Isolation and Genome Sequence of a Novel Phosphate-Solubilizing Rhizobacterium Bacillus altitudinis GQYP101 and Its Effects on Rhizosphere Microbial Community Structure and Functional Traits of Corn Seedling.

Bacillus altitudinis is a widely distributed soil bacterium that has various functional activities, including remediation of contaminated soil, degradation of herbicides, and enhancement of plant growth. B. altitudinis GQYP101 was isolated from the rhizosphere soil of Lycium barbarum L. and demonstrated potential as a plant growth-promoting bacterium. In this work, strain GQYP101 could solubilize phosphorus, and increased the stem diameter, maximum leaf area, and fresh weight of corn in a pot experiment. Nitrogen and phosphorus contents of corn seedlings (aerial part) increased by 100% and 47.9%, respectively, after application of strain GQYP101. Concurrently, nitrogen and phosphorus contents of corn root also increased, by 55.40% and 20.3%, respectively. Furthermore, rhizosphere soil nutrients were altered and the content of available phosphorus increased by 73.2% after application of strain GQYP101. The mechanism by which strain GQYP101 improved plant growth was further investigated by whole genome sequence analysis. Strain GQYP101 comprises a circular chromosome and a linear plasmid. Some key genes of strain GQYP101 were identified that were related to phosphate solubilization, alkaline phosphatase, chemotaxis, and motility. The findings of this study may provide a theoretical basis for strain GQYP101 to enhance crop yield as microbial fertilizer.

Complete Genome Sequence of Bacillus velezensis DSYZ, a Plant Growth-Promoting Rhizobacterium with Antifungal Properties.

Bacillus velezensis DSYZ is a plant growth-promoting rhizobacterium with the capacity to control fungal pathogens. It was isolated from the rhizosphere soil of garlic. Here, we present the complete genome sequence of B. velezensis DSYZ. Several gene clusters that are related to the biosynthesis of antimicrobial compounds were predicted.

Identification of a native promoter PLH-77 for gene expression in Paenibacillus polymyxa.

Paenibacillus polymyxa is a rhizobacterium that has attracted substantial attention due to its ability to produce functional metabolites and promote plant growth. Metabolic and genetic improvements in this species will benefit research and other applications of the bacterium. However, a suitable gene expression system has not been established in this species. In this study, a promoter trap system based on a green fluorescent protein and a chloramphenicol-resistance gene was developed to isolate native promoters of P. polymyxa SC2-M1 to regulate gene expression. Through high-throughput screening, the novel promoter PLH-77 was identified, sequenced, and subsequently characterized. Promoter PLH-77 is a strong, continuous expression system containing the typical -10 and -35 motifs regions. Its effective sequence was evaluated and then cascaded to improve the promotion efficiency. To further verify the existence of PLH-77, a heterogenous xylose isomerase was expressed by PLH-77 in P. polymyxa SC2-M1. In the resulting strain, the amount of xylose consumed was increased by 2.5 g/L during the 78 h fermentation period. Meanwhile, the production levels of lactate and acetate increased. It was confirmed that promoter PLH-77 could effectively mediate gene expression in P. polymyxa SC2-M1 and will further benefit the quantitative monitoring of gene expression in P. polymyxa.

Analysis of the Complete Genome Sequence of Bacillus atrophaeus GQJK17 Reveals Its Biocontrol Characteristics as a Plant Growth-Promoting Rhizobacterium.

Bacillus atrophaeus GQJK17 was isolated from the rhizosphere of Lycium barbarum L. in China, which was shown to be a plant growth-promoting rhizobacterium as a new biological agent against pathogenic fungi and gram-positive bacteria. We present its biological characteristics and complete genome sequence, which contains a 4,325,818 bp circular chromosome with 4,181 coding DNA sequences and a G+C content of 43.3%. A genome analysis revealed a total of 8 candidate gene clusters for producing antimicrobial secondary metabolites, including surfactin, bacillaene, fengycin, and bacillibactin. Some other antimicrobial and plant growth-promoting genes were also discovered. Our results provide insights into the genetic and biological basis of B. atrophaeus strains as a biocontrol agent for application in agriculture.

Complete Genome Sequence of Bacillus velezensis GQJK49, a Plant Growth-Promoting Rhizobacterium with Antifungal Activity.

Bacillus velezensis GQJK49 is a plant growth-promoting rhizobacterium with antifungal activity, which was isolated from Lycium barbarum L. rhizosphere. Here, we report the complete genome sequence of B. velezensis GQJK49. Twelve gene clusters related to its biosynthesis of secondary metabolites, including antifungal and antibacterial antibiotics, were predicted.

A single amino acid mutation in Spo0A results in sporulation deficiency of Paenibacillus polymyxa SC2.

Sporulating bacteria such as Bacillus subtilis and Paenibacillus polymyxa exhibit sporulation deficiencies during their lifetime in a laboratory environment. In this study, spontaneous mutants SC2-M1 and SC2-M2, of P. polymyxa SC2 lost the ability to form endospores. A global genetic and transcriptomic analysis of wild-type SC2 and spontaneous mutants was carried out. Genome resequencing analysis revealed 14 variants in the genome of SC2-M1, including three insertions and deletions (indels), 10 single nucleotide variations (SNVs) and one intrachromosomal translocation (ITX). There were nine variants in the genome of SC2-M2, including two indels and seven SNVs. Transcriptomic analysis revealed that 266 and 272 genes showed significant differences in expression in SC2-M1 and SC2-M2, respectively, compared with the wild-type SC2. Besides sporulation-related genes, genes related to exopolysaccharide biosynthesis (eps), antibiotic (fusaricidin) synthesis, motility (flgB) and other functions were also affected in these mutants. In SC2-M2, reversion of spo0A resulted in the complete recovery of sporulation. This is the first global analysis of mutations related to sporulation deficiency in P. polymyxa. Our results demonstrate that a SNV within spo0A caused the sporulation deficiency of SC2-M2 and provide strong evidence that an arginine residue at position 211 is essential for the function of Spo0A.

[Construction of gene knock-out system for Paenibacillus polymyxa SC2].

OBJECTIVE: To construct an efficient gene knock-out system for Paenibacillus polymyxa SC2. METHODS: Temperature sensitive plasmid pRN5101 was transformed into P. polymyxa SC2 by electrotransformation. A mutant SC2-E was obtained, in which pmxE was disrupted by homologous recombination. To confirm whether pmxE was knocked out, we used antibacterial activity assay and high performance liquid chromatography to analyze the ability of mutants synthesizing polymyxin. RESULTS: We developed an efficient gene knock-out system for P. polymyxa SC2. Plasmid of pRN5101 could replicate at 28 degrees C and suicide at 39 degrees C in SC2. Mutants lost the ability of synthesizing polymyxin, indicating that pmxE gene was successfully knocked out. CONCLUSION: The constructed gene knock-out system for P. polymyxa provides a high-efficiency tool to detect genes function for P. polymyxa.

[Diversity of antagonistic bacteria isolated from rhizosphere of several cash crops].

By adopting antimicrobial spectrum test, BOXAIR-PCR, physiological and biochemical, and 16S rDNA sequencing analysis, this paper analyzed the diversity of 55 antagonistic bacterial strains isolated from the rhizosphere of 10 cash crops. There was a high diversity of the antagonism of the strains. Based on BOXAIR-PCR, all the strains were clustered into 7 groups at the similarity level of 72.1%, and divided into 25 groups at the similarity level of 85.0%. All the strains belonged to Bacillus, Paenibacillus, Brevibacillus, Pseudomonas and Alcaligenes, respectively. The antagonistic bacteria isolated from the rhizosphere had high genetic diversity and high diversity in antagonistic activity.

Complete genome sequence of Paenibacillus polymyxa SC2, a strain of plant growth-promoting Rhizobacterium with broad-spectrum antimicrobial activity.

Paenibacillus polymyxa SC2 is an important plant growth-promoting rhizobacterium (PGPR). Here, we report the complete genome sequence of P. polymyxa SC2. Multiple sets of functional genes have been found in the genome. As far as we know, this is the first complete genome sequence of Paenibacillus polymyxa.

Genetic diversity of siderophore-producing bacteria of tobacco rhizosphere / Diversidade genética de bactérias de rizosfera de tabaco produtoras de sideróforos

The genetic diversity of siderophore-producing bacteria of tobacco rhizosphere was studied by amplifiedribosomal DNA restriction analysis (ARDRA), 16S rRNA sequence homology and phylogenetics analysis methods. Studies demonstrated that 85% of the total 354 isolates produced siderophores in iron limited liquid medium. A total of 28 ARDRA patterns were identified among the 299 siderophore-producing bacterial isolates.The 28 ARDRA patterns represented bacteria of 14 different genera belonging to six bacterial divisions, namely β-, γ-, α-Proteobacteria, Sphingobacteria, Bacilli, and Actinobacteria. Especially, γ- Proteobacteria consisting of Pseudomonas, Enterobacter, Serratia, Pantoea, Erwinia and Stenotrophomonas genus encountered 18 different ARDRA groups. Results also showed a greater siderophore-producing bacterial diversity than previous researches. For example, Sphingobacterium (isolates G-2-21-1 and G-2-27-2), Pseudomonas poae (isolate G-2-1-1), Enterobacter endosymbiont (isolates G-2-10-2 and N-5-10), Delftia acidovorans (isolate G-1-15), and Achromobacter xylosoxidans (isolates N-46-11HH and N-5-20) were reported to be able to produce siderophores under low-iron conditions for the first time. Gram-negative isolates were more frequently encountered, with more than 95% total frequency. For Gram-positive bacteria, the Bacillus and Rhodococcus were the only two genera, with 1.7% total frequency. Furthermore, the Pseudomonas and Enterobacter were dominant in this environment, with 44.5% and 24.7% total frequency, respectively. It was also found that 75 percent of the isolates that had the high percentages of siderophore units (% between 40 and 60) belonged to Pseudomonas. Pseudomonas sp. G-229-21 screened out in this study may have potential to apply to low-iron soil to prevent plant soil-borne fungal pathogen diseases.

A diversidade genética de bactérias de rizosfera de tabaco produtoras de sideróforos foi estudada por meio da técnica de análise de restrição do DNA ribossomal amplificado (ARDRA), homologia de seqüência de 16s rRNA e métodos de análise filogenética. Observou-se que 85% do total de 354 isolados produziram sideróforos em meio liquido com restrição de ferro.Entre os 299 isolados produtores de sideróforos identificou-se 28 padrões ARDRA, que representaram 14 gêneros bacterianos diferentes, pertencentes a seis divisões bacterianas: β-, γ-, α-Proteobacteria, Sphingobacteria, Bacilli e Actinobacteria. γ- Proteobacteria, consistindo de Pseudomonas, Enterobacter, Serratia, Pantoea, Erwinia e Stenotrophomonas, pertenceram a 18 grupos ARDRA. Os resultados também mostraram uma diversidade maior de bactérias produtoras de sideróforos doque a relatada em outros estudos. Por exemplo, Sphingobacterium (isolados G-2-21-1 e G-2-27-2), Pseudomonaspoae (isolado G-2-1-1), Enterobacter endosymbiont (isolados G-2-10-2 e N-5-10), Delftia acidovorans (isolado G-1-15) e Achromobacter xylosoxidans (isolados N-46-1HH e N-5-20), capazes de produzir sideróforos em condições de baixa disponibilidade de ferro, foram relatados pela primeira vez. Isolados Gram negativos foram encontrados com maior freqüência, correspondendo a mais de 95% da freqüência total. Entre as bactérias Gram positivas, foram encontrados apenas os gêneros Bacillus e Rhodococcus, com 1,7% da freqüência total. Além disso, neste ambiente houve predominância de Pseudomonas e Enterobacter, com 44,5% e 24,7% da freqüência total, respectivamente. Verificou-se também que 75% dos isolados com alta porcentagem de unidades de sideróforos (% entre 40 e 60) pertenceram a Pseudomonas. Pseudomonas sp. G- 229-21, selecionado neste estudo, apresenta potencial de aplicação em solos com baixo teor de ferro para prevenção dedoenças fúngicas em plantas.

Genetic diversity of siderophore-producing bacteria of tobacco rhizosphere.

The genetic diversity of siderophore-producing bacteria of tobacco rhizosphere was studied by amplified ribosomal DNA restriction analysis (ARDRA), 16S rRNA sequence homology and phylogenetics analysis methods. Studies demonstrated that 85% of the total 354 isolates produced siderophores in iron limited liquid medium. A total of 28 ARDRA patterns were identified among the 299 siderophore-producing bacterial isolates. The 28 ARDRA patterns represented bacteria of 14 different genera belonging to six bacterial divisions, namely ß-, γ-, α-Proteobacteria, Sphingobacteria, Bacilli, and Actinobacteria. Especially, γ-Proteobacteria consisting of Pseudomonas, Enterobacter, Serratia, Pantoea, Erwinia and Stenotrophomonas genus encountered 18 different ARDRA groups. Results also showed a greater siderophore-producing bacterial diversity than previous researches. For example, Sphingobacterium (isolates G-2-21-1 and G-2-27-2), Pseudomonas poae (isolate G-2-1-1), Enterobacter endosymbiont (isolates G-2-10-2 and N-5-10), Delftia acidovorans (isolate G-1-15), and Achromobacter xylosoxidans (isolates N-46-11HH and N-5-20) were reported to be able to produce siderophores under low-iron conditions for the first time. Gram-negative isolates were more frequently encountered, with more than 95% total frequency. For Gram-positive bacteria, the Bacillus and Rhodococcus were the only two genera, with 1.7% total frequency. Furthermore, the Pseudomonas and Enterobacter were dominant in this environment, with 44.5% and 24.7% total frequency, respectively. It was also found that 75 percent of the isolates that had the high percentages of siderophore units (% between 40 and 60) belonged to Pseudomonas. Pseudomonas sp. G-229-21 screened out in this study may have potential to apply to low-iron soil to prevent plant soil-borne fungal pathogen diseases.

[Screening, identification and antagonistic activity of a siderophore-producing bacteria G-229-21T from rhizosphere of tobacco].

OBJECTIVE: To screen antagonistic bacteria from rhizosphere of tobacco against Phytophthora parasitica var. nicotianae (Breda de Hann) Tucker and to analyze its antagonistic activity. METHODS: The antagonistic bacteria were screened by dual culture with P. parasitica var. nicotianae (Breda de Hann) Tucker on low iron (2 micromol/L FeCl3) Sucrose-L-Asparagine (SA) plate. The chrome azurol S (CAS) assay was used to detect the siderophore production and its affinity to ferric ion. The strain was identified by using morphological, biochemical and physiological characteristics, 16S rRNA sequence homology, phylogenetics and specific species molecular analysis. The siderophore was isolated by column chromatography on Amberlite XAD-2 and analyzed by spectrophotometer. Antagonistic activity of the siderophore was confirmed by incubation of P. parasitica var. nicotianae (Breda de Hann) Tucker in liquid SA medium with different concentration of siderophore and FeCl3. RESULTS: We obtained an antagonistic bacterium G-229-21T against P. parasitica var. nicotianae (Breda de Hann) Tucker. The strain produced high-affinity siderophore under low iron conditions and was identified as Pseudomonas mediterranea. This strain produced carboxylate-type siderophore against P. parasitica var. nicotianae (Breda de Hann) Tucker. The suppression ratio was up to 92.3% under low iron conditions (0.16 micromol/L-10 micromol/L FeCl3). However, it was only 2.0% under Fe-replete conditions (100 micromol/L FeCl3). CONCLUSION: P. mediterranea G-229-21T could produce high-affinity carboxylate-type siderophore against P. parasitica var. nicotianae (Breda de Hann) Tucker under low iron conditions.

[Cloning and functional analysis of cysI gene involved in siderophores biosynthesis in Pseudomonas mosselii E1].

A siderophores-producing strain E1 was isolated from the rhizosphere of cotton. Its 16S rDNA is identical to that of Pseudomonas mosselii sp. nov. at 100% level. The suicide plasmid pRL1063a carrying Tn5-1063 was introduced into E1 by triparental mating and 1000 transposon insertion mutants were subsequently screened using CAS assay. One mutant deficiency in siderophores production was obtained, namely, E1-185. DNA sequences flanking Tn5-1063 of E1-185 was amplified by TAIL-PCR. According to the DNA sequencing results, it is found that Tn5-1063 was inserted into cysI gene. The cysI of E1 is identical to that of Pseudomonas entomophila. L48 at 96% level, and similarity of amino acid sequences of their CysI is 97% . The cysI gene is required for the synthesis of cysteine. However, The ability in siderophores production of E1-185 on the plate of CAS with cysteine was recovered. It is indicated that cysI play an important role during the synthesis of siderophores. It was supposed that cysI is involved in the synthesis of acyl-S-PCPs, which is the key protein in the synthesis pathway of siderophores.