An Analysis of the Complete Nucleotide Sequence of the Haemophilus ducreyi Broad-Host-Range Plasmid pLS88

Dixon, Laurie G.; Albritton, William L.; Willson, Philip

doi:10.1006/plas.1994.1060

Cited by 73 publications

(40 citation statements)

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“…1). This region showed 88 to 91% identity to the origin of replication of Haemophilus ducreyi plasmid, pLS88 (4.8 kb) (14). Similar regions have been described for Pasteurella multocida plasmid pIG1 (5.4 kb) (56), and two plasmids, pYFC1 and pAB2, were isolated from Pasteurella haemolytica (9,55).…”

Section: Resultssupporting

confidence: 68%

“…As described for pLS88 (14) and pIG1 (56), the putative oriV of pVT745 contains stretches of DNA showing strong homologies to portions of the ROB-1-␤-lactamase gene from species such as P. haemolytica, Haemophilus influenzae, and Actinobacillus pleuropneumoniae. Interestingly, an intact bla gene is located just upstream of oriV on pAB2 (55).…”

Section: Resultsmentioning

confidence: 92%

“…These data indicate that there is an evolutionary link between multiple plasmids in the HAP group. Replication of pLS88 seems to be independent of any plasmid-encoded protein (14). Nucleotide sequence analysis performed by Wright et al (56) revealed that the replication regions contained two to three major inverted repeats (IR20, IR16, and IR38).…”

Section: Resultsmentioning

confidence: 99%

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Nucleotide Sequence and Analysis of Conjugative Plasmid pVT745

et al. 2001

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The complete nucleotide sequence and genetic map of pVT745 are presented. The 25-kb plasmid was isolated from Actinobacillus actinomycetemcomitans, a periodontal pathogen. Two-thirds of the plasmid encode functions related to conjugation, replication, and replicon stability. Among potential gene products with a high degree of similarity to known proteins are those associated with plasmid conjugation. It was shown that pVT745 derivatives not only mobilized a coresident nontransmissible plasmid, pMMB67, but also mediated their own conjugative transfer to different A. actinomycetemcomitans strains. However, transfer of pVT745 derivatives from A. actinomycetemcomitans to Escherichia coli JM109 by conjugation was successful only when an E. coli origin of replication was present on the pVT745 construct. Surprisingly, 16 open reading frames encode products of unknown function. The plasmid contains a conserved replication region which belongs to the HAP (Haemophilus-Actinobacillus-Pasteurella) theta replicon family. However, its host range appears to be rather narrow compared to other members of this family. Sequences homologous to pVT745 have previously been detected in the chromosomes of numerous A. actinomycetemcomitans strains. The nature and origin of these homologs are discussed based on information derived from the nucleotide sequence.The gram-negative bacterium Actinobacillus actinomycetemcomitans is a capnophilic coccobacillus. The organism has been associated with several forms of periodontal disease such as localized juvenile periodontitis and rapidly progressive periodontitis, as well as with soft tissue abscesses and endocarditis (58). In a previous study 39 isolates of this periodontal pathogen had been screened for the presence of indigenous plasmids in an effort to evaluate the role(s) of such genetic elements in oral bacteria (32). Three plasmids, pVT736-1 (2 kb), pVT736-2 (Ͼ30 kb), and pVT745 (25 kb), were identified in two strains, suggesting that the occurrence of plasmids in A. actinomycetemcomitans was rare. The ultimate goal was to determine the biological properties of these plasmids, to assess their potential contribution to the pathogenicity of A. actinomycetemcomitans, and to evaluate their usefulness as tools in recombinant DNA technology. Previous work has focused mainly on the characterization of pVT736-1, one of the first rolling circle replicating (RCR) plasmids isolated from gram-negative bacteria (17, 18). It was shown that pVT736-1 was cryptic, that it was not related to RCR plasmids found in gram-positive bacteria, and that it encoded a new type of partitioning system (20).Preliminary characterization suggested that there was no obvious phenotype associated with pVT745 (41). Its size was a strong indication that the plasmid replicated by a theta mechanism rather than by a rolling circle mode. Although pVT745 had been isolated from one strain of A. actinomycetemcomitans (VT745) only, it was demonstrated by Southern hybridization that this plasmid shared sequence homologies with chromos...

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Section: Resultssupporting

confidence: 68%

Section: Resultsmentioning

confidence: 92%

Section: Resultsmentioning

confidence: 99%

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Nucleotide Sequence and Analysis of Conjugative Plasmid pVT745

et al. 2001

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“…pLS88 was used as an E. coli-H. ducreyi shuttle vector (Kan r , Sm r , Sul r ) for cloning and complementation in these studies (6,11). PCR primers 836 and 837 (Table 2), engineered with SacII and PstI restriction sites, were used to amplify the M. catarrhalis lgt3 gene with 265 bp of 5Ј flanking DNA, which was then ligated to SacII/PstI-digested pLS88, to construct plasmid pLS3PR-E (Table 1).…”

Section: Vol 187 2005 Glycosyltransferase Enzymes Essential For Losmentioning

confidence: 99%

Characterization of a Cluster of Three Glycosyltransferase Enzymes Essential for Moraxella catarrhalis Lipooligosaccharide Assembly

Edwards¹,

Allen

Gibson

et al. 2005

J Bacteriol

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Moraxella catarrhalis isolates express lipooligosaccharide (LOS) molecules on their surface, which share epitopes similar to that of the Neisseria and Haemophilus species. These common LOS epitopes have been implicated in various steps of pathogenesis for the different organisms. In this study, a cluster of three LOS glycosyltransferase genes (lgt) were identified in M. catarrhalis 7169, a strain that produces a serotype B LOS. Mutants in these glycosyltransferase genes were constructed, and the resulting LOS phenotypes were consistent with varying degrees of truncation compared to wild-type LOS. The LOS structures of each lgt mutant were no longer detected by a monoclonal antibody (MAb 4G5) specific to a highly conserved terminal epitope nor by a monoclonal antibody (MAb 3F7) specific to the serotype B LOS side chain. Mass spectrometry of the LOS glycoforms assembled by two of these lgt mutants indicated that lgt1 encodes an ␣(1-2) glucosyltransferase and the lgt2 encodes a ␤(1-4) galactosyltransferase. However, these structural studies could not delineate the function for lgt3. Therefore, M. catarrhalis lgt3 was introduced into a defined ␤(1-4) glucosyltransferase Haemophilus ducreyi 35000glu؊ mutant in trans, and monoclonal antibody analysis confirmed that Lgt3 complemented the LOS defect. These data suggest that lgt3 encodes a glucosyltransferase involved in the addition of a ␤(1-4)-linked glucose to the inner core. Furthermore, we conclude that this enzymatic step is essential for the assembly of the complete LOS glycoform expressed by M. catarrhalis 7169.Moraxella catarrhalis is a gram-negative human respiratory pathogen that causes 15 to 20% of acute otitis media in children (56). In addition, this bacterium is responsible for 10 to 35% of lower respiratory infections in adults with chronic obstructive pulmonary disease, the fourth leading cause of death in the United States (20). The emergence of M. catarrhalis as an important human pathogen has occurred in the past 15 years due to the increasing prevalence of ␤-lactamase-positive strains, the high incidence of recurrent infections despite successful antibiotic treatment, and the lack of an effective vaccine (10,39,50,56). Another factor that likely contributes to the persistence of M. catarrhalis disease is the lack of understanding of the basic bacterial factors and mechanisms that promote colonization and survival in the host.Although there have been a number of putative virulence factors described for M. catarrhalis, the actual role of these components in pathogenesis remains largely undefined (13,19,20,31,53). One of the most prominent surface components expressed in the outer membrane of this bacterium is the lipooligosaccharide (LOS) (12). The LOS of M. catarrhalis is similar to the lipopolysaccharide (LPS) of other gram-negative organisms, but it lacks a repeating O antigen (21). Previous studies have suggested that LOS is important for the pathogenesis of other respiratory pathogens, such as Neisseria meningitidis and Haemophilus influenzae, aid...

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“…The isogenic LOS mutants, 35000glu-, 35000hep-, and 35000hepglu-were constructed in our laboratory and have been described previously (13). pLS88 is an H. ducreyi shuttle vector (10). 35000glu-(pGLU), 35000hep-(pHEP), and 35000hepglu-(pLS88HG.13) are complemented strains de-rived from the electroporation of plasmids containing the respective wild-type gene(s) into the respective mutant (13).…”

mentioning

confidence: 99%

Genetic Analysis of a Pyocin-Resistant Lipooligosaccharide (LOS) Mutant of Haemophilus ducreyi : Restoration of Full-Length LOS Restores Pyocin Sensitivity

2001

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DNA sequence and Southern blot analyses were used to determine the genetic defect of a Haemophilus ducreyi pyocin-resistant lipooligosaccharide (LOS) mutant, HD35000R. The region of the HD35000R chromosome containing the suspected mutation was amplified, and sequence analysis detected a 3,189-bp deletion. This deletion resulted in the loss of the entire waaQ gene, another open reading frame that encodes a putative homolog to a hypothetical protein (HI0461) of H. influenzae, the gene encoding an argininosuccinate synthase homolog, and a change in the 3 sequence of the lgtF gene. Southern blot analysis confirmed that no genomic rearrangements had occurred. Isogenic LOS mutants and the respective complemented mutants were evaluated for susceptibility to pyocin C. The mutants expressing truncated LOS were resistant to lysis by pyocin C, and complementation restored sensitivity to the pyocin. We conclude that HD35000R is defective in both glycosyltransferase genes and that pyocin resistance is due to truncation of the full-length LOS molecule.

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An Analysis of the Complete Nucleotide Sequence of the Haemophilus ducreyi Broad-Host-Range Plasmid pLS88

Cited by 73 publications

References 0 publications

Nucleotide Sequence and Analysis of Conjugative Plasmid pVT745

Nucleotide Sequence and Analysis of Conjugative Plasmid pVT745

Characterization of a Cluster of Three Glycosyltransferase Enzymes Essential for Moraxella catarrhalis Lipooligosaccharide Assembly

Genetic Analysis of a Pyocin-Resistant Lipooligosaccharide (LOS) Mutant of Haemophilus ducreyi : Restoration of Full-Length LOS Restores Pyocin Sensitivity

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