scholarly journals Bacteroides fragilis Transfer Factor Tn5520: the Smallest Bacterial Mobilizable Transposon Containing Single Integrase and Mobilization Genes That Function inEscherichia coli

1999 ◽  
Vol 181 (8) ◽  
pp. 2564-2571 ◽  
Author(s):  
Gayatri Vedantam ◽  
Thomas J. Novicki ◽  
David W. Hecht
Keyword(s):  
Antibiotic Resistance ◽  
Sequence Similarity ◽  
Shuttle Vector ◽  
Insertion Site ◽  
Bacteroides Fragilis ◽  
Open Reading Frames ◽  
Transfer Factors ◽  
E Coli ◽  
Isolation And Characterization

ABSTRACT Many bacterial genera, including Bacteroides spp., harbor mobilizable transposons, a class of transfer factors that carry genes for conjugal DNA transfer and, in some cases, antibiotic resistance. Mobilizable transposons are capable of inserting into and mobilizing other, nontransferable plasmids and are implicated in the dissemination of antibiotic resistance. This paper presents the isolation and characterization of Tn5520, a new mobilizable transposon from Bacteroides fragilis LV23. At 4,692 bp, it is the smallest mobilizable transposon reported from any bacterial genus. Tn5520 was captured from B. fragilis LV23 by using the transfer-deficient shuttle vector pGAT400ΔBglII. The termini of Tn5520 contain a 22-bp imperfect inverted repeat, and transposition does not result in a target site repeat. Tn5520 also demonstrates insertion site sequence preferences characterized by A-T-rich nucleotide sequences. Tn5520 has been sequenced in its entirety, and two large open reading frames whose predicted protein products exhibit strong sequence similarity to recombinase-integrase enzymes and mobilization proteins, respectively, have been identified. The transfer, mobilization, and transposition properties of Tn5520 have been studied, revealing that Tn5520mobilizes plasmids in both B. fragilis andEscherichia coli at high frequency and also transposes inE. coli.

scholarly journals Isolation and Characterization of BTF-37: Chromosomal DNA Captured from Bacteroides fragilis That Confers Self-Transferability and Expresses a Pilus-Like Structure in Bacteroides spp. and Escherichia coli

2002 ◽  
Vol 184 (3) ◽  
pp. 728-738 ◽  
Author(s):  
Gayatri Vedantam ◽  
David W. Hecht
Keyword(s):  
Escherichia Coli ◽  
Transfer Factor ◽  
Shuttle Vector ◽  
Bacteroides Fragilis ◽  
Functional Assay ◽  
Broad Host Range ◽  
Chromosomal Dna ◽  
E Coli ◽  
Isolation And Characterization

ABSTRACT We report the isolation and preliminary characterization of BTF-37, a new 52-kb transfer factor isolated from Bacteroides fragilis clinical isolate LV23. BTF-37 was obtained by the capture of new DNA in the nonmobilizable Bacteroides-Escherichia coli shuttle vector pGAT400ΔBglII using a functional assay. BTF-37 is self-transferable within and from Bacteroides and also self-transfers in E. coli. Partial DNA sequencing, colony hybridization, and PCR revealed the presence of Tet element-specific sequences in BTF-37. In addition, Tn5520, a small mobilizable transposon that we described previously (G. Vedantam, T. J. Novicki, and D. W. Hecht, J. Bacteriol. 181:2564–2571, 1999), was also coisolated within BTF-37. Scanning and transmission electron microscopy of Tet element-containing Bacteroides spp. and BTF-37-harboring Bacteroides and E. coli strains revealed the presence of pilus-like cell surface structures. These structures were visualized in Bacteroides spp. only when BTF-37 and Tet element strains were induced with subinhibitory concentrations of tetracycline and resembled those encoded by E. coli broad-host-range plasmids. We conclude that we have captured a new, self-transferable transfer factor from B. fragilis LV23 and that this new factor encodes a tetracycline-inducible Bacteroides sp. conjugation apparatus.

scholarly journals Isolation and Characterization of cLV25, a Bacteroides fragilis Chromosomal Transfer Factor Resembling Multiple Bacteroides sp. Mobilizable Transposons

2002 ◽  
Vol 184 (7) ◽  
pp. 1895-1904 ◽  
Author(s):  
Kathleen A. Bass ◽  
David W. Hecht
Keyword(s):  
Transfer Factor ◽  
Sequence Similarity ◽  
Shuttle Vector ◽  
Bacteroides Fragilis ◽  
Antibiotic Resistance Genes ◽  
Dna Transfer ◽  
Conjugative Plasmid ◽  
Significant Similarity ◽  
Transfer Factors ◽  
Isolation And Characterization

ABSTRACT Horizontal DNA transfer contributes significantly to the dissemination of antibiotic resistance genes in Bacteroides fragilis. To further our understanding of DNA transfer in B. fragilis, we isolated and characterized a new transfer factor, cLV25. cLV25 was isolated from B. fragilis LV25 by its capture on the nonmobilizable Escherichia coli-Bacteroides shuttle vector pGAT400ΔBglII. Similar to other Bacteroides sp. transfer factors, cLV25 was mobilized in E. coli by the conjugative plasmid R751. Using Tn1000 mutagenesis and deletion analysis of cLV25, two mobilization genes, bmgA and bmgB, were identified, whose predicted proteins have similarity to DNA relaxases and mobilization proteins, respectively. In particular, BmgA and BmgB were homologous to MocA and MocB, respectively, the two mobilization proteins of the B. fragilis mobilizable transposon Tn4399. A cis-acting origin of transfer (oriT) was localized to a 353-bp region that included nearly all of the intergenic region between bmgB and orf22 and overlapped with the 3′ end of orf22. This oriT contained a putative nic site sequence but showed no significant similarity to the oriT regions of other transfer factors, including Tn4399. Despite the lack of sequence similarity between the oriTs of cLV25 and Tn4399, a mutation in the cLV25 putative DNA relaxase, bmgA, was partially complemented by Tn4399. In addition to the functional cross-reaction with Tn4399, a second distinguishing feature of cLV25 is that predicted proteins have similarity to proteins encoded not only by Tn4399 but by several Bacteroides sp. transfer factors, including NBU1, NBU2, CTnDOT, Tn4555, and Tn5520.

scholarly journals Isolation and characterization of bacteriophages that infect Citrobacter rodentium, a model pathogen for investigating human intestinal diseases

2018 ◽  
Author(s):  
Carolina M. Mizuno ◽  
Tiffany Luong ◽  
Robert Cedarstrom ◽  
Mart Krupovic ◽  
Laurent Debarbieux ◽  
...  
Keyword(s):  
Antibiotic Resistance ◽  
Phage Therapy ◽  
Limit Of Detection ◽  
Lytic Cycle ◽  
Citrobacter Rodentium ◽  
E Coli ◽  
Isolation And Characterization ◽  
A New Species

AbstractEnteropathogenic Escherichia coli (EPEC) is a major etiology for diarrheal diseases among children. Antibiotics, when used appropriately, are effective; however, their overuse and misuse has led to the rise of antibiotic resistance worldwide. Thus, there are renewed efforts into the development of phage therapy. Due to the drawbacks of EPEC in vivo models, a surrogate is the mouse-restricted gut pathgoen Citrobacter rodentium. In this study, two new phages CrRp3 and CrRp10, which infect C. rodentium, were isolated and characterized. CrRp3 was found to be a new species within the genus Vectrevirus and CrRp10 is a new strain within the genus Tequatrovirus. Neither phage carries known genes associated with bacterial virulence, antibiotic resistance, or lysogeny. CrRp3 and CrRp10 appear to have independently evolved from E. coli phages. CrRp3 appears to be the more ‘potent’ being 24x more likely to find a host cell and has a shorter lytic cycle, while CrRp10 at MOI 0.001 was able to maintain bacterial density below the limit of detection after 18 h. We found that hypoxia (5% O2 and 5% CO2) inhibited CrRp3 ability to reverse exponential bacterial growth. It is unclear whether the subtle characteristic differences between CrRp3 and CrRp10 will influence treatment efficacy in future phage therapy in vivo investigations.

scholarly journals Functional Analysis of Genes in the rfbLocus of Leptospira borgpetersenii Serovar Hardjo Subtype Hardjobovis

2000 ◽  
Vol 68 (7) ◽  
pp. 3793-3798 ◽  
Author(s):  
Dieter M. Bulach ◽  
Thareerat Kalambaheti ◽  
Alejandro de la Peña-Moctezuma ◽  
Ben Adler
Keyword(s):  
Sequence Similarity ◽  
Bacterial Species ◽  
Open Reading Frames ◽  
New Host ◽  
E Coli ◽  
Serovar Typhimurium ◽  
Genes Encoding ◽  
Leptospira Borgpetersenii ◽  
Nucleotide Sugars

ABSTRACT Lipopolysaccharide (LPS) is a key antigen in immunity to leptospirosis. Its biosynthesis requires enzymes for the biosynthesis and polymerization of nucleotide sugars and the transport through and attachment to the bacterial membrane. The genes encoding these functions are commonly clustered into loci; for Leptospira borgpetersenii serovar Hardjo subtype Hardjobovis, this locus, named rfb, spans 36.7 kb and contains 31 open reading frames, of which 28 have been assigned putative functions on the basis of sequence similarity. Characterization of the function of these genes is hindered by the fact that it is not possible to construct isogenic mutant strains in Leptospira. We used two approaches to circumvent this problem. The first was to clone the entire locus into a heterologous host system and determine if a “recombinant” LPS or polysaccharide was synthesized in the new host. The second approach used putative functions to identify mutants in other bacterial species whose mutations might be complemented by genes on the leptospiralrfb locus. This approach was used to investigate the function of three genes in the leptospiral rfb locus and demonstrated function for orfH10, which complemented awbpM strain of Pseudomonas aeruginosa, andorfH13, which complemented an rfbW strain ofVibrio cholerae. However, despite the similarity of OrfH11 to WecC, a wecC strain of E. coli was not complemented by orfH11. The predicted protein encoded byorfH8 is similar to GalE from a number of organisms. ASalmonella enterica serovar Typhimurium strain producing no GalE was used as a background in which orfH8 produced detectable GalE enzyme activity.

Biochemical characterization of the β-1,4-glucuronosyltransferase GelK in the gellan gum-producing strain Sphingomonas paucimobilis A.T.C.C. 31461

2001 ◽  
Vol 358 (2) ◽  
pp. 457-464 ◽  
Author(s):  
Paula VIDEIRA ◽  
Arsénio FIALHO ◽  
Roberto A. GEREMIA ◽  
Christelle BRETON ◽  
Isabel SÁ-CORREIA
Keyword(s):  
Glucuronic Acid ◽  
Biochemical Characterization ◽  
Sequence Similarity ◽  
Enzymic Activity ◽  
Gellan Gum ◽  
Open Reading Frames ◽  
Sphingomonas Paucimobilis ◽  
E Coli ◽  
Repeat Units

Biosynthesis of bacterial polysaccharide-repeat units proceeds by sequential transfer of sugars, from the appropriate sugar donor to an activated lipid carrier, by committed glycosyltransferases (GTs). Few studies on the mechanism of action for this type of GT are available. Sphingomonas paucimobilis A.T.C.C. 31461 produces the industrially important polysaccharide gellan gum. We have cloned the gelK gene from S. paucimobilis A.T.C.C. 31461. GelK belongs to family 1 of the GT classification [Campbell, Davies, Bulone, Henrissat (1997) Biochem. J. 326, 929–939]. Sequence similarity studies suggest that GelK consists of two protein modules corresponding to the -NH2 and -CO2H halves, the latter possibly harbouring the GT activity. The gelK gene and the open reading frames coding for the -NH2 (GelKNH2) and -CO2H (GelKCOOH) halves were overexpressed in Escherichia coli. GelK and GelKNH2 were present in both the soluble and membrane fractions of E. coli, whereas GelKCOOH was only present in the soluble fraction. GelK catalysed the transfer of [14C]glucuronic acid from UDP-[14C]glucuronic acid into a glycolipid extracted from S. paucimobilis or E. coli, even in the presence of EDTA, and the radioactive sugar was released from the glycolipid by β-1,4-glucuronidase. GelK was not able to use synthetic glucosyl derivatives as acceptors, indicating that the PPi-lipid moiety is needed for enzymic activity. Recombinant GelKNH2 and GelKCOOH did not show detectable activity. Based on the biochemical characteristics of GelK and on sequence similarities with N-acetylglucosaminyltransferase, we propose that GT families 1 and 28 form a superfamily.

scholarly journals Isolation and Characterization of Salmonella Jumbo-Phage pSal-SNUABM-04

Viruses ◽  
2020 ◽  
Vol 13 (1) ◽  
pp. 27
Author(s):  
Jun Kwon ◽  
Sang Guen Kim ◽  
Hyoun Joong Kim ◽  
Sib Sankar Giri ◽  
Sang Wha Kim ◽  
...  
Keyword(s):  
Morphological Traits ◽  
Open Reading Frames ◽  
Genome Comparison ◽  
The Novel ◽  
Promising Alternative ◽  
Isolation And Characterization ◽  
Global Issue ◽  
Reading Frames ◽  
Predicted Function

The increasing emergence of antimicrobial resistance has become a global issue. Therefore, many researchers have attempted to develop alternative antibiotics. One promising alternative is bacteriophage. In this study, we focused on a jumbo-phage infecting Salmonella isolated from exotic pet markets. Using a Salmonella strain isolated from reptiles as a host, we isolated and characterized the novel jumbo-bacteriophage pSal-SNUABM-04. This phage was investigated in terms of its morphology, host infectivity, growth and lysis kinetics, and genome. The phage was classified as Myoviridae based on its morphological traits and showed a comparatively wide host range. The lysis efficacy test showed that the phage can inhibit bacterial growth in the planktonic state. Genetic analysis revealed that the phage possesses a 239,626-base pair genome with 280 putative open reading frames, 76 of which have a predicted function and 195 of which have none. By genome comparison with other jumbo phages, the phage was designated as a novel member of Machinavirus composed of Erwnina phages.

scholarly journals Isolation and characterization of escherichia coli in ready-to-eat foods vended in Islamic University, Kushtia

1970 ◽  
Vol 18 ◽  
pp. 99-103 ◽  
Author(s):  
S Biswas ◽  
MAK Parvez ◽  
M Shafiquzzaman ◽  
S Nahar ◽  
MN Rahman
Keyword(s):  
Escherichia Coli ◽  
Pattern Analysis ◽  
University Campus ◽  
Rflp Pattern ◽  
E Coli ◽  
Fish Meat ◽  
Isolation And Characterization ◽  
Food Borne ◽  
Identification And Characterization

Context: Escherichia coli is shed in the feces of warm blooded animals and humans and thus potential for public health. Detection and characterization of E. coli in the ready-to-eat (RTE) foods concerns due to their presence indicates fecal contamination of the food.   Objective: To identify, characterize and RFLP pattern analysis of E. coli isolated from RTE foods vended in Islamic University campus, Kushtia.   Materials and Methods: Fifty samples from four types of consumed foods in six student halls of residence, some temporary restaurants of Islamic University, Kushtia were assessed for bacterial contamination by standard methods. Identification and characterization of E. coli isolates were performed using IMViC tests. Genomic DNA was used to perform RFLP pattern analysis.   Results: Thirty seven out of 50 (74%) examined samples of RTE foods had E. coli contamination. The highest number of E. coli was isolated from vegetable oriented RTE foods (90.90%) and fish, meat and cereals samples were also significantly E. coli positive. RFLP profiling of two E. coli isolates were observed.   Conclusion: The results of this study provide evidence that some RTE foods had unsatisfactory levels of contamination with E. coli. Thus street vended RTE food could be important potential vehicles for food-borne diseases. Molecular characterization may be exploited to identify food borne pathogen among different species.  Keywords: Ready-to-eat foods; Escherichia coli; RFLP pattern DOI: http://dx.doi.org/10.3329/jbs.v18i0.8783 JBS 2010; 18(0): 99-103

scholarly journals Incidence and Characterization of Integrons, Genetic Elements Mediating Multiple-Drug Resistance, in AvianEscherichia coli

1999 ◽  
Vol 43 (12) ◽  
pp. 2925-2929 ◽  
Author(s):  
Lydia Bass ◽  
Cynthia A. Liebert ◽  
Margie D. Lee ◽  
Anne O. Summers ◽  
David G. White ◽  
...  
Keyword(s):  
Antibiotic Resistance ◽  
Resistance Gene ◽  
Multiple Drug Resistance ◽  
Marker Gene ◽  
Clinical Isolates ◽  
Multiple Antibiotic Resistance ◽  
Class 1 Integron ◽  
E Coli ◽  
Class 1

ABSTRACT Antibiotic resistance among avian bacterial isolates is common and is of great concern to the poultry industry. Approximately 36% (n = 100) of avian, pathogenic Escherichia coli isolates obtained from diseased poultry exhibited multiple-antibiotic resistance to tetracycline, oxytetracycline, streptomycin, sulfonamides, and gentamicin. Clinical avian E. coli isolates were further screened for the presence of markers for class 1 integrons, the integron recombinase intI1 and the quaternary ammonium resistance gene qacEΔ1, in order to determine the contribution of integrons to the observed multiple-antibiotic resistance phenotypes. Sixty-three percent of the clinical isolates were positive for the class 1 integron markersintI1 and qacEΔ1. PCR analysis with the conserved class 1 integron primers yielded amplicons of approximately 1 kb from E. coli isolates positive for intI1 andqacEΔ1. These PCR amplicons contained the spectinomycin-streptomycin resistance gene aadA1. Further characterization of the identified integrons revealed that many were part of the transposon Tn21, a genetic element that encodes both antibiotic resistance and heavy-metal resistance to mercuric compounds. Fifty percent of the clinical isolates positive for the integron marker gene intI1 as well as for theqacEΔ1 and aadA1 cassettes also contained the mercury reductase gene merA. The correlation between the presence of the merA gene with that of the integrase and antibiotic resistance genes suggests that these integrons are located in Tn21. The presence of these elements among avianE. coli isolates of diverse genetic makeup as well as inSalmonella suggests the mobility of Tn21 among pathogens in humans as well as poultry.

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