- Volume 139, Issue 6, 1993

Five yeast strains were isolated by enrichment culture on the basis of their ability to grow on mandelate and two of these strains were identified as Rhodotorula glutinis. In addition, a range of yeasts from culture collections was screened for growth on mandelate. The results suggest that mandelate utilization is a widespread but not universal characteristic within the genus Rhodotorula. Several of the yeasts contained an inducible NAD-dependent d(−)-mandelate dehydrogenase and an inducible dye-linked (presumably flavoprotein) l(+)-mandelate dehydrogenase. All the d(−)-mandelate dehydrogenases from the yeasts showed immunological cross-reactivity with each other (as judged by both immunoinhibition and immunoblotting), as did all the yeast l(+)-mandelate dehydrogenases that were tested. Determination of N-terminal amino acid sequences of several bacterial and yeast lactate and mandelate dehydrogenases, together with the evidence from the immunological studies, confirmed and extended previous proposals that there are several major groups of such dehydrogenases: FMN-dependent, membrane-bound l(+)-lactate and l(+)-mandelate dehydrogenases (M r = approx. 44000) in bacteria, mitochondrial flavocytochrome b 2 l(+)-lactate and l(+)-mandelate dehydrogenases (M r = approx. 59000) in yeasts, FAD-dependent, membrane-bound d(−)-lactate and d(−)-mandelate dehydrogenases in bacteria, and soluble NAD-dependent d(−)-mandelate dehydrogenases in both bacteria and yeasts.

A methyltransferase that acts on carminomycin and 13-dihydrocarminomycin, and that is postulated to be the terminal enzyme in the daunomycin biosynthesis pathway, was purified to near-homogeneity from the daunomycin-and baumycin-producing Streptomyces sp. strain C5. The enzyme was obtained in approximately 5% yield with a purification of 114-fold in specific activity over the sample precipitated with 30–50% ammonium sulphate. Polyacrylamide gel electrophoresis under denaturing conditions indicated a subunit M r of about 41000. The enzyme was shown by gel filtration chromatography to have an M r of approximately 166000, suggesting that it is a homotetramer. Kinetic analysis indicated an affinity for S-adenosyl-l-methionine typical of antibiotic methyltransferases; the enzyme had a slightly higher affinity for carminomycin than for 13-dihydrocarminomycin. The reaction product from methylation of carminomycin was confirmed by chromatography and mass spectral analysis to be daunomycin. The purified enzyme did not catalyse methylation of the aglycones carminomycinone or 13-dihydrocarminomycinone. S-Adenosyl-l-homocysteine inhibited the methyltransferase, whereas homocysteine, adenosine, adenine, ε-rhodomycinone, daunomycin, and daunomycinone showed little or no inhibitory activity.

Phosphofructokinase II (PFK II) from Trichoderma reesei was partially purified (247-fold). The calculated K m values for fructose 6-phosphate and ATP were 0·7 mm and 40 μm, respectively. Upon incubation in the presence of [γ-32P]ATP, the enzyme formed a radioactive phosphoprotein with molecular mass of 67 kDa in autoradiography analysis after SDS-PAGE. Upon incubation in the presence of ATP-Mg and the catalytic subunit of cAMP-dependent protein kinase, its activity was not modified. The same result was obtained when a cell-free extract of T. veesei was incubated with ATP-Mg and cAMP. 2,4-Dinitrophenol caused a transient rise in cAMP levels in the fungal cell. These results provide evidence that the fructose 2,6-bisphosphate level in T. reesei is independent of cAMP concentrations and not related to a cAMP-dependent mechanism, but to the availability of substrate fructose 6-phosphate.

A proteinase has been purified from the stipes of senescent sporophores of the mushroom Agaricus bisporus. The proteinase was inhibited by PMSF. It has a broad pH optimum, 6·5–11·5, and a narrow substrate specificity, requiring both a hydrophobic amino acid in the P1 position and a minimum peptide chain length. The apparent molecular mass of the proteinase was 27 kDa when determined by SDS-PAGE and 14·1 kDa when measured by gel filtration. The isoelectric point of the proteinase was 9·0. Polyclonal antibodies have been raised to the proteinase. The proteinase from A. bisporus has similar properties to, and 60% N-terminal sequence identity with, proteinase K from the fungus Tritirachium album.

We investigated the usefulness of rRNA gene restriction fragment length polymorphism (RFLP) for grouping of the Borrelia isolates associated with Lyme disease or from ixodid ticks. Genomic DNA was digested with a restriction enzyme, blotted and hybridized with an rrl (23S rRNA) gene probe. The sizes of the restriction bands showed a good correlation with the genotypes recently proposed, and Borrelia isolates of diverse geographic origin formed four distinct DNA groups. Group I included all of the USA isolates and some European isolates; group II contained European isolates and Asian isolates; group III comprised European and Asian isolates; group IV included Japanese isolates and an eastern Russian isolate. Groups I, II and III corresponded to Borrelia burgdorferi, B. garinii and group VS461, respectively. The RFLPs of Japanese isolates were rather divergent and some of the isolates were quite distinct from the USA and European isolates. RFLP analysis of the rRNA genes and flanking regions, using rrl gene probes as we reported here, may be useful in the taxonomic study of Borrelia.

Previous studies have revealed the occurrence of multiple chromosomal alterations among spontaneous colony form mutants and clinical isolates of Candida albicans. In this report we show that such karyotype alterations are also seen in spontaneous and induced non-germinative mutants of the fungus. To determine if phenotypic changes other than colony form and microscopic morphology accompanied the rearrangements of the electrophoretic karyotype, we studied the following characteristics of the non-germinative and some of the colony form mutants: formation of pseudohyphae, chlamydospore production, germ tube formation, colony morphology, auxotrophy, growth at various temperatures, and colony morphology and pigment formation on selected media (bismuth sulphite and Phloxine B). We established that phenotypic and karyotypic variability among spontaneous, non-germinative mutants was no different than such variability amongspontaneous colony form mutants. Thus, non-germination may represent another phenotypicconsequence of genomic instability in C. albicans. The variability indifferent phenotypic attributes that occurred amongst the mutants was not associated with any given karyotype. Moreover, neither the low nor the high phenotypic variabilities observed were explained by the relatively high number of alterations in a limited number of chromosomes.

A cDNA library was constructed in λgt11 using mRNA from 11-d-old mycelium of Agaricus bisporus. Three clones containing laccase sequence were identified using an affinity-purified anti-laccase antibody. From one of these clones, a 333 bp sequence was used to identify further cDNA clones (including one which is close to full length) and a genomic clone. The coding sequences found were of two similar but not identical versions with differences at 36 out of 520 residues of deduced amino acid sequence. The laccase genes each encode a sequence expressed as a 2·3 kb mRNA, specifying a 520 residue polypeptide including a 19 amino acid residue signal peptide that is absent from the N terminus of the mature (extracellular) protein. The coding sequence of lcc1 is interrupted by 14 short introns. The lcc1 and lcc2 genes are not allelic as they do not segregate in uninucleate spores derived from a four-spored basidium. Comparison of the deduced amino acid sequences with that of the other fungal laccases that have been cloned, and with the very similar ascorbate oxidases from higher plants shows that whilst some sequence is absolutely conserved at and around the amino acid residues involved in copper binding, the overall sequence similarities are low.

A gene which encodes a 35 kDa protein with both carboxymethylcellulase and xylanase activity was cloned from Ruminococcus flavefaciens FD-1 and the nucleotide sequence determined. The FD-1 gene, celE, and the celA gene, which encodes a cellodextrinase, were used as probes to analyse transcription in R. flavefaciens grown under different conditions. Transcription of both genes was induced when cellulose was added to cells growing in cellobiose. This induction continued after cellulose depletion and after cell division had ceased. Transcription of both genes was also induced by cellotriose, although the effect was not as pronounced as induction by cellulose and was greater for the celA gene than for the celE gene.

The xylose isomerase gene from Thermoanaerobacterium saccharolyticum strain B6A-RI was cloned by complementation using Escherichia coli xyl-5 mutant strain HB101. One positive clone was detected and the recombinant plasmid, pZXI6, was isolated. The clone contained the vector pUC18 and an insert fragment of 4·5 kb. The cloned xylose isomerase gene (xylA) was expressed constitutively in E. coli. The gene contained one open reading frame (ORF) of 1317 bp, which corresponds to 439 amino acid residues. The molecular mass of the gene product was calculated to be 50474 Da from the deduced amino acid sequence. A putative promoter region (Pribnow box), TATAATATATAAT, which repeated twice at the −10 region in E. coli, was found 25 bp upstream of the ribosomal binding site. The deduced amino acid sequence of T. saccharolyticum strain B6A-RI xylose isomerase exhibited very high homology to those from Thermoanaerobacterium thermosulfurigenes 4B (formerly Clostridium thermosulfurogenes 4B) and Thermoanaerobacter ethanolicus 39E (formerly Clostridium thermohydrosulfuricum 39E). Codon usage in xynA, xynB and xylA showed a clear propensity for AT-containing isocodons. The native molecular mass of the purified recombinant thermostable xylose isomerase was 200 kDa, and the enzyme was a tetramer comprised of identical subunits. The apparent temperature and pH optima for activity of the cloned xylose isomerase were 80 °C and 7·0 to 7·5, respectively.

Endoxylanase (xynA) and β-xylosidase (xynB) genes from Thermoanaerobacterium saccharolyticum were subcloned from a cosmid clone (pXDM1) to generate pXPH3. The nucleotide sequence of a PstI-HindIII fragment in pXPH3 that contained xynB revealed an open reading frame (ORF) of 1500 bp encoding a 55 kDa protein. Another open reading frame (ORF1) of unknown function was found 21 bp downstream from the first stop codon of xynB. xynB, ORF1 and xynA had the same direction of transcription. xynB from T. saccharolyticum strain B6A-RI exhibited 45 % amino acid similarity, with 18 % amino acid identity to xynA of T. saccharolyticum strain B6A-RI, and 61 % similarity and 37 % identity with the β-xylosidase gene from Caldocellum saccharolyticum. Recombinant β-xylosidase was purified from E. coli (pXPH3) cells. The enzyme was a monomer with a molecular mass of 55 kDa. The specific activity and pH and temperature optima for hydrolysis of p-nitrophenyl-β-d-xylopyranoside (pNPX) were 5·53 U mg−1, 5·5 and 70 °C, respectively. The -xylosidase was stable at 65 °C, but lost activity at 85 °C. The purified enzyme had hydrolytic activity towards xylopentose, xylotriose, xylobiose and pNPX, but had no activity toward xylan.

The gene for the periplasmic β-lactamase of Lysobacter enzymogenes was isolated as part of a 1017 bp EcoRI fragment and the nucleotide sequence of the gene was determined. It has a G + C content of 71·5% and encodes a 27 amino acid signal sequence and the mature β-lactamase of 276 amino acids which has a mass of 29146 Da. The enzyme appears to be unique to L. enzymogenes but its amino acid sequence shows a high degree of homology with the amino acid sequences of the lactamase from Citrobacter diversus and other Class A β-lactamases. The -lactamase gene of L. enzymogenes was expressed in Escherichia coli using pUC118 as the vector. The production of active β-lactamase was highest after the active growth phase of the expression host and reached levels which were about three times higher than those obtained with L. enzymogenes.

The methylenomycin biosynthetic gene cluster was previously shown to be present on the 350 kb giant linear plasmid SCP1. The structures of SCP1 in various Streptomyces coelicolor A3(2) strains were analysed by pulsed field gel electrophoresis (PFGE). S. coelicolor A3(2) strains 1147, M138 and M146, previously genetically characterized as SCP1+, contained the free form of SCP1. S. coelicolor 1984 and 2106, earlier characterized as SCP1′-cysB and a stable cysD donor respectively, were found to contain very large linear plasmids, 550 kb and 1700 kb in size. Strain JCM4979, another SCP1+ derivative of A3(2), had a series of SCP1-related linear plasmids of 390630 kb. The integrated states of SCP1 in S. coelicolor SCP1-NF strains, an NF-like strain and a stable pabA donor were confirmed by PFGE, which revealed a lack of the intact SCP1 termini. In addition, a 70 kb linear plasmid was found in Streptomyces violaceoruber SANK95570, another methylenomycin producer. However, the plasmid was shown by hybridization to a methylenomycin resistance gene not to carry the methylenomycin gene cluster of this strain.

The cryptic plasmid pCI305 from Lactococcus lactis subsp. lactis UC317 was screened for incompatibility with a range of cloning vectors and was found to be incompatible with the theta-replicating plasmids pAM401 and pIL253. In contrast, lactococcal vectors containing the replication functions of the rolling circle replicating plasmids pSH71 and pWV01 were compatible with pCI305. A number of native lactococcal plasmids were also screened for both incompatibility and DNA sequence homology to the pCI305 replicon. A plasmid with high sequence identity was identified which was compatible with pCI305.

A plasmid, designated pFS1, has been identified for the first time in a strain of Bacillus polymyxa. The plasmid is 16 kb in length. It is sensitive to digestion by DNAase I, restriction endonucleases and exonuclease III, but resistant to RNAase A and λ-exonuclease. A restriction map of the plasmid has been constructed, demonstrating its linear nature. The linear DNA molecules of pFS1 are attached at their 5′ ends to a protein which can be cleaved by Pronase. No homology was detected between pFS1 and chromosomal DNAs from a number of different B. polymyxa strains. Also, hybridization between pFS1 and total DNA from B. polymyxa SCE2 plasmid-cured derivatives does not occur. The plasmid seems to be cryptic since B. polymyxa strains harbouring pFS1 and the cured derivatives have the same phenotype.