- Volume 141, Issue 9, 1995

The Thiobacillus ferrooxidans thioredoxin gene, trxA, was isolated by its ability to complement an Escherichia coli gshA trxA mutant which was otherwise unable to grow on minimal medium lacking glutathione. The T. ferrooxidans thioredoxin also enabled the in vivo reduction by E. coli of methionine sulfoxide to methionine, as well as the in vitro reduction of insulin. When present in E. coli, the T. ferrooxidans thioredoxin supported the replication of phage T7, but not the growth of phage M13. The T. ferrooxidans trxA gene was sequenced and the thioredoxin was found to be most like that of E. coli (71% identity) and Chromatium vinosum (70% identity). As in the case of E. coli, the gene was located immediately upstream of the gene for the rho transcriptional terminator. DNA:RNA blot hybridization and primer-extension analysis of the trxA gene in T. ferrooxidans and the cloned gene in E, coli indicated that it was transcribed as an independent unit and that the major transcriptional start sites were the same in both organisms.

An 837 nt long group IA intron was discovered in the Lactobacillus delbrueckii subsp. lactis virulent phage LL-H genome. The LL-H intron conforms well to the secondary structure that is common to all group I introns. The only exception is that the extreme 3' nucleotide of the intron is an A residue instead of the usual G; despite this the intron is efficiently spliced in vivo. This LL-H intron contains an ORF, ORF168, which shows homology with endonucleases encoded by ORFs contained in Bacillus subtilis phage introns. At present, the LL-H intron is the only one found in the phages of lactic acid bacteria and the first one to be found in a phage belonging to the most abundant taxonomic group, group B or Siphoviridae. The LL-H intron interrupts gene terL, the product of which (50.5 kDa, TerL) is significantly homologous to the large subunit of B. subtilis phage SPP1 terminase. The product of the upstream gene, terS of LL-H (15.9 kDa, TerS), shows homology to small subunits of B. subtilis phage terminases.

The ability of the bacteriophage 434 operator/repressor system to function in a eukaryotic cell has been explored. An idealized 434 operator was placed at various positions in the PGK promoter of Saccharomyces cerevisiae: within the upstream activator sequence, close to the TATA box, and downstream of the transcription-initiation site. Expression of the 434 cl gene from a 2 μm-based plasmid resulted in significant repression of gene expression from constructs containing the altered promoters linked to a β-galactosidase reporter gene. Attempts to use a variant of the 434 repressor that has the binding specificity of the P22 repressor (434P22) were unsuccessful, due to a severely inhibitory effect of this gene-product on the growth of the yeast cells.

Two mutants carrying different deletions of the IMP2 coding sequence of Saccharomyces cerevisiae, Ã1, which encodes a protein lacking the last 26 C-terminal amino acids, and Ã2, which completely lacks the coding region, were analysed for derepression of glucose-repressible maltose, galactose, raffinose and ethanol utilization pathways in response to glucose limitation. The role of the IMP2 gene product in the regulation of carbon catabolite repressible enzymes maltase, invertase, alcohol dehydrogenase, NAD-dependent glutamate dehydrogenase (NAD-GDH) and L-lactate:ferricytochrome-c oxidoreductase (L-LCR) was also analysed. The IMP2 gene product is required for the rapid glucose derepression of all above-mentioned carbon source utilization pathways and of all the enzymes except for L-LCR. NAD-GDH is regulated by IMP2 in the opposite way and, in fact, this enzyme was released at higher levels in both imp2 mutants than in the wild-type strain. Therefore, the product of IMP2 appears to be involved in positive and negative regulation. Both deletions result in growth and catalytic defects; in some cases partial modification of the gene product yielded more dramatic effects than its complete absence. Moreover, evidence is provided that the IMP2 gene product regulates galactose- and maltose-inducible genes at the transcriptional level and is a positive regulator of maltase, maltose permease and galactose permease gene expression.

The purT gene from Bacillus subtilis encoding the formate-dependent glycinamide ribonucleotide transformylase T was cloned by functional complementation of an Escherichia coli purN purT double mutant. The nucleotide sequence revealed an open reading frame of 384 amino acids. The purT amino acid sequence showed similarity to the enzyme phosphoribosylaminoimidazole carboxylase encoded by the purK gene but not to the N 10-formyltetrahydrofolate-dependent glycinamide ribonucleotide transformylase N enzyme encoded by the purN gene. The glycinamide ribonucleotide transformylase T level was repressed in cells grown in rich medium compared to minimal-medium-grown cells. However, when the culture entered the stationary-growth phase the enzyme level increased in rich medium and decreased in minimal medium. By comparing the deduced amino acid sequence of the B. subtilis purT gene product with translated nucleotide sequences in various databanks, evidence for the existence of putative purT genes in the Gram-negative bacteria Pasteurella haemolytica and Pseudomonas aeruginosa was obtained.

We report the complete nucleotide sequence and over-expression of the nahOM genes for the acetaldehyde dehydrogenase (acylating) and the 4-hydroxy-2-oxovalerate aldolase from the meta pathway operon of the naphthalene catabolic plasmid pWW60-22 from Pseudomonas sp. NCIMB9816. Additional partial sequence analysis of adjacent DNA shows the gene order within the operon to be nahNLOMK, identical to the order found for the isofunctional genes in the meta pathway operons in the toluene/xylene pathway of TOL plasmid pWWO and the phenol/methylphenol pathway of pVI150. The deduced amino acid sequences of NahO and NahM were significantly homologous to the equivalent enzymes encoded by other Pseudomonas meta pathways, although both were the most divergent in each comparison. The nahOM genes were inserted downstream of the T7 promoter in the expression vector pET3a and similar constructs were also made of the isofunctional regions from pVI150 (dmpFG) and TOL plasmid pDK1 (xylQK). High expression of all three gene pairs was detected by enzyme assays and by SDS-PAGE.

The exact identity of cyanobacteria that have been cultured from symbiotic associations with the water fern Azolla spp., whether they are required in the symbiotic process, and their relationship to the symbiotic species, is a matter of some debate. We have characterized a 6 kb region containing the nifB operon and the nifH gene from cyanobacterium Anabaena azollae 1a, a putative symbiont of Azolla caroliniana. Five complete open reading frames have been sequenced. All are very highly conserved when compared with the corresponding regions of Anabaena sp. PCC 7120, with 93% to 97% identity at the nucleotide level and 93% to almost 100% at the amino-acid level. The intergenic regions, however, are not highly conserved (53-89% identity) when compared to the corresponding regions of Anabaena 7120: the A. azollae genome contains both more copies and more types of short tandemly repeated repetitive sequences than Anabaena 7120. The startpoints of transcription for both the nifB and nifH operons were mapped and found to be the same as those in Anabaena 7120. It was not possible to discern an improved consensus nif promoter sequence, but it was possible to define the likely extent of the promoter to within 40 bases upstream of the transcription start-point.

In the plant pathogen Xanthomonas campestris pv. campestris, fructose is transported by a specific phosphotransferase system (PTS). This PTS involves a multiphosphoryl transfer protein (MTP) encoded by the fruB gene, which was cloned and sequenced. fruB is part of a transcriptional unit together with the fruK gene, coding for 1-phosphofructokinase, which is located upstream from the fruA gene, coding for the fructose-specific permease (EIIB'BCFru). The amino acid sequence of the X. campestris MTP deduced from the fruB sequence shared 46% identical residues with an MTP identified in Rhodobacter capsulatus. The X. campestris MTP (837 amino acid residues) consists of three moieties: a fructose-specific enzyme-IIA-like N-terminal moiety (residues 1-148), followed by an HPr-like moiety (161-251) and an enzyme-l-like C-terminal moiety (274-837). The three domains are separated by two flexible hinge regions rich in proline and alanine residues. The construction of a fruB mutant confirmed the role of the MTP in fructose transport and phosphorylation in X. campestris.

Aspergillus nidulans wild-type and the extreme carbon catabolite derepressed mutant creAd-30 were characterized with respect to enzyme activities, metabolite concentrations and polyol pools all related to glycolysis, after growth on D-glucose. In the creAd-30 strain the enzymes hexokinase and fructose-6-phosphate reductase showed a two- and threefold increase in activity, respectively, whereas phosphofructokinase and pyruvate kinase activity decreased two- and threefold, respectively, in comparison with the wild-type strain. The most notable changes in metabolite concentrations were that fructose 2,6-bisphosphate and fructose 1,6-bisphosphate showed a 2.5-fold increase, whereas both pyruvate and citrate decreased in the creAd-30. Striking differences were found for the polyol concentrations measured for the two strains tested. Intracellular glycerol and arabitol concentrations were 10-fold higher and erythritol fivefold higher in creAd-30, whereas intracellular trehalose and mannitol were both decreased. The total internal polyol concentration appears to be constant at ~ 700 mol (g dry wt)-1. All polyols were also detected in high amounts in the culture filtrate of the creAd-30 mutant strain but no extracellular trehalose was found. The overall production of polyols in this strain was therefore much higher than in the wild-type. The high level of polyols produced and the changes in metabolite concentrations in the creAd-30 strain suggest that the differences in enzyme activities result in an altered flow through glycolysis leading to a more rapid formation of polyols which are subsequently secreted.

Five new mutants of Neurospora crassa that require supplementation with unsaturated fatty acids have been isolated. The mutants, designated pfa, are impaired in the synthesis of the polyunsaturated fatty acids α-linoleic or α-linolenic acid, but are able to synthesize oleic acid. The pfa mutants are thus distinct from previously described ufa mutants, which are unable to synthesize oleic acid. The five pfa mutants map to distinct loci, and have characteristic patterns of incorporation of [14C]acetate and [14C]oleate into their fatty acids.

The activity of haemagglutinins in plasmodia of Physarum polycephalum was measured under different culture conditions. The activity was markedly increased when the plasmodia were incubated in a non-nutrient salt medium. During starvation, significant amounts of haemagglutinins were found in the slime layer on the surface of the plasmodia. An increase in activity was not observed in the presence of actinomycin D or cycloheximide. Under starvation conditions, plasmodia are known to differentiate into either sclerotia (spherules) or fruiting bodies. Acceleration of haemagglutinin synthesis, however, was not always observed during spherulation and fruiting-body formation. Attempts to detect endogenous glycoconjugates that bind to the haemagglutinins were unsuccessful but we found that the haemagglutinins could bind to acidic polysaccharides produced by Escherichia coli K12. The bacterial glycoconjugates were purified and partially characterized. They contained N-acetylhexosamine residues which appeared to be important for binding with the haemagglutinins. It is possible that the haemagglutinins play a physiological role in the interaction with these organisms.

Spiroplasma melliferum cells utilize arginine via the arginine dihydrolase pathway. L-Arginine uptake by intact cells was a saturable process both as a function of time and arginine concentration (Km = 40 μM). Uptake was not affected by pH in the range pH 5.0-8.0, or by L-citrulline, D-arginine, L-histidine or L-canavanine at concentrations tenfold higher than that of L-arginine. In contrast, L-arginine uptake was markedly inhibited by L-ornithine and partially inhibited by L-lysine. Uptake was neither affected by protonophores nor by cation ionophores, but was inhibited by protease treatment or by the sulfhydryl reagents p-chloromercuribenzoate or N-ethylmaleimide. Sealed membrane vesicles prepared by fusing isolated S. melliferum membranes with asolectin-cholesterol vesicles catalysed a rapid exchange (t1/2 = 1 min) between arginine and ornithine. Exchange did not require ATP and could be demonstrated in both directions, i.e. with either arginine or ornithine trapped within vesicles. These observations suggest that the driving forces for arginine uptake by whole cells are the concentration gradients of arginine and ornithine formed by arginine metabolism.

Increasing the expression of various glycolytic operons in Zymomonas mobilis caused a significant decrease rather than increase in the glycolytic flux and growth rate. Because the relative decrease depended on the amount of overexpressed protein, and was independent of which enzyme was overexpressed, we attributed it to a protein burden effect. More specifically, we examined if the decrease in glycolytic flux could be explained by a decreased concentration of other glycolytic enzymes (for which glucokinase was used as a marker enzyme). Using the summation theorem of metabolic control theory we predicted the extent of this protein burden effect. The predictions were in good agreement with the experimental observations. This suggests that the negative flux control is caused either by a simple competition of the overexpressed gene with the expression of all other genes or by simple dilution. Furthermore, we determined the implications of protein burden for the determination of the extent to which an enzyme limits a flux. We conclude that a protein burden can cause a significant underestimation of the flux control coefficient, especially if the enzyme under investigation is a highly expressed enzyme.

‘Sphingomonas (formerly Pseudomonas) elodea’ produces the industrially important polysaccharide gellan when grown in media containing glucose. Glucose catabolic enzymes and enzymes of central carbon metabolism were assayed in crude extracts of glucose-grown cultures of this bacterium. Based on these analyses it was concluded that glucose is converted to either gluconate or glucose 6-phosphate and that both of these products are converted to 6-phosphogluconate, a precursor for the Entner-Doudoroff (ED) and pentose phosphate pathways. Phosphoglucoisomerase (Pgi) activity was detected, but the lack of phosphofructokinase activity indicated that the Embden-Meyerhof glycolytic pathway is non-functional for glucose degradation. Thus, this bacterium utilizes glucose mainly via the ED and pentose phosphate pathways. Enzyme analyses suggested the involvement of glucose-6-phosphate dehydrogenase (Zwf) in glucose utilization and CO2 production. The zwf gene was cloned from ‘S. elodea’ and partially sequenced, and a null zwf mutant was constructed. This mutant exhibited no Zwf activity in in vitro assays, grew normally on glucose minimal medium and accumulated biomass (cells plus gellan) and produced CO2 at the same rates as the parental strain. Potential explanations for this finding are provided. Clones carrying the pgi gene were isolated fortuitously.

Seventy-seven Brucella reference and field strains from different geographic origins and hosts representing the six recognized species and their different biovars were analysed for diversity of their genes encoding the major 25 and 36 kDa outer-membrane proteins (OMPs) by PCR-RFLP. The 25 kDa OMP is encoded by a single gene (omp25) whereas two closely related genes (omp2a and omp2b) encode and potentially express the 36 kDa OMP. Analysis of PCR products of the omp25 gene digested with nine restriction enzymes revealed two species-specific markers, i.e. the absence of the EcoRV site in all Brucella melitensis strains and an ~ 50 bp deletion at the 3' terminal end of the gene in all Brucella ovis strains. Analysis of PCR products of the omp2a and omp2b genes digested with 13 restriction enzymes indicated a greater diversity than the omp25 gene among the six Brucella species and within the Brucella abortus, Brucella suis, B. melitensis and B. ovis species. Greater polymorphism was also detected for the omp2b than for the omp2a gene, especially in B. ovis which seemed to carry two similar (but not identical) copies of omp2a instead of one copy each of omp2a and omp2b for the other Brucella species as was previously suggested by Ficht et al. (1990; Mol Microbiol 4, 1135-1142). Results of PCR-RFLP indicated that distinction can be made between Brucella species and some of their biovars, except between B. canis and B. suis bv. 3 and 4, on the basis of the size and diversity of their major OMP genes, and that it could be of importance for diagnostic, epidemiological and evolutionary study purposes.

Genistein and other flavonoids from host legumes are known to stimulate cells of the nitrogen-fixing soybean symbiont Rhizobium fredii to synthesize Nod factors, which function as signals during nodule initiation. Flavonoids also trigger R. fredii to secrete a set of signal-responsive (SR) proteins into the environment. By insertion mutagenesis, we showed that secretion of SR proteins by this organism has an absolute dependence on the regulatory gene nodD1. We isolated and sequenced nodD1 and nodD2 of R. fredii USDA257 and constructed strains containing additional, plasmid-borne copies of these genes. Extra copies of nodD1 had no effect on secretion of SR proteins, but extra copies of nodD2 rendered the process constitutive. Extracts from seeds of the soybean cultivars McCall and Peking can substitute for purified flavonoids as inducers of SR proteins. The nolXWBTUV locus is known to control cultivar-specific nodulation of McCall soybean in a negative, flavonoid-dependent manner. Inactivation of any of these genes prevented SR proteins from accumulating in culture fluids. Protein secretion in response to host signals was a characteristic of nine out of ten R. fredii strains tested. Immunological probes failed to detect SR3 or SR5 in mature soybean or cowpea nodules. Although the functions of these proteins remain unknown, their potential role in symbiosis is strengthened by the discovery that their accumulation depends on nodD1, nodD2 and nolXWBTUV.