Pyrimidine biosynthetic pathway of Pseudomonas fluorescens

Pyrimidine biosynthesis in Pseudomonas fluorescens strain A126 was investigated. In this study, de novo pyrimidine biosynthetic pathway mutant strains were isolated using both conventional mutagenesis and transposon mutagenesis. The resulting mutant strains were deficient for either aspartate transcarbamoylase, dihydroorotase or orotate phosphoribosyltransferase activity. Uracil, uridine or cytosine could support the growth of every mutant strain selected. In addition, the aspartate transcarbamoylase mutant strains could utilize orotic acid to sustain their growth while the orotidine-5'-monophosphate decarboxylase mutant strains grew slowly upon uridine 5'-monophosphate. The wild-type strain and the mutant strains were used to study possible regulation of de novo pyrimidine biosynthesis in P. fluorescens. Dihydroorotase specific activity more than doubled after the wild-type cells were grown in orotic acid relative to unsupplemented minimal-medium-grown cells. Starving the mutant strains of pyrimidines also influenced the levels of several de novo pyrimidine biosynthetic pathway enzyme activities.     

Identification and characterization of an iron ABC transporter operon in Gluconacetobacter diazotrophicus Pal 5

Gluconacetobacter diazotrophicus is a nitrogen-fixing bacterium and endophyte of sugarcane. We have cloned and sequenced the genes coding for the components of the iron ABC-type acquisition system of G. diazotrophicus. Sequence analysis revealed three ORFs, (feuA, feuB, and feuC) organized as an operon and encoding polypeptides of 346 (38 kDa), 342 (34.2 kDa), and 240 (26 kDa) amino acids, respectively. The deduced translation products of the feu operon showed similarity with a periplasmic solute-binding protein (FeuA), permease (FeuB), and ATPase (FeuC) involved in Fe transport. The role of FeuB in the survival of G. diazotrophicus under iron depletion was evaluated by comparing the ability of wild-type and FeuB-Km^R -mutant strains in a medium without iron supplementation and in a medium containing 2, 2′-dipyridyl (DP). Growth of the mutant was affected in the medium containing DP. The operon was expressed at higher levels in cells depleted for iron than in those that contained the metal. A decrease in nitrogenase activity was observed with the FeuB-Km^R -mutant strain that with the wild-type under iron deficiency conditions, suggesting that the Feu operon play role in Fe nutrition of G. diazotrophicus.     

Cloning and expression in Escherichia coli of apolyurethane-degrading enzyme from Pseudomonasfluorescens

A polyester polyurethane (PU)-degrading enzyme, PU esterase, derived from Pseudomonas fluorescens, a bacterium that utilizes polyester PU as the sole carbon source,was purified to homogeneity as indicated by sodium dodecyl sulfate-polyacrylamide gelelectrophoresis. This enzyme was a soluble, extracellular protein with a molecular mass of 48 kDa and was inhibited by phenylmethylsulfonylfluoride (PMSF). A genomic library of Ps.fluorescens was constructed using the Escherichia coli bacteriophage l vector lZAPII. A recombinant phage exhibiting activity against Impranil DLN was isolated. The geneencoding the polyurethanase (PUase) protein was subcloned into a plasmid expression vectorpT7-6 and expressed in E. coli. Upon expression, the PUase was secreted by the host,displayed esterase activity which was inhibited by PMSF, and in vivo ³⁵S-methionine labeling of the gene product encoded by the open reading frame of the clone insertrevealed a single polypeptide with a molecular mass of 48 kDa.     

Zinc affects siderophore-mediated high affinity iron uptake systems in the rhizosphere Pseudomonas aeruginosa 7NSK2

Zinc concentrations ranging between 0.1 and 1 mm only slightly reduced maximal growth of wild-type Pseudomonas aeruginosa 7NSK2 in iron-limiting casamino acid medium, but had a clear negative effect on the growth of mutant MPFM1 (pyoverdin negative) and especially mutant KMPCH (pyoverdin and pyochelin negative). Production of pyoverdin by wild-type strain 7NSK2 was significantly increased in the presence of 0.5 mm zinc and could not be repressed by iron even at a concentration of 100 m. Siderophore detection via isoelectrofocusing revealed that mutant KMPCH did not produce any siderophores, while mutant MPFM1 overproduced a siderophore with an acidic isoelectric point, most likely pyochelin. Pyochelin production by MPFM1 was stimulated by the presence of zinc in a similar way as pyoverdin for the wild-type. Analysis of outer membrane proteins revealed that three iron regulated outer membrane proteins (IROMPs) (90, 85 and 75 kDa) were induced by iron deficiency in the wild-type, while mutants were found to have altered IROMP profiles. Zinc specifically enhanced the production of a 85 kDa IROMP in 7NSK2, a 75 kDa IROMP in MPFM1 and a 90 kDa IROMP in KMPCH.     

Inducible surface proteins from some strains of Pseudomonas species bacteria while adapting to cobalt

The possibility of increasing resistance of some Pseudomonas strains to cobalt at adaptation to monotonous increasing its concentration was studied. Strains Pseudomonas fluorescens B5242 and Pseudomonas fluorescens B894 are capable to increase its resistance in such conditions via inducible synthesis of protective surface proteins. The molecular masses of such proteins were 55.0; 45.0 and 33.0 kDa for P. fluorescens B5242 strain.     

Trehalose induces antagonism towards Pythium debaryanum in Pseudomonas fluorescens ATCC 17400.

Pseudomonas fluorescens ATCC 17400 shows in vitro activity against Pythium debaryanum under conditions of iron limitation. A lacZ reporter gene introduced by transposon mutagenesis into the P. fluorescens ATCC 17400 trehalase gene (treA) was induced by a factor released by the phytopathogen Pythium debaryanum. The induction of the lacZ gene was lost upon treatment of the Pythium supernatant with commercial trehalase. A trehalose concentration as low as 1 microM could induce the expression of treA. The mutation did not affect the wild-type potential for fungus antagonism but drastically decreased the osmotolerance of the mutant in liquid culture and suppressed the ability of P. fluorescens ATCC 17400 to utilize trehalose as a carbon source. A subsequent transposon insertion in treP, one of the trehalose phosphotransferase genes upstream of treA, silenced the lacZ gene. This double mutant restricted fungal growth only under conditions of high osmolarity, which probably results in internal trehalose accumulation. These data confirm the role of the disaccharide trehalose in osmotolerance, and they indicate its additional role as an initiator of or a signal for fungal antagonism.     

Mutagenesis induced by mono- and bi-functional alkylating agents in yeast mutants sensitive to photo-addition of furocoumarins (pso)

The inactivation and the induction of forward and reverse mutations by a mono- and a bifunctional nitrogen mustard in 3 pso mutants of Saccharomyces cerevisiae, initially selected for their sensitivity to psoralen photo-addition, were compared with that of the wild-type.The pso1-1 mutant was very sensitive to both alkylating agents, and the mutagenecity was abolished. This correlates with the defect in the error-prone repair capacity for lesions induced by psoralen photo-addition and radiations already observed for this mutant. Therefore it appears that the PSO1⁺ gene product acts on a spectrum of DNA lesions.The pso2-1 mutant was highly sensitive to the lethal effect of the bifunctional nitrogen mustard and was only slightly sensitive to the monofunctional one. For both agents a reduction in induced mutagenesis was seen. The same was true for mono- and bifunctional psoralen derivatives. The pso2-1 mutant having the same sensitivity as the wild-type to UV and ionizing radiations, it is suggested that the PSO2⁺ gene product is predominantly necessary for the repair of cross-links irrespective of their molecular nature.In contrast with psoralen photo-induced inactivation the pso3-1 mutant had the same sensitivity as the wild-type to alkylating agents. However, a reduction in induced mutagenesis was seen in both cases. This response was modulated according to dose and type of mutation. Consequently, it appeared that the PSO3⁺ gene product acts specifically on psoralen photo-induced sub-lethal lesions and on a fraction of premutagenic lesions independently of their structure.     

Quinolobactin, a New Siderophore of Pseudomonas fluorescens ATCC 17400, the Production of Which Is Repressed by the Cognate Pyoverdine

Transposon mutant strain 3G6 of Pseudomonas fluorescens ATCC 17400 which was deficient in pyoverdine production, was found to produce another iron-chelating molecule; this molecule was identified as 8-hydroxy-4-methoxy-quinaldic acid (designated quinolobactin). The pyoverdine-deficient mutant produced a supplementary 75-kDa iron-repressed outer membrane protein (IROMP) in addition to the 85-kDa IROMP present in the wild type. The mutant was also characterized by substantially increased uptake of ⁵⁹Fe-quinolobactin. The 75-kDa IROMP was produced by the wild type after induction by quinolobactin-containing culture supernatants obtained from the pyoverdine-negative mutant or by purified quinolobactin. Conversely, adding purified wild-type pyoverdine to the growth medium resulted in suppression of the 75-kDa IROMP in the pyoverdine-deficient mutant; however, suppression was not observed when Pseudomonas aeruginosa PAO1 pyoverdine, a siderophore utilized by strain 3G6, was added to the culture. Therefore, we assume that the quinolobactin receptor is the 75-kDa IROMP and that the quinolobactin-mediated iron uptake system is repressed by the cognate pyoverdine.     

The diversity of lipases from psychrotrophic strains of Pseudomonas : a novel lipase from a highly lipolytic strain of Pseudomonas fluorescens

Strains of Pseudomonas fluorescens and Ps. fragi are the predominant psychrotrophs found in raw milk and may cause spoilage due to the secretion of hydrolytic enzymes such as lipase and protease. The diversity of lipases has been examined in Pseudomonas isolates from raw milk which represent different taxonomic groups (phenons). Significant diversity was found using both DNA hybridization and immunoblotting techniques, which has implications for the development of a diagnostic test. The lipase-encoding gene ( lipA ) was cloned from one strain, C9, of Ps. fluorescens biovar V. In contrast to previously reported lipase sequences from Ps. fluorescens , the gene encodes a lipase of M_r 33 kDa. Alignment of all known Pseudomonas and Burkholderia lipase amino acid sequences indicates the existence of two major groups, one of M_r approximately 30 kDa comprising sequences from Ps. fragi , Ps. aeruginosa , Ps. fluorescens C9 and Burkholderia , and one of approximately 50 kDa comprising Ps. fluorescens lipases. The lipase from C9 does not contain a signal peptide and is presumed to be secreted via a signal peptide-independent pathway. The lipA gene of strain C9 was disrupted by insertional mutagenesis. The mutant retained its lipolytic phenotype, strongly suggesting the presence of a second lipase in this strain.     

Influence of recC and uvrD on ultraviolet-induced mutation to valine resistance in E. coli K12

The production of mutants in E. coli exposed to ultraviolet light is initiated by photochemical reactions, and completed by metabolic processes controlled by recA and other genes. Ultraviolet-induced mutagenesis to valine resistance was measured in cells carrying recC, uvrD, or both recC and uvrD. The spontaneous and UV-induced mutagenesis was slightly greater in those carrying uvrD, as compared to recC or wild-type. At low doses, UV mutagenesis in the recC uvrD double mutant was greater than in either recC or wild-type, and was comparable to that in the uvrD strain, although this double mutant was very UV-sensitive and showed poor survival at doses above 2 J/m².     

Transposon Tn5 mutagenesis of Pseudomonas fluorescens to isolate mutants deficient in antibacterial activity

Abstract Pseudomonas fluorescens was subjected to insertion mutagenesis studies using the transposon Tn5-GM to generate mutants deficient in antibacterial activity minus mutants. The transposon located on the temperature-sensitive plasmid pCHR84 was conjugally transferred into the non-pathogenic pseudomonad using the triparental mating procedure. Random integration of Tn 5 -GM into the chromosome of P. fluorescens was achieved by heat ttreatment of the transformed cells at 42°C. Approximately 2% of transconjugants revealed an auxotrophic phenotype indicating efficient integration of the employed transposon into the chromosome of P. fluorescens . One transposon insertion mutant was obtained showing an antibacterial activity minus phenotype. This mutant (MM-7) was found to be defective in the production of an unidentified antibacterial compound against B. subtilis . These results introduce Tn 5 transposon mutagenesis as a new useful tool for the molecular analysis of P. fluorescens .     

Improvement of alkaline protease production by Penicillium chrysogenum NRRL 792 through physical and chemical mutation,optimization, characterization and genetic variation between mutant and wild-type strains

Penicillium chrysogenum NRRL 792 was exposed successively to gamma radiation (physical mutagen) and ethyl methansulfonate (EMS; chemical mutagen). Gamma mutant G9 produced more alkaline protease than the wild type (62.92 vs. 40.0 U/g, respectively). Subsequent mutagenesis of G9 by EMS resulted in mutant EMS-1, which produced the highest level of enzyme (120.0 U/g). Optimal conditions for alkaline protease production by this mutant fungal strain were examined. The optimized medium was supplemented with 1 % (w/w) casein and 2.5 mM MgSO₄, while the optimal pH and temperature were 9, and 30 °C after 7 days of incubation. The purified mutant alkaline protease from EMS-1 was more stable than that from the wild-type, resulting in the former having a higher pH stability and thermostability. The mutant and wild enzymes were subjected to sodium dodecylsulfate-polyacrylamide gel electrophoresis. The purified mutant enzyme showed two bands with molecular weights of 40 and 65 kDa, while the molecular weight of the purified wild-type enzyme was 66 kDa. Random amplified polymorphic DNA and inter-simple sequence repeat markers were used to identify polymorphism and genetic variations between the mutant and wild-type strains.     

L-(+)-Tartrate dehydratase from Pseudomonas putida is an iron-sulphur enzyme

The enzyme L-(+)-tartrate dehydratase has been isolated from extracts of Pseudomonas putida by a one-step procedure involving dye-ligand chromatography. The enzyme loses activity rapidly in the absence of Fe²⁺; concentrated solutions have a brown colour typical of iron-sulphur proteins. Analysis of iron and acid-labile sulphide indicated 3–5 atoms of each per molecule of 100 kDa. The enzyme's structure consists of four subunits, two each of 23 and 27 kDa.     

Effects of Chemical Speciation on the Mineralization of Organic Compounds by Microorganisms

The mineralization of 1.0 to 100 ng each of four complexing compounds—oxalate, citrate, nitrilotriacetate (NTA), and EDTA—per ml was tested in media prepared in accordance with equilibrium calculations by a computer program so that the H, Ca, Mg, Fe, or Al complex (chemical species) was predominant. Sewage microorganisms mineralized calcium citrate more rapidly than iron, aluminum, or hydrogen citrate, and magnesium citrate was degraded slowest. Aluminum, hydrogen, and iron oxalates were mineralized more rapidly than calcium oxalate, and magnesium oxalate was decomposed slowest. Sewage microorganisms mineralized calcium NTA but not aluminum, magnesium, hydrogen, or iron NTA or any of the EDTA complexes. Pseudomonas sp. mineralized calcium and iron citrates but had no activity on hydrogen, aluminum, or magnesium citrate. Pseudomonas pseudoalcaligenes mineralized calcium, iron, hydrogen, and aluminum citrates but had little activity on magnesium citrate. Pseudomonas alcaligenes used calcium, iron, hydrogen, and aluminum oxalates readily, but it used magnesium oxalate at a slower rate. Listeria sp. destroyed calcium NTA but had no effect on hydrogen, iron, or magnesium NTA. Increasing the Ca concentration in the medium enhanced the breakdown of NTA by Listeria sp. The different activities of the bacterial isolates were not a result of the toxicity of the complexes or the lack of availability of a nutrient element. NTA mineralization was not enhanced by the addition of Ca to Beebe Lake water, but it was enhanced when Ca and an NTA-degrading inoculum were added to water from an oligotrophic lake. The data show that chemical speciation influences the mineralization of organic compounds by naturally occurring microbial communities and by individual bacterial populations.     

Prevalence of Pseudomonas aeruginosa FP plasmids which enhance spontaneous and uv-induced mutagenesis.

From work reported here and from previous studies 16 out of 53 (30%) FP plasmids (i.e. those plasmids that promote host chromosome transfer) of Pseudomonas aeruginosa are found to protect host cells against UV irradiation. 13 of these UV-protecting FP plasmids were tested to determine their mode of DNA repair and were found to contribute to error-prone repair because of their enhancement of UV-induced mutagenesis and in most instances spontaneous mutagenesis as well. Some of these plasmids were tested for their behaviour in a DNA polymerase I deficient (Pol^?) mutant of P. aeruginosa; the remainder could not be tested due to plasmid instability in the Pol^? mutant. 11 of these FP plasmids provided wild-type level of UV protection to the mutant. 4 of the plasmids tested (FP18, FP103, FP109 and FP111) were able to enhance the mutant's ability to host cell reactivate UV irradiated phage, though not to the level of the Pol⁺ parent. The presence of FP18 or FP111 in the Pol^? mutant did not increase polymerase I-like enzymatic activity. It is concluded that the plasmids do not confer a polymerase activity functionally equivalent to host DNA polymerase I. It is possible however, that the plasmids code for another polymerase or for a cofactor which interacts with a host polymerase, as seen by the partial restoration by FP plasmids of host-cell reactivation of UV-irradiated phage in the polymerase I deficient mutant.The mutagenic properties of those FP plasmids tested appears to be nonspecific because of their ability to mutate two host chromosomal genes, trpB1 and leu38 and an R plasmid gene, bla.The implications of the prevalence of FP plasmids in P. aeruginosa which enhance mutagenesis are discussed.     

Mutational analysis of N-glycosylation recognition sites on the biochemical properties of Aspergillus kawachii α-l-arabinofuranosidase 54

A role for N-linked oligosaccharides on the biochemical properties of recombinant α-l-arabinofuranosidase 54 (AkAbf54) defined in glycoside hydrolase family 54 from Aspergillus kawachii expressed in Pichia pastoris was analyzed by site-directed mutagenesis. Two N-linked glycosylation motifs (Asn⁸³–Thr–Thr and Asn²⁰²–Ser–Thr) were found in the AkAbf54 sequence. AkAbf54 comprises two domains, a catalytic domain and an arabinose-binding domain classified as carbohydrate-binding module 42. Two N-linked glycosylation sites are located in the catalytic domain. Asn⁸³, Asn²⁰², and the two residues together were replaced with glutamine by site-directed mutagenesis. The biochemical properties and kinetic parameters of the wild-type and mutant enzymes expressed in P. pastoris were examined. The N83Q mutant enzyme had the same catalytic activity and thermostability as the wild-type enzyme. On the other hand, the N202Q and N83Q/N202Q mutant enzymes exhibited a considerable decrease in thermostability compared to the glycosylated wild-type enzyme. The N202Q and N83Q/N202Q mutant enzymes also had slightly less specific activity towards arabinan and debranched arabinan. However, no significant effect on the affinity of the mutant enzymes for the ligands arabinan, debranched arabinan, and wheat and rye arabinoxylans was detected by affinity gel electrophoresis. These observations suggest that the glycosylation at Asn²⁰² may contribute to thermostability and catalysis.     

Comparisons of Characteristics of Mercury-Tolerant Bacterium Pseudomonas oleovorans G-1 and Its Mercury-Sensitive Mutant Strain

An Hg²⁺-sensitive mutant strain was isolated from an Hg²⁺-tolerant bacterium Pseudomonas oleovorans G-1 strain by mutagenesis with N-methyl-N′-nitro-N-nitrosoguanidine. The Hg²⁺-sensitive mutant strain was about 10-times as sensitive to Hg²⁺ as the parent strain. Moreover, the mutant strain was considerably more sensitive to Cr⁶⁺ than the parent strain, but it did not show an appreciable change in sensitivity to Cd²⁺ and Cu²⁺. The mutant strain was considerably more sensitive to antibiotics achromycin, chloramphenicol and streptomycin than the parent strain. A more rigid structure was observed in the cell envelope of the mutant strain than the parent strain under transmission electron microscope. Higher amounts of DNA but less protein and RNA were found in the mutant strain compared to the parent strain. Disc electrophoretic patterns showed some differences in protein bands between the parent and mutant strain.     

Involvement of Nitrate Reductase and Pyoverdine in Competitiveness of Pseudomonas fluorescens Strain C7R12 in Soil

Involvement of nitrate reductase and pyoverdine in the competitiveness of the biocontrol strain Pseudomonas fluorescens C7R12 was determined, under gnotobiotic conditions, in two soil compartments (bulk and rhizosphere soil), with the soil being kept at two different values of matric potential (−1 and −10 kPa). Three mutants affected in the synthesis of either the nitrate reductase (Nar⁻), the pyoverdine (Pvd⁻), or both (Nar⁻ Pvd⁻) were used. The Nar⁻ and Nar⁻ Pvd⁻ mutants were obtained by site-directed mutagenesis of the wild-type strain and of the Pvd⁻ mutant, respectively. The selective advantage given by nitrate reductase and pyoverdine to the wild-type strain was assessed by measuring the dynamic of each mutant-to-total-inoculant (wild-type strain plus mutant) ratio. All three mutants showed a lower competitiveness than the wild-type strain, indicating that both nitrate reductase and pyoverdine are involved in the fitness of P. fluorescens C7R12. The double mutant presented the lowest competitiveness. Overall, the competitive advantages given to C7R12 by nitrate reductase and pyoverdine were similar. However, the selective advantage given by nitrate reductase was more strongly expressed under conditions of lower aeration (−1 kPa). In contrast, the selective advantage given by nitrate reductase and pyoverdine did not differ in bulk and rhizosphere soil, indicating that these bacterial traits are not specifically involved in the rhizosphere competence but rather in the saprophytic ability of C7R12 in soil environments.