基于基因组的一株土壤固氮菌分离菌株鉴定及其促生作用

[目的] 为获得高效固氮菌株,充分研究利用土壤固氮菌资源。[方法] 选取固氮能力较高的紫色土发育水稻土,采用富集纯化法分离固氮微生物菌株。通过16S rRNA基因系统发育分析和全基因组相关指数比较对新分离菌株进行物种鉴定。采用乙炔还原法和¹⁵N₂示踪法定量测定新分离菌株的固氮能力,通过培养特性和接种效果初步研究固氮菌株的促生作用。[结果] 从紫色土发育水稻土中分离得到1株可在无氮培养基上快速生长的菌株P208。基于16S rRNA基因和基因组92个核心基因的系统发育分析结果表明,新分离菌株P208与Azotobacter chroococcum IAM 12666^T（=ATCC 9043^T）系统发育距离最近（16S rRNA基因相似度为99.79%）。菌株P208与A.chroococcum ATCC 9043^T的基因组平均核苷酸一致性（ANI）、平均氨基酸一致性（AAI）和数字DNA-DNA杂交值（dDDH）高于物种分类阈值（ANI>95%-96%,AAI>95%-96%,dDDH>70%）,最大唯一匹配指数（MUMi）低于物种分类阈值（<0.33）,得出新分离菌株P208为褐球固氮菌（A.chroococcum）。A.chroococcum P208固氮活性为模式菌株A.chroococcum ATCC 9043^T的2.61倍。除固氮能力外,A.chroococcum P208具有IAA生成、溶磷活性和铁载体生成等促进植物生长潜力的培养特性,室内培养条件下接种A.chroococcum P208能够促进水稻、小麦幼苗根系的生长。[结论] 从固氮能力较强的水稻土中分离纯化得到1株具有较强固氮、促生潜力的固氮菌,具有潜在的开发应用价值,可为研究利用生物固氮提供微生物资源。     

Dinitrogen fixation (C2H2 reduction) by bacterial strains at various temperatures

Acetylene-reducing activities (ARA) of strains ofEnterobacter agglomerans, Azospirillum brasilense, Azotobacter chroococcum, and Bacillus, isolated from temperate or tropical soils, were compared at different temperatures to study temperature adaptability. All Enterobacter strains and Bacillus strain C-11-25 reduced C₂H₂ at temperatures as low as 5°C. ARA by Enterobacter strains declined sharply above 30°C but ARA by Bacillus strain C-11-25 continued to increase with an increase in temperature.A. brasilense strain sp 245, isolated from wheat roots in Brazil, reduced more C₂H₂ at lower temperatures than strain Cd, isolated from a Californian soil. Similarly, the temperate strain ofA. chroococcum was a better N₂ fixer than the tropicalA. chroococcum strain at lower temperatures. Tropical strains ofA. brasilense andA. chroococcum reduced more C₂H₂ than temperate strains at higher temperatures. Therefore, it appears that temperate and tropical N₂-fixing organisms adapt themselves to their particular environment and should have more potential to benefit crops grown at the particular temperatures favorable to them. Only Bacillus strain C-11-25 has potential to benefit both temperate and tropical crops because it reduced significant acetylene over a wide temperature range.     

Establishment of phosphate-solubilizing strains of Azotobacter chroococcum in the rhizosphere and their effect on wheat cultivars under green house conditions

A pot experiment was conducted in the green house to investigate the establishment of phosphate solubilizing strains of Azotobacter chroococcum, including soil isolates and their mutants, in the rhizosphere and their effect on growth parameters and root biomass of three genetically divergent wheat cultivars (Triticum aestivum L.). Five fertilizer treatments were performed: Control, 90 kg N ha^—1, 90 kg N + 60 kg P₂O₅ ha^—1, 120 kg N ha^—1 and 120 kg N + 60 kg P₂O₅ ha^—1. Phosphate solubilizing and phytohormone producing parent soil isolates and mutant strains of A. chroococcum were isolated and selected by an enrichment method. In vitro phosphate solubilization and growth hormone production by mutant strains was increased compared with soil isolates. Seed inoculation of wheat varieties with P solubilizing and phytohormone producing A. chroococcum showed better response compared with controls. Mutant strains of A. chroococcum showed higher increase in grain (12.6%) and straw (11.4%) yield over control and their survival (12—14%) in the rhizosphere as compared to their parent soil isolate (P4). Mutant strain M37 performed better in all three varieties in terms of increase in grain yield (14.0%) and root biomass (11.4%) over control.     

Synthesis of poly(3-hydroxyalkanoates) by mutant and recombinant Pseudomonas strains

We have studied the accumulation kinetics and physical characteristics of the poly(3-hydroxyalkanoates) (PHAs) formed by several Pseudomonas strains, mutants and recombinants. Although PHA synthesis generally begins only after an essential nutrient such as N, P, S or Mg becomes limiting, we have identified at least one strain (P. putida KT2442) that begins producing PHA during the exponential growth phase. This PHA is chemically and physically identical to that produced by P. oleovorans GPol, the strain in which we first identified PHA. Analysis of the PHA formed by a mutant strain defective in PHA degradation (P. oleovorans GPo500) revealed that the molecular mass (M_w), the monomer composition and thermal characteristics were similar to that of the PHA of the wild-type parent strain P. oleovorans GPo1. The pha locus of P. oleovorans encodes enzymes that are involved in PHA biosynthesis and degradation. It has been subcloned to study the two PHA polymerases separately in a PHA^– mutant (GPp104) derived from P. putida KT2442. The recombinant strains accumulated lower PHA levels than the wild-type strains, and the M_w of these polymers were lower than those produced by the wild-type P. oleovorans and parent strain. The monomer composition of the two PHAs formed by the two PHA polymerases differed, indicating that the PHA polymerases have different substrate specificities for the incorporation of 3-hydroxyoctanoate and 3-hydroxyhexanoate monomers into PHA. Despite these differences, the PHAs formed were essentially indistinguishable from wild-type PHAs with respect to their thermal characteristics.Correspondence to: B. Witholt     

Nitrogen fixation in strains ofAzotobacter chroococcum bearing deletions of a cluster of genes coding for nitrogenase

Strains of the obligately aerobic nitrogen fixing organismAzotobacter chroococcum were constructed which contained defined chromosomal deletions in which the nitrogenase structural genenifHDK cluster (nifH for the polypeptide of the Fe-protein component of nitrogenase andnifD andnifK for the alpha and beta subunits respectively of the MoFe-protein component of the enzyme) was replaced by a kanamycin resistance gene. N₂ fixation was nevertheless observed in deletion strains though only in a molybdenum-deficient medium or in spontaneously arising tungstate-resistant derivatives. In comparison with the parent strain growing in molybdenum-sufficient medium, diazotrophic growth was slow and the nitrogenase activity in vivo was characterised by disproportionately low rates of C₂H₂-reduction compared to H₂-evolution and relative insensitivity of H₂-evolution to inhibition by C₂H₂. The findings show reiteration of functional structural genes for nitrogenase inA. chroococcum consistent with our previous observation of twonifH genes in this organism and detection in this work of a secondnifK-like sequence in the genomes of both parent and deletion strains whenA. chroococcum nifK DNA was used as a probe.     

Effect of Simazine on the production of lysine and methionine byAzotobacter chroococcum andAzotobacter vinelandii

Summary Production of lysine and methionine byAzotobacter chroococcum strain H23 andA. vinelandii strain ATCC 12837 was studied in chemically-defined medium and dialysed-soil medium, amended with different concentrations of Simazine. Responses on production due to Simazine were different for each strain and were fairly conditioned by culture media composition. Quantitative production of amino acids was significantly affected by the xenobiotic only at higher doses (50–100,g/ml). The effect of Simazine on methionine production by strain H23 was very pronounced when bacteria were grown in dialysed-soil medium, which was specially formulated to reproduce the natural habitat of the organisms.     

Production of microalgae from olive mill wastewater

With olive mill wastewater (alpechin) used as a nutrient medium, the influence of the aeration level and the composition of the culture medium were analysed in relation to the concentration of alpechin and KNO₃ added in a batch culture of the microalgae Chlorella pyrenoidosa and Scenedesmus obliquus. The pH and temperature of the culture were previously fixed at 6.5 and 30°C. As kinetic parameters, the maximum specific growth rate (μ_m) and the biomass productivity (P_max) were determined. The biomass composition obtained was evaluated for chlorophyll and crude protein content as well as fatty-acid composition corresponding to the lipid fraction. To achieve a balanced biomass composition in terms of proteins and lipids, the most suitable conditions included an alpechin concentration of 10%, without the addition of nitrates and with an aeration level of 1 v/v min. Under these conditions, the μ_m values reached with both microalgae were around 0.03h⁻¹.     

Influence of analogue-resistant mutants ofAzotobacter chroococcum solubilizing phosphate on yield and quality of sunflower (Helianthus annuus)

Two phosphate-solubilizing analogue-resistant mutants ofAzotobacter chroococcum showed a positive interactive effect on sunflower (Helianthus annuus). Plant height, leaf area, seed mass, seed yield, protein and oil content increased significantly by inoculation withA. chroococcum mutants. Both levels of fertilizers applied (80N+30P₂O₅ and 80N+60P₂O₅ kg/hm² showed increased seed yield. Likewise, inoculation resulted in a significantly higher yield. Application ofP-solubilizingA. chroococcum strains with the lower fertilizer level showed no significant difference on plant growth parameters when compared with the higher fertilizer level, indicating that inoculation of P-solubilizingAzotobacter strains can reduce the necessary levels of fertilization from 60P₂O₅ to 30P₂O₅ kg/hm².     

Growth enhancement of Sesamum indicum L. by rhizosphere-competent Azotobacter chroococcum AZO2 and its antagonistic activity against Macrophomina phaseolina

Indigenous strains isolated from rhizosphere may contain highly competent genotypes to enhance the plant growth and often perform better than the introduced isolates. The present study deals with the characterisation of plant growth-promoting (PGP) attributes and antagonistic activity of Azotobacter chroococcum AZO2 against Macrophomina phaseolina causing charcoal rot disease and their effect on the growth of sesame (Sesamum indicum L.). Eight strains of Azotobacter were isolated from sesame rhizosphere on nitrogen-free medium, which exhibited significant PGP parameters such as phosphate solubilisation, indole acetic acid and siderophore production. The strain A. chroococcum AZO2 (EU274299) was characterised by 16S rDNA gene sequencing. Amplification of 781 bp nif H gene confirms nitrogenase activity of all the strains. A. chroococcum AZO2 exhibited strong antagonistic activities against M. phaseolina causing 81% colony growth inhibition and resulted in hyphal perforations, empty cell (halo) formation, hyphal twisting, shrinking and lysis of fungal mycelia along with degeneration of sclerotia. A. chroococcum AZO2 produced chitinase that caused degradation and digestion of the cell wall component of M. phaseolina. Different vegetative and reproductive parameters of sesame were found to be enhanced significantly upon application of A. chroococcum AZO2 + half doses of chemical fertilisers. A. chroococcum AZO2 was also found to be an effective root coloniser, plant growth promoter and potential antagonistic bacterium. It can be concluded that A. chroococcum AZO2 strain bears the characteristics of technological applications for inoculant preparation and growth enhancement of sesame besides being utilised as a better PGP bacterium as well as an effective agent for biocontrol of M. phaseolina.     

Effect of some herbicides on the production of lysine byAzotobacter chroococcum

Summary Production of lysine byAzotobacter chroococcum strain H23 was studied in chemically-defined media amended with different concentrations of alachlor, metolachlor, 2,4-D, 2,4,5-T and 2,3,6-TBA. The presence of 5, 10, and 50g/ml of alachlor or 2,3,6-TBA significantly decreased quantitative production of lysine. However, the presence 2,4-D or 2,4,5-T at concentrations of 10 and 50g/ml enhanced the production of lysine. Quantitative production of lysine was not affected as consequence of the addition of metolachlor to the culture medium, showing that the release lysine to the culture media byA. chroococcum was not affected by that herbicide.     

Production of amino acids by Azotobacter vinelandii and Azotobacter chroococcum with phenolic compounds as sole carbon source under diazotrophic and adiazotrophic conditions

Summary. Azotobacter vinelandii strain ATCC 12837 and Azotobacter chroococcum strain H23 (CECT4435) were tested to grow in N-free or NH₄Cl amended chemically defined media, with protocatechuic acid or sodium p-hydroxybenzoate as sole carbon (C) sources at a concentration of 2 mmol/L. Both substrates supported grow at similar rates than bacteria grown in control media amended with 2 mmol/L sodium succinate as C source. The two strains produced aspartic acid, serine, glutamic acid, glycine, hystidine, threonine, arginine, alanine, proline, cysteine, tyrosine, valine, methionine, lysine, isoleucine, leucine and phenylalanine after 72 h of growth in chemically defined media with 2 mmol/L of phenolic compounds or sodium succinate as sole C source amended or unamended with 0.1% (w/v) NH₄Cl. Qualitative and quantitative production of all amino acids was not affected by the use of different C and N substrates.     

Short-term ammonium inhibition of nitrate uptake by Azotobacter chroococcum

Addition of NH₄Cl at low concentrations to Azotobacter chroococcum cells caused an immediate cessation of nitrate uptake activity, which was restored when the added NH ₄ ⁺ was exhausted from the medium or by adding an NH ₄ ⁺ assimilation inhibitor, l-methionine-dl-sulfoximine (MSX) or l-methionine sulfone (MSF). In the presence of such inhibitors the newly-reduced nitrate was released into the medium as NH ₄ ⁺ . When the artificial electron donor system ascorbate/N-methylphenazinium methylsulfate (PMS), which is a respiratory substrate that was known to support nitrate uptake by A. chroococcum while inhibiting glutamine synthetase activity, was the energy source, externally added NH ₄ ⁺ had no effect on nitrate uptake. It is concluded that, in A. chroococcum cells, NH ₄ ⁺ must be assimilated to exert its short-term inhibitory effect on nitrate uptake. A similar proposal was previously made to explain the short-term ammonium inhibition of N₂ fixation in this bacterium.Abbreviations MOPS morpholinopropanesulfonic acid - MSX l-methionine-dl-sulfoximine - PMS N-methylphenazinium methylsulfate - MSF l-methionine sulfone     

Cloning and characterization of hydrogenase genes from Azotobacter chroococcum

Summary Genomic DNA from Azotobacter chroococcum was shown by DNA hybridization to contain sequences homologous to Rhizobium japonicum H₂-uptake (hup) hydrogenase genes carried on the plasmid pHU1. Two recombinant cosmid clones, pACD101 and pACD102, were isolated from a gene library of A. chroococcum by colony hybridization and physically mapped. Each contained approximately 42 kb of insert DNA with approximately 27 kb of overlapping DNA. Further hybridization studies using three fragments from pHU1 (6 kb HindIII, 6.4 kb BglII and 5 kb EcoRI) showed that the hup-specific regions of R. japonicum and A. chroococcum are probably highly conserved. Weak homology to the hydrogenase structural genes from Desulfovibrio vulgaris (Hildenborough) was also observed. A 24 kb BamHI fragment from pACD102 subcloned into a broad host-range vector restored hydrogenase activity to several Hup^- mutants of A. chroococcum.     

Chemical-physical characterization of polyhydroxyalkanoates recovered by means of a simplified method from cultures of <Emphasis Type="Italic">Halomonas campaniensis</Emphasis>

In this study we suggest a simplified and effective method to directly recover polyhydroxyalkanoates (PHAs) from humid biomass of Halomonas campaniensis with no pre-treatment steps. Sodium dodecyl sulphate (SDS) was directly added to dispersed biomass of cultured micro-organism (w/w ratio: 1) in distilled water followed by shaking, heat treatment, and washing steps. The purity of the recovered PHAs synthesized by H. campaniensis was over 95%, regardless of the cell concentrations and the best yield was 12% (w/w) of the cell wet weight when the micro-organism was cultivated in a glucose-based medium or a glucose/propionate-based medium. MS spectroscopy and ¹H, ¹³C-NMR analysis were used to chemically characterize the PHAs; their thermal characteristics were obtained using a differential scanning calorimeter and the average viscosity molecular weight was assessed through specific viscosity measurements. Due to its ease and velocity, our simplified method is suitable for the detection and recovery of PHAs from humid biomasses with high yield and purity. The method, which is quick and at low environmental impact, is very valuable for the simultaneous testing of cultures grown with different inducers for PHAs having particular chemical/physical characteristics.     

Amycolatopsis camponoti sp. nov., new tetracenomycin-producing actinomycete isolated from carpenter ant Camponotus vagus

An actinobacterial strain A23^T, isolated from adult ant Camponotus vagus collected in Ryazan region (Russia) and established as tetracenomycin X producer, was subjected to a polyphasic taxonomic study. Morphological characteristics of this strain included well-branched substrate mycelium and aerial hyphae fragmented into rod-shaped elements. Phylogenetic analyses based on 16S rRNA gene and genome sequences showed that strain A23^T was most closely related to Amycolatopsis pretoriensis DSM 44654^T. Average nucleotide identity and digital DNA–DNA hybridization values between the genome sequences of isolate A23^T and its closest relative, Amycolatopsis pretoriensis DSM 44654^T, were 39.5% and 88.6%, which were below the 70% and 95–96% cut-off point recommended for bacterial species demarcation, respectively. The genome size of the isolate A23^T was 10,560,374 bp with a DNA G?+?C content of 71.2%. The whole-cell hydrolysate contained meso-diaminopimelic acid and arabinose and galactose as main diagnostic sugars as well as ribose and rhamnose. It contained MK-9(H4) as the predominant menaquinone and iso-C_16:0, iso-C_15:0, anteiso-C_17:0 and C_16:0 as the major cellular fatty acids. Diphosphatidylglycerol and phosphatidylethanolamine prevailed among phospholipids. Mycolic acids were not detected. Based on the phenotypic, genomic and phylogenetic data, isolate A23^T represents a novel species of the genus Amycolatopsis, for which the name Amycolatopsis camponoti sp. nov. is proposed, and the type strain is A23^T (=?DSM 111725^T?=?VKM Ac-2882^T).

     

Fuscibacter oryzae gen. nov., sp. nov., a phosphate-solubilizing bacterium isolated from the rhizosphere of rice plant

An ovoid to rod shaped, white to brown pigmented, facultative anaerobic, mesophilic, non-phototrophic, Gram-staining-negative, non-motile, multiply by binary fission designated strain KVB23^T, which was isolated from root of rice plant, near Ilsan, South Korea, was investigated for its taxonomic position by polyphasic approach. Optimal growth was found to occur at 30?C, at pH 6.5 and in the absence of NaCl on R2A. Phylogenetic analysis based on the 16S rRNA gene sequence of strain KVB23^T revealed that it formed a distinct lineage, as a separate deep branch within the family Rhodobacteriaceae, with?<?96.5% sequence similarity to representatives of the genera Rhodobacter, Xinfangfangia, Tabrizicola, Falsirhodobacter, Haematobacter, Paenirhodobacter, Pseudorhodobacter and Pararhodobacter. Based in 16S rRNA sequences strain KVB23^T was most closely related to Tabrizicola fusiformis KCTC 62105 ^T (96.5%) and Rhodobacter thermarum KCTC 52712 ^T (96.2%). The draft genome of strain KVB23^T was 3.80 bp long with a DNA G?+?C content of 63.1%. Genome of strain KVB23^T harboured gene clusters for tryptophan and cobalamin biosynthesis. The strain contained Q-10 as the sole respiratory quinone. The predominant fatty acids were found to consist of C_16:0, C_18:0 and summed feature 8 (comprising C_18:1 ω7c and / or C_18:1 ω6). The polar lipids were identified as diphosphatidylglycerol, phosphatidylethanolamine, seven unidentified phosphoglycolipids, two unidentified aminophosphoglycolipid, one unidentified glycolipid and four unidentified lipids. Phosphate-solubilizing bacteria have the ability to dissolve insoluble phosphates and enhance the soil fertility. Strain KVB23^T can solubilize calcium phosphate tribasic. Phosphate solubilizing and tryptophan biosynthesis property of strain KVB23^T could be a possible factor for the increase in growth of rice plant. Differential phenotypic, chemotaxonomic and genotypic properties, together with the phylogenetic distinctiveness, demonstrated that strain KVB23^T was found to represent a novel genus in the Rhodobacteriaceae family, for which the name Fuscibacter oryzae gen. nov., sp. nov. is proposed, with the type strain KVB23^T(=?KACC 21711 ^T?=?NBRC 114716 ^T).

     

<Emphasis Type="Italic">Shewanella upenei</Emphasis> sp. nov., a lipolytic bacterium isolated from bensasi goatfish <Emphasis Type="Italic">Upeneus bensasi</Emphasis>

A Gram-staining-negative, motile, non-spore-forming and rod-shaped bacterial strain, 20-23R^T, was isolated from intestine of bensasi goatfish, Upeneus bensasi, and its taxonomic position was investigated by using a polyphasic study. Phylogenetic analyses based on 16S rRNA gene sequences revealed that strain 20-23R^T belonged to the genus Shewanella. Strain 20-23R^T exhibited 16S rRNA gene sequence similarity values of 99.5, 99.2, and 97.5% to Shewanella algae ATCC 51192^T, Shewanella haliotis DW01^T, and Shewanella chilikensis JC5^T, respectively. Strain 20-23R^T exhibited 93.1–96.0% 16S rRNA gene sequence similarity to the other Shewanella species. It also exhibited 98.3–98.4% gyrB sequence similarity to the type strains of S. algae and S. haliotis. Strain 20-23R^T contained simultaneously both menaquinones and ubiquinones; the predominant menaquinone was MK-7 and the predominant ubiquinones were Q-8 and Q-7. The fatty acid profiles of strain 20–23R^T, S. algae KCTC 22552^T and S. haliotis KCTC 12896^T were similar; major components were iso-C_15:0, C_16:0, C_16:1 ω7c and/or iso-C_15:0 2-OH and C_17:1 ω8c. The DNA G+C content of strain 20-23R^T was 53.9 mol%. Differential phenotypic properties and genetic distinctiveness of strain 20–23R^T, together with the phylogenetic distinctiveness, revealed that this strain is distinguishable from recognized Shewanella species. On the basis of the data presented, strain 20-23R^T represents a novel species of the genus Shewanella, for which the name Shewanella upenei sp. nov. is proposed. The type strain is 20–23R^T (=KCTC 22806^T =CCUG 58400^T).     

Characterization of medium-chain-length polyhydroxyalkanoate biosynthesis by Pseudomonas mosselii TO7 using crude glycerol

Polyhydroxyalkanoates (PHAs) are biopolyesters produced by microorganisms that are environmentally friendly. PHAs can be used to replace traditional plastic to reduce environmental pollution in various fields. PHA production costs are high because PHA must be produced from a carbon substrate. The purpose of this study was to find the strain that can used the BDF by-product as the sole carbon source to produce high amounts of medium-chain-length PHA. Three isolates were evaluated for potential PHA production by using biodiesel-derived crude glycerol as the sole carbon source. Among them, Pseudomonas mosselii TO7 yielded high PHA content. The PHA produced from P. mosselii TO7 were medium-chain-length-PHAs. The PHA content of 48% cell dry weight in 48 h with a maximum PHA productivity of 13.16 mg PHAs L^?1 h^?1. The narrow polydispersity index value of 1.3 reflected the homogeneity of the polymer chain, which was conducive to industrial applications.     

Actinopolyspora righensis sp. nov., a novel halophilic actinomycete isolated from Saharan soil in Algeria

A novel halophilic actinomycete strain, H23^T, was isolated from a Saharan soil sample collected in Djamâa (Oued Righ region), El-Oued province, South Algeria. Strain H23^T was identified as a member of the genus Actinopolyspora by a polyphasic approach. Phylogenetic analysis showed that strain H23^T had 16S rRNA gene sequence similarities ranging from 97.8 % (Actinopolyspora xinjiangensis TRM 40136^T) to 94.8 % (Actinopolyspora mortivallis DSM 44261^T). The strain grew optimally at pH 6.0–7.0, 28–32 °C and in the presence of 15–25 % (w/v) NaCl. The substrate mycelium was well developed and fragmented with age. The aerial mycelium produced long, straight or flexuous spore chains with non-motile, smooth-surfaced and rod-shaped spores. Strain H23^T had MK-10 (H₄) and MK-9 (H₄) as the predominant menaquinones. The whole micro-organism hydrolysates mainly consisted of meso-diaminopimelic acid, galactose and arabinose. The diagnostic phospholipid detected was phosphatidylcholine. The major cellular fatty acids were anteiso-C_17:0 (37.4 %), iso-C_17:0 (14.8 %), iso-C_15:0 (14.2 %), and iso-C_16:0 (13.9 %). The genotypic and phenotypic data show that the strain represents a novel species of the genus Actinopolyspora, for which the name Actinopolyspora righensis sp. nov. is proposed, with the type strain H23^T (=DSM 45501^T = CCUG 63368^T = MTCC 11562^T).     

The HypB protein from Bradyrhizobium japonicum can store nickel and is required for the nickel-dependent transcriptional regulation of hydrogenase

The HypB protein from Bradyrhizobium japonicum is a metal-binding GTPase required for hydrogenase expression. In-frame mutagenesis of hypB resulted in strains that were partially or completely deficient in hydrogenase expression, depending on the degree of disruption of the gene. Complete deletion of the gene yielded a strain (JHΔEg) which lacked hydrogenase activity under all conditions tested, including the situation as bacteroids from soybean nodules. Mutant strain JHΔ23H lacking only the N-terminal histidine-rich region (38 amino acids deleted, 23 of which are His residues) expressed partial hydrogenase activity. The activity of strain JHΔ23H was low in comparison to the wild type in 10–50 nM nickel levels, but could be cured to nearly wild-type levels by including 50 μM nickel during the derepression incubation. Studies on strains harbouring the hup promoter–lacZ fusion plasmid showed that the complete deletion of hypB nearly abolished hup promoter activity, whereas the histidine deletion mutant had 60% of the wild-type promoter activity in 50 μM NiCl₂. Further evidence that HypB is required for hup promoter-binding activity was obtained from gel-shift assays. HypB could not be detected by immunoblotting when the cells were cultured heterotrophically, but when there was a switch to microaerobic conditions (1% partial pressure O₂, 10% partial pressure H₂) HypB was detected, and its expression preceded hydrogenase synthesis by 3–6 h. ⁶³Ni accumulation by whole cells showed that both of the mutant strains accumulate less nickel than the wild-type strain at all time points tested during the derepression incubation. Wild-type cultures that received nickel during the HypB expression-specific period and were then washed and derepressed for hydrogenase without nickel had activities comparable to those cells that were derepressed for hydrogenase with nickel for the entire time period. In contrast to the wild type, strain JHΔ23H cultures supplied with nickel only during the HypB expression period achieved hydrogenase activities that were 30% of those cultures supplied with nickel for the entire hydrogenase derepression period. These results indicate that the loss of the metal-binding area of HypB causes a decrease in the ability of the cells to sequester and store nickel for later use in one or more hydrogenase expression steps.