- Volume 136, Issue 3, 1990

Previous studies of Haemophilus influenzae documented the importance of several pyridine nucleotide-dependent enzymes in processing extracellular NAD and NMN to satisfy the V-factor growth requirement of the organism. The substrate specificities of two of these enzymes, NMN: ATP adenylyltransferase and NAD kinase, were investigated following partial purification. The ability of the transferase to utilize 3-acetylpyridine mononucleotide and 3-aminopyridine mononucleotide as substrates for the synthesis of the corresponding dinucleotides was demonstrated. The NAD kinase was observed to accept 3-acetylpyridine adenine dinucleotide as a substrate but failed to utilize 3-aminopyridine adenine dinucleotide. The mononucleotides of 3-acetylpyridine and 3-aminopyridine were shown to be as effective as the corresponding dinucleotides in the support of growth and inhibition of growth of H. influenzae, respectively. Inhibition of growth of H. influenzae by submicromolar 3-aminopyridine adenine dinucleotide was shown to occur because 3-aminopyridine mononucleotide was produced from it in reactions catalysed by the H. influenzae periplasmic nucleotide pyrophosphatase. The presence of an additional important pyridine nucleotide-dependent enzyme, NMN glycohydrolase, is also reported.

Biological activities of lipopolysaccharides (LPS) extracted from Bordetella pertussis, B. parapertussis and B. bronchiseptica were compared with those of Escherichia coli LPS. The LPS preparations from B. pertussis showed biological activities comparable to those of E. coli LPS in terms of lethal toxicity in galactosamine-sensitized mice, pyrogenicity in rabbits, mitogenicity in C3H/He spleen cell cultures, macrophage activation, and induction of tumour necrosis factor. All the activities of LPS preparations from B. parapertussis, except mitogenicity, were lower than those of E. coli LPS. LPS from B. parapertussis gave the greatest mitogenic action of all those tested. Biological activities stronger than or comparable to those of E. coli LPS were observed for LPS from B. bronchiseptica.

A high l-asparaginase (l-asparagine amidohydrolase; EC 3.5.1.1) activity was found under conditions of lysine overproduction in cultures of Corynebacterium glutamicum. l-Asparaginase was purified 98-fold by protamine sulphate precipitation, DEAE-Sephacel anion exchange, ammonium sulphate precipitation and Sephacryl S-200 gel filtration. The asparaginase protein was subjected to PAGE under non-denaturing conditions, identified by an in situ reaction and eluted from the gel in an active form. The estimated M r from gel filtration and SDS-PAGE was 80000. The l-asparaginase activity was inhibited by the l-asparagine analogue 5-diazo-4-oxo-l-norvaline. Neither d-asparagine nor l-glutamine was a substrate for the enzyme. l-Asparaginase was produced constitutively; its role may be that of an overflow enzyme, converting excess asparagine into aspartic acid, the direct precursor of lysine and threonine.

Lysobacter enzymogenes produces an inducible β-lactamase and induction with 100 μg ampicillin ml−1 resulted in an increase of more than 100-fold in enzyme activity. Various other β-lactam antibiotics also served as effective inducers. The enzyme was obtained from cells by osmotic shocking to release periplasmic components and it was purified primarily by ion-exchange chromatography and PAGE. The β-lactamase consists of one polypeptide with a molecular mass of about 28 kDa and an isoelectric point greater than 9·6. It is strongly inhibited by p-chloromercuribenzoate and clavulanic acid but not by EDTA. The enzyme readily hydrolyses several penicillins and cephalosporins, but not oxacillin or cloxacillin. The enzyme therefore belongs to group 2b of the bacterial β-lactamases.

A filamentous division mutant, PV302, of Bacillus megaterium QM B1551 was isolated while screening for sporulation-defective mutants after nitrosoguanidine mutagenesis. Both phase-contrast and electron microscopy revealed that the mutant produced small spherical cells as well as filaments. It also accumulated large amounts of poly-β-hydroxybutyrate. Poly-β-hydroxybutyrate accounted for 16% of the dry weight of the mutant strain even after 28 h growth. In comparison to the parental strain, the division mutant also showed both an inability to sporulate and a reduced growth rate. All these phenotypes transduced together. Revertants gained the ability to sporulate, divide, and grow normally. Transductional mapping of the mutation, designated div-1, established a new linkage group for B. megaterium consisting of div-1 and the pyrimidine biosynthesis genes pyrDBCF. The spherical cells were separated from filaments by sucrose gradients and were tested for nucleic acid content and viability. The purified spherical cell fraction contained one-fifth the amount of DNA per mg protein as compared with the filamentous cell fraction and was shown to contain both non-viable minicells and some cells capable of growing after a lag of about 4 h. This suggests that the mutation not only causes defects in septum placement and sporulation, but may possibly affect DNA partitioning.

Shaken cultures of Streptomyces venezuelae ISP5230 in minimal medium with galactose and ammonium sulphate as carbon and nitrogen sources, respectively, showed extensive sporulation after 72 h incubation at 37 °C. The spores formed in these cultures resembled aerial spores in their characteristics. The ability of the spores to withstand lysozyme treatment was used to monitor the progress of sporulation in cultures and to determine the physiological requirements for sporulation. In media containing ammonium sulphate as the nitrogen source, galactose was the best of six carbon sources tested. With galactose S. venezuelae ISP5230 sporulated when supplied with any of several nitrogen sources; however, an excess of nitrogen source was inhibitory. In cultures containing galactose and ammonium sulphate, sporulation was suppressed by a peptone supplement. The onset of sporulation was accompanied by a drop in intracellular GTP content. When decoyinine, an inhibitor of GMP synthase, was added to a medium containing starch and ammonium sulphate, a slight increase in sporulation was seen after 2 d. The suppression of sporulation by peptone in liquid or agar cultures was not reversed by addition of decoyinine. A hypersporulating mutant of S. venezuelae ISP5230 was altered in its ability to assimilate sugars. In cultures containing glucose the mutant sporulated more profusely than did the wild-type and did not acidify the medium to the same extent. However, the suppressive effect of glucose on sporulation was not merely a secondary result of acid accumulation.

Many Escherichia coli K12 strains undergo switching between two forms which differ in a number of surface properties including colony morphology and the ability to auto-aggregate. This paper describes the identification of a gene which appears to play a part in controlling this switching phenomenon. This gene has been designated mor and is located at 89 minutes on the E. coli chromosome map between the argECBH operon and the trmA gene. By manipulation of this gene it is possible to overcome the switching of surface properties and fix a strain in one form or the other. The mor gene has been cloned and its DNA sequence determined. The putative protein sequence shows a high level of homology with four regulatory genes, the ilvY, cysB and lysR genes from E. coli and the metR gene from Salmonella typhimurium. It has also been shown that the mor gene is autoregulated at the transcriptional level.

Mitochondrial DNA (mt DNA) from a patulin producer, Penicillium urticae (synonym P. griseofulvum), was 27·8 kb ± 0·6 kb in size by electron microscopy and 27.2 kb by agarose gel electrophoresis. Restriction endonuclease maps for nine restriction enzymes were constructed, and eleven fragments which covered the total range of the mt DNA were cloned into the Escherichia coli plasmid vector pUC19. Southern analysis of the native genomes of P. urticae and P. chrysogenum with six of the cloned fragments as probes indicated similar genome arrangements as well as similar restriction maps. Both the large and small rRNA genes of P. urticae and P. chrysogenum were located on these restriction maps using Southern hybridization, and the result also supported the similar arrangement. Agarose/formaldehyde gel electrophoresis indicated that the small rRNA was 1·5 kb in size in both species; but, surprisingly, the large rRNA was 4·2 kb in size for P. urticae and 3·5 kb for P. chrysogenum. These sizes were, respectively, 1·1 kb and 0·4 kb larger than those from the very closely related Aspergillus nidulans.

The structural gene for the precursor of the peptide antibiotic nisin was isolated and characterized. As with other lanthionine-containing antibiotics, nisin is synthesized as a pre-propeptide which undergoes post-translational modification to generate the mature antibiotic. The sequence data obtained agreed with those of precursor nisin genes isolated by other workers from different Lactococcus lactis strains. Analysis of regions flanking the precursor nisin gene revealed the presence of a downstream open reading frame that may be involved in maturation of the precursor molecule. Nucleotide sequences characteristic of an IS element were located upstream of the nisin determinant. This element, termed IS904, is present in multiple copies in the genome of L. lactis. The nisin determinant of L. lactis is a component of a large transmissible gene block that also encodes nisin resistance and sucrose-metabolizing genes. Gene probe experiments indicated that the nisin/sucrose gene block was located in the chromosome. Furthermore, the copy of IS904 identified adjacent to the precursor nisin gene lies at, or very close to, one end of this transmissible DNA segment and may play a role in mediating its transfer between strains.

Eighteen related strains of Arthrobacter, Brevibacterium and Corynebacterium were- used as indicator strains in an attempt to isolate corynephages from a large number of soils and from waste-water samples. Although no phages capable of producing plaques were isolated, one of the indicator strains used, Brevibacterium flavum ATCC 14067, was lysogenic for the inducible phage ϕGA1. This phage was not observed to form plaques on any of the strains tested. ϕGA1 is of B1-morphotype with a linear double stranded DNA genome of 48·1 kb with cohesive ends; a restriction map is presented.

Bacteriophage JHJ-1 was isolated from Saccharopolyspora hirsuta strain 367 NRRL 12045 as an endogenous but virulent phage. The plaque size was not self-limiting, since a few p.f.u. could completely lyse a lawn. Electron microscopy showed that this phage belonged to group B of Bradley’s morphological classification. The JHJ-1 genome is a linear DNA molecule of 41·1 kbp with cohesive ends and a G + C content of 68·8–70·0 mol%. The DNA cleavage was established for 12 restriction endonucleases. The host range is apparently very narrow, being limited to two strains of S. hirsuta (NRRL 12045 and NRRL B-5792). However, JHJ-1 did not lytically infect S. hirsuta 367 UC 8106. Phage JHJ-1 was shown, by Southern blot analysis, to lysogenize both S. hirsuta strain 367 UC 8106. It thus appears to behave as a virulent mutant of a temperate phage on one, but not on the other, JHJ-1 lysogen.

Genes encoding extracellular β-lactamases of Streptomyces badius, Streptomyces cacaoi, Streptomyces fradiae and Streptomyces lavendulae were cloned and mapped in Streptomyces lividans. DNA sequence analysis of the β-lactamase genes revealed a high overall G + C content, ranging from 71 to 75 mol%, with a G + C content of 95 mol % at the third position of the codons for all four genes. The primary structure of the β-lactamases including their signal peptides was deduced. The four β-lactamases exhibited homology to each other and to class A β-lactamases from other bacterial genera. We suggest that Streptomyces β-lactamases are representatives of a superfamily of genes, from which class A β-lactamases of Gram-negative bacteria may have evolved.

Protein antigen b (Pab) of Mycobacterium tuberculosis has previously attracted interest because of its immunological and diagnostic relevance. In this study we present evidence that Pab possesses a signal sequence and is secreted from the cytoplasm of M. tuberculosis. The synthesis of Pab is enhanced under phosphate starvation indicating that the protein is involved in phosphate metabolism in M. tuberculosis.

A method was devised for the reproducible isolation of envelopes from Pasteurella haemolytica serotype A2. It was also possible to prepare envelopes from other serotypes of P. haemolytica and Pasteurella multocida using this methodology. Examination of these preparations by SDS-PAGE showed major differences between strains of P. haemolytica and strains of P. multocida which allowed the clear distinction of isolates of these species. Amongst the P. haemolytica serotypes it was possible to distinguish envelope preparations made from A biotype and T biotype organisms easily, but it was not possible to identify individual serotypes from each other. Envelope profiles were sufficiently different between the individual P. multocida serotypes examined to allow each to be identified by its polypeptide profile. Experiments using radiolabelling, antibody absorption, and susceptibility to protease digestion, together with heat modifiability and detergent solubility characteristics indicated that 13 of the envelope proteins were probably surface-located. A high molecular mass immunogenic envelope protein was shown, by immunoblotting, to be present in all strains of P. haemolytica and P. multocida examined.

Abbreviations: CBB, Coomassie Brilliant Blue; KDO, 2-keto-3- deoxyoctonate (3-deoxy-D-mawio-octulosonate); PMSF, phenylmethyl- sulphonyl fluoride; TLCK, V-a-/>-tosyl-L-lysine chloromethyl ketone.