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L.2-Amino-4-methoxy-trans-3-butenoic acid (AMB) is a potent antibiotic and toxin produced by Pseudomonas aeruginosa. Using a novel biochemical assay combined with site-directed mutagenesis in strain PAO1, we have identified a five-gene cluster specifying AMB biosynthesis, probably involving a thiotemplate mechanism. Overexpression of this cluster in strain PA7, a natural AMB-negative isolate, led to AMB overproduction. doi: 10.1128/JB.00492-10

Restricted bioavailability of copper in certain environments can interfere with cellular respiration because copper is an essential cofactor of most terminal oxidases. The global response of the metabolically versatile bacterium and opportunistic pathogen Pseudomonas aeruginosa to copper limitation was assessed under aerobic conditions. Expression of cioAB (encoding an alternative, copper-independent, cyanide-resistant ubiquinol oxidase) was upregulated, whereas numerous iron uptake functions (including the siderophores pyoverdine and pyochelin) were expressed at reduced levels, presumably reflecting a lower demand for iron by respiratory enzymes. Wild-type P. aeruginosa was able to grow aerobically in a defined glucose medium depleted of copper, whereas a cioAB mutant did not grow. Thus, P. aeruginosa relies on the CioAB enzyme to cope with severe copper deprivation. A quadruple cyo cco1 cco2 cox mutant, which was deleted for all known heme-copper terminal oxidases of P. aeruginosa, grew aerobically, albeit more slowly than did the wild type, indicating that the CioAB enzyme is capable of energy conservation. However, the expression of a cioA'-'lacZ fusion was less dependent on the copper status in the quadruple mutant than in the wild type, suggesting that copper availability might affect cioAB expression indirectly, via the function of the heme-copper oxidases.

The signal extracts prepared from culture supernatants of Pseudomonas fluorescens CHA0 and Pseudomonas aeruginosa PAO have stimulated GacA-dependent expression of small RNAs and also of antibiotic compounds in both hosts. Pseudomonas corrugata LMG2172 and Pseudomonas fluorescens SBW25 have produced signal molecules stimulating GacA-controlled antibiotic synthesis in strain CHA0, thus presenting a novel, N-acyl-homoserine lactone-independent type of interspecies communication.

In Pseudomonas aeruginosa, the GacS/GacA two-component system positively controls the quorum-sensing machinery and the expression of extraccelular products via two small regulatory RNAs, RsmY and RsmZ. An rsmY rsmZ double mutant and a gacA mutant were similarly impaired in the synthesis of the quorum-sensing signal N-butanoyl-homoserine lactone, the disulfide bond-forming enzyme DsbA, and the exoproducts hydrogen cyanide, pyocyanin, elastase, chitinase (ChiC), and chitin-binding protein (CbpD). Both mutants showed increased swarming ability, azurin release, and early biofilm development.

In many Gram-negative bacteria, the GacS/GacA two-component system positively controls the expression of extracellular products or storage compounds. In the plant-beneficial rhizosphere bacterium Pseudomonas fluorescens CHA0, the GacS/GacA system is essential for the production of antibiotic compounds and hence for biological control of root-pathogenic fungi. The small (119-nt) RNA RsmX discovered in this study, together with RsmY and RsmZ, forms a triad of GacA-dependent small RNAs, which sequester the RNA-binding proteins RsmA and RsmE and thereby antagonize translational repression exerted by these proteins in strain CHA0. This small RNA triad was found to be both necessary and sufficient for posttranscriptional derepression of biocontrol factors and for protection of cucumber from Pythium ultimum. The same three small RNAs also positively regulated swarming motility and the synthesis of a quorum-sensing signal, which is unrelated to N-acylhomoserine lactones, and which autoinduces the Gac/Rsm cascade. Expression of RsmX and RsmY increased in parallel throughout cell growth, whereas RsmZ was produced during the late growth phase. This differential expression is assumed to facilitate fine tuning of GacS/A-controlled cell population density-dependent regulation in P. fluorescens. GacA | posttranscriptional control

In Pseudomonas aeruginosa, N-acylhomoserine lactone signals regulate the expression of several hundreds of genes, via the transcriptional regulator LasR and, in part, also via the subordinate regulator RhlR. This regulatory network termed quorum sensing contributes to the virulence of P. aeruginosa as a pathogen. The fact that two supposed PAO1 wild-type strains from strain collections were found to be defective for LasR function because of independent point mutations in the lasR gene led to the hypothesis that loss of quorum sensing might confer a selective advantage on P. aeruginosa under certain environmental conditions. A convenient plate assay for LasR function was devised, based on the observation that lasR mutants did not grow on adenosine as the sole carbon source because a key degradative enzyme, nucleoside hydrolase (Nuh), is positively controlled by LasR. The wild-type PAO1 and lasR mutants showed similar growth rates when incubated in nutrient yeast broth at pH 6.8 and 37[degrees]C with good aeration. However, after termination of growth during 30 to 54 h of incubation, when the pH rose to [greater than or equal to] 9, the lasR mutants were significantly more resistant to cell lysis and death than was the wild type. As a consequence, the lasR mutant-to-wild-type ratio increased about 10-fold in mixed cultures incubated for 54 h. In a PAO1 culture, five consecutive cycles of 48 h of incubation sufficed to enrich for about 10% of spontaneous mutants with a [Nuh.sup.-] phenotype, and five of these mutants, which were functionally complemented by [lasR.sup.+], had mutations in lasR. The observation that, in buffered nutrient yeast broth, the wild type and lasR mutants exhibited similar low tendencies to undergo edi lysis and death suggests that alkaline stress may be a critical factor providing a selective survival advantage to lasR mutants.

In the plant-beneficial soil bacterium Pseudomonas fluorescens CHA0, the production of biocontrol factors (antifungal secondary metabolites and exoenzymes) is controlled at a posttranscriptional level by the GacS/ GacA signal transduction pathway involving RNA-binding protein RsmA as a key regulatory, element. This protein is assumed to bind to the ribosome-binding site of target mRNAs and to block their translation. RsmA-mediated repression is relieved at the end of exponential growth by two GacS/GacA-controlled regulatory RNAs RsmY and RsmZ, which bind and sequester the RsmA protein. A gene (rsmE) encoding a 64-aminoacid RsmA homolog was identified and characterized in strain CHA0. Overexpression of rsmE strongly reduced the expression of target genes (hcnA, for a hydrogen cyanide synthase subunit; aprA, for the main exoprotease; and phlA, for a component of 2,4-diacetylphloroglucinol biosynthesis). Single null mutations in either rsmA or rsmE resulted in a slight increase in the expression of hcnA, aprA, and phlA. By contrast, an rsmA rsmE double mutation led to strongly increased and advanced expression of these target genes and completely suppressed a gacS mutation. Both the RsmE and RsmA levels increased with increasing cell population densities in strain CHA0; however, the amount of RsmA showed less variability during growth. Expression of rsmE was controlled positively by GacA and negatively by RsmA and RsmE. Mobility shift assays demonstrated specific binding of RsmE to RsmY and RsmZ RNAs. The transcription and stability of both regulatory. RNAs were strongly reduced in the rsmA rsmE double mutant. In conclusion, RsmA and RsmE together account for maximal repression in the GacS/GacA cascade of strain CHA0.

In Pseudomonas aeruginosa, the antibiotic dihydroaeruginoate (Dha) and the siderophore pyochelin are produced from salicylate and cysteine by a thiotemplate mechanism involving the peptide synthetases PchE and PchF. A thioesterase encoded by the pchC gene was found to be necessary for maximal production of both Dha and pyochelin, but it was not required for Dha release from PchE and could not replace the thioesterase function specified by the C-terminal domain of PchF. In vitro, 2-aminobutyrate, a cysteine analog, was adenylated by purified PchE and PchF proteins. In vivo, this analog strongly interfered with Dha and pyochelin formation in a pchC deletion mutant but affected production of these metabolites only slightly in the wild type. Exogenously supplied cysteine overcame the negative effect of a pchC mutation to a large extent, whereas addition of salicylate did not. These data are in agreement with a role for PchC as an editing enzyme that removes wrongly charged molecules from the peptidyl carrier protein domains of PchE and PchF.

In Pseudomonas aeruginosa, the small RNA-binding, regulatory protein RsmA is a negative control element in the formation of several extracellular products (e.g., pyocyanin, hydrogen cyanide, PA-IL lectin) as well as in the production of N-acylhomoserine lactone quorum-sensing signal molecules. RsmA was found to control positively the ability to swarm and to produce extracellular rhamnolipids and lipase, i.e., functions contributing to niche colonization by P. aeruginosa. An rsmA null mutant was entirely devoid of swarming but produced detectable amounts of rhamnolipids, suggesting that factors in addition to rhamnolipids influence the swarming ability of P. aeruginosa. A small regulatory RNA, rsmZ, which antagonized the effects of RsmA, was identified in P. aeruginosa. Expression of the rsmZ gene was dependent on both the global regulator GacA and RsmA, increased with cell density, and was subject to negative autoregulation. Overexpression of rsmZ and a null mutation in rsmA resulted in quantitatively similar, negative or positive effects on target genes, in agreement with a model that postulates titration of RsmA protein by RsmZ RNA.

Fusaric acid can suppress the gene for 2,4-diacetylphloroglucinol (DAPG) in Pseudomonas fluorescens CHA0. Fusaric acid is produced by Fusarium oxysporum. DAPD is an antimicrobial substance produced by fluorescent pseudomonads that is effective against bacteria, fungi, and helminths.

Research has been conducted on diffusible signaling molecules, autoinducers of the N-acylhomoserine lactone type which control virulence in the human pathogen Pseudomonas aeruginosa. Results indicate that N-acylhomoserine lactone degradation gene introduced into P. aeruginosa can block cell-cell communication but cannot interfere with surface colonization.

Research has been conducted on the rifampin-resistant Pseudomonas fluorescens CHA0iRif. The comparison of this bacterium and its mutants for persistence in different nonsterile soils has been carried out and the results indicate that the mutant functions are necessary for maintaining cell culturability in soil.

Results demonstrate that GacA protein upregulates the expression of regulatory RNA, RsmZ, in Pseudomonas fluorescens CHA0 in response to a bacterial signal. This leads RsmZ to alleviate the respression activity of RsmA on target mRNAs expression.

The sigma factor AlgU of Pseudomonas fluorescens CHA0 appears to protect the bacterium from osmotic and desiccation stress but not from oxidative or heat stress. This bacterium is used as a biocontrol agent of soil fungi.

Research has been conducted on the posttranscriptional control that contributes to the secondary metabolism and virulence determinants regulation. The isolation of the Pseudomonas aeruginosa gene encoding the global translational regulatory protein, the secondary metabolite regulator, has been carried out and examined and the results are presented.

Research demonstrates the involvement of three biosynthetic genes, pchG, pchH, and pchI in pyochelin biosynthesis and translocation in Pseudomonas aeruginosa. Data show that pchG, with its reductase function and in association with the other genes, catalyzes pyochelin formation in vitro from salicylate, L-cysteine, ATP, NADPH, and S-adenosylmethionine.

Research has been conducted on the hcnABC genes that encode hydrogen cyanide synthase in Pseudomonas aeruginosa. The analysis of the P. aeruginosa hcn promoter region has been carried out and the results are discussed.

Results demonstrate that biosynthetic autoinduction of 2,4-diacetylphloroglucinol is inhibited by certain bacterial and fungal metabolites. Data indicate that this control mechanism may balance homeostatically the amount of extracellular metabolites produced in Pseudomonas fluorescens.

An oxygen-sensitive biosensor was developed based on a transcriptional fusion of a modified anaerobic regulation of arginine deiminase and nitrate reductase pathways-dependent arcDABC promoter of Pseudomonas aeruginosa PAO1 to the lacZ structural gene of Escherichia coli. The P fluorescens biosensor CHA900 was then used to analyze oxygen concentrations in a plant-soil microcosm. Results indicate the utility of the biosensor for detecting low oxygen concentrations in the rhizosphere and other soil habitats.

Tandemly repeated insertion sequence IS21, found on a suicide plasmid, was found to promote replicon fusion with bacteriophage lambda in vitro in the presence of adenosine triphosphate. This reaction was catalyzed in a cell extract containing the 45-kDa IstA protein (cointegrase) and the 30-kDa IstB helper protein of IS21 after both proteins had been overexpressed in Escherichia coli. Without IstB, replicon fusion was found to be inefficient and did not produce the 4-bp target duplications common to IS21.

The mechanism by which arginine induces the arginine demnase pathway in Pseudomonas aeruginosa was investigated. Arginine was found to enhance the expression of the arcD operon by two- to threefold. Results also revealed that the arginine effect is essentially mediated by the transcriptional regulator ArgR, which binds to a conserved sequence at the upstream of the ANR binding site in the arcD promoter.

A study was conducted to characterize the hcnABC gene cluster encoding hydrogen cyanide synthase and anaerobic regulation in Pseudomonas fluorescens CHA0. Triparental matings were carried out P. fluorescence recipients with Escherichia coli supporting a mobilized plasmid while the CTAB method was used to carry out small-scale preparations of plasmid DNA from E. coli. Results indicated that the anaerobic regulator ANR was needed for cyanide synthesis in the aerobic strain CHAO.

The pchBA genes of Pseudomonas aeruginosa were sequenced and analyzed to determine the specific salicylate biosynthetic genes that mediate the synthesis of pyochelin from salicylate. Sequencing of the Pseudomonas aeruginosa pchBA operon indicated the presence of two additional genes designated as pchD and pchC. The contiguous genes from the pchDCBA operon also mediated the initial steps of pyochelin synthesis from salicylate. Furthermore, the pchDCBA operon and the adjacent pchR gene were divergently transcribed from a single iron-repressible promoter region.