h3>Abstract</h3> Integrative and conjugative elements (ICEs) are chromosomally encoded mobile genetic elements that can transfer DNA between bacterial strains. Recently, as part of efforts to determine hypothetical gene functions, we have discovered an important regulatory module encoded on an ICE known as TnSmu1 on the <i>Streptococcus mutans</i> chromosome. The regulatory module consists of a cI-like repressor with a helix-turn-helix DNA binding domain <i>immR</i>_Smu (immunity repressor) and a metalloprotease <i>immA</i>_Smu (anti-repressor). It is not possible to create an in-frame deletion mutant of <i>immR</i>_Smu and repression of <i>immR</i>_Smu with CRISPRi causes substantial cell defects. We used a bypass of essentiality (BoE) screen to discover genes that allow deletion of the regulatory module. This revealed that conjugation genes, located within TnSmu1, can restore the viability of an <i>immR</i>_Smu mutant. Deletion of <i>immR</i>_Smu also leads to production of a circular intermediate form of TnSmu1, that is also inducible by the genotoxic agent mitomycin C. To gain further insights into potential regulation of TnSmu1 by ImmR_Smu and broader effects on <i>S. mutans</i> UA159 physiology we used CRISPRi and RNA-seq. Strongly induced genes included all the TnSmu1 mobile element, genes involved in amino acid metabolism, transport systems, and a Type I-C CRISPR-Cas system. Lastly, bioinformatic analysis shows that the TnSmu1 mobile element and its associated genes are well distributed across <i>S. mutans</i> isolates. Taken together, our results show that activation of TnSmu1 is controlled by the <i>immRA</i>_Smu module, and that activation is deleterious to <i>S. mutans</i>, highlighting the complex interplay between mobile elements and their host.

A bistable prokaryotic differentiation system underlying development of conjugative transfer competence.

The mechanisms and impact of horizontal gene transfer processes to distribute gene functions with potential adaptive benefit among prokaryotes have been well documented. In contrast, little is known about the life-style of mobile elements mediating horizontal gene transfer, whereas this is the ultimate determinant for their transfer fitness. Here, we investigate the life-style of an integrative and conjugative element (ICE) within the genus Pseudomonas that is a model for a widespread family transmitting genes for xenobiotic compound metabolism and antibiotic resistances. Previous work showed bimodal ICE activation, but by using single cell time-lapse microscopy coupled to combinations of chromosomally integrated single copy ICE promoter-driven fluorescence reporters, RNA sequencing and mutant analysis, we now describe the complete regulon leading to the arisal of differentiated dedicated transfer competent cells. The regulon encompasses at least three regulatory nodes and five (possibly six) further conserved gene clusters on the ICE that all become expressed under stationary phase conditions. Time-lapse microscopy indicated expression of two regulatory nodes (i.e., bisR and alpA-bisDC) to precede that of the other clusters. Notably, expression of all clusters except of bisR was confined to the same cell subpopulation, and was dependent on the same key ICE regulatory factors. The ICE thus only transfers from a small fraction of cells in a population, with an estimated proportion of between 1.7-4%, which express various components of a dedicated transfer competence program imposed by the ICE, and form the centerpiece of ICE conjugation. The components mediating transfer competence are widely conserved, underscoring their selected fitness for efficient transfer of this class of mobile elements.