Efficient markerless gene deletions in Pseudomonas protegens Pf-5 using a upp-based counterselective system.

OBJECTIVES: Developing a counterselective system for efficient markerless gene deletions in biocontrol strain P. protegens Pf-5. RESULTS: We successfully implemented a markerless deletion of upp in Pf-5 to obtain the 5-FU resistant strain Pf5139. With this strain, we performed markerless gene deletions for each component of Gac/Rsm system and a 17 kb DNA fragment with the deletion ratio of 20 to 50%, and efficiently constructed a strain with triple deletions based on the suicide plasmid pJQ200UPP. In addition, there is no obvious connection between the deleted fragment length and the deletion ratio. CONCLUSION: The upp-based counterselective system in this study is efficient and valuable for markerless gene deletions in Pf-5, indicating that it has great potential in the study of gene function and in the application of genome reduction for Pseudomonas strains.

ArgR directly inhibits lipA transcription in Pseudomonas protegens Pf-5.

ArgR, a transcriptional regulator belonging to the AraC/XylS family, plays a key role in arginine metabolism regulation. ArgR has also been found to repress the transcription of a lipase gene, but its molecular mechanism is still unknown. In this study, we investigated the molecular mechanism acting on the expression of intracellular lipase gene lipA regulated by ArgR in Pseudomonas protegens Pf-5 through knockout and overexpression of argR, detection of DNA-protein interaction in vivo, determining whole-cell lipase activities of various strains derived from Pf-5, and examining ß-galactosidase activities of various lacZ fusions. The results demonstrated that ArgR inhibits lipA expression at the transcriptional level. Further results showed that the inhibition of lipA transcription by ArgR is mediated by binding to the ArgR binding site of lipA promoter to produce steric hindrance, in which the common sequence, TGTCGC is crucial for the ArgR binding. Besides, arginine inhibits lipA expression in both wild-type and argR mutant, and shows a synergistic inhibition on lipA expression when combined with ArgR. To the best of our knowledge, this is the first report on ArgR directly repressing the transcription of a lipase gene.

Microbacterium lacusdiani sp. nov., a phosphate-solubilizing novel actinobacterium isolated from mucilaginous sheath of Microcystis.

A novel actinobacterium, designated strain JXJ CY 01T, was isolated from a mucilaginous sheath of Microcystis aeruginosa FACHB-905 collected from Lake Dianchi, south-west China. Taxonomic position of the isolate was determined by polyphasic approaches. Strain JXJ CY 01T shared 16S rRNA sequence similarities of 98.9 and 98.0% with Microbacterium marinilacus YM11-607T and Microbacterium paludicola US15T, and less than 98% with other members of the genus Microbacterium. The DNA-DNA relatedness values between strains JXJ CY 01T and M. marinilacus JCM 16546T and M. paludicola JCM 14308T were 53.5±1.4 and 53.8±2.1%, respectively. l-Ornithine was detected in the cell wall, and rhamnose, galactose, glucose, arabinose, fucose and mannose as signature sugars in the whole-cell hydrolysates. Other chemotaxonomic characteristics determined were MK-12 and MK-11 as predominant menaquinones, anteiso-C15:0, iso-C16:0, anteiso-C17:0 and iso-C17:0 as major cellular fatty acids (>10%), and diphosphatidylglycerol, phosphatidylglycerol, an unidentified glycolipid and two unidentified phospholipids as the polar lipids. The DNA G+C content was 70.4 mol%. On the basis of the above taxonomic data, strain JXJ CY 01T is determined to represent a novel species of the genus Microbacterium, for which the name Microbacterium lacusdiani sp. nov. is proposed. The type strain is JXJ CY 01T (= KCTC 29655T=DSM 29188T). The type strain JXJ CY 01T can solubilize both insoluble inorganic (calcium phosphate) and organic phosphate (l-α-phosphatidylcholine) and is possibly one of the mechanism for enhancement of growth of M. aeruginosa FACHB-905.

Citricoccus lacusdiani sp. nov., an actinobacterium promoting Microcystis growth with limited soluble phosphorus.

A novel actinobacterium, designated strain JXJ CY 21T, was isolated from the culture mass of Microcystis sp. FACHB-905 collected from Lake Dianchi, South-west China. Polyphasic taxonomic study revealed that the isolate should be a member of the genus Citricoccus. Comparison of the 16S rRNA gene sequence of strain JXJ CY 21T with the available sequences in the GenBank database showed that the strain is closely related to Citricoccus zhacaiensis FS24T (97.8 % similarity), Citricoccus parietis 02-Je-010T (97.7 %), Citricoccus terreus V3M1T (97.6 %), Citricoccus nitrophenolicus PNP1T (97.2 %), Citricoccus alkalitolerans YIM 70010T (97.2 %) and Citricoccus muralis 4-0T (97.0 %). The DNA-DNA hybridization values between strain JXJ CY 21T and the related type strains C. zhacaiensis FS24T and C. parietis 02-Je-010T were 16.0 ± 2.6 and 5.4 ± 1.7 %, respectively. The peptidoglycan in the cell wall was A4α type containing lysine-glutamic acid-glycine. The major respiratory menaquinone was found to be MK-8 (H2) (98.5 %), while the major cellular fatty acids (>10 %) were anteiso-C15:0, iso-C16:0, iso-C15:0 and iso-C14:0. The polar lipids detected were diphosphatidylglycerol, phosphatidylinositol, phosphatidylglycerol, an unidentified phospholipid and an unidentified glycolipid. The DNA G + C content was determined to be 62.7 mol%. Strain JXJ CY 21T can solubilize both insoluble inorganic and organic phosphates up to 24.7 and 1.7 mg/l respectively. This property of the novel actinobacterium acts as a modulator for enhancement of growth of Microcystis sp. FACHB-905 in the lake ecosystem where the amount of soluble phosphate is limited. On the basis of the above taxonomic data, strain JXJ CY 21T represents a novel species of the genus Citricoccus, for which the name Citricoccus lacusdiani sp. nov. is proposed. The type strain is JXJ CY 21T (=KCTC 29653T = DSM 29160T).

Modestobacter lacusdianchii sp. nov., a Phosphate-Solubilizing Actinobacterium with Ability to Promote Microcystis Growth.

A novel actinobacterium, designated strain JXJ CY 19T, was isolated from a culture mat of Microcystis aeruginosa FACHB-905 collected from Dianchi Lake, South-west China. 16S rRNA gene sequences comparison of strain JXJ CY 19T and the available sequences in the GenBank database showed that the strain was closely related to Modestobacter marinus 42H12-1T (99.1% similarity) and Modestobacter roseus KLBMP 1279T (99.0%). The isolate had meso-diaminopimelic in the cell wall with whole-cell sugars of mannose, rhamnose, ribose, glucose, galactose, and arabinose. The menaquinone detected was MK-9(H4), while the major cellular fatty acids include C17:1 ω8c, C15:0 iso, C15:1 iso G and C16:0 iso. The polar lipids were diphosphatidylglycerol, phosphatidylethanolamine, phosphatidylinositol, phosphatidylinositol mannoside and an unidentified phospholipid. The DNA-DNA hybridization values between strains JXJ CY 19T and the closely related type strains Modestobacter marinus CGMCC 4.5581T and Modestobacter roseus NBRC 108673T were determined to be 50.8 ± 0.8% and 44.1 ± 1.7%, respectively. The DNA G+C content was 71.9 mol%. On the basis of the above taxonomic data and differences in physiological characters from the closely related type strains, strain JXJ CY 19T was recognized as a novel species of the genus Modestobacter, for which the name Modestobacter lacusdianchii sp. nov. (JXJ CY 19T = KCTC 39600T = CPCC 204352T) is proposed. The type strain JXJ CY 19T can solubilize calcium phosphate tribasic (Ca3(PO4)2), phytin and L-α-phosphatidylcholine. The phosphate-solubilizing property of the novel actinobacterium could be a possible factor for the increase in growth of Microcystis aeruginosa FACHB-905 in ecosystem where the amount of available soluble phosphate is limited such as Dianchi Lake.

L-valine, an antialgal amino acid from Streptomyces jiujiangensis JXJ 0074(T).

An antialgal compound was isolated from the cultured broth of Streptomyces jiujiangensis JXJ 0074(T) by using bioassay methods. Based on the data of (1)H-NMR, (13)C-NMR, ESI-MS, and thin layer chromatography, the active compound was identified as L-valine, which showed antialgal activity mainly against Microcystis. L-valine exhibited greater antialgal activities than both L-lysine and copper sulfate (CuSO4) did on Microcystis aeruginosa lawn. However, M. aeruginosa recovered growth earlier with higher growth rate in L-valine treatment than in L-lysine treatment. L-valine dissipated completely within 2 days, much quicker than L-lysine (6 days), which resulted in the lysing of more than 80 % M. aeruginosa cells and the release of amount of intracellular microcystin-LR (MC-LR) within 2 days. As a resultant, the extracellular MC-LR content was more than twice of the control from day 1 to 5. Exposure to L-valine significantly promoted the synthesis of MC-LR. L-lysine also promoted the release and synthesis of MC-LR with much lesser efficiency than L-valine. L-valine could damage Microcystis severely, causing perforation and collapse of M. aeruginosa cells and decrease of the chlorophyll. The superoxide dismutase (SOD) activity in L-valine-treated cells of M. aeruginosa initially increased with 32.94 ± 3.37 % higher than the control after 36 h and then decreased quickly. However, the increase rate of superoxide anion radical (O2 (-)) was much higher than that of SOD, which resulted in serious lipid peroxidation and accumulation of malondialdehyde (MDA). To our knowledge, this is the first report showing L-valine active against cyanobacteria.