- Volume 130, Issue 8, 1984

By SDS-PAGE or gel filtration on Sephadex G-25, lipopolysaccharide (LPS) isolated from Vibrio cholerae 569B (Inaba) can be separated into two distinct fractions, one corresponding to smooth LPS and the other to rough LPS. Pulse-labelling of LPS with [14C]glucose showed that the rough form is synthesized first followed by the biosynthesis of the smooth form. A preferential release of the smooth LPS of V. cholerae 569B was also detected during normal growth of cells.

Twenty-six fungal cultures, including ten Caldariomyces (= Leptoxyphium) cultures and five other capnodiaceous fungi, were examined for extracellular chloroperoxidase production on four growth media. Only the Caldariomyces cultures produced enzyme activity and enzyme production was highest on growth in a phytone medium. Chloroperoxidase was partly purified from the ten Caldariomyces cultures. All preparations were glycoproteins, with different carbohydrate content. The absorption spectra of the ten samples were indistinguishable and this was reflected in similar Rz (A 403/A 280) values. SDS-PAGE revealed two major bands of protein in all preparations but in different proportions: staining for carbohydrate confirmed these bands to be glycoproteins. PAGE at pH 3 showed each preparation to be composed of several isoenzymes and these could be grouped according to their migration patterns. Kinetic constants, where determined, and pH optima showed no differences among the ten preparations and all exhibited catalase and peroxidase activities in the same relative proportions to chlorinating activity.

The Calvin cycle enzyme phosphoribulokinase has been localized in terms of catalytic activity and enzyme protein in the cyanobacterium Chlorogloeopsis fritschii. In contrast to the CO2-fixing enzyme of the Calvin cycle, d-ribulose-1,5-bisphosphate carboxylase/oxygenase (RuBisCO), which occurs in the cytoplasm and in the carboxysomes, phosphoribulokinase is essentially (95%) cytoplasmic in extracts from cells grown photoautotrophically to late exponential phase. Total phosphoribulokinase accounted for 0·6% of total cell protein in these cells. Immunochemical identity was found between the phosphoribulokinase located in the cytoplasm and the remaining enzyme (5%) associated with the particulate fraction of the cell obtained following differential centrifugation. On density gradient centrifugation of the particulate fraction into Percoll plus sucrose the RuBisCO activity was located in a carboxysome band in the lower half of the gradient while the phosphoribulokinase activity essentially remained concentrated at the top of the gradient. Comparison of RuBisCO and phosphoribulokinase distributions suggests that the latter is not a carboxysomal enzyme in late exponential phase cells of C. fritschii.

Patterns of morphogenesis in the branching, filamentous cyanobacterium Mastigocladus laminosus were examined by light and electron microscopy. Morphologically different types of filaments possessed, and were restricted to, distinct modes of cell division. Division in narrow and lateral branch filaments was always perpendicular to the long axis of the filament, indicating that these filaments were involved principally with trichome elongation. Division in wide filaments was either diagonal or (more often) parallel to the long axis of the filament, the latter enabling production of lateral branches. Cells of filaments in transition between the narrow and wide forms changed in shape and size, but divided only rarely. Wide cells that produced lateral branches were ultrastructurally identical to the other cells in wide filaments and were not surrounded by sheath material; branches did not arise by germination of akinetes. The morphological and ultrastructural characteristics of successive branch-filament cells (starting proximal to the parental filament) ranged from those typical of wide-filament cells to those typical of narrow-filament cells. Wide cells differed ultrastructurally from narrow cells in containing fewer ribosomes, less nuclear material, and more reserve materials. Wide cells lacked characteristic ultrastructural features of akinetes or other resting cells, appearing instead to be active vegetative forms. Lateral branches were released from parental filaments by deterioration of specific wide-filament cells.

A DNA plasmid, designated pRS64, was detected in three isolates of anastomosis group 4 (AG-4) of Rhizoctonia solani Kühn by biophysical methods. The plasmid was a linear double-stranded DNA with a molecular weight of 1·68 ± 0·06 × 106 or 2617 ± 87 bp. Weakly pathogenic isolates of R. solani, 1668 RI-1, 1271 RI-64 and 1272 RI-1, which showed abnormally slow growth, contained the plasmids, but pathogenic isolates, 1668, 1271 and 1272, showing normal growth, contained no detectable plasmid DNA.

Two recessive mutations of the cellular slime mould Dictyostelium discoideum, phgA1356 and phgB1351, which express altered phagocytic properties, were characterized using parasexual genetic analysis. The phgA locus was assigned to linkage group VII, and the phgB locus to linkage group IV. Complementation analysis of a further twelve independently derived phagocytosis mutants showed that each fell into one of these two complementation groups.

A mutant of Dictyostelium discoideum strain AX2 with altered responses to cyclic AMP has been analysed genetically. The mutant, HG302, aggregates and fruits in the presence of adenosine 3′:5′-cyclic phosphorothioate (cAMPS). This slowly hydrolysed analogue of cyclic AMP inhibits wild-type development by blocking cell differentiation from the growth phase to the aggregation-competent stage. The mutant forms small aggregates in the absence and in the presence of cAMPS, and is further distinguished from the wild type by decreased activities of extracellular phosphodiesterase and phosphodiesterase inhibitor. These phenotypic characteristics were all linked to the cAMPS resistance, which was assigned to linkage group II. Mitotic crossover experiments showed that the cAMPS-resistance mutation, casA1000, maps proximal to the acrA locus in the neighbourhood of whiA. The data suggest that casA1000 acts pleiotropically, and that the product of the wild-type allele is required in a part of the signal-processing pathway common to control of development and regulation of extracellular phosphodiesterase.

Twenty-nine mutants blocked during stage V of sporulation have been isolated following directed mutagenesis of the lys-1 region of the Bacillus subtilis 168 chromosome. All of a sample of eight mutants tested are unaffected in sporulation marker events up to stage IV but did not produce dipicolinic acid. They produced stable ‘phase white’ spores that were released from the mother cell, and were partially resistant to toluene and lysozyme but sensitive to chloroform and heat. Mutation spoVA89, known to be in the lys-1 region, showed similar phenotypic characteristics. Three-factor transformation crosses and recombination indices showed that the new mutations and spoVA89 lie in a single linkage group, which maps between lys-1 and another sporulation locus, spoIIA. The size of the spoV A locus is such that it probably contains several genes, and these may be contiguous with the cluster of genes included within the spoIIA locus.

Plasmids carrying different portions of the polycistronic spoIIA locus, and unable to replicate autonomously in Bacillus subtilis, were able to transform a Spo+ B. subtilis strain, BR 151, for the plasmid-determined chloramphenicol resistance by Campbell-like insertion into the region of homology on the chromosome. Two such plasmids, pPP35 and pPP36, yielded Spo-transformants, indicating that the cloned regions of these plasmids were entirely within the chromosomal spoIIA transcriptional unit. The cloned regions overlapped the end of a known spoIIA cistron, so that the transcriptional unit was larger than this cistron, and was polycistronic. This is the first demonstration of such a polycistronic sporulation transcriptional unit. The DNA sequence of the region has now been determined (given in an accompanying paper) and suggests a transcriptional unit with three open reading frames. Two other plasmids yielded Spo+ transformants of BR 151, and these define the outer limits of the transcriptional unit. The adjacent sporulation locus identified by the spoVA89 mutation was not part of the same transcriptional unit.

A 6·95 kb HindIII-generated DNA fragment from Bacillus subtilis 168 was inserted into the DNA of phage ϕ105DI:lt. The recombinant phage (ϕ105DS1) contained DNA of 33·8 kb as compared with 35·2 kb for ϕ105DI:lt and 39·2 kb for the wild-type phage. In the presence of helper phage, ϕ105DS1 complemented both spoIIA and spoVA mutations in B. subtilis.

We have determined a sequence of 2073 bp from two recombinant plasmids carrying the whole spoIIA locus from Bacillus subtilis, the expression of which is required for spore formation. The sequence contains three long open reading frames (ORFs), each of them being preceded by a ribosome binding site. These three putative proteins (mol. wts 13100, 16300 and 22200) are likely to be expressed and are probably encoded on the same mRNA. The stop codon of ORF1 overlaps with the start codon of ORF2 suggesting that there might be translational coupling between the two ORFs. Although some known promoter sequences were found, the only one upstream from the first open reading frame is about 260 bp from it.

A 1·6 kb fragment of DNA of plasmid pBD64, obtained after partial digestion with HpaII, carrying a chloramphenicol-resistance determinant and a single site for the enzyme Bg/II, was inserted into the genome of defective phage ø105 d/ys. Two types of phage were subsequently isolated and both transduced cells of Bacillus subtilis to chloramphenicol resistance. One type contained 26 kb and the other 32 kb of DNA. Bacillus subtilis chromosomal DNA fragments generated by cleavage with Bg/II were ligated into the unique Bg/II site within the smaller phage genome. A specialized transducing phage was isolated which carried the metC gene on a 6 kb Bg/II fragment. This phage, denoted ø105 d(Cmr met), transduced B. subtilis strain MB79 pheA12 metC3 to Met+ and to chloramphenicol resistance, and the metC3 mutation was complemented in transductants.

Candida albicans requires Mg2+ for germ-tube formation. Mg-deficient media, metal ion chelators and the ionophore A23187 inhibited germ-tube formation. Cell Mg content during exponential yeast-phase growth remained constant but increased throughout germ-tube formation. The onset of germ-tube formation coincided with a sharp peak in Mg concentration within the cells. Yeast-phase cells of strain CA2, which did not form germ-tubes, had a lower Mg content and failed to accumulate Mg when incubated under conditions for germ-tube formation. Mg also increased the uptake and incorporation of N-acetylglucosamine. These findings point to a central regulatory role for Mg in C. albicans morphogenesis.

Nitrogen assimilation during growth of Candida boidinii on methylated amines as sole nitrogen source involves NADP-dependent glutamate dehydrogenase. Changes in enzyme activities during the adaptation of the yeast from growth on ammonium to growth on trimethylamine were examined. No ammonia, dimethylamine or monomethylamine could be detected in the medium during growth on trimethylamine. When two methylated amines were supplied together, they were used simultaneously, although monomethylamine was metabolized more quickly than the others. When cells were grown on a low concentration of ammonium plus higher concentrations of di- or trimethylamine, the ammonium was used first. NADP-dependent glutamate dehydrogenase was the first enzyme to be derepressed, followed by methylamine oxidase and formaldehyde dehydrogenase. Di- and trimethylamine mono-oxygenase activities only appeared when the ammonium concentration fell below 0.5 mM. At this point amine utilization could be detected and no diauxic lag was observed in the growth curve. During growth on limiting ammonium, there was an increase in the activity of methylamine oxidase (150-fold) and catalase (5-fold) in the absence of any amine, but no amine mono-oxygenase activity was detected. Addition of ammonium ions to cultures growing on dimethylamine produced an immediate repression of synthesis of methylamine oxidase, NADP-dependent glutamate dehydrogenase and the two amine mono-oxygenases. An inverse correlation was found between intracellular ammonium concentration and methylamine oxidase activity. Ammonium ions also inhibited the uptake of dimethylamine or trimethylamine by washed suspensions of dimethylamine-grown cells. It is concluded that the control of methylamine oxidase and catalase and (independently) of NADP-dependent glutamate dehydrogenase is by repression of enzyme synthesis by ammonium, while expression of amine mono-oxygenases seems to require the amine to be present in the medium. Formaldehyde and formate dehydrogenases seem also to be induced by their respective substrates.

As the sole source of nitrogen, methylamine supported the growth of a range of species of Rhizobium. The methylamine assimilatory system was inducible in R. leguminosarum MNF3841, whereas the capacity to utilize NH4 + as a nitrogen source was constitutive. An uptake system for [14C]methylamine (methylamine permease) was induced by growth of MNF3841 on methylamine or ethylamine. The uptake was sensitive to 2,4-dinitrophenol, azide and carbonyl cyanide m-chlorophenylhydrazone. The methylamine permease had a K m of 0·035 mm, a V max of 2·2 nmol min−1 (mg protein)−1 and a K i for ammonium of 1·5 mm. Most of the [14C]methylamine accumulated by cells was rapidly incorporated into TCA-insoluble materials. An NH4 +-sensitive methylamine-accumulating system distinct from the methylamine permease was demonstrated in ammonia-limited cells grown in continuous culture. This system, the ammonium permease, had a K m of 0·11 mm (for methylamine), a K i for NH4 + of 0·007 mm and a V max, of 2·5 nmol min−1 (mg protein)−1. Methylamine was accumulated by chemostat-grown, N-limited cells and could exchange with unlabelled methylamine. Treatment with carbonyl cyanide m-chlorophenylhydrazone caused efflux of the accumulated methylamine, whereas high concentrations of NH4 + did not. Thus R. leguminosarum possesses a specific methylamine permease which is quite distinct from the ammonium permease.

The marked copper tolerance of Penicillium ochro-chloron has been confirmed as has its ability to grow in solutions of high salinity. The major low molecular weight organic solutes present in P. ochro-chloron were glycerol, erythritol, arabitol, mannitol, sorbitol and trehalose. Of these, glycerol increased significantly during growth in high concentrations of Na+ or Cu2+, a 15-fold increase, relative to the control, occurring in concentrations of 0.5 M. Cell K+ increased with elevated external Na+ but decreased slightly with elevated external Cu2+. With high external Cu2+ mycelium maintained a constant level of Cu2+, with low levels of Na+ in all treatments. It is concluded that the high concentrations of glycerol induced by high external Na+ or Cu2+ had a significant osmotic effect, allowing growth in media of high osmolality. The exclusion of ions may have a gratuitous role in the copper tolerance of P. ochro-chloron.