- Volume 138, Issue 10, 1992

In the psychrophilic bacterium Vibrio sp. strain ANT-300, the rate of protein degradation in vivo, measured at fixed temperatures, increased with elevation of the growth temperature. A shift in growth temperature induced a marked increase in this rate. Dialysed cell-free extracts hydrolysed exogenous insulin, globin and casein (in decreasing order of activity) but did not hydrolyse exogenous cytochrome c. Cells contained at least seven proteases separated by DEAE-Sephacel chromatography, one of which was an ATP-dependent serine protease. The ATP-dependent proteolytic activity in extracts of cells incubated for 3 h at 16 °C after a shift-up from 0 °C increased to a level 36% and 17% higher than that of cells grown at 0 °C and 13 °C, respectively. A shift-down to 0 °C from 13 °C induced only a slight increase in the proteolytic activity. Extracts of all cells, whether exposed to temperature shifts or not, showed the same temperature dependence with respect to both ATP-dependent and ATP-independent protease activity. In all the extracts these proteases also exhibited the same heat lability. The ATP-dependent protease was inactivated by incubation at temperatures above 25 °C. There was an increase in ATP-independent protease activity during incubation at temperatures between 25 and 30 °C, but a decrease at 35 °C and higher. These results suggest that the marked increases in proteolysis in vivo, caused by a shift in temperature, may result not only from increases in levels of ATP-dependent serine protease(s) but also from increases in the susceptibility of proteins to degradation.

In Escherichia coli, iron assimilation by means of the siderophore enterobactin requires two hydrophobic cytoplasmic membrane proteins, FepD and FepG, which are essential components of a binding-protein-dependent transport system. Such components are typically difficult to detect. Here we report observation of the fepD and fepG gene products in polyacrylamide gels; they appeared as diffuse bands at positions consistent with smaller sizes than those predicted by sequence analysis. Translational coupling was suggested by the lack of a detectable product from the fepG message in the absence of translation of the upstream fepD message. The orientation of FepD/FepG in the membrane was predicted based on their similarities in sequence and hydrophobicity to FhuB.

A plasma membrane fraction was obtained by the combined use of differential centrifugation and aqueous polymer two-phase partitioning techniques. Vanadate-inhibited ATPase and glucan synthase activities were highly enriched in this fraction, although the presence of ATPase activity which was not inhibited by vanadate, nitrate, molybdate, anyimycin A or azide was also detected. Other intracellular membrane marker activities were present at very low or undetectable levels. A further separation step using Percoll density gradient centrifugation resulted in the separation of a fraction which exclusively contained vanadate-inhibited ATPase activity, and was enriched with silicotungstic-acid-staining membrane material. Latency tests performed on the plasma membrane markers showed that the membrane vesicles were in the right-side-out orientation.

The differentiation-specific formation of three isoforms of pectinesterase by the broad bean rust fungus Uromyces viciae-fabae is described. Activity becomes detectable when substomatal vesicles are formed. In crude extracts isoform A contributed 78% of the total pectinesterase activity, and isoforms B and C contributed 20% and 2%, respectively. All three isoforms were found extracellularly in ratios identical to those in extracts. The isoelectric points of the pectinesterase isoforms were 8.4 (A), 5.7 (B), and 4.7 (C) as determined by chromatofocusing. Isoform A had an M r of 33500 and showed a distinct pH optimum at 6.0. The M r of isoform B was 40000; its pH optimum ranged from 5.5 to 7.5. Due to its extremely low activity, isoform C was not further characterized. The possible role of pectinesterases in the infection process of the broad bean rust fungus is discussed.

Cell synchrony was investigated during glycolytic oscillations in starved yeast cell suspensions at cell densities ranging from 2 × 106-5 × 107cells ml-1. Oscillations in NAD(P)H were triggered by inhibition of mitochondrial respiration when intracellular NAD(P)H had reached a steady state after glucose addition. Before macroscopic damping of the oscillations, individual yeast cells oscillated in phase with the cell population. After oscillations had damped out macroscopically, a significant fraction of the cells still exhibited oscillatory dynamics, slightly out-of-phase. At cell concentrations higher than 107cells ml-1the dependence upon cell-density of (i) the damping of glycolytic oscillations and (ii) the amplitude per cell suggested that cell-to-cell interaction occurred. Most importantly, at cell densities exceeding 107cells ml-1the damping was much weaker. A combination of modelling studies and experimental analysis of the kinetics of damping of oscillations and their amplitude, with and without added ethanol, pyruvate or acetaldehyde, suggested that the autonomous glycolytic oscillations of the yeast cells depend upon the balance between oxidative and reductive (ethanol catabolism) fluxes of NADH, which is affected by the extracellular concentration of ethanol. Based on the facts that cell (i) excrete ethanol, (ii) are able to catabolize external ethanol, and (iii) that this catabolism affects their tendency to oscillate, we suggest that the dependence of the oscillations on cell density is mediated through the concentration of ethanol in the medium.

In Saccharomyces cerevisiae cells the actin cytoskeleton is present as actin dots in the bud and around the septum, i.e. in areas of intensive cell wall synthesis, and as actin cables, which are loose bundles along the longitudinal cell axis. However, the apparently asymmetrical pattern of actin no longer persisted after protoplasting, when the cables disappeared and dots were evenly distributed under the whole protoplast surface. This pattern was maintained during regeneration of a new cell wall all over the protoplast surface, thus providing evidence of a relationship between the new wall formation and the presence of a regular arrangement of actin dots. The completed cell wall allowed the protoplast to bud and produce a normal daughter cell. However, before the walled protoplast began to bud, actin dots accumulated at the site of bud emergence and actin cables appeared, extending to the cytoplasm. Later, actin dots accumulated in the growing bud, forming a ring in the neck, and actin cables passed to the bud. Completion of the protoplast-to-cell reversion was preceded by restoration of the normal actin cytoskeleton.

The Bacillus subtilis gene senS, when present in high copy number, stimulates the expression of several extracellular protein genes during the onset of stationary phase, e.g. aprE. A novel integration vector, pINT, was constructed for transcription expression studies; it employed a unique method of promoter insert production for fusion with the lacZ reporter gene. Deletions were made of the 5' flanking region of the aprE promoter to localize the site responsible for SenS-mediated enhancement activity. pINT was used to translationally fuse aprE promoter deletion fragments with the lacZ reporter gene. A site between −177 and −415 with respect to the aprE start site of transcription was found to be required for the maximal SenS-mediated transcription increase from the aprE promoter. A multicopy vector containing the senS coding region without its native negative regulation was highly unstable in B. subtilis; this was due to the expressed senS insert.

The stability of plasmid F'lac in Escherichia coli strain SP45 (a temperature conditional mutant which grows as spherical cells at 42 °C and as a rod at 30 °C) was studied. F'lac elimination was demonstrated when bacteria exposed to subinhibitory concentrations of various chemicals were induced to form filaments. No plasmid loss was found when spherical cells were subjected to the same treatments. Plasmid loss was also observed in dnaA46 and lexA41 mutants when cell filamentation was induced at 42 °C, but not when they were cultured at 30 °C. Nalidixic acid promoted F'lac elimination at 0.25 μg ml-1in a recA13 mutant and at 1.5 μg ml-1in the recA +counterpart. A marked difference was found in the rate of F'lac elimination from thermosensitive DNA gyrase mutants [gyrA43(Ts) and gyrB41(Ts)] between rods and their spherical (rodA51) derivatives growing at semipermissive temperature (36.5 °C). Plasmids carrying the ccd segment of F in DNA gyrase mutants were lost after 2.5 generations from rods and after 6 generation from spherical cells. Plasmid segregation into non-viable minicell-like elements was found after induction of filaments. These data suggest that plasmid stability is correlated with cell shape and that curing is more easily achieved when bacteria can elongate normally.

The nucleotide sequence of the Escherichia coli envM gene was determined. It codes for a protein of 262 amino acids. The sequences of the E. coli and Salmonella typhimurium EnvM proteins are 98% identical. Gene envM is preceded in E. coli by a 43-nucleotide-long structural element, termed ‘box C’, which occurs in several E. coli operons between structural genes. This sequence element is totally absent in S. typhimurium. Gene envM was mapped at coordinate position 1366.8 kb of the physical map of Kohara et al. (Cell, 1987, 50, 495–508). As in S. typhimurium, a Gly for Ser exchange at position 93 of the amino acid sequence leads to a diazaborine-resistant E. coli phenotype. A Ser for Phe exchange at position 241 of the EnvM protein results in a temperature-sensitive growth phenotype. Comparison of the EnvM amino acid sequence with sequences available in databases showed significant homology with the family of short-chain alcohol dehydrogenases.

We report the isolation and characterization of a mutant of Escherichia coli unable to grow aerobically on non-fermentable substrates, except for very slow growth on glycerol. The mutant contains cytochrome oxidases o and d, and grows anaerobically with alternative electron acceptors. Oxygen consumption rates of cell-free extracts were low relative to activities in an isogenic control strain, but were restored in vitro by adding ubiquinone-1 to cell-free extracts. Transformation with a cloned 2.8 kb ClaI-EcoRV fragment of chromosomal DNA restored the ability of this mutant (AN2571) to grow on succinate and also restored cellular quinone levels in this strain. The plasmid also complemented a previously isolated ubiG mutant (AN151) for aerobic growth on succinate. The nucleotide sequence revealed a 0.7 kb portion of gyrA. Unidirectional nested deletions from this fragment and complementation analysis identified an open reading frame encoding a protein with a predicted molecular mass of 26.5 kDa. This gene (ubiG) encodes the enzyme 2-octaprenyl-3-methyl-5-hydroxy-6-methoxy-1,4-benzoquinone methyltransferase, which catalyses the terminal step in the biosynthesis of ubiquinone. The open reading frame is preceded by a putative Shine-Dalgarno sequence and followed by three palindromic unit sequences. Comparison of the inferred amino acid sequence of UbiG with the sequence of other S-adenosylmethionine (AdoMet)-dependent methyltransferases reveals a highly conserved AdoMet-binding region. The cloned 2.8 kb fragment also contains a sequence encoding the C-terminus of a protein with 42–44% identity to fungal acetyl-CoA synthetases.

Methanol oxidation in Methylophaga marina appears to be organized in a way similar to that in other Gram-negative methylotrophs, involving a quinoprotein methanol dehydrogenase (MDH; EC 1.1.99.8) and two soluble cytochromes. The MDH is composed of two different subunits, most probably arranged in an α2β2 structure. The two cytochromes are of the c-type and differ in size (molecular mass 19.5 and 12.5 kDa) and isoelectric point (pI 4.6 and 9.2). The one with the lowest isoelectric point, commonly designated as cytochrome c L, is able to oxidize reduced MDH. Taking advantage of the ability of M. marina, a restricted facultative methylotroph, to utilize fructose as a growth substrate, mutants impaired in methanol utilization were isolated after application of optimal concentrations of ethylmethane sulphonate. Three classes of methanol oxidation mutants were obtained. Class I mutants were affected in a global regulation of the synthesis of both apo-methanol-dehydrogenase and cytochrome c L as well as PQQ (pyrroloquinoline quinone). Class II mutants did not produce an active MDH, but instead a comparable amount of a 65 kDa protein was found in the cell-free extract upon SDS-PAGE. This mutant protein was purified and compared to wild-type MDH. It was located in the periplasm, but unlike MDH it was composed of only two identical large subunits. Each of these subunits was able to bind one molecule of PQQ. An antiserum raised against wild-type MDH did not react with the mutant protein. Conversely, an antiserum raised against mutant protein weakly cross-reacted with wild-type MDH, suggesting that the presence of the β-subunit in MDH dramatically changes its immunochemical behaviour. Three of the class II mutants did not produce PQQ. In the presence of PQQ, partial revertants able to grow on methanol medium were obtained (class III). Class III mutants produced a stable apo-MDH consisting of α- and β-subunits and showing a normal reaction with the anti-MDH serum. Although this apo-MDH can bind PQQ, enzymic activity was not restored in vitro. This suggests that, in addition to apo-MDH and PQQ, other factors are required for the assembly of an enzymically active MDH.

Using two complementary strategies for low-resolution S1 mapping, the global pattern of ϕC31 transcription was studied after induction of thermoinducible ϕC31 lysogens of Streptomyces coelicolor A3(2). A complex pattern of early transcripts was seen, with a peak of abundance at about 10 min post-induction. Nearly all of these transcripts were from DNA located to the right of the c (repressor) gene and to the left of the attP site: a region of about 14 kb. Early transcription was also observed immediately to the left of the c gene. The c gene itself was also induced, with an earlier expression peak (about 5 min post-induction). Primary late transcripts were generally relatively long, but degraded. They apparently corresponded to most of the 18 kb region to the left of the c gene. Some shorter and more persistent late transcripts corresponded to DNA close to or overlapping the cos site. Large late transcripts from a region close to the left-hand end of the ϕC31 genome showed evidence of processing to more stable, smaller RNA species. A failure of older cultures (more than 12 h old) to be induced productively was correlated with a much longer period of early transcription, reduced late transcription, failure to synthesize a major virion protein, and failure to package ϕC31 DNA. Moreover, heat treatment of the older lysogenic cultures did not result in the ϕC31-dependent shut-down of host rRNA transcription previously observed for young cultures (Rodríguez et al., Journal of General Microbiology (1986) 132, 1695–1701; Clayton & Bibb, Molecular Microbiology (1990) 4, 2179–2185).

Motility of the alkalophilic Bacillus sp. C-125, a flagellate bacterium, was demonstrated to be Na+- and pH-dependent. Flagellin protein from this strain was purified to homogeneity and the N-terminal sequence determined. Using the hag gene of Bacillus subtilis as a probe, the hag gene of Bacillus sp. C-125 was identified and cloned into Escherichia coli. Sequencing of this hag gene revealed that it encodes a protein of 272 amino acids (M r 29995). The predicted N terminal sequence of this protein was identical to that determined by N-terminal sequencing of the flagellin protein from strain C-125. The alkalophilic Bacillus sp. C-125 flagellin shares homology with other known flagellins in both the N- and C-terminal regions. The middle portion, however, shows considerable differences, even from that of flagellin from the related species, B. subtilis.

The chromosomal DNAs of eight medically important Candida species, C. albicans, C. stellatoidea, C. tropicalis, C. parapsilosis, C. krusei, C. guilliermondii, C. kefyr and C. glabrata, were analysed by pulsed-field gel electrophoresis under various conditions. The corresponding bands in the gels were assigned by three kinds of DNA probe which hybridized to DNA of all the species: rDNA, TUB2 and PEP4. The best conditions for separating the chromosomal DNAs were investigated and the numbers and molecular sizes of the chromosome bands were determined for each species. The chromosomal DNAs of the species were separated into 5–14 bands ranging in size from 0.5 to 4.5 Mb. Based on the quantification of the chromosome band intensities using a laser fluorescent gel scanner, the chromosome numbers were estimated. The apparent average total number of chromosomes per cell was 16 for C. albicans, 16 for C. stellatoidea, 12 for C. tropicalis, 14 for C. parapsilosis, 8 for C. krusei, 8 for C. guilliermondii, 18 for C. kefyr, and 14 for C. glabrata; the total chromosomal DNA size of each species per cell was calculated at about 31 Mb, 33 Mb, 31 Mb, 26 Mb, 20 Mb, 12 Mb, 29 Mb and 14 Mb, respectively.

Respiratory activities of moderately halophilic bacteria from diverse origins were studied with respect to the requirement for Na+, the site of Na+-dependent reactions in the respiratory chain and the presence of a redox-driven Na+pump. All the moderately halophilic bacteria examined required 1.0–2.0 M-NaCl for optimum growth. The NADH oxidase activity of the respiratory chain was influenced by the method of membrane preparation. When the cells were disrupted by a French press, the presence of 0.2 M-Na2SO4 (or 0.4 M-NaCl) was required to protect against loss of Na+-dependent NADH oxidase activity. Membranes prepared by osmotic lysis retained high Na+-dependent NADH oxidase activity. Of eight moderate halophiles investigated, the six Gram-negative bacteria possessed Na+-dependent NADH oxidase activity. The site of Na+-dependent activation in the respiratory chain was located on the NADH: quinone reductase segment in all these halophiles. Other activities, such as succinate oxidase and the terminal oxidase, showed no specific requirement for Na+. Using inverted membrane vesicles prepared from these halophiles, it was found that membrane potential generation linked to NADH oxidation was not completely dissipated by addition of the protonophore carbonyl cyanide m-chlorophenylhydrazone (CCCP), but was entirely sensitive to CCCP plus the Na+-conducting ionophore monensin. A Na+-dependent NADH oxidase was not detected in membranes from the two Gram-positive halophiles, Marinococcus halophilus and Micrococcus varians subsp. halophilus, that we investigated. Since the membrane potential generated by oxidation of NADH was completely dissipated by CCCP, these halophiles did not have any respiration-driven Na+pumps.

The Escherichia coli uhpT gene encodes an active transport system for sugar phosphates. When the uhpT gene was carried on a multicopy plasmid, amplified levels of transport activity occurred, and growth of these strains was inhibited upon the addition of various sugar phosphates. Two different mechanisms for this growth inhibition were distinguished. Exposure to glucose-6-phosphate, fructose-6-phosphate or mannose-6-phosphate, which enter directly into the glycolytic pathway, resulted in cessation of growth and substantial loss of viability. Cell killing was correlated with the production of the toxic metabolite, methylglyoxal. In contrast, addition of 2-deoxyglucose-6-phosphate, galactose-6-phosphate, glucosamine-6-phosphate or arabinose-5-phosphate, which do not directly enter the glycolytic pathway, resulted in growth inhibition without engendering methylglyoxal production or cell death. Inhibition of growth could result from excessive accumulation of organophosphates in the cell or depletion of inorganic phosphate pools as a result of the sugar-P/Pi exchange process catalysed by UhpT. The phosphate-dependent uptake of glycerol-3-phosphate by the GlpT antiporter was strongly inhibited under conditions of elevated sugar-phosphate transport. There are thus two separate toxic effects of elevated sugar-phosphate transport, one of which was lethal and related to increased flux through glycolysis. It is likely that the control of uhpT transcription by catabolite repression exists to limit the level of UhpT transport activity and thereby prevent the toxic events that result from elevated uptake of its substrates.