- Volume 134, Issue 11, 1988

A recently isolated organism, capable of chemolithoautotrophic growth on dimethyl disulphide, was characterized as a strain of Thiobacillus thioparus. It had DNA with a base composition of 60·5 ± 1·0 mol% G + C, and ubiquinone-8 (UQ-8) as its only respiratory quinone. Its growth in chemostat culture (at a growth rate of 0·07–0·08 h−1) showed yields of 14·4, 11·8 and 2·45 g cell-carbon per mol of dimethyl disulphide (DMDS), dimethyl sulphide (DMS) and thiosulphate, respectively. This is consistent with energy generation from the oxidation of the methyl and the sulphide moieties of DMDS, with oxidation of sulphide to sulphate contributing a yield of about 2·8 g cell-carbon mol−1. From whole organism and cell-free extract studies, DMDS oxidation was shown to proceed by its (NADH-stimulated) reduction to methanethiol (MT), which was oxidized via sulphide, formaldehyde and formate to CO2 and sulphate by MT oxidase, formaldehyde and formate dehydrogenases, and an uncharacterized sulphide-oxidizing system. The MT oxidase had a K m of 9·7 μm and showed substrate inhibition with a K i of about 8 μm. The essential role of catalase during growth on DMDS was shown by the sensitivity of growth on DMDS (but not on thiosulphate) to 3-amino-1,2,4-triazole. Catalase is believed to destroy the peroxide produced by the MT oxidase reaction. DMDS-grown organisms oxidized sulphide, thiosulphate and tetrathionate (with the latter indicated to be an intermediate in thiosulphate oxidation), suggesting the pathway of sulphide oxidation to be similar to that in some other thiobacilli. Carbon assimilation was by the Calvin cycle, with ribulose bisphosphate carboxylase being present in cell-free extracts at a specific activity of 80 nmol CO2 fixed min−1 (mg protein)−1. Hydroxypyruvate reductase (HPR) was not detected at levels sufficient to indicate any role in primary carbon assimilation.

β-Ketoadipate elicits expression of five structural pca genes encoding enzymes that catalyse consecutive reactions in the utilization of protocatechuate by Pseudomonas putida. Three derivatives of P. putida PRS2000 were obtained, each carrying a single copy of Tn5 DNA inserted into a separate region of the genome and preventing expression of different sets of pca genes. Selection of Tn5 in or near the pca genes in these derivatives was used to clone four structural pca genes and to enable their expression as inserts in pUC19 carried in Escherichia coli. Three of the genes were clustered as components of an apparent operon in the order pcaBDC. This observation indicates that rearrangement of the closely linked genes accompanied divergence of their evolutionary homologues, which are known to appear in the order pcaDBC in the Acinetobacter calcoaceticus pcaEFDBCA gene cluster. Additional evidence for genetic reorganization during evolutionary divergence emerged from the demonstration that the P. putida pcaE gene lies more than 15 kilobase pairs (kbp) away from the pcaBDC operon. An additional P. putida gene, pcaR, was shown to be required for expression of the pca structural genes in response to β-ketoadipate. The regulatory pcaR gene is located about 15 kbp upstream from the pcaBDC operon.

No free plasmid has ever been found in the myxobacterium Myxococcus xanthus, but IncP-1 plasmids are able to integrate into the chromosome of this bacterium. The frequency of integration depends greatly upon the structure of the IncP-1 plasmid used. This property has been used to devise new delivery systems for transposon mutagenesis in this species. Plasmids with low integration efficiencies have proved to be efficient donors of Tn5, while plasmids with very high frequencies of integration could be used directly to generate mutations. These vectors have also proved efficient for Tn5 transfer into other species of myxobacteria, which have not so far been susceptible to genetic analysis.

A 7·8 kb plasmid (pQM17) encoding mercury resistance was isolated from two epilithic strains of Acinetobacter calcoaceticus. The plasmid had a broad host range when mobilized by RP1, transferring into Pseudomonas aeruginosa, P. putida, P. fluorescens, Escherichia coli, Proteus vulgaris and Chromobacterium sp. with frequencies ranging from 5·3 × 10−9 to 4·6 × 10−4 per recipient. The plasmid could be transferred into A. calcoaceticus BD413 using intact cells of donor and recipient bacteria (i.e. natural transformation) and there was a broad temperature optimum (14–37 °C) for transformation. Transformation was as efficient in liquid matings as on plates but there was no effect of pH in the range 5·6–7·9. Maximum transformation frequencies were obtained after 24 h on agar plates containing 3·5–10 g C1−1 with donor to recipient ratios ranging from 6 to 415.

A procedure was developed for the isolation of heterocysts from cyanobacterial filaments without recourse to mechanical disruption of the vegetative cells. DNA was then extracted from purified heterocysts by heating with 2% (w/v) SDS at 70 C for 10 min. Following purification, this DNA was used for treatment with a range of restriction endonucleases and the results compared with DNA isolated from vegetative cells. Both heterocyst and vegetative DNAs from Anabaena PCC 7120 and Anabaena CA (ATCC 33047) were cut by XbaI, HindIII, EcoRI, ClaI, HpaII and MspI. However, none of the DNAs were cut by XhoI, SalI or MboI, indicating that the DNA from both organisms is methylated, but that no gross changes in methylation occur during heterocyst formation. Treatment of the DNAs with the former enzymes, followed by separation of the fragments by agarose gel electrophoresis, resulted in most cases in patterns of bands, which allowed a limited comparison of heterocyst and vegetative DNAs. No major differences were seen between the heterocyst and vegetative DNAs of either organism, implying that there are unlikely to be extensive rearrangements or major loss of DNA during heterocyst differentiation.

The extremely halophilic archaebacterium Halococcus morrhuae CCM 537 was found to contain a plasmid, pHM2, of 6·0 kb. A restriction map was constructed. Southern hybridization of pHM2 DNA to DNA extracted from other Halococcus sp. strains revealed the presence of a similar plasmid in one other strain. A plasmid of Halobacterium trapanicum shared common sequences with pHM2. Other plasmids from halobacteria displayed no homology with pHM2. The characterization of pHM2 may make it useful for the development of cloning vectors for extremely halophilic archaebacteria.

pWW53 is a 110 kbp catabolic plasmid which encodes the complete pathway for the utilization of toluene and the xylenes. The upper pathway operon xylCAB is located between two homologous but distinct meta pathway operons, xylDLEGF(I, J, K)H, which are in direct repeat. These have each been cloned on large HindIII restriction fragments HA (17.5 kbp) and HB (15.6 kbp), the restriction sites of which have been mapped. During growth of MT53 on benzoate, mutants which have lost the ability to grow on hydrocarbons such as m-xylene (Mxy−) but which retain the ability to grow on their carboxylic acid metabolites such as m-toluate (Mtol+) take over the culture before ultimately being displaced by plasmid-free strains which are Mxy− Mtol−. The plasmids in the Mxy− Mtol+ mutants are formed by a large deletion between homologous regions of the two duplicate meta pathway operons. This causes the loss of the intervening xylCAB operon and the formation of a hybrid xylDLEGF(I, J, K)H operon, starting with the genes originally on HA and terminating with the genes originally on HB.

Auxotrophs derived by UV or N-methyl-N′-nitro-N-nitrosoguanidine mutagenesis from three North American, three European and four Australian tomato wilt isolates of Verticillium dahliae were used to investigate heterokaryon compatibility. Ninety ‘crosses’ involving race-1 and race-2 isolates were attempted using mixed conidial suspensions and a ‘hanging drop’ technique. Crosses between North American and Australian isolates failed to produce heterokaryons over a 20 d period but within each of these two groups of isolates (with one exception) the frequency of heterokaryon formation was 100%. In crosses involving the three European isolates the frequency of heterokaryon formation varied from 0 to 100%. Two of the European isolates produced heterokaryons both with North American and with Australian isolates. Prototrophic diploid conidia were recovered from 13 of the 30 crosses that produced heterokaryons. The rate of diploid production was greatest in crosses showing 100% compatibility but rare diploids were recovered from two crosses which had produced no observable heterokaryotic growth on minimal medium. The formation of heterozygous diploids between race-1 and race-2 isolates of V. dahliae indicates the potential for the genetic analysis of pathogenicity through the parasexual cycle.

All the single-spore cultures originating from zoospores and oospores of wild-types of Phytophthora parasitica were sensitive to chloramphenicol and streptomycin. Cultures from zoospores and oospores of chloramphenicol-resistant mutants were resistant to chloramphenicol but not streptomycin, while those from zoospores and oospores of streptomycin-resistant mutants were resistant to streptomycin but not chloramphenicol. When chloramphenicol-resistant A1 (6133Cr) of P. parasitica was paired directly with streptomycin-resistant A2 (6134Sr) of the same species, all single-oospore cultures from three crosses were resistant to either chloramphenicol or streptomycin but not both, suggesting absence of genetic exchange in the progeny. Appearance of A2 resistant to chloramphenicol and A1 resistant to streptomycin was due to a mating-type change during oospore formation because oospores produced by A1 through hormone stimulation also gave rise to A2 cultures, and vice versa. Similar results were obtained when reciprocal crosses involving the same direct and membrane-separated pairings between streptomycin-resistant A1 and chloramphenicol-resistant A2 were studied. When A1 wild-type (6133), sensitive to both chloramphenicol and streptomycin, was paired with A2 double mutant 6134CrSr, resistant to chloramphenicol first and streptomycin later, all single-oospore cultures from three crosses were either sensitive to both chloramphenicol and streptomycin or resistant to both antibiotics, indicating that all single-oospore cultures from direct pairing were of uniparental origin. When A2 isolate 6134Sr, resistant to streptomycin, was paired directly with its A1 variant 6134VCr, resistant to chloramphenicol, all single-oospore cultures from three crosses were resistant to either chloramphenicol or streptomycin but not both, indicating an apparent absence of genetic exchange in the progeny. This ruled out the possibility that absence of genetic exchange in the crosses between two different isolates of P. parasitica might be due to genetic divergence between the strains crossed. We interpret the results of this study to show that all progenies from direct pairings between different mating types of P. parasitica were of uniparental origin, although close linkage or mitochondrial location of the markers cannot as yet be eliminated as possible explanations of the apparent absence of genetic exchange. We propose the term hormonal heterothallism to denote this suggested novel phenomenon.

A set of pneumococcal strains containing immediately adjacent or nearby double mutations at the amiA locus, conferring resistance to amethopterin, has been isolated by oligonucleotide site-specific mutagenesis. Repair of these double mutations has been measured by transformation of wild-type strains with DNA extracted from these strains. In several transformations we have observed an inhibition of repair by neighbouring mismatches. This inhibition ranges from mild to severe depending upon the interfering mismatch. Unrepaired mismatches can strongly inhibit repair of an adjacent repairable mutation. This suggests that the repair-complex proteins attach not only to repairable mismatches but also to some mismatches known to escape the repair system.

The mode of action of aqueous garlic extract (AGE) was studied in Candida albicans. The minimum inhibitory concentration (MIC) of AGE against six clinical yeast isolates ranged between 0.8 and 1.6 mg ml−1. Scanning electron microscopy and cell leakage studies showed that garlic treatment affected the structure and integrity of the outer surface of the yeast cells. Growth of C. albicans in the presence of AGE affected the yeast lipid in a number of ways: the total lipid content was decreased; garlic-grown yeasts had a higher level of phosphatidylserines and a lower level of phosphatidylcholines; in addition to free sterols and sterol esters, C. albicans accumulated esterified steryl glycosides; the concentration of palmitic acid (16:0) and oleic acid (18:1) increased and that of linoleic acid (18:2) and linolenic acid (18:3) decreased. Oxygen consumption of AGE-treated C. albicans was also reduced. The anticandidal activity of AGE was antagonized by thiols such as L-cysteine, glutathione and 2-mercaptoethanol. Interaction studies between AGE and thiols included growth antagonism, enzymic inhibition and interference of two linear zones of inhibition. All three approaches suggest that AGE exerts its effect by the oxidation of thiol groups present in the essential proteins, causing inactivation of enzymes and subsequent microbial growth inhibition.

The isolation and identification of cytoplasmic membranes from cyanobacteria is complicated by the presence of multiple photosynthetic membranes (thylakoids), and by a lack of biochemical markers characteristic of the cytoplasmic membranes of heterotrophic pro-karyotes. [3H]Benzylpenicillin was used to label the penicillin-binding proteins (PBPs) in the unicellular cyanobacterium Synechococcus sp. strain R2 (PCC 7942), in spheroplasts and in fractionated cell membranes. Spheroplasts were made with lysozyme/EDTA, and were broken by shaking with glass beads in hypo-osmotic buffer at 4 °C. Sequential glycerol density gradients were then used to purify the membrane fractions. A low-density membrane (LDM) fraction was isolated which, when compared to the thylakoid fraction, was enriched in a unique carotenoid with an absorption maximum at 390 nm and low in chlorophyll a absorption at 678 nm (A 390/A 678 > 10). This fraction had a characteristic polypeptide profile on denaturing polyacrylamide gels (SDS-PAGE), and specific enrichment of PBPs, as detected by fluorography of membrane fractions incubated with [3H]benzylpenicillin followed by SDS-PAGE. The M r values of the Synechococcus PBPs differed from those in Staphylococcus aureus or Escherichia coli membranes. The high content of specific PBPs in LDM suggested that this fraction was highly enriched in cytoplasmic membrane. A membrane fraction with density between that of the thylakoid and LDM fractions, previously considered to be a mixture of these membranes, showed specific enrichment of a PBP of M r 78000. This fraction may contain contact zones between the different membrane types.

Spores of Mucor rouxii had low levels of respiratory activity partially sensitive to cyanide and salicylhydroxamic acid (SHAM). They contained a full set of cytochromes but in low concentrations, especially cytochromes aa 3. Aerobic growth induced a rapid increase in respiratory activity resistant to SHAM but fully sensitive to cyanide, and in the levels of cytochromes, mainly cytochromes aa 3. These processes were subject to catabolite repression. Incubation under anaerobic conditions, independent of cell morphology, favoured the persistence of SHAM-sensitive respiration, and the maintenance of low levels of b and c cytochromes. No cytochromes aa 3 were formed under these conditions. CO-difference spectra suggested the presence of cytochrome o in higher levels in aerobic than in anaerobic cells.

The results of a physiological study of the interaction between NH4Cl, inosine, and the stereoisomers of alanine during germination of spores of Bacillus cereus T are presented. Detailed kinetics for the germination of unheated spores in moderate concentrations of l-alanine (in the absence of auto-inhibition due to alanine racemase) are established, as is the specificity of the stimulatory effect of NH4Cl in relation to other salts, amines, and germinants. The results suggest that NH4Cl and inosine affect an early step in germination closely related to the function of an l-alanine receptor.

The ability of micro-organisms to grow on carbon disulphide (CS2) as a sole source of carbon and energy appears to be very limited: none was obtained from enrichment culture and eight Thiobacillus species could not use it. Thiobacillus thioparus strain TK-m could grow autotrophically on either CS2 or carbonyl sulphide (COS) as sole substrates. Growth yield on CS2 was 7·9 ± 0·9 g cell-carbon (mol CS2)−1, and yields on COS, thiosulphate or thiocyanate were in the range 5·6–6·1. COS was detected as an intermediate during growth on CS2, and there was quantitative conversion of the sulphur of CS2 to sulphate during growth. Aerobic oxidation of CS2 by suspensions of strain TK-m exhibited a K s of 16·5 μm and a V max of 524 nmol O2 consumed min−1 (mg organism-protein)−1. When incubated anaerobically with CS2, strain TK-m sequentially produced COS and H2S. CS2 oxidation is proposed to proceed by its sequential hydrolytic cleavage to COS then H2S, with release of all the carbon as CO2, followed by oxidation of the sulphide to sulphate. This oxidation provides all the energy for growth, which is dependent on the autotrophic fixation of CO2, apparently by means of ribulose bisphosphate carboxylase.