- Volume 139, Issue 12, 1993

Glutamate synthase (GOGAT) from the phototrophic non-sulphur purple bacterium Rhodobacter capsulatus ElFl has been purified to electrophoretic homogeneity by affinity chromatography. The native protein consisted of two different subunits of 175 and 53 kDa and contained 4 mol FAD, 4 mol iron and 4 mol labile sulphide per mol of dimer enzyme. The enzyme used NADPH as the electron donor and was inhibited by iron-chelating and thiol group reagents. GOGAT exhibited NAD(P)H-diaphorase activity which used sodium ferricyanide, cytochrome c and dichlorophenol indophenol as alternative electron acceptors. By contrast, glutaminase activity was not detected in purified GOGAT. The amino acid composition was quite different from that of other bacterial GOGATs, and the protein presented different aggregation states depending on the ionic strength. Two major multimeric active species with Stokes’ radii of 6·18 and 7·32 nm could be separated by gel-filtration of protein solutions made in 0·5 m-KCl, whereas in the absence of salt, the maximal GOGAT activity corresponded to an oligomer with Stokes radius of 6·80 nm. The enzyme exhibited apparent negative cooperativity for glutamine, and was competitively inhibited by l-glutamate and NADP+.

The enzymic activities of the citric acid cycle and the connected pathway of 5-aminolevulinic acid (ALA) formation in the methylotroph Methylophilus methylotrophus (strain AS1) have been studied. The organism has the enzymes required for conversion of pyruvate to 2-oxoglutarate. Of these, isocitrate dehydrogenase is unusual because of its preference of NAD as coenzyme over NADP. In addition, the segment of the cycle that oxidizes 2-oxoglutarate to oxaloacetate is incomplete, lacking 2-oxoglutarate and succinate and malate dehydrogenase activities. Furthermore, alternative routes of 2-oxoglutarate oxidation to succinate are undetectable. The enzymes of the glyoxylate cycle are also absent. This suggests that the cycle in M. methylotrophus has no catabolic role and is purely biosynthetic. We also show that M. methylotrophus uses the C5 pathway of ALA formation. Cell-free extracts can convert glutamate to ALA in an ATP-, NADPH- and tRNA-dependent manner via the intermediate formation of Glu-tRNAGlu and glutamate 1-semialdehyde. Consistent with the absence of a detectable route by which it could synthesize succinate, M. methylotrophus cannot generate ALA from succinyl-CoA and glycine, the pathway found in mammalian cells and yeast.

The mannoprotein components solubilized from the walls of Candida albicans blastoconidia following degradation of the glucan network with β-glucanase (Zymolyase) have higher molecular masses than their probable precursors present in the supernatant of regenerating protoplasts. It therefore appears that the mannoproteins are released from the walls as part of supramolecular complexes. Immunological analysis using both polyclonal and monoclonal antibodies has demonstrated the probable relationship between molecules found in a mixed membrane preparation, those secreted by regenerating protoplasts, and those present in yeast cell walls. Some mannoproteins secreted by protoplasts incubated in the presence of tunicamycin had significantly increased mobility on SDS-PAGE, whereas others were not affected by the treatment. It is therefore possible that two types of mannoproteins are secreted by protoplasts: one carrying N-glycosylated chains (mannan) and one lacking them. All the proteins secreted in the presence of tunicamycin stained with Concanavalin A-peroxidase, demonstrating that they all, including the N-glycosylated ones, carried O-glycosylated sugar residues. Both classes of mannoproteins, secreted independently of each other, were found in the molecular complexes rendered soluble from the wall by Zymolyase digestion. Data obtained with a monoclonal antibody demonstrated the presence of a repeated epitope within one wall protein(s) detectable in a mixed membrane preparation and in the wall complexes released by Zymolyase.

The transport and incorporation of d-glucose into the human pathogen Helicobacter pylori was investigated employing radioactive tracer analysis and 1H and 13C nuclear magnetic resonance spectroscopy. The bacterium was found to utilize d-glucose contrary to the accepted view that it cannot catabolize carbohydrates. Under the experimental conditions employed, the rate of transport of [14C]glucose was 3·24 mmol min−1 (g protein)−1, and the rate of incorporation into the cellular mass was 1·06 μmol h−1 (g protein)−1. The utilization of [13C]glucose showed biphasic characteristics with a slower initial period followed by a phase with a rate of utilization at least an order of magnitude faster. The apparent rates of decline of glucose levels during both phases varied between strains and depended on the growth conditions of the bacteria prior to harvesting. The main product of glucose catabolism was identified as lactate. These findings provide new perspectives into the physiology of H. pylori and have implications for the active search to develop appropriate therapies for the micro-organism.

Cytosolic free calcium concentration [Ca2+]c of protoplasts from Penicillium notatum was measured using the permeant acetoxy ester (quin-2-AM) of the calcium-chelating fluorescent dye quin-2. Low uptake of the ester occurred at pH 5·8–7·0 and its subsequent hydrolysis was low. Uptake was promoted by an external pH of 5·0 and significant hydrolysis to quin-2 achieved by adjustment of the internal pH to 7·2, which was near the optimum of the carboxylic esterases responsible for the hydrolysis. Uptake of Ca2+ was biphasic with the average cell calcium concentration of protoplasts increasing from an initial value of 2 μmol to 50 μmol (kg cell water)−1, during attainment of the steady state after 30 min, at which time [Ca2+]c was unchanged at 20 nm but increased to 182 nm at 2–6 h exposure to 2·5 mm-Ca2+. Broadly similar changes in [Ca2+]c were found in protoplasts derived from mycelium samples exposed to Ca2+ over the same period of time. The location of Ca2+ was determined in subfractionated organelles and characterized using enzyme markers and electron microscopy. In 32 h mycelium preloaded with Ca2+ for 6 h, Ca2+ was located principally in the mitochondria with lower concentrations associated with the endoplasmic reticulum, Golgi, vacuoles and plasma membrane components. Calcium was not released by inositol 1,4,5-trisphosphate or the calcium ionophore A23187 from any subcellular fractions obtained from mycelium on Percoll gradients, nor from preparations of vacuoles or plasmalemma vesicles, except in the case of mitochondria where rapid release of the ion was achieved by the addition of 2–5 μm-A23187. The anti-calmodulin agent calmidazolium (R24571) greatly reduced sporulation when addition preceded that of Ca2+. Calcium-induced cultures showed massive novel protein phosphorylation 2 h after addition of the ion which was virtually eliminated by R24571, whilst 1 h and 4–6 h protein phosphorylations, which were also present to some degree in vegetative controls, were substantially reduced. Two-dimensional SDS-PAGE analysis of phosphoproteins confirmed that Ca2+-induced mycelium had enhanced capacity for calmodulin-mediated phosphorylation relative to corresponding vegetative cells and that complex differential changes in such phosphorylations occurred during Ca2+-induction of the sporulation process.

1,3-β-Glucan synthase activity has been detected in a membrane fraction extracted from the mycelium of the filamentous fungus Aspergillus fumigatus. The enzyme was solubilized by CHAPS and stabilized by filtration on a Bio-gel P30 column. Highest activity was obtained in the early exponential phase of growth. Four factors - GTP, NaF, sucrose and EDTA - added during the extraction procedure, were essential for optimal 1,3-β-glucan synthase activity. The soluble enzyme preparation was photolabelled with 5-azido-[32P]UDP-glucose and 5-125IASA-UDP-glucose which bind covalently to the enzyme after UV irradiation. These UDP-glucose substrate analogues were competitive inhibitors of the enzyme with a K i of 1·42 mm and 0·3 mm for 5-azido-UDP-glucose and 5-ASA-UDP-glucose, respectively (K m for UDP-glucose = 1·9 mm). Potential UDP-glucose-binding polypeptides were identified with molecular masses of 31, 50 and 115 kDa.

SUMMARY: The CWLA lytic amidase of Bacillus subtilis 168 was purified and antisera raised against the purified protein. No expression of cwlA could be demonstrated under any conditions by the use of the antisera and cwlA::lacZ fusion analysis. Two lytic enzymes of apparent molecular masses 34 and 30 kDa (as measured by renaturing SDS-PAGE) were found to be mitomycin C-inducible, the larger of which corresponds to a protein immunologically related to CWLA. Both of these inducible lysins were found to be encoded by prophage PBSX. Prophage SPβ was shown by renaturing SDS-PAGE to produce a 43 kDa lytic enzyme unrelated immunologically to CWLA. The smaller of the two PBSX enzymes was purified and found to be an N-acetylmuramyl-l-alanine amidase of 32 kDa (as measured by SDS-PAGE and Coomassie blue staining) which cross-reacts only weakly with the anti-CWLA sera. The potential origin of cwlA and its possible relationship to the other phage lytic enzymes are discussed.

SUMMARY: For the first time polyphosphate (poly P) granules have been detected in Helicobacter pylori organisms colonizing the gastric antrum as well as in organisms isolated from the same tissue. Poly P granules showed typical sublimation characteristics during exposure to the electron beam and chipped out of ultrathin sectioning. A prominent phosphorus signal was identified using elemental specific electron microscopy such as electron energy loss spectroscopy (EELS) and was localized to at least three different locations: the cytoplasm, the flagellar pole and in association with the cell membrane. Intracytoplasmatic structures had a diameter of 0·05–0·2 μm, whereas the structures near the flagellar pole were much smaller (0·02 μm). The membrane-associated phosphate aggregates were visible only after staining with Pb(NO3)2 or with electron spectroscopic imaging (ESI). Poly P granules seem to be important energy and phosphorus stores and it is thought that they participate in the regulation of various and distinct metabolic processes of H. pylori.

The pea phytoalexin pisatin, at its inhibitory concentration, was shown to have two distinct inhibitory effects on amoebae of the cellular slime mould Dictyostelium discoideum. One effect was cytolytic and was demonstrable even in non-growing cells whereas the second effect was observed only under conditions favourable to growth. Pretreatment with a sublethal concentration of pisatin induced the amoebae to acquire resistance to both these effects. Mutations in nysC that alter membrane sterols and confer resistance to the polyene antibiotics nystatin and pimaricin blocked resistance to the growth-associated inhibitory effect but did not affect acquisition of resistance to the cytolytic effect. The nysB sunD double mutant HK412 displayed a partially constitutive resistance to the cytolytic effect but, like the nysC mutants, was blocked in the acquisition of resistance to the growth-associated inhibitory effect. Pisatin-treated cells incubated in pisatin-free medium lost their ability to grow on pisatin-containing medium much more rapidly than they lost resistance to the cytolytic effect of pisatin. These results suggest that the induction of pisatin resistance may involve the turning-on of independent resistance mechanisms against the two inhibitory effects of pisatin. This could account for our inability to isolate pisatin-resistant mutants in a single step. The Neurospora crassa erg1 and erg3 mutants that have altered membrane sterols and are nystatin resistant displayed sensitivity to pisatin. The pisatin-sensitivity phenotype of the erg mutants was used in selections to identify complementing plasmids from an ordered Neurospora genomic library. The association of pisatin sensitivity with membrane sterol alterations in both D. discoideum and N. crassa supports the hypothesis that mechanisms underlying nondegradative pisatin resistance are evolutionarily conserved.

The Dictyostelium discoideum glycogen phosphorylase-1 (gp-1) exhibits a complex pattern of developmental expression in which differential temporal regulation of enzyme activity, protein levels and mRNA levels is observed. This pattern of expression implies that gp-1 regulation occurs at multiple levels, probably involving both transcriptional and post-transcriptional events. Post-translational control of gp-1 activity, in effect, actually regulates the protein from a developmental perspective. In this report we have examined several facets of this regulation. We show that addition of exogenous cAMP to cells in suspension culture caused changes in gp-1 enzyme activity and mRNA levels that are identical to those observed during normal development, suggesting that cAMP is involved in the regulation of gp-1. Exogenous cAMP could regulate gp-1 mRNA expression at concentrations as low as 1·0 μm. cAMP regulation of gp-1 mRNA appeared to occur through a mechanism that required intracellular cAMP signalling. We identified regions of the promoter necessary for gp-1 expression by using gp-1 promoter deletions to drive the expression of a luciferase reporter gene. Results of these experiments suggested that developmental and cAMP-mediated changes in gp-1 mRNA levels were the result of alterations in transcription. The promoter analysis also suggested that a vegetative specific element is located between −785 and −1894 nucleotides from the transcriptional start site. Elements necessary for maximal developmental and cAMP-mediated expression appear to be located between −1153 and −1894 nucleotides from the cap site. Sequence elements located between −180 and −1153 appear to be required for a basal level of late developmental expression.

A plasmid designated pLPG36 was isolated from the naturally occurring Legionella pneumophila serogroup-1 and purified by CsCl buoyant density centrifugation. A restriction map of this 58 kb plasmid was constructed and provided the basis for cloning four BamHI fragments into the unique BamHI site of pUC18. The four recombinant plasmids were investigated for the mobilization function in Escherichia coli strains. Only one of these, pFLJ2, was mobilized by the IncP plasmids RP4, pRK231 and R702, but not by plasmids pSa, R40a, R387, pN3 or R16. The derivative plasmid pFLJ2 was mobilized more efficiently by R702 than by RP4 or pRK231. By genetic and deletion analysis, the mobilization region of pLPG36 was located to a 6 kb EcoRI fragment of the plasmid.

Nucleotide sequencing revealed that gtaB, the structural gene of UDP-glucose pyrophosphorylase (EC 2.7.7.9), is part of a divergon-like genetic entity. The latter consists of two monocistronic operons gtaB and orfX, transcribed from a 245 bp regulatory region, each encoding an acidic protein with a molecular mass of 33·0 and 42·6 kDa, respectively. gtaB is transcribed from a distal PA promoter, and a proximal PB promoter which is negatively controlled by the Sin protein. Sin-mediated transcriptional attenuation and enhancement of PB and PD, respectively, suggest that these promoters control functions which antagonize each other. Transcription of orfX is mediated by a PA promoter. The regulatory region comprises four ATGAAA hexamers, present as two inverse repeats. Protein GtaB exhibits high homology to analogous prokaryotic enzymes, while OrfX shows 55·4% homology with the product of Escherichia coli o389, which is part of a regulatory unit involved in sugar processing. Mutations gtaB515 and gtaBg100, which define different bacteriophage adsorption patterns, were sequenced. They are transitions leading to substitution of amino acids which occupy conserved positions, and are thus likely to be part of an enzyme active site. The nature of the possible receptors for defective bacteriophages PBSY and PBSZ is discussed.

The spoIIAB gene of Bacillus subtilis encodes an inhibitor of σF, a transcription factor that plays a crucial role in the establishment of prespore-specific gene expression during sporulation. The SpoIIAB protein can probably also inhibit a closely related sigma factor σG, which determines the later phase of prespore-specific transcription. We have isolated two new missense mutations in the spoIIAB gene. spoIIAB28 behaves like the previously described spoIIAB1 mutation, in that it mainly affects the activity of σG. In contrast, the spoIIAB22 mutation seems to be impaired mainly in its ability to inhibit σF. All three missense mutations are clustered in the N-terminal coding region of spoIIAB, suggesting that this region of the protein interacts with the sigma factors. The extreme N-terminal part of SpoIIAB may be specifically concerned with the regulation of σG activity.

Chlorate-resistant mutants of the denitrifying bacterium Thiosphaera pantotropha were generated by transposon Tn5 mutagenesis. One class was deficient in membrane-bound nitrate reductase activity but retained a periplasmic nitrate reductase activity. Using transposon marker rescue it was shown that in one such mutant, M-6, the transposon was inserted in the membrane-bound nitrate reductase β subunit structural gene (termed narH in order to be consistent with the nomenclature of the Escherichia coli major nitrate reductase operon). The translated sequence (total of 106 amino acids) from around the point of transposon insertion showed approximately 90% amino acid identity with the β subunits of the E. coli nitrate reductases. Under anaerobic growth conditions M-6 overproduced the periplasmic nitrate reductase activity allowing anaerobic growth with nitrate as electron acceptor. A regulatory link was inferred between the presence of the membrane-bound nitrate reductase and expression of the periplasmic nitrate reductase. This is the first demonstration of full denitrification in an organism possessing only a periplasmic nitrate reductase.

A gene encoding the extracellular α-amylase of Aeromonas hydrophila MCC-1 was cloned and expressed using its own promoter on the recombinant plasmid pCA101. Subcellular fractionation of Escherichia coli JA221 carrying pCA101 revealed that approximately 60% of the amylase activity was localized in the periplasmic space. The extracellular amylase was purified to homogeneity, identified as an α-type and its amino-terminal sequence was determined. Nucleotide sequence analysis predicted a 443 amino acid ORF and 24 amino acids at the amino terminus of the sequence that are not found in the secreted protein. This 24 amino acid sequence has many of the characteristics common to known signal peptides. The predicted amino acid sequence has considerable similarity with mammalian, invertebrate and Streptomycete α-amylases. Most of the amino acid residues that are involved in catalytic activity, substrate binding and calcium binding in several α-amylases were also present in A. hydrophila α-amylase at the corresponding positions.