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Archives of Microbiology (v.188, #4)

Spore germination in Mortierella alpina is associated with a transient depletion of arachidonic acid and induction of fatty acid desaturase gene expression by C. Lounds; J. Eagles; A. T. Carter; D. A. MacKenzie; D. B. Archer (pp. 299-305).

Mortierella alpina is an oleaginous filamentous fungus whose vegetative mycelium is known to accumulate triglyceride oil containing large amounts of arachidonic acid (ARA 20:4, n − 6). We report that the spores of Mortierella alpina also contain a large proportion of ARA, comprising 50% of total fatty acid. Fatty acid desaturase genes were not expressed in dormant spores but were induced during germination, following a significant drop in the level of ARA (down from 50% of total fatty acid to 12%) prior to germ-tube emergence. We propose that ARA serves as a reserve supply of carbon and energy that is utilised during the early stages of spore germination in Mortierella alpina.

Keywords: Arachidonic acid; Zygomycete; Spore germination

Isolation and structural analysis of bamylocin A, novel lipopeptide from Bacillus amyloliquefaciens LP03 having antagonistic and crude oil-emulsifying activity by Sang-Cheol Lee; Sun-Hee Kim; In-Hye Park; Soo-Yeol Chung; Yong-Lark Choi (pp. 307-312).

Bacillus amyloliquefaciens strain LP03 isolated from soil, produced an antagonistic compound that strongly inhibited the growth of plant-pathogenic fungi and a lipopeptide biosurfactant. Also, isolated strain LP03 had a marked crude oil-emulsifying activity as it developed a clear zone around the colony after incubation for 24 h at 37°C. LP03 was identified as Bacillus amyloliquefaciens by analysis of partial 16 S rRNA gene and partial gyrA gene sequence. The lipopeptide was purified by acid precipitation of cell-free culture broth, extraction of the precipitates with methanol, silica gel column chromatography, and reverse-phase, high-pressure liquid chromatography. The purified biosurfactant was analyzed biochemical structure by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF) and electrospray ionization mass spectrometry/mass spectrometry (ESI-MS/MS). The masses of the two peaks were observed by HPLC chromatography. Their masses were determined to be 1,044 and 1,058 m/z with MALDI-TOF mass spectrometry. As constituents of the peptide and lipophilic part of the m/z 1,022.6, seven amino acids (Glu-Leu-Met-Leu-Pro-Leu-Leu) and β-hydroxy-C13 fatty acid were determined by ESI-MS/MS. The lipopeptide of 1,022.6 Da differed from surfactins in the substitution of leucine, valine and aspartic acid in positions 3, 4, and 5 by methionine, leucine, and proline, respectively. Novel lipopeptide was designated as bamylocin A.

Keywords: Antagonist; Bacillus amyloliquefaciens ; Surfactin; Bamylocin; Lipopeptide; Biosurfactant

Degradation of β-hexachlorocyclohexane by haloalkane dehalogenase LinB from γ-hexachlorocyclohexane-utilizing bacterium Sphingobium sp. MI1205 by Michihiro Ito; Zbyňek Prokop; Martin Klvaňa; Yoshiyuki Otsubo; Masataka Tsuda; Jiří Damborský; Yuji Nagata (pp. 313-325).

The technical formulation of hexachlorocyclohexane (HCH) mainly consists of the insecticidal γ-isomer and noninsecticidal α-, β-, and δ-isomers, among which β-HCH is the most recalcitrant and has caused serious environmental problems. A γ-HCH-utilizing bacterial strain, Sphingobium sp. MI1205, was isolated from soil which had been contaminated with HCH isomers. This strain degraded β-HCH more rapidly than the well-characterized γ-HCH-utilizing strain Sphingobium japonicum UT26. In MI1205, β-HCH was converted to 2,3,5,6-tetrachlorocyclohexane-1,4-diol (TCDL) via 2,3,4,5,6-pentachlorocyclohexanol (PCHL). A haloalkane dehalogenase LinB (LinB_MI) that is 98% identical (seven amino-acid differences among 296 amino acids) to LinB from UT26 (LinB_UT) was identified as an enzyme responsible for the two-step conversion of β-HCH to TCDL. This property of LinB_MI contrasted with that of LinB_UT, which catalyzed only the first step conversion of β-HCH to PCHL. Site-directed mutagenesis and computer modeling suggested that two of the seven different amino acid residues (V134 and H247) forming a catalytic pocket of LinB are important for the binding of PCHL in an orientation suitable for the reaction in LinB_MI. However, mutagenesis also indicated the involvement of other residues for the activity unique to LinB_MI. Sequence analysis revealed that MI1205 possesses the IS6100-flanked cluster that contains two copies of the linB _MI gene. This cluster is identical to the one located on the exogenously isolated plasmid pLB1, suggesting that MI1205 had recruited the linB genes by a horizontal transfer event.

Keywords: Haloalkane dehalogenase; Hexachlorocyclohexane; Environmental pollutant; Sphingomonads

Detection of chromosomally located and plasmid-borne genes on 20 kb DNA fragments in parasporal crystals from Bacillus thuringiensis by Yunjun Sun; Wei Wei; Xuezhi Ding; Liqiu Xia; Zhiming Yuan (pp. 327-332).

The association of 20 kb heterologous DNA fragments with the parasporal crystals from native and recombinant Bacillus thuringiensis strains was analyzed, respectively. The cry2Aa10 gene cloned in plasmid pHC39 was transformed into B. thuringiensis subsp. kurstaki strains Cry¯B and HD73, producing recombinant strains Cry¯B(pHC39) and HD73(pHC39). SDS-PAGE and scanning electron microscopy analyses demonstrated that the recombinant Cry¯B(pHC39) produced cuboidal crystals of Cry2Aa10 protoxin, while recombinant HD73(pHC39) produced both bipyramidal crystals of Cry1Ac1 protoxin and cuboidal crystals of Cry2Aa10 protoxin. Bioassay results proved that recombinant HD73(pHC39) showed higher insecticidal activity to Helicoverpa armigera than Cry¯B(pHC39). It was found that 20 kb DNA fragments were present in bipyramidal and cuboidal crystals from both native and recombinant strains, and the 20 kb heterologous DNAs contained chromosome-specific and resident large plasmid-borne DNA fragments, suggesting the 20 kb heterologous DNA fragment embodied in crystals came randomly from the bacterial chromosomal and plasmid genome. This was the first investigation devoted exclusively on the origin of 20 kb DNA fragments in the parasporal crystals of B. thuringiensis. The data provides a basis for further investigation of the origin of 20 kb DNAs in the crystals and the interaction of DNA and protoxins.

Keywords: Bacillus thuringiensis ; 20 kb DNAs; Crystal; Chromosome and plasmid; Insecticidal activity

Knockout of the gene (ste15) encoding a glycosyltransferase and its function in biosynthesis of exopolysaccharide in Streptomyces sp. 139 by Qing-Li Sun; Ling-Yan Wang; Jun-Jie Shan; Rong Jiang; Lian-Hong Guo; Yang Zhang; Ren Zhang; Yuan Li (pp. 333-340).

Streptomyces sp. 139 produces a novel exopolysaccharide (EPS) designated Ebosin which has antagonistic activity for IL-1R in vitro and remarkable anti-rheumatic arthritis activity in vivo. We previously identified a ste (Streptomyces eps) gene cluster consisting of 27 ORFs responsible for Ebosin biosynthesis. The gene product of ste15 shows high homology to known glycosyltransferases (GTFs). To elucidate its function in Ebosin biosynthesis, the ste15 gene was knocked out with a double crossover via homologous recombination. Our analysis of monosaccharide composition for EPS-m produced by the mutant strain Streptomyces sp. 139 (ste15 ⁻) showed that glucose was significantly diminished compared to its natural counterpart Ebosin. This derivative of Ebosin lost the antagonistic activity for IL-1R in vitro and its molecular mass was smaller than Ebosin. These results have demonstrated that the ste15 gene codes for a GTF for glucose, which is functionally involved in Ebosin biosynthesis.

Keywords: Gene ste15 ; Glycosyltrasferase; Ebosin biosynthesis; Gene knockout; Streptomyces sp. 139

Regulation of expression of Na⁺-translocating NADH:quinone oxidoreductase genes in Vibrio harveyi and Klebsiella pneumoniae by Maria S. Fadeeva; Evgenia A. Yakovtseva; Galina A. Belevich; Yulia V. Bertsova; Alexander V. Bogachev (pp. 341-348).

The expression of genes encoding sodium-translocating NADH:quinone oxidoreductase (Na⁺-NQR) was studied in the marine bacterium Vibrio harveyi and in the enterobacterium Klebsiella pneumoniae. It has been shown that such parameters as NaCl concentration, pH value, and presence of an uncoupler in the growth media do not influence significantly the level of nqr expression. However, nqr expression depends on the growth substrates used by these bacteria. Na⁺-NQR is highly repressed in V. harveyi during anaerobic growth, and nqr expression is modulated by electron acceptors and values of their redox potentials. The latter effect was shown to be independent of the ArcAB regulatory system.

Keywords: Regulation; Sodium translocation; Na⁺-NQR; NADH dehydrogenase; Klebsiella pneumoniae ; Vibrio ; ArcB

Role of nitrite reductase in the ammonia-oxidizing pathway of Nitrosomonas europaea by J. Jason L. Cantera; Lisa Y. Stein (pp. 349-354).

Metabolism of ammonia (NH₃) and hydroxylamine (NH₂OH) by wild-type and a nitrite reductase (nirK) deficient mutant of Nitrosomonas europaea was investigated to clarify the role of NirK in the NH₃ oxidation pathway. NirK-deficient N. europaea grew more slowly, consumed less NH₃, had a lower rate of nitrite (NO₂ ⁻) production, and a significantly higher rate of nitrous oxide (N₂O) production than the wild-type when incubated with NH₃ under high O₂ tension. In incubations with NH₃ under low O₂ tension, NirK-deficient N. europaea grew more slowly, but had only modest differences in NH₃ oxidation and product formation rates relative to the wild-type. In contrast, the nirK mutant oxidized NH₂OH to NO₂ ⁻ at consistently slower rates than the wild-type, especially under low O₂ tension, and lost a significant pool of NH₂OH–N to products other than NO₂ ⁻ and N₂O. The rate of N₂O production by the nirK mutant was ca. three times higher than the wild-type during hydrazine-dependent NO₂ ⁻ reduction under both high and low O₂ tension. Together, the results indicate that NirK activity supports growth of N. europaea by supporting the oxidation of NH₃ to NO₂ ⁻ via NH₂OH, and stimulation of hydrazine-dependent NO₂ ⁻ reduction by NirK-deficient N. europaea indicated the presence of an alternative, enzymatic pathway for N₂O production.

Keywords: Ammonia oxidation; Hydroxylamine oxidation; Nitrifier denitrification; Nitrite reductase; Nitrosomonas europaea ; Nitrous oxide

Diversity and geographical distribution of rhizobia associated with Lespedeza spp. in temperate and subtropical regions of China by Chun Tao Gu; En Tao Wang; Xin Hua Sui; Wen Feng Chen; Wen Xin Chen (pp. 355-365).

Eighty-eight root-nodule isolates from Lespedeza spp. grown in temperate and subtropical regions of China were characterized by a polyphasic approach. Nine clusters were defined in numerical taxonomy and SDS-PAGE analysis of whole cell proteins. Based upon further characterizations of amplified 16S rDNA restriction analysis (ARDRA), PCR-based restriction fragment length polymorphism of ribosomal IGS, 16S rDNA sequence analysis and DNA-DNA hybridization, these isolates were identified as Bradyrhizobium japonicum, B. elkanii, B. yuanmingense, Mesorhizobium amorphae, M. huakuii, Sinorhizobium meliloti and three genomic species related to B. yuanmingense, Rhizobium gallicum and R. tropici. The Bradyrhizobium species and R. tropici-related rhizobia were mainly isolated from the subtropical region and the species of Mesorhizobium, S. meliloti and R. gallicum-related species were all isolated from the temperate region. Phylogenetic analyses of nifH and nodC indicated that the symbiotic genes of distinct rhizobial species associated with Lespedeza spp. might have different origins and there was no evidence for lateral gene transfer of symbiotic genes. The results obtained in the present study and in a previous report demonstrated that Lespedeza spp. are nodulated by rhizobia with diverse genomic backgrounds and these Lespedeza-nodulating rhizobia were not specific to the host species, but specific to their geographic origins.

Keywords: Lespedeza ; Rhizobia; Phylogeny; Diversity; Biogeography

Characterization of a broad range antibacterial substance from a new Bacillus species isolated from Amazon basin by Amanda S. Motta; Fabiana S. Cannavan; Siu-Mui Tsai; Adriano Brandelli (pp. 367-375).

A Bacillus sp. strain producing a bacteriocin-like substance was characterized by biochemical profiling and 16S rDNA sequencing. The phylogenetic analysis indicated that this strain has low sequence similarity with most Bacillus spp., suggesting a new species was isolated. The antimicrobial activity was detected starting at the exponential growth phase, and maximum activity was observed at stationary phase. The substance was inhibitory to a broad range of indicator strains, incluing pathogenic and food spoilage bacteria such as Listeria monocytogenes, B. cereus, Aeromonas hydrophila, Erwinia carotovora, Pasteurella haemolytica, Salmonella Gallinarum, among other. The antibacterial substance was stable over a wide pH range, but it was sensitive to pronase E and lipase. The antibacterial substance was bactericidal and bacteriolytic to L. monocytogenes and B. cereus at 160 AU ml⁻¹. The identification of a broad range bacteriocin-like inhibitory substance active against L. monocytogenes addresses an important aspect of food protection against pathogens and spoilage microorganisms.

Keywords: Amazon; Antimicrobial; Bacillus ; Bacteriocin; Fish bacteria

Identification and characterization of two Streptomyces davawensis riboflavin biosynthesis gene clusters by Simon Grill; Hiroyuki Yamaguchi; Helen Wagner; Laure Zwahlen; Ute Kusch; Matthias Mack (pp. 377-387).

In Streptomyces davawensis roseoflavin is synthesized from GTP and ribulose-5-phosphate through riboflavin. As a first step towards the molecular analysis of flavin metabolism in S. davawensis the genes involved in riboflavin biosynthesis were cloned by hybridization of heterologous probes to a genomic library on a high-density colony-array. The genes ribB (riboflavin synthase, α-chain; EC 2.5.1.9), ribM (putative membrane protein), ribA (bifunctional GTP cyclohydrolase II/3,4-dihydroxy-2-butanone-4-phosphate synthase; EC 3.5.4.25) and ribH (lumazine synthase; EC 2.5.1.9) are organized in an operon-like cluster. Northern blot analysis of this cluster revealed two transcripts of 1.7 and 3.1 kb, respectively. The gene ribB was overexpressed in Escherichia coli. The specific riboflavin synthase activity in a cell-free extract of a recombinant strain was 0.246 nmol mg⁻¹min⁻¹. Overexpression of ribM enhanced the transport of riboflavin in the corresponding recombinant E. coli strain. Furthermore, overexpression of ribM increased roseoflavin sensitivity of E. coli. On another subgenomic fragment a putative S. davawensis ribG gene coding for the missing pyrimidine deaminase/reductase (EC 3.5.4.26 and EC 1.1.1.193) of the riboflavin biosynthetic pathway and ribY coding for a second (monofunctional) GTP cyclohydrolase II were identified.

Keywords: Riboflavin biosynthesis; Streptomyces davawensis ; Roseoflavin

Phosphate sensing in Synechocystis sp. PCC 6803: SphU and the SphS–SphR two-component regulatory system by Waraporn Juntarajumnong; Tripty A. Hirani; Joanne M. Simpson; Aran Incharoensakdi; Julian J. Eaton-Rye (pp. 389-402).

The Pho regulon is controlled by the histidine kinase-response regulator pair SphS–SphR in many cyanobacteria and up-regulation of the Pho regulon can be monitored by measuring alkaline phosphatase activity. However, the mechanism regulating signal transduction between SphS and SphR has not been described. We have created a cyanobacterial strain allowing the introduction of mutations into the transmitter domain of SphS. Mutations at Thr-167, adjacent to the H motif of SphS, introduce elevated alkaline phosphatase activity in the presence of phosphate and an enhancement of alkaline phosphatase activity, when compared to the control strain, in phosphate-limiting media. SphU acts as a negative regulator of the SphS–SphR system in Synechocystis sp. PCC 6803 and we show that constitutive alkaline phosphatase activity in the absence of SphU requires signal transduction through SphS and SphR. However, constitutive activity in the absence of SphU is severely attenuated in the ΔSphU:SphS-T167N mutant. Our data suggest that Thr-167 contributes to the mechanism underlying regulation by SphU. We have also assembled a deletion mutant system allowing the introduction of mutations into SphR and show that Gly-225 and Trp-236, which are both conserved in SphR from cyanobacteria, are essential for activation of the Pho regulon under phosphate-limiting conditions.

Keywords: Alkaline phosphatase; Cyanobacteria; Phosphate sensing; PhoB; PhoR; PhoU; SphR; SphS; SphU

The effect of sulfur compounds on H₂ evolution/consumption reactions, mediated by various hydrogenases, in the purple sulfur bacterium, Thiocapsa roseopersicina by Tatyana V. Laurinavichene; Gábor Rákhely; Kornél L. Kovács; Anatoly A. Tsygankov (pp. 403-410).

The influence of reduced sulfur compounds (including stored S⁰) on H₂ evolution/consumption reactions in the purple sulfur bacterium, Thiocapsa roseopersicina BBS, was studied using mutants containing only one of the three known [NiFe] hydrogenase enzymes: Hox, Hup or Hyn. The observed effects depended on the kind of hydrogenase involved. The mutant harbouring Hox hydrogenase was able to use S₂O ₃ ²⁻ , SO ₃ ²⁻ , S²⁻ and S⁰ as electron donors for light-dependent H₂ production. Dark H₂ evolution from organic substrates via Hox hydrogenase was inhibited by S⁰. Under light conditions, endogenous H₂ uptake by Hox or Hup hydrogenases was suppressed by S compounds. СО₂-dependent H₂ uptake by Hox hydrogenase in the light required the additional presence of S compounds, unlike the Hup-mediated process. Dark H₂ consumption via Hyn hydrogenase was connected to utilization of S⁰ as an electron acceptor and resulted in the accumulation of H₂S. In wild type BBS, with high levels of stored S⁰, dark H₂ production from organic substrates was significantly lower, but H₂S accumulation significantly higher, than in the mutant GB1121(Hox⁺). There is a possibility that H₂ produced via Hox hydrogenase is consumed by Hyn hydrogenase to reduce S⁰.

Keywords: Thiocapsa roseopersicina ; Hox-, Hup-, Hyn-hydrogenase; S⁰ ; Thiosulfate; H₂ evolution/consumption

Knockout of crtB or crtI gene blocks the carotenoid biosynthetic pathway in Deinococcus radiodurans R1 and influences its resistance to oxidative DNA-damaging agents due to change of free radicals scavenging ability by Lei Zhang; Qiao Yang; Xuesong Luo; Chengxiang Fang; Qiuju Zhang; Yali Tang (pp. 411-419).

Deinococcus radiodurans R1, a red-pigmented strain of the extremely radioresistant genus Deinococcus, contains a major carotenoid namely deinoxanthin. The high resistance of this organism against the lethal actions of DNA-damaging agents including ionizing radiation and ultraviolet light (UV) has been widely reported. However, the possible antioxidant role of carotenoids in this strain has not been completely elucidated. In this study, we constructed two colorless mutants by knockout of crtB and crtI genes, respectively. Comparative analysis of the two colorless mutants and the wild type showed that the two colorless mutants were more sensitive to ionizing radiation, UV, and hydrogen peroxide, but not to mitomycin-C (MMC). With electron spin resonance (ESR) and spin trapping techniques, we observed that hydroxyl radical signals occurred in the suspensions of UV irradiated Deinococcus radiodurans cells and the intensity of signals was influenced by carotenoids levels. We further showed that the carotenoid extract from the wild type could obviously scavenge superoxide anions generated by the irradiated riboflavin/EDTA system. These results suggest that carotenoids in D. radiodurans R1 function as free radical scavengers to protect this organism against the deleterious effects of oxidative DNA-damaging agents.

Keywords: Deinococcus radiodurans ; Carotenoids; ESR; Free radical

DNA bipyrimidine photoproduct repair and transcriptional response of UV-C irradiated Bacillus subtilis by Ralf Moeller; Erko Stackebrandt; Thierry Douki; Jean Cadet; Petra Rettberg; Hans-Joachim Mollenkopf; Günther Reitz; Gerda Horneck (pp. 421-431).

Vegetative wild-type and DNA repair-deficient (homologous recombination, recA and nucleotide excision repair, uvrB) Bacillus subtilis cells were exposed to UV-C radiation. Colony formation, DNA bipyrimidine photoproducts and gene expression were measured during cell recovery. Gene expression was measured after 60 min cell recovery where 50% (wild-type), 30% (recA) and 8% (uvrB), respectively, of the UV-C induced DNA photoproducts were repaired. We examined changes in the gene expression following UV exposure in wild-type and both repair-deficient strains. A set of known and unknown genes were found to be significantly up-regulated in wild-type B. subtilis cells, whereas no or lower gene induction was determined for both mutant strains. In addition, the possible roles of newly identified UV-responsive genes are discussed with respect to cellular recovery following exposure to UV irradiation.

Keywords: Bacillus subtilis ; UV-C radiation; DNA repair; Gene expression; DNA photoproducts

Isolation, sequencing and characterization of cluster genes involved in the biosynthesis and utilization of the siderophore of marine fish pathogen Vibrio alginolyticus by Qiyao Wang; Qin Liu; Yue Ma; Lingyun Zhou; Yuanxing Zhang (pp. 433-439).

In fish pathogen Vibrio alginolyticus MVP01, the isolated 11-gene cluster consisted of two divergently transcribed, Fe³⁺ and ferric uptake regulator (Fur) regulated operons, pvsABCDE and psuA-pvuABCDE, sharing high similarity with that related to siderophore biosynthesis and transportation locus in V. parahaemolyticus. Siderophore biosynthesis or utilization was blocked when pvsA and pvsD of the pvsABCDE operon or pvuA, pvuB and pvuE of the psuA-pvuABCDE operon was single-gene in-frame mutated, demonstrating their essential roles for siderophore biosynthesis or utilization in V. alginolyticus MVP01. Addition of the purified siderophore restored the cell growth in siderophore biosynthesis mutants, but not in siderophore uptake mutants.

Keywords: Vibrio alginolyticus ; Siderophore; Biosynthesis; Uptake; Cluster gene; Plasposon

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Archives of Microbiology (v.188, #4)