- Volume 132, Issue 5, 1986

A dipeptidyl aminopeptidase was identified in Streptococcus faecalis JH2SS and was partially purified (approximately 245-fold) by HPLC. Gel filtration chromatography indicated an M r of 140 000. The partially purified enzyme exhibited a requirement for Co2+. The pH optimum for the hydrolysis of l-Val-l-Ala-p-nitroanilide was approximately 9·5. The apparent K m for this substrate was 0·22 mm. The enzyme preferentially hydrolysed X-Ala-Y substrates, but also utilized X-Pro-Y substrates, and therefore is most closely related to the mammalian dipeptidyl aminopeptidase II (EC 3.4.14.-). The enzyme was inhibited by p-chloromercuribenzoate, but not by iodoacetate, N-ethylmaleimide or the serine protease inhibitor phenylmethylsulphonyl fluoride.

The coupling of the quinoprotein glucose dehydrogenase to the electron transport chain has been investigated in Acinetobacter calcoaceticus. No evidence was obtained to support a previous suggestion that the soluble form of the dehydrogenase and the soluble cytochrome b associated with it are involved in the oxidation of glucose. Analysis of cytochrome content, and of reduction of cytochromes in membranes by substrates, and of sensitivity to cyanide indicated that glucose, succinate and NADH are all oxidized by way of the same b-type cytochrome(s) and cytochrome oxidases (cytochrome o and cytochrome d). Mixed inhibition studies [with KCN and hydroxyquinoline N-oxide (HQNO)] showed that the b-type cytochrome(s) formed a binary complex with the o-type oxidase and that there was thus no communication between the electron transport chains at the cytochrome level. Measurements of the reduction of ubiquinone-9 by glucose and NADH, and inhibitor studies using HQNO, indicated that the ubiquinone mediates electron transport from both the glucose and NADH dehydrogenases. In some conditions the quinone pool facilitated communication between the ‘glucose oxidase’ and ‘NADH oxidase’ electron transport chains, but in normal conditions these chains were kinetically distinct.

The flux to arginine was determined in growing mycelium of Neurospora crassa carrying the aga mutation, by measuring the exponential growth rate and the arginine content of the free pool and that in protein. Derepression of the enzymes in the pathway by a factor of about three resulted in a 14% increase in the flux. Using a mutant (cpc-1), which affected the cross-pathway control, the pathway enzymes were found to have about threefold lower activities. This resulted in a 16% decrease in the measured flux. The effect of arginine acting as a feedback inhibitor of acetylglutamate kinase was estimated in an arg-12 mutant by varying the steady-state arginine concentration using histidine as a competitive inhibitor of the citrulline uptake. It is concluded that the feedback loop is mainly responsible for the small response of the flux to the large coordinate changes in the pathway enzymes. The results are discussed in terms of control analysis of metabolic systems.

The alkaline phosphatase (EC 3.1.3.1) associated with the outer membrane of Lysobacter enzymogenes was solubilized from a crude membrane preparation with detergent and purified 219-fold using ion-exchange chromatography and gel filtration. The yield of the purified enzyme was 21% and the specific activity was 438 units mg-1. The enzyme was most active at pH 8·5, readily hydrolysed 5'-, 2'- and 3'-ribose and deoxyribose nucleotides, glucose 6-phosphate, glycerophosphates and p-nitrophenylphosphate and was strongly inhibited by EDTA and 8-hydroxyquinoline. The EDTA-inhibited enzyme could be reactivated to some extent by CoCl2 and more effectively by ZnCl2. The phosphatase was slightly activated by p-nitrophenylphosphate and from kinetic studies using this substrate two K m values, 0·56 x 10-4 m and 3·4 x 10-4 m, were estimated. The enzyme retained activity in the presence of SDS, unless it was heated, and had an apparent M r of 69 000, as determined by SDS-PAGE. Gel filtration data suggested that the native enzyme might consist of at least two subunits. The properties of the enzyme were consistent with the view that it is held in the outer membrane by hydrophobic interactions

Nine oral bacteria, associated with both healthy and diseased sites in the mouth, were grown at D = 0·05 h-1 (mean generation time 13·9 h) in a glucose-limited chemostat. After an initial period of steady-state growth at pH 7·0, pH control was discontinued. The pH then decreased until it stabilized at pH 4·1 after 9 d (16 generations), while the E h rose from -165 mV to +160 mV. The lowering in pH resulted in the composition and metabolism of the flora being altered and in increased bacterial aggregation. At pH 7·0, ‘Streptococcus mitior’, Veillonella alcalescens and S. sanguis were most numerous while at pH 4·1 the counts of all bacteria fell except for Lactobacillus casei, which became predominant. The proportions of S. mutans within the community also increased while S. sanguis was recovered only occasionally and Bacteroides intermedius was not detected below pH 4·6. The survival at pH 4·1 of several other species would not have been predicted from earlier pure culture studies. Relative to pH 7·0, the community growing at pH 4·1 produced more lactic acid, washed cells had a greater glycolytic activity over a wider pH range but amino acid metabolism decreased. In general, when pH control was restored, so were the original patterns of metabolism and bacterial counts, except for B. intermedius, which was still not detected. The inverse relationship between S. sanguis and S. mutans, and the increase in proportions of L. casei and S. mutans during growth in a low pH environment parallel observations made in vivo and suggest that the chemostat can be used as a model for microbial behaviour in dental plaque.

An Enterobacter cloacae strain isolated from the faeces of a child with diarrhoea in Indonesia contained a transferable 216 MDa plasmid, pIN32, exhibiting IncHI2 phenotypic characters, including temperature sensitivity of transfer and the expression of H serotype pili at a repressed level. A derivative plasmid (pIN32-1), which had lost the IncHI2 phenotype, and contained only 60 MDa of the original replicon, was obtained after mating at 37 °C. It was IncFII, showed regions of homology with plasmid R100, determined IncFII serotype conjugative pili constitutively and was transfer-derepressed. After overnight growth at 37 °C in non-selective medium, pIN32 gave rise to another derivative, pIN32-2 (size 184·3 MDa), which retained the IncHI2 phenotype and several other pIN32 characters.

Mutant derivatives of a plasmid, pCF20, which carries the XhoI-D fragment of the TOL plasmid pWW0 have been isolated using Tn5 transposon mutagenesis. Insertion mutations of the xylR and xylS regulatory genes of the catabolic pathway have been isolated and characterized and their ability to induce catechol 2,3-oxygenase activity determined. Analysis of the insertion mutants and also segments of the XhoI-D fragment cloned into plasmid pUC8 in maxicells has identified a 68 kDa polypeptide product encoded by the xylR gene. No clear candidate for the xylS polypeptide was observed. The nucleotide sequence of the xylS region, the intergenic region and part of the xylR region has been determined and open reading frames (ORFs) assigned for both genes. The ORF designated xylS appears capable of encoding a polypeptide of ~ 37 kDa.

Functions encoded by the yeast plasmid pSB3 were analysed in Saccharomyces cerevisiae and Zygosaccharomyces rouxii. The autonomously replicating sequence (ARS) and partitioning mechanisms of pSB3 worked as satisfactorily in Z. rouxii ME3 as in the native host Z. rouxii IFO 1730 (formerly Saccharomyces bisporus var. mellis). The ARS in Z. rouxii ME3 was located within a 168 bp BanII–HindIII fragment spanning part of the inverted repeat (IR) and a unique region contiguous to it. The FLP enzyme (responsible for the intramolecular recombination at IRs) of pSB3 was functional in Z. rouxii ME3. In spite of the similarity of the putative recognition site for the FLP enzyme in pSB3 and pSR1, the FLP enzyme of pSB3 did not recognize the recombination site of pSR1, and the FLP enzyme of pSR1 did not use the recombination site of pSB3. Three transcripts of 3·4, 1·8 and 1·1 kb from pSB3 in Z. rouxii ME3 were identified by Northern blotting; they encompassed the A, B and C genes, respectively. pSB3 contained a region from which no poly(A)-containing RNA was transcribed.

Clostridium acetobutylicum P262 endoglucanase and cellobiase genes, cloned on a 4·9 kb DNA fragment in the recombinant plasmid pHZ100, were expressed from their own promoter in Escherichia coli. Active carboxymethylcellulase and cellobiase enzymes were produced, but there was no degradation of Avicel. The endoglucanase activities observed in cell extracts of E. coli HB101(pHZ100) differed in their pH and temperature optima from those previously reported for C. acetobutylicum P270. Complementation of E. coli arg and his mutations by cloned C. acetobutylicum DNA was also observed.

The mRNA for the adaptive enzyme isocitrate lyase (ICL) from Chlorella fusca has been identified by fractionation of total poly(A)-containing RNA and in vitro translation followed by immune precipitation. The M r of ICL mRNA was approximately 8·0 x 105, which is in good agreement with a previous estimate obtained by in vivo double-labelling.

The growth phase of a bacterial (Salmonella typhimurium) culture was shown to have pronounced effects on the pathogenic properties of the harvested bacteria. Salmonellae obtained from a culture in primary (exponential) growth phase (PP) were more readily cleared from the blood and more readily killed by phagocytes than were salmonellae obtained from a more slowly growing secondary growth phase (SP) culture. PP salmonellae were observed to cause death of mice sooner than SP salmonellae. This appeared to be because the more rapid growth of PP, as compared to SP, salmonellae continued in the liver and spleen for several hours following intravenous injection, and more than compensated for their high in vivo death rate. As a result, within 4 h there were approximately 10-fold more live salmonellae in the spleens and livers of mice that had received PP, as compared to SP, salmonellae. This 10-fold difference was maintained until the death of the mice, indicating that after the first 4 h post-inoculation, the net in vivo growth of the salmonellae was the same regardless of their growth phase in the inoculating culture. This transition between PP and SP salmonellae occurred long before a dense stationary phase culture was obtained. Salmonellae grown in minimal media exhibited the biological properties of SP salmonellae and never entered as rapid a growth phase as did salmonellae in complete media.

Iron restriction was induced in Escherichia coli O111, E. coli O164 and E. coli C by growing the organisms in trypticase soy broth containing ovotransferrin, desferal, EDDA (ethylenediamine-dihydroxyphenylacetic acid) or α,α'-dipyridyl. There were marked qualitative and quantitative differences in the iron regulated outer membrane proteins expressed in the presence of the various iron chelators. Differences in the kinetics of growth were also noted. E. coli C was devoid of a ferric enterobactin iron uptake system.

On SDS-PAGE, solubilized and proteinase K treated preparations of Neisseria gonorrhoeae strain BS4 (agar) showed differences in silver stained lipopolysaccharide (LPS) patterns, before and after induction to resistance to serum killing by incubation for 3 h at 37 °C with low M r fractions from lysates of guinea pig red blood cells. Preparations from the original serum susceptible gonococci and LPS purified from such bacteria showed two components, but the preparations from the serum resistant gonococci were deficient in the higher M r component. Furthermore, on immunoblotting with fresh human serum (FHS), the two LPS components of the susceptible gonococci reacted strongly with IgM. With preparations from the serum resistant gonococci there was no reaction in the area corresponding to the higher M r component and a weaker reaction with the component of low M r. Purified LPS from the susceptible gonococci neutralized the bactericidal activity of FHS against N. gonorrhoeae strain BS4 (agar) probably by reacting with the relevant antibody, since heated FHS was no longer bactericidal when mixed with a source of complement (human placental serum) after prior reaction with the LPS. These neutralization tests coupled with the results of immunoblotting strongly suggest that increased serum resistance is due to the lack of the high M r LPS moiety.

L929 mouse fibroblast cells and J774 macrophage-like cells are both susceptible to persistent infection with the Q fever agent Coxiella burnetii. Previously this laboratory has shown that persistently infected cell populations multiply with unaltered generation times or cell cycle progression. It has also been reported by others and us that highly infected cells typically exhibit one large parasite-containing vacuole. We now report that lightly and heavily infected cells are capable of division and in the process segregate the parasite-containing vacuole into one of the emerging daughter cells; the companion daughter cell emerges parasite-free. This asymmetric division of infected cells, revealed via photomicrography of stained cells, accounts for the appearance of uninfected cells within persistently infected host cell populations that were previously 100% infected. Some of the persistently infected L929 populations were maintained in culture for over two years without the addition of normal cells.