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Archives of Microbiology (v.186, #5)

Characterisation of the laccase-encoding gene abr2 of the dihydroxynaphthalene-like melanin gene cluster of Aspergillus fumigatus by Venelina Sugareva; Albert Härtl; Matthias Brock; Katrin Hübner; Manfred Rohde; Thorsten Heinekamp; Axel A. Brakhage (pp. 345-355).

Aspergillus fumigatus is an important pathogen of the immunocompromised host. Previously, it was shown that the polyketide synthase encoded by the pksP (alb1) gene represents a virulence determinant. pksP is part of a gene cluster involved in dihydroxynaphthalene (DHN)-like melanin biosynthesis. Because a putative laccase-encoding gene (abr2) is also part of the cluster and a laccase was found to represent a virulence factor in Cryptococcus neoformans, here, the Abr2 laccase was characterised. Deletion of the abr2 gene changed the gray-green conidial pigment to a brown color and the ornamentation of conidia was reduced compared with wild-type conidia. In contrast to the white pksP mutant, the susceptibility of the Δabr2 mutant against reactive oxygen species (ROS) was not increased, suggesting that the intermediate of DHN-like melanin produced up to the step catalysed by Abr2 already possesses ROS scavenging activity. In an intranasal mouse infection model, the Δabr2 mutant strain showed no reduction in virulence compared with the wild type. In the Δabr2 mutant, overall laccase activity was reduced only during sporulation, but not during vegetative growth. An abr2p-lacZ gene fusion was expressed during sporulation, but not during vegetative growth confirming the pattern of laccase activity due to Abr2.

Keywords: Dihydroxynaphthalene-like melanin; Aspergillus fumigatus ; Gene cluster; Laccase

Importance of the DsrMKJOP complex for sulfur oxidation in Allochromatium vinosum and phylogenetic analysis of related complexes in other prokaryotes by Johannes Sander; Sabine Engels-Schwarzlose; Christiane Dahl (pp. 357-366).

In the phototrophic sulfur bacterium Allochromatium vinosum, sulfur of oxidation state zero stored in intracellular sulfur globules is an obligate intermediate during the oxidation of sulfide and thiosulfate. The proteins encoded in the dissimilatory sulfite reductase (dsr) locus are essential for the oxidation of the stored sulfur. DsrMKJOP form a membrane-spanning complex proposed to accept electrons from or to deliver electrons to cytoplasmic sulfur-oxidizing proteins. In frame deletion mutagenesis showed that each individual of the complex-encoding genes is an absolute requirement for the oxidation of the stored sulfur in Alc. vinosum. Complementation of the ΔdsrJ mutant using the conjugative broad host range plasmid pBBR1-MCS2 and the dsr promoter was successful. The importance of the DsrMKJOP complex is underlined by the fact that the respective genes occur in all currently sequenced genomes of sulfur-forming bacteria such as Thiobacillus denitrificans and Chlorobaculum tepidum. Furthermore, closely related genes are present in the genomes of sulfate- and sulfite-reducing prokaryotes. A phylogenetic analysis showed that most dsr genes from sulfide oxidizers are clearly separated of those from sulfate reducers. Surprisingly, the dsrMKJOP genes of the Chlorobiaceae all cluster together with those of the sulfate/sulfite-reducing prokaryotes, indicating a lateral gene transfer at the base of the Chlorobiaceae.

Keywords: Allochromatium vinosum ; Sulfur oxidation; Heterodisulfide reductase; DsrMKJOP complex; Horizontal gene transfer

The rice field cyanobacteria Anabaena azotica and Anabaena sp. CH1 express vanadium-dependent nitrogenase by Gudrun Boison; Caroline Steingen; Lucas J. Stal; Hermann Bothe (pp. 367-376).

Anabaena azotica FACHB-118 and Anabaena sp. CH1, heterocystous cyanobacteria isolated from Chinese and Taiwanese rice fields, expressed vanadium-containing nitrogenase when under molybdenum deficiency. This is the second direct observation of an alternative nitrogenase in cyanobacteria. The vanadium nitrogenase-specific genes vnfDG are fused and clustered in a phylogenetic tree next to the corresponding genes of Methanosarcina. The expression of vnfH in cells cultured in Mo-free medium and of nifH in Mo-grown cells was shown for the first time by sequencing cDNA derived from cultures of A. azotica and Anabaena sp. CH1. The vnfH sequences clustered with that of Anabaena variabilis. The vnf genes were strongly transcribed only in cultures grown either in Mo-free medium, or in W-containing medium, but also weakly in Mo-containing medium. NifH was transcribed in all media. On-line measurements of acetylene reduction by Mo-free A. azotica cultures demonstrated that the V-nitrogenase was active. Ethane was formed continuously at a rate of 2.1% of that of ethylene. Acetylene reduction of cultures grown either with or without Mo had a high temperature optimum of 42.5°C. The uptake hydrogenase gene hupL was expressed in Mo-free medium concomitantly with vnfDG in A. azotica, Anabaena sp. CH1, and A. variabilis.

Keywords: Alternative nitrogenase; Vanadium nitrogenase; Cyanobacteria; Tungsten; vnfDG; vnfH; Uptake hydrogenase; Gene expression

The synthesis and role of the mechanosensitive channel of large conductance in growth and differentiation of Bacillus subtilis by Paul G. Wahome; Peter Setlow (pp. 377-383).

A translational lacZ fusion of the Bacillus subtilis mscL gene that encodes the mechanosensitive channel of large conductance (MscL) was expressed at significant levels during log phase growth of B. subtilis, and the level of mscL–lacZ expression was increased 1.5-fold by growth in medium with high salt (1 M NaCl). However, in growth media with either low or high salt, mscL–lacZ expression fell drastically beginning in the late log phase of growth, and fell to even lower levels during sporulation, although a significant amount of β-galactosidase from mscL to lacZ was accumulated in the developing spore. Deletion of mscL had no effect on B. subtilis growth, sporulation or subsequent spore germination. The ΔmscL strain also grew as well as the wild-type parental strain in medium with 1.2 M NaCl. While log phase wild-type cells grown with 1.2 M NaCl survived a rapid 0.9 M osmotic downshift, log phase ΔmscL cells rapidly lost viability and lysed when subjected to this same osmotic downshift. However, by the early stationary phase of growth, ΔmscL cells had become resistant to a 0.9 M osmotic downshift.

Keywords: Bacillus ; Sporulation; Spore germination; Mechanosensitive channels; Osmoregulation

Oxidative inactivation of reduced NADP-generating enzymes in E. coli: iron-dependent inactivation with affinity cleavage of NADP-isocitrate dehydrogenase by Keiko Murakami; Ryoko Tsubouchi; Minoru Fukayama; Tadashi Ogawa; Masataka Yoshino (pp. 385-392).

Treatment of E. coli extract with iron/ascorbate preferentially inactivated NADP-isocitrate dehydrogenase without affecting glucose-6-phosphate dehydrogenase. NADP-Isocitrate dehydrogenase required divalent metals such as Mg²⁺, Mn²⁺or Fe²⁺ ion. Iron/ascorbate-dependent inactivation of the enzyme was accompanied with the protein fragmentation as judged by SDS-PAGE. Catalase protecting the enzyme from the inactivation suggests that hydroxyl radical is responsible for the inactivation with fragmentation. TOF-MS analysis showed that molecular masses of the enzyme fragments were 36 and 12, and 33 and 14 kDa as minor components. Based on the amino acid sequence analyses of the fragments, cleavage sites of the enzyme were identified as Asp307-Tyr308 and Ala282-Asp283, which are presumed to be the metal-binding sites. Ferrous ion bound to the metal-binding sites of the E. coli NADP-isocitrate dehydrogenase may generate superoxide radical that forms hydrogen peroxide and further hydroxyl radical, causing inactivation with peptide cleavage of the enzyme. Oxidative inactivation of NADP-isocitrate dehydrogenase without affecting glucose 6-phosphate dehydrogenase shows only a little influence on the antioxidant activity supplying NADPH for glutathione regeneration, but may facilitate flux through the glyoxylate bypass as the biosynthetic pathway with the inhibition of the citric acid cycle under aerobic growth conditions of E. coli.

Keywords: NADP-isocitrate dehydrogenase; Reactive oxygen species; Affinity cleavage; Metal binding; Iron

Analysis of conserved glutamate residues in Porphyromonas gingivalis outer membrane receptor HmuR: toward a further understanding of heme uptake by Teresa Olczak (pp. 393-402).

The aim of this study was to broaden the current knowledge about the Porphyromonas gingivalis heme receptor HmuR. Site-directed mutagenesis was employed to replace Glu427, Glu448, Glu458 and Glu503 by alanines and to construct a triple Glu427Ala/Glu448Ala/Glu 458Ala mutant. All iron/heme-starved P. gingivalis mutants showed decreased growth recovery when human serum as the iron/heme source was used, hmuR::ermF, hmuR ^E503A and hmuR ^{E427A,E448A,E458A} mutant strains being the most affected. E. coli cells expressing HmuR with mutated glutamate residues bound hemin, hemoglobin and hemin–serum albumin complex with the same efficiency as did the wild-type recombinant protein, suggesting that the residues were not directly involved in heme binding. These data indicate that in addition to two conserved histidine residues (His95 and His434), NPDL and YRAP motifs, conserved glutamate residues are important for HmuR to utilize heme present in serum hemoproteins.

Keywords: Porphyromonas gingivalis ; HmuR; Heme receptor; Outer membrane receptor; Heme transport

Negative control of the high light-inducible hliA gene and implications for the activities of the NblS sensor kinase in the cyanobacterium Synechococcus elongatus strain PCC 7942 by Anthony D. Kappell; Devaki Bhaya; Lorraine G. van Waasbergen (pp. 403-413).

The hliA gene of the cyanobacterium Synechococcus elongatus PCC 7942 is known to be upregulated by high-intensity light through the activity of the NblS sensor kinase. In this work it was found that, within the hliA upstream region, changes to the sequence around −30 to −25 (relative to the transcriptional start site) resulted in elevated hliA expression, implicating this region in negative regulation of the gene. Electrophoretic mobility shift assays performed were consistent with a protein binding this region that acts to keep the gene off in lower light. A reduction in gene dosage of nblS in vivo resulted in enhanced hliA expression, suggesting that negative control of hliA is mediated through NblS. An extended version of the high light regulatory 1 (HLR1) motif (previously described in Synechocystis PCC 6803) was identified within the sequence surrounding −30 to −25 of hliA. The extended HLR1 sequence was found upstream of other NblS-controlled genes from S. elongatus and Synechocystis PCC 6803 and upstream of hli genes from a variety of cyanobacterial and related genomes. These results point to the evolutionary conservation of the HLR1 element and its importance in NblS-mediated signaling and yield new insight into NblS-mediated control of gene expression.

Keywords: Cyanobacteria; High intensity light; hliA gene; HLR1 cis element; NblS sensor kinase

Characterization of the Mn²⁺-stimulated (di)adenosine polyphosphate hydrolase encoded by the Deinococcus radiodurans DR2356 nudix gene by David I. Fisher; Jared L. Cartwright; Alexander G. McLennan (pp. 415-424).

The DR2356 nudix hydrolase gene from Deinococcus radiodurans has been cloned and the product expressed as an 18 kDa histidine-tagged protein. The enzyme hydrolysed adenosine and diadenosine polyphosphates, always generating ATP as one of the initial products. ATP and other (deoxy)nucleoside triphosphates were also substrates, yielding (d)NDP and Pi as products. The DR2356 protein was most active at pH 8.6–9.0 and showed a strong preference for Mn²⁺ as activating cation. Mg²⁺ ions at 15 mM supported only 5% of the activity achieved with 2 mM Mn²⁺. K _m and k _cat values for diadenosine tetra-, penta- and hexaphosphates were 2.0, 2.4 and 1.1 μM and 11.4, 28.6 and 12.0 s⁻¹, respectively, while for GTP they were 20.3 μM and 1.8 s⁻¹, respectively. The K _mfor adenosine 5′-pentaphosphate was <1 μM. Expression analysis showed the DR2356 gene to be induced eight- to ninefold in stationary phase and in cells subjected to slow dehydration plus rehydration. Superoxide (but not peroxide) treatment and rapid dehydration caused a two-to threefold induction. The Mn-requirement and induction in stationary phase suggest that DR2356 may have a specific role in maintenance mode metabolism in stationary phase as Mn²⁺ accumulates.

Keywords: Deinococcus radiodurans ; Nudix; Diadenosine; Nucleotide metabolism; Manganese

Physiological, biochemical, and genetic characterization of an alicyclic amine-degrading Mycobacterium sp. strain THO100 isolated from a morpholine-containing culture of activated sewage sludge by Yong-Hak Kim; Ilnam Kang; Hélène Bergeron; Peter C. K. Lau; Karl-Heinrich Engesser; Sang-Jong Kim (pp. 425-434).

Mycobacterium sp. strain THO100 was isolated from a morpholine-containing culture of activated sewage sludge. This strain was able to utilize pyrrolidine, morpholine, piperidine, piperazine, and 1,2,3,6-tetrahydropyridine as the sole sources of carbon, nitrogen, and energy. The degradation pathway of pyrrolidine as the best substrate for cellular growth was proposed based on the assays of substrate-induced cytochrome P450 and constitutive enzyme activities toward 4-aminobutyric acid (GABA) and succinic semialdehyde (SSA). Its 16S ribosomal RNA gene sequence (16S rDNA) was identical to that of Mycobacterium tokaiense ATCC 27282^T. The morABC genes responsible for alicyclic amine degradation were nearly identical among different species of Mycobacteria. Remarkably, repetitive sequences at the intergenic spacer (IGS) region between morC and orf1’ were detected by comparison of the nearly identical mor gene cluster regions. Considering the strain activity for alicyclic amine degradation, the deleted 65-bp DNA segment did not significantly alter the open reading frames, and the expression and functions of the P450_mor system remained unaltered. In addition, we found a spontaneous deletion of P450_mor from another strain HE5 containing the archetypal mor gene cluster, which indicated a possible occurrence of DNA recombination to rearrange the DNA.

Keywords: Alicyclic amines (AA); Cytochrome P450 monooxygenase (P450); DNA deletion; Mycobacterium ; Phylogeny

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Archives of Microbiology (v.186, #5)

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Archives of Microbiology (v.186, #5)