Genetic and biochemical analyses of pantothenate biosynthesis in Escherichia coli and Salmonella typhimurium.

Pantothenate (pan) auxotrophs of Escherichia coli K-12 and Salmonella typhimurium LT2 were characterized by enzymatic and genetic analyses. The panB mutants of both organisms and the pan-6 ("panA") mutant of S. typhimurium are deficient in ketopantoate hydroxymethyltransferase, whereas the panC mutants lack pantothenate synthetase. panD mutants of E. coli K-12 were previously shown to be deficient in aspartate 1-decarboxylase. All mutants showed only a single enzyme defect. The finding that the pan-6 mutant was deficient in ketopantoate hydroxymethyltransferase indicates that the genetic lesion is a panB allele. The pan-6 mutant therefore is deficient in the utilization of alpha-ketoisovalerate rather than the synthesis of alpha-ketoisovalerate, as originally proposed. The order of the pan genes of E. coli K-12 was determined by phage P1-mediated three-factor crosses. The clockwise order was found to be aceF panB panD panC tonA on the genetic map of E. coli K-12. The three-factor crosses were greatly facilitated by use of a closely linked Tn10 transposon as the outside marker. We also found that supplementation of E. coli K-12 auxotrophs with a high concentration of pantothenate or beta-alanine increased the intracellular coenzyme A level two- to threefold above the normal level. Supplementation with pantoate or ketopantoate resulted in smaller increases.     

A <Emphasis Type="Italic">sirA</Emphasis>-like gene, <Emphasis Type="Italic">sirA2</Emphasis>, is essential for 3-succinoyl-pyridine metabolism in the newly isolated nicotine-degrading <Emphasis Type="Italic">Pseudomonas</Emphasis> sp. HZN6 strain

A novel nicotine-degrading Pseudomonas sp. strain, HZN6, was isolated from a pesticide-wastewater treatment facility in Hangzhou. The strain could grow on nicotine as its sole source of carbon, nitrogen, and energy. The strain’s main intermediate metabolites were determined to be pseudooxynicotine, 3-succinoyl-pyridine (SP), and 6-hydroxy-3-succinoyl-pyridine (HSP). A Tn5 transposon mutant was generated in which the degradation pathway was blocked at the SP. A 4,583-bp DNA fragment flanking the transposon insertion site was obtained through self-formed adaptor PCR and analyzed. The mutant gene orfC displays 89% deduced amino acid sequence identity with the sirA-like gene (sirA2, a sulfurtransferase homologue gene) of Pseudomonas stutzeri A1501. The orfC-disrupted strain lost the ability to degrade SP, and the complementation strains with the orfC from the Pseudomonas sp. HZN6 and the sirA2 (PP_1233) from Pseudomonas putida KT2440 recovered the degradation ability. Though the orfC-disrupted strain also lost the xanthine dehydrogenase activity, the effects of tungsten on the degradation of SP and hypoxanthine revealed that the hydroxylation of SP to HSP was not a xanthine dehydrogenase type. These results demonstrated that the orfC gene was essential for the SP metabolism involved in the nicotine metabolic pathway in the Pseudomonas sp. HZN6 strain. This study might advance the understanding of the nicotine metabolic mechanism in Pseudomonas.     

Isolation,transposon mutagenesis,and characterization of the novel nicotine-degrading strain Shinella sp. HZN7

Nicotine is a significant toxic waste generated in tobacco manufacturing. Biological methods for the degradation of nicotine waste are in high demand. In this study, we report the identification and characterization of the novel nicotine-degrading strain Shinella sp. HZN7. This strain can degrade 500 mg/L nicotine completely within 3 h at 30 °C and pH values of 6.5?～?8.0. The biodegradation of nicotine by Shinella sp. HZN7 involves five intermediate metabolites: 6-hydroxy-nicotine (6HN), 6-hydroxy-N-methylmyosmine, 6-hydroxypseudooxynicotine (6HPON), 6-hydroxy-3-succinoyl-pyridine (HSP), and 2,5-dihydroxypyridine, as detected by ultraviolet spectrophotometry, HPLC, and LC-MS. We generated three mutants, N7-W18, N7-X5, and N7-M17, by transposon mutagenesis, in which the nicotine-degrading pathway terminated at 6HN, 6HPON, and HSP, respectively. The production of the five intermediate metabolites and their order in the degradation pathway were confirmed in the three mutants, indicating that strain HZN7 degrades nicotine via a variant of the pyridine and pyrrolidine pathways. The mutant gene from strain N7-X5, orf2, was cloned by self-formed adaptor PCR, but the nucleotide and amino acid sequence showed no similarity to any gene or gene product with defined function. However, orf2 disruption and complementation suggested that the orf2 gene is essential for the conversion of 6HPON to HSP in strain HZN7. This is the first study to provide genetic evidence for this variant nicotine degradation pathway.     

Isolation and Characterization of Mutants of the Plant Growth-Promoting Rhizobacterium Pseudomonas putida GR12-2 That Overproduce Indoleacetic Acid

Following transposon Tn5 mutagenesis of the plant growth-promoting rhizobacterium Pseudomonas putida GR12-2, mutants that were able to grow in the presence of the tryptophan analog 5-fluorotryptophan were selected. Seven of the 50 5-fluorotryptophan-resistant mutants overproduced the phytohormone indoleacetic acid (IAA). Of these seven mutants, the highest level of IAA was observed with strain P. putida GR12-2/aux1, which produced four times the amount of indoleacetic acid synthesized by the wild-type strain. Strain P. putida GR12-2/aux1, in contrast to the wild type, lost the ability to stimulate the elongation of the roots of canola seedlings under gnotobiotic conditions. The growth rate, siderophore production, and 1-aminocyclopropane-1-carboxylate deaminase activity of mutant strain P. putida GR12-2/aux1 were identical to those of the wild-type strain. The role of IAA in the mechanism of plant growth stimulation by P. putida GR12-2 and other plant growth-promoting rhizobacteria is discussed.     

Chromosomal insertion of the entire Escherichia coli lactose operon,into two strains of Pseudomonas,using a modified mini-Tn5 delivery system

A 12-kb PstI fragment including the entire E. coli lactose operon (lacIPOZYA) was inserted in one copy into the chromosome of Pseudomonas putida, Pseudomonas fluorescens and an E. coli strain with lac⁻ phenotype. This was made possible by improvements of an already existing mini-Tn5 transposon delivery system (de Lorenzo et al., 1990; Herrero et al., 1990), which integrates cloned DNA fragments at random sites on the chromosome of the recipient bacteria in single copies. This has resulted in: (a) the making of two useful low copy-number cloning vectors both with extensive multi-cloning regions flanked by NotI sites needed in the mini-Tn5 delivery system; (b) the generation of E. coli nonlysogenic strains expressing the π protein thus being capable of maintaining and delivering R6K-based mini-Tn5 vectors to other E. coli strains; (c) the successful insertion of the E. coli lactose operon into the P. fluorescens chromosome giving P. fluorescens the ability to grow on lactose; (d) evidence from Southern blotting that contradicts the assumption that the mini-Tn5 delivery system always creates one-copy inserts. These improvements allow insertion of large DNA fragments encoding highly expressed proteins into the chromosome of a large variety of Gram-negative bacteria including E. coli.     

Inactivation of Isocitrate Lyase Leads to Increased Production of Medium-Chain-Length Poly(3-Hydroxyalkanoates) in Pseudomonas putida

Medium-chain-length (mcl) poly(3-hydroxyalkanoates) (PHAs) are storage polymers that are produced from various substrates and accumulate in Pseudomonas strains belonging to rRNA homology group I. In experiments aimed at increasing PHA production in Pseudomonas strains, we generated an mcl PHA-overproducing mutant of Pseudomonas putida KT2442 by transposon mutagenesis, in which the aceA gene was knocked out. This mutation inactivated the glyoxylate shunt and reduced the in vitro activity of isocitrate dehydrogenase, a rate-limiting enzyme of the citric acid cycle. The genotype of the mutant was confirmed by DNA sequencing, and the phenotype was confirmed by biochemical experiments. The aceA mutant was not able to grow on acetate as a sole carbon source due to disruption of the glyoxylate bypass and exhibited two- to fivefold lower isocitrate dehydrogenase activity than the wild type. During growth on gluconate, the difference between the mean PHA accumulation in the mutant and the mean PHA accumulation in the wild-type strain was 52%, which resulted in a significant increase in the amount of mcl PHA at the end of the exponential phase in the mutant P. putida KT217. On the basis of a stoichiometric flux analysis we predicted that knockout of the glyoxylate pathway in addition to reduced flux through isocitrate dehydrogenase should lead to increased flux into the fatty acid synthesis pathway. Therefore, enhanced carbon flow towards the fatty acid synthesis pathway increased the amount of mcl PHA that could be accumulated by the mutant.     

Effect of Overexpression of Actinobacillus succinogenes Phosphoenolpyruvate Carboxykinase on Succinate Production in Escherichia coli

Succinate fermentation was investigated in Escherichia coli strains overexpressing Actinobacillus succinogenes phosphoenolpyruvate carboxykinase (PEPCK). In E. coli K-12, PEPCK overexpression had no effect on succinate fermentation. In contrast, in the phosphoenolpyruvate carboxylase mutant E. coli strain K-12 ppc::kan, PEPCK overexpression increased succinate production 6.5-fold.     

Enhancement of the Potential To Utilize Octopine in the Nonfluorescent Pseudomonas sp. Strain 92

The nonfluorescent Pseudomonas sp. strain 92 requires the presence of a supplementary carbon source for growth on octopine, whereas the spontaneous mutant RB100 has acquired the capacity to utilize this opine as the sole carbon and nitrogen source. Insertional mutagenesis of RB100 with transposon Tn5 generated mutants which were unable to grow on octopine and others which grew slowly on this substrate. Both types of mutants yielded revertants that had regained the ability to utilize octopine. Some of the revertants had lost the transposon, whereas in others the transposon was retained but with rearrangements of the insertion site. Genes of octopine catabolism from strain 92 were cloned on a cosmid vector to generate pK3. The clone pK3 conferred the ability to utilize octopine as the sole carbon and nitrogen source on the host Pseudomonas putida KT2440. Although they conferred an equivalent growth phenotype, the mutant genes carried by RB100 and the cloned genes on pK3 differed in their regulation. Utilization of [¹⁴C]octopine was inducible by octopine in RB100 and was constitutive in KT2440(pK3).     

Correlation Between the Expression of an Escherichia coli Cell Surface Protein and the Ability of the Protein to Bind to Lipopolysaccharide

The ompA gene of Escherichia coli codes for a major protein of the outer membrane. When this gene was moved between various unrelated strains (E. coli K-12 and two clinical isolates of E. coli) by transduction, the gene was expressed very poorly. Recombinants carrying “foreign” genes produced no OmpA protein which could be detected on polyacrylamide gels and became resistant to bacteriophage K3, which uses this protein as receptor. The recombinants were sensitive to host-range mutants of K3, indicating a very low level of OmpA protein was produced. When an E. coli K-12 recombinant carrying an unexpressed foreign ompA allele was subjected to two cycles of selection for an OmpA⁺ phenotype, a mutant strain was obtained which was sensitive to K3 and which expressed nearly normal levels of OmpA protein in the outer membrane. This strain carried mutations in the foreign ompA gene, as indicated both by genetic mapping and the alteration of a peptide in the mutant OmpA protein. The ability of the OmpA protein to bind to lipopolysaccharide (LPS) showed similar strain specificity, and the mutant OmpA protein which was expressed in an unrelated host showed enhanced ability to bind LPS from its new host. Thus, cell surface expression of the ompA gene appears to depend upon the ability of the gene product to bind LPS, suggesting that an interaction between the protein and LPS plays an essential role in biosynthesis of this outer membrane protein.     

Distribution of Bacterial Growth Activity in Flow-Chamber Biofilms

In microbial communities such as those found in biofilms, individual organisms most often display heterogeneous behavior with respect to their metabolic activity, growth status, gene expression pattern, etc. In that context, a novel reporter system for monitoring of cellular growth activity has been designed. It comprises a transposon cassette carrying fusions between the growth rate-regulated Escherichia coli rrnBP1 promoter and different variant gfp genes. It is shown that the P1 promoter is regulated in the same way in E. coli and Pseudomonas putida, making it useful for monitoring of growth activity in organisms outside the group of enteric bacteria. Construction of fusions to genes encoding unstable Gfp proteins opened up the possibility of the monitoring of rates of rRNA synthesis and, in this way, allowing on-line determination of the distribution of growth activity in a complex community. With the use of these reporter tools, it is demonstrated that individual cells of a toluene-degrading P. putida strain growing in a benzyl alcohol-supplemented biofilm have different levels of growth activity which develop as the biofilm gets older. Cells that eventually grow very slowly or not at all may be stimulated to restart growth if provided with a more easily metabolizable carbon source. Thus, the dynamics of biofilm growth activity has been tracked to the level of individual cells, cell clusters, and microcolonies.     

Ribosomal Proteins Involved in the Suppression of Streptomycin Dependence in Escherichia coli

Suppression of streptomycin dependence in Escherichia coli strain K-114, a spectinomycin-sensitive strain, is correlated with modification of 30S ribosomal protein P4, the component modified in spectinomycin-resistant mutants. The mutant is unusual in that reversion from dependence has previously been correlated only with modification in 30S protein P4a. Introduction into K-114 of another mutation conferring spectinomycin resistance results in a further alteration in protein P4.     

omp T: Escherichia coli K-12 structural gene for protein a (3b)

Chromosomal DNA from strain UT400, a previously described deletion mutant of Escherichia coli K-12 that lacks outer membrane protein a, failed to hybridize with plasmid DNA (pGGC110) containing the structural gene for protein a. We designate the genetic locus for protein a, located at approximately 12.5 min of the E. coli chromosome, ompT.     

Growth of a mutant of Escherichia coli K-12 on xylitol by recruiting enzymes for D-xylose and L1,2-propanedol metabolism

Wild type Escherichia coli K-12 cannot grow on xylitol and we have been unsuccessful in isolating a mutant directly which had acquired this new growth ability. However, a mutant had been selected previously for growth on L-1,2-propanediol as the sole source of carbon and energy. This mutant constitutively synthesized a propanediol dehydrogenase. Recently, we have found that this dehydrogenase fortuitously converted xylitol to D-xylose which could normally be metabolized by E. coli K-12. In addition, it was also discovered that the D-xylose permease fortuitously transported xylitol into the cell. A second mutant was thus isolated from the L-1,2-propanediol-growing mutant that was constitutive for enzymes of the D-xylose pathway. This mutant could indeed grow on xylitol as the sole source of carbon and energy, by utilizing the enzymes normally involved in D-xylose and L-1,2-propanediol metabolism.     

pbpB, a gene coding for a putative penicillin-binding protein, is required for aerobic nitrogen fixation in the cyanobacterium Anabaena sp. strain PCC7120

Transposon mutagenesis of Anabaena sp. strain PCC7120 led to the isolation of a mutant strain, SNa1, which is unable to fix nitrogen aerobically but is perfectly able to grow with combined nitrogen (i.e., nitrate). Reconstruction of the transposon mutation of SNa1 in the wild-type strain reproduced the phenotype of the original mutant. The transposon had inserted within an open reading frame whose translation product shows significant homology with a family of proteins known as high-molecular-weight penicillin-binding proteins (PBPs), which are involved in the synthesis of the peptidoglycan layer of the cell wall. A sequence similarity search allowed us to identify at least 12 putative PBPs in the recently sequenced Anabaena sp. strain PCC7120 genome, which we have named and organized according to predicted molecular size and the Escherichia coli nomenclature for PBPs; based on this nomenclature, we have denoted the gene interrupted in SNal as pbpB and its product as PBP2. The wild-type form of pbpB on a shuttle vector successfully complemented the mutation in SNa1. In vivo expression studies indicated that PBP2 is probably present when both sources of nitrogen, nitrate and N₂, are used. When nitrate is used, the function of PBP2 either is dispensable or may be substituted by other PBPs; however, under nitrogen deprivation, where the differentiation of the heterocyst takes place, the role of PBP2 in the formation and/or maintenance of the peptidoglycan layer is essential.     

Reactivating effect of Escherichia coli exometabolites on UV-irradiated cells

Wild-type and mutant (AB 1157 and K-12) strains of Escherichia coli were shown to synthesize the logarithmic growth phase, exometabolites reactivating UV-irradiated cells of producer strains. The exometabolites of the strain K-12 were of protein nature and had a molecular weight of no more than 10 kDa. The reactivating activity of these exometabolites was inversely related to bacterial survival and slightly increased under the influence of stress factors. The reactivating factor of Luteococcus casei had a cross-reactivating and protective effect on UV-irradiated cells of E. coli strain K-12. Due to activation of the reactivating factor after UV irradiation and heating, the cross-protective effect increased more than threefold. The reactivating effect remained unchanged under these conditions. The protein exometabolites of E. coli did not induce cross-stress response in L. casei.     

Influence of periplasmic oxidation of glucose on pyoverdine synthesis in Pseudomonas putida S11

Fluorescent pseudomonads catabolize glucose simultaneously by two different pathways, namely, the oxidative pathway in periplasm and the phosphorylative pathway in cytoplasm. This study provides evidence for the role of glucose metabolism in the regulation of pyoverdine synthesis in Pseudomonas putida S11. We have characterized the influence of direct oxidation of glucose in periplasm on pyoverdine synthesis in P. putida S11. We identified a Tn5 transposon mutant of P. putida S11 showing increased pyoverdine production in minimal glucose medium (MGM). This mutant designated as IST1 had Tn5 insertion in glucose dehydrogenase (gcd) gene. To verify the role of periplasmic oxidation of glucose on pyoverdine synthesis, we constructed mutants S11 Gcd^? and S11 PqqF^? by antibiotic cassette mutagenesis. These mutants of P. putida S11 with loss of glucose dehydrogenase gene (gcd) or cofactor pyrroloquinoline quinone biosynthesis gene (pqqF) showed increased pyoverdine synthesis and impaired acid production in MGM. In minimal gluconate medium, the pyoverdine production of wild-type strain S11 and mutants S11 Gcd^? and S11 PqqF^? was higher than in MGM indicating that gluconate did not affect pyoverdine synthesis. In MGM containing PIPES–NaOH (pH?7.5) buffer which prevent pH changes due to gluconic acid production, strain S11 produced higher amount of pyoverdine similar to mutants S11 Gcd^? and S11 PqqF^?. Therefore, it is proposed that periplasmic oxidation of glucose to gluconic acid decreases the pH of MGM and thereby influences pyoverdine synthesis of strain S11. The increased pyoverdine synthesis enhanced biotic and abiotic surface colonization of the strain S11.     

Dechlorination of Chloral Hydrate Is Influenced by the Biofilm Adhesin Protein LapA in Pseudomonas putida LF54

LapA is the largest surface adhesion protein of Pseudomonas putida that initiates biofilm formation. Here, by using transposon insertion mutagenesis and a conditional lapA mutant, we demonstrate for the first time that LapA influences chloral hydrate (CH) dechlorination in P. putida LF54.