- Volume 141, Issue 9, 1995

Arginine is metabolized by the arginine dihydrolase pathway in Giardia intestinalis trophozoites and is an important metabolic fuel for this parasite. Radiolabelled arginine was used to characterize the transport of arginine into Giardia intestinalis trophozoites. The transporter had a high affinity for arginine (K m 15 üM) and a high activity [V max 76 nmol min-1 (mg protein)-1 at 25 °C]. Substrate specificity studies indicated an absolute requirement for the α-amino and carboxyl groups, but a tolerance for some substitutions in the guanidino group. The use of non-metabolized arginine analogues in combination with HPLC amino acid analysis of intra- and extracellular pools demonstrated that the arginine transporter is an arginine-ornithine antiport. Investigations of the first step of arginine metabolism, involving arginine deiminase, revealed a relatively high affinity for arginine (K m 0.16 mM) and a large maximal velocity [V max 550 nmol min-1 (mg protein)-1 at 37 °C]. Substrate specificity studies showed that the arginine deiminase had a characteristically different substrate recognition profile to that of the arginine transporter. Overall, the combination of the transporter and the deiminase result in very low intracellular arginine concentrations and their properties are consistent with the rapid transport of arginine for metabolism via the arginine dihydrolase pathway.

Copper- and zinc-containing superoxide dismutases ([Cu,Zn]-SODs) are generally considered almost exclusively eukaryotic enzymes, protecting the cytosol and extracellular compartments of higher organisms from damage by oxygen free-radicals. The recent description of a few examples of bacterial forms of the enzyme, located in the periplasm of different Gram-negative micro-organisms, prompted a re-evaluation of this general perception. A PCR-based approach has been developed and used successfully to identify bacterial genes encoding [Cu,Zn]-SOD in a wide range of important human and animal pathogens - members of the Haemophilus, Actinobacillus and Pasteurella (HAP) group, and Neisseria meningitidis. Comparison of [Cu,Zn]-SOD peptide sequences found in Haemophilus ducreyi, Actinobacillus pleuropneumoniae, Actinobacillus actinomycetemcomitans, Pasteurella multocida, and N. meningitidis with previously described bacterial proteins and examples of eukaryotic [Cu,Zn]-SOD has shown that the bacterial proteins constitute a distinct family apparently widely separated in evolutionary terms from the eukaryotic examples. The widespread occurrence of [Cu,Zn]-SOD in the periplasm of bacterial pathogens, appropriately located to dismute exogenously derived superoxide radical anions, suggests that this enzyme may play a role in the interactive biology of organisms with their hosts and so contribute to their capacity to cause disease.

The proteolytic system of lactic acid bacteria has been extensively studied over the past 10 years and peptidases from lactococci are now well known. The situation is, however, different for Streptococcus thermophilus from which only a few peptidases have been purified and characterized. The present work was conducted to characterize an aminopeptidase of S. thermophilus CNRZ 302, called St-PepA, with high specificity for acidic amino acids. St-PepA was purified by a three-step procedure from a spheroblast extract of S. thermophilus CNRZ 302. Its molecular mass was estimated to be 360 kDa by gel filtration and 45 kDa by SDS-PAGE, indicating that it had an octameric structure. Its activity against aspartate-p-nitroanilide was maximal at pH 8.5 and 62 C and highly enhanced by Zn2+, Co2+ and Mg2+. St-PepA was inhibited by metal-chelating reagents and, to a lesser extent, by agents modifying sulfhydryl groups. It showed an activity towards p-nitroanilide derivatives, di-and tripeptides, and larger peptides such as fragment 43-58 of as1-casein. It had a high substrate specificity towards N-terminal acidic amino acid residues but it could also release serine and malic acid, the a-hydroxy acid homologue of aspartic acid. Kinetic studies revealed that the affinity of St-PepA was more than 18-fold higher for aspartic acid-p-nitroanilide (K m = 0.42 mM) than for glutamic acid-p-nitroanilide (K m = 7.65 mM) with a similar V max for both substrates [about 40 mol min-1 (mg enzyme)-1]. St-PepA is heat stable, with a maximal loss of activity of 15% after incubation for 120 min at 50 C and 60C. It is likely to be involved in the nitrogen metabolism of S. thermophilus and in the development of the organoleptic characteristics of cheese and yoghurt.

A mixed membrane fraction (MMF) was isolated from yeast cells of Candida albicans with the ability to synthesize dolichol phosphate mannose (Dol-P-Man) from GDP-Man and dolichol phosphate (Dol-P) and transfer the sugar to proteins. Temperature of incubation (20-37 °C) did not affect the synthesis of Dol-P-Man but protein mannosylation occurred better at physiological temperatures (28 °C and 37 °C). Most of the sugar (87-93 %) in the mannoproteins was O-linked as judged by its release by β-elimination. Mannose was identified as the sole product after this treatment. Following incubation of MMF with the sugar donor, parallel levels of Dol-P-Man and mannosylated proteins were detected up to 30 min. Thereafter, Dol-P-Man levels reached a steady value whereas mannoproteins rapidly accumulated. Lipid-linked oligosaccharides were also detected in incubation mixtures, though in much lower amounts than those of Dol-P-Man or mannoproteins. Dol-P-Man synthase activity increased proportionally in response to increasing concentrations of either of the two enzyme substrates. A K m value of 0·36 μM for GDP-Man was calculated. MMF failed to use exogenous Dol-P-Man for protein glycosylation. Specific inhibition of Dol-P-Man synthesis with amphomycin was concomitant with a parallel decrease in protein mannosylation, indicating that most of the sugar is transferred to protein via the carrier lipid. Results are discussed in terms of the role of Dol-P-Man in protein glycosylation in C. albicans.

In Aspergillus nidulans three alcohol dehydrogenases (ADHs) have been described. ADHI is induced by ethanol and is the physiological enzyme of ethanol utilization, ADHII has not been attributed a function but is repressed by ethanol. The ALCR regulatory protein acts positively to induce ADHI, and negatively in its control of ADHII. ADHIII is specifically induced by anaerobic stress. We have characterized the substrate specificity of these three enzymes by looking at their staining profile on polyacrylamide gels with a range of alcohols. In addition to these enzymes we have observed six other NAD+-dependent ADHs, two of which, propan-2-ol dehydrogenase and pentan-2-ol dehydrogenase, share similar control with ADHII. The inducibility of these enzymes with some alcohols has also been investigated. The profile of ADHs with NADP+ as an electron acceptor is also reported.

The gene encoding a 51 kDa polypeptide of Porphyromonas gingivalis 381 was isolated by immunoblotting using an antiserum raised against P. gingivalis alkaline phosphatase. DNA sequence analysis of a 2.5 kb DNA fragment containing a gene encoding the 51 kDa protein revealed one complete and two incomplete ORFs. Database searches using the FASTA program revealed significant homology between the P. gingivalis 51 kDa protein and the MurC protein of Escherichia coli, which functions in peptidoglycan synthesis. The cloned 51 kDa protein encoded a functional product that complemented an E. coli murC mutant. Moreover, the ORF just upstream of murC coded for a protein that was 31% homologous with the E. coli MurG protein. The ORF just downstream of murC coded for a protein that was 17% homologous with the Streptococcus pneumoniae penicillin-binding protein 2B (PBP2B), which functions in peptidoglycan synthesis and is responsible for antibiotic resistance. These results suggest that P. gingivalis contains a homologue of the E. coli peptidoglycan synthesis gene murC and indicate the possibility of a cluster of genes responsible for cell division and cell growth, as in the E. coli mra region.

Regions of the genes encoding flagellin (flaA), the invasive associated protein (iap), listeriolysin O (hly) and 23S rRNA were sequenced for a range of Listeria monocytogenes isolates of different origin and serotypes. Several nucleotide sequence variations were found in the flaA, iap and hly genes. No differences were found for the rRNA genes, but our approach does not exclude the existence of differences between single copies of these genes. Based on the sequence differences, the L. monocytogenes strains can be divided into three distinct sequence types. Further, the presence of only a small number of sequence differences within each group indicates a strong degree of conservation within the groups. There was a complete correspondence among the groups of strains formed according to the analysis of the flaA, iap and hly genes, and the grouping correlates with serotype, pulsed field gel electrophoretic and multilocus enzyme electrophoretic data. Analysis of the region encoding the threonine-asparagine repeat units in the iap gene revealed some striking features. Sequence type 1 strains were found to have 16-17 repeats, sequence type 2 strains had 16-20 repeats whereas the two sequence type 3 strains analysed had only 11 repeats. Furthermore, within a 19 bp segment there was a 37% difference between the sequences of type 1 and 2 strains and that segment was absent in type 3 strains. Within the threonine-asparagine repeat region the nucleotide differences gave rise to four amino acid changes; however, all were changes among the three amino acids present in the repeat structure indicating a strong selective pressure on the composition of this region.

The stable maintenance and expression of foreign genes in mollicutes (mycoplasmas) have been difficult to achieve due to the lack of suitable vectors. In this paper we show for the first time that a replicating vector can been used to express foreign genes other than antibiotic resistance genes in Acholeplasma laidlawii. Plasmids derived from the lactococcal vector pNZ18 could introduce and maintain four different genes for many generations in A. laidlawii. One of these, encoding the dominant membrane lipoprotein spiralin from the mollicute Spiroplasma citri, was expressed; however, expression was weak, the signal peptide of spiralin was not cleaved and the protein was not covalently modified by fatty acids. This resulted in a hydrophilic character of spiralin and its cytoplasmic localization in A. laidlawii. To increase the expression of foreign genes, random A. laidlawii DNA fragments were cloned into a pNZ18-related plasmid and expression signals were selected using the Bacillus licheniformis a-amylase gene as a probe. Selection was done in Escherichia coli as well as directly in A. laidlawii. Active recombinants from E. coli were also able to express a-amylase activities and an enzyme of native size in A. laidlawii. The highest activity was obtained from a recombinant selected directly in A. laidlawii. This is the first example of a promoter sequence selected in a mollicute. Analysis of the putative promoters in seven clones revealed similar -10 and -35 regions, and similar spacer distances in A. laidlawii, Acholeplasma oculi, Lactococcus and E. coli. Vectors related to pNZ18 should be useful for the genetic analysis of specific A. laidlawii proteins and functions.

Dichelobacter nodosus is the primary pathogen implicated in ovine footrot. In this paper we have delineated a 27 kb locus, termed the virulence-related locus (vrl), that was essentially specific for virulent D. nodosus isolates. The precise ends of this locus were mapped and the sequences of the junction regions from the virulent strain A198 were compared to corresponding sequences from the benign isolate C305. The left end of the vrl locus was located in a sequence similar to that of the small stable 10Sa RNA molecule of Escherichia coli, next to a phage-attachment-site-like sequence, which indicated that the vrl locus might have arisen by the integration of a phage. However, no attachment-like sequence could be found at the right end of the vrl locus. In the chromosome of the benign strain the sequences bordering vrl were not contiguous but were separated by about 3 kb. It was concluded that the divergence of the benign and virulent strains at this locus was a multi-step process. Several potential ORFs were identified at the junction regions but only one ORF, encoding a 126 kDa protein, was expressed in a T7 expression system in E. coli.

A Cowdria ruminantium genomic DNA library was constructed in the expression vector γZAPII, and an immunoreactive clone, designated γCr9.4, was isolated by screening with serum from a C. ruminantium -infected goat. Sequencing of the insert from this clone revealed two open reading frames, encoding peptides of 10462 and 58697 kDa respectively. Database searching indicated that the two genes were homologues of groES and groEL, genes encoding a group of heat shock proteins involved in protein processing, export and assembly. Western blotting experiments showed that the recombinant GroEL protein was recognized by sera raised against four isolates of C. ruminantium which originate from South Africa, West Africa and the Caribbean, but not by antisera to the closely related Ehrlichia species (E. ovina, E. [Cytoecetes] ondiri, E. bovis, E. phagocytophila) of African and European ruminants which can be expected to occur in similar geographical areas to C. ruminantium. This suggests that this protein may be useful in development of serodiagnostic tests for C. ruminantium infection which are not subject to cross-reactions with antibodies to Ehrlichia species. The cloning and expression of the GroE operon will also facilitate further study of the roles of the GroE proteins in the immune response to C. ruminantium.

Using the phoA gene fusion methodology adapted to mycobacteria, several Mycobacterium tuberculosis DNA fragments encoding exported proteins were recently identified. In this paper, the molecular cloning, genomic positioning, nucleotide sequence determination and transcriptional start site mapping of a new M. tuberculosis gene, identified by this methodology, are reported. This gene was called erp (for exported repetitive protein) and has a sequence similar to that of the Mycobacterium leprae 28 kDa antigen irg gene M. tuberculosis erp gene contains a putative iron box close to the mapped transcriptional start site. The predicted Erp protein displays a typical N-terminal signal sequence, a hydrophobic domain at the C-terminus and harbours repeated amino acid motifs. These structural features are reminiscent of cell-wall-associated surface proteins from Gram-positive bacteria. We found that these repeats are conserved among M. tuberculosis isolates, and are absent from the published M. leprae irg gene sequence. In addition to being present in M. leprae, erp sequences were found in other members of the M. tuberculosis complex, but not in other mycobacteria tested. These results suggest that erp might encode a cell surface component shared by major pathogenic mycobacteria.

The Random Amplified Polymorphic DNA (RAPD) technique was used in the identification of a species-specific fragment of Mycobacterium bovis. A fragment of approximately 500 bp was amplified from the genome of 15 different M. bovis strains, including M. bovis BCG Pasteur, but was shown to be absent in 26 different mycobacteria and 20 different clinical isolates of Mycobacterium tuberculosis. When the fragment was used as a probe in a Southern blot analysis, several radioactive bands common to M. tuberculosis and M. bovis were observed. However, this fragment hybridized specifically to a 2900 bp EcoRI fragment in the M. bovis genome, but failed to hybridize in either M. tuberculosis or M. avium chromosomal DNA. Based on a partial nucleotide sequence of the 500 bp fragment, two oligonucleotide primers were designed and a PCR assay was developed. Using purified mycobacterial DNA samples, only M. bovis and M. bovis BCG rendered a unique amplification band. This PCR assay is able to detect down to 10 fg purified M. bovis DNA, which corresponds roughly to two bacilli. The assay is also useful for identifying the bacilli directly from uncultured biological samples, such as milk.

To facilitate the differential identification of the genus Streptomyces, the 16S rRNA genes of 17 actinomycetes were sequenced and screened for the existence of streptomycete-specific signatures. The 16S rDNA of the Streptomyces strains and Amycolatopsis orientalis subsp. lurida exhibited 95-100% similarity, while that of the 16S rDNA of Actinoplanes utahensis showed only 88% similarity to the streptomycete 16S rDNAs. Potential genus-specific sequences were found in regions located around nucleotide positions 120, 800 and 1100. Several sets of primers derived from these characteristic regions were investigated as to their specificity in PCR-mediated amplifications. Most sets allowed selective amplification of the streptomycete rDNA sequences studied. RFLPs in the 16S rDNA permitted all strains to be distinguished.

The ardA gene of I1 plasmid Collb-P9 was previously shown to alleviate DNA restriction by type I enzymes and to promote conjugative transmission of the unmodified plasmid to a restricting host. To clarify the ecological role of ardA, its distribution was determined on plasmids from 23 incompatibility groups using hybridization to the coding sequence as an assay. Hybridizing sequences, shown by nucleotide sequencing to have at least 60% identity with ardA, were detected on plasmids belonging to the I complex (IncB, I1 and K), the F complex (IncFV) and the IncN group. The ardA homologues were found to specify an antirestriction phenotype which was enhanced by genetic derepression of the plasmid transfer system. ardA loci map in plasmid leading regions but show no consistent association with a particular type of origin-of-transfer or a leading region gene of the ssb (single-stranded DNA-binding protein), psiB (plasmid SOS inhibition) and hok (host killing) families. It may be significant that ardA + plasmids are authentic enterobacterial plasmids and that type I restriction systems are associated historically with members of the Enterobacteriaceae.