- Volume 131, Issue 3, 1985

Extracts of Micromonospora inyoensis contain aminoglycoside acetyltransferase activity not previously encountered among actinomycetes. Neomycin and its derivatives (excluding butirosin) are good substrates as is the novel aminoglycoside apramycin, whereas the gentamicins and kanamycins are utilized poorly if at all. Sisomicin, which is produced by M. inyoensis, is not acetylated. When neomycin was modified by extracts of M. inyoensis the product, most probably 3-N-acetylneomycin, was inactive against cell-free protein synthesis.

An l-aromatic-amino-acid aminotransferase (AT-IA) and an l-aspartate aminotransferase (AT-IB) were partially purified from Brevibacterium linens 47 grown on l-phenylalanine as sole nitrogen source and some properties were examined. Both enzymes were active with l-aromatic amino acids and l-aspartate. AT-IA showed 6 to 12 times higher affinity and slightly higher V max for the aromatic amino acids than for aspartate. AT-IB had higher affinity for tryptophan and tyrosine than for aspartate. However, this enzyme showed 6 to 10 times higher V max for the latter than for the aromatic amino acid substrates. Both enzymes had similar pH (8·5-9·0) and temperature (37–40 °C) optima, but they differed in their molecular weights (126000 for IA and 81000 for IB) and markedly in their thermostability. At 50 °C, AT-IB was 14 times more rapidly inactivated and its inactivation rate also increased more rapidly (z-value, 7·9 °C) as the temperature increased than AT-IA (z-value, 15·5 °C). The cofactor pyridoxal-5′-phosphate was tightly bound to both enzymes. Two enzymes co-eluted on DEAE-Trisacryl M column chromatography at pH 7·5 and could be separated by chromatography on a hydroxyapatite (HA-Ultrogel) column at the same pH. Chromatography on hydroxyapatite of AT-I from cells grown on l-phenylalanine and on ammonium sulphate revealed that AT-IA was present only in phenylalanine grown cells. AT-IB was present in both extracts at similar levels, suggesting that it is constitutive. The inducibility of IA suggested that it has an in vivo catabolic role. AT-IA is probably the key enzyme for the utilization of the aromatic amino acids as sole ritrogen sources in B. linens 47.

A strain of Pseudomonas syringae was isolated from garden soil for its ability to utilize propan-2-yl sulphate as sole source of carbon and energy. Growth was accompanied by disappearance of the ester from culture fluids and formation of stoicheiometric amounts of inorganic sulphate. However, enzyme activity capable of desulphating propan-2-yl sulphate was not detected under a wide range of assay conditions, either intracellular or extracellular, or at different phases of batch culture. No organic radioactive metabolites were detected during growth on propan-2-yl [35S]sulphate or [1, 3-14C]propan-2-yl sulphate alone, but when the latter compound was supplemented with unlabelled lactate, small amounts of 14C-labelled lactate were detected. This and the induction by propan-2-yl sulphate of a specific d-lactate-2-sulphatase led to a proposed pathway in which propan-2-yl sulphate undergoes a stereospecific oxidation to d-lactate-2-sulphate before rapid desulphation to d-lactate and inorganic sulphate.

Mutants of the obligatory aerobic yeast Rhodotorula glutinis were selected after treatment of wild-type cells with N-methyl-N′-nitro-N-nitrosoguanidine on glycerol/glutamic acid medium containing 1%2-deoxy-d-glucose. Rates of d-glucose transport in the mutants were about one-tenth that of the wild-type, whereas the transport of d-fructose and d-xylose was unaffected. However, glucose transport in the mutants was inhibited by uncouplers (for example carbonyl cyanide m-chlorophenylhydrazone) and, therefore, remained an energy-dependent process. One of the mutants, M8. was chosen for further characterization of the transport and metabolism of hexoses. Biochemical analysis of the hexose-ATP-kinase activities revealed the absence of glucokinase (EC 2.7.1.2) activity in M8. Thus, the phosphorylation capacity was reduced to one-tenth to one-fifteenth that of the wild-type, resulting in accumulation of d-glucose which, in turn, slowed down net glucose uptake. With the help of the mutant, the stoicheiometry of the H+/glucose symport in R. glutinis was determined to be one proton per molecule of d-glucose transported.

Extracellular 1,3-α-d-glucan synthase (sucrose: 1,3-α-d-glucan 3-α-d-glucosyltransferase, EC 2.4.1. –) of Streptococcus mutans HS6 (serotype a) was purified from culture supernatant by ultrafiltration, DEAE-Sepharose chromatography and preparative isoelectric focusing. The enzyme had a molecular weight of 158000 by SDS-PAGE and an isoelectric point of pH 5·2. The specific activity of the enzyme was 48·3 i.u. (mg protein)–1. The K m for sucrose was 1·2 mm and the activity was optimal at pH 6·0. The enzyme activity was stimulated about 20-fold in the presence of dextran T10. Glucan was synthesized de novo from sucrose by the enzyme and characterized as a linear 1,3-α-d-glucan by GC–MS.

The quinoxaline chromophores of the antibiotics produced by Streptomyces triostinicus are derived from tryptophan. Protoplasts of this organism made novel products when they were incubated with dl-5-fluorotryptophan or dl-6-fluorotryptophan. When added to batch cultures of the organism. dl-5-fluorotryptophan. at concentrations as low as 10 μm, inhibited both mycelial growth and triostin production, but gave rise to novel products. These have been characterized, using fast atom bombardment mass spectrometry, as novel triostins in which one or both of the quinoxaline rings contain an atom of fluorine. The chromatographic properties of the triostins arising from the incorporation of dl-5-fluorotryptophan are very similar to those of triostins containing chlorine or bromine at position 6 of the quinoxaline ring; they are clearly different from those having a chlorine atom at position 7. Accordingly, it is suggested that the carbon atom at position 5 of the indole ring of tryptophan ends up at position 6 of the quinoxaline ring system in triostins A and C.

The addition of tribromo-, tetrabromo- and chloromethanes to cultures of Aspergillus parasiticus strongly stimulated aflatoxin biosynthesis. The presence of phenobarbital in the cultures enhanced this effect. Following the addition of 14CC14 to a culture, after 15 d incubation, 8·6% of the total radioactivity was recovered from the mycelium, and 4·7% from the culture filtrate. Some 0·4% of the total radioactivity was found in microsomes, indicating their involvement in CC14 activation and biotransformation.

The distribution of lipid fatty acids was studied in marine sediment slurries in which sulphate reduction had been stimulated by the addition of acetate, propionate, lactate or hydrogen. Acetate was directly oxidized to carbon dioxide at the expense of sulphate, propionate was incompletely oxidized to acetate at the expense of sulphate, and lactate was largely fermented (approximately 75%) to propionate and acetate, with the propionate subsequently utilized for sulphate reduction. Changes in the lipid fatty acids within these slurries were interpreted by comparison with the lipid fatty acid profiles of pure cultures of Desulfovibrio desulfuricans, Desulfobacter sp. and Desulfobulbus sp. Desulfobacter sp. seems to be the main acetate-utilizing sulphate-reducing bacterium, as in both the slurry and the original sediment the lipid fatty acids in the C12–C18 range (where bacterial contributions would be expected) were dominated by even chain fatty acids similar to the lipid fatty acid distribution of Desulfobacter sp. Desulfobulbus sp. appears to be responsible for propionate oxidation and also for hydrogen consumption as its biomarker fatty acid n-C17:1 was increased significantly by the addition of either propionate or hydrogen. Surprisingly, the biomarker for Desulfovibrio desulfuricans (br-C17:1) was not stimulated in any of the sediment slurries. The results demonstrate that there are at least two functional groups of sulphate-reducing bacteria in marine systems, and that lipid fatty acid analysis is a useful technique for investigating bacterial distributions within a complex sedimentary environment.

The interrelation of propionate and succinate metabolism in anoxic sewage sludge and in two different anoxic lake sediments was studied. The mechanism of propionate degradation and the kinetics of propionate and succinate metabolism were analysed using specifically labelled 14C tracers and gas chromatography-gas proportional counting techniques. The labels of [1-14C]propionate and [1,4-14C]succinate were transformed almost exclusively to carbon dioxide whereas the label of [3-14C]propionate was transformed to equal amounts of methane and carbon dioxide, indicating a randomizing pathway of propionate degradation. The pool sizes of propionate and succinate (0·3–2·3 μmol 1–1) were similar to each other in, but were both different between, each of the three environments studied. The turnover times of succinate were shorter than those of propionate, and the C-1 label of propionate and the C-1 and C-4 labels of succinate were metabolized far faster than the respective C-3 and C-2 and C-3 labels. These results indicate that propionate, although formed via succinate, is also degraded via succinate in the anoxic environments studied, and that succinate metabolism is at least as important as propionate metabolism in anaerobic degradation processes.

Pool sizes, turnover times and turnover rates of ethanol and acetate in anoxic sediments of Lake Mendota and Knaack Lake and in anoxic sewage digestor sludge were determined by gas chromatography-gas proportional counting techniques. Ethanol accounted for 6, 14 and 2·5% of the total carbon flux to methane in these environments, respectively. The distribution of labelled carbon in the methane and carbon dioxide fractions obtained during incubation of the anoxic materials with C-1 and C-2 labelled acetate and ethanol revealed a significantly higher degree of randomization with ethanol than with acetate tracers. HPLC analysis of sediment pore water preparations revealed that labelled acetate and propionate were formed as intermediates of labelled ethanol degradation, whereas no labelled butyrate was detected. Addition of hydrogen to Knaack Lake sediment samples inhibited ethanol degradation drastically and led to a significant accumulation of labelled butyrate. The above findings together with the results of most probable number enumerations of anaerobic ethanol-degrading bacteria indicate that propionate-forming bacteria contributed significantly to ethanol degradation in Knaack Lake sediment and in sewage sludge.

Candida albicans undergoes yeast to mycelial conversion under both in vivo and in vitro conditions but the relative pathogenicity of the two forms of growth is still unknown. By adapting a recently developed 51Cr radiolabel release assay, we have quantified the killing ability of different murine effector cell populations for the hyphal form of C. albicans. Up to 50% of specific 51Cr release from the mycelial form could be detected after incubation for only 1 h, with no requirement for opsonization, provided that appropriate effector : target cell ratios were used. The specific 51Cr release correlated well with viability, as assessed by dye exclusion tests, and with pathogenicity potential in cyclophosphamide-immunodepressed mice. Comparison of the activity of different murine effectors against yeast and hyphal forms showed that hyphal forms were killed by murine effectors to a similar, if not greater, extent than yeast forms. In particular, thioglycollate-induced murine polymorphonuclear neutrophils were able to kill hyphal cells extracellularly and without an opsonic requirement.

SUMMARY: Studies on the pathogenesis of Haemophilus influenzae b infection have used bacteria grown in vitro which are relatively serum-sensitive (using serum devoid of anticapsular antibody) compared to organisms taken from infected hosts. We compared the virulence of relatively serum-sensitive and serum-factor induced serum-resistant H. influenzae b by inoculating rats with organisms having one or the other phenotype. The serum-resistant phenotype was more virulent following intraperitoneal or intravenous inoculation; however, there was no difference in the incidence of colonization or bacteraemia following intranasal inoculation. Furthermore, organisms colonizing the pharynx of rats had the serum-resistant phenotype. Thus, different phenotypes of the same strain of H. influenzae b differed in virulence following parenteral, but not intranasal, inoculation of bacteria. This could be explained by a change from serum-sensitive to serum-resistant phenotype shortly after entering the nasopharynx. The phenotype of micro-organisms grown in vitro may differ from organisms in infected individuals and these differences may be of critical importance in studies of immunity to infection and the pathogenesis of infection.

The determinant(s) of gonococcal resistance to killing by human phagocytes has been extracted from outer membrane vesicles (OMV) of a phagocyte-resistant strain, BS4 (agar), with sodium cholate (1%, w/v). The extracts, like the OMV, nullified the effect of antiserum raised against whole BS4 (agar) to promote intracellular killing of the latter by human peripheral blood phagocytes. Fractionation of the extract on Sephadex G75 produced an active fraction with much less protein and lipopolysaccharide (LPS) than in the original extract. Furthermore, crude LPS prepared from the resistant gonococci was inactive. These results imply that the factor(s) promoting intracellular resistance is a protein. SDS-PAGE of the active fraction suggested that the factor was not a principal outer membrane protein nor one of three proteins previously thought to be associated with resistance. In contrast to a similar preparation from a phagocyte-susceptible strain, BSSH, the active fraction from BS4 (agar) showed faintly staining proteins in the regions of 20 and 60 kDal. When eluted from the gels, the former but not the latter neutralized the above effect of antisera, thus associating the 20 kDal protein(s) with resistance to intracellular killing.

Three stilbene phytoalexins, elicited by slicing and incubating imbibed peanut kernels under aerobic conditions, inhibited spore germination and hyphal extension of Aspergillus flavus with ED50 values in the range 4·9–12·8 μg ml–1. Phytoalexin yield was dependent on cultivar, conditions and duration of incubation after slicing, and crop history. The yield of phytoalexin from ten cultivars studied, after slicing and incubating at 25 °C for 24 h, ranged from 28 to 935 μg per g fresh weight and was negatively correlated with dry kernel colonization by A. flavus [r = –0·868 when plotted as In (phytoalexin concn) against In (percentage peanut colonization)]. When the incubation period was extended to 96 h there was no such correlation. Reduced phytoalexin yields were obtained when sliced kernels of one cultivar studied were incubated in water or at 37 °C, and no phytoalexin was obtained when the slices were incubated under nitrogen gas or frozen before aerobic incubation. Drought stress during pod development in four cultivars studied reduced phytoalexin yields of sliced kernels incubated at 25 °C for 24 h by 17–65% compared with non-stressed controls.

Serovars jequitaia and tororò of Leptospira biflexa were cultured in the presence of homologous factor serum containing factorial antibodies (FcAbs) to their major antigens. After 39 serial passages they were then re-tested to determine whether their major antigens had remained unchanged. It was found that each parent strain had been replaced by an antigenic variant. The disappearance of each parent strain and its replacement by an antigenic variant was attributed to the selective conditions imposed by FcAbs. The antigenic variants behaved like true mutants. They lacked the major serovar antigens of the parent strains and had acquired some major antigens similar to those of two different serovars, one of which belonged to the same serogroup as the parent strain and the other to a different serogroup.

A comparison of the major antigens of the parent strains with those of their antigenic variants indicated that factorial antibodies may be used selectively to obtain antigenic variants with a predefined pattern of major antigens.