- Volume 130, Issue 2, 1984

Colletotrichum lindemuthianum race ? secreted two forms of pectin lyase, having pI values of 8·2 and 9·7, when grown in culture with sodium polypectate or isolated Phaseolus vulgaris hypocotyl cell walls as the main source of carbon. A single form of polygalacturonase was also present in both media. A pectin lyase, with a pI value of 9·7 was detected in necrotic P. vulgaris tissue infected with race ?, but polygalacturonase activity was absent.

Chitin synthase has been characterized from the stipes of Coprinus cinereus by a number of techniques. In the absence of digitonin, on gel filtration columns and during electrophoresis the enzyme showed properties consistent with having molecular weights from 1.5 105 to several million, suggesting its reversible aggregation into large multimolecular units. Gel chromatography in buffers containing digitonin gave highly reproducible results, and when followed by anion-exchange chromatography gave preparations with very high activity [e.g. 3.4 mol substrate incorporated min-1 (mg protein)-1] and apparent molecular weight 8.0 105. The best purification (140-fold) was achieved by gel chromatography followed by copper chelate affinity chromatography, giving a nearly pure enzyme preparation of activity 4.7 mol substrate incorporated min-1 (mg protein)-1, which showed only one band of molecular weight 6.7 104 on SDS-polyacrylamide electrophoresis. These purified preparations were free of the nucleoside diphosphatase and protease activities that were present in the early stages of purification.

The activities of the enzymes ethanolamine ammonia-lyase, CoA-dependent and CoA-independent aldehyde dehydrogenases, and isocitrate lyase were assayed in Escherichia coli which had been grown on various sources of carbon and nitrogen. Induction of ethanolamine ammonia-lyase and of maximal levels of both aldehyde dehydrogenases required the concerted effects of ethanolamine and vitamin (or coenzyme) B12. Molecular exclusion chromatography revealed that, in the absence of one or both co-inducers, two repressible isoenzymes of CoA-dependent aldehyde dehydrogenase (mol. wts 900000 and 120000) were produced, these being replaced by two inducible isoenzymes (mol. wts 520000 and 370000) in the presence of both co-inducers. A similar inducible repressible series of isoenzymes was also observed for CoA-independent aldehyde dehydrogenase. No evidence was found for structural relationships between ethanolamine ammonia-lyase, CoA-dependent aldehyde dehydrogenase and CoA-independent aldehyde dehydrogenase, but mutant and physiological studies demonstrated that the induction of the first two enzymes is under common control. Evidence is presented for the operation of a previously unreported pathway of ethanolamine metabolism in E. coli.

Preliminary studies demonstrated that Clostridium sporogenes synthesized isoleucine by a pathway not involving threonine or threonine dehydratase. Radiotracer experiments with cells grown in a defined carbohydrate-free medium showed that radioactivity from [U-14C]serine, [3-14C]pyruvate, [14C]NaHCO3 and [1-], [2-] and [3-14C]propionate was incorporated into isoleucine. Conversely, there was no detectable incorporation of 14C into isoleucine during growth with [U-14C]glutamate, [U-14C]threonine, [U-14C]valine, [U-14C]leucne or [U-14C]methionine. Crude extracts of the bacteria grown in a minimal medium contained levels of a-acetohydroxyacid synthase activities comparable to those in Escherichia coli K12 grown in minimal medium. Stepwise degradation of isoleucine obtained from C. sporogenes grown in the presence of specifically-labelled precursors indicated that C. sporogenes can make isoleucine via the reductive carboxylation of propionate to yield a-oxobutyrate, which is metabolized to isoleucine in the classical fashion. Isoleucine was also formed by C. sporogenes via the reductive carboxylation of a-methylbutyrate to α-oxo-β-methylvalerate.

A mixed culture of Aspergillus nidulans (GH79) and Aspergillus flavus (CMI 91019B) produced two antibiotics, designated VI and VII, which were not elaborated when either fungus was grown alone. Chemical and spectroscopic analysis of VI, the major component, indicated that this compound was identical to hydroxyaspergillic acid. The minor component. VII, was produced in too low a yield for its identity to be established. However, partial characterization suggests that this antibiotic also belongs to the aspergillic acid group of mycotoxins.

All the enzymes of the tricarboxylic acid cycle have now been demonstrated in extracts of Mycobacterium leprae grown in armadillo liver. Many were also present in homogenates of host-tissue, but biochemical evidence is presented which indicates that all enzymes detected in extracts from M. leprae were authentic bacterial enzymes. Further evidence for a complete tricarboxylic acid cycle in M. leprae was obtained by first establishing that citrate could be taken up and catabolized by whole M. leprae organisms, then showing that oxidation of radioisotopically labelled pyruvate to CO2 by suspensions of M. leprae was stimulated by adding unlabelled citrate. Control of tricarboxylic acid cycle activity in M. leprae by the inactivation of fumarase by a protease is speculated upon.

Lipopolysaccharide and protein material were released from cells of Desulfovibrio vulgaris on treatment with 10 mm-EDTA. Preincubation with up to 15 mm-ferrous ions caused increased removal of lipopolysaccharide from bacteria grown in iron-limited medium, but preincubation with calcium, magnesium, or zinc ions was unable to induce this response. Ferrous ions appear to have an important role in the stabilization in vivo of the outer membrane of D. vulgaris. The selective interaction between ferrous ions and D. vulgaris lipopolysaccharide may play a part in iron uptake.

The site selection for bud and germ tube emergence has been studied in the dimorphic yeast Candida albicans. The fluorescent dye Calcofluor was used to stain bud scars. Two different modes of site selection were observed in yeasts. On cells cultivated at 23–28 °C, buds emerged primarily at one end of the cell at adjacent sites suggesting a type of persistent memory. Sites selected on yeasts growing at 37 °C were non-adjacent and scattered over the cell. The pattern of bud emergence during resumption of budding from stationary phase, or after temperature shift between conditions, was not a simple transition between modes. During these transitions the budding pattern sequentially passed through non-adjacent and adjacent budding modes before establishing the pattern characteristic of the growth temperature. Germ tubes emerged in a non-adjacent pattern from cells grown to stationary phase at 28 °C. The higher frequency of non-adjacent emergence and the absence of sequential pattern changing in site selection for germ tubes suggested that non-adjacent site selection for germ tubes and buds might not be the same.

Amensal indirect interactions between a Klebsiella pneumoniae microcin-producing strain and several Escherichia coli strains, all of intestinal origin, were studied. Mixed batch cultures of both microcin-producing and microcin-sensitive strains showed that microcin production and excretion into the medium allowed the producer strain to prevail over sensitive strains, even when initial competition conditions were highly unfavourable for the producer. Mixed cultures also showed the production of a microcin-antagonist by the same microcin-producing strain when the nutrients in the medium had been depleted. The antagonist apparently promoted the viability of sensitive cells already damaged by microcin. These results have likely ecological implications.

The plasmid content of 14 colicinogenic strains of Escherichia coli has been examined. Four strains contained miniplasmids (1.2-2.0 kb). Small plasmids (4-7 kb) were detected in all the strains specifying group A colicins (colicins A, E1, E2, E3 and K; group I plasmids). Larger plasmids (55-130 kb) were detected in seven out of nine strains specifying group B colicins (colicins B, H, Ia, Ib, M, V and S1; group II plasmids). DNA-DNA hybridization with group II plasmids showed a wide variation in the degree of DNA sequence homology among its members. In contrast little (if any) DNA sequence homology was detected with the group I plasmids when the same group II plasmid DNAs were used as hybridization probes. The results of DNA-DNA hybridization studies with the two small group II plasmids (pcolG-CA46 and pcolD-CA23) suggested that these plasmids are equivalent to deleted forms of larger group II plasmids. Our hybridization data thus support the division of colicin plasmids into the two groups (I and II) that have been previously defined from genetic and physiological studies.

A recombinant cosmid clone was isolated from a library created from cosmid pQB79-1 and Bacillus subtilis DNA, and a 15 kb BamHI fragment derived from the cloned insert was transferred to the vector pHV33. The recombinant clone, pRC12, was capable of complementing eight auxotrophic markers in the spoIIA-tyrA region of the B. subtilis chromosome (map positions 205-210). It also complemented eight of nine markers in the spoIIA locus. The exception, spoIIA176, is the most distal marker from lysine. Although pRC12 failed to complement sporulation defects in spoVA or spoIVA (spoIIA +) strains, subclones of pRC12, lacking a functional spoIIA gene, did complement these mutations. pRC12 inhibited sporulation in a spo + recE strain, possibly due to the presence of multiple functional spoIIA genes. Both the original cosmid and pRC12 were unstable in Escherichia coli and B. subtilis. Antibiotic selection of the vector resulted in extensive deletion of the insert, while selection for insert function in B. subtilis invariably led to loss of the chloramphenicol resistance vector function.

We analysed the physical structure of the DNA of øFSW, which is a temperate phage of Lactobacillus casei S-1. A circular restriction map of the virion DNA has been constructed with three restriction endonucleases, BamHI, SalI and XhoI. Other data indicated that the phage genome was circularly permuted. In lysogens, the DNA of the prophage was found to be linearized at a specific site and integrated into a specific locus of the host genome, with the same orientation in each case, as evidenced by Southern filter hybridization. We compared the physical structure of øFSW with its three virulent mutants. One of them had a restriction map indistinguishable from that of øFSW and two of them contained host-derived DNA sequence(s) in a specific region of the øFSW genome (V-region). The prophage integration site was mapped on a different segment of the phage genome to the V-region. Derivation of virulent mutants from øFSW is discussed in relation to the physical structure of the phage genome.

Rhizobium leguminosarum accumulates fructose by an active process sensitive to azide, 2,4-dinitrophenol and carbonyl cyanide m-chlorophenylhydrazone. The fructose is not phosphorylated during transport. Sorbose and glucose interfere with fructose uptake. Inside the cell fructose is metabolized via fructose 6-phosphate; there is no evidence for an alternative metabolic route via sorbitol to glucose or via sorbitol 6-phosphate to fructose 6-phosphate. Tn5-induced mutants lacking fructokinase failed to grow on fructose, mannitol or sorbitol and grew slowly on sucrose; growth was normal on all other single carbon sources tested. Growth of these mutants on a range of carbon sources was retarded by added fructose. Revertants which had regained the capacity to utilize fructose all had an unstable fructokinase which could be partially stabilized by fructose.

Rhizobium leguminosarum metabolizes sugars via the Entner-Doudoroff and pentose phosphate pathways but does not have a functional Embden-Meyerhof pathway. Although some sugar catabolizing enzymes are constitutive, activities of the ‘Entner-Doudoroff’ enzymes vary with the carbon source. Bacteroids have complete pathways for sugar catabolism even though the specific activities of some enzymes, e.g., glucokinase, are lower than in free-living cells. Tn5-induced mutants lacking glucokinase, fructokinase and pyruvate dehydrogenase have been isolated. Although these mutants are unable to utilize sugars, they all nodulate peas and fix N2. The capacity to utilize particular C6 and C12 sugars is apparently not essential for bacteroid development or the establishment of effective N2 fixation.

The surface charge against pH behaviour of spores of Clostridium perfringens type A was determined using a colloid titration method in the pH range 4–9. The native spores as well as various ionic forms of the spores loaded with divalent cations were found to be negatively charged. A positive colloid, MGCh, inhibited the ionic germination of the spores and this inhibition was highly dependent on pH. However, another positive colloid, GCh, did not inhibit the germination. Chemical modification of carboxyl groups on the spore surface by use of a water-soluble carbodiimide with two nucleophiles, glycine ethyl ester and taurine, caused spores not only to have little or no colloidal charge but also to lose the ability to germinate. The modification of carboxyl groups in the spore coat protein also resulted in elimination of, or marked reduction in its negative charge. These results suggest that carboxyl groups in the spore coat protein are the major negatively charged species on the spore surface. The role of surface charge in the ionic germination of the spores is discussed.