Actinobacillus pleuropneumoniae hlyX gene homology with the fnr gene of Escherichia coli.

The hlyX gene from Actinobacillus pleuropneumoniae, which confers a hemolytic phenotype on Escherichia coli, was sequenced, and its role in regulation of gene expression was investigated. No similarity was found between the hlyX sequence and sequences of known hemolysin or cytotoxin genes. However, the hlyX sequence was very similar to that of the fnr gene of Escherichia coli which encodes the global regulatory protein, FNR. Comparison of the deduced amino acid sequence of the hlyX gene product (HlyX) with that of FNR revealed a high degree of well-aligned sequence correlation throughout the polypeptide chain. For example, 23 of 24 amino acids in the DNA-binding region of FNR are identical in the corresponding region of HlyX. Four cysteine residues in the amino-terminal region are also conserved. The promoter region of hlyX is very similar to that of fnr. It has a putative -10 sequence which closely resembles the E. coli -10 consensus sequence. This sequence is overlapped by a potential operator which is very similar to the FNR-binding-site consensus sequence. Functional homology between HlyX and FNR was also demonstrated. Plasmids carrying hlyX complemented the nutritional lesion of an fnr deletion strain of E. coli. These data suggest that HlyX may regulate, rather than mediate, hemolytic activity in E. coli, but the possibility that HlyX is both a regulator of gene expression and a hemolysin cannot be excluded.     

Application of multivariate analyses of enzymic data to classification of members of the Actinobacillus-Haemophilus-Pasteurella group.

Outer membrane vesicles and fragments from Actinobacillus actinomycetemcomitans, Actinobacillus lignieresii, Actinobacillus ureae, Haemophilus aphrophilus, Haemophilus paraphrophilus, Haemophilus influenzae, Haemophilus parainfluenzae, Pasteurella haemolytica, and Pasteurella multocida were isolated and examined semiquantitatively for 19 enzyme activities by using the API ZYM micromethod. The enzyme contents of vesicles and fragments were compared with the enzyme contents of whole cells of the same organisms. Enzymic data were analyzed by using principal-component analysis and soft independent modeling of class analogy. This technique allowed us to distinguish among the closely related organisms A. actinomycetemcomitans, H. aphrophilus, and H. paraphrophilus. A. actinomycetemcomitans was divided into two groups of strains. A. lignieresii fell outside or on the border of the A. actinobacillus class. A. ureae, H. influenzae, H. parainfluenzae, P. haemolytica, and P. multocida fell outside the A. actinomycetemcomitans, H. aphrophilus, and H. paraphrophilus classes.     

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.     

Multivariate analyses of carbohydrate data from lipopolysaccharides of Actinobacillus (Haemophilus) actinomycetemcomitans, Haemophilus aphrophilus, and Haemophilus paraphrophilus

The taxonomic distinction between Actinobacillus (Haemophilus) actinomycetemcomitans and Haemophilus aphrophilus and the taxonomic distinction between H. aphrophilus and Haemophilus paraphrophilus have been questioned. This study was done to determine whether multivariate statistical analyses of carbohydrate data from lipopolysaccharides could be used to distinguish between these closely related species. Lipopolysaccharides were extracted with phenol-water and purified. Carbohydrates were assessed by using gas chromatography and gas chromatography-mass spectrometry after methanolysis and derivatization with trifluoroacetic acid anhydride. The lipopolysaccharides from all of the species contained rhamnose, fucose, galactose, glucose, L-glycero-D-mannoheptose, and glucosamine plus galactosamine, but in varying amounts. A. actinomycetemcomitans and H. paraphrophilus also contained D-glycero-D-mannoheptose, while H. aphrophilus did not. Sample- and variable-oriented principal-component analyses of the carbohydrate data clearly distinguished among A. actinomycetemcomitans, H. aphrophilus, and H. paraphrophilus. Soft independent modelling of class analogy showed that no sample in the A. actinomycetemcomitans class fell within the 95% confidence limits of the H. aphrophilus class. H. paraphrophilus fell outside both classes.     

Periodontopathic micro-organisms in the rice rat (Oryzomys palustris)

The rice rat spontaneously develops periodontal disease, and this process can be accelerated if the animal is placed on a high sucrose diet. As the aetiological agent(s) is unknown, this study was undertaken to determine the micro-organisms associated with bone loss. Animals at weaning were placed on either a high sucrose diet or on regular rat chow and were sampled periodically for a variety of micro-organisms. Bacteroides spp., spirochetes, and Actinobacillus actinomycetemcomitans were not isolated from any animals, but fusiform-like organisms and Capnocytophaga spp. were isolated occasionally. An unidentified organism which had characteristics of both A. actinomycetemcomitans and Haemophilus aphrophilus was isolated from all animals at every sampling. Whether this organism is the primary aetiological agent of periodontal disease in the rice rat remains to be determined. Over the 12 week experimental period the animals on the high sucrose diet had significantly more exposed root surface than animals on regular rat chow.     

Characterization of two genes encoding ferritin-like protein in Actinobacillus actinomycetemcomitans.

Two genes encoding ferritin-like protein, designated afnA and afnB, were identified in the upstream region of actX on the Actinobacillus actinomycetemcomitans chromosomal DNA. The actX has been reported to be a regulatory gene homologous to the Escherichia coli fnr, which controls the growth and virulence of A. actinomycetemcomitans under anaerobic conditions. The afnB located 340 bp-upstream from the actX, and the afnA located just 15 bp-upstream from afnB. The afnA and afnB encoded 161 and 165 amino acid residues, respectively, which were similar to ferritin-like proteins of other microorganisms. Western immunoblotting using rabbit antiserum against E. coli ferritin showed these two proteins, which are reactive with the serum with 19-kDa molecular masses, are produced from A. actinomycetemcomitans. The N-terminal amino acid sequences of the two proteins were consequent with those deduced from afnA and afnB. Northern hybridization revealed that the afnA and afnB constituted a bicistronic operon and the accumulation of afnA and afnB mRNA was upregulated under aerobic conditions. These findings suggested that the operon was regulated by the presence of oxygen. The two ferritin-like proteins may have important roles in the adaptation of A. actinomycetemcomitans to oxidative environmental changes.     

Staphylococcus haemolyticus lipase: biochemical properties, substrate specificity and gene cloning

Streptococcus pneumoniae is a Gram-positive bacterium which is naturally resistant to tellurite. In this study, we cloned and sequenced a homologue of the Escherichia coli tellurite resistance gene tehB from S. pneumoniae. It encoded a protein of 284 amino acids which is 86 residues longer than E. coli TehB, but similar in size to Haemophilus influenzae TehB and Eikenella corrodens hemagglutinin (Hag1) as well as homologues from Actinobacillus actinomycetemcomitans, Neisseria gonorrhoeae and Neisseria meningitidis. The S. pneumoniae TehB displayed 46-58% identity (52-68% similarity) to these proteins. The results in this study showed that the S. pneumoniae tehB alone not only conferred on E. coli high level resistance to tellurite, but also caused filamentous morphology in E. coli.     

Recombinant expression and redox properties of triheme c membrane-bound quinol peroxidase

The qpo gene of Aggregatibacter actinomycetemcomitans encodes a triheme c -containing membrane-bound enzyme, quinol peroxidase (QPO) that catalyzes peroxidation reaction in the respiratory chain and uses quinol as the physiological electron donor. The QPO of A. actinomycetemcomitans is the only characterized QPO, but homologues of the qpo gene are widely distributed among many gram-negative bacteria, including Haemophils ducreii, Bacteroides fragilis , and Escherichia coli . One-third of the amino acid sequence of QPO from the N-terminal end is unique, whereas two-thirds of the sequence from the C-terminal end exhibits high homology with the sequence of the diheme bacterial cytochrome c peroxidase. In order to obtain sufficient protein for biophysical studies, the present study aimed to overproduce recombinant QPO (rQPO) from A. actinomycetemcomitans in E. coli . Coexpression of qpo with E. coli cytochrome c maturation ( ccm ) genes resulted in the expression of an active QPO with a high yield. Using purified rQPO, we determined the midpoint reduction potentials of the three heme molecules.     

Multivariate analysis of quantitative chemical and enzymic characterization data in classification of Actinobacillus, Haemophilus and Pasteurella spp

Chemotaxonomic data for strains of Actinobacillus, Haemophilus and Pasteurella spp. were analysed using three multivariate statistical strategies: principal components, partial least squares discriminant, and soft independent modelling of class analogy. The species comprised Actinobacillus actinomycetemcomitans. Haemophilus aphrophilus, H. paraphrophilus, H. influenzae, Pasteurella multocida, P. haemolytica and P. ureae. Strains were characterized by cell sugar and fatty acid composition, lysis kinetics during EDTA and EDTA plus lysozyme treatment, and methylene blue reduction. In total 23 quantitative variables were compiled from chemotaxonomic analyses of 25 strains. A. actinomycetemcomitans and H. aphrophilus formed distinct classes which differed from those of H. paraphrophilus, H. influenzae and Pasteurella spp. All characterization variables, except those describing fatty acid content, contributed significantly to inter-species discrimination.     

Vertebral osteomyelitis due to coccobacilli of the HB group.

Three cases of pyogenic vertebral osteomyelitis occurred in which unusual, fastidious, Gram negative coccobacilli belonging to the "HB" group were isolated. The organisms were Haemophilus aphrophilus in case 1, intermediate between H aphrophilus and Actinobacillus actinomycetemcomitans in case 2, and Eikenella corrodens in case 3. All HB bacteria are sensitive to a wide range of antibiotics.     

Differentiation between Actinobacillus (Haemophilus) actinomycetemcomitans, Haemophilus aphrophilus and Haemophilus paraphrophilus by multilocus enzyme electrophoresis

Genetic relationships among isolates assigned to Actinobacillus actinomycetemcomitans, Haemophilus aphrophilus and H. paraphrophilus were determined by analysis of electrophoretically demonstrable allelic variation in 14 structural genes encoding metabolic enzymes. Among the 51 isolates analysed there were 25 electrophoretic types (ETs), among which mean genetic diversity per locus was 0.753. Cluster analysis of ETs demonstrated one well-defined group of 11 ETs representing solely the genotypes of all 17 isolates assigned to A. actinomycetemcomitans. The remaining 14 ETs represented the genotypes of the 34 isolates of H. aphrophilus and H. paraphrophilus. With the exception of ATCC 13252, all strains of H. aphrophilus were closely related, whereas strains assigned to H. paraphrophilus included distantly related lineages, some of which were similar to those of H. aphrophilus and should be assigned to this species. Thus, the study showed that there is no significant overall genetic similarity between A. actinomycetemcomitans and the two Haemophilus spp.     

Identification of a plasmid-encoded gene from Haemophilus ducreyi which confers NAD independence

Members of the family Pasteurellaceae are classified in part by whether or not they require an NAD supplement for growth on laboratory media. In this study, we demonstrate that this phenotype can be determined by a single gene, nadV, whose presence allows NAD-independent growth of Haemophilus influenzae and Actinobacillus pleuropneumoniae. This gene was cloned from a 5.2-kb plasmid which was previously shown to be responsible for NAD independence in Haemophilus ducreyi. When transformed into A. pleuropneumoniae, this cloned gene allowed NAD-independent growth on complex media and allowed the utilization of nicotinamide in place of NAD on defined media. Sequence analysis revealed an open reading frame of 1,482 bp that is predicted to encode a protein with a molecular mass of 55,619 Da. Compared with the sequence databases, NadV was found to have significant sequence homology to the human pre-B-cell colony-enhancing factor PBEF and to predicted proteins of unknown function identified in the bacterial species Mycoplasma genitalium, Mycoplasma pneumoniae, Shewanella putrefaciens, Synechocystis sp., Deinococcus radiodurans, Pasteurella multocida, and Actinobacillus actinomycetemcomitans. P. multocida and A. actinomycetemcomitans are among the NAD-independent members of the Pasteurellaceae. Homologues of NadV were not found in the sequenced genome of H. influenzae, an NAD-dependent member of the Pasteurellaceae, or in species known to utilize a different pathway for synthesis of NAD, such as Escherichia coli. Sequence alignment of these nine homologues revealed regions and residues of complete conservation that may be directly involved in the enzymatic activity. Identification of a function for this gene in the Pasteurellaceae should help to elucidate the role of its homologues in other species.