Gene cloning, structural gene and promoter identification, and active assay of the phosphatidylcholine synthase of <i>Pseudomonas</i> sp. strain 593

He, Huoguang; Wu, Bin; Xiong, Min; Li, Yang; Wu, Wenhua; Wang, Xingguo

doi:10.1139/w11-073

Cited by 3 publications

(1 citation statement)

References 20 publications

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“…Pseudomonas sp. 593 was previously isolated from soil for other research purposes in our laboratory [36,37]. Sequence analysis of 16S rRNA gene revealed that this soil bacterium belongs to Pseudomonas genus, but further classification has not been done.…”

Section: Soil Bacterium Pseudomonas Sp 593 Does Not Elicit Hypersensitive Responses In Tobacco and Soybean Leavesmentioning

confidence: 99%

A SOIL-DWELLING Pseudomonas SP.593 HARBORING AN EXOGENOUS hrpZ GENE ELICITS A TYPICAL HYPERSENSITIVE RESPONSE IN TOBACCO AND SOYBEAN LEAVES

Long¹,

He²,

Xiong³

et al. 2013

Int J of Micr Res

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Pseudomonas sp.593 is a soil-dwelling bacterium unable to elicit any hypersensitive response (HR) in tobacco or soybean. The hrpZ gene, encoding an abundant Type III secretion system (T3SS) dependent protein, was cloned from a phytopathogen Pseudomonas syringae pv. syringae Van Hall CFCC 1336. The HrpZ harpin expressed in E. coli was purified to homogeneity, and used to raise polyclonal anti-HrpZ rabbit serum. The cloned hrpZ gene was then introduced into the soil bacterium Pseudomonas sp.593 via transformation of the plasmid pMEK-hrpZ. Western blotting and HR assay showed that the hrpZ-transformed Pseudomonas sp.593 was not only able to secret HrpZ harpin but also elicited a strong HR reaction in tobacco and soybean just as P. syringae did. In addition, bacterial cells were able to grow and multiply in the HR zone. Our results demonstrate that an avirulent strain can become a virulent-like pathogen, or a pathogen strain can broaden its host range once a single exogenous hrpZ gene cloned from a phytopathogen is introduced into a bacterium displaying phylogenetic diversity

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Section: Soil Bacterium Pseudomonas Sp 593 Does Not Elicit Hypersensitive Responses In Tobacco and Soybean Leavesmentioning

confidence: 99%

A SOIL-DWELLING Pseudomonas SP.593 HARBORING AN EXOGENOUS hrpZ GENE ELICITS A TYPICAL HYPERSENSITIVE RESPONSE IN TOBACCO AND SOYBEAN LEAVES

Long¹,

He²,

Xiong³

et al. 2013

Int J of Micr Res

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Phosphatidylcholine synthesis is essential for HrpZ harpin secretion in plant pathogenic Pseudomonas syringae and non-pathogenic Pseudomonas sp. 593

Xiong

Long²,

He³

et al. 2014

Microbiological Research

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Pseudomonas syringae pv. syringae van Hall is important phytopathogenic bacterium of stone fruit trees, and able to elicit hypersensitive response (HR) in nonhost plants. The HrpZ, secreted via type III secretion system (T3SS) to the extracellular space of the plant, is a T3SS-dependent protein and a sole T3SS effector able to induce the host defense response outside host cells. We deleted the phosphatidylcholine synthase gene (pcs) of P. syringae pv. syringae van Hall CFCC 1336, and found that the 1336 pcs(-) mutant was unable to synthesize phosphatidylcholine and elicit a typical HR in soybean. Further studies showed that the 1336 pcs(-) mutant was unable to secrete HrpZ harpin but could express HrpZ protein in cytoplasm as effectively as the wild type. To confirm if phosphatidylcholine affects HrpZ harpin secretion, we introduced the hrpZ gene into the soil-dwelling bacterium Pseudomonas sp. 593 and the 593 pcs(-) mutant, which were unable to express HrpZ harpin and elicit HR in tobacco or soybean. Western blotting and HR assay showed that the 593H not only secreted HrpZ harpin but also caused a strong HR in tobacco and soybean. In contrast, the 593 pcs(-)H only expressed HrpZ protein in its cytoplasm at the wild type level, but did not secrete HrpZ harpin or elicit HR reaction. Our results demonstrate that phosphatidylcholine is essential for the secretion of HrpZ harpin in P. syringae pv. syringae van Hall and other Pseudomonas strains.

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Phosphatidylcholine affects the secretion of the alkaline phosphatase PhoA in Pseudomonas strains

Liu

Long

You

et al. 2016

Microbiological Research

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Pseudomonas aeruginosa ATCC 27853 and Pseudomonas sp. 593 use the phosphatidylcholine synthase pathway (Pcs-pathway) for the biosynthesis of phosphatidylcholine (PC). Both bacterial strains contain the phoA and lapA genes encoding alkaline phosphatases (ALP) and display strong ALP activities. The PhoA and LapA enzymes are thought to be independently secreted via the Xcp and Hxc type II secretion system (T2SS) subtypes, in which the Hxc system may act as a complementary mechanism when the Xcp pathway becomes limiting. Inactivation of the pcs gene in both bacteria abolished PC synthesis and resulted in approximately 50% less ALP activity in the cell-free culture. Analysis by western blotting showed that LapA protein content in the wild type and the pcs- mutant was unchanged in the cytoplasmic, periplasmic or extracellular protein fractions. In contrast, the PhoA protein in the pcs- mutant was less prevalent among extracellular proteins but was more abundant in the periplasmic protein fraction compared to the wild type. Semi- quantitative reverse transcriptase PCR showed that phoA, lapA and 12 xcp genes were equally expressed at the transcriptional level in both the wild types and the pcs- mutants. Our results demonstrate that the absence of PC in bacterial membrane phospholipids does not interfere with the transcription of the phoA and lapA genes but primarily affects the export of PhoA from the cytoplasm to the extracellular environment via the Xcp T2SS.

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Gene cloning, structural gene and promoter identification, and active assay of the phosphatidylcholine synthase of Pseudomonas sp. strain 593

Cited by 3 publications

References 20 publications

A SOIL-DWELLING Pseudomonas SP.593 HARBORING AN EXOGENOUS hrpZ GENE ELICITS A TYPICAL HYPERSENSITIVE RESPONSE IN TOBACCO AND SOYBEAN LEAVES

A SOIL-DWELLING Pseudomonas SP.593 HARBORING AN EXOGENOUS hrpZ GENE ELICITS A TYPICAL HYPERSENSITIVE RESPONSE IN TOBACCO AND SOYBEAN LEAVES

Phosphatidylcholine synthesis is essential for HrpZ harpin secretion in plant pathogenic Pseudomonas syringae and non-pathogenic Pseudomonas sp. 593

Phosphatidylcholine affects the secretion of the alkaline phosphatase PhoA in Pseudomonas strains

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