- Volume 133, Issue 7, 1987

SUMMARY: On the basis of information from computer-assisted sequence comparison of the Mycoplasma pneumoniae 16S ribosomal RNA (rRNA) sequences with sequences from various other mycoplasmal and bacterial species, we constructed M. pneumoniae-specific oligonucleotide probes complementary to variable regions in the 16S rRNA molecule. Using a DNA/RNA dot blot hybridization procedure, it was possible to detect less than 1 × 103mycoplasmas. This test is a most sensitive assay for species-specific detection of bacteria. It can easily be adapted for detection and identification of other bacterial species and may have wide medical and industrial application.

SUMMARY: Immune precipitation patterns of Mycobacterium intracellulare, M. phlei and M. smegmatis were analysed by selective enzyme staining procedures in order to characterize individual mycobacterial antigens. Enzyme activity was shown in eight precipitinogens of M. intracellulare, seven of M. phlei, and six of M. smegmatis. The identification of mycobacterial precipitinogens as enzymes is important since only a few mycobacterial antigens have been functionally characterized.

SUMMARY: Cuticular extracts of Acyrthosiphon pisum, the pea aphid, stimulated the formation of germ-tubes of ballistospores of aggressive strains of Conidiobolus obscurus, but showed no influence on those of non-aggressive strains. Aggressive strains were separated into two groups: (a) those stimulated by both (i) water- or alcohol-soluble cuticular extracts originating from honeydew and (ii) lipidic extracts of the cuticle, and (b) those stimulated by (ii) only. The hydrocarbon and polar fractions of the cuticular lipids were the most active. These results obtained with C. obscurus are compared to the effect of cuticular compounds on spore germination in other entomopathogenic fungi.

SUMMARY: Of 40 polyploid strains of Saccharomyces cerevisiae screened for growth on D-mannitol (5%, w/v), half grew well (5-20 mg dry biomass ml−1). Certain of these strains were unable to grow on low concentrations of mannitol (1-2%, w/v) and others, initially unable to grow on mannitol, exhibited long-term adaptation to growth. An NAD+-dependent D-mannitol dehydrogenase (EC 1.1.1.67) was detected in mannitol-grown yeast. Growth was dependent on mitochondrial function and was obligately aerobic. Measurement of products of metabolism and respiratory activity indicated that growth on mannitol allows catabolite derepression.

SUMMARY: Following transfer to medium lacking a nitrogen source, cells of Synechocystis PCC 6803 continued to divide, giving a doubling of cell number after 40 h. Phycocyanin degradation commenced immediately after the transfer, with a rapid phase lasting 5 h in which 50% of the phycocyanin disappeared and a slow second phase in which the phycocyanin content decreased to 10% of its initial value by 24 h. In the presence of glucose, a utilizable carbon source for this facultatively heterotrophic cyanobacterium, phycocyanin was degraded initially at a rate 60% of that observed in the absence of the sugar and proteolysis was almost completely inhibited after 6 h when only 27% of the phycocyanin had been lost; cell division ceased at this time. Photosynthetic O2 evolution decreased rapidly in the presence of glucose and the cells consumed O2 in the light after 7 h, indicative of a switch to oxidative metabolism; net O2 uptake in the absence of glucose occurred only after approximately 25 h. Inhibition of phycocyanin degradation by glucose required metabolism of the sugar, probably via the oxidative pentose phosphate cycle, and appeared to result from irreversible inactivation of the protease.

SUMMARY: When presented with an equimolar mixture of ammonia and L-glutamate, batch cultures of Rhizobium leguminosarum MNF3841 and ‘R. trifolii’ (R. leguminosarum biovar trifolii) TA1 used ammonia at a significantly faster rate than L-glutamate. The cowpea Rhizobium strain NGR234, however, used L-glutamate at a marginally faster rate than ammonia. R. leguminosarum MNF3841 also grew faster on ammonia than on L-glutamate as the nitrogen source.

Chemostat cultures of R. leguminosarum MNF3841 limited for phosphate did not release excess ammonia when grown on mannitol/L-glutamate, showing that L-glutamate catabolism was tightly regulated to meet the cells' nitrogen requirement. Further, the rate of consumption of ammonia was similar to that for L-glutamate when either was supplied as the sole nitrogen source. However, with L-histidine or L-alanine as a nitrogen source, large quantities of excess ammonia were released. When chemostat cultures of R. leguminosarum MNF3841 were supplied with an equimolar mixture of ammonia and L-glutamate, 89-100% of the nitrogen consumed was ammonia. Similarly, with mixtures of L-glutamate/L-histidine or L-glutamate/L-alanine, almost no L-glutamate was consumed, a result attributable to the release of excess ammonia from either L-histidine or L-alanine. The use of 14C-labelled fructose or L-glutamate showed that the intra- and extracellular L-glutamate pools were isolated, indicating that the ammonia preference must be exerted by a restriction in L-glutamate transport. L-Glutamate transport rates were low in chemostats containing L-glutamate/NH4Cl, an effect attributable in part to a significant repression of synthesis of the L-glutamate transport system by ammonia.

SUMMARY: A bacterium capable of growth on trans-2-butene was isolated from soil and identified as a Nocardia sp. The isolate also grew on propane, butane, pentane and hexane; gaseous and volatile 1-alkenes did not support growth. Intact cells grown on trans-2-butene or butane oxidized all n-alkanes, 1-alkenes, 2-alkenes and alcohols tested. Simultaneous adaptation studies, inhibitor experiments and measurements of enzyme activities in crude extracts indicated a degradative route of trans-2-butene via crotonic acid and of butane via butyric acid. The key enzymes in the proposed pathways were induced by growth on either trans-2-butene or butane. The induction of these enzymes and the substrate specificities of the enzymes suggest a relation between trans-2-butene and butane degradation.

SUMMARY: Bacillus subtilis mutants with altered penicillin-binding proteins (PBPs), or altered expression of PBPs, were isolated by screening for changes in susceptibility to β-lactam antibiotics. Mutations affecting only PBPs 2a, 2b and 3 were isolated. Cell shape and peptidoglycan metabolism were examined in representative mutants. Cells of a PBP 2a mutant (UB8521) were usually twisted whereas PBP 2b (UB8524) and 3 (UB8525) mutants produced helices, particularly after growth at 41 °C. The PBP 2a mutant (UB8521) had a higher peptidoglycan synthetic activity than its parent strain whereas the opposite applied to the PBP 2b mutant UB8524. The PBP 3 mutant (UB8525) had a similar peptidoglycan synthetic activity to that of the parent strain when grown at 37 °C, but 40% higher activity after growth at 41°C. The PBP 2a mutant (UB8521) exhibited the same wall thickening activity as the parent, but the PBP 2b and 3 mutants (UB8524 and UB8525) were partially defective in this respect. The changes in the susceptibility of PBP 2a, 2b and 3 mutants to β-lactam antibiotics imply that these PBPs are killing targets, consistent with the fact that these PBPs are also important for shape determination and peptidoglycan synthesis.

SUMMARY: The synthesis, cross-linking and O-acetylation of gonococcal peptidoglycan in growing cells were studied by following incorporation of radioactive glucosamine and separation of the SDS-insoluble peptidoglycan into uncross-linked (monomer) and cross-linked (dimer and oligomer) fragments. Cultures to which penicillin or piperacillin at concentrations near the minimum growth inhibitory concentration (MIC) had been added 20 min before the radioactive label were compared with controls. The β-lactams affected the early stage of cross-linking (up to 3 min) but had no effect thereafter. The deficit of cross-linking, however, did not recover. The O-acetylation, particularly of the monomer fraction, was decreased by β-lactams even at concentrations that had no effect on culture optical density, viable counts or overall peptidoglycan synthesis. These effects on O-acetylation occurred mainly after the first 3 min of incorporation, rather than before. O-Acetylation of the oligomer fraction was also followed. Here penicillin led to increased levels of O-acetylation during the early stages of incorporation but the final value was never exceeded; indeed at higher drug concentrations the later stages of O-acetylation of oligomers were inhibited (e.g. almost completely at 2·5 × MIC).

SUMMARY: A catechol-type siderophore was secreted by Azospirillum brasilense in the growth medium when the cells became iron-deficient. The siderophore, which we call spirilobactin, was purified and a partial characterization of its structure by hydrolysis revealed that it contained 2,3-dihydroxybenzoic acid, ornithine and serine in equimolar amounts. A high-affinity iron transport system capable of 59Fe uptake from a 59Fe(III)-spirilobactin complex was also induced in A. brasilense grown under iron deficiency. Kinetic studies on the iron uptake system revealed that it was carrier-mediated, with K m and V max values of 0·23 μm and 0·27 nmol Fe min−1(mg cell protein)−1, respectively. Dependence of iron uptake on an energized membrane, its insensitivity to arsenate and inhibition by protonophores suggest that transport is driven by the proton gradient across the membrane. Iron-deficient Azospirillum lipoferum transported 59Fe from the 59Fe(III)-spirilobactin complex (at a rate 3-fold lower than that of A. brasilense) but A. amazonense failed to show uptake of iron under the same conditions.

SUMMARY: Cell envelopes (i.e. unfractionated inner and outer membranes) were obtained from Providencia stuartii by following procedures previously applied to the isolation of envelopes from Escherichia coli. The P. stuartii envelopes contained known inner membrane enzymes that included a variety of dehydrogenases and ATPase. The catalytic activity of the ATPase depended upon the concentration of magnesium ions, the substrate (ATP) level and the ratio of magnesium ions to ATP. Cell envelopes from P. stuartii were further fractionated to recover inner and outer membrane polypeptides by treatment with the detergent Sarkosyl. Proteins from the periplasmic region were recovered by a simple osmotic shock procedure also previously applied to E. coli. The purity of the various P. stuartii cell envelope fractions was assessed by a combination of techniques that included one- and two-dimensional gel electrophoresis of proteins, enzyme assays and detection of penicillin-binding proteins.

SUMMARY: Proline accumulation in Escherichia coli is mediated by three proline porters. Proline catabolism is effected by proline porter I (PPI) and proline/Δ1-pyrroline carboxylate dehydrogenase. Proline did not accumulate cytoplasmically when E. coli was subjected to osmotic stress in minimal salts medium. Although PPI is induced when proline is provided as carbon or nitrogen source, its activity decreased following growth of the bacteria in minimal salts medium of high osmotic strength. Proline dehydrogenase was induced by proline in low or high osmotic strength media. Proline porter II (PPII) was both activated and induced in osmotically stressed bacteria, though the dependencies of the two responses on medium osmolarity differed. Osmotic downshift during the transport measurement decreased the uptake of proline, serine and glutamine by bacteria cultured in media of high osmotic strength. Thus, while osmotic upshift caused specific activation of PPII, osmotic downshift caused a non-specific reduction in amino acid uptake. Glycine betaine inhibited the uptake of [14C]proline via PPII and PPIII but not via PPI. The dependence of that inhibition on glycine betaine concentration was similar when PPII was uninduced, induced or activated by osmotic stress, or induced by amino acid limited growth. Thus PPII and PPIII, not PPI, contribute to the mechanism of osmoprotection by proline and glycine betaine. The tendency for exogenous proline to accumulate in the cytoplasm of bacteria exposed to osmotic stress would, however, be countered by increased proline catabolism.

SUMMARY: Growth rates of Vibrio costicola showed a broad optimum between 0·8 and 1·5 M-NaCl, and there was no growth above 3·3 M-NaCl in a peptone-based medium. The minimum requirement of 0·5 M-NaCl for growth in NaCl alone was reduced to 0·3 M-NaCl when the total solute concentration was raised to 0·5 to 1·0 M equivalent with sucrose or glycerol. Compared with equivalent NaCl concentrations, higher concentrations of sucrose were more inhibitory to growth, whereas glycerol had less effect. Increasing the medium NaCl concentration suddenly by 2- or 3-fold with either a constant starting, or final, salt concentration showed that, after the shift-up, the lag in growth, the rate of growth, and the inhibition of phospholipid synthesis depended both on the final NaCl concentration and the magnitude of the shift in salinity. The time-courses of phospholipid synthesis following a 2-or 3-fold shift-up in NaCl or sucrose media were very similar and exhibited a relative increase in phosphatidylglycerol synthesis over that of phosphatidylethanolamine. This ‘switch-over’ was not seen following shift-up in glycerol media when there was also a stimulation, rather than inhibition, of phospholipid synthesis. It is concluded that during phenotypic haloadaptation of V. costicola, osmotic effects play a significant part in the sensing of and response to raised external salinity.

SUMMARY: A mathematical model describing the instability of plasmids in micro-organisms has been developed. The model is based on the assumption that the overall causes of plasmid instability are described by the segregational instability of the plasmid, R (i.e. the rate at which plasmid-free cells are generated from plasmid-bearing cells), and the growth rate difference, dμ (i.e. the difference in growth rate between plasmid-free and plasmid-bearing cells). A method for determining the values of R and dμ (accompanied by 95% confidence limits) for any plasmid-bearing micro-organism is described. This method is based on the observation that, depending on the plasmid, various exponential patterns of plasmid instability are observed. The stability of Escherichia coli 1B373(pMG169), where dμ >> R, and E. coli RV308(pHSG415), where R >> dμ, are analysed in order to demonstrate the method.

SUMMARY: The stability of a low-copy-number plasmid, pHSG415, in Escherichia coli, was investigated in batch and continuous culture. The plasmid was unstable in batch culture, but was significantly stabilized by growth in continuous culture with phosphate, nitrogen or potassium limitation. However, the plasmid was very unstable when grown in continuous culture with sulphate limitation. These results contrast with those obtained with multicopy plasmids such as pBR322, which is particularly unstable in carbon- or phosphate-limited continuous culture. The effect of growth rate on the stability of E. coli(pHSG415) grown in continuous culture with glucose limitation was also investigated. The plasmid was significantly more stable in cells grown at higher growth rates. The segregational instability (R) of the plasmid and the difference in growth rate between plasmid-free and plasmid-bearing cells (dμ) were calculated for each condition using the method of Cooper et al. (accompanying paper: Journal of General Microbiology 133, 1871-1880). It was found that the primary cause of the loss of pHSG415 from the cell population was the segregational instability of the plasmid.