- Volume 138, Issue 9, 1992

Xylose reductase (EC 1.1.1.21) from the yeast Pichia stipitis NRC 2548 was purified to homogeneity via a two-step protocol using anion-exchange and gel-filtration chromatography. The pH-activity profile revealed the presence of two ionizable groups with pK app values of 5.8 and 8.1, suggesting the catalytic involvement of histidyl and thiol groups, respectively. Additional evidence supporting the involvement of these residues was provided by the use of group-specific inhibitors. The enzyme was rapidly inactivated in a pseudo-first order manner by the thiol-specific modifier p-chloromercuriphenylsulphonate (PMBS) and analysis of the order-of-reaction suggested that one essential cysteine residue was modified to effect inactivation. Treatment of the enzyme with another thiol-specific modifier, 5,5′-dithiobis-(2-nitrobenzoic acid) (DTNB), showed that modification of one cysteine per monomer led to 90% loss of activity, further supporting the existence of one essential cysteine residue. Inactivation by PMBS was reversed by adding 1 mM-β-mercaptoethanol. Inactivation of xylose reductase by the histidine-specific modifier diethylpyrocarbonate (DEP) followed a pseudo-first order process, and analysis of the order-of-reaction suggested that one essential histidine residue was modified to effect inactivation. Treatment of DEP-inactivated enzyme with 0.2 M-neutral hydroxylamine resulted in the recovery of 45% of enzyme activity. Protection of xylose reductase from PMBSand DEP-inactivation was provided by NADPH and NADH but not by NADP+, D-xylose or DL-glyceraldehyde. This suggests that the essential histidine and cysteine residues may be involved with binding of cofactor by the P. stipitis xylose reductase.

In extracts from vegetative Dictyostelium discoideum V12 the basal NAD-dependent glutamate dehydrogenase (NAD-GDH) activity was low, but it increased on standing at 4°C. When 0.1 mM-AMP was included in the assay mix, enzyme activity was stimulated nearly 30-fold. As the extract was allowed to age, the enzyme rapidly lost its ability to be stimulated by AMP. The response of NAD-GDH to AMP was also dependent on the stage of morphogenesis. The ratios of NAD-GDH activity assayed with and without AMP (+AMP/-AMP ratios) in freshly prepared extracts from cells at 0, 4, 8 and 12 h of development were similar, but declined later in morphogenesis. The + AMP/-AMP ratio decreased sharply during activation at 4°C in extracts from cells at 0, 4, 16 and 20 h of development. By contrast, extracts from cells starved for 8 and 12 h remained more responsive to AMP throughout activation. Analysis of Western blots showed that vegetative NAD-GDH did not undergo any detectable proteolytic cleavage during 96 h of activation at 4°C. Also, no change in molecular mass appeared to take place within the cells until culmination (20–24 h), when some breakdown products appeared. Activation of NAD-GDH also occurred in D. discoideum strains NC4 and AX3, and in D. mucoroides. In addition, the enzyme from these four strains was stimulated by AMP and the + AMP/-AMP ratio declined with similar kinetics during activation. The enzyme from Polysphondylium violaceum was not activated on standing, but it was stimulated by AMP. The effect of activation of NAD-GDH is discussed in relation to a postulated catabolic role for this enzyme.

Three alcohol dehydrogenases have been identified in Acinetobacter calcoaceticus sp. strain HO1-N: an NAD+-dependent enzyme and two NADP+-dependent enzymes. One of the NADP+-dependent alcohol dehydrogenases was partially purified and was specific for long-chain substrates. With tetradecanol as substrate an apparent K m value of 5.2 μM was calculated. This enzyme has a pI of 4.5 and a molecular mass of 144 kDa. All three alcohol dehydrogenases were constitutively expressed. Three aldehyde dehydrogenases were also identified: an NAD+-dependent enzyme, an NADP+-dependent enzyme and one which was nucleotide independent. The NAD+-dependent enzyme represented only 2% of the total activity and was not studied further. The NADP+-dependent enzyme was strongly induced by growth of cells on alkanes and was associated with hydrocarbon vesicles. With tetradecanal as substrate an apparent K m value of 0.2 μM was calculated. The nucleotide-independent aldehyde dehydrogenase could use either Würster’s Blue or phenazine methosulphate (PMS) as an artificial electron acceptor. This enzyme represents approximately 80% of the total long-chain aldehyde oxidizing activity within the cell when the enzymes were induced by growing the cells on hexadecane. It is particulate but can be solubilized using Triton X-100. The enzyme has an apparent K m of 0.36 mM for decanal.

The glycerolipid and protein compositions of the outer and inner membranes of Rhizobium meliloti were studied. The wild-type R. meliloti strain Rm41 was shown to contain three phospholipids characteristic for most Gram-negative bacteria, phosphatidylethanolamine, phosphatidylglycerol and diphosphatidylglycerol, this last compound being concentrated into the inner membrane. As in several bacteria interacting with plants, the presence of phosphatidylcholine, phosphatidyl-N-monomethylethanolamine and phosphatidyl-N,N-dimethylethanolamine was also demonstrated. Induction of the nod genes by luteolin did not affect the lipid composition and no difference in lipid composition was found between the wild-type strain and a number of Nod−and Fix−mutants tested. Protein analysis of the inner and outer membranes showed that they exhibit very different patterns with several bands specific for one or the other membrane. A Nod−mutant carrying a large deletion in the symbiotic megaplasmid pRme41b showed differences in the protein patterns even before induction by luteolin, indicating that this megaplasmid codes for several membrane proteins. When the nod genes of strain Rm41 were induced by luteolin, two new bands at around 60 kDa and 44 kDa appeared in both the outer and the inner membranes. By using a strain overexpressing the nod genes and the technique of immunoblotting with antibodies against NodC, it was confirmed that the 44 kDa band corresponded to the NodC protein. This protein was not found in a nodC:: Tn5 mutant. This work represents the first step in the characterization of modifications induced by luteolin treatment at the membrane level.

A bacteriocin produced by Pediococcus acidilactici has been purified to homogeneity by a rapid and simple four-step purification procedure which includes ammonium sulphate precipitation, chromatography with a cation-exchanger and Octyl Sepharose, and reverse-phase chromatography. The purification resulted in an approximately 80000-fold increase in the specific activity and about a 6-fold increase in the total activity. The amino acid composition and sequencing data indicated that the bacteriocin contained 43–44 amino acid residues. The predicted M r and isoelectric point of the bacteriocin are about 4600 and 8.6, respectively. Comparing the amino acid sequence of this bacteriocin with the sequences of leucocin A-UAL 187, sakacin P and curvacin A (bacteriocins produced by Leuconostoc gelidum, Lactobacillus sake and Lactobacillus curvatus, respectively) revealed that all four bacteriocins had in their N-terminal region the sequence Tyr-Gly-Asn-Gly-Val-Xaa-Cys, indicating that this concensus sequence is of fundamental importance for this group of bacteriocins. The bacteriocin from P. acidilactici and sakacin P were very similar, having at least 25 common amino acid residues. The sequence similarity was greatest in the N-terminal half of the molecules – 17 of the first 19 residues were common – indicating the fundamental importance of this region. Leucocin A-UAL 187 and curvacin A had, respectively, at least 16 and 13 amino acid residues in common with the bacteriocin from P. acidilactici.

The cell cycle of Chlamydomonas eugametos was synchronized at low cell densities by growing cells under conditions of alternating light and dark periods. Synchronization of the cell cycle was demonstrated by determining cell size, cytokinesis, and release of daughter cells. The microscopic data were in agreement with the accumulation patterns of mRNAs for ribulose bisphosphate carboxylase/oxygenase small subunit, histone H4 and β-tubulin. Cells of C. eugametos secrete a sticky material which enables cells to adhere to a solid substrate during cell division. The secretion of this material was also cell-cycle-regulated and preceded cytokinesis. Recently, it was reported that in synchronous exponential-phase cultures of high cell densities, young daughter cells temporarily behave like gametes during the cell cycle. However, under our growth conditions this was not observed. First, daughter cells did not agglutinate with normal gametes of the opposite mating type. Second, extraction of growing cells did not release any agglutinin activity as measured both by an in vitro agglutination assay and by an agglutinin-specific monoclonal antibody. We conclude therefore that gametogenesis in C. eugametos is not necessarily a normal phase of the cell cycle.

The rfb (O antigen) gene cluster of a group C1 Salmonella enterica strain was sequenced; it comprised seven open reading frames which precisely replaced the 16 open reading frames of a group B strain. Two genes of the mannose biosynthetic pathway were present: rfbK (phosphomannomutase) had a G + C content of 0.61 and had only 40% identity to rfbK of group B but was very similar to cpsG of the capsular polysaccharide pathway with 96% identity, whereas rfbM [guanosine diphosphomannose (GDP-Man) pyrophosphorylase] had a G + C content of 0.39. Other genes had G + C contents ranging from 0.24 to 0.28. rfbM(C1) and rfbM(B) had 60% identity, which is much less than expected within a species, but nonetheless indicates a much more recent common ancestor than for rfbK. The other genes showed much lower or no similarity to rfb genes of other S. enterica strains. It appears that the gene cluster evolved outside of Salmonella in a species with low G + C content: the rfbM gene presumably derives from that period whereas the rfbK gene appears to have arisen after transfer of the cluster to S. enterica by duplication of the S. enterica cpsG gene, presumably replacing an rfbK gene of low G + C content.

Growth of Saccharomyces cerevisiae on D-glucono-δ-lactone (δgl) was found to be associated with a specific coordinate induction of the synthesis of two enzymes of the oxidative pentose phosphate pathway – 6-phosphogluconate dehydrogenase and 6-phosphogluconolactonase – together with that of a third enzyme, gluconokinase. The gnd1 mutation, responsible for an approximately 80% loss of 6-phosphogluconate dehydrogenase activity and the inability of the cells to grow on δgl, completely abolished the induction of all three enzymes, while the gnd2 mutation affected this only partially. One class of gnd1 revertants, selected for growth on δgl, was found to have recovered normal dehydrogenase activity and the ability to synthesize the three enzymes when induced by δgl. Another class of δgl-positive revertants possessed constitutively elevated levels of gluconokinase. In contrast, glucose-positive revertants of gnd1, with restored constitutive dehydrogenase activity, continued to remain deficient in induction of the three enzymes and also failed to grow on δgl. Induction of 6-phosphogluconate dehydrogenase activity was associated with increased transcription of the gene coding for the major isoenzyme; the transcript remained undetectable in the gnd1 mutant. Induction of these specific enzymes thus appears to be essential for growth of S. cerevisiae on δgl.

Using segments of the Escherichia coli rpoB and rpoC genes as heterologous probes, we have identified and cloned an 8.3 kb PstI fragment from the Staphylococcus aureus genome containing the rpoB and ropC genes, which respectively encode the β and β’ subunits of DNA-directed RNA polymerase. This region is almost certainly equivalent to the rif locus, located near to fus at interval 12/13 on the S. aureus linkage map. Limited DNA sequencing revealed the gene order rpoB-rpoC (transcribed from left to right) and identified the rplL gene, encoding ribosomal protein L7/L12, upstream of rpoB. This and other evidence suggests that the rpoBC genes of S. aureus form part of a large gene cluster encoding components of the transcription and translation apparatus which is well-conserved in other eubacteria.

RNA polymerase was isolated from Micromonospora echinospora and from Streptomyces lividans. In vitro transcription of a DNA fragment containing multiple tandem promoters from Micromonospora followed the pattern of expression observed previously for in vivo studies. RNA polymerase was prepared from cultures of Micromonospora that were harvested during the growing phase and during the stationary phase. Promoters that were utilized in Micromonospora only during the stationary phase were utilized in vitro only when RNA polymerase was purified from a stationary-phase culture, and not when RNA polymerase was purified from growing cells.

A repeated sequence, named RPS1, approximately 2 kb in size, is found mainly in chromosome 6, the second most variable chromosome among the eight chromosomes of Candida albicans. Most of the RPS1 units of chromosome 6 seem to be located within a single region of about 100 kb in strain FC18. In both strains FC18 and NUM812, a part of RPS1 is apparently tandemly repeated. A unit of RPS1 has been cloned and sequenced. It consists of 2114 bp and has a GC content of 40 mol%. The repeat unit contains smaller repeats of about 80–170 bp which are called REP1, REP2, REP3, REP4 and REP5; REP2 is duplicated. The small repeats are classified into two groups by their homology. One comprises REP1, REP2 and REP5, and the other REP3 and REP4. They are termed the REP1 and REP3 families, respectively. The two families both contain a common 29 bp sequence, called COM29. The dispersed repetitive sequence RPS1 may be involved in chromosomal rearrangements and may in part explain chromosome polymorphism in C. albicans. The origin of RPS1 was not determined.

The regulatory unit of Bacillus subtilis strain 168 encompassing the structural genes of the N-acetylmuramoyl-L-alanine amidase and of its modifier has been sequenced, and found to be a divergon consisting of divergently transcribed operons lytABC and lytR. Proteins LytA, LytB and LytC are endowed with export signal peptides. Mature LytA is a 9.4 kDa, highly acidic polypeptide whose deduced amino acid sequence points to a lipoprotein. LytB and LytC, the modifier and the amidase, are highly basic. After cleavage of the signal sequence their molecular masses are 74.1 and 49.9 kDa, respectively. These two proteins share considerable homology in their N-terminal moieties and have three GSNRY consensus motifs, characteristic of nearly all amidases. The C-terminal moiety of LytB exhibits homology to the product of spollD. LytR is a 35 kDa protein which acts as an attenuator of the expression of both lytABC and lytR operons. Transcription of the lytABC operon proceeds from two promoters: PD, identified as P28–7 (Gilman et al., 1984), and an upstream PA. The former only is subject to LytR attenuation. Translational initiation of lytB and lytC is directed by UUG start codons, suggesting that lytA, B and C undergo coupled translation. Transcription of lytR is initiated at two start sites, one of which corresponds to a highly intense PA promoter whereas the other does not seem to share much homology with any of the known promoter consensus sequences. Both promoters are attenuated by LytR. It is confirmed that the synthesis of the amidase is controlled at least in part by SigD, i.e. that it belongs to the fla regulon and that its activity, or part of it, is co-regulated with flagellar motility. The role of the mutations conferring the Sin, Fla and Ifm phenotypes in the expression of the lytABC operon is discussed.

Approximately 50% (15/28) of a selection of oral isolates of Candida albicans from separate individuals infected with the human immunodeficiency virus (HIV) exhibited low susceptibility to ketoconazole as determined by hyphal elongation assessment. Nine of these isolates exhibited colony morphology variation or switching at 37°C, of which six expressed low ketoconazole susceptibility. To determine whether colony morphology variation could give rise to derivatives with reduced azole susceptibility, several high-frequency switching variants of three HIV-patient isolates were recovered and assessed. All but one of the variants expressed similar azole susceptibility profiles to their respective parental strains. However, the C. albicans derivative 132ACR expressed significantly reduced susceptibility to ketoconazole in comparison to its parental strain 132A. In whole cells, on the basis of total growth the switched derivative 132ACR was markedly less susceptible than its parental isolate 132A to ketoconazole at 10 μM. A much smaller difference was observed with fluconazole at 10 μM, with the switched derivative 132ACR exhibiting a threefold lower susceptibility compared with the parental isolate 132A. The incorporation of [14C]acetate in control and azole-treated cells of both organisms was higher for the parental strain. When cell lysates of strain 132A and its derivative 132ACR were incubated with [14C]mevalonic acid and ketoconazole, the IC50 for 14C-label incorporation into C-4 demethyl sterols was fivefold higher for lysates of the switched derivative 132ACR compared with those of the parental strain 132A. With fluconazole the IC50 value for the derivative 132ACR was 25-fold higher than for strain 132A. The 14-sterol demethylase of the switched derivative 132ACR was possibly less sensitive to azole inhibition than that of the enzyme of strain 132A. These studies indicated that colony morphology variation in vitro can generate derivatives with stable, reduced azole susceptibility without prior exposure to azoles.

A low-molecular-mass cytotoxin produced by Klebsiella oxytoca isolated previously from patients with antibiotic-associated haemorrhagic enterocolitis was purified, and its biological and chemical properties were elucidated. The toxin inhibited the syntheses of DNA and RNA by HEp-2 cells dose-dependently, whereas protein synthesis was only slightly inhibited, as measured by the incorporation of radioactive precursors. When synchronously cultured HEp-2 cells were examined in the presence of cytotoxin, inhibition of DNA synthesis occurred promptly within 5 h, but cell-rounding, the earliest visible morphological change, was not observed until 6 h after exposure. The intracellular levels of ATP decreased with an approximately similar time course. These results suggest that cytotoxicity toward HEp-2 cells is primarily due to the inhibitory effect of the cytotoxin on nucleic acid synthesis, possibly on DNA synthesis. Cell rounding and cell death were induced even in the absence of the cytotoxin after incubation with the cytotoxin for 6 h. The cytotoxin was heat-labile, cytotoxic activity decreasing to 50% of the initial level on heating at 70°C for 20 min. Plasmids were extracted from three strains of K. oxytoca producing the cytotoxin and analysed by agarose gel electrophoresis. Two strains possessed plasmids of different sizes, but one strain possessed no plasmid, indicating that the cytotoxin is probably chromosomally encoded. Analysis by NMR and FAB-mass-spectrometry revealed that the molecular mass of the cytotoxin should be 217.1062 Da (exact mass), its molecular formula being C8H15O4N3.

Genomes of 55 Dutch porcine Serpulina (Treponema) hyodysenteriae and non-pathogenic Serpulina isolates were characterized by restriction endonuclease analysis (REA) and DNA hybridization. The Dutch porcine isolates were compared with American Type Culture Collection (ATCC) strains of S. hyodysenteriae and S. innocens and isolates of S. hyodysenteriae with known serotypes (reference strains). REA of the Dutch S. hyodysenteriae isolates resulted in two main patterns, while the non-pathogenic isolates had many distinct REA patterns, all different from the S. hyodysenteriae strains. The S. hyodysenteriae reference strains all had distinct REA patterns, different from the Dutch strains. Upon Southern hybridization with a S. hyodysenteriae DNA fragment encoding a flagellar protein, all S. hyodysenteriae strains could be divided in two groups. The non-pathogenic Serpulina strains had many distinct hybridization patterns and hybridized less intensely. Upon hybridization with a S. hyodysenteriae DNA fragment encoding a haemolysin, DNA of all S. hyodysenteriae strains reacted in the same band. DNA of non-pathogenic Dutch Serpulina strains and S. innocens did not hybridize. It was concluded that there are two main genotypes of S. hyodysenteriae in the Netherlands. This could be of importance for recombinant DNA vaccine development.