- Volume 129, Issue 1, 1983

Incorporation of sodium [1-14C]acetate into the heterocyst-specific glycolipids and glycerolipids of the cyanobacterium Anabaena cylindrica during induced heterocyst differentiation indicated that (1) the activity of key enzymes involved specifically in the biosynthesis of the very long chain polyhydroxyalkanes and hydroxyfatty acid moieties of the heterocyst glycolipid fraction is stimulated during heterocyst differentiation, and (2) in mature heterocysts, formation of the characteristic very long chain heterocyst glycolipids essentially ceases.

The activity of proteinases in mycelial extracts of Aspergillus nidulans increased during storage. The rate of activation increased with temperature. Three separate proteinase activities, differing in their electrophoretic mobilities on polyacrylamide gels, were readily detected at pH 6.5. Inhibitory activity, effective against all three proteinase activities, was also detected in fractions prepared from fresh mycelial extracts. The inhibitory factor(s) were heat-stable and non-dialysable. The inhibitory activity was lost during storage of mycelial extracts. It is proposed that the inhibitory factor(s) are digested by the proteinases during storage.

A comparative electron microscopic study was made of virulent ovine strains, benign ovine strains, bovine strains and culture variants of Bacteroides nodosus using negative staining, thin section and freeze-fracture etch techniques. The plasma membrane, peptidoglycan layer and outer membrane structures were similar in all the organisms, but there were marked differences in the presence of pili, diffuse polar material and additional layer. The variations in these surface structures were examined in relation to the virulence and immunoprotection of B. nodosus towards foot-rot in sheep. Only organisms with abundant pili caused virulent foot-rot; diffuse polar material and perhaps the additional layer may also be associated with virulence, but conclusive evidence was lacking. It appeared that pili and one or more unknown cell components, possibly diffuse polar material but not the additional layer, were necessary for immunoprotection.

Addition of the antifungal antibiotic papulacandin B to an exponential culture of Geotrichum lactis inhibited incorporation of glucose into the alkali-insoluble and alkali-soluble glucan fractions of the hyphal wall, although the rate of growth was practically unaltered. Synthesis of other cell wall components (i.e. galactomannan and chitin) was not affected. Papulacandin B also induced the proliferation of branches along the hyphae which continued to branch dichotomously resulting in a ‘colonial’ pattern of growth. Aculeacin A, another antifungal antibiotic that inhibited β-glucan synthesis also caused morphological alterations similar to those described for papulacandin B. Inhibition of β-glucan synthesis and the altered growth pattern persisted for several hours after removal of the antibiotic. Recovery of β-glucan synthesis and restoration of the normal pattern of growth occurred simultaneously. Growth of G. lactis in l-sorbose medium also led to inhibition of β-glucan synthesis and dichotomous branching.

The rate of release of Fe(II) from anoxic lake sediments was lower in the presence than in the absence of nitrate. The reduction of Fe(III) by the sediments had a temperature optimum of 30 C and was inhibited by HgCl2, suggesting that the process was largely biological in nature. Of the iron sources tested with cultures of anaerobic iron-reducing bacteria, FeCl3 was the most readily reduced and goethite the least. Reduction was faster in the presence of a chelating agent and was suppressed by the addition of NO- 3, ClO- 3, MnO2, Mn2O3 and O2. An iron-reducing chemoorganotroph, tentatively identified as a member of the genus Vibrio, was isolated. Physical contact between the bacterium and iron particles was essential to ensure maximum rates of Fe(III) reduction but > 30% of the activity appeared to be associated with extracellular components. Although Fe(III) reduction by whole cells and cell-free extracts was decreased in the presence of electron transport inhibitors, the molar growth yield of the organism was unaffected by the presence of Fe(III). It is assumed that the organism used the Fe(III) as a hydrogen sink. A second organism, an anaerobic facultative chemolithotroph, appeared to conserve energy by the reduction of Fe(III). Biomass yield (measured as ATP) was greater in the presence of Fe(III), and the organism was able to use H2 as a source of reducing power.

Enrichments were established to examine the potential of Blelham Tarn profundal sediment to metabolize benzoate to CH4 and CO2. Longadaptation times were required before benzoate-dependent CH4 production occurred, though both increased inoculum size and prior methanogenic adaption to aliphatic fatty acids reduced the adaptation time. Benzoate was metabolized according to the stoichiometry: 4C6H5COOH + 18H2O 15CH4 + 13CO2. The optimum temperature for CH4 production from benzoate in the enrichments was 37°C irrespective of the enrichment temperature. Methanogenic benzoate degradation was associated with a particulatefloc in the enrichments and Methanobacterium soehngenii was tentatively identified as an important constituent of this floc by scanning electron microscopy. Anaerobic benzoate fermentation was observed after 4 h in undiluted sediment by the use of [14C]benzoate, and the temperature optimum for 14C-labelled gas formation was 28°C. The 14CH4: 14CO2 ratio indicated that methanogenic fermentation of benzoate was occurring in situ. CO2 became the main gaseous product from [14C]benzoate when sulphate was added to sediment, and 20 mm-molybdate reversed this effect. Methanogenesis was slightly inhibited by addition of 20 mm-molybdate. Methanogenic benzoate fermentation in sediments was found to be inhibited by H2.

DNA colony hybridization was found to have a high generic and strain specificity for Rhizobium trifolii. We have assessed the use of this technique to determine occupancy of nodules by strains of Rhizobium and found that its high degree of specificity allowed successful identification of rhizobia from nodules. Other techniques currently available for determining the Rhizobium strain involved in forminsg a particular nodule lack specificity or interfere with symbiotic capabilities of the rhizobia.

Ecological factors that control the establishment of cellulolytic bacteria and ciliate protozoa in the lamb rumen were studied in meroxenic animals. Axenic lambs received dilutions of rumen liquor from either conventional lambs and sheep (pool A) or meroxenic lambs (pool B).

The total number of bacteria established in the rumen was between 109 and 5 × 1010 g−1. In lambs inoculated with dilutions (10−6, 10−7, 10−8) of pool A, cellulolytic bacteria did not become established. However, subsequent inoculation with Bacteroides succinogenes, resulted in colonization in lambs that had received 10−6 and 10−7 dilutions of pool A. However, B. succinogenes became established in only one of three lambs that received the 10−8 dilution. Similar results were obtained for the protozoan Entodinium sp.

With pool B, lambs were inoculated earlier and cellulolytic bacteria were established directly from the 10−6 and 10−7 inocula. Polyplastron multivesiculatum establishment occurred readily when inoculated into the lambs which had received the 10−6 dilution of pool B.

Results obtained in this study suggest that establishment of cellulolytic bacteria and protozoa requires an abundant and complex flora and is favoured by early animal inoculation.

Amongst forty wild strains of Escherichia coli, sixteen utilized galactitol (as did K12) and seven utilized ribitol (as did C) of which six utilized d-arabitol; none utilized all three polyols.

Transduction of genes for ribitol utilization (rtl +) to strains able to utilize galactitol (gat +), whether K12 or wild strains, using wild strains and E. coli C as donors always resulted in loss of the galactitol phenotype. The genes for d-arabitol use (atl +) were always cotransduced with rtl + in interstrain crosses. We confirm and extend the mapping of gat + ( Lengeler, 1977 ) and rtl + atl + ( Reiner, 1975 ) in their respective hosts, K12 and C, by showing both regions to be 50% cotransducible with metG and 3% cotransducible with fpk. In reciprocal transductions, gat + replaced rtl + atl +. In partial diploids, rtl + atl + and gat + regions did not interfere with each other’s expression.

Transfer of rtl + from an Rtl+ Atl− donor by R plasmid (pE10)-mediated conjugation, gave Gat− transconjugants of K12 in which rtl + and a kanamycin resistance gene were 100% cotransducible in the metG region of the chromosome.

It is suggested that the rtl + atl + and gat + genes (or parts of them) act as alternative, or mutually exclusive, regions in the chromosome. Possible reasons for the existence of alternative characters are discussed.

Chromosomal genes conferring resistance to trimethoprim were transferred from three independently isolated thy + clinical strains of Escherichia coli to Escherichia coli K12 by using P1 transduction. Trimethoprim-resistant transductants were obtained less frequently than transductants of other chromosomal markers, suggesting that there were problems related to the expression of the trimethoprim resistance genes in E. coli K12. Mapping studies revealed that one of the resistance determinants was located at a similar position on the chromosome (1 min) to the fol-type mutations previously described in E. coli K12. The two remaining resistance determinants mapped at separate positions between 2·5 and 3 min on the chromosome. The presence of one of these determinants reduced the efficiency with which either donor or recipient cells carrying it could participate in conjugation mediated by the sex factor F and also resulted in phenotypic interaction with the azi gene. The mechanisms of trimethoprim resistance in the three clinical E. coli isolates studied were more complex and diverse than was expected from previous studies of E. coli K12 mutants.

The isolation and properties of nitrofurantoin- and nitrofurazone-resistant mutants have been compared. Nitrofurantoin-resistant mutants were easier to obtain and showed a higher resistance level. There was some cross-resistance at lower drug concentrations but not at higher levels. There was no difference in the UV resistance of most of the mutants obtained although a recB strain, AB2470, did yield nitrofurantoin-resistant mutants with increased UV resistance. This was however the only repair-defective strain which could be mutated to nitrofurantoin resistance. It is suggested that there is a mechanism for nitrofurantoin resistance in Escherichia coli K12 which does not confer resistance to nitrofurazone and which may be associated with the repair of damaged DNA. This mechanism is in addition to that which also confers resistance to nitrofurazone.

Pseudomonas sp. MT14, isolated from soil as a result of its ability to utilise m-toluate as sole source of carbon, contains two large catabolic plasmids. One of these, pWW14, is a TOL plasmid of about 270 kb which determines the ability to grow on toluene, m-xylene and p-xylene as sole carbon sources. The other, pWW17, which is about 280 kb, determines both the ability to grow on phenylacetate and resistance to mercury salts. Growth of MT14 on benzoate results in the segregation of derivatives in which either (i) one or both plasmids are completely lost, or (ii) one or both plasmids have undergone a large deletion which results in a change in the phenotype determined by that plasmid. Deletion of about 100 kb from pWW14 results in a regulatory mutation which allows its host to grow on m-xylene but not on its metabolite m-toluate (B3 mutants). Deletion of about 95 kb from pWW17 results in loss of the ability of its host to grow on phenylacetate but the mercury resistance is not lost.

The isolation of a stable, chlorophyll-less Mendelian mutant (y-y) of Chlamydomonas reinhardtii is described and its growth, carotenoid spectra and ultrastructure are compared to chlorophyll-containing strains. Unlike most chlorophyll-less mutants, y-y did not die in the light but grew as bright yellow colonies. It was unable to grow in a mineral medium with CO2 and it required acetate for growth. When compared to green C. reinhardtii strains, y-y grew slower in the light but at about the same rate in the dark. Light-grown and dark-grown y-y cells had no detectable chlorophyll and similar amounts of total carotenoids, which were 20–35% of the values for the green strains. The only noticeable difference in ultrastructure between y-y and the green cells was in the inner chloroplast membranes. In the light and dark y-y lacked lamellae, although the outer chloroplast membrane was retained.

This paper describes an experimental system to study interactions between porcine enterotoxigenic Escherichia coli (ETEC) and porcine intestinal epithelial cells in vitro at the molecular level. Radiolabeled bacteria or bacterial membrane fractions were incubated with brush borders prepared from purified epithelial cells, which were then washed repeatedly. The bacterial components removed by washing or retained by the brush borders were analysed to determine their composition and source. For this it was necessary to develop a minimal medium in which attachment factors of porcine ETEC could be radiolabelled. Furthermore, an improved method for the isolation of porcine intestinal epithelial cells was developed, since other procedures did not yield sufficiently pure preparations. The resulting method was rapid and yielded large quantities of viable epithelial cells, free from crypt cells and contaminating intestinal contents. Finally, we adapted existing procedures to isolate brush borders from these epithelial cells with special emphasis on the removal of nuclear and cytosolic material and on the isolation of morphologically intact brush borders. Using this system, mixtures of bacterial cytoplasmic and outer membranes were incubated with brush borders. Cytoplasmic membranes were easily removed by washing, while the outer membranes were not.