- Volume 137, Issue 1, 1991

Summary: Oscillatoria spp. UCSB8 and UCSB25 are both capable of aerobic N2 fixation. The optimum temperature for C2H2 reduction was 22 °C for Oscillatoria sp. UCSB8 and 35 °C for Oscillatoria sp. UCSB25, whilst the optimum temperature for growth on N2 was 25 °C and 30 °C, respectively. In Oscillatoria sp. UCSB25, but not in UCSB8, inhibition of N2 fixation may limit diazotrophic growth at temperatures above 35 °C. When grown under alternating 12 h light and 12 h darkness, both isolates reduced C2H2 predominantly in the dark and both were capable of N2 fixation and photoheterotrophic growth in the presence of 20 μm-DCMU to inhibit photosystem II activity. Under these conditions, the best exogenous carbon source for Oscillatoria sp. UCSB8 was glucose, whilst that for Oscillatoria sp. UCSB25 was fructose. In Oscillatoria sp. UCSB8, exogenous glucose was catabolized mainly through the oxidative pentose phosphate pathway. Although cultures grown photoheterotrophically showed higher specific activities of nitrogenase than photoautotrophic cultures, they grew more slowly. Furthermore, cultures grown photoheterotrophically under alternating light and darkness reduced C2H2 both in the light and in the dark, but the highest rates of C2H2 reduction were observed in the dark. This cyclic pattern of N2 fixation was independent of photosystem II activity.

Summary: A novel acid cellulase (endo-1,4-β-glucanase, EC 3.2.1.4) was found in a culture of Bacillus sp. KSM-330 isolated from soil. One-step chromatography on a column of CM-Bio-Gel A yielded a homogeneous enzyme, as determined by silver staining of both sodium dodecyl sulphate (SDS) and nondenaturing gels. The enzyme had a molecular mass of 42 kDa, as determined by SDS-polyacrylamide gel electrophoresis. The isoelectric point was higher than pH 10. The N-terminal amino acid sequence of the enzyme was Val-Ala-Lys-Glu-Met-Lys-Pro-Phe-Pro-GIn-GIn-Val-Asn-Tyr-Ser-Gly-Ile-Leu-Lys-Pro. This enzyme had an optimum pH for activity of 5·2, being active over an extremely narrow range of pH values, from 4·2 to 6·9; below and above these pH values no activity was detectable. The optimum temperature at pH 5·2 was around 45 °. The enzyme efficiently hydrolysed carboxymethylcellulose (CMC) and lichenan, but more crystalline forms of cellulose, curdlan, laminarin, 4-nitrophenyl-β-D-glucopyranoside and 4-nitrophenyl-β-d-cellobioside were barely hydrolysed. The enzymic activity was inhibited by Hg2+ but was not affected by other inhibitors of thiol enzymes, such as 4-chloromercuribenzoate, N-ethylmaleimide and monoiodoacetate. N-Bromosuccinimide abolished the enzymic activity, and CMC protected the enzyme from inactivation by this tryptophan-specific oxidant. It is suggested that a tryptophan residue(s) is involved in the mechanism of action of the Bacillus cellulase and that the inhibition of enzymic activity by Hg2+ is ascribable to interactions with the tryptophan residue(s) rather than with thiol group(s).

A dipeptidyl aminopeptidase catalysing hydrolysis of X-prolyl amidomethylcoumarin (AMC) substrates has been purified from Lactococcus lactis subsp. lactis H1. The active enzyme has a molecular mass of approximately 150 kDa, a subunit molecular mass of 82 to 83 kDa and is inhibited by the serine protease inhibitor phenylmethylsulphonyl fluoride. The K m and k cat values for five different dipeptidyl AMC substrates (Gly-Pro-; Leu-Pro-; Lys-Pro-; Phe-Pro- and Glu-Pro-AMC) are similar except for the K m value for Glu-Pro-AMC, which is about threefold higher than that for the other substrates. The enzyme also catalyses hydrolysis of X-Ala-AMC substrates but with much lower k cat and higher K m values than the corresponding X-Pro-AMC substrates. The β-casein-derived heptapeptides Lys-Ala-Val-Pro-Tyr-Pro-Gln and Tyr-Pro-Phe-Pro-Gly-Pro-Ile were hydrolysed, but bradykinins with N-terminal sequences Arg-Pro-Pro- and Lys-Pro-Pro-were not. Dipeptidyl aminopeptidase specific activity is the same in a plasmid-free strain of L. lactis subsp. lactis H1 and in the wild-type, indicating that the enzyme is chromosomally encoded.

The obligately methylotrophic bacterium Methylophilusmethylotrophus hydrolyses acetamide and acrylamide using a cytoplasmic amidase. In previous work, continuous culture was used to isolate spontaneous mutants which overexpressed either the wild-type amidase (strain MM6) or a mutant amidase with an apparently higher K cat (strain MM8). We now report that NTG mutagenesis of strain MM8 followed by acrylamide-limited growth at low dilution rate (D 0·025 h-1; 37 °C) led to the selection of a strain which continued to overexpress the amidase, but which exhibited an unexpectedly low amidase activity and a greatly decreased K m for acrylamide (strain MM15). Amidases from the wild-type and mutant strains were purified and shown to be homotetramers (subunit M r 38000, pI 4·1). The N-terminal amino acid sequence of the wild-type enzyme was 90% homologous with the aliphatic amidase from Pseudomonas aeruginosa, and Southern blotting using an oligonucleotide probe for this region showed that overexpression of the enzyme in the mutant strains was not due to gene amplification. Compared with the wild-type and MM6 enzymes, the MM8 enzyme exhibited a threefold higher K m and a slightly lower K m for acrylamide, whereas the MM15 enzyme exhibited a similar K cat and an eightfold lower K m for acrylamide. The MM15 enzyme also reacted more extensively with the thiol group reagent DTNB, had a significantly lower sedimentation coefficient and exhibited a more relaxed substrate specificity, all of which were compatible with a looser tetrameric structure. It was also much more susceptible than the other three enzymes to inactivation by high temperature or by freezing and thawing (MM15»MM8>MM6/wild-type), both of which variably dissociated the enzyme into inactive dimers and monomers. The amidase activity of strain MM15 was almost 15-fold higher following growth at 25 °C than at 37 °C, since at this lower temperature the enzyme exhibited a similar K cat to the MM8 enzyme and was not significantly dissociated. However, as strain MM15 readily outgrew the organism from which it was derived (strain MM8) during acrylamide-limited continuous culture at 37 °C, it is clear that under these conditions a low K m was a greater selective advantage than a high K cat.

Triton X-100 (TX-100) extraction of Spiroplasma citri plasma membrane solubilized two types of ATPase differing in their pH of maximum activity. The activity measured at pH 8·5 was inhibited by vanadate and the activity measured at pH 6·5 was not. The vanadate-sensitive ATPase had a relatively basic isoelectric point (8·65) and therefore could be separated from the vanadate-insensitive ATPase using chromatofocusing. Elution of the TX-100 membrane extract in a pH gradient from 9 to 6 generated two peaks of ATPase activity: one in the acidic range, composed of an F0F1-type ATPase, and one in the basic range, corresponding to the vanadate-sensitive activity. Electrophoretic analysis of proteins from the latter peak revealed one major polypeptide of 37 kDa. This peptide was shown to correspond to spot A37 in a two-dimensional protein map of S. citri. Using the gene for the kdp-operon of Escherichia coli as a probe in heterologous hybridization, sequences were detected in the genomic DNA of S. citri, suggesting that a gene coding for an enzyme related to this P-type ATPase is present in the S. citri genome. We therefore postulate the presense of two distinct kinds of ATPase in S. citri: one of the F-type which is resistant to vanadate inhibition, and one, probably of the P-type, which is vanadate-sensitive.

The polar lipids and their fatty acid components in Pseudomonas caryophylli, Pseudomonas gladioli and Pseudomonas pickettii have been identified. In addition to diphosphatidylglycerol and phosphatidylglycerol (a trace only for P. pickettii), all three species contained two forms of phosphatidylethanolamine differing in the presence or absence of α-hydroxy fatty acids. This seems to be a distinctive feature of species in Pseudomonas RNA homology group II. Also, P. caryophylli and P. gladioli (but not P. pickettii) produced two forms of ornithine amide lipid, differing in the nature (hydroxy or non-hydroxy) of the ester-linked fatty acid. In all three species, the major non-hydroxy acids were hexadecanoic acid, a hexadecenoic acid, an octadecenoic acid, and cyclopropane derivatives of the monoenoic acids. The α-hydroxy acids were the derivatives of the same components, while the amide-linked acid of the ornithine amide lipids was mainly or entirely 3-hydroxyhexadecanoic acid. The possible taxonomic implications of the data are discussed.

Summary: Root surface colonization by Azospirillum brasilense Cd of tomato, pepper and cotton plants under normal growth conditions and soybean plants under normal and water-stress conditions was monitored by scanning electron microscopy and bacterial counts. A. brasilense Cd was capable of efficiently colonizing the elongation and root-hair zones of all four plant species tested. In these zones, the bacteria mainly colonized the root surface (tomato, soybean), root-hairs (pepper), or both (cotton), by single cells (tomato, soybean), micro-aggregates (pepper), or a combination of the two (cotton). All inoculated plants demonstrated (i) larger amounts of mucigel-like substance on the root surface than non-inoculated plants and (ii) fibrillar material which anchored the bacterial cells to the root surface and established connections between cells within bacterial aggregates. On non-water-stressed soybean plants, most A. brasilense Cd cells in the rhizosphere occurred as vibroid forms whereas those on water-stressed plants (wilting) were cyst-like. A lower rhizosphere bacterial population was observed on water-stressed plants. When water-stress conditions were eliminated, the bacterial cells reverted to the vibroid form and a concomitant increase in the bacterial population was observed. It is suggested that cyst-like formation is a natural response for A. brasilense Cd in the rhizosphere of water-stressed plants.

Summary: The 2·6 kb ColE7-K317 plasmid was mapped and the DNA fragments of the colicin E7 operon subcloned into pUC18 and pUC19. The size of the functional colicin E7 operon deduced by subcloning was 2·3 kb. The colicin E7 gene product was purified by carboxymethylcellulose chromatography. Both colicin E7 and E9 were demonstrated to exhibit a non-specific DNAase-type activity by in vitro biological assay. The molecular mass of colicin E7 was 61 kDa, as determined by SDS-PAGE. From DNA sequence data, the estimated sizes of the E7 immunity protein and the E7 lysis protein were 9926 Da and 4847 Da, respectively. Comparison of restriction maps and DNA sequence data suggests that ColE7 and ColE2 are more closely related than other E colicin plasmids.

Summary: The most common effect of transposon insertion is the inactivation of genes. However, in some cases, transposons can activate in cis the expression of genes in the neighbourhood of their integration site. We previously described an insertion of the transposon Tn917 into the Bacillus subtilis sacXY locus. sacX and sacY encode respectively a negative and a positive regulator involved in induction by sucrose of the exoenzyme levansucrase. Data in this paper show that the Tn917 insertion had two effects: it inactivated sacX and it increased the transcription of sacY. The latter effect involved one or several elements internal to the transposon.

Summary: Engineered variants of the transposon Tn917 have been widely used to obtain insertion mutations and transcriptional fusions in Bacillus subtilis and other Gram-positive bacteria. We have developed a novel Tn917-based methodology useful for isolation and characterization of mutants resulting from gene over-expression. A Tn917 variant was constructed which contains a strong out-facing promoter near one end, able to promote transcription of genes in the vicinity of its insertion target. This transposon, designated Tn917PF1, was tested in model conditions. Three Tn917PF1 mutants of B. subtilis, with phenotypes presumed to result from gene over-expression, were analysed. Their phenotypes were shown to be due to transcription from the transposon promoter. In one mutant the promoter activated a deg gene, probably degQ. The other two contained different insertions decryptifying a B. subtilis gene encoding β-galactosidase.

Summary: The serC-aroA operon of Salmonella gallinarum was isolated from a gene library using a labelled oligonucleotide probe and by complementation of an aroA Escherichia coli strain. The nucleotide sequence of a 2·6 kbp fragment was determined. The predicted amino acid sequence of the aroA gene product was compared to the equivalent sequence from ten other organisms. Computer-generated evolutionary trees clearly divide the eleven sequences into four different groups: Gram-negative bacteria, Gram-positive bacteria, fungi and plants. These trees depict a close evolutionary relationship between the sequences from Gram-negative bacteria and higher plants.

Two distinct xylanase genes (designated xynA and xynB) were subcloned in pUC13 from non-homologous restriction fragments of Ruminococcus flavefaciens 17 DNA originally isolated in λ EMBL3. The products of the two genes showed similar pH optima for hydrolysis of oat spelt xylan (around 5·5) and had little or no activity against carboxymethylcellulose. Trace activities against p-nitrophenyl (pNP) cellobioside and pNP-xyloside were detected in clones containing xynA, but not in one harbouring xynB. The xylanase associated with clones carrying xynA produced mainly xylobiose and xylose from xylan and did not give hydrolysis of xylobiose, while that encoded by xynB produced mainly xylobiose and higher xylo-oligosaccharides from xylan. There was evidence of increased expression, at the RNA level, of these two genes, and of another cloned region encoding multiple activities including xylanase, in R. flavefaciens 17 grown with xylan, as compared with cellobiose, as energy source. Total cell-associated xylanase and β-xylosidase activities, and supernatant xylanase activity, were shown to be similarly induced in xylan-grown R. flavefaciens, 17.

Summary: Two pectinesterase-positive Escherichia coli clones, differing in expression levels, were isolated from a genomic library of Pseudomonas solanacearum. Both clones contained a common DNA fragment which included the pectinesterase-encoding region. The different expression levels found with the two clones could be ascribed to different positioning of the pectinesterase gene with respect to a vector promoter. Restriction analysis, subcloning, and further exonuclease deletion mapping revealed that the genetic information for pectinesterase was located within a 1·3 kb fragment. A protein of 41 to 42 kDa was expressed from this fragment. Nucleotide sequence analysis of the respective region disclosed an open reading frame of 1188 bp. The deduced polypeptide had a calculated molecular mass of 41 004 Da, which is consistent with the determined size of the pectinesterase protein. The predicted amino acid sequence showed significant homology to pectinesterases from Erwinia chrysanthemi and tomato. In cultures of E. coli clones up to 30% of total pectinesterase activity was transported into the medium. However, no significant pectinesterase activity could be detected in the periplasm.

Summary: Two β-lactamase genes present in the chromosome of Yersinia enterocolitica have been cloned individually into the plasmid pACY184 and expressed in Escherichia coli. The gene for broad-spectrum β-lactamase I (‘A’) was cloned from a strain belonging to the O:3 serotype, and the gene for (cephalosporinase) β-lactomase II (‘B’) was cloned from a strain of the O:5b serotype. The properties of the β-lactamases expressed in E. coli are similar to those previously described in Y. enterocolitica.

The aroD gene from Salmonella typhi, encoding 5-dehydroquinate hydrolyase (3-dehydroquinase), has been cloned into Escherichia coli and the DNA sequence determined. The aroD gene was isolated from a cosmid gene bank by complementation of an S. typhimurium aroD mutant. Analysis of the DNA sequence revealed the presence of an open reading frame capable of encoding a protein of 252 amino acids with a calculated M r of 27706. Comparison of the deduced S. typhi 3-dehydroquinase protein sequence with that elucidated for E. coli revealed 69 % homology. Alignment of the S. typhi sequence and equivalent Aspergillus nidulans and Saccharomyces cerevisiae sequences showed that homology was lower, at 24%, but still significant. Use of a minicell expression system demonstrated that a polyclonal antibody raised against E. coli 3-dehydroquinase cross-reacted with its S. typhi counterpart.

Summary: Two chromosomal fragments of the severe strain (SAY) of the western aster yellows mycoplasma-like organism (MLO) were cloned in Escherichia coli. These fragments were used to probe Southern blots of DNA extracted from plants infected with geographically and pathologically diverse MLOs. The two SAY probes hybridized with some, but not all, of the virescence-inducing MLOs examined, indicating that some of these MLOs are genetically related. Comparisons of restriction fragment length polymorphisms of MLO DNA established additional relationships within this group. The SAY-MLO probes did not hybridize with decline-inducing MLOs, plant pathogenic spiroplasmas, or healthy plants.

Summary: UV irradiation treatment of the asexual yeast Candida tropicalis gave rise to morphological mutants exhibiting at least four different types of abnormal colonies on glucose-containing solid medium. These mutants were named according to their colony morphologies: ‘doughnut’, ‘frilly’, ‘echinoid’ and ‘walnut’ mutants. The doughnut mutant produced a wrinkled colony with a hollow in its central region that was rich in filamentous pseudohyphal cells. With increased incubation time, the colony gradually changed to a reticulate shape. The parent strain, which normally produced smooth colonies, gave similar colonies to those of the doughnut mutant when grown in medium containing oleic acid as carbon source. Both the frilly and the walnut mutants produced pseudohyphal cells in a similar fashion to the doughnut mutant. The echinoid mutant produced an echinulate colony morphology with aerial hyphae and contained true hyphal cells as well as pseudohyphal ones. Pulsed-field gel electrophoresis showed that the echinoid and frilly mutants had different karyotypes from that of their parent strain, suggesting the occurrence of chromosomal rearrangements associated with these morphological mutations.