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1.
Anim Microbiome ; 6(1): 36, 2024 Jun 25.
Article in English | MEDLINE | ID: mdl-38918824

ABSTRACT

Mounting evidence of the occurrence of direct and indirect interactions between the human blood fluke, Schistosoma mansoni, and the gut microbiota of rodent models raises questions on the potential role(s) of the latter in the pathophysiology of hepatointestinal schistosomiasis. However, substantial differences in both the composition and function between the gut microbiota of laboratory rodents and that of humans hinders an in-depth understanding of the significance of such interactions for human schistosomiasis. Taking advantage of the availability of a human microbiota-associated mouse model (HMA), we have previously highlighted differences in infection-associated changes in gut microbiota composition between HMA and wildtype (WT) mice. To further explore the dynamics of schistosome-microbiota relationships in HMA mice, in this study we (i) characterize qualitative and quantitative changes in gut microbiota composition of a distinct line of HMA mice (D2 HMA) infected with S. mansoni prior to and following the onset of parasite egg production; (ii) profile local and systemic immune responses against the parasite in HMA as well as WT mice and (iii) assess levels of faecal inflammatory markers and occult blood as indirect measures of gut tissue damage. We show that patent S. mansoni infection is associated with reduced bacterial alpha diversity in the gut of D2 HMA mice, alongside expansion of hydrogen sulphide-producing bacteria. Similar systemic humoral responses against S. mansoni in WT and D2 HMA mice, as well as levels of faecal lipocalin and markers of alternatively activated macrophages, suggest that these are independent of baseline gut microbiota composition. Qualitative comparative analyses between faecal microbial profiles of S. mansoni-infected WT and distinct lines of HMA mice reveal that, while infection-induced alterations of the gut microbiota composition are highly dependent on the baseline flora, bile acid composition and metabolism may represent key elements of schistosome-microbiota interactions through the gut-liver axis.

2.
Clin Microbiol Infect ; 26(4): 492-498, 2020 Apr.
Article in English | MEDLINE | ID: mdl-31525517

ABSTRACT

OBJECTIVES: Clostridium difficile is a major global human pathogen divided into five clades, of which clade 3 is the least characterized and consists predominantly of PCR ribotype (RT) 023 strains. Our aim was to analyse and characterize this clade. METHODS: In this cohort study the clinical presentation of C. difficile RT023 infections was analysed in comparison with known 'hypervirulent' and non-hypervirulent strains, using data from the Netherlands national C. difficile surveillance programme. European RT023 strains of diverse origin were collected and whole-genome sequenced to determine the genetic similarity between isolates. Distinctive features were investigated and characterized. RESULTS: Clinical presentation of C. difficile RT023 infections show severe infections akin to those seen with 'hypervirulent' strains from clades 2 (RT027) and 5 (RT078) (35%, 29% and 27% severe CDI, respectively), particularly with significantly more bloody diarrhoea than RT078 and non-hypervirulent strains (RT023 8%, other RTs 4%, p 0.036). The full genome sequence of strain CD305 is presented as a robust reference. Phylogenetic comparison of CD305 and a further 79 previously uncharacterized European RT023 strains of diverse origin revealed minor genetic divergence with >99.8% pairwise identity between strains. Analyses revealed distinctive features among clade 3 strains, including conserved pathogenicity locus, binary toxin and phage insertion toxin genotypes, glycosylation of S-layer proteins, presence of the RT078 four-gene trehalose cluster and an esculinase-negative genotype. CONCLUSIONS: Given their recent emergence, virulence and genomic characteristics, the surveillance of clade 3 strains should be more highly prioritized.


Subject(s)
Clostridioides difficile/classification , Clostridioides difficile/pathogenicity , Clostridium Infections/microbiology , Adolescent , Adult , Aged , Aged, 80 and over , Bacterial Typing Techniques , Child , Child, Preschool , Clostridium Infections/epidemiology , Cohort Studies , Diarrhea/microbiology , Female , High-Throughput Nucleotide Sequencing , Hospitals/statistics & numerical data , Humans , Infant , Male , Middle Aged , Multilocus Sequence Typing , Netherlands/epidemiology , Phylogeny , Ribotyping , Sentinel Surveillance , Young Adult
3.
Clin Microbiol Infect ; 25(7): 904.e1-904.e7, 2019 Jul.
Article in English | MEDLINE | ID: mdl-31130255

ABSTRACT

OBJECTIVES: Clostridioides difficile infection (CDI) has become the main cause of nosocomial infective diarrhoea. To survey and control the spread of different C. difficile strains, there is a need for suitable rapid tests. The aim of this study was to identify peptide/protein markers for the rapid recognition of C. difficile strains by matrix-assisted laser desorption/ionization mass spectrometry (MALDI-MS). METHODS: We analysed 44 well-characterized strains, belonging to eight different multi-locus sequence types (MLST), using ultrahigh-resolution Fourier transform ion cyclotron resonance (FTICR) MS. The amino acid sequence of two peptide markers specific for MLST-1 and MLST-11 strains was elucidated by MALDI-TOF-MS/MS. The investigation of 2689 C. difficile genomes allowed the determination of the sensitivity and specificity of these markers. C18-solid-phased extraction was used to enrich the MLST-1 marker. RESULTS: Two peptide markers (m/z 4927.81 and m/z 5001.84) were identified and characterized for MLST-1 and MLST-11 strains, respectively. The MLST-1 marker was found in 786 genomes of which three did not belong to MLST-1. The MLST-11 marker was found in 319 genomes, of which 14 did not belong to MLST-11. Importantly, all MLST-1 and MLST-11 genomes were positive for their respective marker. Furthermore, a peptide marker (m/z 5015.86) specific for MLST-15 was found in 59 genomes. We translated our findings into a fast and simple method that allowed the unambiguous identification of the MLST-1 marker on a MALDI-TOF-MS platform. CONCLUSIONS: MALDI-FTICR MS-based peptide profiling resulted in the identification of peptide markers for C. difficile MLST-1 and MLST-11.


Subject(s)
Clostridioides difficile/classification , Multilocus Sequence Typing , Peptides/genetics , Bacterial Typing Techniques , Biomarkers/analysis , Clostridium Infections/diagnosis , Clostridium Infections/microbiology , Genome, Bacterial , Humans , Sensitivity and Specificity , Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization
4.
Nature ; 553(7688): 291-294, 2018 01 18.
Article in English | MEDLINE | ID: mdl-29310122

ABSTRACT

Clostridium difficile disease has recently increased to become a dominant nosocomial pathogen in North America and Europe, although little is known about what has driven this emergence. Here we show that two epidemic ribotypes (RT027 and RT078) have acquired unique mechanisms to metabolize low concentrations of the disaccharide trehalose. RT027 strains contain a single point mutation in the trehalose repressor that increases the sensitivity of this ribotype to trehalose by more than 500-fold. Furthermore, dietary trehalose increases the virulence of a RT027 strain in a mouse model of infection. RT078 strains acquired a cluster of four genes involved in trehalose metabolism, including a PTS permease that is both necessary and sufficient for growth on low concentrations of trehalose. We propose that the implementation of trehalose as a food additive into the human diet, shortly before the emergence of these two epidemic lineages, helped select for their emergence and contributed to hypervirulence.


Subject(s)
Clostridioides difficile/drug effects , Clostridioides difficile/pathogenicity , Clostridium Infections/epidemiology , Clostridium Infections/microbiology , Dietary Sugars/pharmacology , Trehalose/pharmacology , Virulence/drug effects , Animals , Bacterial Proteins/genetics , Bacterial Proteins/metabolism , Clostridioides difficile/genetics , Clostridioides difficile/metabolism , Dietary Sugars/administration & dosage , Dietary Sugars/metabolism , Female , Gastrointestinal Microbiome , Humans , Male , Mice , Mice, Inbred C57BL , Multigene Family , Phosphoenolpyruvate Sugar Phosphotransferase System/genetics , Phosphoenolpyruvate Sugar Phosphotransferase System/metabolism , Point Mutation , Repressor Proteins/genetics , Repressor Proteins/metabolism , Ribotyping , Trehalose/administration & dosage , Trehalose/metabolism
5.
J Clin Microbiol ; 56(3)2018 03.
Article in English | MEDLINE | ID: mdl-29237792

ABSTRACT

The emergence of Clostridium difficile as a significant human diarrheal pathogen is associated with the production of highly transmissible spores and the acquisition of antimicrobial resistance genes (ARGs) and virulence factors. Unlike the hospital-associated C. difficile RT027 lineage, the community-associated C. difficile RT078 lineage is isolated from both humans and farm animals; however, the geographical population structure and transmission networks remain unknown. Here, we applied whole-genome phylogenetic analysis of 248 C. difficile RT078 strains from 22 countries. Our results demonstrate limited geographical clustering for C. difficile RT078 and extensive coclustering of human and animal strains, thereby revealing a highly linked intercontinental transmission network between humans and animals. Comparative whole-genome analysis reveals indistinguishable accessory genomes between human and animal strains and a variety of antimicrobial resistance genes in the pangenome of C. difficile RT078. Thus, bidirectional spread of C. difficile RT078 between farm animals and humans may represent an unappreciated route disseminating antimicrobial resistance genes between humans and animals. These results highlight the importance of the "One Health" concept to monitor infectious disease emergence and the dissemination of antimicrobial resistance genes.


Subject(s)
Animals, Domestic/microbiology , Clostridium Infections/transmission , Communicable Diseases, Emerging/transmission , Drug Resistance, Bacterial/genetics , Zoonoses/transmission , Animals , Clostridioides difficile/classification , Clostridioides difficile/genetics , Clostridioides difficile/isolation & purification , Clostridium Infections/microbiology , Communicable Diseases, Emerging/microbiology , Genome, Bacterial/genetics , Humans , Phylogeography , Zoonoses/microbiology
6.
BMC Pregnancy Childbirth ; 17(1): 439, 2017 12 28.
Article in English | MEDLINE | ID: mdl-29282072

ABSTRACT

BACKGROUND: Few data are available to guide biological sample collection around the time of birth for large-scale birth cohorts. We are designing a large UK birth cohort to investigate the role of infection and the developing immune system in determining future health and disease. We undertook a pilot to develop methodology for the main study, gain practical experience of collecting samples, and understand the acceptability of sample collection to women in late pregnancy. METHODS: Between February-July 2014, we piloted the feasibility and acceptability of collecting maternal stool, baby stool and cord blood samples from participants recruited at prolonged pregnancy and planned pre-labour caesarean section clinics at University College London Hospital. Participating women were asked to complete acceptability questionnaires. RESULTS: Overall, 265 women were approached and 171 (65%) participated, with ≥1 sample collected from 113 women or their baby (66%). Women had a mean age of 34 years, were primarily of white ethnicity (130/166, 78%), and half were nulliparous (86/169, 51%). Women undergoing planned pre-labour caesarean section were more likely than those who delivered vaginally to provide ≥1 sample (98% vs 54%), but less likely to provide maternal stool (10% vs 43%). Pre-sample questionnaires were completed by 110/171 women (64%). Most women reported feeling comfortable with samples being collected from their baby (<10% uncomfortable), but were less comfortable about their own stool (19% uncomfortable) or a vaginal swab (24% uncomfortable). CONCLUSIONS: It is possible to collect a range of biological samples from women around the time of delivery, and this was acceptable for most women. These data inform study design and protocol development for large-scale birth cohorts.


Subject(s)
Feces , Fetal Blood , Maternal Serum Screening Tests/methods , Patient Acceptance of Health Care , Pregnancy, Prolonged/diagnosis , Preoperative Care/methods , Specimen Handling/methods , Adult , Blood Specimen Collection/methods , Blood Specimen Collection/psychology , Cesarean Section , Feasibility Studies , Female , Humans , Longitudinal Studies , Maternal Serum Screening Tests/psychology , Pilot Projects , Pregnancy , Pregnancy, Prolonged/psychology , Preoperative Care/psychology , Specimen Handling/psychology , United Kingdom
8.
J Clin Microbiol ; 55(3): 865-876, 2017 03.
Article in English | MEDLINE | ID: mdl-28031436

ABSTRACT

The diarrheal pathogen Clostridium difficile consists of at least six distinct evolutionary lineages. The RT017 lineage is anomalous, as strains only express toxin B, compared to strains from other lineages that produce toxins A and B and, occasionally, binary toxin. Historically, RT017 initially was reported in Asia but now has been reported worldwide. We used whole-genome sequencing and phylogenetic analysis to investigate the patterns of global spread and population structure of 277 RT017 isolates from animal and human origins from six continents, isolated between 1990 and 2013. We reveal two distinct evenly split sublineages (SL1 and SL2) of C. difficile RT017 that contain multiple independent clonal expansions. All 24 animal isolates were contained within SL1 along with human isolates, suggesting potential transmission between animals and humans. Genetic analyses revealed an overrepresentation of antibiotic resistance genes. Phylogeographic analyses show a North American origin for RT017, as has been found for the recently emerged epidemic RT027 lineage. Despite having only one toxin, RT017 strains have evolved in parallel from at least two independent sources and can readily transmit between continents.


Subject(s)
Clostridioides difficile/classification , Clostridioides difficile/genetics , Clostridium Infections/microbiology , Clostridium Infections/veterinary , Genetic Variation , Phylogeny , Ribotyping , Animals , Clostridioides difficile/isolation & purification , Clostridium Infections/epidemiology , Genome, Bacterial , Global Health , Humans , Molecular Epidemiology , Polymerase Chain Reaction , Sequence Analysis, DNA
9.
J Clin Microbiol ; 53(10): 3141-7, 2015 Oct.
Article in English | MEDLINE | ID: mdl-26179308

ABSTRACT

Clostridium difficile remains the leading cause of nosocomial diarrhea worldwide, which is largely considered to be due to the production of two potent toxins: TcdA and TcdB. However, PCR ribotype (RT) 017, one of five clonal lineages of human virulent C. difficile, lacks TcdA expression but causes widespread disease. Whole-genome sequencing was applied to 35 isolates from hospitalized patients with C. difficile infection (CDI) and two environmental ward isolates in London, England. The phylogenetic analysis of single nucleotide polymorphisms (SNPs) revealed a clonal cluster of temporally variable isolates from a single hospital ward at University Hospital Lewisham (UHL) that were distinct from other London hospital isolates. De novo assembled genomes revealed a 49-kbp putative conjugative transposon exclusive to this hospital clonal cluster which would not be revealed by current typing methodologies. This study identified three sublineages of C. difficile RT017 that are circulating in London. Similar to the notorious RT027 lineage, which has caused global outbreaks of CDI since 2001, the lineage of toxin-defective RT017 strains appears to be continually evolving. By utilization of WGS technologies to identify SNPs and the evolution of clonal strains, the transmission of outbreaks caused by near-identical isolates can be retraced and identified.


Subject(s)
Clostridioides difficile/classification , Clostridium Infections/epidemiology , Cross Infection/epidemiology , Diarrhea/epidemiology , Disease Outbreaks , Enterotoxins/deficiency , Ribotyping , Bacterial Toxins , Clostridioides difficile/genetics , Clostridioides difficile/isolation & purification , Clostridium Infections/microbiology , Cluster Analysis , Cross Infection/microbiology , Diarrhea/microbiology , Genome, Bacterial , Humans , London/epidemiology , Molecular Epidemiology , Phylogeny , Polymerase Chain Reaction , Polymorphism, Single Nucleotide , Sequence Analysis, DNA
10.
Euro Surveill ; 19(45): 20954, 2014 Nov 13.
Article in English | MEDLINE | ID: mdl-25411691

ABSTRACT

Farm animals are a potential reservoir for human Clostridium difficile infection (CDI), particularly PCR ribotype 078 which is frequently found in animals and humans. Here, whole genome single-nucleotide polymorphism (SNP) analysis was used to study the evolutionary relatedness of C. difficile 078 isolated from humans and animals on Dutch pig farms. All sequenced genomes were surveyed for potential antimicrobial resistance determinants and linked to an antimicrobial resistance phenotype. We sequenced the whole genome of 65 C. difficile 078 isolates collected between 2002 and 2011 from pigs (n = 19), asymptomatic farmers (n = 15) and hospitalised patients (n = 31) in the Netherlands. The collection included 12 pairs of human and pig isolates from 2011 collected at 12 different pig farms. A mutation rate of 1.1 SNPs per genome per year was determined for C. difficile 078. Importantly, we demonstrate that farmers and pigs were colonised with identical (no SNP differences) and nearly identical (less than two SNP differences) C. difficile clones. Identical tetracycline and streptomycin resistance determinants were present in human and animal C. difficile 078 isolates. Our observation that farmers and pigs share identical C. difficile strains suggests transmission between these populations, although we cannot exclude the possibility of transmission from a common environmental source.


Subject(s)
Clostridioides difficile/classification , Clostridioides difficile/genetics , Polymorphism, Single Nucleotide/genetics , Ribotyping , Swine Diseases/microbiology , Animal Husbandry , Animals , Anti-Bacterial Agents , Clostridioides difficile/isolation & purification , Clostridium Infections/epidemiology , Clostridium Infections/microbiology , Clostridium Infections/veterinary , Genome-Wide Association Study , Genotype , Humans , Netherlands/epidemiology , Polymerase Chain Reaction/methods , Polymerase Chain Reaction/veterinary , Sus scrofa , Swine
11.
Euro Surveill ; 18(4): 20381, 2013 Jan 24.
Article in English | MEDLINE | ID: mdl-23369393

ABSTRACT

Molecular typing is an essential tool to monitor Clostridium difficile infections and outbreaks within healthcare facilities. Molecular typing also plays a key role in defining the regional and global changes in circulating C. difficile types. The patterns of C. difficile types circulating within Europe (and globally) remain poorly understood, although international efforts are under way to understand the spatial and temporal patterns of C. difficile types. A complete picture is essential to properly investigate type-specific risk factors for C. difficile infections (CDI) and track long-range transmission. Currently, conventional agarose gel-based polymerase chain reaction (PCR) ribotyping is the most common typing method used in Europe to type C. difficile. Although this method has proved to be useful to study epidemiology on local, national and European level, efforts are made to replace it with capillary electrophoresis PCR ribotyping to increase pattern recognition, reproducibility and interpretation. However, this method lacks sufficient discriminatory power to study outbreaks and therefore multilocus variable-number tandem repeat analysis (MLVA) has been developed to study transmission between humans, animals and food. Sequence-based methods are increasingly being used for C. difficile fingerprinting/typing because of their ability to discriminate between highly related strains, the ease of data interpretation and transferability of data. The first studies using whole-genome single nucleotide polymorphism typing of healthcare-associated C. difficile within a clinically relevant timeframe are very promising and, although limited to select facilities because of complex data interpretation and high costs, these approaches will likely become commonly used over the coming years.


Subject(s)
Clostridioides difficile/genetics , DNA, Bacterial/genetics , Minisatellite Repeats/genetics , Molecular Epidemiology/methods , Multilocus Sequence Typing/methods , Clostridium Infections/epidemiology , Clostridium Infections/microbiology , Clostridium Infections/transmission , DNA Fingerprinting/methods , Disease Outbreaks/prevention & control , Europe/epidemiology , Humans , Molecular Epidemiology/trends , Multilocus Sequence Typing/trends , Polymerase Chain Reaction/methods , Polymorphism, Restriction Fragment Length , Population Surveillance/methods , Ribotyping
12.
FEMS Microbiol Lett ; 224(1): 1-15, 2003 Jul 15.
Article in English | MEDLINE | ID: mdl-12855161

ABSTRACT

The F sex factor of Escherichia coli is a paradigm for bacterial conjugation and its transfer (tra) region represents a subset of the type IV secretion system (T4SS) family. The F tra region encodes eight of the 10 highly conserved (core) gene products of T4SS including TraAF (pilin), the TraBF, -KF (secretin-like), -VF (lipoprotein) and TraCF (NTPase), -EF, -LF and TraGF (N-terminal region) which correspond to TrbCP, -IP, -GP, -HP, -EP, -JP, DP and TrbLP, respectively, of the P-type T4SS exemplified by the IncP plasmid RP4. F lacks homologs of TrbBP (NTPase) and TrbFP but contains a cluster of genes encoding proteins essential for F conjugation (TraFF, -HF, -UF, -WF, the C-terminal region of TraGF, and TrbCF) that are hallmarks of F-like T4SS. These extra genes have been implicated in phenotypes that are characteristic of F-like systems including pilus retraction and mating pair stabilization. F-like T4SS systems have been found on many conjugative plasmids and in genetic islands on bacterial chromosomes. Although few systems have been studied in detail, F-like T4SS appear to be involved in the transfer of DNA only whereas P- and I-type systems appear to transport protein or nucleoprotein complexes. This review examines the similarities and differences among the T4SS, especially F- and P-like systems, and summarizes the properties of the F transfer region gene products.


Subject(s)
Conjugation, Genetic/physiology , Escherichia coli/genetics , F Factor/genetics , F Factor/metabolism , Amino Acid Sequence , Molecular Sequence Data , Phylogeny
13.
Mol Microbiol ; 42(3): 705-15, 2001 Nov.
Article in English | MEDLINE | ID: mdl-11722736

ABSTRACT

Conjugal transfer of IncHI plasmid DNA between Gram-negative bacteria is temperature sensitive, as mating is optimal between 22 degrees C and 30 degrees C but is inhibited at 37 degrees C. R27, isolated from Salmonella enterica serovar Typhi, is an IncHI1 plasmid of 180 kbp that has been sequenced completely. The gene encoding green fluorescent protein (GFP) was inserted into R27 in frame with trhC. TrhC is a mating pair formation (Mpf) protein that is essential for plasmid transfer and H-pilus production. Fluorescence microscopy allowed visualization of the TrhC-GFP fusion protein, and Escherichia coli cells were examined for the subcellular localization and temperature-dependent production of TrhC-GFP. At 27 degrees C, TrhC-GFP was found at the periphery of cells as discrete foci, indicating an association of TrhC within protein complexes in the bacterial cell membrane, whereas at 37 degrees C, little fluorescence was detected. These foci probably represent the intracellular position of protein complexes involved in conjugative transfer, as the formation of foci was dependent upon the presence of other Mpf proteins. During temperature shift experiments from 37 degrees C to 27 degrees C, a long lag period was required for generation of GFP foci. Conversely, during short shifts from 27 degrees C to 37 degrees C, the GFP foci remained stable. These results suggest that the expression of transfer genes in the Tra2 region of R27 is temperature dependent. Subcellular localization of TrhC was verified by cellular fractionation. Expression patterns of TrhC-GFP were confirmed with immunoblot analysis and reverse transcriptase-polymerase chain reaction (RT-PCR). These results allow us to propose mechanisms to explain the temperature-sensitive transfer of R27.


Subject(s)
Bacterial Proteins/metabolism , Carrier Proteins/metabolism , Conjugation, Genetic , Plasmids/genetics , Salmonella typhi/genetics , Bacterial Proteins/genetics , Carrier Proteins/genetics , Cell Membrane/metabolism , Fimbriae, Bacterial/metabolism , Green Fluorescent Proteins , Luminescent Proteins/genetics , Luminescent Proteins/metabolism , Recombinant Fusion Proteins/genetics , Recombinant Fusion Proteins/metabolism , Salmonella typhi/metabolism , Subcellular Fractions/metabolism , Temperature
14.
Nucleic Acids Res ; 28(10): 2177-86, 2000 May 15.
Article in English | MEDLINE | ID: mdl-10773089

ABSTRACT

Salmonella typhi, the causative agent of typhoid fever, annually infects 16 million people and kills 600 000 world wide. Plasmid-encoded multiple drug resistance in S. typhi is always encoded by plasmids of incompatibility group H (IncH). The complete DNA sequence of the large temperature-sensitive conjugative plasmid R27, the prototype for the IncHI1 family of plasmids, has been compiled and analyzed. This 180 kb plasmid contains 210 open reading frames (ORFs), of which 14 have been previously identified and 56 exhibit similarity to other plasmid and prokaryotic ORFs. A number of insertion elements were found, including the full Tn 10 transposon, which carries tetracycline resistance genes. Two transfer regions, Tra1 and Tra2, are present, which are separated by a minimum of 64 kb. Homologs of the DNA-binding proteins TlpA and H-NS that act as temperature-regulated repressors in other systems have been located in R27. Sequence analysis of transfer and replication regions supports a mosaic-like structure for R27. The genes responsible for conjugation and plasmid maintenance have been identified and mechanisms responsible for thermosensitive transfer are discussed.


Subject(s)
Drug Resistance, Multiple/genetics , R Factors/chemistry , Salmonella typhi/genetics , Amino Acid Sequence , Base Sequence , Conjugation, Genetic , DNA Nucleotidyltransferases/chemistry , DNA Nucleotidyltransferases/genetics , Deoxyribonuclease I/chemistry , Deoxyribonuclease I/genetics , Molecular Sequence Data , Open Reading Frames , Sequence Alignment , Sequence Homology, Amino Acid , Temperature
15.
Plasmid ; 43(3): 235-9, 2000 May.
Article in English | MEDLINE | ID: mdl-10783303

ABSTRACT

An analysis of the complete nucleotide sequence of the composite tetracycline-resistance transposon Tn10 (9147 bp) from the Salmonella typhi conjugative plasmid R27 is presented. A comparison of the protein sequences from IS10-right and IS10-left transposases has identified four amino acid differences. These residues appear to play an important role in normal transposase function and may account for the differences in exhibited transposition activities. The tetracycline determinants encoded by this version of Tn10 share >99% identity with those of Tn10(R100), demonstrating the conservation that exists between these transposons. A previously uncharacterized approximately 3000-bp region of Tn10 contains four putative open reading frames. One of these open reading frames shares 55% identity with the glutamate permease protein sequence from Haemophilus influenzae although it was unable to complement an Escherichia coli glutamate permease mutant, with which it shares 51% identity. The three remaining putative open reading frames are arranged as a discrete genetic unit adjacent to the glutamate permease homolog and are transcribed in the opposite direction. Two of these open reading frames are homologous with Bacillus subtilis proteins of unknown functions while the other has no homologs in the database. The presence of an aminoacyl-tRNA synthetase class II motif in one of these open reading frames in combination with the glutamate permease homolog allows us to postulate that this region of Tn10 could once have played a role in amino acid metabolism.


Subject(s)
Amino Acid Transport Systems, Acidic , DNA Transposable Elements , Membrane Transport Proteins/genetics , Tetracycline Resistance/genetics , Amino Acid Sequence , Escherichia coli/genetics , Escherichia coli Proteins , Genetic Complementation Test , Membrane Transport Proteins/metabolism , Molecular Sequence Data , Mutation , Plasmids/genetics , Sequence Analysis, DNA , Sequence Homology, Amino Acid , Symporters
16.
Plasmid ; 41(3): 207-18, 1999 May.
Article in English | MEDLINE | ID: mdl-10366526

ABSTRACT

IncHI1 plasmids are one of the few plasmids known to mediate multiple antibiotic resistance in Salmonella typhi. These plasmids are temperature-sensitive for transfer and R27 is the prototype plasmid. DNA sequencing within the Tra2 region of R27, encoding genes involved in mating pair formation, identified trhC encoding the TrhC protein of 90,000 Da, which was visualized using an in vitro transcription/translation system. Expression of the TrhC protein also identified two smaller protein products of approximately 23 and 25 kDa which are predicted to be protease digestion products. The migration of these smaller products changed when the reactions were run at 28 vs 37 degrees C. The TrhC protein contained a putative nucleotide triphosphate-binding protein and exhibited sequence similarities with several other proteins implicated in bacterial conjugation, including TraC (F), TraB (pKM101), VirB4 (Ti), TrbE (RP4). Phylogenetic analysis showed TrhC was most closely related to TraC. Mini-Tn10 insertions in trhC generated transfer defective mutants, and no pili were specified by the trhC mutants. The trhC gene appeared to be a hot spot for transposon insertion as 37.5% mapped into this open reading frame. One trhC mutant, pDT2990, was able to be complemented by a cloned trhC gene giving a transfer frequency of 1 x 10(-3) transconjugants per recipient in an 18-h mating, whereas the wild-type transfer frequency of R27 was 1 x 10(-2) transconjugants per recipient.


Subject(s)
Genes, Bacterial , Plasmids/genetics , Salmonella typhi/genetics , Amino Acid Sequence , Bacterial Proteins/genetics , Bacterial Proteins/metabolism , Base Sequence , Binding Sites/genetics , Carrier Proteins/genetics , Carrier Proteins/metabolism , DNA Primers/genetics , DNA, Bacterial/genetics , Gene Transfer Techniques , Molecular Sequence Data , Mutagenesis, Insertional , Nucleotides/metabolism , Phylogeny , Restriction Mapping , Salmonella typhi/metabolism , Sequence Homology, Amino Acid
17.
Plasmid ; 41(3): 226-39, 1999 May.
Article in English | MEDLINE | ID: mdl-10366528

ABSTRACT

In this study, the DNA sequence of one of the transfer regions of the IncHI1 plasmid R27 was determined. This region, which corresponds to coordinates 0-40 on the R27 map has been called the Tra2 region, and is believed to be involved in mating pair formation. DNA sequence analysis of the transfer region identified 11 open reading frames which showed similarities to the transfer genes from other conjugative systems. The R27 transfer genes appear to most closely resemble the genes from the F plasmid and Sphingomonas aromaticivorans plasmid pNL1, both within the individual genes and in the overall gene order. The Tra2 region is also distinct in that replication, partitioning, and stability genes are found in the middle of the transfer region. The R27 Tra2 region also contains a gene, trhF, which appears to be related to the TraF genes of Agrobacterium and Rhizobium species. This, along with the temperature-sensitive transfer system found in both H plasmids and Agrobacterium, leads to the speculation that the R27 transfer region evolved from both ancestral F-like and P-like plasmids.


Subject(s)
Plasmids/genetics , Agrobacterium tumefaciens/genetics , Amino Acid Sequence , Bacterial Proteins/genetics , Base Sequence , Cloning, Molecular , Conjugation, Genetic , DNA Primers/genetics , Genes, Bacterial , Molecular Sequence Data , Mutagenesis, Insertional , Restriction Mapping , Rhizobium/genetics , Sequence Homology, Amino Acid , Zymomonas/genetics
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