- Volume 149, Issue 8, 2003

DNA ligase IV is thought to be involved in DNA double-strand break repair and DNA non-homologous end-joining pathways, but these mechanisms are still unclear. To investigate the roles of DNA ligase IV from a biologically functional viewpoint, the authors studied its relationship to meiosis in a basidiomycete, Coprinus cinereus, which shows a highly synchronous meiotic cell cycle. The C. cinereus cDNA homologue of DNA ligase IV (CcLIG4) was successfully cloned. The 3·2 kb clone including the ORF encoded a predicted product of 1025 amino acid residues with a molecular mass of 117 kDa. A specific inserted sequence composed of 95 amino acids rich in aspartic acid and glutamic acid could be detected between tandem BRCT domains. The inserted sequence had no sequence identity with other eukaryotic counterparts of DNA ligase IV or with another aspartic acid and glutamic acid rich sequence inserted in C. cinereus proliferating cell nuclear antigen (CcPCNA), although the length and the percentages of aspartic and glutamic acids were similar. In addition, the recombinant CcLIG4 protein not only showed ATP-dependent ligase activity, but also used (dT)16/poly(dA) and (dT)16/poly(rA) as substrates, and had double-strand ligation activity, like human DNA ligase IV. Northern hybridization analysis and in situ hybridization indicated that CcLIG4 was expressed not only at the pre-meiotic S phase but also at meiotic prophase I. Intense signals were observed in leptotene and zygotene. Based on these observations, the possible role(s) of C. cinereus DNA ligase IV during meiosis are discussed.

Fungal laccase gene transcription is strongly induced by copper ions; notably, some laccase promoters contain multiple putative metal-responsive elements (MREs). Previously, it has been demonstrated that the Pleurotus ostreatus laccase genes poxc and poxa1b are transcriptionally induced by copper, and several putative MREs were found in the promoter regions of these genes, which extend for about 400 nt upstream of the start codon (ATG). Identification of MRE sequences, which are protected by protein binding in the poxc and poxa1b promoter regions, has been achieved by footprinting analyses. Electromobility shift assays led to the evaluation of the ability of the identified MREs to bind protein(s), and the role of specific nucleotides of these elements in complex formation has also been analysed. The formation of complexes between analysed MREs and fungal proteins requires the absence of metal ions. Proteins extracted from fungus grown in copper-depleted medium are able to form complexes with MREs, whilst proteins extracted from fungus grown in copper-containing medium are able to form complexes only in the presence of a metal chelator. Moreover, copper-depleted proteins are unable to form complexes when copper or zinc ions are added. UV-cross-linking analyses led to the determination of the molecular masses of the MRE-binding proteins. In the poxa1b promoter, a GC-rich region, homologous to the core binding site for transcription factor Sp1, decreases the binding affinity of the adjacent MRE, affecting its interactions with fungal protein factors.

The PII signal transduction proteins GlnB and GlnK are uridylylated/deuridylylated in response to the intracellular glutamine level, the primary signal of the cellular nitrogen status. Furthermore, GlnB was shown to be allosterically regulated by 2-oxoglutarate, and thus GlnB was suggested to integrate signals of the cellular carbon and nitrogen status. Receptors of GlnB signal transduction in Escherichia coli are the NtrB/NtrC two-component system and GlnE, an enzyme which adenylylates/deadenylylates glutamine synthetase. In this study, the authors investigated the effect of different carbon sources on the expression of the NtrC-dependent genes glnA and glnK and on the uridylylation status of GlnB and GlnK. With glutamine as nitrogen source, high levels of glnA and glnK expression were obtained when glucose was used as carbon source, but expression was strongly decreased when the cells were grown with poor carbon sources or when cAMP was present. This response correlated with the uridylylation status of GlnB, suggesting that the carbon/cAMP effect was mediated through GlnB uridylylation, a conclusion that was confirmed by mutants of the PII signalling pathway. When glutamine was replaced by low concentrations of ammonium as nitrogen source, neither glnAglnK expression nor GlnB uridylylation responded to the carbon source or to cAMP. Furthermore, glutamine synthetase could be rapidly adenylylated in vivo by the external addition of glutamine; however, this occurred only when cells were grown in the presence of cAMP, not in its absence. Together, these results suggest that poor carbon sources, through cAMP signalling, favour glutamine uptake. The cellular glutamine signal is then transduced by uridylyltransferase and GlnB to modulate NtrC-dependent gene expression.

The pnp gene, encoding the enzyme polynucleotide phosphorylase (PNPase), was overexpressed in the actinomycin producer Streptomyces antibioticus. Integration of pIJ8600, bearing the thiostrepton-inducible tipA promoter, and its derivatives containing pnp into the S. antibioticus chromosome dramatically increased the growth rate of the resulting strains as compared with the parent strain. Thiostrepton induction of a strain containing pJSE340, bearing pnp with a 5′-flanking region containing an endogenous promoter, led to a 2·5–3 fold increase in PNPase activity levels, compared with controls. Induction of a strain containing pJSE343, with only the pnp ORF and some 3′-flanking sequence, led to lower levels of PNPase activity and a different pattern of pnp expression compared with pJSE340. Induction of pnp from pJSE340 resulted in a decrease in the chemical half-life of bulk mRNA and a decrease in poly(A) tail length as compared to RNAs from controls. Actinomycin production decreased in strains overexpressing pnp as compared with controls but it was not possible to attribute this decrease specifically to the increase in PNPase levels. Overexpression of pnp had no effect on ppGpp levels in the relevant strains. It was observed that the 3′-tails associated with RNAs from S. antibioticus are heteropolymeric. The authors argue that those tails are synthesized by PNPase rather than by a poly(A) polymerase similar to that found in Escherichia coli and that PNPase may be the sole RNA 3′-polynucleotide polymerase in streptomycetes.

The aminocoumarin antibiotic clorobiocin contains a 5-methylpyrrole-2-carboxylic acid unit, attached via an ester bond to the 3-OH group of the deoxysugar moiety. To investigate candidate genes responsible for the formation of this ester bond, a gene inactivation experiment was carried out in the clorobiocin producer Streptomyces roseochromogenes var. oscitans DS 12.976. An in-frame deletion was created in the coding sequence of the gene cloN2. The production of secondary metabolites in the wild-type and in the cloN2 mutant was analysed. The wild-type showed clorobiocin as the main product, whereas the cloN2 mutant accumulated a new aminocoumarin derivative, novclobiocin 104, lacking the pyrrole moiety at the 3-OH of the deoxysugar. In addition, free pyrrole-2-carboxylic acid accumulated in the culture extract of the cloN2 mutant. The structures of the metabolites were confirmed by NMR and LC-MS analysis. Clorobiocin production was successfully restored in the cloN2 mutant by introducing a replicative plasmid containing the cloN2 sequence. These results prove an involvement of cloN2 in the formation of the ester bond between the pyrrole moiety and the deoxysugar in clorobiocin biosynthesis. Furthermore, they indicate that the C-methylation at position 5 of the pyrrole moiety occurs after the attachment of pyrrole-2-carboxylic acid unit to the deoxysugar moiety.

The authors have previously reported the identification of novel signal peptides (SPs) from Lactococcus lactis using transposon insertion. Of these, SP310 caused the highest level of secretion. However, the levels were lower than those obtained using the signal peptide from Usp45 (SPUSP), the major secreted lactococcal protein. In this study, site-directed mutagenesis of signal peptide SP310 was used to investigate the effect of amino acid alterations on lactococcal secretion and to improve secretion efficiency. Several mutated SPs caused higher secretion. This increase in secretion was due to modifications in the cleavage region. In fermenter experiments, the signal peptide SP310mut2 resulted in an extracellular Staphylococcus aureus nuclease (Nuc) yield which was 45 % higher than that with the natural SP310. Surprisingly, increasing the hydrophobicity of the hydrophobic core or increasing the number of positively charged amino acids in the N-terminal region of SP310 decreased secretion. High extracellular yields of Nuc resulted from more efficient secretion, as strains with less efficient SPs accumulated more intracellular SP-Nuc precursor.

In most bacteria, nitrogen metabolism is tightly regulated and PII proteins play a pivotal role in the regulatory processes. Rhodobacter capsulatus possesses two genes (glnB and glnK) encoding PII-like proteins. The glnB gene forms part of a glnB–glnA operon and the glnK gene is located immediately upstream of amtB, encoding a (methyl-) ammonium transporter. Expression of glnK is activated by NtrC under nitrogen-limiting conditions. The synthesis and activity of the molybdenum and iron nitrogenases of R. capsulatus are regulated by ammonium on at least three levels, including the transcriptional activation of nifA1, nifA2 and anfA by NtrC, the regulation of NifA and AnfA activity by two different NtrC-independent mechanisms, and the post-translational control of the activity of both nitrogenases by reversible ADP-ribosylation of NifH and AnfH as well as by ADP-ribosylation independent switch-off. Mutational analysis revealed that both PII-like proteins are involved in the ammonium regulation of the two nitrogenase systems. A mutation in glnB results in the constitutive expression of nifA and anfA. In addition, the post-translational ammonium inhibition of NifA activity is completely abolished in a glnB–glnK double mutant. However, AnfA activity was still suppressed by ammonium in the glnB–glnK double mutant. Furthermore, the PII-like proteins are involved in ammonium control of nitrogenase activity via ADP-ribosylation and the switch-off response. Remarkably, in the glnB–glnK double mutant, all three levels of the ammonium regulation of the molybdenum (but not of the alternative) nitrogenase are completely circumvented, resulting in the synthesis of active molybdenum nitrogenase even in the presence of high concentrations of ammonium.

Bacterial modular polyketide synthase (PKS) genes are commonly associated with another gene that encodes a thioesterase II (TEII) believed to remove aberrantly loaded substrates from the PKS. Co-expression of the Saccharopolyspora erythraea ery-ORF5 TEII and eryA genes encoding 6-deoxyerythronolide B synthase (DEBS) in Streptomyces hosts eliminated or significantly lowered production of 8,8′-deoxyoleandolide [15-nor-6-deoxyerythronolide B (15-nor-6dEB)], which arises from an acetate instead of a propionate starter unit. Disruption of the TEII gene in an industrial Sac. erythraea strain caused a notable amount of 15-norerythromycins to be produced by utilization of an acetate instead of a propionate starter unit and also resulted in moderately lowered production of erythromycin compared with the amount produced by the parental strain. A similar behaviour of the TEII gene was observed in Escherichia coli strains that produce 6dEB and 15-methyl-6dEB. Direct biochemical analysis showed that the ery-ORF5 TEII enzyme favours hydrolysis of acetyl groups bound to the loading acyl carrier protein domain (ACPL) of DEBS. These results point to a clear role of the TEII enzyme, i.e. removal of a specific type of acyl group from the ACPL domain of the DEBS1 loading module.

Ruminal bacteria of the genus Prevotella play a crucial role in peptide breakdown in the rumen, a component of protein catabolism that leads to the inefficient use of dietary protein by ruminant animals. This is the first report of the cloning of a peptidase gene from a ruminal bacterium. Part of the dipeptidyl peptidase type IV (DPP-IV) gene from Prevotella albensis M384T was cloned using degenerate primers designed from conserved regions found within other known DPP-IV sequences. Flanking regions were determined by genomic walking. The DPP-IV gene was expressed in Escherichia coli. The cloned enzyme required a free N terminus and catalysed the removal of X-Pro dipeptide from proline-containing oligopeptides, where proline was the second residue from the N terminus. It was inhibited by serine protease inhibitors and the substrate analogue for mammalian DPP-IV, diprotin A. The properties of the cloned enzyme were similar to those of the native form in P. albensis and, in general, DPP-IVs from other organisms. The enzyme contained a conserved motif which is associated with the S9 class of prolyl oligopeptidases. The DPP-IV gene appeared not to be part of a contiguous operon. Regions with similarity to other putative promoters of Prevotella spp. were also identified. Construction of a phylogenetic tree demonstrated that the DPP-IV of P. albensis clusters with other DPP-IVs found in bacteria of the Cytophaga–Flexibacter–Bacteroidaceae (CFB) phylum, which are more closely related to eukaryotic DPP-IVs than the DPP-IV-like enzyme (PepX) of the lactic acid bacteria.

In vitro polymerization of the essential bacterial cell division protein FtsZ, in the presence of GTP, is rapid and transient due to its efficient binding and hydrolysis of GTP. In contrast, the in vivo polymeric FtsZ structure which drives cell division – the Z-ring – is present in cells for extended periods of time whilst undergoing constant turnover of FtsZ. It is demonstrated that dynamic polymerization of Escherichia coli FtsZ in vitro is sensitive to the ratio of GTP to GDP concentration. Increase of GDP concentration in the presence of a constant GTP concentration reduces both the duration of FtsZ polymerization and the initial light-scattering maximum which occurs upon addition of GTP. It is also demonstrated that by use of a GTP-regeneration system, polymers of FtsZ can be maintained in a steady state for up to 85 min, while preserving their dynamic properties. The authors therefore present the use of a GTP-regeneration system for FtsZ polymerization as an assay more representative of the in vivo situation, where FtsZ polymers are subject to a constant, relatively high GTP to GDP ratio.

Treatment of bacterial cultures with chelating agents such as 2,2′-dipyridyl (DPD) induces expression of iron-regulated genes. It is known that in the γ-Proteobacteria, the Fur protein is the major regulator of genes encoding haem- or haemoglobin-binding proteins. Electrophoretic analysis of outer-membrane proteins of the γ-proteobacterium Pasteurella multocida has revealed the induction of two proteins of 60 and 40 kDa in DPD-treated cultures in both wild-type and fur-defective strains. These two proteins have the same N-terminal amino acid sequence, which identifies this protein as the product of the PM0592 ORF. Analysis of the sequence of this ORF, which encodes a protein of 60 kDa, revealed the presence of a hexanucleotide (AAAAAA) at which a programmed translational frameshift can occur giving rise to a 40 kDa protein. Analyses conducted in Escherichia coli, using the complete PM0592 ORF and a derivative truncated at the hexanucleotide position, have shown that both polypeptides bind haemin. For this reason, the PM0592 ORF product has been designated HbpA (for haemin-binding protein). Expression studies using both RT-PCR and lacZ fusions, as well as electrophoretic profiles of outer-membrane protein composition, have demonstrated that the hbpA gene is negatively regulated by iron, manganese and haemin through a fur-independent pathway. Despite the fact that serum of mice infected with P. multocida contained antibodies that reacted with both the 60 and 40 kDa products of the hbpA gene, these proteins did not offer protection when used in immunization assays against this micro-organism.

After screening of a Candida albicans genome database, the product of an ORF (IPF 3054) that has 62 % homology with Saccharomyces cerevisiae Ssr1p, an internal cell-wall protein, was identified and named CaSsr1p. The deduced amino acid sequence shows that CaSsr1p contains an N-terminal hydrophobic signal peptide, is rich in Ser and Thr amino acids and has a potential glycosylphosphatidylinositol-attachment signal. CaSsr1p is released following degradation of isolated cell walls by zymolyase (mainly a 1,3-β-glucanase) and therefore seems to be covalently linked to the β-glucan of the cell walls. Both disruption and overexpression of the CaSSR1 gene caused an increased sensitivity to calcofluor white, Congo red and zymolyase digestion. These results suggest that CaSsr1p has a structural role associated with the cell-wall β-glucan.