Abstract
Extracytoplasmic function (ECF) sigma factors are members of cell-surface signaling systems, abundant in the opportunistic pathogen Pseudomonas aeruginosa. Twenty genes coding for ECF sigma factors are present in P. aeruginosa sequenced genomes, most of them being part of TonB systems related to iron uptake. In this work, poorly characterized sigma factors were overexpressed in strain PA14, in an attempt to understand their role in the bacterium´s physiology. Cultures overexpressing SigX displayed a biphasic growth curve, reaching stationary phase earlier than the control strain, followed by subsequent growth resumption. During the first stationary phase, most cells swell and die, but the remaining cells return to the wild type morphology and proceed to a second exponential growth. This is not due to compensatory mutations, since cells recovered from late time points and diluted into fresh medium repeated this behavior. Swollen cells have a more fluid membrane and contain higher amounts of shorter chain fatty acids. A proteomic analysis was performed to identify differentially expressed proteins due to overexpression of sigX, revealing the induction of several fatty acid synthesis (FAS) enzymes. Using qRT-PCR, we showed that at least one isoform from each of the FAS pathway enzymes were upregulated at the mRNA level in the SigX overexpressing strain thus pointing to a role for this ECF sigma factor in the FAS regulation in P. aeruginosa.
Highlights
Alternative sigma factors of the extracytoplasmic function (ECF) family are members of cell-surface signaling systems, which include regulators that modulate ECF sigma function and proteins responsible for sensing environmental cues [1,2,3]
From the twenty genes in the PA14 genome coding for ECF sigma factors, twelve are predicted or proven to be part of systems involved in siderophore uptake, reflecting the importance of iron in the bacterial metabolism [1,9,10,11,12,13]
In E. coli, this step is catalyzed by FabH, but, in P. aeruginosa, it was recently shown that FabY acts preferentially in the condensation of acetyl-CoA with malonyl-acyl carrier protein (ACP) to initiate the fatty acid synthesis (FAS) pathway, instead of the well characterized FabH [28]. β-oxoacyl-ACP is reduced in the first reaction of the FAS cycle by FabG in a NADPH-dependent reaction, generating β-hydroxyacyl-ACP, which has its hydroxyl group removed by FabA or FabZ (3-hydroxyacyl-ACP-dehydratases), resulting in a double bond, which is reduced in a NADHdependent reaction catalyzed by FabI or FabV in P. aeruginosa (NADH-dependent enoyl-ACP-reductases)
Summary
Alternative sigma factors of the extracytoplasmic function (ECF) family are members of cell-surface signaling systems, which include regulators that modulate ECF sigma function and proteins responsible for sensing environmental cues [1,2,3]. Β-oxoacyl-ACP is reduced in the first reaction of the FAS cycle by FabG (βoxoacyl-ACP-reductase) in a NADPH-dependent reaction, generating β-hydroxyacyl-ACP, which has its hydroxyl group removed by FabA or FabZ (3-hydroxyacyl-ACP-dehydratases), resulting in a double bond, which is reduced in a NADHdependent reaction catalyzed by FabI or FabV in P. aeruginosa (NADH-dependent enoyl-ACP-reductases) At this point, the cycle begins again with the condensation of acyl-ACP with another malonyl-ACP group, but the successive condensation reactions are catalyzed by either FabB (β-oxoacyl-ACPsynthase I) or FabF (β-oxoacyl-ACP-synthase II) (reviewed by Chan and Vogel [26]) (Figure 1). We found that overexpression of the ECF sigma factor SigX results in growth and morphology changes in P. aeruginosa PA14, and we show that high levels of SigX favor the upregulation of a set of FAS enzymes, altering lipid composition and membrane fluidity
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