- Volume 134, Issue 12, 1988

The electron transfer system that transports electrons from formate to nitrogenase was examined in extracts of the obligate methanotroph Methylosinus trichosporium OB3b. By supplementing a crude nitrogenase extract with NAD, formate dehydrogenase, NADH-ferredoxin reductase and FMN (an FDH cofactor), an electron transport system was established that coupled formate oxidation to nitrogenase-dependent acetylene reduction. The ferredoxin dependence of this reaction was demonstrated by its severe inhibition by antibodies to ferredoxin. The reaction sequence is as follows: formate → formate dehydrogenase → NADH → NADH-ferredoxin reductase → ferredoxin → nitrogenase.

The presence of multiple bromoperoxidases in extracts of Streptomyces griseus Tü 6 was detected. The enzyme pattern varied with the age of the culture. A haem-type bromoperoxidase (BPO 2) was always present. Additionally three nonhaem-type bromoperoxidases (BPO 1a, 1b and 3) were detected and purified to homogeneity. The M r of non-denatured BPO 1a was 70000 ± 10000 and those of BPO 1b and 3 were 90000 ± 5000. BPO 1a and 1b were dimers with subunit M r values of 34000 and 43000, respectively. BPO 3 was a trimer with a subunit M r of 31000. The enzymes differed in their isoelectric points, heat stability, and K m values. In immunodiffusion experiments BPO 1a and 3 showed partial identity with the nonhaem-type bromoperoxidase from Streptomyces aureofaciens. The nonhaem-type BPO 1a, 1b and 3 had neither peroxidase nor catalase activity.

The yeast Candida parapsilosis possesses two routes of electron transfer from exogenous NAD(P)H to oxygen. Electrons are transferred either to the classical cytochrome pathway at the level of ubiquinone through an NAD(P)H dehydrogenase, or to an alternative pathway at the level of cytochrome c through another NAD(P)H dehydrogenase which is insensitive to antimycin A. Analyses of mitoplasts obtained by digitonin/osmotic shock treatment of mitochondria purified on a sucrose gradient indicated that the NADH and NADPH dehydrogenases serving the alternative route were located on the mitochondrial inner membrane. The dehydrogenases could be differentiated by their pH optima and their sensitivity to amytal, butanedione and mersalyl. No transhydrogenase activity occurred between the dehydrogenases, although NADH oxidation was inhibited by NADP+ and butanedione. Studies of the effect of NADP+ on NADH oxidation showed that the NADH:ubiquinone oxidoreductase had Michaelis-Menten kinetics and was inhibited by NADP+, whereas the alternative NADH dehydrogenase had allosteric properties (NADH is a negative effector and is displaced from its regulatory site by NAD+ or NADP+).

Valine dehydrogenase (VDH) was purified to homogeneity from cell-free extract of Streptomyces fradiae, which produces tylosin. The enzyme was purified 1508-fold in a 17·7% yield using a combination of hydrophobic chromatography and ion-exchange fast protein liquid chromatography. The M r of the native enzyme was determined to be 218000 and 215000, by equilibrium ultracentrifugation and size-exclusion high-performance liquid chromatography, respectively. The enzyme is composed of 12 subunits of M r 18000. Using analytical isoelectric focusing the isoelectric point of VDH was found to be 4·7. Oxidative deamination of l-valine was optimal at pH 10·6. Reductive amination of 2-oxoisovalerate was optimal at pH 8·8. The Michaelis constants (K m) were 1 mm for l-valine and 0·029 mm for NAD+. K m values for reductive amination were 0·80 mm for 2-oxoisovalerate, 0·050 mm for NADH and 22 mm for NH+ 4.

Substitutions of an aspartate or an arginine residue for the glycine residue at position 366 of the mature part of Bacillus subtilis levansucrase were obtained by mutagenesis. Quantitative estimation from immunoblot analysis showed that the two transient membrane forms of the modified proteins were present in the membrane at the same level as that of the wild-type protein. The proteolytic processing, which was previously shown to be the first step of the levansucrase secretion process, was not affected in these modified proteins. Results from pulse-chase experiments showed that the half-times for secretion of the modified levansucrases into the culture medium were nearly the same as that of the wild-type protein, but the amount of the modified proteins secreted was significantly reduced. Purified samples of the modified enzymes were subtilisin insensitive and possessed enzyme activities very similar to those of the wild-type enzyme. The results suggest that the 366 site probably belongs to a functional domain of the protein which could play an important role in the second step of the levansucrase secretion process.

A small collection of clostridia was surveyed for type II restriction endonucleases. Enzymes were detected in two organisms. Clostridium pasteurianum ATCC 6013 contains an isoschizomer of ThaI (FnuDII) [5′-CGCG-3′] and preliminary evidence suggests that cleavage generates bluntended fragments. Clostridium thermohydrosulfuricum DSM 568 contains an isoschizomer of MboI (Sau3A) [5′-GATC-3′] that is inactive on dam methylated substrates. The DNA of this latter organism shows dam methylation.

Pseudomonas fluorescens subsp. cellulosa was shown to express extracellular xylanases. Genes encoding these enzymes were isolated from a gene library of P. fluorescens subsp. cellulosa DNA, constructed in bacteriophage λ47.1. One of the phages (PXC) that expressed xylanase also conferred the ability to hydrolyse carboxymethylcellulose. An 11·8 kb HindIII DNA restriction fragment and a 6·2 kb EcoRI DNA fragment were subcloned from two distinct xylanase-expressing phages, into pUC18, to yield recombinant plasmids pGHJ4 and pGHJ5 respectively. Cells of Escherichia coli harbouring either of these two plasmids, or plasmid pJHH1 (comprising the cellulase gene from PXC, previously cloned on a 7·3 kb partial EcoRI DNA fragment in pUC18), expressed xylanase activity. The positions of the xylanase genes in the recombinant plasmids were elucidated by subcloning and transposon mutagenesis. In pJHH1 the xylanase gene was adjacent to the DNA region encoding the endoglucanase. The polysaccharide-degrading genes in pJHH1 were transcribed from different promotors. Hybridization studies revealed that the xylanase genes encoded by pGHJ4 and pGHJ5 showed strong homology. All three cloned enzymes cleaved p-nitrophenyl β-d-glucopyranoside and 4-methylumbelliferyl β-d-cellobioside. Xylan and glucose did not affect expression of xylanase in E. coli strains harbouring pJHH1, pGHJ4 or pGHJ5.

Two extragenic suppressor mutations, sur0B20 and sur0F1, which restore the sporulation of spo0B or spo0F mutants of Bacillus subtilis to the wild-type level, were obtained. These suppressor mutations were located in the spo0A gene. Their location is close to that of the sof-1 mutation, which suppresses spo0B, spo0E and spo0F mutations. However, spo0 strains bearing the sur0B20 mutation differed in several phenotypic characteristics from spo0 mutants bearing the sof-1 suppressor. Nucleotide sequence analysis revealed that the sur0B20 and sur0F1 mutations resulted in Glu14 to Val and Asn12 to Lys conversion, respectively, in the spo0A gene. This result indicates that sur0B20 is a new suppressor of spo0B and spo0F mutations, whereas sur0F1 is identical to sof-1.

A new ‘Bacillus natto’ gene, senN, that regulates the expression of several extracellular proteins in B. subtilis has been cloned and sequenced. senN codes for a small, highly basic protein with an amino acid sequence different from the products coded by the regulatory genes sacQ, sacV, prtR and hpr. SenN stimulates gene expression at the transcriptional level. A putative homologous locus has been detected in the B. subtilis chromosome by Southern blotting.

We have previously shown that some strains of Neisseria cinerea can serve as recipients in conjugation (Con+) with Neisseria gonorrhoeae while others cannot (Con-). To determine if a replication defect contributes to the inability of certain strains of N. cinerea to serve as recipients in conjugation, we attempted to introduce a naturally occurring gonococcal β-lactamase plasmid into N. cinerea by transformation. Various methods were employed, and all proved unsuccessful. Since specific sequences are required for DNA uptake in transformation of N. gonorrhoeae, we constructed a number of hybrid plasmids containing N. cinerea chromosomal DNA inserted into the N. gonorrhoeae/Escherichia coli β-lactamase shuttle vector, pLES2. When nine randomly selected plasmids with inserts were used to transform an N. cinerea strain which did not accept the gonococcal β-lactamase plasmid by conjugation, transformants were observed with four of the hybrid plasmids. The presence of one of the hybrid plasmids, pCAG9, in transformants was confirmed by agarose gel electrophoresis, Southern hybridization, and β-lactamase production. When an N. gonorrhoeae donor strain containing pCAG9 was used in conjugation with several N. cinerea strains, only those strains that were previously shown to act as recipients could accept and maintain pCAG9. The ability of pCAG9 and the other three hybrid plasmids to transform Con− strains demonstrates that the β-lactamase plasmid can replicate in Con− strains, and, therefore, the Con− phenotype is due to a block in some other stage of the conjugation process.

Little is known concerning the mechanism involved in cell contact between the donor and recipient during conjugation in Neisseria gonorrhoeae. The formation of stable mating pairs during conjugation in Escherichia coli appears to require a specific protein as well as LPS in the outer membrane of the recipient cell. To attempt to identify the cell surface components necessary for conjugation in the neisseriae, we began a comparison of the outer membrane of Neisseria cinerea strains that can (Con+) and cannot (Con−) serve as recipients in conjugation with N. gonorrhoeae. There were no differences in outer-membrane protein profiles on SDS-polyacrylamide gel electrophoresis between Con+ and Con− strains that could be correlated with the ability to conjugate. However, whole outer membrane isolated from Con+ strains specifically inhibited conjugation while those from Con− strains did not. Proteolytic cleavage of outer-membrane proteins by trypsin, pronase or α-chymotrypsin abolished the inhibitory effect of Con+ outer membranes, suggesting that these outer membranes contained a protease-sensitive protein(s) involved in conjugation. Although periodate oxidation of Con+ outer-membrane carbohydrates did not abolish the inhibitory action of these membranes, purified LPS from both Con+ and Con− strains inhibited conjugation when added at low concentrations. These results suggest that conjugation requires the presence of a specific conjugal receptor that consists of both LPS and one or more outer-membrane proteins. Both Con+ and Con− strains contain the necessary LPS, but only Con+ strains contain the required protein(s).

Agrobacterium radiobacter NCIB 11883 was grown in glucose-limited continuous culture at low dilution rate. Whole cells transported glucose using an energy-dependent mechanism which exhibited an accumulation ratio > 2000. Three major periplasmic proteins were purified and their potential role as glucose-binding proteins (GBP) were investigated using equilibrium dialysis. Two of these, GBP1 (M r 36500) and GBP2 (M r 33500), bound d-glucose with high affinity (K D 0·23 and 0·07 μm respectively), whereas the third protein (M r 30500) showed no binding ability. Competition experiments using various analogues showed that those which differed from glucose at C-6 (e.g. 6-chloro-6-deoxy-d-glucose and 6-deoxy-d-glucose) variably decreased the binding of glucose to both GBP1 and GBP2, whereas those which differed at C-4 (e.g. d-galactose) were only effective with GBP1. The rate of glucose uptake and the concentration of the glucose-binding proteins increased in parallel during prolonged growth under glucose-limitation due to the emergence of new strains in which GBP1 (e.g. strain AR18) or GBP2 (e.g. strain AR9), but not both, was hyperproduced and accounted for at least 27% of the total cell protein. It is concluded that A. radiobacter synthesizes two distinct periplasmic binding proteins which are involved in glucose transport, and that these proteins are maximally derepressed during growth under glucose limitation.

Agrobacterium radiobacter NCIB 11883 was grown in ammonia-limited continuous culture at low dilution rate with glucose as the carbon source. Under these conditions the organism produced an extracellular succinoglucan polysaccharide and transported glucose using the same periplasmic glucose-binding proteins (GBP1 and GBP2) as during glucose-limited growth. Transition from glucose- to ammonia-limited growth was accompanied by a very rapid decrease in glucose uptake capacity, whereas the glucose-binding proteins were diluted out much more slowly (t 1/2 approximately 1 h and 14 h respectively). Although the rate of glucose uptake and the concentrations of GBP1 and GBP2 were much lower during ammonia limitation, the activities of enzymes involved in the early stages of glucose metabolism and in the production of succinoglucan precursors were essentially unchanged. Glucose transport was also investigated in two new strains of A. radiobacter which had been isolated following prolonged growth under glucose limitation. Glucose uptake by strain AR18 was significantly less repressed during ammonia limitation compared with either the original parent strain or strain AR9, and this was reflected both in its relatively high concentration of GBP1 and in its significantly higher rate of succinoglucan synthesis. Flux control analysis using 6-chloro-6-deoxy-d-glucose as an inhibitor of glucose transport showed that the latter was a major kinetic control point for succinoglucan production. It is concluded that glucose uptake by A. radiobacter, particularly via the GBP1-dependent system, is only moderately repressed during ammonia-limited growth and that the organism avoids the potentially deleterious effects of accumulating excess glucose by converting the surplus into succinoglucan.

The fungicide Captan inhibited the growth of Azospirillum brasilense at a concentration of 25 μg ml−1. When cysteine and glutathione were added to the medium they removed the toxicity of the fungicide. A spontaneous mutant was isolated which was able to grow and fix nitrogen in the presence of 100 μg Captan ml−1. Characterization of the mutant indicated very high levels of glutathione and glutathione transferase activity as compared to the parent strain. The role of these cellular components in the mechanism of resistance to Captan is discussed; the involvement of a selenium-independent glutathione peroxidase appears essential to Captan resistance in the mutant strain.

Bacillus stearothermophilus IC3 degraded the meta cleavage product of catechol, 2-hydroxymuconic semialdehyde, to pyruvate and acetaldehyde via the 4-oxalocrotonate pathway. The pathway was identical to those previously delineated in several mesophilic organisms. However, all the enzymes showed activity at 55 °C and other properties (substrate specificities and effects of metal ions) also differed from those displayed by the mesophilic enzymes. All enzymes of this meta cleavage pathway, except the 2-hydroxy-6-oxohepta-2,4-dienoate hydrolase and 4-hydroxy-2-oxovalerate aldolase activities, were induced by growth on phenol.