Sanfeng Chen

Isolation and identification of nitrogen‐fixing bacilli from plant rhizospheres in Beijing region

Aims:  To isolate and identify nitrogen‐fixing bacilli from the plant rhizospheres in Beijing region of China.

Colonization of maize and rice plants by strain Bacillus megaterium C4.

Bacillus megaterium C4, a nitrogen-fixing bacterium, was marked with the gfp gene. Maize and rice seedlings were inoculated with the, GFP-labeled B. megaterium C4 and then grown in gnotobiotic condition. Observation by confocal laser scanning microscope showed that the GFP-labeled bacterial cells infected the maize roots through the cracks formed at the lateral root junctions and then penetrated into cortex, xylem, and pith, and that the bacteria migrated slowly from roots to stems and leaves. The bacteria were mainly located in the intercellular spaces, although a few bacterial cells were also present within the xylem vessels, root hair cells, epidermis, cortical parenchyma, and pith cells. In addition, microscopic observation also revealed clearly that the root tip in the zone of elongation and differentiation and the junction between the primary and the lateral roots were the two sites for the bacteria entry into rice root. Therefore, we conclude that this Gram-positive nitrogen-fixer has a colonization pattern similar to those of many Gram-negative diazotrophs, such as Azospirillun brasilense Yu62 and Azoarcus sp. As far as we know, this is the first detailed report of the colonization pattern for Gram-positive diazotrophic Bacillus.

Strain improvement for enhanced production of cellulase in Trichoderma viride.

The filamentous fungi Trichoderma species produce extracellular cellulase. The current study was carried out to obtain an industrial strain with hyperproduction of cellulase. The wild-type strain, Trichoderma viride TL-124, was subjected to successive mutagenic treatments with UV irradiation, low-energy ion beam implantation, atmospheric pressure non-equilibrium discharge plasma (APNEDP), and N-methyl-N′-nitro-N-nitrosoguanidine to generate about 3000 mutants. Among these mutants, T. viride N879 strain exhibited the greatest relevant activity: 2.38-fold filter paper activity and 2.61-fold carboxymethyl cellulase, 2.18-fold β-glucosidase, and 2.27-fold cellobiohydrolase activities, compared with the respective wild-type activities, under solid-state fermentation using the inexpensive raw material wheat straw as a substrate. This work represents the first application of APNEDP in eukaryotic microorganisms.

Characterization and analysis of nifH genes from Paenibacillus sabinae T27

Paenibacillus sabinae T27 (CCBAU 10202=DSM 17841) is a gram-positive, spore-forming diazotroph with high nitrogenase activities. Three nifH clusters were cloned from P. sabinae T27. Phylogenetic analysis revealed that NifH1, NifH2 and NifH3 cluster with Cyanobacterium. Each of the coding regions of nifH1, nifH2 and nifH3 from P. sabinae T27 under the control of the nifH promoter of Klebsiella pneumoniae could partially restore nitrogenase activity of K. pneumoniae nifH(-) mutant strain 1795, which has no nitrogenase activity. This suggests that the three nifH genes from P. sabinae T27 have some function in nitrogen fixation. RT-PCR showed that all three nifH genes were expressed under nitrogen-fixing growth conditions. Using promoter vectors which have promoterless lacZ gene, three putative promoter regions of nifH genes were identified.

Identification of nitrogen-fixing Paenibacillus from different plant rhizospheres and a novel nifH gene detected in the P. stellifer

A total of 534 isolates were selectively obtained from different plant rhizospheres based on their growth on nitrogen-free medium and their resistance to 80°C for 15 min. Of the 534 isolates, 23 isolates had nifH gene and exhibited nitrogenase activities. Based on 16S rDNA sequence, G + C content assay and DNA-DNA hybridization, the 23 isolates which divided into four monophyletic clusters were all belonged to the Paenibacillus genus. nifH gene deduced amino acid alignment aLnalysis revealed that cluster I, including 15 isolates, showed the highest NifH identity with Paenibacillus genus; while cluster II identified as P. stellifer by DNA-DNA hybridization was consistent with four uncultured bacterial clones. This study suggested that the nitrogen-fixing Paenibacillus were distributed in various ecosystems and prevalent in different plant rhizospheres. It was the first demonstration that nitrogen fixation existed in P. jamilae and P. stellifer. In eight isolates identified as P. stellifer species, a novel nifH gene was detected in Paenibacillus.

A hybrid two-component system protein from Azospirillum brasilense Sp7 was involved in chemotaxis.

We here report the sequence and functional analysis of org35 of Azospirillum brasilense Sp7, which was originally identified to be able to interact with NifA in yeast-two-hybrid system. The org35 encodes a hybrid two-component system protein, including N-terminal PAS domains, a histidine kinase (HPK) domain and a response regulator (RR) domain in C-terminal. To determine the function of the Org35, a deletion-insertion mutant in PAS domain [named Sp7353] and a complemental strain Sp7353C were constructed. The mutant had reduced chemotaxis ability compared to that of wild-type, and the complemental strain was similar to the wild-type strain. These data suggested that the A. brasilense org35 played a key role in chemotaxis. Variants containing different domains of the org35 were expressed, and the functions of these domains were studied in vitro. Phosphorylation assays in vitro demonstrated that the HPK domain of Org35 possessed the autokinase activity and that the phosphorylated HPK was able to transfer phosphate groups to the RR domain. The result indicated Org35 was a phosphorylation-communicating protein.

cDNA-AFLP analysis of differential gene expression related to cell chemotactic and encystment of Azospirillum brasilense.

Our previous study indicated org35 was involved in chemotaxis and interacted with nitrogen fixation transcriptional activator NifA via PAS domain. In order to reveal the role of org35 in nitrogen regulation, the downstream target genes of org35 were identified. We here report differentially expressed genes in org35 mutants comparing with wild type Sp7 by means of cDNA-AFLP. Four up-regulated transcript-derived fragments (TDFs) homologues of chemotaxis transduction proteins were found, including CheW, methyl-accepting chemotaxis protein and response regulator CheY-like receiver. Three distinct TDFs (AB46, AB58 and AB63) were similar to PHB de-polymerase C-terminus, cell shape-determining protein and flagellin domain protein. And 11 TDFs showed similarities with signal transduction proteins, including homologous protein of the nitrogen regulation protein NtrY and nitrate/nitrite response regulator protein NarL. These data suggested that the Azospirillum brasilense org35 was a multi-effecter and involved in chemotaxis, cyst development and regulation of nitrogen fixation.

Diversity of nifH gene sequences in the sediments of South China Sea

In order to contribute to knowledge about structure of marine diazotrophic communities in the sediments of South China Sea, the molecular diversity of the nifH gene, which encodes the Fe protein of the nitrogenase complex, was assessed by polymerase chain reaction (PCR) amplification using PolF/R primers, followed by cloning and sequencing. Sequences of nifH genes were amplified from environmental deoxyribonucleic acid (DNA) samples collected during three stations including shallow sea (75 m, station L10), shelf (450 m, station L2) and deep sea (1000 m, station L21), and covering an area between 17 to 19°N and 111 to 119°E. Samples from shallow sea contained β-, and δproteobacteria; the shelf contained α-, β-, δproteobacteria; the deep sea contained α-, δ-, γproteobacteria, firmicutes, and green nonsulfur (GNS) bacterium. These results suggested that diazotroph was significant component potentially contributing to nitrogen fixation in South China Sea.

A CheR/CheB fusion protein is involved in cyst cell development and chemotaxis in Azospirillum brasilense Sp7.

We here report the sequence and functional analysis of cstB of Azospirillum brasilense Sp7. The predicted cstB contains C-terminal two PAS domains and N-terminal part which has similarity with CheB-CheR fusion protein. cstB mutants had reduced swarming ability compared to that of A. brasilense wild-type strain, implying that cstB was involved in chemotaxis in A. brasilense. A microscopic analysis revealed that cstB mutants developed mature cyst cells more quickly than wild type, indicating that cstB is involved in cyst formation. cstB mutants were affected in colony morphology and the production of exopolysaccharides (EPS) which are essential for A. brasilense cells to differentiate into cyst-like forms. These observations suggested that cstB was a multi-effector involved in cyst development and chemotaxis in A. brasilense.

Cloning, Sequencing, and Characterization of the Azospirillum brasilense fhuE Gene

The fhuE gene of Escherichia coli encodes the FhuE protein, which is a receptor protein in the coprogen-mediated siderophore iron-transport system. A fhuE gene homologue from Azospirillum brasilense, a nitrogen-fixing soil bacterium that lives in association with the roots of cereal grasses, was cloned, sequenced, and characterized. The A. brasilense fhuE encodes a protein of 802 amino acids with a predicted molecular weight of approximately 87 kDa. The deduced amino-acid sequence showed a high level of homology to the sequences of all the known fhuE gene products. The fhuE mutant was sensitive to iron starvation and defective in coprogen-mediated iron uptake. The mutant failed to express one membrane protein of approximately 78 kDa that was induced by iron starvation in the wild type. Complementation studies showed that the A. brasilense fhuE gene, when present on a low-copy number plasmid, could restore the functions of the mutant. Mutation in fhuE gene did not affect nitrogen fixation.