An ABC transporter is essential for resistance to the antitumor agent mithramycin in the producerStreptomyces argillaceus

The organization of the phosphate-specific transport (pst) operon inPseudomonas aeruginosa has been determined. The gene order of thepst operon ispstC, pstA, pstB, phoU, and a well-conserved Pho box sequence (16/18 bases identical) exists in the promoter region. The most striking difference from the knownEscherichia coli pst operon is the lack of thepstS gene encoding a periplasmic phosphate (P_i)-binding protein. Even though the threepst genes were absolutely required for P_i-specific transport, expression of thepst operon at high levels did not increase P_i uptake inP. aeruginosa. DNA sequences for thepstB andphoU genes have been determined previously. The newly identifiedpstC andpstA genes encode possible integral membrane proteins of 677 amino acids (M _r 73 844) and 513 amino acids (M _r 56 394), respectively. The amino acid sequences of PstC and PstA predict that these proteins contain a long hydrophilic domain not seen in theirE. coli counterparts. A chromosomal deletion of the entirepst operon renderedP. aeruginosa unable to repress P_i taxis under conditions of P_i excess. ThephoU andpstB genes are essential for repressing P_i taxis. However, mutants lacking either PstC or PstA alone were able to repress P_i taxis under conditions of P_i excess.     

The Integration Host Factor (IHF) Affects the Expression of the Phosphate-Binding Protein and of Alkaline Phosphatase in Escherichia coli

The genes encoding alkaline phosphatase (phoA) and the inducible inorganic phosphate transport system Pst (pstS,C,A,B,U) belong to the PHO regulon. Mutants of Escherichia coli lacking the global regulatory protein integration host factor (IHF) show an increased level of alkaline phosphatase and a decreased level of Pst. IHF binds weakly but specifically to a DNA fragment containing the promoter region of the pst operon but does not bind to a fragment that includes the promoter region of phoA. It is proposed that IHF is a positive regulator of the pst operon and as such controls indirectly the expression of phoA. Received: 4 May 1998 / Accepted: 19 August 1998     

Regulation of the phosphate regulon of Escherichia coli: Characterization of the promoter of the pstS gene

Summary The pstS gene belongs to the phosphate regulon whose expression is induced by phosphate starvation and regulated positively by the PhoB protein. The phosphate (pho) box is a consensus sequence shared by the regulatory regions of the genes in the pho regulon. We constructed two series of deletion mutations in a plasmid in vitro, with upstream and downstream deletions in the promoter region of pstS, which contains two pho boxes in tandem, and studied their promoter activity by connecting them with a promoterless gene for chloramphenicol acetyltransferase. Deletions extending into the upstream pho box but retaining the downstream pho box greatly reduced promoter activity, but the remaining activity was still regulated by phosphate levels in the medium and by the PhoB protein, indicating that each pho box is functional. No activity was observed in deletion mutants which lacked the remaining pho box or the-10 region. Therefore, the pstS promoter was defined to include the two pho boxes and the-10 region. The PhoB protein binding region in the pstS regulatory region was studied with the deletion plasmids by a gelmobility retardation assay. The results suggest the protein binds to each pho box on the pstS promoter. A phoB deletion mutant was constructed, and we demonstrated that expression of pstS was strictly dependent on the function of the PhoB protein.     

Employment of a Promoter-Swapping Technique Shows that PhoU Modulates the Activity of the PstSCAB2 ABC Transporter in Escherichia coli

Expression of the Pho regulon in Escherichia coli is induced in response to low levels of environmental phosphate (P_i). Under these conditions, the high-affinity PstSCAB₂ protein (i.e., with two PstB proteins) is the primary P_i transporter. Expression from the pstSCAB-phoU operon is regulated by the PhoB/PhoR two-component regulatory system. PhoU is a negative regulator of the Pho regulon; however, the mechanism by which PhoU accomplishes this is currently unknown. Genetic studies of phoU have proven to be difficult because deletion of the phoU gene leads to a severe growth defect and creates strong selection for compensatory mutations resulting in confounding data. To overcome the instability of phoU deletions, we employed a promoter-swapping technique that places expression of the phoBR two-component system under control of the P_tac promoter and the lacO^ID regulatory module. This technique may be generally applicable for controlling expression of other chromosomal genes in E. coli. Here we utilized P_phoB::P_tac and P_pstS::P_tac strains to characterize phenotypes resulting from various ΔphoU mutations. Our results indicate that PhoU controls the activity of the PstSCAB₂ transporter, as well as its abundance within the cell. In addition, we used the P_phoB::P_tac ΔphoU strain as a platform to begin characterizing new phoU mutations in plasmids.     

Induction of the pho regulon and polyphosphate synthesis against spermine stress in Pseudomonas aeruginosa

Growth of Pseudomonas aeruginosa on spermine requires a functional γ‐glutamylpolyamine synthetase PauA2. Not only subjected to growth inhibition by spermine, the pauA2 mutant became more sensitive to β‐lactam antibiotics in human serum. To explore PauA2 as a potential target of drug development, suppressors of the pauA2 mutant, which alleviated toxicity, were isolated from selection plates containing spermine. These suppressors share common phenotypic changes including delayed growth rate, retarded swarming motility, and pyocyanin overproduction. Genome resequencing of a representative suppressor revealed a unique C₅₉₉T mutation at the phoU gene that results in Ser₂₀₀Leu substitution and a constitutive expression of the Pho regulon. Identical phenotypes were also observed in a ΔpauA2ΔphoU double knockout mutant and complemented by the wild‐type phoU gene. Accumulation of polyphosphate granules and spermine resistance in the suppressor were reversed concomitantly when expressing exopolyphosphatase PPX from a recombinant plasmid, or by the introduction of deletion alleles in pstS pstC for phosphate uptake, phoB for Pho regulation, and ppk for polyphosphate synthesis. In conclusion, this study identifies polyphosphate accumulation due to an activated Pho regulon and phosphate uptake by the phoU mutation as a potential protection mechanism against spermine toxicity.     

Identification of a Streptococcus pneumoniae Gene Locus Encoding Proteins of an ABC Phosphate Transporter and a Two-Component Regulatory System

The Escherichia coli Pst system belongs to the family of ABC transporters. It is part of a phosphate (PHO) regulon which is regulated by extracellular phosphate. Under conditions of phosphate limitation, the response regulator PhoB is phosphorylated by the histidine kinase PhoR and binds to promoters that share a consensus PHO box. Under conditions of phosphate excess, PhoR, Pst, and PhoU downregulate the PHO regulon. Screening of a library of pneumococcal mutants with defects in exported proteins revealed a putative two-component regulatory system, PnpR-PnpS, and a downstream ABC transporter, similar to the Pst system in E. coli including a gene encoding a PhoU protein. Similar to E. coli, mutagenesis of the ATP-binding cassette gene, pstB, resulted in decreased uptake of phosphate. The effects of the loss of the pneumococcal Pst system extended to decreased transformation and lysis. Withdrawal of phosphate led to transformation deficiency in the parent strain R6x but not to penicillin tolerance, suggesting that reduced bacterial death was independent of phosphate. None of these phenotypes was observed in the pneumococcal loss-of-function mutant phoU. By using a lacZ reporter construct, it was demonstrated that expression of the two-component regulatory system PnpR-PnpS was not influenced by different concentrations of phosphate. These results suggest a more complex role of the Pst system in pneumococcal physiology than in that of E. coli.     

Cyanophage host-derived genes reflect contrasting selective pressures with depth in the oxic and anoxic water column of the Eastern Tropical North Pacific

Cyanophages encode host-derived genes that may increase their fitness. We examined the relative abundance of 18 host-derived cyanophages genes in metagenomes and viromes along depth profiles from the Eastern Tropical North Pacific Oxygen Deficient Zone (ETNP ODZ) where Prochlorococcus dominates a secondary chlorophyll maximum within the ODZ. Cyanophages at the oxic primary chlorophyll maximum encoded genes related to light and phosphate stress (psbA, psbD and pstS in T4-like and psbA in T7-like), but the proportion of cyanophage with these genes decreased with depth. The proportion of cyanophage with purine biosynthesis genes increased with depth in T4-like, but not T7-like cyanophages. No additional host-derived genes were found in deep T7-like cyanophages, suggesting that T4-like and T7-like cyanophages have different host-derived gene acquisition strategies, possibly linked to their different genome packaging mechanisms. In contrast to the ETNP, in the oxic North Atlantic T4-like cyanophages encoded psbA and pstS throughout the euphotic zone. Differences in pstS between the ETNP and the North Atlantic stations were consistent with differences in phosphate concentrations in those regimes. We suggest that the low proportion of cyanophage with psbA within the ODZ reflects the stably stratified low-light conditions occupied by their hosts, a Prochlorococcus ecotype endemic to ODZs.     

Impact of MtrA on phosphate metabolism genes and the response to altered phosphate conditions in Streptomyces

Phosphate metabolism is known to be regulated by the PhoPR regulatory system in Streptomyces and some other bacteria. In this study, we report that MtrA also regulates phosphate metabolism in Streptomyces. Our data showed that, in Streptomyces coelicolor, MtrA regulates not only phosphate metabolism genes such as phoA but also phoP under different phosphate conditions, including growth on rich complex media without added inorganic phosphate and on defined media with low or high concentrations of inorganic phosphate. Cross-regulation was also observed among mtrA, phoP and glnR under these conditions. We demonstrated both in vitro and in vivo binding of MtrA to the promoter regions of genes associated with phosphate metabolism and to the intergenic region between phoR and phoU, indicating that these phosphate metabolism genes are targets of MtrA. We further showed that MtrA in S. lividans and S. venezuelae has detectable regulatory effects on expression of phosphate metabolism genes. Additionally, the MtrA homologue from Corynebacterium glutamicum bound predicted MtrA sites of multiple phosphate metabolism genes, implying its potential for regulating phosphate metabolism in this species. Overall, our findings support MtrA as a major regulator for phosphate metabolism in Streptomyces and also potentially in other actinobacteria.     

Promotion, termination, and anti-termination in the rpsU-dnaG-rpoD macromolecular synthesis operon of E. coli K-12

Summary The regulatory regions for the rpsU-dnaG-rpoD macromolecular synthesis operon have been fused to a structural gene whose product is readily assayed (the Cm^r structural gene coding for chloramphenicol acetyl transferase, CAT). The promoters (P₁, P₂, P₃, P_a, P_b, P_hs) for the macromolecular synthesis operon have different strengths as shown by their relative abilities to drive expression of the CAT gene. Promoter occlusion by P₁ can be demonstrated within this operon. Regions 5kb upstream have a profound effect on operon gene expression. There is a thermoinducible promoter located within the dnaG structural gene. One of the macromolecular synthesis operon promoters is under lexA control. Although the operon structure allows coordinate expression of rpsU, dnaG and rpoD these additional features suggest that expression of individual genes can be independently regulated in response to altered growth conditions.Abbreviations Ap^r ampicillin resistance - CAT chloramphenicol acetyl transferase - Cm^r chloramphenicol resistance - kb kilobase pair - orf open reading frame - P promoter - T terminator - Tc^r tetracycline resistance     

The effect of temperature and pH gradients on Lactobacillus rhamnosus gene expression of stress-related genes

In this study, Lactobacillus rhamnosus, a renowned probiotic, was cultivated in fluctuating environment. Base gradients caused by a pH control in an industrial process and temperature gradients caused by uneven heating were simulated with a scale-down method. A pH gradient was created in a plug flow reactor (PFR). Expression of pH stress-related genes (atpA, aldB, cfa, groEL, hrcA and pstS) were studied as a relative gene expression study using ldhD as a reference gene. Expression measurements were carried out with the TRAC method. The responses of groEL, hrcA and atpA genes to temperature and pH changes were observed. The expression of phosphate uptake system-related pstS gene was induced almost linearly in the chemostat cultivation experiments when the base gradient in the PFR was increased. Correlations between the results from gene expression studies and freeze stability or acid stress survival were studied. However, by measuring the expression of these genes, we were not able to predict eventual freeze stability or survival from the acid stress test.