Expression analysis of the Pseudomonas aeruginosa AlgZR two-component regulatory system

Pseudomonas aeruginosa virulence components are subject to complex regulatory control primarily through two-component regulatory systems that allow for sensing and responding to environmental stimuli. In this study, the expression and regulation of the P. aeruginosa AlgZR two-component regulatory sy...

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Published in:	Journal of bacteriology Vol. 197; no. 4; pp. 736 - 748
Main Authors:	Pritchett, Christopher L, Little, Alexander S, Okkotsu, Yuta, Frisk, Anders, Cody, William L, Covey, Christopher R, Schurr, Michael J
Format:	Journal Article
Language:	English
Published:	United States American Society for Microbiology 15-02-2015
Subjects:	Alginates - metabolism Bacteria Bacterial Proteins - genetics Bacterial Proteins - metabolism Bacteriology Base Sequence Biosynthesis Fusion Gene expression Gene Expression Regulation, Bacterial Molecular Sequence Data Polymerase chain reaction Promoter Regions, Genetic Pseudomonas aeruginosa Pseudomonas aeruginosa - genetics Pseudomonas aeruginosa - metabolism Trans-Activators - genetics Trans-Activators - metabolism Transcription factors Transcription Initiation Site Transcription, Genetic
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Summary:	Pseudomonas aeruginosa virulence components are subject to complex regulatory control primarily through two-component regulatory systems that allow for sensing and responding to environmental stimuli. In this study, the expression and regulation of the P. aeruginosa AlgZR two-component regulatory system were examined. Primer extension and S1 nuclease protection assays were used to identify two transcriptional initiation sites for algR within the algZ coding region, and two additional start sites were identified upstream of the algZ coding region. The two algR transcriptional start sites, RT1 and RT2, are directly regulated by AlgU, consistent with previous reports of increased algR expression in mucoid backgrounds, and RpoS additionally plays a role in algR transcription. The expression of the first algZ promoter, ZT1, is entirely dependent upon Vfr for expression, whereas Vfr, RpoS, or AlgU does not regulate the second algZ promoter, ZT2. Western blot, real-time quantitative PCR (RT-qPCR), and transcriptional fusion analyses show that algZR expression is Vfr dependent. The algZ and algR genes also are cotranscribed in both nonmucoid and mucoid backgrounds. Furthermore, algZR was found to be cotranscribed with hemCD by RT-PCR. RT-qPCR confirmed that hemC transcription in the PAO1 ΔalgZ mutant was 40% of the level of the wild-type strain. Taken together, these results indicate that algZR transcription involves multiple factors at multiple start sites that control individual gene expression as well as coexpression of this two-component system with heme biosynthetic genes.
Bibliography:	ObjectType-Article-1 SourceType-Scholarly Journals-1 ObjectType-Feature-2 content type line 23 Citation Pritchett CL, Little AS, Okkotsu Y, Frisk A, Cody WL, Covey CR, Schurr MJ. 2015. Expression analysis of the Pseudomonas aeruginosa AlgZR two-component regulatory system. J Bacteriol 197:736–748. doi:10.1128/JB.02290-14. Present address: Anders Frisk, Pelham, New York, USA; William L. Cody, University of Dallas, Irving, Texas, USA. C.L.P., A.S.L., and Y.O. contributed equally to the manuscript.
ISSN:	0021-9193 1098-5530
DOI:	10.1128/JB.02290-14