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1.
PLoS Genet ; 20(3): e1011088, 2024 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-38437248

RESUMO

Bacterial type IV secretion systems (T4SSs) are a versatile family of macromolecular translocators, collectively able to recruit diverse DNA and protein substrates and deliver them to a wide range of cell types. Presently, there is little understanding of how T4SSs recognize substrate repertoires and form productive contacts with specific target cells. Although T4SSs are composed of a number of conserved subunits and adopt certain conserved structural features, they also display considerable compositional and structural diversity. Here, we explored the structural bases underlying the functional versatility of T4SSs through systematic deletion and subunit swapping between two conjugation systems encoded by the distantly-related IncF plasmids, pED208 and F. We identified several regions of intrinsic flexibility among the encoded T4SSs, as evidenced by partial or complete functionality of chimeric machines. Swapping of VirD4-like TraD type IV coupling proteins (T4CPs) yielded functional chimeras, indicative of relaxed specificity at the substrate-TraD and TraD-T4SS interfaces. Through mutational analyses, we further delineated domains of the TraD T4CPs contributing to recruitment of cognate vs heterologous DNA substrates. Remarkably, swaps of components comprising the outer membrane core complexes, a few F-specific subunits, or the TraA pilins supported DNA transfer in the absence of detectable pilus production. Among sequenced enterobacterial species in the NCBI database, we identified many strains that harbor two or more F-like plasmids and many F plasmids lacking one or more T4SS components required for self-transfer. We confirmed that host cells carrying co-resident, non-selftransmissible variants of pED208 and F elaborate chimeric T4SSs, as evidenced by transmission of both plasmids. We propose that T4SS plasticity enables the facile assembly of functional chimeras, and this intrinsic flexibility at the structural level can account for functional diversification of this superfamily over evolutionary time and, on a more immediate time-scale, to proliferation of transfer-defective MGEs in nature.


Assuntos
Fator F , Sistemas de Secreção Tipo IV , Sistemas de Secreção Tipo IV/genética , Sistemas de Secreção Tipo IV/química , Sistemas de Secreção Tipo IV/metabolismo , Proteínas de Fímbrias/genética , Plasmídeos/genética , DNA Bacteriano , Proteínas de Bactérias/metabolismo
2.
J Biopharm Stat ; 34(1): 78-89, 2024 Jan 02.
Artigo em Inglês | MEDLINE | ID: mdl-36710402

RESUMO

In vitro dissolution profile has been shown to be correlated with the drug absorption and has often been considered as a metric for assessing in vitro bioequivalence between a test product and corresponding reference one. Various methods have been developed to assess the similarity between two dissolution profiles. In particular, similarity factor f2 has been reviewed and discussed extensively in many statistical articles. Although the f2 lacks inferential statistical properties, the estimation of f2 and its various modified versions were the most widely used metric for comparing dissolution profiles. In this paper, we investigated performances of the naive f2 estimate method, bootstrap f2 confidence interval method and bias corrected-accelerated (BCa) bootstrap f2 confidence interval method for comparing dissolution profiles. Our studies show that naive f2 estimate method and BCa bootstrap f2 confidence interval method are unable to control the type I error rate. The bootstrap f2 confidence interval method can control the type I error rate under a specific level. However, it will cause great conservatism on the power of the test. To solve the potential issues of the previous methods, we recommended a bootstrap bias corrected (BC) f2 confidence interval method in this paper. The type I error rate, power and sensitivity among different f2 methods were compared based on simulations. The recommended bootstrap BC f2 confidence interval method shows better control of type I error than the naive f2 estimate method and BCa bootstrap f2 confidence interval method. It also provides better power than the bootstrap f2 confidence interval method.


Assuntos
Fator F , Humanos , Solubilidade , Equivalência Terapêutica , Viés
3.
Nucleic Acids Res ; 51(17): 8925-8933, 2023 09 22.
Artigo em Inglês | MEDLINE | ID: mdl-37592747

RESUMO

Bacterial conjugation was first described by Lederberg and Tatum in the 1940s following the discovery of the F plasmid. During conjugation a plasmid is transferred unidirectionally from one bacterium (the donor) to another (the recipient), in a contact-dependent manner. Conjugation has been regarded as a promiscuous mechanism of DNA transfer, with host range determined by the recipient downstream of plasmid transfer. However, recent data have shown that F-like plasmids, akin to tailed Caudovirales bacteriophages, can pick their host bacteria prior to transfer by expressing one of at least four structurally distinct isoforms of the outer membrane protein TraN, which has evolved to function as a highly sensitive sensor on the donor cell surface. The TraN sensor appears to pick bacterial hosts by binding compatible outer membrane proteins in the recipient. The TraN variants can be divided into specialist and generalist sensors, conferring narrow and broad plasmid host range, respectively. In this review we discuss recent advances in our understanding of the function of the TraN sensor at the donor-recipient interface, used by F-like plasmids to select bacterial hosts within polymicrobial communities prior to DNA transfer.


Assuntos
Bactérias , Conjugação Genética , Plasmídeos , Bactérias/genética , Bactérias/metabolismo , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , DNA Bacteriano/genética , Fator F/genética , Proteínas de Membrana/genética , Plasmídeos/genética
4.
Microb Genom ; 9(7)2023 07.
Artigo em Inglês | MEDLINE | ID: mdl-37471138

RESUMO

Extraintestinal pathogenic Escherichia coli (ExPEC) are the most frequent cause of urinary tract infections (UTIs) globally. Most studies of clinical E. coli isolates are selected based on their antimicrobial resistance (AMR) phenotypes; however, this selection bias may not provide an accurate portrayal of which sequence types (STs) cause the most disease. Here, whole genome sequencing (WGS) was performed on 320 E. coli isolates from urine samples sourced from a regional hospital in Australia in 2006. Most isolates (91%) were sourced from patients with UTIs and were not selected based on any AMR phenotypes. No significant differences were observed in AMR and virulence genes profiles across age sex, and uro-clinical syndromes. While 88 STs were identified, ST73, ST95, ST127 and ST131 dominated. F virulence plasmids carrying senB-cjrABC (126/231; 55%) virulence genes were a feature of this collection. These senB-cjrABC+ plasmids were split into two categories: pUTI89-like (F29:A-:B10 and/or >95 % identity to pUTI89) (n=73) and non-pUTI89-like (n=53). Compared to all other plasmid replicons, isolates with pUTI89-like plasmids carried fewer antibiotic resistance genes (ARGs), whilst isolates with senB-cjrABC+/non-pUTI89 plasmids had a significantly higher load of ARGs and class 1 integrons. F plasmids were not detected in 89 genomes, predominantly ST73. Our phylogenomic analyses identified closely related isolates from the same patient associated with different pathologies and evidence of strain-sharing events involving isolates sourced from companion and wild animals.


Assuntos
Infecções por Escherichia coli , Escherichia coli Extraintestinal Patogênica , Infecções Urinárias , Animais , Escherichia coli , Virulência/genética , Antibacterianos/farmacologia , Fator F , Genótipo , Farmacorresistência Bacteriana/genética , Austrália , Genômica
5.
J Bacteriol ; 205(4): e0006123, 2023 04 25.
Artigo em Inglês | MEDLINE | ID: mdl-36988519

RESUMO

Horizontal gene transfer via conjugation plays a major role in bacterial evolution. In F-like plasmids, efficient DNA transfer is mediated by close association between donor and recipient bacteria. This process, known as mating pair stabilization (MPS), is mediated by interactions between the plasmid-encoded outer membrane (OM) protein TraN in the donor and chromosomally-encoded OM proteins in the recipient. We have recently reported the existence of 7 TraN sequence types, which are grouped into 4 structural types, that we named TraNα, TraNß, TraNγ, and TraNδ. Moreover, we have shown specific pairing between TraNα and OmpW, TraNß and OmpK36 of Klebsiella pneumoniae, TraNγ and OmpA, and TraNδ and OmpF. In this study, we found that, although structurally similar, TraNα encoded by the Salmonella enterica pSLT plasmid (TraNα2) binds OmpW in both Escherichia coli and Citrobacter rodentium, while TraNα encoded by the R100-1 plasmid (TraNα1) only binds OmpW in E. coli. AlphaFold2 predictions suggested that this specificity is mediated by a single amino acid difference in loop 3 of OmpW, which we confirmed experimentally. Moreover, we show that single amino acids insertions into loop 3 of OmpK36 affect TraNß-mediated conjugation efficiency of the K. pneumoniae resistance plasmid pKpQIL. Lastly, we report that TraNß can also mediate MPS by binding OmpK35, making it the first TraN variant that can bind more than one OM protein in the recipient. Together, these data show that subtle sequence differences in the OM receptors can impact TraN-mediated conjugation efficiency. IMPORTANCE Conjugation plays a central role in the spread of antimicrobial resistance genes among bacterial pathogens. Efficient conjugation is mediated by formation of mating pairs via a pilus, followed by mating pair stabilization (MPS), mediated by tight interactions between the plasmid-encoded outer membrane protein (OMP) TraN in the donor (of which there are 7 sequence types grouped into the 4 structural isoforms α, ß, γ, and δ), and an OMP receptor in the recipient (OmpW, OmpK36, OmpA, and OmpF, respectively). In this study, we found that subtle differences in OmpW and OmpK36 have significant consequences on conjugation efficiency and specificity, highlighting the existence of selective pressure affecting plasmid-host compatibility and the flow of horizontal gene transfer in bacteria.


Assuntos
Escherichia coli , Fator F , Escherichia coli/genética , Escherichia coli/metabolismo , Sequência de Aminoácidos , Conjugação Genética , Plasmídeos/genética , Proteínas de Membrana/metabolismo , Isoformas de Proteínas/genética , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo
6.
Microbiol Spectr ; 10(6): e0255422, 2022 12 21.
Artigo em Inglês | MEDLINE | ID: mdl-36409140

RESUMO

Lower urinary tract, renal, and bloodstream infections caused by phylogroup B2 extraintestinal pathogenic Escherichia coli (ExPEC) are a leading cause of morbidity and mortality. ST1193 is a phylogroup B2, multidrug-resistant sequence type that has risen to prominence globally, but a comprehensive analysis of the F virulence plasmids it carries is lacking. We performed a phylogenomic analysis of ST1193 (n = 707) whole-genome sequences from EnteroBase using entries with comprehensive isolation metadata. The data set comprised isolates from humans (n = 634 [90%]), including 339 (48%) from extraintestinal infection sites, and isolates from companion animals, wastewater, and wildlife. Phylogenetic analyses combined with gene detection and genotyping resolved an ST1193 clade structure segregated by serotype and F plasmid carriage. Most F plasmids fell into one of three related plasmid subtypes: F-:A1:B10 (n = 444 [65.97%]), F-:A1:B1 (n = 84 [12.48%]), and F-:A1:B20 (n = 80 [11.89%]), all of which carry the virulence genes cjrABC colocalized with senB (cjrABC-senB), a trademark signature of F29:A-:B10 subtype plasmids (pUTI89). To examine the phylogenetic relationship of these plasmids with pUTI89, complete sequences of F-:A1:B1 and F-:1:B20 plasmids were resolved. Unlike pUTI89, the most dominant and widely disseminated F plasmid that carries cjrABC-senB, F plasmids in ST1193 often carry a complex resistance region with an integron truncation (intI1Δ745) signature embedded within a structure assembled by IS26. Plasmid analysis shows that ST1193 has F plasmids that carry cjrABC-senB and ARG-encoding genes but lack tra regions and are likely derivatives of pUTI89. Further epidemiological investigation of ST1193 should seek to confirm its presence in human-associated environments and identify any potential agricultural links, which are currently lacking. IMPORTANCE We have generated an updated ST1193 phylogeny using publicly available sequences, reinforcing previous assertions that Escherichia coli ST1193 is a human-associated lineage, with many examples sourced from human extraintestinal infections. ST1193 from urban-adapted birds, wastewater, and companion animals are frequent, but isolates from animal agriculture are notably absent. Phylogenomic analysis identified several clades segregated by serogroup, all noted to carry highly similar F plasmids and antimicrobial resistance (AMR) signatures. Investigation of these plasmids revealed virulence regions with similarity to pUTI89, a key F virulence plasmid among dominant pandemic extraintestinal pathogenic E. coli lineages, and encoding a complex antibiotic resistance structure mobilized by IS26. This work has uncovered a series of F virulence plasmids in ST1193 and shows that the lineage mimics the host range and virulence attributes of other E. coli strains that carry pUTI89. These observations have significant ramifications for epidemiological source tracking of emerging and established pandemic ExPEC lineages.


Assuntos
Infecções por Escherichia coli , Escherichia coli Extraintestinal Patogênica , Animais , Humanos , Escherichia coli , Filogenia , Virulência/genética , Fator F , Infecções por Escherichia coli/epidemiologia , Infecções por Escherichia coli/veterinária , Antibacterianos , Águas Residuárias , Pandemias , Plasmídeos/genética , Escherichia coli Extraintestinal Patogênica/genética , Fatores de Virulência/genética
7.
Plasmid ; 123-124: 102652, 2022.
Artigo em Inglês | MEDLINE | ID: mdl-36228885

RESUMO

Two phylogenetically distantly-related IncF plasmids, F and pED208, serve as important models for mechanistic and structural studies of F-like type IV secretion systems (T4SSFs) and F pili. Here, we present the pED208 sequence and compare it to F and pUMNF18, the closest match to pED208 in the NCBI database. As expected, gene content of the three cargo regions varies extensively, although the maintenance/leading regions (MLRs) and transfer (Tra) regions also carry novel genes or motifs with predicted modulatory effects on plasmid stability, dissemination and host range. By use of a Cre recombinase assay for translocation (CRAfT), we recently reported that pED208-carrying donors translocate several products of the MLR (ParA, ParB1, ParB2, SSB, PsiB, PsiA) intercellularly through the T4SSF. Here, we extend these findings by reporting that pED208-carrying donors translocate 10 additional MLR proteins during conjugation. In contrast, two F plasmid-encoded toxin components of toxin-antitoxin (TA) modules, CcdB and SrnB, were not translocated at detectable levels through the T4SSF. Remarkably, most or all of the pED208-encoded MLR proteins and CcdB and SrnB were translocated through heterologous T4SSs encoded by IncN and IncP plasmids pKM101 and RP4, respectively. Together, our sequence analyses underscore the genomic diversity of the F plasmid superfamily, and our experimental data demonstrate the promiscuous nature of conjugation machines for protein translocation. Our findings raise intriguing questions about the nature of T4SS translocation signals and of the biological and evolutionary consequences of conjugative protein transfer.


Assuntos
Escherichia coli , Sistemas de Secreção Tipo IV , Sistemas de Secreção Tipo IV/genética , Plasmídeos/genética , Escherichia coli/genética , Fator F , Análise de Sequência , Conjugação Genética , Proteínas de Bactérias/metabolismo
8.
Res Vet Sci ; 150: 98-106, 2022 Dec 05.
Artigo em Inglês | MEDLINE | ID: mdl-35809419

RESUMO

CpxAR is a global regulatory protein and has important roles in plasmid mating. However, except for traJ, the regulatory effect of CpxAR on other tra genes is unclear. The aim of this study was to explore the effects of CpxAR on conjugative transfer of the epidemic plasmid pEC011 (IncFII replicon) in Escherichia coli. The plasmid mating frequencies were significantly higher for the single deletion mutant strain FΔcpxR than for the parental strain F25922. Additionally, expression levels of traM, traJ and traY in FΔcpxR were significantly higher than those in F25922. Further investigations revealed that His6-CpxR protein could directly bind to the traM, traJ and traY promoter regions with the binding sites of 5'-TTTACATT-3' (PM), 5'-ATAAGAAT-3' (PJ), and 5'-AATTTTAT-3' (PY), respectively. Taken together, our results demonstrate that CpxAR can downregulate the expression of traM, traJ and traY by directly binding to the CpxR box-like sites of promoters, thus significantly reducing the mating rates of IncFII replicon plasmid pEC011.


Assuntos
Proteínas de Bactérias/metabolismo , Proteínas de Escherichia coli , Animais , Proteínas da Membrana Bacteriana Externa/genética , Proteínas da Membrana Bacteriana Externa/metabolismo , Proteínas de Bactérias/genética , Sequência de Bases , Escherichia coli/genética , Escherichia coli/metabolismo , Proteínas de Escherichia coli/genética , Proteínas de Escherichia coli/metabolismo , Fator F , Plasmídeos/genética , Regiões Promotoras Genéticas , Replicon
9.
Nat Microbiol ; 7(7): 1016-1027, 2022 07.
Artigo em Inglês | MEDLINE | ID: mdl-35697796

RESUMO

Bacterial conjugation mediates contact-dependent transfer of DNA from donor to recipient bacteria, thus facilitating the spread of virulence and resistance plasmids. Here we describe how variants of the plasmid-encoded donor outer membrane (OM) protein TraN cooperate with distinct OM receptors in recipients to mediate mating pair stabilization and efficient DNA transfer. We show that TraN from the plasmid pKpQIL (Klebsiella pneumoniae) interacts with OmpK36, plasmids from R100-1 (Shigella flexneri) and pSLT (Salmonella Typhimurium) interact with OmpW, and the prototypical F plasmid (Escherichia coli) interacts with OmpA. Cryo-EM analysis revealed that TraNpKpQIL interacts with OmpK36 through the insertion of a ß-hairpin in the tip of TraN into a monomer of the OmpK36 porin trimer. Combining bioinformatic analysis with AlphaFold structural predictions, we identified a fourth TraN structural variant that mediates mating pair stabilization by binding OmpF. Accordingly, we devised a classification scheme for TraN homologues on the basis of structural similarity and their associated receptors: TraNα (OmpW), TraNß (OmpK36), TraNγ (OmpA), TraNδ (OmpF). These TraN-OM receptor pairings have real-world implications as they reflect the distribution of resistance plasmids within clinical Enterobacteriaceae isolates, demonstrating the importance of mating pair stabilization in mediating conjugation species specificity. These findings will allow us to predict the distribution of emerging resistance plasmids in high-risk bacterial pathogens.


Assuntos
Proteínas de Bactérias , Conjugação Genética , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Escherichia coli/genética , Escherichia coli/metabolismo , Fator F , Porinas/genética , Porinas/metabolismo , Especificidade da Espécie
10.
Mol Microbiol ; 117(5): 1275-1290, 2022 05.
Artigo em Inglês | MEDLINE | ID: mdl-35434837

RESUMO

F plasmids circulate widely among the Enterobacteriaceae through encoded type IV secretion systems (T4SSF s). Assembly of T4SSF s and associated F pili requires 10 VirB/VirD4-like Tra subunits and eight or more F-specific subunits. Recently, we presented evidence using in situ cryoelectron tomography (cryoET) that T4SSF s undergo structural transitions when activated for pilus production, and that assembled pili are deposited onto alternative basal platforms at the cell surface. Here, we deleted eight conserved F-specific genes from the MOBF12C plasmid pED208 and quantitated effects on plasmid transfer, pilus production by fluorescence microscopy, and elaboration of T4SSF structures by in situ cryoET. Mutant phenotypes supported the assignment of F-specific subunits into three functional Classes: (i) TraF, TraH, and TraW are required for all T4SSF -associated activities, (ii) TraU, TraN, and TrbC are nonessential but contribute significantly to distinct T4SSF functions, and (iii) TrbB is essential for F pilus production but not for plasmid transfer. Equivalent mutations in a phylogenetically distantly related MOB12A F plasmid conferred similar phenotypes and generally supported these Class assignments. We present a new structure-driven model in which F-specific subunits contribute to distinct steps of T4SSF assembly or activation to regulate DNA transfer and F pilus dynamics and deposition onto alternative platforms.


Assuntos
Proteínas de Escherichia coli , Fator F , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Conjugação Genética , Escherichia coli/genética , Proteínas de Escherichia coli/metabolismo , Fímbrias Bacterianas/genética , Fímbrias Bacterianas/metabolismo , Plasmídeos/genética , Sistemas de Secreção Tipo IV/genética , Sistemas de Secreção Tipo IV/metabolismo
11.
mSystems ; 7(1): e0121221, 2022 02 22.
Artigo em Inglês | MEDLINE | ID: mdl-35076267

RESUMO

Escherichia coli sequence type 95 (ST95) is an extraintestinal pathogenic E. coli (ExPEC) renowned for its ability to cause significant morbidity and mortality in humans and poultry. A core genome analysis of 668 ST95 isolates generated 10 clades (A to J), 5 of which are reported here for the first time. F plasmid replicon sequence typing showed that almost a third (178/668 [27%]) of the collection carry pUTI89 (F29:B10) and were restricted to clade A and a sublineage of clade B. In contrast, almost half (328/668 [49%]) of the collection across multiple clades harbor ColV plasmids (multiple F types). Strikingly, ST95 lineages with pUTI89 were almost exclusively from humans, while ColV+ ST95 lineages were sourced from poultry and humans. Clade I was notable because it comprises temporally and geographically matched ColV+ isolates sourced from human and retail poultry meat, suggesting interspecies transmission via food. Clade F contained ST95 isolates of bovine origin, none of which carried ColV or pUTI89 plasmids. Remarkably, an analysis of a cohort of 34,176 E. coli isolates comprising 2,570 sequence types mirrored what was observed in ST95: (i) pUTI89 was overwhelmingly linked to E. coli sourced from humans but almost entirely absent from 13,027 E. coli isolates recovered from poultry, pigs, and cattle, and (ii) E. coli isolates harboring ColV plasmids were from multiple sources, including humans, poultry, and swine. Overall, our data suggest that F plasmids influence E. coli host range, clade structure, and zoonotic potential in ST95 and ExPEC more broadly. IMPORTANCE E. coli ST95 is one of five dominant ExPEC lineages globally and noted for causing urinary tract and bloodstream infections and neonatal meningitis in humans and colibacillosis in poultry. Using high-resolution phylogenomics, we show that F replicon sequence type is linked to ST95 clade structure and zoonotic potential. Specifically, human centric ST95 clades overwhelmingly harbor F29:B10 (pUTI89) plasmids, while clades carrying both human- and poultry-sourced isolates are typically ColV+ with multiple replicon types. Importantly, several clades identified clonal ColV+ ST95 isolates from human and poultry sources, but clade I, which housed temporally and spatially matched isolates, provided the most robust evidence. Notably, patterns of association of F replicon types with E. coli host were mirrored within a diverse collection of 34,176 E. coli genomes. Our studies indicate that the role of food animals as a source of human ExPEC disease is complex and warrants further investigation.


Assuntos
Escherichia coli Extraintestinal Patogênica , Fator F , Humanos , Animais , Bovinos , Suínos , Escherichia coli , Especificidade de Hospedeiro , Zoonoses , Resistência Microbiana a Medicamentos
12.
Plasmid ; 119-120: 102618, 2022.
Artigo em Inglês | MEDLINE | ID: mdl-35077724

RESUMO

Plasmids exhibit great diversity of gene content and host ranges and are famous for quick adaptation to the genetic background of the bacterial host cell. In addition to observing ever evolving plasmids, some plasmids have conserved backbones: a stable core composition and arrangement of genes in addition to variable regions. There are a few reports of extremely conserved plasmids. Here we report the complete sequence of pRK100 plasmid - a large, well-characterized conjugative F-like plasmid found in an Escherichia coli strain isolated from a urinary tract infection patient in 1990. The sequence shows that the 142 kb-long pRK100 plasmid is nearly identical to plasmids circulating in distant geographical locations and found in different host E. coli strains between 2007 and 2017. We also performed additional functional characterization of pRK100. Our results showed that pRK100 does not have a strong pathogenicity phenotype in porcine primary bladder epithelial cell culture. Moreover, the conjugation of pRK100 seems to strongly depend on recipient characteristics. These observations and identification of the pRK100 plasmid in different strain genotypes leave the extreme sequence conservation and broad distribution of this plasmid unexplained.


Assuntos
Infecções por Escherichia coli , Proteínas de Escherichia coli , Animais , Conjugação Genética , Escherichia coli/genética , Infecções por Escherichia coli/microbiologia , Proteínas de Escherichia coli/genética , Fator F , Humanos , Plasmídeos/genética , Suínos
13.
Nat Commun ; 13(1): 379, 2022 01 19.
Artigo em Inglês | MEDLINE | ID: mdl-35046412

RESUMO

Bacterial type IV secretion systems (T4SSs) are largely responsible for the proliferation of multi-drug resistance. We solved the structure of the outer-membrane core complex (OMCCF) of a T4SS encoded by a conjugative F plasmid at <3.0 Å resolution by cryoelectron microscopy. The OMCCF consists of a 13-fold symmetrical outer ring complex (ORC) built from 26 copies of TraK and TraV C-terminal domains, and a 17-fold symmetrical central cone (CC) composed of 17 copies of TraB ß-barrels. Domains of TraV and TraB also bind the CC and ORC substructures, establishing that these proteins undergo an intraprotein symmetry alteration to accommodate the C13:C17 symmetry mismatch. We present evidence that other pED208-encoded factors stabilize the C13:C17 architecture and define the importance of TraK, TraV and TraB domains to T4SSF function. This work identifies OMCCF structural motifs of proposed importance for structural transitions associated with F plasmid dissemination and F pilus biogenesis.


Assuntos
Farmacorresistência Bacteriana Múltipla , Fator F/metabolismo , Sistemas de Secreção Tipo IV/química , Membrana Celular/química , Escherichia coli/metabolismo , Proteínas de Escherichia coli/química , Proteínas de Escherichia coli/metabolismo , Modelos Moleculares , Sistemas de Secreção Tipo IV/ultraestrutura
14.
Plasmid ; 119-120: 102617, 2022.
Artigo em Inglês | MEDLINE | ID: mdl-35041919

RESUMO

The rapid emergence and spread of antibiotic resistance is a growing global burden. Antibiotic resistance is often associated with large single or low copy number plasmids, which rely upon cytoskeletal proteins for their stable maintenance. While the mechanism of plasmid partitioning has been well established for the R plasmids, the molecular details by which the F plasmid is maintained is only beginning to emerge. The partitioning function of the F plasmid depends upon a ParA/ MinD family of proteins known as SopA. SopA, by virtue of its ATP-dependent non-specific DNA binding activity and association with the bacterial nucleoid, drives the segregation of the F plasmid into the daughter cells. This function further depends upon the stimulation of the ATPase activity of SopA by the SopBC complex. Here, we report that several residues in the last C-terminal helix in SopA play a crucial but distinct role in SopA function and plasmid maintenance. While the deletion of the last five residues in SopA does not affect its ability to bind the nucleoid or SopB, they severely affect the plasmid partitioning function. Further, we show that while mutations in certain polar residues in the C-terminal helix only mildly affect its localisation to the nucleoid, others cause defects in nsDNA binding and disrupt plasmid maintenance functions.


Assuntos
Proteínas de Escherichia coli , Fator F , Proteínas de Bactérias/metabolismo , Escherichia coli/genética , Escherichia coli/metabolismo , Proteínas de Escherichia coli/genética , Proteínas de Escherichia coli/metabolismo , Plasmídeos/genética
15.
mBio ; 12(4): e0162921, 2021 08 31.
Artigo em Inglês | MEDLINE | ID: mdl-34253063

RESUMO

Bacterial type IV secretion systems (T4SSs) mediate the conjugative transfer of mobile genetic elements (MGEs) and their cargoes of antibiotic resistance and virulence genes. Here, we report that the pED208-encoded T4SS (TrapED208) translocates not only this F plasmid but several plasmid-encoded proteins, including ParA, ParB1, single-stranded DNA-binding protein SSB, ParB2, PsiB, and PsiA, to recipient cells. Conjugative protein translocation through the TrapED208 T4SS required engagement of the pED208 relaxosome with the TraD substrate receptor or coupling protein. T4SSs translocate MGEs as single-stranded DNA intermediates (T-strands), which triggers the SOS response in recipient cells. Transfer of pED208 deleted of psiB or ssb, which, respectively, encode the SOS inhibitor protein PsiB and single-stranded DNA-binding protein SSB, elicited a significantly stronger SOS response than pED208 or mutant plasmids deleted of psiA, parA, parB1, or parB2. Conversely, translocation of PsiB or SSB, but not PsiA, through the TrapED208 T4SS suppressed the mating-induced SOS response. Our findings expand the repertoire of known substrates of conjugation systems to include proteins with functions associated with plasmid maintenance. Furthermore, for this and other F-encoded Tra systems, docking of the DNA substrate with the TraD receptor appears to serve as a critical activating signal for protein translocation. Finally, the observed effects of PsiB and SSB on suppression of the mating-induced SOS response establishes a novel biological function for conjugative protein translocation and suggests the potential for interbacterial protein translocation to manifest in diverse outcomes influencing bacterial communication, physiology, and evolution. IMPORTANCE Many bacteria carry plasmids and other mobile genetic elements (MGEs) whose conjugative transfer through encoded type IV secretion systems (T4SSs), or "mating" channels, can lead to a rapid intra- and interspecies proliferation of genes encoding resistance to antibiotics or heavy metals or virulence traits. Here, we show that a model IncF plasmid-encoded T4SS translocates not only DNA but also several proteins intercellularly. The repertoire of translocated proteins includes the plasmidic SOS inhibitor protein PsiB, single-stranded DNA-binding protein SSB, and several partitioning proteins. We demonstrate that intercellular transmission of PsiB and SSB suppresses the SOS response, which is triggered in recipient cells upon acquisition of the single-stranded DNA transfer intermediate during mating. Our findings identify a new biological function for conjugative protein translocation in mitigating potentially deleterious consequences to plasmid and genome integrity resulting from SOS-induced recombination and mutation events.


Assuntos
Conjugação Genética , Escherichia coli/genética , Fator F/genética , Plasmídeos/genética , Resposta SOS em Genética , Sistemas de Secreção Tipo IV/metabolismo , Proteínas de Bactérias/metabolismo , DNA Bacteriano/metabolismo , Escherichia coli/metabolismo , Translocação Genética , Sistemas de Secreção Tipo IV/genética
16.
Nucleic Acids Res ; 49(15): 8407-8418, 2021 09 07.
Artigo em Inglês | MEDLINE | ID: mdl-33907814

RESUMO

In bacterial synthetic biology, whole genome transplantation has been achieved only in mycoplasmas that contain a small genome and are competent for foreign genome uptake. In this study, we developed Escherichia coli strains programmed by three 1-megabase (Mb) chromosomes by splitting the 3-Mb chromosome of a genome-reduced strain. The first split-chromosome retains the original replication origin (oriC) and partitioning (par) system. The second one has an oriC and the par locus from the F plasmid, while the third one has the ori and par locus of the Vibrio tubiashii secondary chromosome. The tripartite-genome cells maintained the rod-shaped form and grew only twice as slowly as their parent, allowing their further genetic engineering. A proportion of these 1-Mb chromosomes were purified as covalently closed supercoiled molecules with a conventional alkaline lysis method and anion exchange columns. Furthermore, the second and third chromosomes could be individually electroporated into competent cells. In contrast, the first split-chromosome was not able to coexist with another chromosome carrying the same origin region. However, it was exchangeable via conjugation between tripartite-genome strains by using different selection markers. We believe that this E. coli-based technology has the potential to greatly accelerate synthetic biology and synthetic genomics.


Assuntos
Cromossomos Bacterianos/genética , Escherichia coli/genética , Fator F/genética , Genoma Bacteriano/genética , Replicação do DNA/genética , Escherichia coli/crescimento & desenvolvimento , Origem de Replicação/genética , Biologia Sintética/tendências , Vibrio/genética
17.
J Membr Biol ; 254(3): 243-257, 2021 06.
Artigo em Inglês | MEDLINE | ID: mdl-33427942

RESUMO

Stable maintenance and partitioning of the 'Fertility' plasmid or the F plasmid in its host Escherichia coli require the function of a ParA superfamily of proteins known as SopA. The mechanism by which SopA mediates plasmid segregation is well studied. SopA is a nucleoid-binding protein and binds DNA in an ATP-dependent but sequence non-specific manner. ATP hydrolysis stimulated by the binding of the SopBC complex mediates the release of SopA from the nucleoid. Cycles of ATP-binding and hydrolysis generate an ATPase gradient that moves the plasmid through a chemophoresis force. Nucleoid binding of SopA thus assumes a central role in its plasmid-partitioning function. However, earlier work also suggests that the F plasmid can be partitioned into anucleate cells, thus implicating nucleoid independent partitioning. Interestingly, SopA is also reported to be associated with the inner membrane of the bacteria. Here, we report the identification of a possible membrane-targeting sequence, a predicted amphipathic helix, at the C-terminus of SopA. Molecular dynamics simulations indicate that the predicted amphipathic helical motif of SopA has weak affinity for membranes. Moreover, we experimentally show that SopA can associate with bacterial membranes, is detectable in the membrane fractions of bacterial lysates, and is sensitive to the membrane potential. Further, unlike the wild-type SopA, a deletion of the C-terminal 29 amino acids results in the loss of F plasmids from bacterial cells.


Assuntos
Proteínas de Escherichia coli , Fator F , Proteínas de Bactérias/metabolismo , Escherichia coli/genética , Escherichia coli/metabolismo , Proteínas de Escherichia coli/genética , Proteínas de Escherichia coli/metabolismo , Plasmídeos/genética
18.
Genes (Basel) ; 11(11)2020 10 22.
Artigo em Inglês | MEDLINE | ID: mdl-33105635

RESUMO

Bacterial conjugation, also referred to as bacterial sex, is a major horizontal gene transfer mechanism through which DNA is transferred from a donor to a recipient bacterium by direct contact. Conjugation is universally conserved among bacteria and occurs in a wide range of environments (soil, plant surfaces, water, sewage, biofilms, and host-associated bacterial communities). Within these habitats, conjugation drives the rapid evolution and adaptation of bacterial strains by mediating the propagation of various metabolic properties, including symbiotic lifestyle, virulence, biofilm formation, resistance to heavy metals, and, most importantly, resistance to antibiotics. These properties make conjugation a fundamentally important process, and it is thus the focus of extensive study. Here, we review the key steps of plasmid transfer by conjugation in Gram-negative bacteria, by following the life cycle of the F factor during its transfer from the donor to the recipient cell. We also discuss our current knowledge of the extent and impact of conjugation within an environmentally and clinically relevant bacterial habitat, bacterial biofilms.


Assuntos
Biofilmes/crescimento & desenvolvimento , Farmacorresistência Bacteriana/genética , Fator F/genética , Transferência Genética Horizontal/genética , Bactérias Gram-Negativas/genética , DNA Bacteriano/genética , Fator F/fisiologia , Fímbrias Bacterianas/metabolismo
19.
J Mol Graph Model ; 101: 107723, 2020 12.
Artigo em Inglês | MEDLINE | ID: mdl-32927271

RESUMO

One promising strategy to combat antimicrobial resistance is to use bacteriophages that attach to the sex pili produced by transmissible antimicrobial resistance (AMR) plasmids, infect AMR bacteria and select for loss of the AMR plasmids, prolonging the life of existing antimicrobials. The maturation protein of the bacteriophage MS2 attaches to the pili produced by Incompatibility group F plasmid-containing bacteria. This interaction initiates delivery of the viral genetic material into the bacteria. Using protein-protein docking we constructed a model of the F pilus comprising a trimer of subunits binding to the maturation protein. Interactions between the maturation protein and the F pilus were investigated using molecular dynamics simulations. In silico alanine scanning and in silico single-point mutations were explored, with the longer term aim of increasing the affinity of the maturation protein to other Incompatibility group pili, without reducing the strength of binding to F pilin. We report our computational findings on which residues are required for the maturation protein and F pilin to interact, those which had no effect on the interaction and the mutations which led to a stronger interaction.


Assuntos
Proteínas de Escherichia coli , Pili Sexual , Escherichia coli/genética , Proteínas de Escherichia coli/genética , Fator F/genética , Levivirus/genética
20.
mSphere ; 5(4)2020 08 05.
Artigo em Inglês | MEDLINE | ID: mdl-32759337

RESUMO

The evolution and propagation of antibiotic resistance by bacterial pathogens are significant threats to global public health. Contemporary DNA sequencing tools were applied here to gain insight into carriage of antibiotic resistance genes in Escherichia coli, a ubiquitous commensal bacterium in the gut microbiome in humans and many animals, and a common pathogen. Draft genome sequences generated for a collection of 101 E. coli strains isolated from healthy undergraduate students showed that horizontally acquired antibiotic resistance genes accounted for most resistance phenotypes, the primary exception being resistance to quinolones due to chromosomal mutations. A subset of 29 diverse isolates carrying acquired resistance genes and 21 control isolates lacking such genes were further subjected to long-read DNA sequencing to enable complete or nearly complete genome assembly. Acquired resistance genes primarily resided on F plasmids (101/153 [67%]), with smaller numbers on chromosomes (30/153 [20%]), IncI complex plasmids (15/153 [10%]), and small mobilizable plasmids (5/153 [3%]). Nearly all resistance genes were found in the context of known transposable elements. Very few structurally conserved plasmids with antibiotic resistance genes were identified, with the exception of an ∼90-kb F plasmid in sequence type 1193 (ST1193) isolates that appears to serve as a platform for resistance genes and may have virulence-related functions as well. Carriage of antibiotic resistance genes on transposable elements and mobile plasmids in commensal E. coli renders the resistome highly dynamic.IMPORTANCE Rising antibiotic resistance in human-associated bacterial pathogens is a serious threat to our ability to treat many infectious diseases. It is critical to understand how acquired resistance genes move in and through bacteria associated with humans, particularly for species such as Escherichia coli that are very common in the human gut but can also be dangerous pathogens. This work combined two distinct DNA sequencing approaches to allow us to explore the genomes of E. coli from college students to show that the antibiotic resistance genes these bacteria have acquired are usually carried on a specific type of plasmid that is naturally transferrable to other E. coli, and likely to other related bacteria.


Assuntos
Farmacorresistência Bacteriana Múltipla/genética , Escherichia coli/genética , Fator F/genética , Simbiose , Antibacterianos/farmacologia , Escherichia coli/efeitos dos fármacos , Infecções por Escherichia coli/microbiologia , Transferência Genética Horizontal , Genoma Bacteriano , Humanos , Testes de Sensibilidade Microbiana , Análise de Sequência de DNA , Adulto Jovem
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