- Volume 139, Issue 1, 1993

GDP-mannose dehydrogenase (GMD) is a key regulatory enzyme and the committal step in alginate biosynthesis. In this study, a metabolic approach has been used to investigate GMD activity in non-mucoid and isogenically related mucoid strains of Pseudomonas aeruginosa. Intracellular concentrations of GDP-mannose and GDP-mannuronate have been quantified using HPLC separation methods, and their concentrations have been related to GMD activity and total alginate production. In all strains of P. aeruginosa tested, GDP-mannose accumulated particularly during the exponential phase of growth in batch culture; the GDP-mannose concentrations in mucoid strains were significantly lower compared with isogenic non-mucoid strains. The product of GMD activity, GDP-mannuronate, was detectable only in mucoid strains, albeit at low but relatively constant levels irrespective of growth phase. The GDP-mannose concentrations in mucoid strains were always significantly greater than those of GDP-mannuronate, indicating that GMD is a rate-limiting enzyme in the biosynthesis of alginate. Significant GMD activity and extracellular alginate production were detected only in mucoid strains. The metabolic data reported here, together with previous genetic studies, provide strong evidence that GMD is the key regulatory enzyme controlling alginate biosynthesis in mucoid strains of P. aeruginosa.

Protein kinase C (PKC) from Pleurotus ostreatus mycelium (Basidiomycotina) was purified by affinity chromatography using activated agarose (Affigel 15) coupled with the protein kinase inhibitor N-(2-aminoethyl)-5-isoquinolinesulphonamide hydrochloride. Using buffer containing 2·0 M-arginine. PKC activity eluted as a single peak corresponding to a protein with apparent molecular mass of approximately 68000 Da as estimated by SDS-PAGE. The enzyme was not stimulated by Ca2+ alone, and activation by phosphatidylserine (PS) was significant only at 10−6 M-Ca2+. However, the activity was enhanced by the addition of 1,2-dioctanoyl-sn-glycerol in the presence of PS and Ca2+. Additionally, although sensitivity was lower than for mammalian PKC, the enzyme was activated by a tumour-promoting phorbol ester, phorbol-12-myristate 13-acetate (PMA). The saturation concentration of PMA was 50 g ml−1, which was 5 × 103 times the value for mammalian PKC.

Agaricus bisporus secretes abundant laccase activity into the medium during mycelial growth. SDS-PAGE analysis of extracellular laccase protein, purified from compost extract, showed a predominant band of 65 kDa molecular mass, together with lesser amounts of smaller polypeptides. The main polypeptide was purified electrophoretically. Amino acid sequence analysis of the N-terminal region of the main polypeptide was used to specify the sequence of a 15-residue chemically synthesized peptide (N-terminal peptide). Rabbit antibodies were raised against pure laccase, electrophoretically purified main polypeptide and the synthetic N-terminal peptide. Electrophoretically purified main polypeptide antibody was further purified by affinity chromatography on laccase-CNBr-Sepharose. Western blot analysis showed that the antigenic behaviour of laccase in compost extract, culture filtrate from malt-extract culture, and the purified enzyme from both sources, differed. The patterns of bands revealed are most simply explained by generation of (proteolytically) partially cleaved enzyme molecules in the culture medium, possibly combined with differences in extent of glycosylation. [35S]Methionine incorporation and immunoprecipitation were used to follow laccase synthesis in cultures grown on malt extract. After short-term labelling, a single polypeptide of 68 kDa apparent molecular mass was immunoprecipitated from both mycelial extracts and the culture medium. When poly(A)-containing RNA from malt-extract-grown mycelium was translated in vitro in rabbit reticulocyte lysate, a single polypeptide of about 57 kDa molecular mass was immunoprecipitated, consistent with the previously measured carbohydrate content of 15% for the pure enzyme. After treatment with N-glycanase, the polypeptide showed an increase in mobility during SDS-PAGE consistent with a reduction in molecular mass of about 5 kDa, indicating about equal amounts of N- and O-linked carbohydrate. C-terminal labelling of pure laccase was attempted by transpeptidation with carboxypeptidase Y. Although some minor bands were labelled, the main polypeptide was not, indicating that the C-terminus of the enzyme may be blocked.

The structure and function of the N-linked carbohydrate chains in laccase III, one of the ligninolytic glycoenzymes from the white-rot fungus Coriolus versicolor, have been partially characterized using endoglycosidases (Endo F and Endo H) and the N-asparagine-specific inhibitor, tunicamycin. In the presence of 10 μg tunicamycin ml−1 laccase and proteinase activities in culture filtrates of C. versicolor were measured over 3 weeks. Laccase activity was slightly decreased by the addition of tunicamycin, whereas proteinase activity was increased. The N-linked carbohydrate chains were not necessary for laccase secretion and activity. Endo-glycosidase digestion showed that laccase III contained at least four N-linked carbohydrate chains, of which two were high-mannose type or hybrid type and two were complex type. Judging from the differences in the resistance of the native and the carbohydrate-depleted laccase to proteolytic digestion and high temperature, the four N-linked carbohydrate chains have important protective functions against proteolytic attack and elevated temperature.

Flagellin from Bacillus thuringiensis subspecies alesti strain Bt75 was isolated both from the culture medium and from flagella. Two protein forms with molecular masses close to 32 kDa were obtained fromflagella; one form was identical to the flagellin purified from the culture medium. The N-terminal amino acid sequences were identical for both forms. Two genes coding for flagellin have been identified in B. thuringiensis subsp. alesti. The flaB gene was cloned and sequenced in its entire length. The clone containing the flaA gene was incomplete. Both genes were expressed in the mid-exponential growth phase.The flaB gene was flanked by long (355 bp) direct repeats protruding into the coding region in both th N- and C-terminal parts of the gene. DNA sequences related to the flaB gene were found in most other B. thuringiensis subspecies, and in two of them, subsp. kurstaki and subsp. entomocidus, such sequences were present in multiple copies.

Three additional alleles of the outB gene of Bacillus subtilis, whose activity is required for spore outgrowth, were identified. The nucleotide sequence of three mutant genes was determined. Analyses of dominance-recessivity showed that the wild-type allele is dominant over the mutant ones. When the outB gene was placed under the control of the inducible spac-1 promoter, the presence of IPTG was necessary to obtain normal growth. The results suggested that the outB gene is required for growth of B. subtilis. Expression of outB from the sporulation promoter spoIID negatively affected subsequent spore outgrowth, without altering vegetative growth and sporulation.

The gene nprM encoding the calcium-dependent extracellular proteinase from Bacillus megaterium ATCC 14581 was cloned in the vector pBR322 and expressed in Escherichia coli HB101. The DNA sequence of the cloned 3·7 kb fragment revealed only one open reading frame consisting of1686 bp with a coding capacity of 562 amino acid residues. A predicted Shine-Dalgarno (SD) sequence was observed 9 bp upstream from the presumptive translation startsite (ATG). A possible promoter sequence (TAGACG for the −35 region and TATAAT for the −10 region) was found about 69 bp upstreamof the ATG start site. The deduced amino acid sequence exhibited a 24 amino acid residue signal peptide and an additional polypeptide ‘pro’ sequence of 221 amino acids preceding the putative mature protein of 317 amino acidresidues. Amino acid sequence comparison revealed 84·5% homology between the mature protein and that of a thermolabile neutral protease from B. cereus. It also shares 73% homology with the thermostable neutral proteases of B. thermoproteolyticus and B. stearothermophilus. The zinc-binding sites and the catalytic residues are completely conserved in allfour proteases. NprM has a temperature optimum of 58 °C, a pH optimum of between 6·4 and 7·2, and is stimulated by calcium ions and inhibited by EDTA. These results indicate that the enzyme is a neutral (metallo-) protease.

The recA gene of Pseudomonas fluorescens OE 28.3 was isolated by complementation of the Fec− phenotype of recombinantlambda EMBL3 phages in a RecA− Escherichia coli strain. The subcloned recA restored resistance to UV and methyl methanesulphonate (MMS) exposure in recA mutants of E. coli. DNA sequence analysis showed that the coding region of the P. fluorescens gene, specifying a protein of 352 amino acid residues, waspreceded by an SOS box highly similar to those of Pseudomonas aeruginosa and Azotobacter vinelandii. The deduced amino acid sequence displayed highest homology to the RecA proteins from P. aeruginosa (87.8% identity) and A. vinelandii (84.3% identity). In both the regulatory region and the structural gene, a relatively high degree of sequence divergence from the Pseudomonas cepacia gene was observed. A mutant of P. fluorescens was constructed by inserting a kanamycin resistance cassette into its recA gene. This mutant exhibited an increased sensitivity to UV irradiation and MMS, and was strongly impaired in homologous recombinational activity.

The Clostridium acetobutylicum eglA gene, encoding a β-1,4-endoglucanase (EG), was shown to be a useful reporter gene for the study of gene expression in Bacteroides fragilis. The eglA reporter gene has the advantages that it can be easily identified in both Escherichia coli and B. fragilis on agar media containing carboxymethylcellulose, and EG production can be rapidly quantified in liquid medium. Since the B. fragilis glutamine synthetase (GS) is inactivated in permeabilized cells and cell extracts, the eglA reporter gene was used to study the regulation of GS production in B. fragilis. Gene fusions containing the GS glnA promoter region fused to the promoterless eglA gene showed that glnA expression was regulated by nitrogen in B. fragilis at the transcriptional level. A glnA upstream region containing a near-perfect direct repeat sequence was essential for efficient GS expression and for regulation by nitrogen.

Twenty-eight genetic loci have been physically mapped to specific large restriction fragments of the Streptococcus mutans GS-5 chromosome by hybridization with probes of cloned genes or, for transposon-generated amino acid auxotrophs, with probes for Tn916. In addition, restriction fragments generated by one low-frequency-cleavage enzyme were used as probes to identify overlapping fragments generated by other restriction enzymes. The approach allowed construction of a low resolution physical map of the S. mutans GS-5 genome using restriction enzymes ApaI (5′-GGGCC/C), SmaI (5′-CCC/GGG), and NotI (5′-GC/GGCCGC).

We have analysed the genes borne on a 6·0 kb Hin dIII fragment cloned from the chromosome of Clostridium botulinum type E strain Mashike. This fragment, cloned within plasmid pU9EMH, contains part of the structural gene for botulinum toxin type E neurotoxin as well as the entire structural gene for a nontoxic component of botulinum type E progenitor neurotoxin gene, ent-120. ent-120 is transcribed in the same direction as the neurotoxin gene and consists of one open reading frame encoding 1162 amino acid residues. Western blotting with anti-nontoxic component sera demonstrates that ent-120 encodes a protein of 120 kDa which forms part of the nontoxic component. ent-120 is homologous to an analogous gene found in botulinum type C strains (69.3% identity at the nucleotide level and 56.1% at the amino acid level). Two stretches of amino acids at the N-terminus of the ent-120 protein are highly homologous to amino acid sequences within the type E neurotoxin. The stop codon of the ent-120 gene is situated 27 nucleotides upstream from the start codon of the neurotoxin gene.

By selecting for growth of Escherichia coli mutant strains in the absence of the required amino acid, clones were found in a Brucella abortus library carrying genes for glutamylphosphate reductase (proA) and β-isopropylmalate dehydrogenase (leuB). These clones hybridized to unique fragments in a genomic digest of B. abortus DNA. The proA-complementing DNA was found in a region of 1·3 kb, which directed the synthesis of a protein of 48000 Da with a pI of 6·3 in maxicells. The leuB-complementing activity was in a region of 1·4 kb and directed synthesis of a protein of 46000 Da with a pI of 5·9.

Previously reported cell fractionation experiments have yielded conflicting information on the cellular localization of the DnaK heat shock protein of Escherichia coli. Here we used immunogold labelling of ultra-thin sections to determine the localization of DnaK in unstressed cells at 30 °C as well as in heat-shocked cells. In cells grown at 30 °C, gold particles were found predominantly in the cytoplasm, indicating that the majority of the DnaK molecules are cytoplasmic; however, a fraction of the gold particles was located in proximity to the membranes, raising the possibility that a subpopulation of DnaK proteins is membrane-associated. Heat shock of the cells did not induce detectable relocalization of DnaK.

Gene deletions of the puc operon of Rhodobacter capsulatus showed that the pucC and pucE genes, but not pucD, were required for formation of wild-type levels of the LHII complex. Deletion of pucC or pucE also impaired photosynthetic growth. The effects of pucC deletion were suppressed by secondary mutations that mapped outside the puc operon. Fusion of a lacy′Z gene to pucE′ showed that most of pucE transcription originated from upstream of pucB. RNA blot analysis revealed a 2·4 kb transcript that hybridized to probes specific for the pucBA, pucC and pucDE regions, indicating that some puc operon messages extend from just before the pucB gene to just after the pucE gene.

A cDNA clone derived from the altA locus, encoding one of several αtubulins in Physarum, was sequenced and used to determine the developmental and cell cycle expression patterns of its corresponding gene. The predicted amino acid sequence of the altA gene product, α1A-tubulin, is 92% identical to the other known Physarum α-tubulins, α1B and α2B, which are products of two tightly linked genes at the altB locus. The nucleotide sequence of the altA coding region is 82% identical to the two altB genes. Expression of the altA gene was found in all three cell types examined-amoeba, flagellate and plasmodium-but at substantially different levels in each. The peak level of altA message detected in flagellates was 14-fold higher than in amoebae, while the peak level in plasmodia was 5-fold lower than in amoebae. The expression pattern of altA and the predicted amino acid sequence of the α-tubulin it encodes suggest that α1A is the substrate for post-translational acetylation, giving rise to the α3-tubulin isoform found specifically in amoebae and flagellates. Northern blot analysis of plasmodial RNA samples from specific times in the cell cycle showed that the level of altA message varies over the cell cycle in a pattern similar to transcripts from other tubulin genes, with a peak at mitosis and little or no message detected during most of interphase.

Staurosporine is a strong inhibitor of protein kinases. At low concentrations, this antibiotic inhibited the growth of both protoplasts and mycelium of Pleurotus ostreatus. Its minimum inhibitory concentrations were 6·5 M and 9·6 M for protoplasts and mycelium, respectively. At a lower concentration (0·54 µM), it caused swelling of hyphal tips and subapical regions while some hyphae became stubby and bulbous. The phenomena were compared with the morphological effects of polyoxin D, which is a competitive inhibitor of chitin synthase. The results suggest the involvement ofprotein kinases(s) in regulation of apical growth of fungi.