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Characterization ofthe ECF sigma fator σx overexpression in Pseudomonas aeruginosa PA14.

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Author(s):
Ana Laura Boechat Borges
Total Authors: 1
Document type: Doctoral Thesis
Press: São Paulo.
Institution: Universidade de São Paulo (USP). Conjunto das Químicas (IQ e FCF) (CQ/DBDCQ)
Defense date:
Examining board members:
Regina Lúcia Baldini; Marcelo Brocchi; Frederico José Gueiros Filho; José Freire da Silva Neto; Aline Maria da Silva
Advisor: Regina Lúcia Baldini
Abstract

Pseudomonas aeruginosa is a very versatile gammaproteobacteria, able to colonize different environments and to infect phylogenetically distinct hosts, including immunocompromised humans. The extracytoplasmic function sigma factors (ECFs) are members of cell signaling systems (CSS), abundant in P. aeruginosa. Twenty genes coding for ECF sigma factors are present in the sequenced genomes of P. aeruginosa, most of them being part of TonB systems related to iron uptake. In this work, six poorly characterized sigma factors were overexpressed in strain PA14 from an arabinose inducible promoter to investigate their role in the expression of the two-component systems PvrSR and RcsCB, which regulates CupD fimbria, and their influence in P. aeruginosa cultures growth. None of the tested sigma factors led to two-component systems upregulation and overexpression of five of them caused no change in the growth profile, but pyocyanin production was altered in PA14_55550 overexpression and PA14_26600 and PA14_46810 overexpression led to a slight increase in biofilm initiation in PA14. By the other side, cultures overexpressing σx (ALB04) presented an altered lipopolysaccharide profile and a biphasic growth curve, reaching an early stationary phase followed by a growth resuming untill a second stationary phase. During the early stationary phase, most cells swells and dies, but the remaining cells return to wild type morphology and proceed to the second exponential phase of growth. This is not due to compensatory mutations, since cells collected from late points of the curve and diluted in fresh medium repeat this behavior. Although studies with strain PAO1 associate σx with transcription of oprF, encoding the major nonspecific porin of Pseudomonas, under our experiments conditions with PA14, this porin expression is not induced by σx overexpression. Thus, the effects observed in this overexpression cannot be attributed to OprF. Transcription of oprF in PA14 proved to be mainly controlled by the σ70-dependent promoter region instead of the σx-dependent promoter region reported in the literature. Proteomic analyses were performed to investigate the elements involved in these effects of σx overexpression, which revealed the induction of several enzymes involved in fatty acids biosynthesis. Cells overexpressing σx exhibit a greater proportion of hexadecanoic (C16) and hexadecenoic (C16: 1) acids and anisotropy data show higher fluidity of the membrane (s). This work is the first report of an ECF sigma factor involved in lipid biosynthesis in P. aeruginosa. (AU)

FAPESP's process: 09/03528-0 - Investigation of the signals that activate the expression of CupD fimbria and caracterization of SigX role and regulation in Pseudomonas aeruginosa PA14
Grantee:Ana Laura Boechat Borges
Support Opportunities: Scholarships in Brazil - Doctorate