Evidence for two phosphonate degradative pathways in Enterobacter aerogenes.

We screened mini-Mu plasmid libraries from Enterobacter aerogenes IFO 12010 for plasmids that complement Escherichia coli phn mutants that cannot use phosphonates (Pn) as the sole source of phosphorus (P). We isolated two kinds of plasmids that, unexpectedly, encode genes for different metabolic pathways. One kind complements E. coli mutants with both Pn transport and Pn catalysis genes deleted; these plasmids allow degradation of the 2-carbon-substituted Pn alpha-aminoethylphosphonate but not of unsubstituted alkyl Pn. This substrate specificity is characteristic of a phosphonatase pathway, which is absent in E. coli. The other kind complements E. coli mutants with Pn catalysis genes deleted but not those with both transport and catalysis genes deleted; these plasmids allow degradation of both substituted and unsubstituted Pn. Such a broad substrate specificity is characteristic of a carbon-phosphorus (C-P) lyase pathway, which is common in gram-negative bacteria, including E. coli. Further proof that the two kinds of plasmids encode genes for different pathways was demonstrated by the lack of DNA homology between the plasmids. In particular, the phosphonatase clone from E. aerogenes failed to hybridize to the E. coli phnCDEFGHIJKLMNOP gene cluster for Pn uptake and degradation, while the E. aerogenes C-P lyase clone hybridized strongly to the E. coli phnGHIJKLM genes encoding C-P lyase but not to the E. coli phnCDE genes encoding Pn transport. Specific hybridization by the E. aerogenes C-P lyase plasmid to the E. coli phnF, phnN, phnO, and phnP genes was not determined. Furthermore, we showed that one or more genes encoding the apparent E. aerogenes phosphonatase pathway, like the E. coli phnC-to-phnP gene cluster, is under phosphate regulon control in E. coli. This highlights the importance of Pn in bacterial P assimilation in nature.     

Mapping and molecular cloning of the phn (psiD) locus for phosphonate utilization in Escherichia coli.

The Escherichia coli phn (psiD) locus encodes genes for phosphonate (Pn) utilization, for phn (psiD) mutations abolish the ability to use as a sole P source a Pn with a substituted C-2 or unsubstituted hydrocarbon group such as 2-aminoethylphosphonate (AEPn) or methylphosphonate (MPn), respectively. Even though the E. coli K-12 phosphate starvation-inducible (psi) phn (psiD) gene(s) shows normal phosphate (Pi) control, Pn utilization is cryptic in E. coli K-12, as well as in several members of the E. coli reference (ECOR) collection which are closely related to K-12. For these bacteria, an activating mutation near the phn (psiD) gene is necessary for growth on a Pn as the sole P source. Most E. coli strains, including E. coli B, are naturally Phn+; a few E. coli strains are Phn- and are deleted for phn DNA sequences. The Phn+ phn(EcoB) DNA was molecularly cloned by using the mini-Mu in vivo cloning procedure and complementation of an E. coli K-12 delta phn mutant. The phn(EcoB) DNA hybridized to overlapping lambda clones in the E. coli K-12 gene library (Y. Kohara, K. Akiyama, and K. Isono, Cell 50:495-508, 1987) which contain the 93-min region, thus showing that the phn (psiD) locus was itself cloned and verifying our genetic data on its map location. The cryptic phn(EcoK) DNA has an additional 100 base pairs that is absent in the naturally Phn+ phn(EcoB) sequence. However, no gross structural change was detected in independent Phn+ phn(EcoK) mutants that have activating mutations near the phn locus.     

A Col E1 hybrid plasmid containing Escherichia coli genes complementing d-xylose negative mutants of Escherichia coli and Salmonella typhimurium

The Clarke and Carbon bank of Col El - Escherichia coli DNa hybrid plasmids was screened for complementation of d-xylose negative mutants of E. coli. Of several obtained, the smallest, pRM10, was chosen for detailed study. Its size was 16 kilobases (kb) and that of the insert was 9.7 kg. By transformation or F'-mediated conjugation this plasmid complemented mutants of E. coli defective in either D-xylose isomerase or D-xylulose kinase activity, or both. The activity of D-xylulose kinase in E. coli transformants which bear an intact chromosomal gene for this enzyme was greater than that for the host, due to a gene dosage effect. The plasmid also complemented D-xylose negative mutants of Salmonella typhimurium by F'-mediated conjugation between E. coli and S. typhimurium. Salmonella typhimurium mutants complemented were those for D-xylose isomerase and for D-xylulose kinase in addition to pleiotropic D-xylose mutants which were defective in a regulatory gene of the D-xylose operon. In addition, the plasmid complemented the glyS mutation in E. coli and S. typhimurium. The glyS mutant of E. coli was temperature sensitive, indicating that the plasmid carried the structural gene for glycine synthetase. The glyS mutation in E. coli maps at 79 min, as do the xyl genes. The behaviour of the plasmid is consistent with the existence of a d-xylose operon in E. coli. The data also suggest that the plasmid carries three of the genes of this operon, specifically those for D-xylose isomerase, D-xylulose kinase, and a regulatory gene.     

The role of three-tandem Pho Boxes in the control of the C-P lyase operon in a thermophilic cyanobacterium

Cyanobacteria have an inherited advantage in phosphonate phytoremediation. However, studies on phosphonate metabolism in cyanobacteria are rare and mostly focus on physiology and ecology. Here, C-P lyase gene cluster regulation in an undomesticated thermophilic Synechococcus OS-B′ was examined in Synechocystis sp. PCC6803, a unicellular cyanobacterial model. Phylogenetic and cluster synteny analysis of C-P lyase genes revealed a closer relationship between Syn OS-B′ and Thermus thermophilus, than with other cyanobacteria. Pho boxes were identified in the 5′-end-flanking region of the C-P lyase gene cluster, through which the downstream gene expression was regulated in a phosphate concentration-dependent manner. Unexpectedly, the phosphate concentration that thoroughly inhibited Pho boxes was almost two orders of magnitude higher than that of any natural or anthropogenic wastewater reported so far. The Pho boxes mediated regulation was achieved through the Pho regulon two-component system, and the absence of either SphS or SphR ablated the cell's ability to sense ambient phosphate changes. The three tandems of Pho boxes maintained inequivalent roles, of which the third tandem was not essential; however, it played a role in adjusting Pho boxes response in both positive and negative manner under phosphorus limitation.     

Involvement of the phosphate regulon and the psiD locus in carbon-phosphorus lyase activity of Escherichia coli K-12.

Escherichia coli K-12 can readily mutate to use methylphosphonic acid as the sole phosphorus source by a direct carbon-to-phosphorus (C-P) bond cleavage activity that releases methane and Pi. The in vivo C-P lyase activity is both physiologically and genetically regulated as a member of the phosphate regulon. Since psiD::lacZ(Mu d1) mutants cannot metabolize methylphosphonic acid, psiD may be the structural gene(s) for C-P lyase.     

A Salmonella typhimurium genetic locus which confers copper tolerance on copper-sensitive mutants of Escherichia coli.

Three distinct clones from a Salmonella typhimurium genomic library were identified which suppressed the copper-sensitive (Cu(s)) phenotype of cutF mutants of Escherichia coli. One of these clones, pCUTFS2, also increased the copper tolerance of cutA, -C, and -E mutants, as well as that of a lipoprotein diacylglyceryl transferase (lgt) mutant of E. coli. Characterization of pCUTFS2 revealed that the genes responsible for suppression of copper sensitivity (scs) reside on a 4.36-kb DNA fragment located near 25.4 min on the S. typhimurium genome. Sequence analysis of this fragment revealed four open reading frames (ORF120, ORF627, ORF207, and ORF168) that were organized into two operons. One operon consisted of a single gene, scsA (ORF120), whereas the other operon contained the genes scsB (ORF627), scsC (ORF207), and scsD (ORF168). Comparison of the deduced amino acid sequences of the predicted gene products showed that ScsB, ScsC, and ScsD have significant homology to thiol-disulfide interchange proteins (CutA2, DipZ, CycZ, and DsbD) from E. coli and Haemophilus influenzae, to an outer membrane protein (Com1) from Coxiella burnetii, and to thioredoxin and thioredoxin-like proteins, respectively. The two operons were subcloned on compatible plasmids, and complementation analyses indicated that all four proteins are required for the increased copper tolerance of E. coli mutants. In addition, the scs locus also restored lipoprotein modification in lgt mutants of E. coli. Sequence analyses of the S. typhimurium scs genes and adjacent DNAs revealed that the scs locus is flanked by genes with high homology to the cbpA (predicted curved DNA-binding protein) and agp (acid glucose phosphatase) genes of E. coli located at 22.90 min (1,062.07 kb) and 22.95 min (1,064.8 kb) of the E. coli chromosome, respectively. However, examination of the E. coli chromosome revealed that these genes are absent at this locus and no evidence has thus been obtained for the occurrence of the scs locus elsewhere on the genome.     

Molecular genetics of carbon-phosphorus bond cleavage in bacteria

Phosphonates (Pn) are a large class of organophosphorus molecules that have direct carbon-phosphorus (C - P) bonds in place of the carbon-oxygen-phosphorus ester bond. In bacteria two pathways exist for Pn breakdown for use as a P source: the phosphonatase and C - P lyase pathways. These pathways differ both in regard to their substrate specificity and their cleavage mechanism. The phosphonatase pathway acts on the natural Pn -aminoethylphosphonate(AEPn). In a two-step process it leads to cleavage of the C - P bond by a hydrolysis reaction requiring an adjacent carbonyl group. In contrast the C - P lyase pathway has a broad substrate specificity. It leads to cleavage of substituted Pn (such as AEPn) as well as unsubstituted Pn by a mechanism involving redox or radical chemistry. Due to its broad substrate specificity, the C - P lyase pathway is generally thought to be responsible for the breakdown of Pn herbicides (such as glyphosate) by bacteria. As a way to gain a more in-depth understanding of these Pn degradative pathways, their respective genes have been isolated and characterized. In the absence of a biochemical assay for the C - P lyase pathway such molecular approaches have been especially valuable. The roles of individual genes have been inferred from DNA sequence analysis and mutational effects. Genes for the C - P lyase pathway exist in a fourteen-gene operon that appears to encode both a binding protein-dependent Pn transporter and a C - P lyase. Genes for the phosphonatase pathway also exist in a gene cluster containing Pn uptake and degradative genes. A combination of biochemistry, molecular biology, and molecular genetics approaches has provided more detailed understanding of the mechanisms of C - P bond cleavage. Such basic information may provide a new handle for improvement of Pn degradation capabilities in bacteria, or in other cells in which the respective genes may be introduced and expressed.Abbreviations AEPn -aminoethylphosphonate - C carbon - kbp kilobase pair - kDa kilodalton - MPn methylphosphonate - P phosphorus - P _i inorganic phosphate - Pn phosphonate - psi phosphate starvation inducible     

Molecular genetic studies of a 10.9-kb operon in Escherichia coli for phosphonate uptake and biodegradation

Bacteria that use phosphonates as a phosphorus source must be able to break the stable carbon-phosphorus bond. In Escherichia coli phosphonates are broken down by a C-P lyase that has a broad substrate specificity. Evidence for a lyase is based on in vivo studies of product formation because it has been proven difficult to detect the activity in vitro. By using molecular genetic techniques, we have studied the genes for phosphonate uptake and degradation in E. coli, which are organized in an operon of 14 genes, named phnC to phnP. As expected for genes involved in P acquisition, the phnC-phnP operon is a member of the PHO regulon and is induced many hundred-fold during phosphate limitation. Three gene products (PhnC, PhnD and PhnE) comprise a binding protein-dependent phosphonate transporter, which also transports phosphate, phosphite, and certain phosphate esters such as phosphoserine; two gene products (PhnF and PhnO) may have a role in gene regulation; and nine gene products (PhnG, PhnH, PhnI, PhnJ, PhnK, PhnL, PhnM, PhnN, and PhnP) probably comprise a membrane-associated C-P lyase enzyme complex. Although E. coli can degrade many different phosphonates, the ability to use certain phosphonates appears to be limited by the specificity of the PhnCDE transporter and not by the specificity of the C-P lyase.     

Regulation of components of the Pseudomonas aeruginosa phosphate-starvation-inducible regulon in Escherichia coli

Plasmids pPBP and pRS-XP containing the cloned genes for the Pseudomonas aeruginosa phosphate-starvation-inducible periplasmic phosphate-binding protein and outer membrane porin P (oprP), respectively, were introduced into various Escherichia coli Pho-regulon regulatory mutants. Using Western immunoblots and specific antisera, the production of both gene products was observed to be under the control of regulatory elements of the E. coli Pho regulon. Sequencing of the region upstream of the translational start site of the oprP gene revealed a 'Pho box' with strong homology to the E. coli consensus 'Pho box', the putative binding site of the PhoB activator. Since P. aeruginosa and E. coli belong to different families and have quite different GC contents, these data suggest strong evolutionary conservation of regulatory elements of the Pho regulon.     

Identification of the enzymatic reactions encoded by the purG and purI genes of Escherichia coli

The chromosomal locations of the genes purG and purI on the Escherichia coli linkage map are the opposites of those of Salmonella typhimurium. By methods which permit the identification of lesions in any of the five early enzymes of the purine de novo pathway, the gene-enzyme relationships of the purG and purI loci have been reevaluated in these two organisms. The results demonstrate that the relative locations of the genes encoding the two enzymes (phosphoribosylformylglycinamidine synthetase and phosphoribosylaminoimidazole synthetase) are similar in the two organisms. The gene products have been correctly determined in S. typhimurium. The gene products currently listed for the loci in E. coli are incorrect. The E. coli purG locus is equivalent to the S. typhimurium purI locus, and the E. coli purI locus is equivalent to the S. typhimurium purG locus.     

Mapping and characterization of the nad genes in Salmonella typhimurium LT-2.

An ampicillin enrichment technique was used to isolate 39 nicotinic acid-requiring mutants of Salmonella typhimurium LT-2. Using interrupted-mating and transductional mapping procedures, three loci, designated nadA, nadB, and nadC, were identified. These loci mapped at 33, 82, and 6 min, respectively, on the S. typhimurium linkage map. The arrangement of the loci on the Salmonella linkage map corresponded closely to the nadA, nadB, and nadC loci on the Escherichia coli K-12 linkage map, indicating that the de novo pathway to nicotinamide adenine dinucleotide and the genes governing the enzymes involved in this pathway in S. typhimurium are very similar to those in E. coli. Evidence is also presented which indicates that the product of the nadC locus in S. typhimurium LT-2 is the enzyme quinolinic acid phosphoribosyltransferase. All nadC mutants of S. typhimurium secreted between 2 and 8 mumol of quinolinic acid per 100 ml of secretion medium. In addition, none of the nadC mutants isolated were able to grow in 10(-3) M quinolinic acid, whereas all nadA and nadB mutants of S. typhimurium grew well in the presence of quinolinic acid. Transductional crosses between nadB mutants provided evidence suggestive of more than one locus in the nadB region.     

Genetic analysis of the genes involved in synthesis of the lipopolysaccharide core in Escherichia coli K-12: three operons in the rfa locus.

The region of the Escherichia coli K-12 chromosome encoding the enzymes responsible for the synthesis of responsible for the synthesis of the lipopolysaccharide (LPS) core has been cloned in vivo by using a mini-Mu vector. This region, formerly known as the rfa locus, comprises 18 kb of DNA between the markers tdh and rpmBG. Results of in vitro mutagenesis of this region with MudII1734 indicate the presence of at least 17 open reading frames or genes, a number considerably higher than expected on the basis of genetic and biochemical studies. Specific insertions in different genes have been recombined into the chromosome, and the mutations have been phenotypically characterized. Complementation analysis indicates that these genes are arranged in three different operons transcribed in opposite directions. A detailed physical map of this region has been constructed on the basis of complementation analysis, fusion protein data, and phenotypic characterizations. Additionally, the role of some genes in the synthesis of LPS has been defined by complementation analysis with known Salmonella typhimurium LPS mutants. The genetic organization of this locus seems to be identical in E. coli K-12 and S. typhimurium.