- Volume 138, Issue 4, 1992

Summary: TDP-d-glucose 4,6-dehydratase, which converts TDP-d-glucose to TDP-d-4-keto-6-deoxyglucose, was purified to near-homogeneity from the daunorubicin and baumycin-producing organism Streptomyces sp. C5 (968-fold purification with a 41% recovery), and from the daunorubicin producer Streptomyces peucetius ATCC 29050 (1000-fold purification with a 37% recovery). The TDP-d-glucose 4,6-dehydratases from Streptomyces sp. C5 and S. peucetius were determined by SDS-PAGE and HPLC gel filtration to be homodimers with subunit relative molecular masses of 39000 and 36000, respectively. For the enzymes from both organisms, negligible activity was observed in the absence of added NAD+, or when ADP-glucose, ADP-mannose, GDP-mannose, UDP-glucose or UDP-galactose was substituted for TDP-d-glucose as substrate. For the enzyme from Streptomyces sp. C5, the K'm values for NAD+ and TDP-d-glucose were 19·2 μm and 31·3 μm, respectively. The V'max for TDP-d-glucose was 309 nmol min−1 (mg protein)−1. For the S. peucetius enzyme, the K'm values for NAD+ and TDP-d-glucose were 20·1 μm 34·7 μm, respectively. V max values were 180 nmol min−1 (mg protein)−1 for NAD+ and 201 nmol min−1 (mg protein)−1 for TDP-d-glucose. TDP was a good inhibitor of TDP-d-glucose 4,6-dehydratase from both organisms. The N-terminal amino acid sequence of the TDP-d-glucose 4,6-dehydratase from S. peucetius and from the erythromycin producer, Saccharopolyspora erythraea, were similar, whereas the enzyme from Streptomyces sp. C5 contained a different N-terminal amino acid sequence from either of the other two enzymes.

Summary: The pyruvate dehydrogenase complex from the thermophilic bacterium Thermus aquaticus was purified by Triton X-100 extraction and chromatography on phenyl-Sepharose CL-4B and HPLC-hydroxyapatite. The electrophoretic pattern of the purified enzyme complex was similar to that of the enzyme complex from Bacillus subtilis, with four bands: the α-chain (M r 39600) and β-chain (M r 37500) of the pyruvate dehydrogenase component, the dihydrolipoamide acetytransferase component (M r 58500) and the dihydrolipoamide dehydrogenase component (M r 53900). Antibodies against the purified T. aquaticus pyruvate dehydrogenase complex cross-reacted with the enzyme complex from B. subtilis and, to a minor extent, with that from bovine heart. No cross-reactivity could be observed with the enzyme complex from Escherichia coli. The T. aquaticus enzyme complex had a temperature maximum at 72°C. 2-Oxobutyrate was a poor substrate and other 2-oxoacids were competitive inhibitors of the overall reaction. Long-chain 2-oxoacids showed a greater inhibitory effect, possibly caused by hydrophobic interactions. GTP inhibited the enzyme activity. Regulation of the pyruvate dehydrogenase complex from T. aquaticus by allosteric mechanisms or by reversible phosphorylation could not be demonstrated.

Summary: Pyruvate decarboxylase from the obligate anaerobe Sarcina ventriculi was purified eightfold. The subunit M r was 57000 ± 3000 as estimated from SDS-PAGE, and the native M r estimated by gel filtration on a Superose 6 column was 240000, indicating that the enzyme is a tetramer. The M r values are comparable to those for pyruvate decarboxylase from Zymomonas mobilis and Saccharomyces cerevisiae, which are also tetrameric enzymes. The enzyme was oxygen stable, and had a pH optimum within the range 6·3-6·7. It displayed sigmoidal kinetics for pyruvate, with a S 0·5 of 13 mm, kinetic properties also found for pyruvate decarboxylase from yeast and differing from the Michaelis-Menten kinetics of the enzyme from Z. mobilis. No activators were found. p-Chloromercuri-benzoate inhibited activity and the inhibition was reversed by the addition of dithiothreitol, indicating that cysteine is important in the active site. The N-terminal amino acid sequence of pyruvate decarboxylase was more similar to the sequence of S. cerevisiae than Z. mobilis pyruvate decarboxylase.

Summary: Phospholipase activities releasing fatty acyl moieties from phosphatidylcholine and phosphatidylethanolamine and lysophospholipase activity releasing fatty acid from lyso-phosphatidylcholine were detected in both Mycobacterium microti and Mycobacterium avium. Fatty acyl groups were released from both the 1- and 2-positions of phosphatidylcholine. Generally, phospholipase activities of M. avium were cryptic while phospholipase activities of M. microti were located on the bacterial surface. However, intact M. microti did not release fatty acids from phospholipids faster than M. avium. Neither Mycobacterium secreted acyl-hydrolysing phospholipase activity. All phospholipase activities were stimulated by including phospholipids in growth media: generally, cell extracts contained 6- to 15-fold higher specific activities than extracts from mycobacteria grown in media without added phospholipid. However, not all phospholipase activities were stimulated to the same degree in any given set of conditions, suggesting the existence of more than one phospholipase gene in each Mycobacterium.

Summary: The growth and survival of two genetically engineered micro-organisms (GEMs), Pseudomonas putida strain PaW8 containing either marker plasmid pLV1013 (IncQ) or pLV1016 (IncP), both of which contain the xylE gene, were investigated in soils ranging in texture from a sandy loam to a peat. Viable GEMs were enumerated over a period of 89 d. Analysis of the decay kinetics indicated that GEM survival, as monitored by the persistence of the xylE gene, was biphasic in the sandy and clay loams. Both organisms showed evidence of initial growth in the peat soil prior to a rapid death phase. The results indicated that soil texture has an effect on the survival of genetically engineered P. putida, death decreasing with increasing clay content.

Summary: The nucleotide sequence of the proBA operon from a proline-hyperproducing mutant of Serratia marcescens was determined. Two base substitutions were found: one in the proB structural gene, coding for γ-glutamyl kinase (GK), and a second one in the promoter region of the operon. The former base substitution led to a change of the predicted amino acid at position 117 from an alanine to a valine in GK. This mutation rendered GK 700-fold less sensitive to proline-mediated feedback inhibition than the wild-type enzyme. The other base substitution, a transversion from a G-C to an A-T, was located in the spacer region between the ‘---35’ and ‘---10’ sequences of the promoter, and it increased the transcriptional activity of this operon fourfold. Both these two base substitutions, which were acquired at the step of selecting mutants resistant to a toxic proline analogue, 3,4-dehydroproline, confer upon cells a high proline productivity and an increased osmotolerance.

Summary: An esterase gene (estA) from a lipolytic psychrotroph (Pseudomonas sp. LS107d2), was cloned in Escherichia coli and its nucleotide sequence was determined, revealing an ORF encoding a polypeptide of 389 amino acid residues, with a molecular mass of 42276 Da. Labelling of plasmid-encoded proteins with [35S)methionine, using the maxicell procedure, gave a single polypeptide of molecular mass 42 kDa, consistent with that calculated from the ORF. Colonies of E. coli cells containing estA produced a clear halo when grown on solid media containing tributyrin; no clearance was produced when cells were grown on media containing triolein. Extracts of cells containing estA also hydrolysed water-soluble nitrophenol esters, but were unable to cleave water-insoluble substrates. The preference for water-soluble substrates indicates that the gene product is an esterase.

Summary: The proteinase genes from Lactococcus lactis subsp. lactis UC317 were identified on a plasmid, pCI310, which is a deletion derivative of a cointegrate between pCI301, the 75 kb Lac Prt plasmid from UC317 and the 38·5 kb cryptic plasmid from that strain. The prt genes were cloned using a replacement cloning strategy whereby fragments from pCI310 were exchanged with the equivalent fragments in pNZ521, which contains the cloned proteinase genes from L. lactis subsp. lactis SK112. This generated two plasmids which encoded a cell-envelope-associated and a secreted proteinase, respectively. Specific regions of the UC317 structural prtP gene known to encode seven of the amino acids essential for substrate cleavage specificity were sequenced and compared with the known sequences of prt genes from L. lactis strains SK112, Wg2 and NCD0763. In spite of various differences that were detected in the nucleotide sequence of this region, it appears that these seven amino acids in strains UC317 and NCD0763 are identical, and represent a combination of three of the amino acids from SK112 and four from Wg2. These results indicate that the UC317 proteinase is a natural hybrid of the SK112 and Wg2 proteinases.

Summary: A marine Vibrio (designated Vibrio sp. 60) that is related to Vibrio anguillarum was used as a host for a plasmid that encodes the non-toxic B subunit (EtxB) of Escherichia coli heat-labile enterotoxin. Expression of EtxB in Vibrio sp. 60 resulted in the efficient and selective secretion of the B subunit into the extracellular growth medium. This indicated that Vibrio sp. 60, which does not normally produce cholera-like enterotoxins, nonetheless possesses a secretory machinery that permits these toxins to be translocated across its cytoplasmic and outer membranes. Expression of EtxB in a sec mutant of Vibrio sp. 60 (MVT1192), which had previously been shown to be defective in the secretion of several extracellular proteins, resulted in approximately 95% of the B subunit remaining entrapped within the periplasm of the bacterial cell envelope. This implies that the mutation in MVT1192 defines a locus that determines a common step in the secretion of extracellular proteins, including oligomeric toxins.

Summary: The lipoprotein I gene (oprI) of Pseudomonas aeruginosa PAO1 was cloned and sequenced. A high degree of homology was found between our cloned PAO1 gene sequence and two published oprI sequences. Specific oligonucleotides were designed to amplify the oprI gene by the polymerase chain reaction (PCR). The potential of either the complete gene sequence or the specific oligonucleotide primers as a tool for rapid strain identification was directly assessed against bacterial colonies by PCR or against purified genomic DNA by Southern blot analysis, using a number of representative strains within the Pseudomonadaceae. The oprI gene was found to be well conserved within RNA group I.

Summary: The genetic analysis of 16 recessive pea mutants in Aspergillus nidulans deficient in the metabolism of protocatechuic acid (PCA) has revealed seven functional genes. The seven gene loci are distributed over three chromosomes: pcaA, pcaC and pcaD on linkage group II; pcaE, pcaF and pcaG on group V, and pcaB on group VIII, where it shows linkage [recombination frequency (RF) = 6·9%] to the qut gene cluster controlling the degradation of quinic acid to PCA. Only two of the pea gene loci are closely linked: pcaE and pcaG (RF = 0·8%). The properties of the qut and pea mutants clearly demonstrate the separate identity and regulation of the converging pathways from quinate or benzoate to PCA, which in turn is oxidatively degraded through β-ketoadipate to TCA intermediates. Similarly, the mutants are not affected in the metabolism of salicylate to catechol and its oxidation to β-ketoadipate, although two genes (pcaA and pcaF) are required for the further metabolism of β-ketoadipate. Catechol dioxygenase is induced by growth in the presence of salicylate, and PCA dioxygenase by benzoate or quinate. Three groups of pea mutants (pcaB, pcaD and pcaE) are deficient in the induction of PCA oxygenase and accumulate PCA when grown in the presence of quinate or benzoate. All three pcaE mutants and the single pcaA strain totally lack PCA oxygenase activity, while a single pcaB mutant strain has properties tentatively suggesting a positive role in the induction of the PCA pathway.

Summary: SDS extracts of whole bacteria, representing five species and 15 serovars of Listeria, were analysed by SDS-PAGE and by immunoblotting with serum directed against whole formalin-treated L. monocytogenes. Profiles of L. monocytogenes were very different from those of other species of Listeria (i.e. L. innocua, L. welshimeri, L. seeligeri and L. ivanovii). This low degree of similarity between species was found even in the case of common serovars. Within the species L. monocytogenes, protein patterns were characterized, on the one hand, by a high degree of homogeneity between all strains of the same serovar and, on the other hand, by large differences between serovars, especially between sv. 1/2 and 4b. Thus we have identified major, surface-located protein antigens, specific for L. monocytogenes, either common to all serovars (64 and 68 kDa) or characteristic of certain serovars: 98 kDa for sv. 1/2 and 3; 76 and 78 kDa for sv. 4b, 4d and 4e; and 80 and 100 kDa for sv. 4a and 4c. Moreover, some of these bands (68 and 98 kDa) might be related to virulence, since differences were noticed between the profiles of haemolytic L. monocytogenes sv. 1/2a differing only in their virulence for immunocompromised mice. All these results confirmed, for the first time, the classification of Listeria obtained previously by genomic studies. They should help in the identification of new virulence factors and the development of easier and more specific methods of detection and identification.

Summary: Lactobacillus plantarum strain 4B2, which exhibits a strong autoaggregating phenotype, receives the broad-host-range plasmid pAMβ1 with conjugation efficiencies as high as 10−2 transconjugants per donor using solid matings; broth matings also occur, but at low transfer frequencies. Filter-sterilized spent supernatant of this strain contains a 32 kDa protein that promotes aggregation, and consequently a high frequency of conjugation, in lactic acid bacteria containing α-1,2-glucose-substituted lipoteichoic or teichoic acids. It appears, therefore, that the substituted lipoteichoic or teichoic acids act as receptors for the aggregation-promoting protein.

Summary: 3 5S-labelling experiments and Western blot analysis were used to investigate methionine induction of tyrosinase synthesis in Streptomyces antibioticus. De novo synthesis of the enzyme occurred as a function of time and methionine concentration. Induction appeared to be relatively specific for methionine and closely related analogues. Under the conditions used, the enzyme was secreted rapidly, with little intracellular accumulation. Upon induction in the absence of Cu2+, apotyrosinase was synthesized at 70% of the level in control cultures provided with the cation. Inhibitor studies showed that both transcriptional and translational events are required for tyrosinase induction. Deletions in the ORF 438 region of the mel operon suggest that this sequence has a role in the phenotypic expression of tyrosinase.