Cloning and characterization of a gene encoding a secreted tripeptidyl aminopeptidase from Streptomyces lividans 66.

The gene encoding a tripeptidyl aminopeptidase (Tap) from Streptomyces lividans was cloned by using a simple agar plate activity assay. Overexpression of the cloned gene results in the production of a secreted protein which has an apparent subunit molecular weight of 55,000 and is responsible for the major amino-terminal degradative activity in culture broths of S. lividans strains. A DNA sequence analysis revealed a potential protein-encoding region of the size expected to encode the observed protein, which contained a sequence that exhibited significant homology around a putative active site serine residue observed for lipases, esterases, and acyl transferases. Preceding the amino terminus of the secreted protein was a predicted signal peptide of 36 amino acids followed by a tripeptide, which could be autocatalytically removed from a secreted Tap precursor. The transcriptional start site for the gene was mapped by primer extension. Mutant strains of S. lividans lacking detectable Tap activity were able to grow and sporulate normally. Cross-species hybridization experiments showed that DNA homologs of the tap gene are present in most of the Streptomyces strains tested.

Regulation of gene expression by the HlyX protein of Actinobacillus pleuropneumoniae.

The hlyX gene of the swine pathogen Actinobacillus pleuropneumoniae and the fnr gene of Escherichia coli encode very similar proteins. The hlyX gene is able to complement delta fnr mutations and will permit the growth of E. coli fnr strains in nitrate minimal salts medium under anoxic conditions. In addition, the hlyX gene product appears to induce the expression of a latent haemolytic activity as evidenced by the presence of a strong zone of haemolysis around E. coli (hlyX+) colonies grown on bovine or ovine blood. In this study, the ability of the hlyX gene product to induce haemolytic activity and regulate expression of frdA and its own gene was examined in the presence of various carbon sources and in the presence and absence of iron; fnr was included for comparison. The HlyX protein was able to induce the synthesis of the latent E. coli haemolytic activity only under anoxic conditions. Haemolytic activity was highest during the late exponential phase and then levelled off in the stationary phase. The hlyX gene product was able to activate the expression of a phi (frdA'-lacZ) in E. coli JRG1787 (delta fnr); however, the level of expression depended on carbon source, growth phase and copy number. Like fnr, the hlyX gene product appeared to affect its own synthesis but the nature and extent of regulation depended not only on the presence of oxygen but also on growth conditions.

The aminopeptidase N-encoding pepN gene of Streptomyces lividans 66.

The gene (pepN) encoding an aminopeptidase N (PepN) has been cloned from Streptomyces lividans. This was done using either leucine-beta-naphthylamide or arginine-beta-naphthylamide in a liquid overlayer on colonies growing on agar medium to screen for overproduction of the ability to hydrolyse the substrates. The nucleotide sequence of pepN was determined and shown to encode a 95-kDa protein, which displayed significant homology to PepN proteins from other organisms. Analysis of the overproduced proteinase confirmed that this protein was located intracellularly as a monomeric active species. PepN is a metallo-exopeptidase cleaving next to Leu, Arg and Lys in peptide-bond-containing substrates.

Cloning and characterization of btr, a Bordetella pertussis gene encoding an FNR-like transcriptional regulator.

To determine whether hemolytic factors other than the bifunctional hemolysin-adenylate cyclase toxin (cyclolysin) are expressed by Bordetella pertussis, a gene library was constructed from a virulent strain of B. pertussis, BP504, transformed into nonhemolytic Escherichia coli, and screened on blood agar plates. A strongly hemolytic colony which contained the plasmid pHLY1A was isolated. Nucleotide sequencing of pHLY1A revealed an open reading frame that could encode a 27-kDa protein. No similarity was detected between the deduced amino acid sequence of this open reading frame and those of any known bacterial cytolysins. However, significant homology was detected with FNR of E. coli and several other transcriptional regulators including HylX from Actinobacillus pleuropneumoniae, which can also confer a hemolytic phenotype on E. coli. An fnr mutant of E. coli, JRG1728, could be complemented by pHLY1A. Thus, the B. pertussis transcriptional regulator-like gene and the protein which it encoded were named btr and BTR, respectively. A BTR-deficient B. pertussis strain, BJB1, was constructed. The btr::kan mutation had no effect on the expression of hemolytic activity or on phase variation. Northern (RNA) blotting revealed that btr expression was not regulated by the BvgAS two-component sensor-regulator. On the basis of sequence similarity to FNR-like transcriptional regulators and the ability to complement an anaerobically deficient E. coli strain (JRG1728) in growing anaerobically, BTR may regulate B. pertussis gene expression in response to changes in oxygen levels or to changes in the redox potential of the bacterial environment. Its role in virulence remains to be determined.

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.