- Volume 150, Issue 8, 2004

The killer toxin secreted by Kluyveromyces phaffii (KpKt) is active against spoilage yeast under winemaking conditions and thus has potential applications in the biocontrol of undesired micro-organisms in the wine industry. Biochemical characterization and N-terminal sequencing of the purified toxin show that KpKt is a glycosylated protein with a molecular mass of 33 kDa. Moreover, it shows 93 % and 80 % identity to a β-1,3-glucanase of Saccharomyces cerevisiae and a β-1,3-glucan transferase of Candida albicans, respectively, and it is active on laminarin and glucan, thus showing a β-glucanase activity. Competitive inhibition of killer activity by cell-wall polysaccharides suggests that glucan (β-1,3 and β-1,6 branched glucans) represents the first receptor site of the toxin on the envelope of the sensitive target. Flow cytometry analysis of the sensitive target after treatment with KpKt and K1 toxin of S. cerevisiae, known to cause loss of cell viability via formation of pores in the cell membrane, suggests a different mode of action for KpKt.

The Gram-positive soil bacterium and cellulose degrader Streptomyces reticuli synthesizes the mycelium-associated enzyme CpeB, which displays haem-dependent catalase and peroxidase activity, as well as haem-independent manganese-peroxidase activity. Downstream of the cpeB gene, a so far unknown gene was identified. The new gene and its mutated derivatives were cloned in Escherichia coli as well as in Streptomyces lividans and a gene-disruption mutant within the chromosome of the original S. reticuli host was constructed, comparative physiological, biochemical and immunological studies then allowed the deduction of the following characteristics of the novel gene product. (i) The protein was found extracellularly; the substitution of twin arginines within the signal peptide abolished its secretion. (ii) The highly purified protein interacted specifically with haem and hence was designated HbpS (haem-binding protein of Streptomyces). (iii) HbpS contained three histidine residues surrounded by hydrophobic amino acids; one of them was located within the motif LX3THLX10AA, which is related to the motif within the yeast cytochrome c peroxidase LX2THLX10AA whose histidine residue interacts with haem. (iv) The addition of haemin (Fe3+ oxidized form of haem) to the Streptomyces cultures led to enhanced levels of HbpS which correlated with increased haemin-resistance. (v) The presence of HbpS increased synthesis of the highly active catalase-peroxidase CpeB containing haem. In this process HbpS could act as a chaperone that binds haem and then delivers it to the mycelium-associated CpeB; HbpS could also interact with membrane-associated proteins involved in a signal transduction cascade regulating the expression of cpeB. (vi) HbpS shared varying degrees of amino acid identities with bacterial proteins of so far unknown function. This report contributes to the elucidation of the biological function of these proteins.

In order to assimilate iron, Acinetobacter baumannii ATCC 19606T produces a siderophore named acinetobactin (Ab) that is composed of equimolar quantities of 2,3-dihydroxybenzoic acid (DHBA), l-threonine and N-hydroxyhistamine. Application of the Fur titration assay system to A. baumannii genomic libraries, followed by further cloning of the regions surrounding the candidate genes, led to the identification of the Ab cluster, which harbours the genetic determinants necessary for the biosynthesis and transport of the siderophore. However, an entA homologue essential for DHBA biosynthesis was not found in this cluster. Functions of potential biosynthetic genes inferred by homology studies suggested that the precursors, DHBA, l-threonine and N-hydroxyhistamine, are linked in steps resembling those of bacterial non-ribosomal peptide synthesis to form Ab. Genes responsible for the two-step biosynthesis of N-hydroxyhistamine from histidine were also identified in this cluster. Their genetic organization suggests that five genes involved in the transport system of ferric Ab into the cell cytosol form an operon. Construction of disruptants of some selected genes followed by phenotypic analysis supported their predicted biological functions. Interestingly, three additional genes probably involved in the intracellular release of iron from ferric Ab and the secretion of nascent Ab are contained in this cluster. Primer extension and RT-PCR analyses suggested that the Ab cluster, which includes 18 genes, is organized in seven transcriptional units originating from respective Fur-regulated promoter-operator regions.

This study reports, for the first time, purification and biochemical characterization of a cyanophycin synthetase from a non-cyanobacterial strain. Cyanophycin synthetase of Acinetobacter calcoaceticus strain ADP1 was purified 69-fold from recombinant Escherichia coli by two chromatographic steps and one novel affinity step utilizing the Mg2+-dependent binding of the enzyme to cyanophycin. Unlike cyanobacterial cyanophycin synthetases characterized so far, the purified enzyme from A. calcoaceticus strain ADP1 did not accept lysine as an alternative substrate to arginine. The apparent K m-values for arginine (47 μM) and aspartic acid (240 μM) were similar to those of known cyanophycin synthetases from cyanobacteria, but this enzyme had a slightly higher affinity for aspartic acid. In addition, the two different ATP-binding sites of the enzyme were characterized independently of each other with respect to K m values for ATP. The ATP-binding site responsible for the addition of arginine was found to have a much higher affinity for ATP (38 μM) than that responsible for the addition of aspartate (210 mM). Furthermore, the binding of the enzyme to the two possible forms of cyanophycin granule polypeptide (CGP), CGP-Asp and CGP-Arg, was studied. While both forms bound around 30–40 % of the enzyme activity present under the assay conditions, binding was Mg2+-dependent in the case of CGP-Asp. Two-dimensional gel electrophoresis revealed that both forms of cyanophycin were equally abundant in cyanophycin-accumulating cells of A. calcoaceticus ADP1.

During phosphate starvation, Bacillus subtilis regulates genes in the PhoP regulon to reduce the cell's requirement for this essential substrate and to facilitate the recovery of inorganic phosphate from organic sources such as teichoic and nucleic acids. Among the proteins that are highly induced under these conditions is PstS, the phosphate-binding lipoprotein component of a high-affinity ABC-type phosphate transporter. PstS is encoded by the first gene in the pst operon, the other four members of which encode the integral membrane and cytoplasmic components of the transporter. The transcription of the pst operon was analysed using a combination of methods, including transcriptional reporter gene technology, Northern blotting and DNA arrays. It is shown that the primary transcript of the pst operon is processed differentially to maintain higher concentrations of PstS relative to other components of the transporter. The comparative studies have revealed limitations in the use of reporter gene technology for analysing the transcription of operons in which the messenger RNA transcript is differentially processed.

The ftsH gene of Mycobacterium smegmatis SN2 (MsftsH) was cloned from two independent partial genomic DNA libraries and characterized, along with the identification of ephA and folE as the neighbouring upstream and downstream genes respectively. The genomic organization of the MsftsH locus was found to be identical to that of the Mycobacterium tuberculosis ftsH gene (MtftsH) and similar to that of other bacterial genera, but with divergence in the upstream region. The MsftsH gene is 2·3 kb in size and encodes the AAA (ATPases Associated with diverse cellular Activities) family Zn2+-metalloprotease FtsH (MsFtsH) of 85 kDa molecular mass. This was demonstrated from the expression of the full-length recombinant gene in Escherichia coli JM109 cells and from the identification of native MsFtsH in M. smegmatis SN2 cell lysates by Western blotting with anti-MtFtsH and anti-EcFtsH antibodies respectively. The recombinant and the native MsFtsH proteins were found localized to the membrane of E. coli and M. smegmatis cells respectively. Expression of MsFtsH protein in E. coli was toxic and resulted in growth arrest and filamentation of cells. The MsftsH gene did not complement lethality of a ΔftsH3 : : kan mutation in E. coli, but when expressed in E. coli cells, it efficiently degraded conventional FtsH substrates, namely σ 32 protein and the protein translocase subunit SecY, of E. coli cells.

Mu transposition complexes were used for transposon mutagenesis of Pseudomonas aeruginosa strain PA68. Mu DNA transposition complexes were assembled with MuA transposase and an artificial mini-Mu transposon in vitro, and introduced into Pseudomonas aeruginosa by electroporation. Eight mutants deficient in twitching motility were isolated. Southern blotting confirmed that the insertions had occurred as single events. DNA sequencing of the region flanking the insertion in the twitching-motility mutants revealed that the mini-Mu transposon had inserted into six different genes, PAO171, PA1822, PAO413, PA4959, PA4551 and PA5040. Four of these have previously been proven to be needed for twitching motility, whereas the PA1822 and PA0171 genes have not previously been shown to be required for twitching motility. The twitching-motility defect in the PA1822 mutant was partially complemented by providing the PA1822 gene in trans, and the defect in the PA0171 mutant was fully complemented when PA0171 was provided. A PA0171 mutant and a PA1822 mutant were constructed by gene replacement in the P. aeruginosa PAO1 strain. These mutants were deficient in twitching motility, showing that both the PA1822 and the PA0171 gene are involved in twitching motility.

The cytotoxin CytK produced by Bacillus cereus is believed to be involved in food-borne diseases. The transcriptional activity of the cytK promoter region in a food-poisoning strain was studied using a reporter gene and compared with that in the reference B. cereus strain ATCC 14579. In the food-poisoning strain, cytK is more strongly transcribed, possibly explaining the pathogenicity. The global regulator PlcR in B. cereus controls several putative virulence factors. It was found that PlcR regulates cytK in this clinical strain despite a mismatch in the PlcR recognition site, as currently defined. This suggests that the PlcR box consensus should be reconsidered and that the PlcR regulon might be larger than suspected. It is also shown that the high level of cytK transcription is not caused by a modification in the PlcR recognition site.

Phycobilisomes (PBS) are the major light-harvesting complexes of cyanobacteria. These usually blue-coloured multiprotein assemblies are rapidly degraded when the organisms are starved for combined nitrogen. This proteolytic process causes a colour change of the cyanobacterial cells from blue-green to yellow-green (‘bleaching’). As is well documented for the unicellular, non-diazotrophic cyanobacteria Synechococcus elongatus PCC 7942 and Synechocystis sp. PCC 6803, a gene termed nblA plays a key role in PBS degradation. Filamentous, diazotrophic cyanobacteria like Anabaena adapt to nitrogen deprivation by differentiation of N2-fixing heterocysts. However, during the first hours after nitrogen deprivation all cells degrade their PBS. When heterocysts mature and nitrogenase becomes active, vegetative cells resynthesize their light-harvesting complexes while in heterocysts the phycobiliprotein content remains very low. Expression and function of nblA in Anabaena sp. PCC 7120 was investigated. This strain has two nblA homologous genes, one on the chromosome (nblA) and one on plasmid delta (nblA-p). Northern blot analysis indicated that only the chromosomal nblA gene is up-regulated upon nitrogen starvation. Mutants with interrupted nblA and nblA-p genes, respectively, grew on N2 and developed functional heterocysts. Mutant ΔnblA-p behaved like the wild-type. However, mutant ΔnblA was unable to degrade its PBS, which was most obvious in non-bleaching heterocysts. The results show that NblA, encoded by the chromosomal nblA gene, is required for PBS degradation in Anabaena but is not essential for heterocyst differentiation.

The antifungal polyketide soraphen A is produced by the myxobacterium Sorangium cellulosum So ce26. The slow growth, swarming motility and general intransigence of the strain for genetic manipulations make industrial strain development, large-scale fermentation and combinatorial biosynthetic manipulation of the soraphen producer very challenging. To provide a better host for soraphen A production and molecular engineering, the biosynthetic gene cluster for this secondary metabolite was integrated into the chromosome of Streptomyces lividans ZX7. The upstream border of the gene cluster in Sor. cellulosum was defined by disrupting sorC, which is proposed to take part in the biosynthesis of methoxymalonyl-coenzyme A, to yield a Sor. cellulosum strain with abolished soraphen A production. Insertional inactivation of orf2 further upstream of sorC had no effect on soraphen A production. The genes sorR, C, D, F and E thus implicated in soraphen biosynthesis were then introduced into an engineered Str. lividans strain that carried the polyketide synthase genes sorA and sorB, and the methyltransferase gene sorM integrated into its chromosome. A benzoate-coenzyme A ligase from Rhodopseudomonas palustris was also included in some constructs. Fermentations with the engineered Str. lividans strains in the presence of benzoate and/or cinnamate yielded soraphen A. Further feeding experiments were used to delineate the biosynthesis of the benzoyl-coenzyme A starter unit of soraphen A in the heterologous host.

The genome of Bacillus subtilis, like those of some other AT-rich Gram-positive bacteria, has the uncommon feature of containing several copies of arrangements in which the genes encoding two-component and cognate ABC transporter systems are adjacent. As the function of one of these systems, the product of the yxd locus, is still unknown, it was analysed further in order to get some clues on the physiological role of the gene products it encodes. The yxdJ gene was shown to encode a DNA-binding protein that directly controls transcription of the neighbouring operon encoding the ABC transporter YxdLM. Primer extension and DNase protection experiments allowed precise definition of the yxdLM transcription start and controlling region. Two putative direct repeats were identified that are proposed to be the YxdJ response regulator binding sites. Whole-cell transcriptome analyses revealed that the YxdJ regulon is extremely restricted. In addition to the yxdJKLMyxeA operon, only a few genes involved in modifications of the bacterial cell wall were shown to be regulated by YxdJ.

A cluster of multicopper oxidase genes (mco1, mco2, mco3, mco4) from the lignin-degrading basidiomycete Phanerochaete chrysosporium is described. The four genes share the same transcriptional orientation within a 25 kb region. mco1, mco2 and mco3 are tightly grouped, with intergenic regions of 2·3 and 0·8 kb, respectively, whereas mco4 is located 11 kb upstream of mco1. All are transcriptionally active, as shown by RT-PCR. Comparison of cDNAs and the corresponding genomic sequences identified 14–19 introns within each gene. Based on homology and intron composition, two subfamilies of mco sequences could be identified. The sequences have copper-binding motifs similar to ferroxidase proteins, but different from fungal laccases. Thus, these sequences constitute a novel branch of the multicopper oxidase family. Analysis of several cDNA clones obtained from poly(A) RNA revealed the presence of transcripts of various lengths. Splice variants from mco2, mco3 and mco4 were characterized. They generally exhibited the presence of one to five introns, whereas other transcripts lacked some exons. In all cases, the presence of introns leads to frame shifts that give rise to premature stop codons. In aggregate, these investigations show that P. chrysosporium possesses a novel family of multicopper oxidases which also feature clustering and incomplete processing of some of their transcripts, a phenomenon referred to in this paper as ‘altered splicing’.