scholarly journals Complete Nucleotide Sequence of the Conjugative Tetracycline Resistance Plasmid pFBAOT6, a Member of a Group of IncU Plasmids with Global Ubiquity

2004 ◽  
Vol 70 (12) ◽  
pp. 7497-7510 ◽  
Author(s):  
Glenn Rhodes ◽  
Julian Parkhill ◽  
Christine Bird ◽  
Kerrie Ambrose ◽  
Matthew C. Jones ◽  
...  
Keyword(s):  
Secretory Pathway ◽  
Transfer Functions ◽  
Genetic Load ◽  
Tetracycline Resistance ◽  
Complete Sequence ◽  
Pair Formation ◽  
Broad Host Range ◽  
Mating Pair ◽  
Associated Bacteria ◽  
Type Iv

ABSTRACT This study presents the first complete sequence of an IncU plasmid, pFBAOT6. This plasmid was originally isolated from a strain of Aeromonas caviae from hospital effluent (Westmorland General Hospital, Kendal, United Kingdom) in September 1997 (G. Rhodes, G. Huys, J. Swings, P. McGann, M. Hiney, P. Smith, and R. W. Pickup, Appl. Environ. Microbiol. 66:3883-3890, 2000) and belongs to a group of related plasmids with global ubiquity. pFBAOT6 is 84,748 bp long and has 94 predicted coding sequences, only 12 of which do not have a possible function that has been attributed. Putative replication, maintenance, and transfer functions have been identified and are located in a region in the first 31 kb of the plasmid. The replication region is poorly understood but exhibits some identity at the protein level with replication proteins from the gram-positive bacteria Bacillus and Clostridium. The mating pair formation system is a virB homologue, type IV secretory pathway that is similar in its structural organization to the mating pair formation systems of the related broad-host-range (BHR) environmental plasmids pIPO2, pXF51, and pSB102 from plant-associated bacteria. Partitioning and maintenance genes are homologues of genes in IncP plasmids. The DNA transfer genes and the putative oriT site also exhibit high levels of similarity with those of plasmids pIPO2, pXF51, and pSB102. The genetic load region encompasses 54 kb, comprises the resistance genes, and includes a class I integron, an IS630 relative, and other transposable elements in a 43-kb region that may be a novel Tn1721-flanked composite transposon. This region also contains 24 genes that exhibit the highest levels of identity to chromosomal genes of several plant-associated bacteria. The features of the backbone of pFBAOT6 that are shared with this newly defined group of environmental BHR plasmids suggest that pFBAOT6 may be a relative of this group, but a relative that was isolated from a clinical bacterial environment rather than a plant-associated bacterial environment.

scholarly journals Genetic and Sequence Analysis of the pTiC58 trb Locus, Encoding a Mating-Pair Formation System Related to Members of the Type IV Secretion Family

1998 ◽  
Vol 180 (23) ◽  
pp. 6164-6172 ◽  
Author(s):  
Pei-Li Li ◽  
Dawn M. Everhart ◽  
Stephen K. Farrand
Keyword(s):  
Sequence Analysis ◽  
Pair Formation ◽  
Type Iv Secretion ◽  
Ti Plasmid ◽  
Open Reading Frames ◽  
Conjugal Transfer ◽  
Mating Pair ◽  
Secretion Systems ◽  
Type Iv ◽  
And Function

ABSTRACT Conjugal transfer of pTiC58 requires two regions, trawhich contains the oriT and several genes involved in DNA processing and a region of undefined size and function that is located at the 2-o’clock position of the plasmid. Using transposon mutagenesis with Tn3HoHo1 and a binary transfer system, we delimited this second region, called trb, to an 11-kb interval between the loci for vegetative replication and nopaline catabolism. DNA sequence analysis of this region identified 13 significant open reading frames (ORFs) spanning 11,003 bp. The first, encodingtraI, already has been described and is responsible for the synthesis of Agrobacterium autoinducer (AAI) (I. Hwang, P.-L. Li, L. Zhang, K. R. Piper, D. M. Cook, M. E. Tate, and S. K. Farrand, Proc. Natl. Acad. Sci. USA 91:4639–4643, 1994). Translation products of the next 11 ORFs showed similarities to those of trbB, -C, -D,-E, -J, -K, -L,-F, -G, -H, and -I of the trb region of the octopine-type Ti plasmid pTi15955 and of the tra2 core region of RP4. In RP4, these genes encode mating-pair formation functions and are essential for the conjugal transfer of the IncP plasmid. Each of the trb gene homologues is oriented counterclockwise on the Ti plasmid. Expression of these genes, as measured by using the lacZ fusions formed by Tn3HoHo1, required the traI promoter and the transcriptional activator TraR along with its coinducer, AAI. While related to that of RP4, the trb system of pTiC58 did not allow propagation of the trb-specific bacteriophages PRD1, PRR1, and Pf3. The products of several trb genes of the Ti plasmid are similar to those of other loci that encode DNA transfer or protein secretion systems, all of which are members of the type IV secretion family.

scholarly journals Cooperativity between KorB and TrbA Repressors of Broad-Host-Range Plasmid RK2

2001 ◽  
Vol 183 (3) ◽  
pp. 1022-1031 ◽  
Author(s):  
Malgorzata Zatyka ◽  
Lewis Bingle ◽  
Anthony C. Jones ◽  
Christopher M. Thomas
Keyword(s):  
Host Range ◽  
Pair Formation ◽  
Broad Host Range ◽  
Conjugative Transfer ◽  
Mating Pair ◽  
Transcription Start ◽  
Broad Host Range Plasmid ◽  
Plasmid Genes ◽  
High Degree ◽  
Plasmid Rk2

ABSTRACT The KorB and TrbA proteins of broad-host-range plasmid RK2 are key regulators of the plasmid genes required for conjugative transfer.trbBp is the primary promoter responsible for expression of mating pair formation genes. We show that despite the targets for KorB and TrbA at trbBp being about 165 bp apart, 189 bp upstream of the transcription start point and overlapping the −10 region, respectively, these two proteins show up to 10-fold cooperativity for the repression of trbBp. Deletion analysis of TrbA showed that the C-terminal domain (CTD), which has a high degree of sequence conservation with the CTD of KorA, is required for this cooperativity with KorB. Western blotting demonstrated that the apparently mutual enhancement of repression is not due simply to elevation of repressor level by the presence of the second protein, suggesting that the basis for cooperativity is interaction between KorB and TrbA bound at their respective operators.

scholarly journals The IncP-6 Plasmid Rms149 Consists of a Small Mobilizable Backbone with Multiple Large Insertions

2005 ◽  
Vol 187 (14) ◽  
pp. 4728-4738 ◽  
Author(s):  
Anthony S. Haines ◽  
Karen Jones ◽  
Martin Cheung ◽  
Christopher M. Thomas
Keyword(s):  
Transfer Functions ◽  
Complete Sequence ◽  
Broad Host Range ◽  
Replication Proteins ◽  
Incompatibility Group ◽  
E Coli ◽  
Mobilizable Plasmid ◽  
Reference Information ◽  
Broad Host Range Plasmid ◽  
Single Polypeptide

ABSTRACT Plasmid Rms149, the archetype of Pseudomonas plasmid incompatibility group IncP-6, was identified in Pseudomonas aeruginosa as an agent conferring resistance to streptomycin, sulfanilamide, gentamicin, and carbenicillin in 1975. It has been classed as a broad-host-range plasmid due to its ability to replicate in both Escherichia coli (where it is designated IncG) and Pseudomonas species, although both species are γ-proteobacteria. To provide reference information on this Inc group, we have determined the complete sequence of Rms149 and found that, although the genome comprises 57,121 bp, it is essentially a small mobilizable plasmid carrying multiple mobile elements, which make up 79% (>45 kb) of its genome. A replicon has been identified which encodes a single polypeptide with moderate identity to other replication proteins. The region encoding this protein can replicate in Pseudomonas putida and E. coli. This sequence is directly downstream of a putative partitioning region highly similar to that of pRA2. A functional IncQ-type mobilization region is also present. Thus, the backbone appears to be a novel combination of modules already identified in other plasmid systems. Analysis of the segments that fall outside this core of stable inheritance and transfer functions show that this plasmid has been subject to multiple insertion events and that the plasmid appears to carry a considerable load of DNA that no longer should be phenotypically advantageous. The plasmid therefore functions not just as a vehicle for spread of selective traits but also as a store for DNA that is not currently under selection.

scholarly journals A Type II Protein Secretory Pathway Required for Levansucrase Secretion by Gluconacetobacter diazotrophicus

2004 ◽  
Vol 186 (15) ◽  
pp. 5031-5039 ◽  
Author(s):  
Juan G. Arrieta ◽  
Mailin Sotolongo ◽  
Carmen Menéndez ◽  
Dubiel Alfonso ◽  
Luis E. Trujillo ◽  
...  
Keyword(s):  
Codon Usage ◽  
Outer Membrane ◽  
Gene Cluster ◽  
Secretory Pathway ◽  
Cosmid Library ◽  
Polymorphism Analysis ◽  
Type Ii ◽  
Gluconacetobacter Diazotrophicus ◽  
Type Iv ◽  
Geographical Regions

ABSTRACT The endophytic diazotroph Gluconacetobacter diazotrophicus secretes a constitutively expressed levansucrase (LsdA, EC 2.4.1.10) to utilize plant sucrose. LsdA, unlike other extracellular levansucrases from gram-negative bacteria, is transported to the periplasm by a signal-peptide-dependent pathway. We identified an unusually organized gene cluster encoding at least the components LsdG, -O, -E, -F, -H, -I, -J, -L, -M, -N, and -D of a type II secretory system required for LsdA translocation across the outer membrane. Another open reading frame, designated lsdX, is located between the operon promoter and lsdG, but it was not identified in BLASTX searches of the DDBJ/EMBL/GenBank databases. The lsdX, -G, and -O genes were isolated from a cosmid library of strain SRT4 by complementation of an ethyl methanesulfonate mutant unable to transport LsdA across the outer membrane. The downstream genes lsdE, -F, -H, -I, -J, -L, -M, -N, and -D were isolated through chromosomal walking. The high G+C content (64 to 74%) and the codon usage of the genes identified are consistent with the G+C content and codon usage of the standard G. diazotrophicus structural gene. Sequence analysis of the gene cluster indicated that a polycistronic transcript is synthesized. Targeted disruption of lsdG, lsdO, or lsdF blocked LsdA secretion, and the bacterium failed to grow on sucrose. Replacement of Cys162 by Gly at the C terminus of the pseudopilin LsdG abolished the protein functionality, suggesting that there is a relationship with type IV pilins. Restriction fragment length polymorphism analysis revealed conservation of the type II secretion operon downstream of the levansucrase-levanase (lsdA-lsdB) locus in 14 G. diazotrophicus strains representing 11 genotypes recovered from four different host plants in diverse geographical regions. To our knowledge, this is the first report of a type II pathway for protein secretion in the Acetobacteraceae.

scholarly journals A Type IV-Secretion-Like System Is Required for Conjugative DNA Transport of Broad-Host-Range Plasmid pIP501 in Gram-Positive Bacteria

2007 ◽  
Vol 189 (6) ◽  
pp. 2487-2496 ◽  
Author(s):  
Mohammad Y. Abajy ◽  
Jolanta Kopeć ◽  
Katarzyna Schiwon ◽  
Michal Burzynski ◽  
Mike Döring ◽  
...  
Keyword(s):  
Host Range ◽  
Sequence Similarity ◽  
Type Iv Secretion ◽  
Conjugative Plasmid ◽  
Computer Assisted ◽  
Transcriptional Level ◽  
Broad Host Range ◽  
Gram Positive ◽  
Gram Positive Bacteria ◽  
Type Iv

ABSTRACT Plasmid pIP501 has a very broad host range for conjugative transfer among a wide variety of gram-positive bacteria and gram-negative Escherichia coli. Functionality of the pIP501 transfer (tra) genes in E. coli was proven by pIP501 retrotransfer to Enterococcus faecalis (B. Kurenbach, C. Bohn, J. Prabhu, M. Abudukerim, U. Szewzyk, and E. Grohmann, Plasmid 50:86-93, 2003). The 15 pIP501 tra genes are organized in a single operon (B. Kurenbach, J. Kopeć, M. Mägdefrau, K. Andreas, W. Keller, C. Bohn, M. Y. Abajy, and E. Grohmann, Microbiology 152:637-645, 2006). The pIP501 tra operon is negatively autoregulated at the transcriptional level by the conjugative DNA relaxase TraA. Three of the 15 pIP501-encoded Tra proteins show significant sequence similarity to the Agrobacterium type IV secretion system proteins VirB1, VirB4, and VirD4. Here we report a comprehensive protein-protein interaction map of all of the pIP501-encoded Tra proteins determined by the yeast two-hybrid assay. Most of the interactions were verified in vitro by isolation of the protein complexes with pull-down assays. In conjunction with known or postulated functions of the pIP501-encoded Tra proteins and computer-assisted prediction of their cellular location, we propose a model for the first type IV-secretion-like system encoded by a conjugative plasmid from gram-positive bacteria.

scholarly journals Genetic Elements Carrying erm(B) in Streptococcus pyogenes and Association with tet(M) Tetracycline Resistance Gene

2007 ◽  
Vol 51 (4) ◽  
pp. 1209-1216 ◽  
Author(s):  
Andrea Brenciani ◽  
Alessandro Bacciaglia ◽  
Manuela Vecchi ◽  
Luca A. Vitali ◽  
Pietro E. Varaldo ◽  
...  
Keyword(s):  
Streptococcus Pyogenes ◽  
Tetracycline Resistance ◽  
Complete Sequence ◽  
M Gene ◽  
Genetic Elements ◽  
Conjugative Transposons ◽  
Dna Insertion ◽  
Methylase Gene ◽  
Genotypic Characteristics ◽  
M Sequences

ABSTRACT This study was directed at characterizing the genetic elements carrying the methylase gene erm(B), encoding ribosome modification-mediated resistance to macrolide, lincosamide, and streptogramin B (MLS) antibiotics, in Streptococcus pyogenes. In this species, erm(B) is responsible for MLS resistance in constitutively resistant isolates (cMLS phenotype) and in a subset (iMLS-A) of inducibly resistant isolates. A total of 125 erm(B)-positive strains were investigated, 81 iMLS-A (uniformly tetracycline susceptible) and 44 cMLS (29 tetracycline resistant and 15 tetracycline susceptible). Whereas all tetracycline-resistant isolates carried the tet(M) gene, tet(M) sequences were also detected in most tetracycline-susceptible isolates (81/81 iMLS-A and 7/15 cMLS). In 2 of the 8 tet(M)-negative cMLS isolates, erm(B) was carried by a plasmid-located Tn917-like transposon. erm(B)- and tet(M)-positive isolates were tested by PCR for the presence of genes int (integrase), xis (excisase), and tndX (resolvase), associated with conjugative transposons of the Tn916 family. In mating experiments using representatives of different combinations of phenotypic and genotypic characteristics as donors, erm(B) and tet(M) were consistently cotransferred, suggesting their linkage in individual genetic elements. The linkage was confirmed by pulsed-field gel electrophoresis and hybridization studies, and different elements, variably associated with the different phenotypes/genotypes, were detected and characterized by amplification and sequencing experiments. A previously unreported genetic organization, observed in all iMLS-A and some cMLS isolates, featured an erm(B)-containing DNA insertion into the tet(M) gene of a defective Tn5397, a Tn916-related transposon. This new element was designated Tn1116. Genetic elements not previously described in S. pyogenes also included Tn6002, an unpublished transposon whose complete sequence is available in GenBank, and Tn3872, a composite element resulting from the insertion of the Tn917 transposon into Tn916 [associated with a tet(M) gene expressed in some cMLS isolates and silent in others]. The high frequency of association between a tetracycline-susceptible phenotype and tet(M) genes suggests that transposons of the Tn916 family, so far typically associated solely with a tetracycline-resistant phenotype, may be more widespread in S. pyogenes than currently believed.

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