- Volume 136, Issue 7, 1990

A conjugation system for mapping the chromosome of Pseudomonas syringae pv. syringae PS224 has been developed using the IncP-10 plasmid R91-5; pMO22, a Tn501-loaded derivative of R91-5; and pMO75, R91-5 loaded with Tn5. Nine different donor origins were identified with R91-5 and pMO22. By insertion of Tn5 into various sites of the chromosome, an additional six donor origins were available using pMO75 as the donor plasmid. In all, 36 markers were located on three linkage groups. Many donor strains were unstable and the limited availability of stable donor strains has limited the extent to which markers have been located. This instability of donor strains is in marked contrast to the highly stable donor strains found in P. putida using the same plasmids. As in P. aeruginosa and P. putida, auxotrophic markers in P. syringae do not show the clustering of related markers found in enterobacteria.

We have identified an insertion sequence, IS116, present in Streptomyces clavuligerus at one copy per genome. The element was discovered as a 1·4 kb insertion into the multicopy plasmid pIJ702 after propagation in S. clavuligerus. The nucleotide sequence of IS116 and the flanking sequences from pIJ702 have been determined. The junctions with pIJ702 show no target site duplication and there are no inverted repeats at the ends of the element. One putative coding open reading frame of 1197 bp was identified which would code for a protein product of 399 amino acids. This protein resembles deduced integrase/transposase proteins specified by three other transposable elements of actinomycetes: IS110 and the mini-circle from Streptomyces coelicolor A3(2), and - most particularly - IS900 of Mycobacterium paratuberculosis. Two regions that are relatively conserved among these gene products show features found in similar positions in many reverse transcriptases. IS116 and IS900 are also closely similar in their general organization and (apparently) in their insertion site specificity, whereas IS110 and the mini-circle are quite different in these features.

Five classes of mutants of Methylophilus methylotrophus which are defective in methanol oxidation (Mox-) have been isolated, partially characterized, and complemented using a Sau3A genomic library. Two classes of mutants were defective in production of the a subunit of methanol dehydrogenase, another two classes in the production of both the a subunit and cytochrome c L, while the fifth class produced the a subunit, but did not contain an active methanol dehydrogenase, nor cytochrome c L. The genes defective in these mutants were linked.

The role of Ca2+ in the maintenance of apical dominance in Neurospora crassa was investigated. In the presence of the calcium-channel blocker verapamil (1 mm), wild-type hyphal tips demonstrated enhanced branching which led to a fan-like pattern of growth, similar to that seen in certain of the morphological mutants of N. crassa such as ‘frost’ and ‘spray’. In verapamil-treated hyphae, unlike untreated controls, Ca2+ was not observed in hyphal tips by fluorescence microscopy and the exaggerated branching pattern could be corrected by the addition of 10 mm-Ca2+. Studies using the morphological mutants ‘frost’ and ‘spray’, which grow typically on minimal medium with a branching pattern quite similar to verapamil-treated wild-type, also failed to demonstrate Ca2+ in hyphal tips. Exogenously added Ca2+ (50–500 mm) almost completely corrected the abnormal branching seen in these mutants, converting them to an essentially wild-type appearance. These observations suggest that low Ca2+ levels, induced in the wild-type by verapamil, and constitutive in the mutants, are responsible for the abnormal branching patterns.

Pseudomonas aeruginosa PAO lacks UV-inducible Weigle reactivation and Weigle mutagenesis of UV-damaged bacteriophages. This lack of UV-inducible, error-prone DNA repair appears to be due to the absence of efficiently expressed umuDC-like genes in this species. When the P. aeruginosa recA gene is introduced into a recA(Def) mutant of Escherichia coli K12, the P. aeruginosa recA gene product is capable of mediating UV-induced mutagenesis, indicating that it could participate in a recA-lexA-like regulatory network and function in inducible DNA repair pathways if such existed in P. aeruginosa. The presence of the IncP9, UV-resistance plasmid R2 in RecA+ strains of P. aeruginosa PAO allows UV-inducible, mutagenic DNA repair of UV-irradiated bacteriophages. R2 also greatly stimulates the ability of UV radiation to induce mutagenesis of the bacterial chromosome. When R2 is introduced into P. aeruginosa strains containing either the recA908 or recA102 mutation, plasmid-mediated UV resistance and Weigle reactivation are not observed. These observations suggest that the increased protection afforded to P. aeruginosa by R2 is derived from a RecA-mediated, DNA-damage-inducible, error-prone DNA repair system which complements the lack of a chromosomally encoded umuDC-like operon.

The yeast Metschnikowia reukaufii could not utilize ethanol as a growth substrate, although ethanol stimulated cellular O2 consumption and the reduction of intracellular nicotinamide adenine nucleotides. Also, ethanol inhibited cell growth in glucose-containing medium. The effect was stronger when the K+ concentration in the growth medium was lowered from 5.8 to 0.6 mM. Addition of glucose to an aerated cell suspension caused an initial H+ efflux and K+ influx in a ratio of approximately 1:1. This was followed by a phase of continuing extracellular acidification without any measurable uptake of K+. In contrast, in cells energized by glucose, ethanol stimulated K+ efflux; concomitantly, H+ extrusion was markedly lowered by ethanol. The rates of H+ extrusion correlated with the intracellular level of glucose 6-phosphate and not of ATP. It is concluded that there is a regulatory interaction, though not by a direct effect, between glucose 6-phosphate and the plasma-membrane ATPase. Ethanol appears to activate electron transfer from cytosolic NADH to O2 by a pathway independent of the mitochondrial respiratory chain.

A glycerol-kinase-deficient mutant of Aspergillus niger was isolated. Genetic analysis revealed that the mutation is located on linkage group VI. The phenotype of this mutant differed from that of a glycerol kinase mutant of Aspergillus nidulans in its ability to utilize dihydroxyacetone (DHA). The weak growth on glycerol of the A. niger glycerol kinase mutant showed that glycerol phosphorylation is an important step in glycerol catabolism. The mutant could still grow normally on DHA because of the presence of a DHA kinase. This enzyme, probably in combination with an NAD+-dependent glycerol dehydrogenase, present only in the mutant, is responsible for the weak growth of the mutant on glycerol. Enzymic analysis of both the mutant and the parental strain showed that at least three different glycerol dehydrogenases were formed under different physiological conditions: the NAD+-dependent enzyme described above, a constitutive NADP+-dependent enzyme and a d-glyceraldehyde-specific enzyme induced on d-galacturonate. The glycerol kinase mutant showed impaired growth on d-galacturonate.

Addition of choline (20 mm) or Tween 80 (0·06%) to the culture medium of Trichoderma reesei QM 9414 increased (a) the secretion of protein under both carbon-catabolite-repressed and -derepressed conditions, and (b) cellulase secretion under carbon-catabolite-derepressed conditions. In contrast, no stimulation by choline or Tween 80 was observed with the hypersecretory strain T. reesei RUT C-30. In view of the obligatory role of O-glycosylation in protein secretion by this fungus, an investigation was made into the effects on this process of choline and Tween 80. A membrane preparation was isolated from both strains of T. reesei and used to assay enzymes involved in O-glycosylation. Significant differences were observed with respect to the activity of dolichol phosphate mannose (Dol-P-Man) synthase only. Strain QM 9414, grown on media supplemented with choline or Tween 80 exhibited a two- to threefold higher activity of Dol-P-Man synthase compared to a control lacking these supplements. This stimulatory effect was observed during growth under both carbon-catabolite-repressed and -derepressed conditions. In contrast, strain RUT C-30 exhibited decreased activities of Dol-P-Man synthase when grown in media supplemented with choline. Choline had no effect on Dol-P-Man synthase in vitro, whereas Tween 80 decreased the activity. Thus the effect of Tween 80 or choline on protein secretion by T. reesei may be due to a stimulation of formation and/or activity of Dol-P-Man synthase, thereby elevating the level of O-glycosylation and protein secretion.

Lactoferrin and transferrin have antimicrobial activity against selected Gram-negative bacteria, but the mechanism of action has not been defined. We studied the ability of lactoferrin and transferrin to damage the Gram-negative outer membrane. Lipopolysaccharide release by the proteins could be blocked by concurrent addition of Ca2+ and Mg2+. Addition of Ca2+ also blocked the ability of lactoferrin to increase the susceptibility of Escherichia coli to rifampicin. Transferrin, but not lactoferrin, increased susceptibility of Gram-negative bacteria to deoxycholate, with reversal of sensitivity occurring with exposure to Ca2+ or Mg2+. In transmission electron microscopy studies polymyxin B caused finger-like membrane projections, but no morphological alterations were seen in cells exposed to EDTA, lactoferrin or transferrin. These data provide further evidence that lactoferrin and transferrin act as membrane-active agents with the effects modulated by Ca2+ and Mg2+.

Cyclic AMP (cAMP)- and Ca2+-dependent protein kinase activities of the fungus Metarhizium anisopliae were sought in extracts of ungerminated conidia, germinating conidia and mycelium, as well as in purified plasma membranes from mycelium. Ungerminated conidia contained a Ca2+/calmodulin-dependent protein kinase capable of phosphorylating multiple endogenous proteins and involved in triggering germination. cAMP-dependent protein kinase activity was not detected in ungerminated conidia in spite of the presence of cAMP in these conidia and the pre-germination synthesis of two cAMP-binding proteins. Most phosphorylation events in crude mycelial extracts were Ca2+-dependent but H-series inhibitors of cAMP-dependent kinases selectively repressed phosphorylation of a 27 kDA protein. Plasma membranes from mycelium contained a Ca2+-independent but H-8-sensitive protein kinase with multiple endogenous substrates for phosphorylation. 8-Azido[32P]cAMP bound selectively to a 52 kDa membrane protein indicative of a single cAMP-binding protein. Plasma membranes contained a phosphatase which rapidly (< 1 min) and selectively dephosphorylated a polypeptide of 15·5 kDa, thus being suited to cause rapid and reversible changes in membrane function. Membranes also contained an adenylate cyclase apparently involved in transmembrane signalling reactions, since mechanical or chemical treatments which stress the fungus caused rapid increases in intracellular levels of cAMP. Reconstitution experiments with a homogenate from a crisp-1 mutant of Neurospora crassa suggested G-protein regulation of Metarhizium plasmalemma adenylate cyclase.

Salt-induced change in protein synthesis were investigated in the cyanobacterium Synechocystis sp. PCC 6803. Immediately after a salt shock of 684 mm-NaCl, total protein synthesis was almost completely blocked. Then, parallel to the accumulation of the osmoprotective compound glucosylglycerol, protein synthesis recovered gradually but remained diminished. The activation of glucosylglycerol synthesis was not inhibited by chloramphenicol at concentrations which totally inhibited protein synthesis. The qualitative protein composition of salt-shocked and control cells was similar. However, the rates of synthesis of single proteins were altered in cells shocked for 10 h and adapted to high salt conditions. Using two-dimensional gel electrophoresis, proteins were found which were synthesized at enhanced rates after adding salt.

Activities of the enzymes which are involved in the poly-β-hydroxybutyrate (PHB) cycle, in both synthesis and degradation reactions, were assayed in crude extracts of Azospirillum brasilense cells containing different amounts of PHB. The enzymes of the PHB cycle, of both the synthesis and the degradation process, were more active in PHB-rich cells than in PHB-poor cells. During 96 h of starvation of cells suspended in phosphate buffer, enzymes of the PHB cycle were more active in PHB-rich cells. There was a peak of activity of hydroxybutyrate dehydrogenase (BOHB-DH), β-ketothiolase and thiophorase after 24 h of starvation, due to polymer degradation. During the following hours of starvation there was a decrease in the activity of these enzymes. After 24 h of starvation the activity of acetoacetyl-CoA reductase dropped to a minimum level, because the cells could not synthesize PHB under these conditions. The specific activities of BOHB-DH, β-ketothiolase and thiophorase were higher in A. brasilense cells which were grown under low oxygen tension and consequently accumulated high levels of PHB, than in cells grown under high oxygen tension, with a low PHB content. Similarities to the pathway of PHB biosynthesis and degradation and its control in Azotobacter beijerinckii are described.

Techniques are described which allow mutated populations of Candida cloacae to be enriched efficiently (up to 167-fold in one round of enrichment) for mutants deficient in the alkane degradation pathway (Alk−). Such mutants, as well as being of scientific importance in studies of the degradation pathway, are also of commercial interest because several of the degradative intermediates are of value to the chemical industry. The Alk− mutants were readily isolated by their inability to grow on agar plates supplied with hexadecane as sole carbon source. A total of 288 Alk− mutants were isolated from, effectively, 4 x 106 mutagen-treated cells. They were further characterized by replica-plating using palmitic acid (PA) or acetate (Ac) as sole carbon source. Preliminary screening studies showed that of the 84 Alk− PA− Ac+ mutants, most could accumulate dicarboxylic acids from hexadecane and palmitic acid and at least one mutant also produced 3-hydroxyhexadecanedioic acid. Of the 80 mutants characterized as Alk− PA+, 16 produced small amounts of hexadecanol.

The expression of a strongly immunomodulatory mannoprotein complex (GMP) in the different forms of growth of the human commensal and opportunistic pathogen Candida albicans was studied using a monoclonal antibody (mAb AF1) directed against an oligosaccharide epitope of GMP. Immunofluorescence revealed that the surface of the yeast cells was highly reactive with mAb AF1, but that the reactivity was greatly reduced or disappeared during mycelial conversion. This modulation was shared by a number of strains of C. albicans, and was not solely a temperature- or nutrition-dependent phenomenon. Hypha-deficient strains (A12 and CA2) did not show variations of surface fluorescence under environmental conditions which were permissive for hyphal conversion (incubation in N-acetylglucosamine or Lee’s medium, at 37 °C). GMP extracts from yeast and mycelial forms of the fungus were separated into three chromatographically distinct, high molecular mass mannoprotein fractions (F1, F2 and F3), which were tested individually by indirect ELISA for mAb AF1 recognition. All yeast-derived constituents and two (F2 and F3) of the hyphal mannoproteins were recognized by the mAb. The low or absent reactivity of the F1 constituent from hyphal cells was confirmed by immunoblots. Irrespective of their source (yeast or mycelial), all fractions reacted to a similar extent with a polyclonal anti-Candida serum. Overall, the data suggest changes in epitope specificity and/or confinement of reactive constituents in the inner wall layers as possible mechanisms of modulated expression of mAb AF1-reactive epitope during mycelial conversion.

A Bacillus subtilis gerC spore germination mutant demonstrating a temperature-sensitive response to l-alanine as germinant has been characterized in detail. The gerC58 mutation is 50% cotransformed with aroB in the gene order gerC-aroB-trpC. The mutation is responsible for a severe growth defect which is manifest at all growth temperatures and is most extreme on rich media. A second, unlinked, mutation in the original strain suppressed this growth defect, but spores of the suppressed strain failed to germinate in alanine at 42 °C. As this germination defect is dependent on the presence of the gerC58 allele, it is likely to be the direct result of a mutant gerC protein. The gerC gene therefore appears to have a role in both spore germination and vegetative cell growth. A gene library of BclI-digested B. subtilis chromosomal DNA was constructed in phage vector 𝜙105J27. A derivative containing the gerC region was obtained by complementation of the growth defect of an unsuppressed gerC58 strain. This phage contained a 6·3 kb insert of bacterial DNA, which is above the reported packaging limit of the phage. It failed to form visible plaques, although it could be handled as a prophage and sufficient phage particles be isolated to allow characterization of the insert. A deletion derivative generated in vitro and carrying only 2·9 kb of insert DNA also complemented the gerC defect. This gerC locus is the second locus to be implicated in alanine-stimulated germination. The first, gerA, is a developmentaly controlled operon whose gene products are present only in the spore. This study of gerC, in contrast, reveals a role in spore germination for a normally essential vegetative protein.

Monoclonal antibody CMA134.1 reacted with a protein antigen of apparent molecular mass 22 kDa from Mycobacterium bovis and Mycobacterium tuberculosis, and with an apparently 24 kDa antigen of Mycobacterium kansasii, but not with other mycobacteria or related species. This antibody was used to screen a gene library of M. bovis in λgt11 and identified a recombinant clone that expressed a protein with an apparent molecular mass of 19–20 kDa. Gene expression occurred from the lac promoter in λgt11, but used an unidentified vector promoter, possibly that of the replication primer RNA, in the final plasmid construct. The sequence of an 840 bp fragment was determined and shown to code for a product of 15 kDa. This sequence is identical to that, independently determined, of a gene from M. tuberculosis, usually referred to as the 19 kDa antigen. The reasons for the apparent size discrepancies are discussed.

A 2·1 kbp DNA fragment from Rhodococcus strain ATCC 21145 gave rise to the production of blue and pink pigments in Escherichia coli when cloned downstream of a strong promoter. The sequence of this DNA fragment contains a single open reading frame with a putative ribosome-binding site, potentially coding for a single protein of M r 42 560. Deletion analysis and in vitro transcription-translation experiments support the hypothesis that pigment production in E. coli is due to a single enzyme whose catalytic activity is still unknown. This small pigment gene may become useful for the development of a new generation of chromogenic cloning vectors which do not require expensive substrates for the detection of gene expression.

Intact cells of Comamonas percolans (NCTC 1937) after growth under O2-limited conditions in batch culture exhibit, in addition to b- and c-type cytochromes, an unusual pigment absorbing variably at 588 to 594 nm in reduced minus oxidized difference spectra at room temperature. CO difference spectra suggest ligand binding by this pigment and also reveal a prominent 448 nm absorption minimum and CO-binding b- and/or c-type cytochromes. Although the 588 to 594 nm-absorbing component is reminiscent of ‘cytochrome a 1’, claimed to be a terminal oxidase in some bacteria, O2-limited cells of C. percolans contain no detectable haem A. In contrast, cells grown under O2-sufficient conditions exhibit a membrane-bound cytochrome aa 3 and contain haem A. Low-temperature photodissociation studies of O2-sufficient cells show cytochrome aa 3 to be functional in CO and O2 binding and suggest that aa 3 is the terminal oxidase of a cytochrome b- and c-containing aerobic respiratory chain. Analogous studies of O2-limited cells reveal a component absorbing, in its unliganded state, at 448 nm. Exposure of such CO-liganded cells to white actinic light is followed by oxidation of b- and/or c-type cytochromes and, although the functional oxidase has not been identified, we conclude that C. percolans does not utilize a cytochrome oxidase of the a 1 type.

A tetracycline resistance (Tcr) determinant from Clostridium difficile strain 630 was cloned into the Escherichia coli plasmid vector pUC13. The resulting plasmid pPPM20, containing an insert of 3.4 kbp, was mapped and a 1·1 kbp SacI-HindIII fragment wholly within the Tcr gene was identified. Dot-blot hybridization studies with the 1·1 kbp fragment showed that the Tcr gene belonged to hybridization class M. Tcr could be transferred between C. difficile strains and to Bacillus subtilis at a frequency of 10−7 per donor cell. The element could be returned from B. subtilis to C. difficile at a frequency of 10−8 per donor cell. This is the first demonstration of C. difficile acting as a recipient in intergeneric crosses. DNA from C. difficile transconjugants digested with EcoRV always has two hybridizing fragments of 9·5 and 11·0 kbp when probed with pPPM20. DNA from B. subtilis transconjugants digested with EcoRV produced one hybridizing band of variable size when probed with pPPM20. The behaviour of the element was reminiscent of the conjugative transposons. Therefore we compared the element to the conjugative transposon Tn916. The HincII restriction maps of the two elements differed and no hybridization was detected to oligonucleotides directed to the ends of Tn916. However, the elements do have some sequence homology, detected by hybridization analysis.

The development cycle of the temperate actinophage VWB was investigated. Adsorption of most phage particles occurred within 30 min and the adsorption constant was 0·6 × 10−8 ml min1. The latent and rise periods were 140 and 100 min, respectively, and the burst size was estimated to be 130–250 p.f.u. Although phage VWB could infect only Streptomyces venezuelae ETH 14630 (ATCC 40755), of six different S. venezuelae strains tested, phage DNA could be introduced by transfection into most non-infectible strains. Upon transfection, phage DNA was propagated in these non-infectible strains and phage particles were released. In addition, the transfected strains could be lysogenized. By comparison of restriction fragments of VWB DNA, either free or integrated in the chromosomal DNA of the S. venezuelae ETH 14630 lysogen, the attachment site was localized. PAGE of the phage proteins revealed at least 17 different proteins with three major bands estimated as 16·5, 27·2 and 43 kDa in size. The N-terminal amino acid sequence of these supposed major head and tail proteins was determined. The corresponding DNA sequences on the phage genome were localized using oligonucleotides synthesized on the basis of the N-terminal amino acid sequences. The genes coding for the major structural proteins were shown to be clustered, as has been observed for other bacteriophages.