- Volume 143, Issue 8, 1997

Summary: It was recently shown that the nematode-infecting fungus Arthrobotrys oligospora contains a saline-soluble lectin (designated AOL) that is a member of a novel family of fungal lectins sharing similar primary sequences and binding specificities. During saprophytic growth in liquid cultures, levels of AOL and AOL mRNA were found to vary depending on the growth phase of the mycelium and the carbon/nitrogen (C/N) ratio of the medium. AOL was not detected in young mycelium. In older mycelium (stationary growth phase) grown in media with low C/N ratios (1 or 6), AOL comprised 5-20% of the total amount of saline-soluble proteins present in the mycelium. Neither the lectin nor its transcript was detected in mycelia grown in medium with higher C/N ratios (≥150). Under conditions of nitrogen starvation, AOL was preferentially degraded in relation to the total amount of saline-soluble proteins present in the mycelium. During the infection of nematodes, the level of AOL protein and AOL mRNA increased significantly once the nematodes had been penetrated and digested. Large amounts of AOL accumulated in the trophic hyphae growing inside the nematode as visualized by immunofluorescence microscopy. Later, AOL labelling was detected outside the digested nematodes, preferentially in strands of aggregated hyphae and in newly developed trap cells. Electron microscopy showed that AOL was localized to the cytoplasm and the nucleus of both vegetative mycelium and trap cells, and in the trophic hyphae growing inside the infected nematodes. These results indicate that AOL functions as a storage protein during both saprophytic and parasitic growth.

Summary: In cells of the yeast Saccharomyces cerevisiae, trehalase activation, repression of CTT1 (catalase), SSA3 (Hsp70) and other STRE-controlled genes, feedback inhibition of cAMP synthesis and to some extent induction of ribosomal protein genes is controlled by the Ras-adenylate cyclase pathway and by the fermentable-growth-medium-induced pathway (FGM pathway). When derepressed cells are shifted from a non-fermentable carbon source to glucose, the Ras-adenylate cyclase pathway is transiently activated while the FGM pathway triggers a more lasting activation of the same targets when the cells become glucose-repressed. Activation of the FGM pathway is not mediated by cAMP but requires catalytic activity of cAMP-dependent protein kinase (cAPK; Tpk1, 2 or 3). This study shows that elimination of Sch9, a protein kinase with homology to the catalytic subunits of cAPK, affects all target systems in derepressed cells in a way consistent with higher activity of cAPK in vivo. In vitro measurements with trehalase and kemptide as substrates confirmed that elimination of Sch9 enhances cAPK activity about two- to threefold, in both the absence and presence of cAMP. In vivo it similarly affected the basal and final level but not the extent of the glucose-induced responses in derepressed cells. The reduction in growth rate caused by delation of SCH9 is unlikely to be responsible for the increase in cAPK activity since reduction of growth rate generally leads to lower cAPK activity in yeast. On the other hand, deletion of SCH9 abolished the responses of the protein kinase A targets in glucose-repressed cells. Re-addition of nitrogen to cells starved for nitrogen in the presence of glucose failed to trigger activation of trehalase, caused strongly reduced and aberrant repression of CTT1 and SSA3, and failed to induce the upshift in RPL25 expression. From these results three conclusions can be drawn: (1) Sch9 either directly or indirectly reduces the activity of protein kinase A; (2) Sch9 is not required for glucose-induced activation of the Rasadenylate cyclase pathway; and (3) Sch9 is required for nitrogen-induced activation of the FGM pathway. The latter indicates that Sch9 might be the target of the FGM pathway rather than cAPK itself.

Summary: The effect of the addition of trimethylamine N-oxide (TMAO) in the growth medium on Escherichia coli anaerobic fermentative and respiratory pathways was examined. Formate dehydrogenase H (FDH-H) activity was totally repressed by the addition of 40 mM TMAO, whereas the overall hydrogenase (HYD) activity was reduced by 25%. Accordingly, expression of lacZ operon fusions with the fdhF and hycB structural genes specifying FDH-H and HYD3 was reduced sevenfold and eightfold, respectively, leading to suppression of an active formate hydrogenlyase system. In contrast, global respiratory formate-dependent phenazine methosulphate reductase (FDH-PMS) activity, which consists of both the major anaerobic FDH-N enzyme and the aerobic FDH-Z isoenzyme, was increased approximately twofold. This was corroborated by a 2.5-fold stimulation of the sole fdoG-uidA transcriptional fusion which reflects the synthesis of the respiratory aerobic FDH-Z enzyme. In fdhD, fdhE or torA mutants lacking either FDH-PMS activity or TMAO reductase (TOR) activity, the formate hydrogenlyase pathway was no longer inhibited by TMAO. In addition, introduction of 30 mM formate in the growth medium was found to relieve the repressive effect of TMAO in the wild-type strain. When TMAO was added as terminal electron acceptor a significant enhancement of anaerobic growth was observed with the wild-type strain and the fdoG mutant. It was associated with the concomitant suppression of the formate hydrogenlyase enzymes. This was in contrast to the fdnG and torA mutants whose growth pattern and fermentative enzymes remained unaffected. Taken together, these results strongly suggest that formate-dependent reduction of TMAO via FDH-N and TOR reduces the amount of formate available for induction of the formate hydrogenlyase pathway.

Summary: By using MudJ (Kan, lac)-directed operon fusion technology, mutants of Salmonella typhi whose gene expression is induced under anaerobic growth conditions were isolated. Characterization of their phenotypes and regulatory properties revealed that two of the mutants were unable to use nitrate as a terminal electron acceptor in the absence of oxygen, suggesting that they were defective in nitrate reductase activity. Anaerobic induction of these fusions did not further increase in response to nitrate. Strains carrying an additional mutation in oxrA were constructed. They showed a lower level of β-galactosidase expression both aerobically and anaerobically; however, the ratios of anaerobic induction remained unaltered. These MudJ insertions mapped to the 17-19 min region of the chromosome. Based upon their phenotypes and mapping, one of the mutants probably possessed a modC (chlD):: MudJ insertion and the other a moaA (chlA):: MudJ insertion. A third mutant was unable to use either nitrate or fumarate as a terminal electron acceptor. All three mutants showed a reduced ability to enter into and proliferate within HEp-2 epithelial cells. The oxrA mutation enhanced entry and proliferation of both the wild-type cells and the three mutants. Taken together, these results suggest that anaerobic respiration plays a role in S. typhi invasiveness.

Summary: Cytochrome bd’ has been implicated in having an important role in microaerobic nitrogen fixation in the enteric bacterium Klebsiella pneumoniae, where it is expressed under all conditions that permit diazotrophy. In this paper the sequence of the genes encoding this terminal oxidase (cydAB) of Klebsiella pneumoniae and the characterization of a cyd mutant are reported. The deduced amino acid sequences support the proposal that His 19, His 186 and Met 393 provide three of the four axial ligands to the Fe of the three haems in the oxidase complex. The nitrogen-fixing ability of the mutant was severely impaired in the presence of low concentrations of oxygen compared with the wild-type bacterium. Only the wild-type organism was capable of microaerobic nitrogenase activity supported by fermentation products. It is proposed that formate dehydrogenase-O may be involved in supplying electrons to a respiratory chain terminated by the bd-type oxidase, which would remove inhibitory oxygen and supply ATP for nitrogenase activity.

Summary: The effects of specific growth rate and specific nutrient uptake rate on morphological differentiation of Streptomyces albidoflavus SMF301 were determined in chemostat cultures. Production of three types of proteases: chymotrypsin-like protease (CTP), trypsin-like protease (TLP) and metalloprotease (MTP) were analysed in relation to mycelium growth and spore formation. Production of CTP was closely linked to mycelium growth, whereas spore formation, TLP synthesis and MTP synthesis were inversely related to growth. Evaluation of various kinetic parameters [specific production rates of spores (q spo), TLP (q TLP), MTP (q MTP) and CTP (q CTP)] showed that mycelium growth rate and CTP production were optimal at 0.1 h−1, but submerged spore formation, TLP production and MTP production were optimal at 0.025 h−1. Changes in specific nutrient uptake rates [glucose (q glu), ammonium ion (q amn) and phosphate (q pho)] affected sporulation and protease production; limitation of carbon, nitrogen and phosphate stimulated spore, TLP and MTP production.

Summary: A-factor (2-isocapryloyl-3R-hydroxymethyl-γ-butyrolactone), produced in a growth-dependent manner, switches on secondary metabolite formation and morphological differentiation in Streptomyces griseus, presumably by binding to the A-factor receptor protein (ArpA)-DNA complex and releasing the repression caused by ArpA. In the A-factor-deficient mutant strain S. griseus HH1 a large deletion includes afsA which is required for A-factor production. Growth and aerial mycelium formation of strain HH1 on media containing high concentrations of sucrose, sorbitol, mannitol, KCI or NaCI was disturbed by the presence of a large amount of A-factor supplied either exogenously or by a high-copy-number plasmid carrying afsA. This disturbance did not occur on media of normal osmolality and was observed only when A-factor was supplied during the very early stage of growth, about 8 h after inoculation. In addition, neither the wild-type strain nor S. griseus KM7 defective in ArpA exhibited the disturbance. These observations suggest that the presence of a large amount of A-factor during the very early stage of growth, probably during the A-factor-sensitive stage, triggered abrupt and disordered expression of some genes. The effect was apparently mediated through ArpA in the A-factor regulatory cascade and disturbed the physiology of strain HH1 under high osmolality. A gene that suppressed the disturbance was identified 5.5 kb upstream of the afsA locus in the wild-type strain. The gene, named sgaA, encoded a protein of 264 aa with a calculated molecular mass of 28 kDa.

Summary: Rhizobium (Sinorhizobium) meliloti strains capable of dissimilatory nitrous oxide reduction (Nos+) carry a nosRZDFY gene cluster on a 10.1 kb EcoRI fragment of the nod megaplasmid near the fixGHIS genes. These nos genes are arranged in three complementation groups and the 10.1 kb EcoRI fragment is sufficient to confer Nos activity to R. meliloti strains lacking such activity. An overlapping HindIII fragment containing the nosRZDFY genes but missing a 0.6 kb Hin dIII-EcoRI downstream segment was found incapable of imparting Nos activity to strains unable to reduce nitrous oxide, suggesting the presence of other nos gene(s) in this region. Tn5 introduced near the HindIII site resulted in mutants with a Nos− phenotype. Complete sequence analysis of nosY showed that it was well-conserved with respect to that of Pseudomonas stutzeri. Two previously unreported genes downstream of nosY in R. meliloti were also revealed. Contiguous with nosY was a sequence showing 63% identity with the ORFL protein of P. stutzeri. It appeared to be in the same operon as nosDFY and was predicted to encode a membrane lipoprotein similar to the putative NosL of P. stutzeri. Unlike the latter protein, however, amino acid sequences typical of metal-binding sites and cysteine residues indicative of the active site of protein disulphide isomerase were absent in the predicted NosL of R. meliloti. The Tn5 mutations resulting in a Nos− phenotype were localized within a 966 nucleotide gene 31 nucleotides downstream of nosDFYL with the same orientation. The new gene, nosX, was determined to be in a separate complementation group. It encoded a periplasmic protein with homology in the C-terminal domain with RnfF of Rhodobacter capsulatus and with a hypothetical Escherichia coli protein, YOJK. It was concluded that there are seven genes constituting the nos cluster in R. meliloti. They are organized in four complementation groups and in the same orientation, spanning a distance of about 9 kb on the nod megaplasmid.

Summary: Genetic diversity among 60 British Pasteurella trehalosi isolates representing the four recognized capsular serotypes, T3, T4, T10 and T15, and recovered predominantly from sheep suffering from systemic pasteurellosis, was estimated by analysing electrophoretically demonstrable allelic variation at structural genes encoding 19 enzymes. Thirteen of the loci were polymorphic and 20 distinctive multilocus genotypes (electrophoretic types, ETs) were identified. The population structure of P. trehalosi is clonal and its genetic diversity is limited compared with most other pathogenic bacteria. ETs represent clones, and isolates of the same ET were generally associated with the same combination of serotype, LPS type and outer-membrane protein (OMP) type. The genetic diversity of isolates within each of the capsular serotypes varied. Serotype T10 was represented by 18 isolates in two related ETs and exhibited little diversity. By contrast, serotype T15 was represented by 18 isolates in nine ETs and was almost as diverse as the species as a whole. Serotype T4 was represented by 18 isolates in five ETs and was less diverse than serotype T15. Although serotype T3 was more diverse than serotype T15 it was represented by only three isolates. With the exception of the T10 isolates and those recovered from healthy sheep, 35 disease isolates belonged to 16 ETs, each of which was represented by only one to four isolates. The fact that a high proportion of disease is caused by a relatively large number of clones suggests that P. trehalosi is essentially an opportunistic pathogen. In addition to having the same capsular structure, isolates belonging to the two T10 clones were characterized by possession of similar, if not identical, O-antigens (LPS types 2 and 4). The occurrence of 18 serotype T10 isolates in only two ETs suggests that the T10 capsule and type 2/4 O-antigen confer enhanced virulence on members of these two clones. Multilocus enzyme electrophoresis (MLEE) had greater resolving power than did capsule/LPS/OMP analysis, being able to distinguish 20 rather than 14 sub-divisions within P. trehalosi. The technique demonstrated genetic identity or non-identity among strains of the same or different serotypes from different geographic localities within the UK and was a useful epidemiological tool.

Summary: In the framework of the international project aimed at the sequencing of the Bacillus subtilis genome, five DNA fragments in the region between rrnB (275° and pai (284°) were cloned by inverse and combinatorial long-range PCR and their nucleotide sequences were determined and analysed. Together these sequences constituted a contig of 62229 bp. On the basis of the position of NotI and SfiI restriction sites, the orientation and order of known genetic markers was determined to be pai (284°) - degQ comQ comP comAA comAB - pbpD - kapB kinB patB - mcpB tlpA mcpA tlpB - rrnB (275°). Fifty-four ORFs were detected. Thirteen of these coincided with known B. subtilis genes, and 41 new ORFs were found. Of the predicted new gene products, 12 showed no significant similarity to other known proteins, whereas ten showed strong similarity to proteins of other organisms with unknown function. Nineteen predicted proteins showed strong similarity to known proteins of other organisms, for instance a Na+/H+ antiporter system of Bacillus alcalophilus, a sugar transport system found in Mycoplasma genitalium, NADH-dependent butanol dehydrogenase of Clostridium acetobutylicum, glucose-6-phosphate isomerase A of B. subtilis, exo-1,4-α-glucosidase activity of Bacillus stearothermophilus and L-rhamnose isomerase of Escherichia coli.

Summary: A 32 kb nucleotide sequence in the region of the lincomycin-resistance gene, located from 22° to 25° on the Bacillus subtilis chromosome, was determined. Among 32 putative ORFs identified, four [lipA for lipase, natA, natB and yzaE (renamed yccK)] have already been reported, although the functions of NatA, NatB and YccK remain to be characterized. Six putative products were found to exhibit significant similarity to known proteins in the databases, namely L-asparaginase precursor, protein aspartate phosphatase, x-glucosidase, two tellurite-resistance proteins and a hypothetical protein from B. subtilis. The region of the tellurite-resistance gene, consisting of seven ORFs, seems to correspond to an operon. The products of 14 ORFs exhibited considerable or limited similarity to known proteins. The sequenced region seems to be rich in membrane proteins, since at least 16 gene products appeared to contain membrane-spanning domains. The site of the lin-2 mutation (two nucleotide replacements) was mapped and identified by sequencing. This site is located between a putative promoter and the SD sequence of ImrA (yccB)[a putative repressor of the Imr operon, which consists of ImrA and ImrB (yccA)]. LmrB is a homologue of proteins involved in drug-export systems and seems likely to be the protein responsible for resistance to lincomycin.

Summary: In this study a description is given of the sequence and analysis of 52 kb from the 1.1 Mb genome of Rickettsia prowazekii, a member of the α-Proteobacteria. An investigation was made of nucleotide frequencies and amino acid composition patterns of 41 coding sequences, distributed in 10 genomic contigs, of which 32 were found to have putative homologues in the public databases. Overall, the coding content of the individual contigs ranged from 59 to 97%, with a mean of 81%. The genes putatively identified included genes involved in the biosynthesis of nucleotides, macromolecules and cell wall structures as well as citric acid cycle component genes. In addition, a putative identification was made of a member of the regulatory response family of two-component signal transduction systems as well as a gene encoding haemolysin. For one gene, the homologue of metK, an internal stop codon was discovered within a region that is otherwise highly conserved. Comparisons with the genomic structures of Escherichia coli, Haemophilus influenzae and Bacillus subtilis have revealed several atypical gene organization patterns in the R. prowazekii genome. For example, R. prowazekii was found to have a unique arrangement of genes upstream of dnaA in a region that is highly conserved among other microbial genomes and thought to represent the origin of replication of a primordial replicon. The results presented in this paper support the hypothesis that the R. prowazekii genome is a highly derived genome and provide examples of gene order structures that are unique for the Rickettsia.