Biomedical subjects

K F Novak

Publications and source records attributed to K F Novak.

6 recordsLinked to original sources

PubMed2001

Actinobacillus actinomycetemcomitans harbours type IV secretion system genes on a plasmid and in the chromosome.

Nine contiguous genes encoding a potential type IV secretion system have been identified in the chromosome of Actinobacillus actinomycetemcomitans strain VT747 and on a plasmid (pVT745) in strain VT745. Seven of these genes encode predicted proteins that share significant homology with type IV secretion proteins in Bordetella pertussis (ptl operon), Brucella melitensis biovar suis and Agrobacterium tumefaciens (virB operons), where they are involved in protein secretion, pathogen intracellular survival and multiplication, and DNA transport, respectively. Results of previous studies have demonstrated that pVT745 is a conjugative plasmid and that a secondary plasmid, pMMB67, can be mobilized from strain VT745. Given these results, it was hypothesized that (1) the type IV secretion genes on pVT745 are responsible for these two functions and (2) the type IV VT747 chromosomal genes also play a role in the transport of DNA. Wild-type and mutant strains of VT745 were evaluated for their conjugative abilities. Wild-type mating efficiency was 10(-6) transconjugants per donor, while the mutant strain yielded no transconjugants. Wild-type VT745 harbouring a co-resident plasmid, pMMB67, mobilized pMMB67 at a frequency of 10(-6), while VT747 was unable to mobilize this plasmid. These results support the hypothesis that the plasmid-encoded type IV secretion system on pVT745 is involved in DNA transport. However, the chromosomally encoded secretion system may not play a role in DNA transport in strain VT747. While the precise function of these chromosomal genes in strain VT747 has not been determined, Northern blot analyses demonstrated that these genes are expressed in both ACT: actinomycetemcomitans strains VT745 and VT747.

Aggregatibacter actinomycetemcomitans↗

PubMed2001

Nucleotide sequence and analysis of conjugative plasmid pVT745.

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.

Aggregatibacter actinomycetemcomitans↗

PubMed2000

Molecular characterization and functional analysis of a secA gene homolog in Actinobacillus actinomycetemcomitans.

Results of Southern blot analyses and polymerase chain reaction revealed that the Gram-negative pathogen, Actinobacillus actinomycetemcomitans, harbored DNA homologous to the secA gene of Escherichia coli. In E. coli, the secA gene product is essential for translocation of proteins across the inner membrane via the Sec system. This A. actinomycetemcomitans secA homolog was cloned and its nucleotide sequence determined. Amino acid sequence analysis of the cloned gene revealed significant homology to the SecA proteins of Haemophilus influenzae, E. coli, Caulobacter crescentus and Bacillus subtilis. Although the cloned gene did not complement a temperature sensitive mutation in the E. coli secA gene, strains harboring the cloned gene did produce a protein that cross-reacted with anti-SecA antibody. In addition, the cloned gene did restore sensitivity to sodium azide in an E. coli azide mutant. These data support the hypothesis that A. actinomycetemcomitans may use a system similar to the Sec system of E. coli to transport proteins across the cytoplasmic membrane, but suggest that the A. actinomycetemcomitans gene product may require genera-specific Sec proteins to complement some Sec mutations in E. coli.

Adenosine Triphosphatases↗

PubMed1998

Functional analysis of pVT745, a plasmid from Actinobacillus actinomycetemcomitans.

Plasmid pVT745 is a 25.1-kb replicon isolated from Actinobacillus actinomycetemcomitans strain VT745. A previous report described the hybridization of pVT745 in 5 strain-specific patterns to chromosomal DNA from 15 other A. actinomycetemcomitans strains. However, pVT745 does not share homology with the chromosome of the strain from which it was isolated, VT745. It was hypothesized that the shared areas of homology might represent insertion sequence elements and/or transposons possibly encoding resistance to one or more antibiotics. An antibiogram of strain VT745 demonstrated that this strain was uniformly susceptible to all antibiotics examined. Because insertion sequence elements and transposons are mobile genetic elements, a series of cell passaging experiments, followed by Southern hybridization was conducted in a attempt to detect transposition of pVT745 homologous DNA within the chromosomes of several A. actinomycetemcomitans strains. The results of these experiments suggested stability of the homologous DNA both within the chromosome and on the plasmid. It was also possible that pVT745 represented a lysogenic bacteriophage. Phage induction experiments were conducted under conditions that induced a previously described A. actinomycetemcomitans lysogenic phage, but no phage could be induced from strain VT745. Attempts to obtain isolates of VT745 cured of pVT745 were also unsuccessful.

Aggregatibacter actinomycetemcomitans↗

PubMed1996

Early-onset periodontitis.

The group of periodontal diseases known as the early-onset periodontal diseases are defined by the age of onset of periodontal destruction, distribution of lesions, association of disease with specific microbial infections, and identification of characteristic alterations, in the host response. Significant progress has recently been made in our understanding of the etiology of juvenile and rapidly progressive periodontitis. Considerable evidence points to a familial pattern of disease; both localized and generalized forms of disease may be observed in the same family. The exact mode of inheritance remains unclear, and disease may be the result of a complex interplay between genetically determined alterations of the host response and a specific bacterial challenge. Both neutrophil function and immunoglobulin response are altered and appear to be characteristic of an immunologic hyperresponsiveness. Bacterial colonization by Actinobacillus actinomycetemcomitans (serotype b) and/or Porphyromonas gingivalis appears to be the primary initiator of disease. Evidence suggests that lesion distribution may be a function of the nature of the infecting agent and the characteristics of the immune response.

Adolescent↗

PubMed1994

Characterization of plasmid pVT745 isolated from Actinobacillus actinomycetemcomitans.

A 25.1-kb plasmid, pVT745, was isolated from Actinobacillus actinomycetemcomitans strain VT745. This plasmid hybridized under stringent conditions to chromosomal DNA from 15 A. actinomycetemcomitans isolates obtained from geographically diverse regions of the United States. Southern blot analyses of HincII-digested A. actinomycetemcomitans genomic DNA revealed five strain-specific patterns of hybridization, which clearly indicated that intact pVT745 was not inserted into any of the genomes. Plasmid pVT745 was digested with BamHI and PstI into three, non-overlapping fragments of approximately 7.0, 8.0, and 10.0 kb. The fragments were cloned in Escherichia coli on the low copy number vector pGB2. Although these three fragments exhibited no cross-hybridization, genomic DNA from isolates representing each of the strain-specific patterns hybridized with two or three of the cloned fragments. The results suggest that two or more unique sequences within pVT745 also may be present in genomic DNA obtained from various strains of A. actinomycetemcomitans.

Aggregatibacter actinomycetemcomitans↗