Paenibacillus caui sp. nov., a nitrogen-fixing species isolated from the rhizosphere soil of a peach tree.

A nitrogen-fixing, endospore-forming, motile, rod-shaped, facultative aerobic bacterium, designated 81-11T, was isolated from rhizosphere soil of a peach tree collected from Handan, Hebei, PR China. From the comparison of 16S rRNA gene sequence, the strain is most closely related to Paenibacillus phoenicis DSM 27463T (96.9 %) and Paenibacillus faecis DSM 23593T (96.7 %). The genome size of strain 81-11T was 4.4 Mb, comprising 4879 predicted genes with a DNA G+C content of 50.0 mol%. The average nucleotide identity values of genome sequences between the novel isolate and the type strains of related species P. phoenicis DSM 27463T and P. faecis DSM 23593T were 71.8 and 72.1 %, respectively. The major cellular fatty acids were anteiso-C15 : 0(47.8 %), iso-C16 : 0 (15.5 %) and iso-C15 : 0 (13.0 %). Menaquinone-7 was the major respiratory quinone. The polar lipids contained phosphatidylethanolamine, phosphatidylglycerol, diphosphatidylglycerol, aminophospholipid, aminoglycopid, unknown polar lipids and unidentified aminophosphoglycolipid. Based on phylogenetic, genomic and phenotypic characteristics, strain 81-11T was classified as a novel species within the genus Paenibacillus, for which the name Paenibacillus caui sp. nov. is proposed. The type strain of Paenibacillus caui is 81-11T (=JCM 34618T=CGMCC 1.18907T).

Paenibacillus maysiensis sp. nov., a Nitrogen-Fixing Species Isolated from the Rhizosphere Soil of Maize.

A novel bacterium SX-49T with nitrogen-fixing capability was isolated from the rhizosphere soil of maize. Phylogenetic analysis of nifH gene fragment and 16S rRNA gene sequence revealed that the strain SX-49T is a member of the genus Paenibacillus. Values of 16S rRNA gene sequence similarity were highest between SX-49T and P. jamilae DSM 13815T (97.0%), P. brasiliensis DSM 14914T (97.8%), P. polymyxa DSM 36T (97.5%), and P. terrae DSM 15891T (98.8%). The similarity between SX-49T and other Paenibacillus species was < 97.0%. DNA-DNA hybridization values between strain SX-49T and the four type strains were P. jamilae DSM 13815T: 40.6%, P. brasiliensis DSM 14914T: 27.9%, P. polymyxa DSM 36T: 29.2%, and P. terrae DSM 15891T: 66.4%. The DNA G+C content of SX-49T was 46.4 mol%. The predominant fatty acids were anteiso-C15:0, C16:0 and iso-C16:0. The predominant isoprenoid quinone was MK-7. The genome contains 5628 putative protein-coding sequences (CDS), 6 rRNAs and 56 tRNAs. The phenotypic and genotypic characteristics, DNA-DNA relatedness, and genome features suggest that SX-49T represents a novel species of the genus Paenibacillus, and the name Paenibacillus maysiensis sp. nov. is proposed.

Genome-wide transcriptome profiling of nitrogen fixation in Paenibacillus sp. WLY78.

BACKGROUND: Diazotrophic (nitrogen-fixing) Gram-positive and endospore-formed Paenibacillus spp. have potential uses as a bacterial fertilizer in agriculture. The transcriptional analysis of nitrogen fixation in Paenibacillus is lacking, although regulation mechanisms of nitrogen fixation have been well studied in Gram-negative diazotrophs. RESULTS: Here we report a global transcriptional profiling analysis of nitrogen fixation in Paenibacillus sp. WLY78 cultured under N2-fixing condition (without O2 and NH4(+)) and non-N2-fixing condition (air and 100 mM NH4(+)). The nif (nitrogen fixation) gene operon composed of 9 genes (nifBHDKENXhesAnifV) in this bacterium was significantly up-regulated in N2-fixing condition compared to non-N2-fixing condition, indicating that nif gene transcription is strictly controlled by NH4(+) and O2. qRT-PCR confirmed that these nif genes were differently expressed. Non-nif genes specifically required in nitrogen fixation, such as mod, feoAB and cys encoding transporters of Mo, Fe and S atoms, were coordinately transcribed with nif genes in N2-fixing condition. The transcript abundance of suf operon specific for synthesis of Fe-S cluster was up-regulated in N2-fixing condition, suggesting that Sul system, which takes place of nifS and nifU, plays important role in the synthesis of nitrogenase. We discover potential specific electron transporters which might provide electron from Fe protein to MoFe protein of nitrogenase. The glnR whose predicted protein might mediate nif transcription regulation by NH4(+) is significantly up-regulated in N2-fixing condition. The transcription levels of nitrogen metabolism and anaerobic respiration were also analyzed. CONCLUSIONS: The nif gene operon (nifBHDKENXhesAnifV) in Paenibacillus sp. WLY78 is significantly up-regulated in N2-fixing condition compared to non-N2-fixing condition. Non-nif genes specifically required in nitrogen fixation were also significantly up-regulated in N2-fixing condition. Fur and Fnr which are involved in anaerobic regulation and GlnR which might mediate nif gene transcription regulation by NH4(+) were significantly up-regulated in N2-fixing condition. This study provides valuable insights into nitrogen fixation process and regulation in Gram-positive firmicutes.

Using synthetic biology to increase nitrogenase activity.

BACKGROUND: Nitrogen fixation has been established in protokaryotic model Escherichia coli by transferring a minimal nif gene cluster composed of 9 genes (nifB, nifH, nifD, nifK, nifE, nifN, nifX, hesA and nifV) from Paenibacillus sp. WLY78. However, the nitrogenase activity in the recombinant E. coli 78-7 is only 10 % of that observed in wild-type Paenibacillus. Thus, it is necessary to increase nitrogenase activity through synthetic biology. RESULTS: In order to increase nitrogenase activity in heterologous host, a total of 28 selected genes from Paenibacillus sp. WLY78 and Klebsiella oxytoca were placed under the control of Paenibacillus nif promoter in two different vectors and then they are separately or combinationally transferred to the recombinant E. coli 78-7. Our results demonstrate that Paenibacillus suf operon (Fe-S cluster assembly) and the potential electron transport genes pfoAB, fldA and fer can increase nitrogenase activity. Also, K. oxytoca nifSU (Fe-S cluster assembly) and nifFJ (electron transport specific for nitrogenase) can increase nitrogenase activity. Especially, the combined assembly of the potential Paenibacillus electron transporter genes (pfoABfldA) with K. oxytoca nifSU recovers 50.1 % of wild-type (Paenibacillus) activity. However, K. oxytoca nifWZM and nifQ can not increase activity. CONCLUSION: The combined assembly of the potential Paenibacillus electron transporter genes (pfoABfldA) with K. oxytoca nifSU recovers 50.1 % of wild-type (Paenibacillus) activity in the recombinant E. coli 78-7. Our results will provide valuable insights for the enhancement of nitrogenase activity in heterogeneous host and will provide guidance for engineering cereal plants with minimal nif genes.

Cohnella capsici sp. nov., a novel nitrogen-fixing species isolated from Capsicum annuum rhizosphere soil, and emended description of Cohnella plantaginis.

A novel bacterial strain designated YN-59(T) was isolated from Capsicum annuum rhizosphere soil in China. The isolate was found to be aerobic, Gram-positive, rod-shaped and to form ellipsoidal or oval spores positioned centrally in swollen sporangia. On the basis of 16S rRNA gene sequence analysis, the isolated strain YN-59 was determined to be related to members of genus Cohnella. High levels of 16S rRNA gene sequence similarity were found between strain YN-59 and Cohnella plantaginis DSM 25424(T) (98.5 %) and Cohnella ginsengisoli DSM18997(T) (97.3 %); the 16S rRNA gene sequence similarities between strain YN-59 and the other strains recognized members of the genus Cohnella were below 97 %. The DNA-DNA hybridization values of strain YN-59 with C. plantaginis DSM 25424(T) and C. ginsengisoli DSM18997(T) were 44.2 ± 8.4 and 28.8 ± 5.8 %, respectively. The DNA G + C content of strain YN-59(T) was determined to be 59.32 mol %. The major isoprenoid quinone was identified as MK-7 and the predominant fatty acids as anteiso-C15:0 (45.32 %), iso-C16:0 (19.19 %), iso-C15:0 (9.65 %) and C16:0 (8.91 %). The polar lipids of strain YN-59(T) were found to consist of diphosphatidylglycerol, phosphatidylethanolamine and phosphatidylglycerol; several unidentified phospholipids were also detected. The diagnostic diamino acid in the cell wall was identified as meso-diaminopimelic. On the basis of its phenotypic and genotypic characteristics and levels of DNA-DNA hybridization, strain YN-59(T) is considered to represent a novel species of the genus Cohnella, for which the name Cohnella capsici sp. nov. (type strain YN-59(T) = CGMCC 1.12046(T) = JCM 19168(T)) is proposed.

Influence of different factors on the nitrogenase activity of the engineered Escherichia coli 78-7.

The engineered Escherichia coli 78-7 is a derivative of E. coli JM109 carrying a nitrogen fixation (nif) gene cluster composed of nine genes (nifB, nifH, nifD, nifK, nifE, nifN, nifX, hesA and nifV) and its own σ(70)-dependent nif promoter from a gram-positive bacterium Paenibacillus sp. WLY78. The physiological and biochemical characteristics of the engineered E. coli 78-7 were analyzed by using Biolog GEN III MicroPlate, with E. coli JM109 and JM109/pHY300PLK (E. coli JM109 carrying empty vector) as controls. Analysis of 94 phenotypic tests: 71 carbon source utilization assays and 23 chemical sensitivity tests showed that the engineered E. coli 78-7, E. coli JM109 and JM109/pHY300PLK gave similar patterns of utilization of various substrates as carbon and energy sources. Furthermore, the effect of carbon source, nitrogen source, culture temperature on the nitrogenase activity of the engineered E. coli 78-7 was investigated. Our study demonstrates that the nif capacity of E. coli 78-7 was affected significantly by the different culture condition. The significant nitrogenase activity of E. coli 78-7 were obtained when cells were cultivated in the medium containing 4 g/l glucose (carbon source) and 2 mM glutamate (nitrogen source) and at 30 °C.

Paenibacillus beijingensis sp. nov., a nitrogen-fixing species isolated from wheat rhizosphere soil.

A novel nitrogen-fixing bacterium, BJ-18(T), was isolated from wheat rhizosphere soil. Strain BJ-18(T) was observed to be Gram-positive, facultatively anaerobic, motile and rod-shaped (0.4-0.9 µm × 2.0-2.9 µm). Phylogenetic analysis based on a partial nifH gene sequence and an assay for nitrogenase activity showed its nitrogen-fixing capacity. Phylogenetic analysis based on full 16S rRNA gene sequences suggested that strain BJ-18(T) is a member of the genus Paenibacillus. High similarity of 16S rRNA gene sequence was found between BJ-18(T) and Paenibacillus peoriae DSM 8320(T) (99.05 %), Paenibacillus jamilae DSM 13815(T) (98.86 %), Paenibacillus brasiliensis DSM 13188(T) (98.55 %), Paenibacillus polymyxa DSM 36(T) (98.74 %), Paenibacillus terrae DSM 15891(T) (97.99 %) and Paenibacillus kribbensis JCM 11465(T) (97.92 %), whereas the similarity was below 96.0 % between BJ-18(T) and the other Paenibacillus species. DNA-DNA relatedness between strain BJ-18(T) and P. peoriae DSM 8320(T), P. jamilae DSM 13815(T), P. brasiliensis DSM 13188(T), P. polymyxa DSM 36(T), P. kribbensis JCM 11465(T) and P. terrae DSM 15891(T) was determined to be 43.6 ± 2.7, 34.2 ± 5.3, 47.9 ± 6.6, 36.8 ± 3.5, 27.4 ± 4.3 and 23.6 ± 4.1 % respectively. The DNA G+C content of BJ-18(T) was determined to be 45.8 mol %. The major fatty acid was identified as anteiso-C15:0 (67.1 %). The polar lipids present in strain BJ-18(T) were identified as diphosphatidylglycerol, phosphatidyl methylethanolamine, phosphatidylethanolamine and phosphatidylglycerol. The phenotypic and genotypic characteristics, and DNA-DNA relatedness data, suggest that BJ-18(T) represents a novel species of the genus Paenibacillus, for which the name Paenibacillus beijingensis sp. nov. (Type strain BJ-18(T)=DSM25425(T)=CGMCC 1.12045(T)) is proposed.

Gracilibacillus kekensis sp. nov., a moderate halophile isolated from Keke Salt Lake.

A novel moderately halophilic bacterium, designated strain K170(T), was isolated from Keke Salt Lake in Qinghai, China. The strain grew with 0-22 % (w/v) NaCl, at 4-50 °C and at pH 6-11, with optimum growth in 3 % (w/v) NaCl, at 40 °C and at pH 8. The predominant respiratory quinone was menaquinone 7 (MK-7). The polar lipids included diphosphatidylglycerol, phosphatidylglycerol, unidentified phospholipids, aminolipids and glycolipids. The major cellular fatty acids were anteiso-C(15 : 0), iso-C(15 : 0) and anteiso-C(17 : 0). The DNA G+C content was 35.8 mol%. Phylogenetic analysis based on the full-length 16S rRNA gene sequence revealed that strain K170(T) was a member of the genus Gracilibacillus. High levels of 16S rRNA gene sequence similarity were found between strain K170(T) and Gracilibacillus boraciitolerans DSM 17256(T) (97.3 %) and Gracilibacillus thailandensis JCM 15569(T) (97.1 %). 16S rRNA gene sequence similarities between strain K170(T) and the type strains of other recognized members of the genus Gracilibacillus were below 97 %. The DNA-DNA hybridization values of strain K170(T) with G. boraciitolerans DSM 17256(T) and G. thailandensis JCM 15569(T) were 21.9 % and 34.3 %, respectively. On the basis of these results, strain K170(T) is considered to represent a novel species of the genus Gracilibacillus, for which the name Gracilibacillus kekensis sp. nov. is proposed; the type strain is K170(T) ( = CGMCC 1.10681(T) = DSM 23178(T)).

Cohnella plantaginis sp. nov., a novel nitrogen-fixing species isolated from plantain rhizosphere soil.

A novel bacterium capable of fixing nitrogen was isolated from plantain rhizosphere soil in China. The isolate, designated YN-83(T), is Gram-positive, aerobic, motile and rod-shaped (0.4-0.6 µm × 1.9-2.6 µm). Phylogenetic analysis based on the 16S rRNA gene sequence revealed that strain YN-83(T) was a member of the genus Cohnella. High similarity of 16S rRNA gene sequence was found between YN-83(T) and Cohnella ginsengisoli DSM18997(T) (97.99%), whereas the similarity was below 96.0% between YN-83(T) and the other Cohnella species. DNA-DNA relatedness between strain YN-83(T) and C. ginsengisoli DSM18997(T) was 27.4 ± 6.2%. The DNA G+C content of strain YN-83(T) was 59.3 mol%. The predominant isoprenoid quinone was MK-7 and the major fatty acids were anteiso-C(15:0) (44.3%), iso-C(15:0) (11.3%), iso-C(16:0) (18.6%) and C(16:0) (7.7%). The polar lipids of strain YN-83(T) consist of diphosphatidylglycerol, phosphatidylethanolamine, phosphatidylglycerol, lyso- phosphatidylglycerol, phosphatidylinositol and phosphatidylinositol mannosides. On the basis of phenotypic and chemotaxonomic properties, 16S rRNA gene sequence, G+C content and DNA-DNA hybridization, strain YN-83(T) represents a novel species of the genus Cohnella, for which the name Cohnella plantaginis sp. nov. (type strain YN-83(T) = DSM 25424(T) = CGMCC 1.12047(T)) is proposed.

Paenibacillus taohuashanense sp. nov., a nitrogen-fixing species isolated from rhizosphere soil of the root of Caragana kansuensis Pojark.

A nitrogen-fixing bacterium, designated strain gs65(T), was isolated from a rhizosphere soil sample of Caragana kansuensis Pojark. Phylogenetic analysis based on a fragment of the nifH gene and the full-length 16S rRNA gene sequence revealed that strain gs65(T) is a member of the genus Paenibacillus. High levels of 16S rRNA gene similarity were found between strain gs65(T) and Paenibacillus borealis DSM 13188(T) (97.5 %), Paenibacillus odorifer ATCC BAA-93(T) (97.3 %), Paenibacillus durus DSM 1735(T) (97.0 %) and Paenibacillus sophorae DSM23020(T) (96.9 %). Levels of 16S rRNA gene sequence similarity between strain gs65(T) and the type strains of other recognized members of the genus Paenibacillus were below 97.0 %. Levels of DNA-DNA relatedness between strain gs65(T) and P. borealis DSM 13188(T), P. odorifer ATCC BAA-93(T) (97.3 %), P. durus DSM 1735(T) and P. sophorae DSM23020(T) were 35.9, 38.0, 34.2 and 35.5 % respectively. The DNA G+C content of strain gs65(T) was determined to be 51.6 mol%. The major fatty acids were found to be iso-C(14:0), anteiso-C(15:0) and iso-C(16:0). On the basis of its phenotypic characteristics and levels of DNA-DNA hybridization, strain gs65(T) is considered to represent a novel species of the genus Paenibacillus, for which the name Paenibacillus taohuashanense sp. nov. is proposed. The type strain is gs65(T) (=CGMCC 1.12175(T) = DSM 25809(T)).

Metagenome cloning and functional analysis of Na⁺/H⁺ antiporter genes from Keke Salt Lake in China.

Na⁺/H⁺ antiporters are ubiquitous membrane proteins and play a central role in cell homeostasis including pH regulation, osmoregulation, and Na⁺/Li⁺ tolerance in bacteria. The microbial communities in extremely hypersaline soil are an important resource for isolating Na⁺/H⁺ antiporter genes. A metagenomic library containing 35,700 clones was constructed by using genomic DNA obtained from the hypersaline soil samples of Keke Salt Lake in Northwest of China. Two Na⁺/H⁺ antiporters, K1-NhaD, and K2-NhaD belonging to NhaD family, were screened and cloned from this metagenome by complementing the triple mutant Escherichia coli strain KNabc (nhaA⁻, nhaB⁻, chaA⁻) in medium containing 0.2 M NaCl. K1-NhaD and K2-NhaD have 75.5% identity at the predicted amino acid sequence. K1-NhaD has 78% identity with Na⁺/H⁺ antiporter NhaD from Halomonas elongate at the predicted amino acid sequence. The predicted K1-NhaD is a 53.5 kDa protein (487 amino acids) with 13 transmembrane helices. K2-NhaD has 73% identity with Alkalimonas amylolytica NhaD. The predicted K2-NhaD is a 55 kDa protein (495 amino acids) with 12 transmembrane helices. Both K1-NhaD and K2-NhaD could make the triple mutant E. coli KNabc (nhaA⁻, nhaB⁻, chaA⁻) grow in the LBK medium containing 0.2-0.6 M Na⁺ or with 0.05-0.4 M Li⁺. Everted membrane vesicles prepared from E. coli KNabc cells carrying K1-NhaD or K2-NhaD exhibited Na⁺/H⁺ and Li⁺/H⁺ antiporter activities which were pH-dependent with the highest activity at pH 9.5. Little K⁺/H⁺ antiporter activity was also detected in vesicles form E. coli KNabc carrying K1-NhaD or K2-NhaD.

Pseudacidovorax intermedius NH-1, a novel marine nitrogen-fixing bacterium isolated from the South China Sea.

We have taxonomically and phylogenetically characterized a novel nitrogen-fixing bacterial strain, NH-1, which was recently isolated from surface sediments of the South China Sea. The presence of the nifH gene was determined by PCR amplification. The strain NH-1 was found to belong to the genus Pseudacidovorax based on phenotypic characterization, 16S rDNA sequencing, G+C content and DNA-DNA hybridization. Isolate NH-1 was identified as Pseudacidovorax intermedius. In addition, we investigated the links between environmental factors and the nitrogenase activity of NH-1. We found that the nitrogen fixation capacity of NH-1 varied strongly when cells were grown with different ammonium ion and oxygen concentrations, amino acids and carbohydrates. This is the first report of the isolation of Pseudacidovorax from the ocean and the first study to explore the effects of different culture conditions on the nitrogenase activities of the isolate. This study provides evidence that marine nitrogen-fixing microorganisms are far more diverse than currently recognized.

Paenibacillus sophorae sp. nov., a nitrogen-fixing species isolated from the rhizosphere of Sophora japonica.

A nitrogen-fixing bacterium, designated strain S27(T), was isolated from rhizosphere soil of Sophora japonica. Phylogenetic analysis based on a fragment of the nifH gene and the full-length 16S rRNA gene sequence revealed that strain S27(T) is a member of the genus Paenibacillus. High levels of 16S rRNA gene sequence similarity were found between strain S27(T) and Paenibacillus durus DSM 1735(T) (97.3 %), Paenibacillus sabinae DSM 17841(T) (96.9 %), Paenibacillus forsythiae DSM 17842(T) (96.7 %) and Paenibacillus zanthoxyli DSM 18202(T) (96.6 %). However, DNA-DNA hybridization values between strain S27(T) and the four type strains were 37.64 %, 23.12 %, 25.6 % and 34.99 %, respectively. Levels of 16S rRNA gene sequence similarity between strain S27(T) and the type strains of other recognized members of the genus Paenibacillus were below 96.5 %. The DNA G+C content of strain S27(T) was 46.0 mol%. The major fatty acids were anteiso-C(15 : 0), C(16 : 0) and iso-C(16 : 0). The major isoprenoid quinone was MK-7. On the basis of its phenotypic characteristics and DNA-DNA hybridization results, strain S27(T) is considered to represent a novel species of the genus Paenibacillus, for which the name Paenibacillus sophorae sp. nov. is proposed. The type strain is S27(T) ( = CGMCC 1.10238(T)  = DSM 23020(T)).

Paenibacillus jilunlii sp. nov., a nitrogen-fixing species isolated from the rhizosphere of Begonia semperflorens.

A nitrogen-fixing bacterium, designated strain Be17(T), was isolated from rhizosphere soil of Begonia semperflorens planted in Beijing Botanical Garden, PR China. Phylogenetic analyses based on a segment of the nifH gene sequence and a full-length 16S rRNA gene sequence revealed that strain Be17(T) was a member of the genus Paenibacillus. High levels of 16S rRNA gene sequence similarity were found between strain Be17(T) and Paenibacillus graminis RSA19(T) (97.9 %), Paenibacillus sonchi LMG 24727(T) (97.8 %), Paenibacillus riograndensis CECT 7330(T) (96.2 %) and Paenibacillus borealis DSM 13188(T) (96.1 %), respectively. Levels of 16S rRNA gene sequence similarity between strain Be17(T) and the type strains of other recognized members of the genus Paenibacillus were below 96.0 %. However, the DNA-DNA hybridization values between strain Be17(T) and P. graminis RSA19(T), P. sonchi LMG 24727(T) and P. riograndensis CECT 7330(T) were 47.9 %, 38.7 % and 37.5 %, respectively. The DNA G+C content of strain Be17(T) was 52.9 mol%. The major fatty acid component of strain Be17(T) was anteiso-branched C(15 : 0) (30.92 %). The major isoprenoid quinone was MK-7. The cell-wall peptidoglycan contained meso-diaminopimelic acid as the diagnostic diamino acid. On the basis of its phenotypic characteristics, 16S rRNA gene sequences, DNA G+C content, DNA-DNA relatedness, chemotaxonomic properties and nifH gene sequence, strain Be17(T) represents a nitrogen-fixing strain of a novel species of the genus Paenibacillus, for which the name Paenibacillus jilunlii sp. nov. is proposed. The type strain is Be17(T) ( = CGMCC 1.10239(T) = DSM 23019(T)).

Rhombencephalitis as an initial manifestation of primary Sjögren's syndrome: a case report and review of the literatures.

PURPOSE: We present a case report and a comprehensive review of the literature concerning aseptic meningoencephalitis and Sjögren's syndrome (SS). CASE REPORT: We report a 44-year-old woman of primary SS with initial presentation of aseptic meningoencephalitis and a reversible magnetic resonance image (MRI) lesion in the medulla. The diagnosis of primary SS based on ocular dryness, lacrimal hyposecretion, secretory and excretory dysfunction from sialocintigraphy, and positive anti-SS-A antibodies. Corticosteroid and cyclophosphamide therapies reversed the neurological deficits and the MRI lesion. CONCLUSION: Primary SS may have variable manifestations in the central nervous system which may precede the classic sicca symptoms. SS should be investigated in cases of aseptic meningoencephalitis even without clinical signs of xerostomia or xerophthalmia. MRI is useful in demonstrating brain lesions and in evaluating treatment efficacy of the SS.

Salinicoccus kekensis sp. nov., a novel alkaliphile and moderate halophile isolated from Keke Salt Lake in Qinghai, China.

A novel alkaliphilic and moderate halophilic bacterium, designated strain K164(T), was isolated from Keke Salt Lake in Qinghai, China. The strain grew with 2.0-20.0% (w/v) NaCl, at 4-50 degrees C and pH 6.5-11.5, with an optimum of 8% (w/v) NaCl, 37degrees C and pH 10, respectively. The predominant respiratory quinone was menaquinone 6 (MK-6) and the major polar lipid was phosphatidylethanolamine. The major cellular fatty acids were anteiso-C(15:0) and iso-C(15:0). The genomic DNA G+C content was 50.16 mol. Phylogenetic analysis based on the full-length 16S rRNA gene sequence revealed that strain K164(T) was a member of the genus Salinicoccus. Strain K164(T) showed the highest similarity (98.4%) with Salinicoccus alkaliphilus AS 1.2691(T) and below 97% similarity with other recognized members of the genus in 16S rRNA gene sequence. Level of DNA-DNA relatedness between strain K164(T) and Salinicoccus alkaliphilus AS 1.2691(T) was 20.1%. On the basis of its phenotypic characteristics and the level of DNA-DNA hybridization, strain K164(T) is considered to represent a novel species of the genus Salinicoccus, for which the name Salinicoccus kekensis sp. nov. is proposed. The type strain is K164(T) (=CGMCC 1.10337(T) = DSM 23173(T)).

Expression and functional analysis of aminotransferase involved in indole-3-acetic acid biosynthesis in Azospirillum brasilense Yu62.

In this study, atrC (a novel gene from Azospirillum brasilense identified in our laboratory) was expressed in Escherichia coli, and SDS-PAGE analysis of the expressed AtrC revealed the apparent molecular weight of 45 kD. When analyzed under non-denaturing PAGE conditions and using L-tryptophan as a substrate, the purified AtrC protein exhibited aminotransferase activity, while crude protein extracts from A. brasilense Yu62 showed two activity bands with molecular masses estimated as 44 and 66 kD. Thus, we deduced that AtrC protein is identical to the 44 kD band of crude protein extracts. The optimal temperature and pH for the catalytic activity of the purified AtrC are 30 degrees C and pH 7.0, respectively.

Microbial production of medium-chain-length 3-hydroxyalkanoic acids by recombinant Pseudomonas putida KT2442 harboring genes fadL, fadD and phaZ.

Monomers of microbial polyhydroxyalkanoates, mainly 3-hydroxyhexanoic acid (3HHx) and 3-hydroxyoctanoic acid (3HO), were produced by overexpressing polyhydroxyalkanoates depolymerase gene phaZ, together with putative long-chain fatty acid transport protein fadL of Pseudomonas putida KT2442 and acyl-CoA synthetase (fadD) of Escherichia coli MG1655 in P. putida KT2442. FadL(Pp), which is responsible for free fatty acid transportation from the extracellular environment to the cytoplasm, and FadD(Ec), which activates fatty acid to acyl-CoA, jointly reinforce the fatty acid beta-oxidation pathway. Pseudomonas putida KT2442 (pYZPst01) harboring polyhydroxyalkanoates depolymerase gene phaZ of Pseudomonas stutzeri 1317 produced 1.37 g L(-1) extracellular 3HHx and 3HO in shake flask studies after 48 h in the presence of sodium octanoate as a sole carbon source, while P. putida KT2442 (pYZPst06) harboring phaZ(Pst), fadD(Ec) and fadL(Pp) achieved 2.32 g L(-1) extracellular 3HHx and 3HO monomer production under the same conditions. In a 48-h fed-batch fermentation process conducted in a 6-L fermentor with 3 L sodium octanoate mineral medium, 5.8 g L(-1) extracellular 3HHx and 3HO were obtained in the fermentation broth. This is the first time that medium-chain-length 3-hydroxyalkanoic acids (mcl-3HA) were produced using fadL(Pp) and fadD(Ec) genes combined with the polyhydroxyalkanoates depolymerase gene phaZ.

Expression of ethylene-forming enzyme (EFE) of Pseudomonas syringae pv. glycinea in Trichoderma viride.

The efe gene encoding an ethylene-forming enzyme from Pseudomonas syringae pv. glycinea has been expressed for the first time under the control of Trichoderma reesei cbh1 promoter in Trichoderma viride. Reverse transcription polymerase chain reaction analysis showed that transformant Y2 produced mRNA of the efe gene. Southern blot analysis showed that there was one copy of efe gene which was integrated into the chromosomal DNA of T. viride. Ethylene production by transformant Y2 was efficiently induced by cellulose, while very low level of ethylene was produced when sodium carboxymethyl cellulose or lactose was used as carbon source. Peptone exerted a much greater stimulatory effect on ethylene production. A high level of ethylene was produced when transformant Y2 was cultured in solid fermentation medium containing wheat straw, indicating that plant wastes could be directly converted to ethylene by the recombinant filamentous fungus.

Isolation of transposon mutants from Azospirillum brasilense Yu62 and characterization of genes involved in indole-3-acetic acid biosynthesis.

The molecular genetics of indole-3-acetic (IAA) synthesis and regulation in Azospirillum brasilense was investigated in this study. Tn5 mutagenesis was performed and five mutants with decreased IAA production were isolated. Five Tn5-inserted genes from these mutants were cloned and sequenced. Four genes were reported for the first time to be involved in IAA production, namely, atrA, ftsA, omaA and aldA that code for GntR-family transcriptional regulator, iron-binding protein component of ABC-type Fe(3+) transport system, outer membrane protein, and aldehyde dehydrogenase, respectively. In addition, two genes atrB and atrC, with predicted proteins that showed high homology to aminotransferases, were cloned from the downstream of atrA in this bacterium. Studies also showed that complementation of atrA, ftsA and omaA were able to restore the IAA production of the corresponding IAA(-) mutants. Comparison of Fe(3+) concentrations in culture supernatants of the wild-type strain, the ftsA mutant and the complemented strain revealed that the iron-uptake ability of the ftsA mutant was highly reduced. This result also points to the necessity of iron as a metal ion in IAA synthesis. Statistical analysis showed no significant difference in the IAA accumulated in cells between the omaA mutant and the wild-type strain, suggesting the omaA might not affect IAA secretion but be involved in IAA production in other unknown ways.