- Volume 150, Issue 2, 2004

The entomopathogenic fungus Paecilomyces fumosoroseus has been successfully used in the control of several insect pests. Asexually produced spores (conidia) are the means for dispersal and transmission of the entomopathogen; upon contact with the insect cuticle they germinate and penetrate the host. In model fungal systems it has been found that phototropism, resetting of the circadian rhythm, the induction of carotenogenesis and the development of reproductive structures are controlled by blue light. The effect of light quality on conidial yield of P. fumosoroseus was investigated. Incubation in total darkness resulted in continued vegetative growth and lack of reproductive structures. In contrast, growth of the fungus in continuous illumination or under a night–day regime resulted in prolific formation of conidiophores bearing abundant mature conidia. Conidiation was photoinduced in competent mycelia by a single pulse of blue light and colonies were competent only after they had grown at least 72 h under total darkness. The fluence–response curves generated with blue light indicated that the minimal fluence required for the photomorphogenetic response was 180 μmol m−2 and the half-maximal response was at 400 μmol m−2. A fluence of 540 μmol m−2 was enough to saturate the system, inducing the maximum production of 2·12×108 conidia per colony. Higher light intensities markedly decreased conidiation, suggesting the occurrence of a process of adaptation. The authors propose the existence of a dual light-perception system with at least two photoreceptors in P. fumosoroseus, one promoting and one inhibiting conidiation.

Anabaena sp. PCC 7120 was mutagenized by transposon Tn5-1087b, generating a mutant whose heterocysts lack the envelope polysaccharide layer. The transposon was located between nucleotides 342 and 343 of alr0117, a 918 bp gene encoding a histidine kinase for a two-component regulatory system. Complementation of the mutant with a DNA fragment containing alr0117 and targeted inactivation of the gene confirmed that alr0117 is involved in heterocyst development. RT-PCR showed that alr0117 was constitutively expressed in the presence or absence of a combined-nitrogen source. hepA and patB, the two genes turned on during wild-type heterocyst development, were no longer activated in an alr0117-null mutant. The two-component signal transduction system involving alr0117 may control the formation of the envelope polysaccharide layer and certain late events essential to the function of heterocysts.

Phospholipids are an important component of bacterial membranes. Borrelia burgdorferi differs from many other bacteria in that it contains only two major membrane phospholipids: phosphatidylglycerol (PG) and phosphatidylcholine (PC). B. burgdorferi appears to lack enzymes required for synthesis of PC through the well-described methylation pathway. However, B. burgdorferi does contain a gene (BB0249) with significant identity to a recently described phosphatidylcholine synthase gene (pcs) of Sinorhizobium meliloti. B. burgdorferi also contains a gene (BB0721) with significant identity to the gene (pgs) encoding phosphatidylglycerolphosphate synthase, an enzyme in the synthetic pathway of PG. Activity of BB0249 was confirmed by cloning the gene into Escherichia coli, which does not produce PC. Transformation with a plasmid carrying BB0249 resulted in production of PC by E. coli, but only in the presence of exogenously supplied choline, as would be predicted for a Pcs. Because loss of Pgs activity is lethal to E. coli, activity of BB0721 was confirmed by the ability of BB0721 to complement an E. coli Pgs− mutant. A plasmid containing BB0721 was transformed into a Pgs− mutant of E. coli containing a copy of the native gene on a temperature-regulated plasmid. The temperature-regulated plasmid was exchanged for a plasmid containing BB0721 and it was shown that BB0721 was able to replace the lost Pgs function and restore bacterial growth. This study has established the existence and function of two critical enzymes in the synthesis of PC and PG in B. burgdorferi. Understanding of the biosynthetic pathways of PC and PG in B. burgdorferi is the first step in delineating the role of these phospholipids in the pathogenesis of Lyme disease.

Biotin has a profound effect on the metabolism of rhizobia. It is reported here that the activities of the biotin-dependent enzymes acetyl-coenzyme A carboxylase (ACC; EC 6.4.1.2) and propionyl-coenzyme A carboxylase (PCC; EC 6.4.1.3) are present in all species of the five genera comprising the Rhizobiaceae which were examined. Evidence is presented that the ACC and PCC activities detectable in Rhizobium etli extracts are catalysed by a single acyl-coenzyme A carboxylase. The enzyme from R. etli strain 12-53 was purified 478-fold and displayed its highest activity with propionyl-CoA as substrate, with apparent K m and V max values of 0·064 mM and 2885 nmol min−1 (mg protein)−1, respectively. The enzyme carboxylated acetyl-CoA and butyryl-CoA with apparent K m values of 0·392 and 0·144 mM, respectively, and V max values of 423 and 268 nmol min−1 (mg protein)−1, respectively. K+, or Cs+ markedly activated the enzyme, which was essentially inactive in their absence. Electrophoretic analysis indicated that the acyl-CoA carboxylase was composed of a 74 kDa biotin-containing α subunit and a 45 kDa biotin-free β subunit, and gel chromatography indicated a total molecular mass of 620 000 Da. The strong kinetic preference of the enzyme for propionyl-CoA is consistent with its participation in an anaplerotic pathway utilizing this substrate.

The Gram-positive bacterium Bacillus subtilis contains two respiratory oxidases of the haem-copper superfamily: cytochrome aa 3, which is a quinol oxidase, and cytochrome caa 3, which is a cytochrome c oxidase. Cytochrome c oxidase uniquely contains a di-copper centre, CuA. B. subtilis CtaG is a membrane protein encoded by the same gene cluster as that which encodes the subunits of cytochrome c oxidase. The role of B. subtilis CtaG and orthologous proteins present in many other Gram-positive bacteria has remained unexplored. The sequence of CtaG is unrelated to that of CtaG/Cox11p of proteobacteria and eukaryotic cells. This study shows that B. subtilis CtaG is essential for the formation of active cytochrome caa 3 but is not required for assembly of the core subunits I and II with haem in the membrane and it has no role in the synthesis of active cytochrome aa 3. B. subtilis YpmQ, a homologue to Sco1p of eukaryotic cells, is also a membrane-bound cytochrome c oxidase-specific assembly factor. Properties of CtaG- and YpmQ-deficient mutants were compared. Cells lacking YpmQ showed a low cytochrome c oxidase activity and this defect was suppressed by the supplementation of the growth medium with copper ions. It has previously been proposed that YpmQ/Sco1p is involved in synthesis of the CuA centre. The results of this study are consistent with this proposal but the exact role of YpmQ in assembly of cytochrome c oxidase remains to be elucidated.

In bacteria, SsrA, a highly conserved RNA molecule, functions in a mechanism meant to rescue stalled ribosomes. In this process, a peptide tag encoded by SsrA is cotranslationally added to truncated polypeptides, thereby targeting these molecules for proteolytic degradation, at least when they stay inside the cell. This study examined the fate of two extracellular proteins that were tagged by the SsrA system of Bacillus subtilis. Gene constructs encoding human interleukin-3 (hIL-3) fused to a signal peptide and B. subtilis α-amylase, both lacking an in-frame stop codon, were used as models to achieve ribosome stalling and activation of the SsrA system. Introduction of these gene constructs into B. subtilis led to tagging of the gene products by SsrA RNA. The tagged protein products bound to antibodies that were raised against the proteolysis tag encoded by B. subtilis SsrA [(A)GKTNSFNQNVALAA]. The apolar C-terminal SsrA-tag does not function as a specific signal for proteolytic degradation of SsrA-tagged amylase directly after trans-translation or during the secretion process. Also, SsrA-tagged amylase appeared to be very stable once outside the cell. In contrast, hIL-3 molecules tagged with the native, apolar SsrA-tag were considerably less stable than hIL-3 molecules that received a negatively charged control tag. Not one specific protease, but several non-specific proteases seem to play a role in the rapid degradation of SsrA-tagged hIL-3. The polarity of the C-terminus of heterologous hIL-3 protein proved to be an important determinant for protein stability when produced by B. subtilis. As observed previously in Escherichia coli and B. subtilis, SsrA tagging also occurs frequently in normally growing Gram-positive bacilli and it appears that intracellular proteins are the predominant natural substrates of SsrA.

A gene cluster containing a gene for maleylacetate reductase (EC 1.3.1.32) was cloned from Ralstonia eutropha 335T (DSM 531T), which is able to utilize 4-fluorobenzoate as sole carbon source. Sequencing of this gene cluster showed that the R. eutropha 335T maleylacetate reductase gene, macA, is part of a novel gene cluster, which is not related to the known maleylacetate-reductase-encoding gene clusters. It otherwise comprises a gene for a hypothetical membrane transport protein, macB, possibly co-transcribed with macA, and a presumed regulatory gene, macR, which is divergently transcribed from macBA. MacA was found to be most closely related to TftE, the maleylacetate reductase from Burkholderia cepacia AC1100 (62 % identical positions) and to a presumed maleylacetate reductase from a dinitrotoluene catabolic gene cluster from B. cepacia R34 (61 % identical positions). By expressing macA in Escherichia coli, it was confirmed that macA encodes a functional maleylacetate reductase. Purification of maleylacetate reductase from 4-fluorobenzoate-grown R. eutropha 335T cells allowed determination of the N-terminal sequence of the purified protein, which was shown to be identical to that predicted from the cloned macA gene, thus proving that the gene is, in fact, recruited for growth of R. eutropha 335T with this substrate.

The Escherichia coli ndh gene encodes NADH dehydrogenase II, a primary dehydrogenase used during aerobic and nitrate respiration. The anaerobic transcription factor FNR represses ndh expression by binding at two sites centred at −94·5 and −50·5. In vivo transcription studies using promoter fusions with 5′ deletions confirmed that both FNR sites are required for maximum repression under anaerobic conditions. The histone-like protein Fis binds to three sites [centred at −123 (Fis I), −72, (Fis II) and +51 (Fis III)] in the ndh promoter. Using ndh : : lacZ promoter fusions carrying 5′ deletions, or replacement mutations it is shown that Fis III is a repressing site and that Fis I and II are activating sites, with the greatest contribution from Fis II. Deletion of the C-terminal domain of the RNA polymerase α-subunit abolished Fis-mediated activation of ndh expression, suggesting that ndh has a Class I Fis-activated promoter. In accordance with the established pattern of Fis synthesis, ndh transcription was greatest during exponential growth. Thus, it is suggested that Fis enhances ndh expression during periods of rapid growth, by acting as a Class I activator, and that the binding of tandem FNR dimers represses ndh expression by preventing interaction of the RNA polymerase α-subunit with DNA and Fis.

To better understand the biology and the virulence determinants of the two major mycobacterial human pathogens Mycobacterium tuberculosis and Mycobacterium leprae, their genome sequences have been determined recently. In silico comparisons revealed that among the 1439 genes common to both M. tuberculosis and M. leprae, 219 genes code for proteins that show no similarity with proteins from other organisms. Therefore, the latter ‘core’ genes could be specific for mycobacteria or even for the intracellular mycobacterial pathogens. To obtain more information as to whether these genes really were mycobacteria-specific, they were included in a focused macro-array, which also contained genes from previously defined regions of difference (RD) known to be absent from Mycobacterium bovis BCG relative to M. tuberculosis. Hybridization of DNA from 40 strains of the M. tuberculosis complex and in silico comparison of these genes with the near-complete genome sequences from Mycobacterium avium, Mycobacterium marinum and Mycobacterium smegmatis were undertaken to answer this question. The results showed that among the 219 conserved genes, very few were not present in all the strains tested. Some of these missing genes code for proteins of the ESAT-6 family, a group of highly immunogenic small proteins whose presence and number is variable among the genomically highly conserved members of the M. tuberculosis complex. Indeed, the results suggest that, with few exceptions, the ‘core’ genes conserved among M. tuberculosis H37Rv and M. leprae are also highly conserved among other mycobacterial strains, which makes them interesting potential targets for developing new specific anti-mycobacterial drugs. In contrast, the genes from RD regions showed great variability among certain members of the M. tuberculosis complex, and some new specific deletions in Mycobacterium canettii, Mycobacterium microti and seal isolates were identified and further characterized during this study. Together with the distribution of a particular 6 or 7 bp micro-deletion in the gene encoding the polyketide synthase pks15/1, these results confirm and further extend the revised phylogenetic model for the M. tuberculosis complex recently presented.

The Gram-positive soil bacterium Bacillus subtilis responds to oxidative stress by the activation of different cellular defence mechanisms. These are composed of scavenging enzymes as well as protection and repair systems organized in highly sophisticated networks. In this study, the peroxide and the superoxide stress stimulons of B. subtilis were characterized by means of transcriptomics and proteomics. The results demonstrate that oxidative-stress-responsive genes can be classified into two groups. One group encompasses genes which show similar expression patterns in the presence of both reactive oxygen species. Examples are members of the PerR and the Fur regulon which were induced by peroxide and superoxide stress. Similarly, both kinds of stress stimulated the activation of the stringent response. The second group is composed of genes primarily responding to one stimulus, like the members of the SOS regulon which were particularly upregulated in the presence of peroxide, and many genes involved in sulfate assimilation and methionine biosynthesis which were only induced by superoxide. Several genes encoding proteins of unknown function could be assigned to one of these groups.

Candida albicans displays a high degree of specificity in selecting and adhering to targets in vivo. The features of target recognition are poorly understood and likely to involve more than the mere chemical composition of the ligand. Using an adherence assay in which protein and peptide ligands are covalently coupled to magnetic beads, the authors have previously described a new adherence mechanism in C. albicans, henceforth referred to as SRS (stable, reversible, specific) adherence. It was previously demonstrated that C. albicans and Saccharomyces cerevisiae expressing agglutinin-like sequence 5 protein (Als5p, previously referred to as Ala1p or Ala1/Als5p) adhere to peptides containing patches of threonine, serine and alanine residues when these are located in the free end of immobilized peptides. The interaction with protein ligands in SRS adherence predominantly involves the formation of hydrogen bonds. Accordingly, this interaction may occur (1) to the peptide backbone of the protein ligand or (2) to the amino acid side chain with an appropriate functional group. Evidence is provided that the primary interaction occurs with the peptide backbone and the secondary interaction occurs with the side chain. The primary interaction with the peptide backbone is sufficient for adherence to occur, whereas the secondary interaction with a side chain possessing an appropriate functional group stabilizes the interaction. In agreement with these results, it is also demonstrated that proteins lacking secondary and tertiary structure, wherein the peptide backbone is sterically accessible, interact with C. albicans and S. cerevisiae expressing Als5p. C. albicans Als proteins are resistant to denaturation by harsh conditions that kill the yeast cells. The proposed interactions in SRS adherence have striking similarities with those of the molecular chaperone Hsp70, which specifically binds to non-native proteins and resists denaturation.

Expression of the major virulence cluster in Listeria monocytogenes is positively regulated by the transcription factor PrfA and is influenced by several environmental factors, including the presence of readily metabolized carbohydrates such as cellobiose and glucose. Although little is understood about the mechanisms through which environmental factors influence expression of the PrfA regulon, evidence for structural and functional similarities of PrfA to the CRP-FNR family of regulatory proteins suggests the possibility that PrfA activity could be modulated by a small molecule ligand. The identity of components of the PrfA-associated regulatory pathway was sought through the isolation of mutants that exhibit high levels of PrfA-controlled gene expression in the presence of cellobiose or glucose. Here are described the properties and preliminary genetic analysis in two different genetic loci, gcr and csr, both unlinked by general transduction to the major virulence cluster. A mutation in gcr deregulates the expression of PrfA-controlled genes in the presence of several repressing sugars and other environmental conditions, a phenotype similar to that of a G145S substitution in PrfA itself. A mutation in the csr locus, within csrA, results in a cellobiose-specific defect in virulence gene regulation. Gene products encoded by the csr locus share homology with proteins involved in the sensing and transport of β-glucosides in other bacteria. Mutations in both gcr and csr are required for full relief of cellobiose-mediated repression of the PrfA regulon. These results suggest the existence of two semi-independent pathways for cellobiose-mediated repression and further reconcile conflicting reports in previous literature concerning the repressive effects of carbohydrates on virulence gene expression in L. monocytogenes.