- Volume 140, Issue 10, 1994

New mutants of Neurosporacrassa having the ufaphenotype have been isolated. Two of these mutants, like previously identified ufamutants, require an unsaturated fatty acid for growth and are almost completely blocked in the de novosynthesis of unsaturated fatty acids. The new mutations map to a different chromosomal location than previously characterized ufamutations. This implies that at least one additional genetic locus controls the synthesis of unsaturated fatty acids in Neurospora.

The white-rot basidiomycete Ceriporiopsis subvermisporaproduces two families of ligninolytic enzymes, namely manganese-dependent peroxidases (MnPs) and laccases, when growing in liquid cultures of defined composition. In medium containing 11 p.p.m. of Mn(II), up to seven isoenzymes of MnP and four isoenzymes of laccase were resolved by isoelectrofocusing (IEF), with pl values in the range 4.10-4.60 and 3.45-3.65, respectively. Occasionally, a fifth laccase isoform of pl 4.70 was also detected. In cultures with 25 and 40 p.p.m. of Mn(II). mainly the MnPs with higher pl values are produced. The isoenzyme pattern of MnP is not altered throughout the growth period of the fungus. MnP and laccase are also produced by C. subvermisporawhen growing on wood chips of Pinus radiata.Highest levels of both enzymes were obtained during the first week of incubation. A second peak of MnP activity was observed during the fourth week, whereas very low levels of laccase were extracted from the chips after the second week of growth. IEF analysis showed that the pl values of these laccases are similar to those of laccases produced in liquid cultures, being in the range 3.45-3.65. In contrast, four isoforms of MnP were resolved during the first week of incubation on wood chips, with pl values of 4.40, 4.17, 4.04 and 3.53. This profile underwent a transition during the second week of growth, at the end of which isoforms of MnP with pl values of 3.53, 3.40, 3.30 and 3.20 were resolved by IEF. Immunoblotting studies showed that the molecular mass of MnP isoenzymes from liquid cultures was about 52.5 kDa, whereas the molecular masses of MnPs extracted from wood varied from 52.5 kDa to 62.5 kDa upon ageing of the cultures. The amino terminal sequences of seven MnP isoenzymes were determined. The consensus sequences of MnPs from liquid and solid cultures were clearly distinct, although both showed homology to MnPs from related white-rot fungi.

The cells of the fungus Microbotryum violaceumproduce many long, fine surface hairs that are similar in size and morphology to bacterial pili or fimbriae. These fungal fimbriae are assembled from 74 kDa glycoprotein subunits. We now present evidence that these fimbriae also have a RNA component. Isopycnic centrifugation of fimbriae in caesium chloride produced one band at a density intermediate to that of protein and nucleic acid. The absorbance spectrum of the intact fimbriae was consistent with that of a nucleoprotein. After extraneous RNAs were enzymically removed from the purified fimbrial preparation, disruption of the fibrils resulted in the release of not only the 74 kDa glycoprotein subunits, but also a 30 base single-stranded RNA species. To our knowledge, this is the first example of extracellular RNA as a component of a surface appendage.

The nematophagous fungus Verticillium chlamydosporiumsecreted several proteases in submerged culture in which soya peptone was the sole carbon and nitrogen source. One protease, VCP1 (M r33000, pl 10.2), was purified 14-fold from culture filtrates to apparent homogeneity using preparative isoelectric focusing in free solution, and shown to rapidly hydrolyse the chymotrypsin substrate Suc-(Ala)2-Pro-Phe-pNA and elastin. VCP1 had a K mfor Suc-(Ala)2-Pro-Phe-pNA of 4.3 × 10-5M and a k catof 5.8 s-1. It was highly sensitive to PMSF and TPCK, but only moderately sensitive to chicken egg-white and soya bean trypsin inhibitors. VCP1 degraded a wide range of polymeric substrates, including Azocoll, hide protein, elastin, casein and albumin, and accounted for most of the non-specific protease activity detected in culture filtrates. The purified enzyme hydrolysed proteins in situfrom the outer layer of the egg shell of the host nematode Meloidogyne incognitaand exposed its chitin layer. VCP1 was secreted by several isolates of V. chlamydosporiumand V. lecanii, pathogens of nematodes and insects respectively, but not plant-pathogenic species of Verticillium.These observations suggest that VCP1 or similar enzyme(s) may play a role in the infection of invertebrates.

The effect of chemical modification of aromatic and basic residues of the Bacillus thuringiensisinsecticidal CrylA(c) toxin on toxicity and receptor binding was studied. Modification of four or more of the 43 arginine residues resulted in abolition of toxicity towards Manduca sextaand Pieris brassicaein vivoand a Choristoneura fumiferanacell line in vitro.Modification of seven or more of the 27 tyrosine residues resulted in reduction of toxicity. Upon modification of most of the 10 tryptophan residues, toxicity was reduced. Modification of histidine residues had no effect on toxicity. A quantitative binding assay was developed and optimized to compare the binding of derivatives with native toxin to M. sextabrush border membrane vesicles. The reduction or abolition of toxicity observed upon selective modification of tyrosine or arginine residues was reflected in the binding abilities of the derivatives. However a non-toxic derivative, modified at tryptophan residues, retained its ability to bind vesicles. These results suggest that tyrosine and arginine residues are involved in the binding interaction of toxin with receptor but tryptophan residues are involved in some subsequent step.

Three nitrogen-fixing bacteria, Azotobacter vinelandii, Beijerinckia indicaand Beijerinckia mobilis, were shown to contain large amounts of triterpenoids of the hopane series. In A. vinelandii, the major compound was a novel bacteriohopanepentol ether accompanied by a similar bacteriohopanetetrol derivative: in both compounds, the hopanoids are linked via an ether bond to a carbapseudopentose moiety often found in bacterial hopanoids. In the two Beijerinckiaspecies, diplopterol and 2β-methyldiplopterol were accumulated in much larger amounts than those usually recorded in hopanoid-producing eubacteria, while 2β-methyldiploptene was isolated for the first time from B. mobilis.Whereas in B. mobilisaminobacteriohopanetriol was the only C35hopanoid, the simultaneous presence of bacteriohopanetetrol and aminobacteriohopanetriol in B. indicais a rather unusual feature.

Evidence for several steps in luteolin uptake by Rhizobium melilotiis presented. The first step is highly reversible and strongly pH dependent absorption, greatly enhanced at acidic pH. Studies with liposomes suggest that this step consists of a passive dissolution of the flavonoid in its non-ionized state in the lipidic matrix of the membrane. This first step is enhanced in bacteria killed by high temperature. The second step, a slower and more stable accumulation, is not pH dependent. The nature of the sites involved in this more stable binding remains to be determined. Low temperature and treatment with the metabolic inhibitor potassium cyanide resulted in an increase of luteolin uptake and a decrease of extrusion of flavonoids, suggesting an active extrusion process. Studies with spheroplasts or isolated membranes showed that under physiological conditions most of the luteolin was entrapped in the outer membrane while only a small amount was kept in the cytoplasmic membrane. Arguments are presented supporting the view that R. melilotihas developed a mechanism to keep luteolin in its membranes which helps avoid the toxic effect of the flavonoid on the respiratory chain by trapping most of it in the outer membrane and to maintain the nodoperon in a transcriptionally active form in conditions where this strong inducer is no longer exuded by the host plant.

An agar plating technique was developed in which the activation of expressior of a Thiobacillus ferrooxidans nifH-lacZgene fusion was used to isolate the ntrBCgenes from a T. ferrooxidansgene library. An Escherichia coli ntrCmutant containing the nifH-lacZfusion was transformed and plated on a low-nitrogen medium so that on flooding with ONPG, the production of yellow colonies indicated the presence of the cloned T. ferrooxidans ntrBCgenes. A 4.47 kb region from the T. ferrooxidanschromosome was sequenced. Analysis of the sequence revealed that the ntrBand ntrCgenes were closely linked to a third ORF of unknown function. Analysis of the 900 bp region upstream of the T. ferrooxidans ntrBCgenes and Southern hybridization experiments confirmed that in T. ferrooxidansATCC 33020, the glnAand ntrBCgenes are unlinked. Expression of the T. ferrooxidans nifH-lacZfusion in E. coliwas activated in the presence of the T. ferrooxidans ntrBCgenes and regulated by nitrogen.

The agarase gene (dagA) of Streptomyces coelicolorA3(2) is transcribed from four promoters that are recognized by at least three, and probably four, different RNA polymerase holoenyzmes, each containing a different factor. S1nuclease protection studies revealed that transcription from all four promoters is induced by the products of agar hydrolysis and strongly repressed by glucose. Mutants deficient in glucose kinase activity were defective in glucose repression of all four promoters. Mutants were isolated or identified in which transcription from all four promoters had become inducer-independent (i.e. constitutive), establishing the existence of a repressor gene for dagAthat does not appear to be located within 9 kb of the structural gene. The cloned dagAgene was also constitutively expressed in the closely related strain Streptomyces lividans, which does not normally make agarase and which presumably lacks the repressor gene. Glucose was still able to repress dagAtranscription even under conditions of constitutive expression, suggesting that glucose kinase does not mediate its effect via inducer exclusion. Relative differences in the use of the four promoters were not detected during different stages of growth of surface-grown cultures, although dagAtranscription appeared to peak during the production of aerial mycelium.

The ccpAmutations alsA1(alsA1is allelic to ccpA) and ccpA::Tn917completely abolished catabolite repression of gluconate kinase and sorbitol dehydrogenase synthesis in Bacillus subtilis, whereas they only partially affected the catabolite repression of inositol dehydrogenase, histidase and xylose isomerase synthesis. The alsA1mutation also partially affected catabolite repression of sporulation. Analysis of revertants from the alsA1mutant by direct sequencing indicated that this mutation comprises a base substitution of guanine at nucleotide −14 to adenine within the Shine-Dalgarno sequence of the ccpAgene (ccpAtranslation starts at nucleotide +1). A 1.37 kb EcoRI fragment carrying the ccpAgene was cloned into Escherichia coliplasmid pUC19 and B. subtilisplasmid pUB110, resulting it plasmids pCCPA19 and pCCPA110, respectively. The ccpAgene carried in pCCPA110 complemented the alsA1mutation. Western blotting revealed that the level of the CcpA protein in B. subtiliscells, which seemed to be constitutively synthesized, was approximately 10 times lower for the alsA1mutant than for the wild-type. The CcpA protein synthesized by either E. colicells bearing pCCPA19 or B. subtiliscells bearing pCCPA110 was purified to over 90% homogeneity; the latter cells were grown in the presence of glucose The molecular mass of the protein purified from E. coliwas 74 kDa, suggesting that this protein exists as a dimer because its subunit molecular mass was 38 kDa as determined by SDS-PAGE. Gel retardation analysis indicated that the purified CcpA protein in both cases did not bind to the cissequence for catabolite repression of the gntoperon, but it bound non-specifically to DNA.

Plasmids conferring mupirocin resistance were prepared from isolates of Staphylococcus aureusobtained from four patients in the same ward. The plasmids are related and in all of them the gene conferring mupirocin resistance (mupA) is flanked by copies of IS257in direct repeat. In two plasmids mupA and IS257have been duplicated and in one of these plasmids (pJ3358) a small pT181-like plasmid conferring tetracycline resistance is present flanked by copies of IS257.Filter mating with a strain containing pJ3358 as donor and selection on tetracycline sometimes resulted in transfer of the pT181-like plasmid containing a copy of IS257.Analysis showed that the pT181-like plasmid with the insertion of IS257is present in high copy number and that the IS257element is inserted in the copy number control region of the plasmid.

Bacteriophage ø393-A2, isolated from an artisanal cheese whey sample, is a temperate phage able to generate stable lysogens through integration of its DNA into the bacterial genome. One-step growth kinetics of its lytic development revealed eclipse and latent periods of 100 and 140 min, respectively, with a burst size of about 200 p.f.u. per infected cell. ϕ393-A2 virions have an isometric head and a long, non-contractile tail terminating in a baseplate. The capsid is composed of two major and at least nine minor structural polypeptides. The phage genome consists of a double-stranded DNA molecule of 44 kbp bearing 3′-protruding cohesive ends. A physical map of the phage DNA has been constructed for six restriction enzymes. The whole ϕ393-A2 genome has been cloned in Escherichiacoli using plasmid- and phage-derived cloning vectors.

Cell extracts of Lactobacillus delbrueckiisubsp. lactisDSM 7290 were found to exhibit unique peptolytic ability against unusual β-alanyl-dipeptides. In order to clone the gene encoding this activity, designated pepV, a gene library of strain DSM 7290 genomic DNA, prepared in the low-copy-number plasmid pLG339, was screened for heterologous expression in Escherichia coli.Recombinant clones harbouring pepVwere identified by their ability to allow the utilization of carnosine (β-alanyl-histidine) as a source of histidine by the E. colimutant strain UK197 (pepD, hisG). Complementation was observed in a colony harbouring a recombinant plasmid (pKV101), carrying pepV.A 2.4 kb fragment containing pepVwas subcloned and its nucleotide sequence revealed an open reading frame (ORF) of 1413 nucleotides, corresponding to a protein with predicted molecular mass of 51998 Da. A single transcription initiation site 71 bp upstream of the ATG translational start codon was identified by primer extension. No significant homology was detected between pepVor its deduced amino acid sequence with any entry in the databases. The only similarity was found in a region conserved in the ArgE/DapE/CPG2/YscS family of proteins. This observation, and protease inhibitor studies, indicated that pepVis of the metalloprotease type. A second ORF present in the sequenced fragment showed extensive homology to a variety of amino acid permeases from E. coliand Saccharomyces cerevisiae.