bInorganic polyphosphate (polyP) is a linear polymer composed of several molecules of orthophosphate (P i ) linked by energyrich phosphoanhydride bonds. In Pseudomonas aeruginosa, P i is taken up by the ABC transporter Pst, encoded by an operon consisting of five genes. The first four genes encode proteins involved in the transport of P i and the last gene of the operon, phoU, codes for a protein which exact function is unknown. We show here that the inactivation of phoU in P. aeruginosa enhanced P i removal from the medium and polyP accumulation. The phoU mutant also accumulated high levels of the alarmone guanosine tetraphosphate (ppGpp), which in turn increased the buildup of polyP. In addition, phoU inactivation had several pleiotropic effects, such as reduced growth rate and yield and increased sensitivity to antibiotics and stresses. However, biofilm formation was not affected by the phoU mutation. Inorganic phosphate (polyP) is an important macronutrient for all living beings, making up to 3% of bacterial dry weight. To cope with orthophosphate (P i ) shortage in natural environments, bacteria activate several systems associated with the process of uptake and assimilation of P i and organophosphates (phosphate esters and phosphonates). Most of these genes, collectively known as the PHO regulon, are strongly expressed under P i limitation (1, 2, 3). Transcription of the PHO regulon genes in Escherichia coli is controlled by the two-component system PhoB-PhoR. PhoR is the histidine kinase that phosphorylates PhoB which in turn activates the transcription of the PHO regulon genes by interacting with the PHO-box, a consensus sequence present at single or multiple copies at the Ϫ35 region of all PHO promoters (2). One of the most well-studied components of the PHO regulon is the pst operon. The four proximal genes of the operon, pstS, pstC, pstA, and pstB, encode a high-affinity P i transport system. PstS is a periplasmic P i -binding protein that captures and transfers P i molecules to the channel formed by the integral proteins PstC and PstA in the cytoplasmic membrane. PstB is an ATPase that provides the energy for transport (4). The last gene of the operon, phoU, encodes a protein whose exact function is unknown. All five proteins encoded by the pst operon act as repressors of the PHO regulon under conditions of P i excess. The mechanism of repression is still unclear, but null mutations in any pst gene (phoU included) result in the constitutive expression of the entire regulon (2).Pseudomonas aeruginosa is a gammaproteobacterium found in natural water reservoirs, soil, plants, and animals. In humans, it is an opportunistic pathogen, causing infections in immunocompromised and cystic fibrosis patients and is also associated with severe burn infections. P. aeruginosa carries a PHO regulon similar to that of E. coli, but it is not as well characterized. An in silico study predicted the presence of 73 PHO-boxes in the chromosome of strain PAO1 (5). The architectures of the pst operon of P. aeruginosa and E. c...

Enteropathogenic Escherichia coli (EPEC) is a known causative agent of diarrhea in children. In the process of colonization of the small intestine, EPEC synthesizes two types of adhesins, the bundle-forming pilus (BFP) and intimin. The BFP pilus is an adhesin associated with the initial stages of adherence of EPEC to epithelial cells, while the outer membrane protein intimin carries out the intimate adherence that takes place at the third stage of infection. BFP is encoded by the bfp operon located in plasmid EAF, present only in typical EPEC isolates, while eae, the gene that encodes intimin is situated in the LEE, a chromosomal pathogenicity island. Transcription of bfp and eae is regulated by the products of the perABC operon, also present in plasmid EAF. Here we show that deletion of relA, that encodes a guanosine penta and tetraphosphate synthetase impairs EPEC adherence to epithelial cells in vitro. In the absence of relA, the transcription of the regulatory operon perABC is reduced, resulting in lower levels of BFP and intimin. Bacterial adherence, BFP and intimin synthesis and perABC expression are restored upon complementation with the wild-type relA allele.

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Phosphate homeostasis is tightly regulated in bacteria. Phosphate scarcity is overcome by inducing the expression of genes associated with the scavenging of phosphate and phosphate-containing molecules, while phosphate surplus is stored in the form of polyphosphate (polyP). Regulation of the genes involved in polyP metabolism was investigated. Knockout of the most distal gene of the pstSCAB-phoU operon that encodes a Pi-transport system results in large accumulation of polyphosphate (polyP). Here, we show that the phoU mutation differentially affects the transcription of ppk and ppx, that respectively, encode a polyP kinase and a polyP exopolyphosphatase, by increasing the former and reducing the latter, further contributing the accumulation of polyP. We also show that ppk forms an operon with the upstream gene hemB and that neither ppk nor ppx positively respond to Pi starvation. Furthermore, a putative PHO-box sequence in ppx regulatory region did not show a strong affinity for the PHO response regulator PhoB, while the promoter of hemB does not carry a PHO-box sequence. Altogether, the data indicate that the main genes involved in polyP metabolism, ppk and ppx, are differentially regulated in the absence of phoU, but neither gene belongs to the PHO regulon.

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