Pseudomonas fluorescens 2-79 produces the broad-spectrum antibiotic phenazine-1-carboxylic acid (PCA), which is active against a variety of fungal root pathogens. In this study, seven genes designated phzABCDEFG that are sufficient for synthesis of PCA were localized within a 6.8-kbBglII-XbaI fragment from the phenazine biosynthesis locus of strain 2-79. Polypeptides corresponding to allphz genes were identified by analysis of recombinant plasmids in a T7 promoter/polymerase expression system. Products of thephzC, phzD, and phzE genes have similarities to enzymes of shikimic acid and chorismic acid metabolism and, together with PhzF, are absolutely necessary for PCA production. PhzG is similar to pyridoxamine-5′-phosphate oxidases and probably is a source of cofactor for the PCA-synthesizing enzyme(s). Products of thephzA and phzB genes are highly homologous to each other and may be involved in stabilization of a putative PCA-synthesizing multienzyme complex. Two new genes, phzXand phzY, that are homologous to phzA andphzB, respectively, were cloned and sequenced from P. aureofaciens 30-84, which produces PCA, 2-hydroxyphenazine-1-carboxylic acid, and 2-hydroxyphenazine. Based on functional analysis of the phz genes from strains 2-79 and 30-84, we postulate that different species of fluorescent pseudomonads have similar genetic systems that confer the ability to synthesize PCA.

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Bacteriophage capsids constitute icosahedral shells of exceptional stability that protect the viral genome. Many capsids display on their surface decoration proteins whose structure and function remain largely unknown. The decoration protein pb10 of phage T5 binds at the centre of the 120 hexamers formed by the major capsid protein. Here we determined the 3D structure of pb10 and investigated its capsid-binding properties using NMR, SAXS, cryoEM and SPR. Pb10 consists of an α-helical capsid-binding domain and an Ig-like domain exposed to the solvent. It binds to the T5 capsid with a remarkably high affinity and its binding kinetics is characterized by a very slow dissociation rate. We propose that the conformational exchange events observed in the capsid-binding domain enable rearrangements upon binding that contribute to the quasi-irreversibility of the pb10-capsid interaction. Moreover we show that pb10 binding is a highly cooperative process, which favours immediate rebinding of newly dissociated pb10 to the 120 hexamers of the capsid protein. In extreme conditions, pb10 protects the phage from releasing its genome. We conclude that pb10 may function to reinforce the capsid thus favouring phage survival in harsh environments.

The protein expression profiles of Rhizobium leguminosarum strains in response to specific genetic perturbations in exopolysaccharide (EPS) biosynthesis genes were examined using two-dimensional gel electrophoresis. Lesions in either pssA, pssD, or pssE of R. leguminosarum bv. viciae VF39 or in pssA of R. leguminosarum bv. trifolii ANU794 not only abolished the capacity of these strains to synthesize EPS but also had a pleiotropic effect on protein synthesis levels. A minimum of 22 protein differences were observed for the two pssA mutant strains. The differences identified in the pssD and pssE mutants of strain VF39 were a distinct subset of the same protein synthesis changes that occurred in the pssA mutant. The pssD and pssE mutant strains shared identical alterations in the proteins synthesized, suggesting that they share a common function in the biosynthesis of EPS. In contrast, a pssC mutant that produces 38% of the EPS level of the parental strain showed no differences in its protein synthesis patterns, suggesting that the absence of EPS itself was contributing to the changes in protein synthesis and that there may be a complex interconnection of the EPS biosynthetic pathway with other metabolic pathways. Genetic complementation of pssA can restore wild-type protein synthesis levels, indicating that many of the observed differences in protein synthesis are also a specific response to a dysfunctional PssA. The relevance of these proteins, which are grouped as members of the pssA mutant stimulon, remains unclear, as the majority lacked a homologue in the current sequence databases and therefore possibly represent a novel functional network(s). These findings have illustrated the potential of proteomics to reveal unexpected higher-order processes of protein function and regulation that arise from mutation. In addition, it is evident that enzymatic pathways and regulatory networks are more interconnected and more sensitive to structural changes in the cell than is often appreciated. In these cases, linking the observed phenotype directly to the mutated gene can be misleading, as the phenotype could be attributable to downstream effects of the mutation.Soil bacteria belonging to the genera Rhizobium, Sinorhizobium, Bradyrhizobium, Mesorhizobium, and Azorhizobium (collectively termed rhizobia) are able to infect the roots of leguminous plants in a host-specific way and stimulate the formation of nodules. Inside these nodules, rhizobia differentiate into bacteroids that reduce atmospheric nitrogen to ammonia, which is used by the plant. One major characteristic of many rhizobia is the production of large amounts of acidic exopolysaccharide (EPS) molecules which serve a variety of roles in free-living rhizobia and in the establishment of symbiosis.EPS forms a biofilm layer on the cell surface which is thought to contribute to the following processes: cellular protection against environmental stresses, attachment to surfaces, nutrient gathering, and the preferential absorption of plant secreted flavonoids along th...

The Pseudomonas fluorescens 23F phosphonoacetate hydrolase gene (phnA) encodes a novel carbon-phosphorus bond cleavage enzyme whose expression is independent of the phosphate status of the cell. Analysis of the regions adjacent to the phosphonoacetate hydrolase structural gene (phnA) indicated the presence of five open reading frames (ORFs). These include one (phnR) whose putative product shows high levels of homology to the LysR family of positive transcriptional regulators. Its presence was shown to be necessary for induction of the hydrolase activity. 2-Phosphonopropionate was found to be an inducer (and poor substrate) for phosphonoacetate hydrolase. Unlike phosphonoacetate, which is also an inducer of phosphonoacetate hydrolase, entry of 2-phosphonopropionate into cells appeared to be dependent on the presence of a gene (phnB) that lies immediately downstream of phnA and whose putative product shows homology to the glycerol-3-phosphate transporter. RNA analysis revealed transcripts for the phnAB and phnR operons, which are transcribed divergently; the resulting mRNAs overlapped by 29 nucleotide bases at their 5 ends. Transcripts of phnAB were detected only in cells grown in the presence of phosphonoacetate, whereas transcripts of phnR were observed in cells grown under both induced and uninduced conditions. The expression of three additional genes found in the phnA region did not appear necessary for the degradation of phosphonoacetate and 2-phosphonopropionate by either Pseudomonas putida or Escherichia coli cells.Organophosphonates are a group of compounds of biogenic and xenobiotic origins that are characterized by possession of a direct carbon-phosphorus bond. Two different routes of C-P bond cleavage, each inducible only under conditions of phosphorus limitation, have been demonstrated when such molecules serve as the sole phosphorus source for microbial growth. "C-P lyase" is the trivial name given to an enzyme complex which catalyzes the cleavage of the C-P bond of both substituted and unsubstituted phosphonates by a mechanism which may involve redox or radical chemistry (27). By contrast, phosphonoacetaldehyde hydrolase ("phosphonatase" [EC 3.11.1.1]) is active in vitro and hydrolytically cleaves only the substituted C-P bond of phosphonoacetaldehyde to yield acetaldehyde and P i (10). Since bacterial cleavage of the C-P bond by both C-P lyase(s) and phosphonatase is under control of the pho regulon (10) and hence occurs only under conditions of P i limitation, organophosphonates generally fail to serve as sources of carbon for microbial growth because the excess of P i released during the catabolism of their carbon skeletons serves to repress and/or inhibit their further mineralization. Genetic analysis of C-P bond cleavage by the C-P lyase and phosphonatase pathways has been reported (10,18,26), and the phosphonatase gene of Pseudomonas aeruginosa has been characterized (5).We have recently reported the purification (15) and molecular cloning (14) of phosphonoacetate (PA) hydrolase (EC 3.11.1.2), ...