- Volume 142, Issue 3, 1996

Lethal toxin from Bacillus anthracisis composed of protective antigen (PA) and lethal factor (LF). Anti-PA mAbs that neutralized lethal toxin activity, either in vivo or in vitro, identified three non-overlapping antigenic regions on PA. Two distinct antigenic regions were recognized by the four mAbs that neutralized lethal toxin activity by inhibiting the binding of 125I-LF to cell-bound PA. Mapping showed that one mAb, 1G3PA63, recognized an epitope on a 17 IcDa fragment located between amino acid residues Ser-168 and Phe-314. The three other mAbs, 2D3PA, 2D5PA and 10D2PA, recognized an epitope between amino acids Ile-581 and Asn-601. A single antigenic region was recognized by the three mAbs, 3B6PA, 14B7PA and 10E10PA63, that inhibited binding of 125I-PA to cells. This region was located between amino acids Asp-671 and lle-721. These results confirm previously defined functional domains of PA and suggest that LF may interact with two different sites on PA to form lethal toxin.

In Streptomyces coelicolor A3(2), polyketides are made from malonyl-CoA, which is presumed to be derived from acetyl-CoA by the action of acetyl-CoA carboxylase (ACC). No ACC activity was found in cell-free extracts of S. coelicolor. However, propionyl-CoA carboxylase (PCC) activity was detected at substantial levels. Fixation of CO2 by ACC and PCC occurs by covalent bonding of CO2 to a biotin-containing protein. Most bacteria have a single small biotinylated protein of approximately 22 kDa, but S. coelicolor contains three larger biotin-containing proteins (approximately 145,88 and 70 kDa). To determine which biotinylated protein was associated with PCC activity, the enzyme was purified and shown to comprise an α subunit (biotin-containing) of 88 kDa and a β subunit of 66 kDa. The N-terminal sequences of these proteins were determined and, using an oligonucleotide probe, the gene for the α subunit (pccA) was cloned.

The magnitude of the proton gradient (Δμ̄H+ ) driving solute accumulation in Saccharomyces Cerevisiae has long been in doubt, principally because of the lack of an agreed method for assaying its electrical component, the membrane potential (Δψ). In the present work, the size of the cytosine gradient (Δμ̄cyt) that the yeast generated was used as a measure of the driving gradient (Δμ̄H+ ). The selected yeast lacked cytosine deaminase and overexpressed cytosine permease, a 1 H+Zcytosine system, Δμ̄cyt, assayed in washed cell suspensions fermenting glucose and containing 0·5 or 50 mM KCI, was about 260 mV at pH 4 or 5, falling to about 194 mV at pH 7. As a first estimate, –Δμ̄H+ was thus at least as large at the respective pH value. A 20 mM solution of the lipophilic cation tetraphenylphosphonium lowered Δμ̄cyt to a value roughly equal to the magnitude of the pH gradient (ΔpH). A mathematical model was used to correct the first estimates of Δμ̄H+ for the effect of cytosine leakage outside the symport. In such a system, Δμ̄cyt cannot exceed the equivalent ratio V max/K m L, where V max and K m are kinetic parameters of the symport and L is the rate coefficient for leakage. The feasibility of assaying Δμ̄H+ depends on it not being much larger than that ratio. The model was tested successfully against observations made with yeast preparations depleted of ATP. After correction, –Δμ̄H+ during fermentation was estimated to be up to 25 mV larger than Δμ̄cyt and at least 70 mV larger than previous estimates in the literature involving lipophilic cations. From a knowledge of ΔpH, Δψ was in turn deduced and compared with the maximum methylamine gradient (Δμ̄M) the yeast formed. The results supported the claim in the literature that, at acid pH, Δμ̄M is a measure of Δψ.

The Gibberella fujikuroi niaD gene, encoding nitrate reductase, has been isolated and used to develop an efficient homologous transformation system. A cosmid vector designated pGFniaD was generated based on niaD selection and shown to give comparable transformation efficiencies. Using pGFniaD, a genomic library was prepared and used for genetic transformations, giving frequencies of up to 200 transformants per μg DNA. Of 15 transformants analysed by Southern blots, six showed homologous integration whilst the remaining nine integrated at heterologous sites, indicating that the vector may be used reliably for both types of integration. The system therefore may be used both for self-cloning of gibberellin biosynthetic genes on the basis of complementation of defective mutants, and also for gene disruption experiments. Electrophoretic karyotype determination suggested at least 11 chromosomes ranging from 2 to 6 Mb, the total genome size being at least 37 Mb. The niaD gene was assigned to chromosome V by Southern blot analysis. The niaD gene is interrupted by one intron, and remarkably the promoter sequence, but not the 3′ untranslated sequence, is highly homologous to that of the corresponding Fusarium oxysporum gene. This situation appears to be unique with respect to the promoter regions of corresponding genes in related species of filamentous fungi.

Since polyamines (PAs) play a potential role in the regulation of growth and developmental processes in a wide variety of organisms, we have examined the influence of the PAs putrescine (Put) and spermidine (Spd) and the PA biosynthetic inhibitors α-difluoromethylornithine (DFMO), α-difluoromethylarginine (DFMA), methylglyoxal bis-(guanylhydrazone) (MGBG) and cyclohexylamine (CHA), singly and in combinations on microcycle conidiation (MC) in Aspergillus flavus. The exogenous application of the diamine Put (concentrations ranging from 0·1 to 5 mM) caused a sharp decline of MC in a dose-dependent fashion, but induced vegetative growth. However, the triamine Spd (0·1-5 mM) had a minimal effect on MC and induced a shift from MC to normal conidiation. PA inhibitors, especially DFMO, MGBG and CHA, produced greater inhibition of MC and complete inhibition of MC was observed at 5 mM of these inhibitors. DFMA even at 5 mM had only a weak inhibitory effect on MC. DFMO also inhibited conidial germination and germ tube growth. MGBG and CHA, while having an inhibitory effect on MC, induced vegetative growth. The inhibitory effect of PA inhibitors was partially reversed by exogenous Put or Spd, with Spd being more effective than Put. The analysis of free PA levels during various phases of MC revealed that undifferentiated spores contained a high Put/Spd ratio and there was a dramatic decrease in Put/Spd ratio before and during microcycle conidiophore maturity. The change in spermine titres could not be detected. These observations imply that Put is essential for vegetative growth, while Spd is involved in MC, and that a low Put/Spd ratio seems to be important for spore differentiation to MC.

The 40 kDa secreted aspartyl proteinase (Sapt1) of Candida tropicalis is a pepsin-like enzyme encoded by the SAPT1 gene. According to the deduced amino acid sequence, Sapt1 has a putative preproregion of 60 amino acids preceding the mature enzyme. Maturation and processing of Sapt1 was analysed in C. tropicalis and Saccharomyces cerevisiae strains expressing wild-type or mutated forms of SAPT1. In S. cerevisiae the glycosylated 46 kDa proenzyme was converted to the mature 40 kDa form of Sapt1 by KEX2-dependent proteolytic cleavage following the Lys59-Arg60 sequence. The replacement of Lys59-Arg60 by Lys59-Gly60 revealed that the precursor can be processed by an autocatalytic cleavage. This alternative processing pathway to produce mature Sapt1 is less efficient than the Kex2-mediated pathway. Finally, it was shown that in C. tropicalis and S. cerevisiae the removal of the proregion was a prerequisite for the secretion of Sapt1.

A 40 kb genomic clone and 2·3 kb EcoRI subclone that rescued the DNA repair and recombination defects of the Aspergillus nidulans nuvA11 mutant were isolated and the subclone sequenced. The subclone hybridized to a cosmid in a chromosome-specific library confirming the assignment of nuvA to linkage group IV and indicating its closeness to bimD. Amplification by PCR clarified the relative positions of nuvA and bimD. A region identified within the subclone, encoding a C3HC4 zinc finger motif, was used as a probe to retrieve a cDNA clone. Sequencing of this clone showed that the nuvA gene has an ORF of 1329 bp with two introns of 51 bp and 60 bp. Expression of nuvA appears to be extremely low. The putative NUVA polypeptide has two zinc finger motifs, a molecular mass of 48906 Da and has 39% identity with the Neurospora crassa uvs-2 and 25% identity with the Saccharomyces cerevisiae RAD18 translation products. Although mutations in nuvA, uvs-2 and RAD18 produce similar phenotypes, only the nuvA11 mutation affects meiotic recombination. A role for nuvA in both DNA repair and genetic recombination is proposed.

Genomic DNA libraries of Streptomyces venezuelae ISP5230 and of a mutant blocked at the chlorination step of chloramphenicol biosynthesis were probed by hybridization with a synthetic oligonucleotide corresponding to the N-terminal amino acid sequence of a bromoperoxidase-catalase purified from the wild-type strain. Hybridizing fragments obtained from the two strains were cloned and sequenced. Analysis of the nucleotide sequences demonstrated that the fragments contained the same 1449 bp open reading frame with no differences in nucleotide sequence. The deduced polypeptide encoded 483 amino acids with a calculated M r of 54200; the N-terminal sequence was identical to that of the bromoperoxidase-catalase purified from wild-type S. venezuelae. Comparison of the amino acid sequence predicted for the cloned bromoperoxidase-catalase gene (bca) with database protein sequences showed a significant similarity to a group of prokaryotic and eukaryotic catalases, but none to other peroxidases or haloperoxidases. Replacement of the bca gene in the wild-type strain of S. venezuelae with a copy disrupted by insertion of a DNA fragment encoding apramycin resistance did not prevent chloramphenicol production. The results suggest that the role of the enzyme in S. venezuelae is related to its activity as a catalase rather than as a halogenating agent.

Mycobacterium smegmatis has two rRNA (rrn) operons designated rrnA f and rrnB f. Appropriate restriction fragments of genomic DNA containing sequences immediately upstream from the 16S rRNA genes were cloned. We now report the nucleotide sequence of 552 bp upstream from the 5′-end of the Box AL antitermination element of the leader region of the rrnA f operon. The 5′-end of this segment of DNA was found to comprise 113 codons of an ORF encoding a protein which is significantly similar to UDP-N-acetylglucosamine 1-carboxyvinyl-transferase (EC 2.5.1.7), which is important to cell wall synthesis. A homologous ORF is located immediately upstream from the single rrn (rrnA s) operons of Mycobacterium tuberculosis and Mycobacterium leprae. Primer-extension analysis of the RNA fraction of M. smegmatis revealed four products which were related to transcription start points; the rrnB f operon appears to have a single promoter whereas the rrnA f operon has three (P1, P2 and P3). Analysis of M. tuberculosis RNA revealed two products corresponding to transcripts directed by promoters homologous with P1 and P3 of the rrnA f of M. smegmatis. Thus, the promoter and upstream regions of the rrnA f operon of M. smegmatis and the rrnA s operon of M. tuberculosis are homologous. The presence of P2 in M. smegmatis and its absence from M. tuberculosis is attributable to insertions/deletions of 97 bp.

A 4·0 kb region of Methylobacterium extorquens AM1 DNA which complements three mutants unable to convert acetyl-CoA to glyoxylate (and therefore defective in the assimilation of methanol and ethanol) has been isolated and sequenced. It contains two ORFs and the 3′-end of a third one. The mutations in all three mutants mapped within the first ORF, which was designated meaA ; it encodes a protein having similarity with methylmalonyl- CoA mutase. However, methylmalonyl-CoA mutase was measured in extracts of one of the mutants and the specific activity was found to be similar to that in extracts of wild-type cells. Furthermore, although the predicted meaA gene product has the proposed cobalamin-binding site, it does not contain a highly conserved sequence (RIARNT) which is present in all known methylmalonyl- CoA mutases; meaA may therefore encode a novel vitamin-B12-dependent enzyme. The predicted polypeptide encoded by the second ORF did not have similarity with any known proteins. The partial ORF encoded a protein with similarity with the 3-oxoacyl-[acyl-carrier-protein] reductases; it was not essential for growth on methanol or ethanol.

The anaerobic transcriptional regulator ANR induces the arginine deiminase and denitrification pathways in Pseudomonas aeruginosa during oxygen limitation. The homologous activator FNR of Escherichia coli , when introduced into an anr mutant of P. aeruginosa , could functionally replace ANR for anaerobic growth on nitrate but not for anaerobic induction of arginine deiminase. In an FNR-positive E. coli strain, the ANR-dependent promoter of the arcDABC operon, which encodes the enzymes of the arginine deiminase pathway, was not expressed. To analyse systematically these distinct induction patterns, a lacZ promoter-probe, broad-host-range plasmid containing various –40 regions (the ANR/FNR recognition sequences) and –10 promoter sequences was constructed. These constructs were tested in P. aeruginosa and in E. coli expressing either ANR or FNR. In conjunction with the consensus –10 hexamer of E. coli σ 70 RNA polymerase (TATAAT), the consensus FNR site (TTGAT .…ATCAA) was recognized efficiently by ANR and FNR in both hosts. By contrast, when promoters contained the Arc box (TTGAC…. ATCAG), which is found in the arcDABC promoter, or a symmetrical mutant FNR site (CTGAT…. ATCAG), ANR was a more effective activator than was FNR. Conversely, an extended 22 bp, fully symmetrical FNR site allowed better activation with FNR than with ANR. Combination of the arc promoter –10 sequence (CCTAAT) with the Arc box or the consensus FNR site resulted in good ANR-dependent expression in P. aeruginosa but gave practically no expression in E. coli , suggesting that RNA polymerase of P. aeruginosa differs from the E. coli enzyme in –10 recognition specificity. In conclusion, ANR and FNR are able to activate the RNA polymerases of P. aeruginosa and E. coli when the –40 and –10 promoter elements are identical or close to the E. coli consensus sequences.

We have studied the mode of recombination of six insertions during genetic transformation of Streptococcus pneumoniae. The six heterologous insertions are located at the same site in the ami locus of the pneumococcal chromosome; insertion sizes range from 4 to 1374 bp. With respect to single-point markers we found that the number of transformants in one-point crosses is reduced, while the number of wild-type transformants in two-point crosses is drastically increased, what we call hyper-recombination. The magnitude of the shift is correlated with the size of the insert. This effect could result either from a special repair pathway of multibase heteroduplexes or from the exclusion of multibase heterologous insertions out of the pairing synapsis. To test these hypotheses we have used insertions in two kinds of three-point crosses. The repair model predicts that the excess of wild-type transformants remains in one set of crosses but is suppressed in the second set. The results we obtained are reversed, ruling out the hypothesis of a repair process, but in agreement with predictions based on the exclusion model. Moreover, we have re-examined the situation of deletions, our previous results suggesting that deletions were likely to be converted at the heteroduplex step. Genetic evidence we obtained in this work no longer supports this hypothesis. Thus, long heterologous insertions are partly excluded at the pairing step.

In order to analyse the involvement of the cAMP pathway in the regulation of gene expression in Saccharomyces cerevisiae, we have examined the effect of cAMP on protein synthesis by using two-dimensional gel electrophoresis. cAMP had only a minor effect on the protein pattern of cells growing exponentially on glucose. However, it interfered with the changes in gene expression normally occurring upon glucose exhaustion in yeast cultures, maintaining a protein pattern typical of cells growing on glucose. This effect was accompanied by a delay before growth recovery on ethanol. We propose a model in which the cAMP-signalling pathway has a role in the maintenance of gene expression, rather than in the determination of a specific programme. A decrease of cAMP would then be required for metabolic transitions such as the diauxic phase.

Decanoic acid, a lipophilic agent, inhibited in vitro the plasma membrane H+-ATPase of Saccharomyces cerevisiae grown in YPD medium. Conversely, when decanoic acid (35 μM) was present in the growth medium, the measured H+-ATPase activity was four times higher than that of control cells. K m, and pH and orthovanadate sensitivity were the same for the two growth conditions, which indicated that H+-ATPase activation was not due to conformational changes in the enzyme. The activation process was not entirely reversible which showed that plasma membrane H+-ATPase activation is due to several mechanisms. 1,6-diphenyl-1,3,5-hexatriene anisotropy performed on protoplasts from cells grown in YPD revealed that as decanoic acid concentration was increased, anisotropy significantly decreased, i.e. membrance fluidity increased. Cells grown in media containing decanoic acid exhibited greater membrane fluidity compared with control cells. Furthermore, these cells did not show any fluidifying effect when increased concentrations of decanoic acid were added. Chemical analysis of cell membrane lipid composition revealed a modification in the distribution of the phospholipid fatty acids and sterols in cells grown in the presence of 35 μM decanoic acid compared with control cells. Our results support the view that the plasma membrane H+-ATPase activation induced by decanoic acid is correlated with an alteration in membrane lipid constituents.

The Pseudomonas syringae cyclic lipodepsipeptide syringomycin inhibits the growth of Saccharomyces cerevisiae. A novel yeast gene, SYR2 was found to complement two syringomycin-resistant S. cerevisiae mutants. SYR2 was cloned, sequenced, and shown to encode a 349 amino acid protein located in the endoplasmic reticulum. SYR2 was identical to SUR2 which is involved in survival during nutritional starvation. Gene disruption or overexpression of SYR2 did not affect cell viability or ergosterol levels, but did influence cellular phospholipid levels. The findings suggest that phospholipids are important for the growth inhibitory action of syringomycin.

For survival, pathogenic organisms such as Candida albicans must possess an efficient mechanism for acquiring iron in the iron-restricted environment of the human body. C. albicans can use iron from a variety of sources found within the host. However, it is not clear how biologically active ferrous iron is obtained from these sources. One strategy adopted by some organisms is to reduce iron extracellularly and then specifically transport the ferrous iron into the cell. We have shown that clinical isolates of C. albicans do have a cell-associated ferric-reductase activity. The determination of ferric-reductase activity of cells growing exponentially in either low- or high-iron media over a period of time indicated that C. albicans reductase activity is induced when in low-iron conditions. Moreover, we have demonstrated that C. albicans reductase activity is also regulated in response to the growth phase of the culture, with induction occurring upon exit from stationary phase and maximal levels being reached in early exponential stage irrespective of the iron content of the medium. These results suggest that C. albicans reductase activity is regulated in a very similar manner to the Saccharomyces cerevisiae ferric-reductase. Iron reduction and uptake in S. cerevisiae are closely connected to copper reduction, and possibly copper uptake. In this report we show that iron and copper reduction also appear to be linked in C. albicans. The ferric-reductase activity is negatively regulated by copper. Moreover, quantitative cupric-reductase assays indicated that C. albicans is capable of reducing copper and that this cupric-reductase activity is negatively regulated by both iron and copper. This is the first report that C. albicans has an iron- and copper-mediated ferric-reductase activity.

Chlorate-resistant, highly branched (colonial) mutants and auxotrophic mutants were used to study the nuclear distribution, morphology and growth of heterokaryons of the Quorn® myco-protein fungus, Fusarium graminearum A3/5. The results showed that for several complementary homokaryons, even a strong selective pressure was insufficient to maintain heterokaryons in a ‘balanced’ condition (i.e. exhibiting a wild-type or near wild-type phenotype). Furthermore, the margins of heterokaryotic colonies generally contained nuclei from only one of the parental homokaryons, indicating imperfect nuclear mixing within the mycelium. These observations suggest that recessive, colonial mutants may appear during Quorn® myco-protein production following shear-induced separation of hyphal fragments which contain a sufficiently high ratio of colonial : wild-type nuclei for the colonial phenotype to be expressed.

In Escherichia coli, cAMP levels vary with the carbon source used in the culture medium. These levels are dependent on the cellular concentration of phosphorylated EnzymeIIAglc, a component of the glucose-phosphotransferase system, which activates adenylate cyclase (AC). When cells are grown on glucose 6-phosphate (Glc6P), the cAMP level is particularly low. In this study, we investigated the mechanism leading to the low cAMP level when Glc6P is used as the carbon source, i.e. the mechanism preventing the activation of AC by phosphorylated EnzymellAglc. Glc6P is transported via the Uhp system which is inducible by extracellular Glc6P. The Uhp system comprises a permease UhpT and three proteins UhpA, UhpB and UhpC which are necessary for uhpT gene transcription. Controlled expression of UhpT in the absence of the regulatory proteins (UhpA, UhpB and UhpC) allowed us to demonstrate that (i) the Uhp regulatory proteins do not prevent the activation of AC by direct interaction with EnzymellAglc and (ii) an increase in the amount of UhpT synthesized (corresponding to an increase in the amount of Glc6P transported) correlates with a decrease in the cAMP level. We present data indicating that Glc6P per se or its degradation is unlikely to be responsible for the low cAMP level. It is concluded that the level of cAMP in the cell is determined by the flux of Glc6P through UhpT.

Inducer expulsion, a phenomenon in which rapidly metabolizable sugars cause cytoplasmic dephosphorylation and efflux of pre-accumulated sugar-phosphates (sugar-P), has been documented for Streptococcus pyogenes, Streptococcus bovis, and Lactococcus lactis but not for other Gram-positive bacteria. Using intact cells and membrane vesicles, we show that Enterococcus faecalis exhibits both inducer exclusion and inducer expulsion, and that the latter phenomenon is dependent on the metabolite-activated ATP-dependent HPr(Ser) kinase that phosphorylates Ser-46 in HPr of the phosphotransferase system. A small, heat-stable, membrane-associated, HPr(Ser-P)-activated sugar-P phosphatase (Pase II), previously identified only in Lc. lactis, is shown to be present in extracts of Enterococcus faecalis and Streptococcus pyogenes but not in those of Staphylococcus aureus, Streptococcus mutans, Streptococcus salivarius or Bacillis subtilis, organisms that do not exhibit the inducer expulsion phenomenon. Further, Lactobacillus brevis an organism that exhibits inducer expulsion by a different mechanism, also apparently lacks Pase II. The results reveal that Pase II is present in those organisms that exhibit the coupled sugar-P hydrolysis/expulsion mechanism but not those that lack this mechanism. They provide correlative evidence that Pase II initiates inducer expulsion in species of enterococci, streptococci and lactococci.

The behavioural response of Rhodobacter Sphaeroides to temporal changes in the concentration of chemoeffectors, and to stimuli affecting electron transport, was analysed using tethered cells. Populations of photosynthetically grown tethered cells of R. sphaeroides showed a transient response, a stop followed by adaptation, to a stepwise reduction in the concentration of chemoattractants (such as organic acids or sugars) and terminal electron acceptors. A step-down response was also measured in free swimming cells to a reduction in light intensity. As this response appears to apply to all effectors this suggests that there is a sensory pathway in anaerobically grown R. sphaeroides which responds primarily to a reduction in a stimulus. R. sphaeroides therefore responds when moving down a concentration gradient. This is the inverse of the classical Escherichia coli–Salmonella typhimurium model of bacterial sensory behaviour in which bacteria respond primarily when there is an increase in an attractant concentration, i.e. when moving up a gradient. R. sphaeroides does show a chemokinetic response to an increase in concentration of a limited number of compounds but this response is sustained and accompanied by an increase in the rate of flagellar rotation and therefore not simply equivalent to the transient increase in smooth swimming measured in E. coli on addition of an attractant.