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985300.9572The master activator of IncA/C conjugative plasmids stimulates genomic islands and multidrug resistance dissemination. Dissemination of antibiotic resistance genes occurs mostly by conjugation, which mediates DNA transfer between cells in direct contact. Conjugative plasmids of the IncA/C incompatibility group have become a substantial threat due to their broad host-range, the extended spectrum of antimicrobial resistance they confer, their prevalence in enteric bacteria and their very efficient spread by conjugation. However, their biology remains largely unexplored. Using the IncA/C conjugative plasmid pVCR94ΔX as a prototype, we have investigated the regulatory circuitry that governs IncA/C plasmids dissemination and found that the transcriptional activator complex AcaCD is essential for the expression of plasmid transfer genes. Using chromatin immunoprecipitation coupled with exonuclease digestion (ChIP-exo) and RNA sequencing (RNA-seq) approaches, we have identified the sequences recognized by AcaCD and characterized the AcaCD regulon. Data mining using the DNA motif recognized by AcaCD revealed potential AcaCD-binding sites upstream of genes involved in the intracellular mobility functions (recombination directionality factor and mobilization genes) in two widespread classes of genomic islands (GIs) phylogenetically unrelated to IncA/C plasmids. The first class, SGI1, confers and propagates multidrug resistance in Salmonella enterica and Proteus mirabilis, whereas MGIVmi1 in Vibrio mimicus belongs to a previously uncharacterized class of GIs. We have demonstrated that through expression of AcaCD, IncA/C plasmids specifically trigger the excision and mobilization of the GIs at high frequencies. This study provides new evidence of the considerable impact of IncA/C plasmids on bacterial genome plasticity through their own mobility and the mobilization of genomic islands.201425340549
997910.9565Type II and IV toxin-antitoxin systems coordinately stabilize the integrative and conjugative element of the ICESa2603 family conferring multiple drug resistance in Streptococcus suis. Integrative and conjugative elements (ICEs) play a vital role in bacterial evolution by carrying essential genes that confer adaptive functions to the host. Despite their importance, the mechanism underlying the stable inheritance of ICEs, which is necessary for the acquisition of new traits in bacteria, remains poorly understood. Here, we identified SezAT, a type II toxin-antitoxin (TA) system, and AbiE, a type IV TA system encoded within the ICESsuHN105, coordinately promote ICE stabilization and mediate multidrug resistance in Streptococcus suis. Deletion of SezAT or AbiE did not affect the strain's antibiotic susceptibility, but their duple deletion increased susceptibility, mainly mediated by the antitoxins SezA and AbiEi. Further studies have revealed that SezA and AbiEi affect the genetic stability of ICESsuHN105 by moderating the excision and extrachromosomal copy number, consequently affecting the antibiotic resistance conferred by ICE. The DNA-binding proteins AbiEi and SezA, which bind palindromic sequences in the promoter, coordinately modulate ICE excision and extracellular copy number by binding to sequences in the origin-of-transfer (oriT) and the attL sites, respectively. Furthermore, AbiEi negatively regulates the transcription of SezAT by binding directly to its promoter, optimizing the coordinate network of SezAT and AbiE in maintaining ICESsuHN105 stability. Importantly, SezAT and AbiE are widespread and conserved in ICEs harbouring diverse drug-resistance genes, and their coordinated effects in promoting ICE stability and mediating drug resistance may be broadly applicable to other ICEs. Altogether, our study uncovers the TA system's role in maintaining the genetic stability of ICE and offers potential targets for overcoming the dissemination and evolution of drug resistance.202438640137
987420.9565Genomic islands related to Salmonella genomic island 1; integrative mobilisable elements in trmE mobilised in trans by A/C plasmids. Salmonella genomic island 1 (SGI1), an integrative mobilisable element (IME), was first reported 20 years ago, in the multidrug resistant Salmonella Typhimurium DT104 clone. Since this first report, many variants and relatives have been found in Salmonella enterica and Proteus mirabilis. Thanks to whole genome sequencing, more and more complete sequences of SGI1-related elements (SGI1-REs) have been reported in these last few years among Gammaproteobacteria. Here, the genetic organisation and main features common to SGI1-REs are summarised to help to classify them. Their integrases belong to the tyrosine-recombinase family and target the 3'-end of the trmE gene. They share the same genetic organisation (integrase and excisionase genes, replicase module, SgaCD-like transcriptional activator genes, traN, traG, mpsB/mpsA genes) and they harbour AcaCD binding sites promoting their excision, replication and mobilisation in presence of A/C plasmid. SGI1-REs are mosaic structures suggesting that recombination events occurred between them. Most of them harbour a multiple antibiotic resistance (MAR) region and the plasticity of their MAR region show that SGI1-REs play a key role in antibiotic resistance and might help multiple antibiotic resistant bacteria to adapt to their environment. This might explain the emergence of clones with SGI1-REs.202133582118
987530.9564Antibiotic Resistance in Vibrio cholerae: Mechanistic Insights from IncC Plasmid-Mediated Dissemination of a Novel Family of Genomic Islands Inserted at trmE. Cholera remains a formidable disease, and reports of multidrug-resistant strains of the causative agent Vibrio cholerae have become common during the last 3 decades. The pervasiveness of resistance determinants has largely been ascribed to mobile genetic elements, including SXT/R391 integrative conjugative elements, IncC plasmids, and genomic islands (GIs). Conjugative transfer of IncC plasmids is activated by the master activator AcaCD whose regulatory network extends to chromosomally integrated GIs. MGIVchHai6 is a multidrug resistance GI integrated at the 3' end of trmE (mnmE or thdF) in chromosome 1 of non-O1/non-O139 V. cholerae clinical isolates from the 2010 Haitian cholera outbreak. In the presence of an IncC plasmid expressing AcaCD, MGIVchHai6 excises from the chromosome and transfers at high frequency. Herein, the mechanism of mobilization of MGIVchHai6 GIs by IncC plasmids was dissected. Our results show that AcaCD drives expression of GI-borne genes, including xis and mobI(M) , involved in excision and mobilization. A 49-bp fragment upstream of mobI(M) was found to serve as the minimal origin of transfer (oriT) of MGIVchHai6. The direction of transfer initiated at oriT was determined using IncC plasmid-driven mobilization of chromosomal markers via MGIVchHai6. In addition, IncC plasmid-encoded factors, including the relaxase TraI, were found to be required for GI transfer. Finally, in silico exploration of Gammaproteobacteria genomes identified 47 novel related and potentially AcaCD-responsive GIs in 13 different genera. Despite sharing conserved features, these GIs integrate at trmE, yicC, or dusA and carry a diverse cargo of genes involved in phage resistance.IMPORTANCE The increasing association of the etiological agent of cholera, Vibrio cholerae serogroup O1 and O139, with multiple antibiotic resistance threatens to deprive health practitioners of this effective tool. Drug resistance in cholera results mainly from acquisition of mobile genetic elements. Genomic islands conferring multidrug resistance and mobilizable by IncC conjugative plasmids were reported to circulate in non-O1/non-O139 V. cholerae clinical strains isolated from the 2010 Haitian cholera outbreak. As these genomic islands can be transmitted to pandemic V. cholerae serogroups, their mechanism of transmission needed to be investigated. Our research revealed plasmid- and genomic island-encoded factors required for the resistance island excision, mobilization, and integration, as well as regulation of these functions. The discovery of related genomic islands carrying diverse phage resistance genes but lacking antibiotic resistance-conferring genes in a wide range of marine dwelling bacteria suggests that these elements are ancient and recently acquired drug resistance genes.202032848007
997740.9563IncC conjugative plasmids and SXT/R391 elements repair double-strand breaks caused by CRISPR-Cas during conjugation. Bacteria have evolved defence mechanisms against bacteriophages. Restriction-modification systems provide innate immunity by degrading invading DNAs that lack proper methylation. CRISPR-Cas systems provide adaptive immunity by sampling the genome of past invaders and cutting the DNA of closely related DNA molecules. These barriers also restrict horizontal gene transfer mediated by conjugative plasmids. IncC conjugative plasmids are important contributors to the global dissemination of multidrug resistance among pathogenic bacteria infecting animals and humans. Here, we show that IncC conjugative plasmids are highly resilient to host defence systems during entry into a new host by conjugation. Using a TnSeq strategy, we uncover a conserved operon containing five genes (vcrx089-vcrx093) that confer a novel host defence evasion (hde) phenotype. We show that vcrx089-vcrx090 promote resistance against type I restriction-modification, whereas vcrx091-vcxr093 promote CRISPR-Cas evasion by repairing double-strand DNA breaks via recombination between short sequence repeats. vcrx091, vcrx092 and vcrx093 encode a single-strand binding protein, and a single-strand annealing recombinase and double-strand exonuclease related to Redβ and λExo of bacteriophage λ, respectively. Homologous genes of the integrative and conjugative element R391 also provide CRISPR-Cas evasion. Hence, the conserved hde operon considerably broadens the host range of large families of mobile elements spreading multidrug resistance.202032556263
301950.9557Identification and Characterization of New Resistance-Conferring SGI1s (Salmonella Genomic Island 1) in Proteus mirabilis. Salmonella genomic island 1 (SGI1) is a resistance-conferring chromosomal genomic island that contains an antibiotic resistance gene cluster. The international spread of SGI1-containing strains drew attention to the role of genomic islands in the dissemination of antibiotic resistance genes in Salmonella and other Gram-negative bacteria. In this study, five SGI1 variants conferring multidrug and heavy metal resistance were identified and characterized in Proteus mirabilis strains: SGI1-PmCAU, SGI1-PmABB, SGI1-PmJN16, SGI1-PmJN40, and SGI1-PmJN48. The genetic structures of SGI1-PmCAU and SGI1-PmABB were identical to previously reported SGI1s, while structural analysis showed that SGI1-PmJN16, SGI1-PmJN40, and SGI1-PmJN48 are new SGI1 variants. SGI1-PmJN16 is derived from SGI1-Z with the MDR region containing a new gene cassette array dfrA12-orfF-aadA2-qacEΔ1-sul1-chrA-orf1. SGI1-PmJN40 has an unprecedented structure that contains two right direct repeat sequences separated by a transcriptional regulator-rich DNA fragment, and is predicted to form two different extrachromosomal mobilizable DNA circles for dissemination. SGI1-PmJN48 lacks a common ORF S044, and its right junction region exhibits a unique genetic organization due to the reverse integration of a P. mirabilis chromosomal gene cluster and the insertion of part of a P. mirabilis plasmid, making it the largest known SGI1 to date (189.1 kb). Further mobility functional analysis suggested that these SGIs can be excised from the chromosome for transfer between bacteria, which promotes the horizontal transfer of antibiotic and heavy metal resistance genes. The identification and characterization of the new SGI1 variants in this work suggested the diversity of SGI1 structures and their significant roles in the evolution of bacteria.201830619228
985460.9545Unraveling the regulatory network of IncA/C plasmid mobilization: When genomic islands hijack conjugative elements. Conjugative plasmids of the A/C incompatibility group (IncA/C) have become substantial players in the dissemination of multidrug resistance. These large conjugative plasmids are characterized by their broad host-range, extended spectrum of antimicrobials resistance, and prevalence in enteric bacteria recovered from both environmental and clinical settings. Until recently, relatively little was known about the basic biology of IncA/C plasmids, mostly because of the hindrance of multidrug resistance for molecular biology experiments. To circumvent this issue, we previously developed pVCR94ΔX, a convenient prototype that codes for a reduced set of antibiotic resistances. Using pVCR94ΔX, we then characterized the regulatory pathway governing IncA/C plasmid dissemination. We found that the expression of roughly 2 thirds of the genes encoded by this plasmid, including large operons involved in the conjugation process, depends on an FlhCD-like master activator called AcaCD. Beyond the mobility of IncA/C plasmids, AcaCD was also shown to play a key role in the mobilization of different classes of genomic islands (GIs) identified in various pathogenic bacteria. By doing so, IncA/C plasmids can have a considerable impact on bacterial genomes plasticity and evolution.201526442183
987270.9545pCTX-M3-Structure, Function, and Evolution of a Multi-Resistance Conjugative Plasmid of a Broad Recipient Range. pCTX-M3 is the archetypic member of the IncM incompatibility group of conjugative plasmids (recently referred to as IncM2). It is responsible for the worldwide dissemination of numerous antibiotic resistance genes, including those coding for extended-spectrum β-lactamases and conferring resistance to aminoglycosides. The IncM plasmids acquired during evolution diverse mobile genetic elements found in one or two multiple resistance regions, MRR(s), grouping antibiotic resistance genes as well as mobile genetic elements or their remnants. The IncM plasmids can be found in bacteria inhabiting various environments. The information on the structure and biology of pCTX-M3 is integrated in this review. It focuses on the functional modules of pCTX-M3 responsible for its replication, stable maintenance, and conjugative transfer, indicating that the host range of the pCTX-M3 replicon is limited to representatives of the family Enterobacteriaceae (Enterobacterales ord. nov.), while the range of recipients of its conjugation system is wide, comprising Alpha-, Beta-, and Gammaproteobacteria, and also Firmicutes.202133925677
985680.9544The extended regulatory networks of SXT/R391 integrative and conjugative elements and IncA/C conjugative plasmids. Nowadays, healthcare systems are challenged by a major worldwide drug resistance crisis caused by the massive and rapid dissemination of antibiotic resistance genes and associated emergence of multidrug resistant pathogenic bacteria, in both clinical and environmental settings. Conjugation is the main driving force of gene transfer among microorganisms. This mechanism of horizontal gene transfer mediates the translocation of large DNA fragments between two bacterial cells in direct contact. Integrative and conjugative elements (ICEs) of the SXT/R391 family (SRIs) and IncA/C conjugative plasmids (ACPs) are responsible for the dissemination of a broad spectrum of antibiotic resistance genes among diverse species of Enterobacteriaceae and Vibrionaceae. The biology, diversity, prevalence and distribution of these two families of conjugative elements have been the subject of extensive studies for the past 15 years. Recently, the transcriptional regulators that govern their dissemination through the expression of ICE- or plasmid-encoded transfer genes have been described. Unrelated repressors control the activation of conjugation by preventing the expression of two related master activator complexes in both types of elements, i.e., SetCD in SXT/R391 ICEs and AcaCD in IncA/C plasmids. Finally, in addition to activating ICE- or plasmid-borne genes, these master activators have been shown to specifically activate phylogenetically unrelated mobilizable genomic islands (MGIs) that also disseminate antibiotic resistance genes and other adaptive traits among a plethora of pathogens such as Vibrio cholerae and Salmonella enterica.201526347724
998790.9534Four genes essential for recombination define GInts, a new type of mobile genomic island widespread in bacteria. Integrases are a family of tyrosine recombinases that are highly abundant in bacterial genomes, actively disseminating adaptive characters such as pathogenicity determinants and antibiotics resistance. Using comparative genomics and functional assays, we identified a novel type of mobile genetic element, the GInt, in many diverse bacterial groups but not in archaea. Integrated as genomic islands, GInts show a tripartite structure consisting of the ginABCD operon, a cargo DNA region from 2.5 to at least 70 kb, and a short AT-rich 3' end. The gin operon is characteristic of GInts and codes for three putative integrases and a small putative helix-loop-helix protein, all of which are essential for integration and excision of the element. Genes in the cargo DNA are acquired mostly from phylogenetically related bacteria and often code for traits that might increase fitness, such as resistance to antimicrobials or virulence. GInts also tend to capture clusters of genes involved in complex processes, such as the biosynthesis of phaseolotoxin by Pseudomonas syringae. GInts integrate site-specifically, generating two flanking direct imperfect repeats, and excise forming circular molecules. The excision process generates sequence variants at the element attachment site, which can increase frequency of integration and drive target specificity.201728393892
9978100.9534Pathogen-encoded Rum DNA polymerase drives rapid bacterial drug resistance. The acquisition of multidrug resistance by pathogenic bacteria is a potentially incipient pandemic. Horizontal transfer of DNA from mobile integrative conjugative elements (ICEs) provides an important way to introduce genes that confer antibiotic (Ab)-resistance in recipient cells. Sizable numbers of SXT/R391 ICEs encode a hypermutagenic Rum DNA polymerase (Rum pol), which has significant homology with Escherichia coli pol V. Here, we show that even under tight transcriptional and post-transcriptional regulation imposed by host bacteria and the R391 ICE itself, Rum pol rapidly accelerates development of multidrug resistance (CIPR, RifR, AmpR) in E. coli in response to SOS-inducing Ab and non-Ab external stressors bleomycin (BLM), ciprofloxacin (CIP) and UV radiation. The impact of Rum pol on the rate of acquisition of drug resistance appears to surpass potential contributions from other cellular processes. We have shown that RecA protein plays a central role in controlling the ability of Rum pol to accelerate antibiotic resistance. A single amino acid substitution in RecA, M197D, acts as a 'Master Regulator' that effectively eliminates the Rum pol-induced Ab resistance. We suggest that Rum pol should be considered as one of the major factors driving development of de novo Ab resistance in pathogens carrying SXT/R391 ICEs.202439413207
58110.9533A Conserved Basal Transcription Factor Is Required for the Function of Diverse TAL Effectors in Multiple Plant Hosts. Many Xanthomonas bacteria use transcription activator-like effector (TALE) proteins to activate plant disease susceptibility (S) genes, and this activation contributes to disease. We recently reported that rice basal transcription factor IIA gamma subunit, OsTFIIAγ5, is hijacked by TALE-carrying Xanthomonas oryzae infecting the plants. However, whether TFIIAγs are also involved in TALE-carrying Xanthomonas-caused diseases in other plants is unknown. Here, molecular and genetic approaches were used to investigate the role of TFIIAγs in other plants. We found that TFIIAγs are also used by TALE-carrying Xanthomonas to cause disease in other plants. The TALEs of Xanthomonas citri pv. citri (Xcc) causing canker in citrus and Xanthomonas campestris pv. vesicatoria (Xcv) causing bacterial spot in pepper and tomato interacted with corresponding host TFIIAγs as in rice. Transcriptionally suppressing TFIIAγ led to resistance to Xcc in citrus and Xcv in pepper and tomato. The 39th residue of OsTFIIAγ5 and citrus CsTFIIAγ is vital for TALE-dependent induction of plant S genes. As mutated OsTFIIAγ5(V 39E), CsTFIIAγ(V 39E), pepper CaTFIIAγ(V 39E), and tomato SlTFIIAγ(V 39E) also did not interact with TALEs to prevent disease. These results suggest that TALE-carrying bacteria share a common mechanism for infecting plants. Using TFIIAγ(V 39E)-type mutation could be a general strategy for improving resistance to TALE-carrying pathogens in crops.201729163628
9846120.9531Integrative Conjugative Elements (ICEs) of the SXT/R391 family drive adaptation and evolution in γ-Proteobacteria. Integrative Conjugative Elements (ICEs) are mosaics containing functional modules allowing maintenance by site-specific integration and excision into and from the host genome and conjugative transfer to a specific host range. Many ICEs encode a range of adaptive functions that aid bacterial survival and evolution in a range of niches. ICEs from the SXT/R391 family are found in γ-Proteobacteria. Over 100 members have undergone epidemiological and molecular characterization allowing insight into their diversity and function. Comparative analysis of SXT/R391 elements from a wide geographic distribution has revealed conservation of key functions, and the accumulation and evolution of adaptive genes. This evolution is associated with gene acquisition in conserved hotspots and variable regions within the SXT/R391 ICEs catalysed via element-encoded recombinases. The elements can carry IS elements and transposons, and a mutagenic DNA polymerase, PolV, which are associated with their evolution. SXT/R391 ICEs isolated from different niches appear to have retained adaptive functions related to that specific niche; phage resistance determinants in ICEs carried by wastewater bacteria, antibiotic resistance determinants in clinical isolates and metal resistance determinants in bacteria recovered from polluted environments/ocean sediments. Many genes found in the element hotspots are undetermined and have few homologs in the nucleotide databases.202436634159
9855130.9530Conjugative IncC Plasmid Entry Triggers the SOS Response and Promotes Effective Transfer of the Integrative Antibiotic Resistance Element SGI1. The broad-host-range IncC plasmid family and the integrative mobilizable Salmonella genomic island 1 (SGI1) and its derivatives enable the spread of medically important antibiotic resistance genes among Gram-negative pathogens. Although several aspects of the complex functional interactions between IncC plasmids and SGI1 have been recently deciphered regarding their conjugative transfer and incompatibility, the biological signal resulting in the hijacking of the conjugative plasmid by the integrative mobilizable element remains unknown. Here, we demonstrate that the conjugative entry of IncC/IncA plasmids is detected at an early stage by SGI1 through the transient activation of the SOS response, which induces the expression of the SGI1 master activators SgaDC, shown to play a crucial role in the complex biology between SGI1 and IncC plasmids. Besides, we developed an original tripartite conjugation approach to directly monitor SGI1 mobilization in a time-dependent manner following conjugative entry of IncC plasmids. Finally, we propose an updated biological model of the conjugative mobilization of the chromosomal resistance element SGI1 by IncC plasmids. IMPORTANCE Antimicrobial resistance has become a major public health issue, particularly with the increase of multidrug resistance (MDR) in both animal and human pathogenic bacteria and with the emergence of resistance to medically important antibiotics. The spread between bacteria of successful mobile genetic elements, such as conjugative plasmids and integrative elements conferring multidrug resistance, is the main driving force in the dissemination of acquired antibiotic resistances among Gram-negative bacteria. Broad-host-range IncC plasmids and their integrative mobilizable SGI1 counterparts contribute to the spread of critically important resistance genes (e.g., extended-spectrum β-lactamases [ESBLs] and carbapenemases). A better knowledge of the complex biology of these broad-host-range mobile elements will help us to understand the dissemination of antimicrobial resistance genes that occurred across Gammaproteobacteria borders.202336472437
302140.9529Characterization of Acr2, an H-NS-like protein encoded on A/C2-type plasmids. Conjugation plays an important role in the horizontal movement of DNA between bacterial species and even genera. Large conjugative plasmids in Gram-negative bacteria are associated with multi-drug resistance and have been implicated in the spread of these phenotypes to pathogenic organisms. A/C plasmids often carry genes that confer resistance to multiple classes of antibiotics. Recently, transcription factors were characterized that regulate A/C conjugation. In this work, we expanded the regulon of the negative regulator Acr2. We developed an A/C variant, pARK01, by precise removal of resistance genes carried by the plasmid in order to make it more genetically tractable. Using pARK01, we conducted RNA-Seq and ChAP-Seq experiments to characterize the regulon of Acr2, an H-NS-like protein. We found that Acr2 binds several loci on the plasmid. We showed, in vitro, that Acr2 can bind specific promoter regions directly and identify key amino acids which are important for this binding. This study further characterizes Acr2 and suggests its role in modulating gene expression of multiple plasmid and chromosomal loci.201627492737
9845150.9529Mobile antibiotic resistance encoding elements promote their own diversity. Integrating conjugative elements (ICEs) are a class of bacterial mobile genetic elements that disseminate via conjugation and then integrate into the host cell genome. The SXT/R391 family of ICEs consists of more than 30 different elements that all share the same integration site in the host chromosome but often encode distinct properties. These elements contribute to the spread of antibiotic resistance genes in several gram-negative bacteria including Vibrio cholerae, the agent of cholera. Here, using comparative analyses of the genomes of several SXT/R391 ICEs, we found evidence that the genomes of these elements have been shaped by inter-ICE recombination. We developed a high throughput semi-quantitative method to explore the genetic determinants involved in hybrid ICE formation. Recombinant ICE formation proved to be relatively frequent, and to depend on host (recA) and ICE (s065 and s066) loci, which can independently and potentially cooperatively mediate hybrid ICE formation. s065 and s066, which are found in all SXT/R391 ICEs, are orthologues of the bacteriophage lambda Red recombination genes bet and exo, and the s065/s066 recombination system is the first Red-like recombination pathway to be described in a conjugative element. Neither ICE excision nor conjugative transfer proved to be essential for generation of hybrid ICEs. Instead conjugation facilitates the segregation of hybrids and could provide a means to select for functional recombinant ICEs containing novel combinations of genes conferring resistance to antibiotics. Thus, ICEs promote their own diversity and can yield novel mobile elements capable of disseminating new combinations of antibiotic resistance genes.200920019796
9816160.9529A two-component system serves as a central hub for connecting energy metabolism and plasmid dissemination in bacteria. Mobile genetic elements such as conjugative plasmids play a key role in the acquisition of antibiotic resistance by pathogenic bacteria. Resistance genes on plasmids can be transferred between bacteria using specialized conjugation machinery. Acinetobacter baumannii, the most common bacterium associated with nosocomial infections, harbors a large conjugative plasmid that encodes a type IV secretion system (T4SS). Feng et al. recently found that the A. baumannii T4SS is specialized for plasmid transfer, suggesting that it may be involved in multidrug resistance (Z. Feng, L. Wang, Q. Guan, X. Chu, and Z.-Q. Luo, mBio e02276-23, 2023, https://doi.org/10.1128/mbio.02276-23), T4SS-encoding genes are shown to be controlled by a versatile GacA/S two-component regulatory system. GacA/S is also found to regulate genes involved in central metabolism. The coordinated regulation of metabolism and plasmid conjugation may be a bacterial strategy for adapting to selective pressure from antibiotics.202338032214
3007170.9528Analysis of the complete nucleotide sequence of an Actinobacillus pleuropneumoniae streptomycin-sulfonamide resistance plasmid, pMS260. pMS260 is an 8.1-kb non-conjugative but mobilizable plasmid that was isolated from Actinobacillus pleuropneumoniae and encodes streptomycin (SM) and sulfonamide (SA) resistances. The analysis of the complete nucleotide sequence of the plasmid revealed a high degree of similarity between pMS260 and the broad-host-range IncQ family plasmids. pMS260 had a single copy of an origin of vegetative replication (oriV). This sequence was identical to a functional oriV of the IncQ-like plasmid pIE1130 that had been exogenously isolated from piggery manure. However, pMS260 did not carry the second IncQ plasmid RSF1010-like oriV region present in pIE1130. A pIE1130-identical transfer origin was also found in pMS260. In addition, the deduced amino acid sequences from 10 open reading frames identified in pMS260 were entirely or nearly identical to those from genes for the replication, mobilization, and SM-SA resistance of pIE1130, indicating that pMS260 belongs to the IncQ-1 gamma subgroup. pMS260 is physically indistinguishable from pIE1130 apart from two DNA regions that contain the chloramphenicol and kanamycin resistance genes (catIII and aphI, respectively) and the second oriV-like region of pIE1130. The codon bias analysis of each gene of pIE1130 and the presence of potential recombination sites in the sulII-strA intergenic regions suggest that pIE1130 seems to have acquired the catIII and aphI genes more recently than the other genes of pIE1130. Therefore, pMS260 may be the ancestor of pIE1130. Information regarding the broad-host-range replicon of pMS260 will be useful in the development of genetic systems for a wide range of bacteria including A. pleuropneumoniae.200414711528
9817180.9528Common regions e.g. orf513 and antibiotic resistance: IS91-like elements evolving complex class 1 integrons. The ability of bacteria to procure, sometimes rearrange, and evince acquired DNA continues to impress us-even more so if this genetic plasticity involves the sequestering of antibiotic resistance genes. The acquisition of genes in bacteria is often facilitated by transposons, integrons and archetype insertion elements. Recently however, a new element, 'orf513', has been increasingly associated with class 1 integrons. Moreover, these 'complex' class 1 integrons can potentially mediate resistance to chloramphenicol, trimethoprim, aminoglycosides and tetracycline and may carry a range of beta-lactamase genes as well as the qnrA gene. Elements such as 'orf513' demonstrate IS91-like characteristics and will mobilize adjacent DNA via a process called rolling circle replication, and thus we have renamed them 'insertion sequence CRs' (ISCRs) to appropriately reflect their structure-function properties. In this article, we provide a brief description of these new and clinically important mobile elements, and how they are able to mobilize antibiotic resistance genes.200616751201
397190.9528PCR-targeted Streptomyces gene replacement identifies a protein domain needed for biosynthesis of the sesquiterpene soil odor geosmin. Streptomycetes are high G+C Gram-positive, antibiotic-producing, mycelial soil bacteria. The 8.7-Mb Streptomyces coelicolor genome was previously sequenced by using an ordered library of Supercos-1 clones. Here, we describe an efficient procedure for creating precise gene replacements in the cosmid clones by using PCR targeting and lambda-Red-mediated recombination. The cloned Streptomyces genes are replaced with a cassette containing a selectable antibiotic resistance and oriT(RK2) for efficient transfer to Streptomyces by RP4-mediated intergeneric conjugation. Supercos-1 does not replicate in Streptomyces, but the clones readily undergo double-crossover recombination, thus creating gene replacements. The antibiotic resistance cassettes are flanked by yeast FLP recombinase target sequences for removal of the antibiotic resistance and oriT(RK2) to generate unmarked, nonpolar mutations. The technique has been used successfully by >20 researchers to mutate around 100 Streptomyces genes. As an example, we describe its application to the discovery of a gene involved in the production of geosmin, the ubiquitous odor of soil. The gene, Sco6073 (cyc2), codes for a protein with two sesquiterpene synthase domains, only one of which is required for geosmin biosynthesis, probably via a germacra-1 (10) E,5E-dien-11-ol intermediate generated by the sesquiterpene synthase from farnesyl pyrophosphate.200312563033