Microbiology - Volume 136, Issue 7, 1990

Summary: 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.

Summary: Incubation of the plant pathogenic bacterium Xanthomonas campestris pv. manihotis with either l-[3,4-14C]methionine or [3,4-14C]KMBA (2-keto-4-methylthiobutyric acid) led to the production of [14C]MTPA (3-methylthiopropionic acid) and [14C]MTAA (3-methylthioacrylic acid). When an excess of non-radioactive KMBA was present in the medium, formation of [14C]KMBA from l-[3,4-14C]methionine was observed. Conversely, [14C]methionine accumulated in a trapping pool of non-radioactive methionine as a result of biotransformation of [3,4-14C]KMBA. Aminooxyacetic acid, a transaminase inhibitor, suppressed totally the formation of [14C]MTPA from l-[3,4-14C]methionine but not from [3,4-14C]KMBA. Metabolism of l-[1-14C]methionine liberated 14CO2 but did not produce [14C]MTPA. These results demonstrate that methionine is catabolized by X. campestris pv. manihotis into MTPA via transamination and subsequent decarboxylation of the intermediate α-keto acid KMBA.

Summary: 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.

Summary: Short exposure (up to 3 h) of phosphate-starved mycelium from Phytophthora palmivora to the antifungal agent potassium phosphonate resulted in decreased levels of NAD, ATP, and a number of compounds tentatively identified as polyphosphorylated nucleotides (“alarmones”). ADP, AMP and adenosine levels were not increased, as would be expected if phosphorylation were the site of inhibition. Pyrophosphate levels, however, were raised. The incorporation of [32P]phosphate into phospholipids and other macromolecules was not affected during this short exposure. Together, these results suggest that adenylate synthesis may be a primary site of action of phosphonate in the fungus.

Summary: 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.

Summary: A mutant of Trichoderma reesei QM 9414 - T. reesei M8 - was isolated after γ-irradiation. It did not form β-glucosidase during growth on glycerol or glucose, but secreted β-glucosidase upon growth on cellobiose. The mutant was also able to grow on cellulose and secrete β-glucosidase, but with a longer lag. β-Glucosidase activity could be induced in the mutant in a replacement medium by cellobiose and β-methyl d-glucoside. Mycelia lacking β-glucosidase were able to take up both these inducers immediately, indicating the presence of a constitutive permease. The mutant produced cellulases upon growth on lactose and – after a lag – on cellulose. Mycelia of T. reesei M8 pregrown on glycerol or cellobiose could be induced by sophorose to produce cellobiohydrolase I in a replacement system. The findings show that (i) cellobiose and other β-linked disaccharides can be taken up by T. reesei without prior hydrolysis, and (ii) that cellobiose most probably induces β-glucosidase formation during growth on cellulose.

Summary: 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.

Summary: The regulatory mechanism for glucoamylase synthesis in Aspergillus terreus strain 4 was studied. Synthesis of the enzyme was induced in the presence of maltose and other carbohydrates containing maltose units. Smaller amounts of enzyme were produced in the presence of glucose or other monosaccharides like sorbose and xylose. Enzyme production was maximal with 0.5% starch as the carbohydrate source. At higher concentrations of starch, production of the enzyme was less. In glucose medium, both extracellular and intracellular glucoamylase activities were very low. When starch medium was supplemented with glucose, the extracellular enzyme activity decreased with increasing concentration of added glucose. Intracellular amylase was also detected. There was no appreciable change in the level of this enzyme activity in the presence of added glucose. Further, when glucose was added to the growing culture after 2 d in starch medium there was a marked decrease in glucoamylase synthesis, which was also proportional to the amount of glucose added. Enzyme synthesis was resumed as soon as the glucose concentration in the culture medium fell below a critical level. All these observations strongly suggest that in A. terreus strain 4, extracellular glucoamylase synthesis is subject to catabolite repression.

Summary: The temperature and pH optima, and the temperature and pH stability, of crude and purified enzymes of the cellulase complex of the cellulolytic ascomycete fungus Neurospora crassa were investigated. The effects of some non-ionic surfactants and fatty acids on the production/release of enzymes of the cellulase complex were also examined. For the different enzymes of the complex, activity maxima occurred between pH 4.0 and 7.0, with pH 5.0 being close to optimal for stability of all. Temperature optima for activity ranged between 45 and 65 °C, with the stability optimum between 45 and 50 °C. The presence of C18 fatty acids and surfactants resulted in increased production of both endoglucanase and exoglucanase in the medium. Oleic acid was the most effective fatty acid tested, and Tween 80 the most effective surfactant. Oleic acid had no detectable effect on production of β-glucosidase, and Tween 80 actually reduced its production.

Summary: Growth of the yeast Pichia pastoris IFP 206 in methanol- and glucose-containing media led respectively to very and weakly flocculent cells. Mannans from both kinds of cells were extracted and compared. Chemical analysis and molecular mass estimation showed some differences between the mannans from very and weakly flocculent cells, especially in quantitative amino acid content. 1H NMR analysis showed that both kinds of mannan contained α-1,2 and β-1,2 linkages. Two acetolysis conditions, combined with 1H NMR analysis, revealed that mannans from both kinds of cells were composed of mannose, mannobiose, mannotriose, mannotetraose and mannopentaose side-chains with the following respective structures: Man; Manα1.2Man; Manα1.2Manα1.2Man; Manβ1.2Manα1.2Man; Manβ1.2Manβ1.2Manα1.2Man; Manα1.2Manβ1.2Manβ1.2Manα1.2Man. Additionally the β-1,2 linkages of the non-reducing terminal residues of the mannotetraose were shown to be acetolysislabile. The mannans from very flocculent cells were richer in mannopentaose than the mannans from weakly flocculent cells. According to these results, the extended conformations in the branching moieties of the mannan could be the basis of the higher degree of flocculation of the methanol-grown cells.

Summary: 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.

Summary: Methods have been developed for chemical transformation and electro-transformation (electroporation) of vegetative cells of Bacillus anthracis with supercoiled plasmid DNA. Chemical transformation was dependent on incubation in Tris/HCl with osmotic support and transformation with plasmid DNA was effected by treatment with polyethylene glycol 3350. Maximum transformation frequencies were 3.8 x 10−5 transformant c.f.u. per viable c.f.u. (1 x 103 c.f.u. per μg DNA). Optimal frequencies were pH dependent and were affected by growth-medium composition. Transformation was not observed with linear or multimeric plasmid DNA. Electro-transformation of B. anthracis using high field intensity electroporation was dependent on the composition of both the growth medium and the electroporation buffer. Maximum electro-transformation frequencies were 5.3 x 10−4 c.f.u. per viable c.f.u. (2.6 x 104 c.f.u. per μg DNA). The use of early exponential phase cells was critical to both procedures and the maximum efficiency (c.f.u. per μg DNA) of each system was strain dependent under the conditions described.

Summary: Different cloning vectors (pC194, pBC16, pUB110, pBD10, pBD8, pAMβ1) and Bacillus anthracis plasmid pX02 were introduced into B. anthracis by a transformation method. To induce an artificial competence state for uptake of isolated plasmid DNA, the cultures were treated with glycine, to reduce cross-linking of peptidoglycan, followed by freezing and thawing. The procedure is extremely rapid and relatively efficient (maximum transformation efficiency about 103 c.f.u. per λg DNA) and allows different cloning vectors with molecular masses ranging from 1.8 to 17.7 MDa to be introduced into B. anthracis.

Summary: A DNA fragment containing the flaD locus of Bacillus subtilis, which had been cloned into plasmid pAC3, was subcloned into an M13 phage and sequenced. The sequence contained five open reading frames (ORFs), of which ORF2 was the flaD gene. Unexpectedly, the sequence of the flaD locus was identical to that of sin [sporulation inhibition gene; Gaur, N. K., Dubnau, E.&Smith, I. (1986). Journal of Bacteriology 168, 860–869]. A B. licheniformis homologue (flaL) of the B. subtilis flaD locus was cloned into pUC19 and identified by colony hybridization. The B. licheniformis DNA was subcloned and sequenced. Two ORFs (ORF1, or L-ORF1; and ORF2, orflaL) were detected, encoding 58 and 111 amino acid residues, respectively. These are almost identical in length to ORF1 (D-ORF1; 57 amino acids) and flaD (111 amino acids) on the fragment of B. subtilis DNA. The overall interspecies differences between the nucleotide sequences of D-ORF1 and L-ORF1, and those offlaD and flaL, were 42% and 11%, respectively, and the differences in the predicted amino acid sequences were 50% and 7%, respectively. The regions 3” of the ORFs (flaL and flaD) in both species resemble rho-independent terminators of transcription. The characteristics of the amino acid sequences are also discussed.

Summary: 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.

Summary: 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.

Summary: 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.

Summary: IncP plasmids were transferred by conjugation from Escherichia coli to gas-vacuolate strains of Ancylobacter aquaticus, between strains of A. aquaticus, and from A. aquaticus to E. coli. These plasmids would also mobilize the chromosome of A. aquaticus. In this way, A. aquaticus genes could complement mutations in either A. aquaticus or E. coli. A locus involved in gas vesicle production was co-transferred with rifampicin resistance in matings between strains of A. aquaticus.

Summary: A gene for alkaline cellulase from the alkalophilic Bacillus sp. KSM-635 was cloned into the HindIII site of pBR322 and expressed in Escherichia coli HB101. Although the recombinant plasmid contained two HindIII inserts of 2.6 kb and 4.0 kb, the inserts were found to be contiguous in the Bacillus genome by hybridization analysis. Nucleotide sequences of a 2.4 kb region which was indispensable for the production of cellulase, and the flanking, 1.1 kb region, were determined. There was an open reading frame (ORF) of 2823 bp in the 3498 bp sequence determined, which encoded 941 amino acid residues. Two putative ribosome-binding sites and a δ43-type, promoter-like sequence were found upstream from an initiation codon in the ORF. The deduced amino-terminal sequence resembles the signal peptide of extracellular proteins. A region of amino acids, 249 to 568, of the deduced amino acid sequence of the cellulase from this organism is homologous with those of alkaline and neutral enzymes of other micro-organisms, but nine amino acid residues were found to be conserved only in the alkaline enzymes.

Summary: 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 Bic/I-digested B. subtilis chromosomal DNA was constructed in phage vector ø105J27. 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.