- Volume 134, Issue 3, 1988

The origin of aflatoxin G1 was studied using mutant strains of Aspergillus parasiticus blocked early in the pathway and by tracing 14C-labelled aflatoxin B1 (AFB1) in wild-type A. flavus and A. parasiticus strains. Sterigmatocystin (ST) was a precursor of AFB1, AFG1 and AFG2 in the four mutants examined. The identity of AFG1 was confirmed by mass spectrometry. No evidence for conversion of AFB1 to AFG1 was found. A rigorously controlled study of conversions of radioactivity based on preparative thin-layer chromatography of aflatoxins demonstrated that low levels of aflatoxin interconversions previously reported in the literature might actually be artifacts.

Levels of the polyamines spermidine and putrescine and the major intracellular thiols glutathione (GSH), glutathionylspermidine (GSH-SPD) and dihydrotrypanothione [bis-(glutathionyl)spermidine); T[SH]2] were measured by high performance liquid chromatography throughout the growth cycle of the insect trypanosomatid Crithidia fasciculata. The amount of total spermidine, putrescine and glutathione (free and conjugated to spermidine) was found to be elevated during growth. Of the total spermidine, 30 to 50% was found conjugated to glutathione during the exponential growth phase, increasing to 60 to 70% at stationary phase. T[SH]2 was the principal intracellular thiol during exponential growth (12·1 to 17·4 nmol per 108 cells), whereas GSH-SPD was the major thiol in stationary phase (26.2 nmol per 108 cells). GSH levels changed little during the growth cycle and represented a constant proportion (10 to 12%) of the total intracellular glutathione. On dilution of stationary phase cells into fresh medium, a rapid decrease in GSH-SPD levels was observed to be associated with synthesis of T[SH]2. This process reached 90% completion by 15 min, with steady state achieved by 120 min. As the total spermidine and glutathione pools did not increase during this interval, it could be calculated that this rapid redistribution of metabolites resulted in the release of 13 nmol per 108 cells unconjugated spermidine without de novo synthesis. This mechanism for rapidly elevating the intracellular concentration of free spermidine may be advantageous to this organism in rapidly adapting to favourable growth conditions.

Toluene-treated cells of Selenomonas ruminantium HD4 used phosphoenolpyruvate (PEP) to phosphorylate glucose and sucrose. Glucose activity was constitutive, while the phosphorylation of sucrose was inducible. Competition experiments indicated that separate phosphotransferase (PTS) enzymes II were present for glucose and sucrose, but it appeared that maltose was hydrolysed by an inducible extracellular maltase and then transported by the glucose PTS. S. ruminantium HD4 grew more slowly on maltose than glucose or sucrose and the specific activity of maltase was rate limiting. The maltase was competitively inhibited by glucose and sucrose. Xylose was not phosphorylated by PEP or ATP, and its uptake was inhibited by the protonophore carbonyl cyanide m-chlorophenylhydrazone (CCCP), and by chlorhexidine diacetate. The absence of PEP-dependent phosphorylation and the effects of CCCP suggested that xylose was transported by an active transport mechanism.

The sheaths of two species of cyanobacteria, Calothrix parietina (two strains) and C. scopulorum (one strain), were studied. Their fine structure showed osmiophilic fibres running parallel to the cell surface. Enriched sheath fractions were obtained from both species in high yields in the phenol/water interphase after hot phenol/water extraction of cell homogenates. The purified sheaths of the two species had similar chemical compositions, with about 50% (of sheath dry weight) neutral sugars (galactose, glucose, mannose, xylose, arabinose, rhamnose, fucose, a 2-O-methylhexose and an unidentified 2-O-methyl sugar), 5% amino acids and small amounts of glucosamine and galacturonic acid. The sheath composition was independent of changes in the iron or phosphate content of the culture medium. The sheath was insoluble in water, and its chemical composition remained essentially unchanged on application of drastic extraction methods, including boiling in 2% (w/v) SDS. The sheath bound heavy metals (up to at least 0.7% of sheath dry weight) with the effectiveness (measured as absolute quantities) Fe > Zn > Cu > Ni > Mn > Mo > Co. Ni, Cu, Zn and Fe were highly enriched relative to their concentration in the culture medium. The concentration factors for Mo. Mn, Ca, Na and K were low.

We compared four decontamination methods for the isolation of mycobacteria from soil specimens. Different media were used: Lwenstein-Jensen, Ogawa and various modified Ogawa media. Statistical analysis demonstrated that the best results (low contamination and high positivity rates) were obtained when the specimens were incubated in trypticase soy broth, treated with solutions containing malachite green and cycloheximide, then decontaminated with sodium hydroxide and inoculated onto Ogawa media. The lowest contamination rates were obtained with Ogawa medium containing 500 g cycloheximide ml-1. The use of these techniques is proposed for the isolation of mycobacteria from heavily contaminated clinical specimens as well as from soil.

Plasmid DNA from an avian strain of Chlamydia psittaci was purified and estimated to be 7·9 kb in size using restriction endonuclease analysis. A 5·9 kb fragment of this plasmid was cloned, mapped and used to screen a range of chlamydial strains. Hybridizing DNA was absent from ovine abortion and arthritis isolates and also from the Cal 10 strain but related sequences were detected in C. psittaci strains of feline pneumonitis, guinea-pig inclusion conjunctivitis, ovine conjunctivitis and C. trachomatis serovar L2. The plasmid DNA from the feline strain was shown to have a distinct restriction endonuclease profile. Similar plasmid sequences were detected in all avian isolates tested: thus the clone may have a useful diagnostic role for the detection of the pathogen in its natural host and in zoonotic episodes.

Determinants of tetracycline resistance have been cloned from two different tetracycline-producing industrial strains of Streptomyces into Streptomyces lividans using the plasmid vector pUT206. Three plasmids, pUT250 and pUT260 with a 9·5 and a 7·5 kb insert respectively of Streptomyces rimosus DNA, and pUT270 with a 14·0 kb insert of Streptomyces aureofaciens DNA, conferring resistance to tetracycline, have been isolated. By in vitro sub-cloning, a similar fragment of 2·45 kb containing the tetracycline resistance gene (tet347) was further localized on these plasmids. The S. rimosus gene has been cloned into Escherichia coli and expressed under the control of λpL or Lpp promoters. Differential protein extraction of E. coli cells revealed the presence of an additional membrane-embedded protein in tetracycline-resistant cells. On the basis of available restriction endonuclease maps, the tet347 gene is probably identical to the tetB gene from S. rimosus recently identified by T. Ohnuki and co-workers as responsible for the reduced accumulation of tetracycline. The nucleotide sequence of a 2052 bp DNA fragment containing the TcR structural gene from S. rimosus has been determined. The amino acid sequence of the tet347 protein (M r 35818) deduced from the nucleotide sequence shows a limited but significant homology to other characterized tetracycline transport acting determinants from pathogenic bacteria.

The subcellular location of LamB-LacZ hybrid proteins in the Escherichia coli K12 strains pop3234 and pop3299 was investigated by immunocytochemical detection and protease-accessibility experiments. Induction of the synthesis of the hybrid proteins resulted in the appearance of membrane-like structures within the cytoplasm of the cells. Labelling of ultrathin cryosections of the cells with anti-β-galactosidase or anti-LamB protein serum and protein-A-gold complexes revealed that the hybrid proteins were associated with these membrane-like structures or accumulated within the cytoplasm. Protease-accessibility experiments confirmed this localization. Moreover, when low quantities of hybrid proteins were produced, i.e. in uninduced pop3234 cells or in induced pop3299 cells, the hybrid proteins were accessible to trypsin from the periplasmic side of the inner membrane, leaving protected fragments with an apparent M r of 83000. Apparently, these hybrid proteins are partly translocated through the inner membrane, resulting in membrane-spanning forms of the proteins.

The construction of a shuttle vector for inducible gene expression allowing fast and easy cloning in Escherichia coli and subsequent transformation of Bacillus subtilis is presented. The expression is based on the regulation of the tac promoter by the Lac repressor which was assayed with the xylE gene from Pseudomonas putida as a marker gene. The laclq gene, transcribed by the strong spo promoter, allowed full repression of the weak tac promoter.

The transciption of genes that function in N2 fixation (nif) and nitrogen assimilation (glnII) in Bradyrhizobium japonicum is coordinately induced in response to O2 limitation as well as symbiotic development. We have determined the relative steady-state mRNA levels for the nifH, nifDK and glnII transcription units in bradyrhizobial cells grown under a variety of levels of aerobiosis and in cells isolated from soybean root nodules. All three transcripts are found in cells grown in a rich medium sparged with O2 concentrations of 5 % (v/v) or less. This expression is qualitatively similar to that observed for Bradyrhizobium during symbiotic development. Potential physiological mechanisms for the coordinate control of these genes are discussed.

True protoplasts of the red alga Porphyridium cruentum and the halophilic green algae Dunaliella bardawil and Dunaliella salina were induced by treatment with pectinase and cellulysin. Protoplasts of P. cruentum and the Dunaliella spp. were fused by polyethylene glycol treatment and then allowed to regenerate on selection media. Fusants that were essentially Porphyridium in nature were generated between P. cruentum and D. bardawil and D. salina; these tolerated a salinity of up to 3·5 m for the former and 2·5 m for the latter, indicating that they were hybrid strains, acquiring the osmotolerance from the Dunaliella spp. A Dunaliella-like clone was isolated from protoplast fusion between P. cruentum and D. salina, with altered antibiotic sensitivity. The acquired resistance to penicillin and erythromycin appears to be the result of a genetic transfer from P. cruentum.

A new class of glycerol non-utilizing mutants, designated glcC, has been isolated. The glcC gene was mapped in linkage group VI and mutants were found to complement the reference strains glcA1 (linkage group V) and glcB33 (linkage group I) in diploids. The new mutants were unable to grow on glycerol. However, in contrast to the glcA and glcB phenotype these mutants did grow well on dihydroxyacetone and d-galacturonate. By in vivo 13C NMR spectroscopy it was shown that the glcC mutant did not take up glycerol but did take up dihydroxyacetone. The latter substrate was converted intracellularly into glycerol which was then catabolized as normal.

The complete nucleotide sequence of a naturally occurring Staphylococcus aureus plasmid, pT48 (from S. aureus strain T48), has been determined. The 2475 bp plasmid confers inducible resistance to macrolide-lincosamide-streptogramin B (MLS) type antibiotics. It is similar to the constitutive MLS resistance plasmid, pNE131, from Staphylococcus epidermidis and shows homology with S. aureus plasmids pSN2 and pE194. It contains a palA structure homologous to that on S. aureus plasmid pT181. The open reading frame, ORF B, within the pSN2 homologous region has a frameshifted C-terminus, relative to pNE131, resulting in a smaller, 158 amino acid putative polypeptide. The pE194 homologous region has the ermC resistance determinant and retains the leader region, deleted in pNE131, required for inducible expression of an adenine methylase.

Another naturally occurring S. aureus strain, J74, shows constitutive resistance to erythromycin and contains a small plasmid, pJ74, which is similar to pNE131 but with a different deletion in the leader sequence. The results are consistent with the translational attenuation model for ermC expression.

We report the nucleotide sequence of a 1·45 kb segment containing the eta gene, coding for staphylococcal exfoliative toxin A (ETA), isolated from the recombinant plasmid pETA-J3. The coding region of 840 bp specified a polypeptide of 280 amino acid residues which included a putative 38 residue signal sequence. The amino acid composition deduced from the structural gene was in agreement with the results of peptide analysis of the ETA molecule reported by others. The sequence of the 35 N-terminal amino acid residues of ETA derived from Staphylococcus aureus strain ZM was also consistent with that deduced from the DNA sequencing.

Operon fusions of the lacZ gene to two different genes of the cysteine regulon controlled by the cysB regulatory protein were isolated. The fusion strains were used for selection of cysB constitutive mutants. Three cysBc alleles have been characterized and cloned into multicopy plasmids.