Cobalt-activated aminopeptidase fromStreptococcus sanguis NCTC10904

An aminopeptidase fromStreptococcus sanguis NCTC 10904 was isolated, purified, and characterized. The enzyme was produced constitutively and could be isolated from the cytoplasmic fraction of lysed cells. Its substrate profile indicated that it is primarily a leucyl aminopeptidase, but with a substrate spectrum including lysyl- and arginyl-peptides. The subunit molecular weight of the enzyme was approximately 74,000, but an octomeric form also was prominent, as indicated by gel filtration separations of active enzymes. The optimal temperature for activity was 32°C, and the optimal pH value was about 7.0. The enzyme showed cooperative kinetics and was activated by Co²⁺. The regulation of synthesis and the characteristics of the enzyme suggest that it may serve a regulatory function rather than just a nutritional function.     

Genome and proteome of Campylobacter jejuni bacteriophage NCTC 12673

Campylobacter jejuni continues to be the leading cause of bacterial food-borne illness worldwide, so improvements to current methods used for bacterial detection and disease prevention are needed. We describe here the genome and proteome of C. jejuni bacteriophage NCTC 12673 and the exploitation of its receptor-binding protein for specific bacterial detection. Remarkably, the 135-kb Myoviridae genome of NCTC 12673 differs greatly from any other proteobacterial phage genome described (including C. jejuni phages CP220 and CPt10) and instead shows closest homology to the cyanobacterial T4-related myophages. The phage genome contains 172 putative open reading frames, including 12 homing endonucleases, no visible means of packaging, and a putative trans-splicing intein. The phage DNA appears to be strongly associated with a protein that interfered with PCR amplification and estimation of the phage genome mass by pulsed-field gel electrophoresis. Identification and analyses of the receptor-binding protein (Gp48) revealed features common to the Salmonella enterica P22 phage tailspike protein, including the ability to specifically recognize a host organism. Bacteriophage receptor-binding proteins may offer promising alternatives for use in pathogen detection platforms.     

Characteristics of protease synthesis in Bacteroides splanchnicus NCTC 10825

Studies to determine the physiological and nutritional characteristics of protease synthesis by Bacteroides splanchnicus NCTC 10825 showed that the proteases were constitutive and cell-associated during exponential growth in batch culture. As growth slowed and the bacteria entered the stationary phase, proteases that had accumulated intracellularly were released into the culture media. In continuous cultures, [dilution rate (D)=0.03 h^–1 to D=0.29 h^–1], protease activity was completely cell-bound and maximal during nitrogen-limited growth at high dilution rates. The proteases hydrolysed a relatively restricted range of protein substrates including casein, azocasein and gelatine (comparative maximum rates of hydrolysis were 1.0, 4.1 and 2.7 units mg^–1 protein respectively). B. splanchnicus proteases exhibited arylamidase activities against leucine p-nitroanilide, valylalanine p-nitroanilide and glycylproline p-nitroanilide. Inhibition experiments indicated that the bacterium produced a mixture of serine, thiol and, possibly, metalloproteases. Protease activities were affected by reducing agents and divalent metal ions. Mercaptoethanol at 1 mm was slightly stimulatory; however, dithiortheitol and dithioerythritol (each 10 mm) respectively inhibited protease activities by 91% and 100%. Calcium ions (5 mm) stimulated protease activity by 30%, whereas Mn²⁺ and Mg²⁺ had little or no effect. Protease and arylamidase activities had neutral to alkaline pH optima. Together, these results show that with respect to the types of protease formed and the physiology of the process, B. splanchnicus proteolysis is similar in many respects to that occurring in species belonging to the B. fragilis group. Correspondence to: G. T. Macfarlane     

Infection of Streptococcus oralis NCTC 11427 by pneumococcal phages

We have found a group of pneumococcal bacteriophages (Cp-1, Cp-7) that can successfully infect and replicate in Streptococcus oralis, whereas Dp-1 was unable to infect this species. We have also developed conditions that allowed transfection of S. oralis using Dp-1 DNA. Our results support the direct involvement of the phage-coded lysins in the liberation of the phage progeny from infected S. oralis cells. Since S. oralis and S. pneumoniae are bacteria that share the same ecological niche in humans, the availability of the system described here should allow to extend our current studies on the modular organization of the lytic enzymes and might serve as a tool to study the evolutionary relationships between host and parasite.     

d-Amino acid dehydrogenase from Helicobacter pylori NCTC 11637

Helicobacter pylori is a microaerophilic bacterium, associated with gastric inflammation and peptic ulcers. d-Amino acid dehydrogenase is a flavoenzyme that digests free neutral d-amino acids yielding corresponding 2-oxo acids and hydrogen. We sequenced the H. pylori NCTC 11637 d-amino acid dehydrogenase gene, dadA. The primary structure deduced from the gene showed low similarity with other bacterial d-amino acid dehydrogenases. We purified the enzyme to homogeneity from recombinant Escherichia coli cells by cloning dadA. The recombinant protein, DadA, with 44 kDa molecular mass, possessed FAD as cofactor, and showed the highest activity to d-proline. The enzyme mediated electron transport from d-proline to coenzyme Q₁, thus distinguishing it from d-amino acid oxidase. The apparent K _m and V _max values were 40.2 mM and 25.0 μmol min⁻¹ mg⁻¹, respectively, for dehydrogenation of d-proline, and were 8.2 μM and 12.3 μmol min⁻¹ mg⁻¹, respectively, for reduction of Q₁. The respective pH and temperature optima were 8.0 and 37°C. Enzyme activity was inhibited markedly by benzoate, and moderately by SH reagents. DadA showed more similarity with mammalian d-amino acid oxidase than other bacterial d-amino acid dehydrogenases in some enzymatic characteristics. Electron transport from d-proline to a c-type cytochrome was suggested spectrophotometrically.     

Occurrence of free ceramides in Bacteroides fragilis NCTC 9343.

The occurrence of free ceramides at high concentrations was demonstrated in the chloroform-methanol extractable lipids of Bacteroides fragilis NCTC 9343. The long-chain bases were isolated from the free ceramides and identified as branched and normal saturated dihydroxy bases with carbon chains consisting of 17, 18, and 19 atoms. The major fatty acid was 3-hydroxy 15-methylhexadecanoic acid. The major molecular species of the ceramides were identified by gas chromatography-mass spectrometry and gas chromatography of the cleaved products as LCB-d-iso17: 0-3-OH iso17: 0 FA, LCB-d-anteiso17: 0-3-OH iso17: 0 FA, LCB-d-iso18: 0-3-OH iso17: 0 FA, and LCB-d-anteiso19: 0-3-OH iso17: 0 FA.     

Draft Genome Sequence of Corynebacterium diphtheriae Biovar Intermedius NCTC 5011

We report an annotated draft genome of the human pathogen Corynebacterium diphtheriae bv. intermedius NCTC 5011. This strain is the first C. diphtheriae bv. intermedius strain to be sequenced, and our results provide a useful comparison to the other primary disease-causing biovars, C. diphtheriae bv. gravis and C. diphtheriae bv. mitis. The sequence has been deposited at DDBJ/EMBL/GenBank with the accession number AJVH01000000.     

The susceptibility to ultrasonic disintegration ofKlebsiella pneumoniae NCTC 418

Summary The resistance to ultrasonic disintegration of cells ofKlebsiella pneumoniae grown at various dilution rates in continuous culture decreased with increasing cell size. Whilst this effect could be related to the cell wall content of the specimens, no direct relationship between the cell wall strength and the rigidity-conferring peptidoglycan was observed.     

Bacteriocin properties of Lactobacillus fermenti, Lactobacillus brevis and Lactobacillus buchneri

Four bacteriocins of L. fermenti, 3 bacteriocins of L. brevis and 1 bacteriocin of L. buchneri were studied with respect to morphology of the inhibition growth zones of the indicator strains, capacity for diffusion through cellophane, sensitivity to high temperature, bacterial proteases, trypsin, chymotrypsin, pepsin, papain, nucleases and lysozyme. According to the differences in their properties the bacteriocins were classified as belonging to 8 types, including 4 types of L. fermenti bacteriocins and 3 types of L. brevis bacteriocins.     

Complete Genome Sequence of a Variant of Campylobacter jejuni NCTC 11168

Campylobacter jejuni NCTC 11168 is widely used in research, but at least two variants have been reported. The available genome was sequenced from a variant which later showed a different phenotype and gene expression profile. Here we present the complete genome sequence of a second variant of C. jejuni NCTC 11168.     

Gluconate metabolism of Klebsiella pneumoniae NCTC 418 grown in chemostat culture

The metabolism of gluconate by Klebsiella pneumoniae NCTC 418 was studied in continuous culture. Under all gluconate-excess conditions at low culture pH values (pH 4.5–5.5) the majority (70–90%) of the gluconate metabolized was converted to 2-oxogluconate via gluconate dehydrogenase (GADH), although specific 2-oxogluconate production rates under potassium-limited conditions were significantly lower than under other gluconate-excess conditions. At high culture pH values, metabolism shifted towards production of acetate. Levels of GADH were highest at low culture pH values and synthesis was stimulated by the presence of (high concentrations of) gluconate. An increase in activity of the tricarboxylic acid cycle was accompanied by a decrease in GADH activity in vivo and in vitro, suggesting that the GADH serves a role as an alternative energy-generating system. Anaerobic 2-oxogluconate production was found to be possible in the presence of nitrate as electron acceptor. Levels of gluconate kinase were highest when K. pneumoniae was grown under gluconate-limited conditions. Under carbon-excess conditions, levels of this enzyme correlated with the intracellular catabolic flux.Abbreviations GADH gluconate dehydrogenase (EC 1.1.99.3) - GAK gluconate kinase (EC 2.7.1.12) - GDH glucose dehydrogenase (EC 1.1.99.17) - PQQ pyrroloquinoline quinone [2,7,9-tricarboxy-1-H-pyrrolo (2,3-f) quinoline-4,5-dione] - TCA trichloroacetic acid     

The complete genome sequence and analysis of Corynebacterium diphtheriae NCTC13129

Corynebacterium diphtheriae is a Gram-positive, non-spore forming, non-motile, pleomorphic rod belonging to the genus Corynebacterium and the actinomycete group of organisms. The organism produces a potent bacteriophage-encoded protein exotoxin, diphtheria toxin (DT), which causes the symptoms of diphtheria. This potentially fatal infectious disease is controlled in many developed countries by an effective immunisation programme. However, the disease has made a dramatic return in recent years, in particular within the Eastern European region. The largest, and still on-going, outbreak since the advent of mass immunisation started within Russia and the newly independent states of the former Soviet Union in the 1990s. We have sequenced the genome of a UK clinical isolate (biotype gravis strain NCTC13129), representative of the clone responsible for this outbreak. The genome consists of a single circular chromosome of 2 488 635 bp, with no plasmids. It provides evidence that recent acquisition of pathogenicity factors goes beyond the toxin itself, and includes iron-uptake systems, adhesins and fimbrial proteins. This is in contrast to Corynebacterium’s nearest sequenced pathogenic relative, Mycobacterium tuberculosis, where there is little evidence of recent horizontal DNA acquisition. The genome itself shows an unusually extreme large-scale compositional bias, being noticeably higher in G+C near the origin than at the terminus.     

Teichoic acid content in different lineages of Staphylococcus aureus NCTC8325

A series of mec transformants of Staphylococcus aureus strain NCTC8325 were analysed for alterations in wall teichoic acid and lipoteichoic acid. Although the methicillin resistance determinant alters the autolytic behaviour of S. aureus, it had no effects on the cellular content, chain length, and alanine substitution of the lipoteichoic acid, or on the wall teichoic acid content and composition. However, independently of the presence or absence of the methicillin resistance determinant, level of methicillin resistance, or autolytic behaviour, a correlation was found between a 25% reduced cell wall phosphate content and either loss of prophages φ11 and 13 or a 30-kb deletion in the chomosmal SmaI-F fragment adjacent to the prophage φ11 attachment site. Received: 23 February 1998 / Accepted: 15 May 1998     

The recovery of Arcobacter butzleri NCTC 12481 from various temperature treatments

AIMS: The aim of this study was to determine the growth and survival characteristics for Arcobacter butzleri NCTC 12481. METHODS AND RESULTS: The temperature and pH growth ranges were 15-39 degrees C and pH 6.0-8.0, as determined using impedance microbiology. The maximum specific growth rate was 00.57 h(-1) at 30 degrees C, pH 7.0. Arcobacter butzleri harvested from the exponential phase was more resistant to heat treatment than stationary phase cells (D55 1.1 and 0.4 min, respectively). Fluorescent dye uptake, and the release of UV-absorbing material, increased in heat-treated cells. After 21 d storage at 4 and -20 degrees C, A. butzleri was recovered on blood agar, but not on the isolation media CAT or CCDA. CONCLUSION: Arcobacter butzleri cells from the exponential phase were less heat sensitive than those from the stationary phase. The organism was able to survive cold storage for at least 3 weeks. SIGNIFICANCE AND IMPACT OF THE STUDY: The growth and survival characteristics have been quantified thus providing a greater understanding of this newly emerging pathogen.     

Role of membrane fluidity in pressure resistance of Escherichia coli NCTC 8164

The relationship among growth temperature, membrane fatty acid composition, and pressure resistance was examined in Escherichia coli NCTC 8164. The pressure resistance of exponential-phase cells was maximal in cells grown at 10 degrees C and decreased with increasing growth temperatures up to 45 degrees C. By contrast, the pressure resistance of stationary-phase cells was lowest in cells grown at 10 degrees C and increased with increasing growth temperature, reaching a maximum at 30 to 37 degrees C before decreasing at 45 degrees C. The proportion of unsaturated fatty acids in the membrane lipids decreased with increasing growth temperature in both exponential- and stationary-phase cells and correlated closely with the melting point of the phospholipids extracted from whole cells examined by differential scanning calorimetry. Therefore, in exponential-phase cells, pressure resistance increased with greater membrane fluidity, whereas in stationary-phase cells, there was apparently no simple relationship between membrane fluidity and pressure resistance. When exponential-phase or stationary-phase cells were pressure treated at different temperatures, resistance in both cell types increased with increasing temperatures of pressurization (between 10 and 30 degrees C). Based on the above observations, we propose that membrane fluidity affects the pressure resistance of exponential- and stationary-phase cells in a similar way, but it is the dominant factor in exponential-phase cells whereas in stationary-phase cells, its effects are superimposed on a separate but larger effect of the physiological stationary-phase response that is itself temperature dependent.