Recombinant and wild-type Pseudomonas aureofaciens strains in soil: survival, respiratory activity and effects on nodulation of whitebean Phaseolus vulgaris L. by Rhizobiutn species

Survival and respiratory activity of a genetically engineered Pseudomonas aureofaciens Ps3732RNL11 were compared to the parental wild-type P. aureofaciens Ps3732RN in loam and sandy loam soils over 17- and 28-day periods. Survival and respiratory activity of P. aureofaciens Ps3732RNL11 was not statistically significantly different from that of P. aureofaciens Ps3732RN. Soil texture had an effect on respiratory activity; carbon dioxide evolution was significantly higher in the sandy loam soil. This effect was observed on days 2, 10 and 18 but not on day 24. The presence of P. aureofaciens Ps3732RNL11 and Ps3732RN did not significantly affect growth of whitebean ( Phaseolus vulgaris L.) in vermiculite, loam, or sandy loam soils. There was no significant difference (95% level) in numbers of nodules produced in the presence of P. aureofaciens Ps3732RNL11 and Ps3732RN as a result of the symbiotic relationship between Rhizobium phaseoli and the whitebean roots in vermiculite. Enumeration of nodules on whitebean roots in loam and sandy loam soils was not conducted due to difficulties in removing intact roots from the soils.     

Molecular properties of elongation factor Tu fromStreptomyces aureofaciens andEscherichia coli

Some molecular properties of the elongation factor Tu of protein synthesis purified in an aggregated state from gram-positive Streptomyces aureofaciens were studied and compared with those of Tu from gram-negative Escherichia coli. Electrofocussing under reducing conditions showed that the molecule of EF-Tu from S. aureofaciens has an isoelectric point shifted more to the acidic side compared with EF-Tu from E. coli. A comparison of amino acid composition revealed minor differences in the content of several amino acids in the two factors and showed that EF-Tu from S. aureofaciens contains four half-cystines per molecule. Under denaturing conditions only two mercapto groups reacted with 5,5'-dithiobis(2-nitrobenzoic acid). Limited tryptic digestion of aggregated EF-Tu from S. aureofaciens yields six fragments: the four main fragments are of a similar size as those of the E. coli factor. All fragments detected after trypsin digestion of S. aureofaciens EF-Tu were immunologically cross-reactive with antibodies against E. coli EF-Tu. However, even after 2 h of the reaction there still remains a small part of streptomycete factor uncleaved, which documents high resistance of aggregated EF-Tu towards trypsin.     

Fate of the biological control agent Pseudomonas aureofaciens TX-1 after application to turfgrass

The fate and impact of Pseudomonas aureofaciens TX-1 following application as a biocontrol agent for fungi in turfgrass were studied. The organism was applied with a modified irrigation system by using a preparation containing 1 x 10(6) P. aureofaciens TX-1 CFU ml(-1) about 100 times between May and August. We examined the impact of this repeated introduction of P. aureofaciens TX-1 (which is known to produce the antimicrobial compound phenazine-1-carboxylic acid) on the indigenous microbial community of the turfgrass system and on establishment of introduced bacteria in the soil system. A PCR primer-DNA hybridization probe combination was developed to accurately monitor the fate of P. aureofaciens TX-1 following application in irrigation water. To assess the impact of frequent P. aureofaciens TX-1 applications on the indigenous bacterial community, turfgrass canopy, thatch, and rhizosphere samples were obtained during the growing season from control and treated plots and subjected to DNA extraction procedures and denaturing gradient gel electrophoresis (DGGE). PCR amplification and hybridization of extracted DNA with the P. aureofaciens TX-1-specific primer-probe combination revealed that P. aureofaciens TX-1 not only became established in the rhizosphere and thatch but also was capable of overwintering. Separation of PCR-amplified partial 16S rRNA genes by DGGE showed that the repeated application of P. aureofaciens TX-1 in irrigation water resulted in transient displacement of a leaf surface bacterial community member. There was no obvious alteration of any dominant members of the thatch and rhizosphere microbial communities.     

Genetic Recombination in Crosses Between Streptomyces aureofaciens and Streptomyces rimosus

Polsinelli, M. (University of Pavia, Pavia, Italy), and Maria Beretta. Genetic recombination in crosses between Streptomyces aureofaciens and Streptomyces rimosus. J. Bacteriol. 91:63-68. 1966.-Biochemical mutants were obtained from Streptomyces rimosus and S. aureofaciens by ultraviolet irradiation. Crosses were performed between auxotrophic strains of S. rimosus and S. aureofaciens with positive results. Data are reported which indicate that the interaction observed in some crosses is due to gene recombination.     

Molecular cloning and sequencing of a non-haem bromoperoxidase gene from Streptomyces aureofaciens ATCC 10762.

A bromoperoxidase gene (bpoT), recently cloned from Streptomyces aureofaciens Tü24, was used as a probe in Southern blot hybridization of total DNA from S. aureofaciens ATCC 10762. A single SstI fragment of 5.4 kb was detected, which was cloned via an enriched gene library into Escherichia coli. The functional bromoperoxidase gene was located on a 2.1 kb BamHI-HindIII fragment by subcloning into S. lividans TK64, using the multicopy plasmid pIJ486. The enzyme was overproduced in S. lividans TK64 (up to 30,000 times compared to S. aureofaciens ATCC 10762) and showed the same electrophoretic and immunological properties as the bromoperoxidase BPO-A2 purified from S. aureofaciens ATCC 10762. DNA sequence analysis revealed an open reading frame encoding a predicted polypeptide with the same M(r) and N-terminal amino acid sequence as the purified subunit of BPO-A2.     

Molecular cloning and high-level expression of a bromoperoxidase gene from Streptomyces aureofaciens Tü24.

A bromoperoxidase gene was cloned from Streptomyces aureofaciens Tü24 into Streptomyces lividans TK64 by using the promoter-probe vector pIJ486. Subcloning of DNA from the original, unstable clone allowed the gene to be localized to a 1.7-kilobase (kb) fragment of DNA. Southern blotting showed that the cloned 1.7-kb insert hybridized to a 4.3-kb fragment in an SstI digest of S. aureofaciens Tü24 total DNA. The 1.7-kb insert was shown to code for a protein with the electrophoretic properties of the subunits of the nonheme bromoperoxidase isolated from S. aureofaciens Tü24. The protein produced by S. lividans TK64 transformed with pHM621, which contained an 8.0-kb insert, was shown to be identical to the S. aureofaciens Tü24 bromoperoxidase in terms of its electrophoretic mobility on denaturing and nondenaturing polyacrylamide gels and its NH2-terminal amino acid sequence. The bromoperoxidase was overproduced (up to 180 times) by S. lividans TK64 containing pHM621. Based on the heat stability of the S. aureofaciens Tü24 bromoperoxidase, a new and simple purification procedure with very high yields was developed.     

Purification, characterization and comparison of two non-haem bromoperoxidases from Streptomyces aureofaciens ATCC 10762.

Two non-haem bromoperoxidases (BPO 1 and BPO 2) were purified from the 7-chlorotetracycline-producing strain Streptomyces aureofaciens ATCC 10762. Both enzymes showed azide-insensitive brominating activity, and bromide-dependent peroxidase activity. BPO 1 was a dimer (Mr 65,000) with subunits of identical size (Mr 31,000). The pI was estimated to be 4.5. The enzyme did not cross-react with antibodies raised against the non-haem bromoperoxidase (Mr 90,000) from S. aureofaciens Tü24, a strain that also produces 7-chlorotetracycline. The Mr of BPO 2 was estimated to be 90,000. The enzyme had three identical subunits (Mr 31,000), and its isoelectric point was 3.5, identical with that of the bromoperoxidase from S. aureofaciens Tü24. Moreover, BPO 2 was immunologically identical with the bromoperoxidase from S. aureofaciens Tü24, although both it and BPO 1 could be distinguished electrophoretically from the latter bromoperoxidase.     

Effect of natural and genetically modified rhizospheric Pseudomonas aureofaciens bacteria on accumulation of arsenic by plants

Gene constructions rendering bacteria resistant to arsenic and capable of dissolving phosphates and/or arsenates were created by cloning ars operon and the gene of citrate synthase from a chromosome of the strain Pseudomonas aeruginosa PAO1. Genetically modified variants of the strain Pseudomonas aureofaciens BS1393 have been constructed, which are resistant to high concentrations of arsenic and dissolve poorly soluble phosphates and/or arsenates. Recombinant strains P. aureofaciens BS1393(pUCP22::arsRBC) and P. aureofaciens BS1393(pUCP22::gltA) exerted positive effects on the survival of sorgo (Sorghum saccharatum L.) and its ability to accumulate arsenic.     

透明颤菌血红蛋白基因表达对金色链霉菌生长代谢的影响

利用四环素抗性基因启动子在金色链霉菌中表达透明颤菌血红蛋白基因。在1m3发酵罐中研究了工程菌株的生长代谢特性。在溶解氧充足的条件下,透明颤菌血红蛋白表达,对金色链霉菌生长代谢未产生明显影响,工程菌株与参比菌株的生长代谢特性基本一致,工程菌株和参比菌株金霉素最终浓度分别为22905u/mL、22896u/mL。在低溶解氧条件下,透明颤菌血红蛋白的表达,可促进金色链霉菌菌体生长、菌丝活力保持和金霉素的合成：工程菌菌体浓度比参比菌株高5％～10％,产物合成提高11.4％。     

Post-translational modification(s) and cell distribution of<Emphasis Type="Italic">Streptomyces aureofaciens</Emphasis> translation elongation factor Tu overproduced in<Emphasis Type="Italic">Escherichia coli</Emphasis>

We cloned EF-Tu from Streptomyces aureofaciens on a pET plasmid and overproduced it using the T7 RNA polymerase system in Escherichia coli. Streptomyces EF-Tu represented more than 40% of the total cell protein and was stored mostly in inclusion bodies formed apically at both ends of E. coli cells. Analysis of the inclusion bodies by transmission and scanning electron microscopy did not reveal any internal or surface ultrastructures. We developed the method for purification of S. aureofaciens EF-Tu from isolated inclusion bodies based on the ability of the protein to aggregate spontaneously. EF-Tu present in inclusion bodies was not active in GDP binding. Purified protein showed a similar charge heterogeneity as EF-Tu isolated from the mycelium of S. aureofaciens and all of the isoforms reacted with EF-Tu antibodies. All isoforms also reacted with monoclonal antibodies against O-phosphoserine and O-phosphothreonine.     

Tryptophan 7-halogenase from Pseudomonas aureofaciens ACN strain: gene cloning and sequencing and the enzyme expression

The gene of tryptophan 7-halogenase was isolated from the Pseudomonas aureofaciens ACN strain producing pyrrolnitrin, a chlorocontaining antibiotic, and sequenced. A high homology degree (over 95%) was established for the genes and the corresponding halogenases from P. aureofaciens ACN and P. fluorescens BL915. The tryptophan 7-halogenase gene was amplified by PCR, and the corresponding enzyme was expressed in Escherichia coli cells using the pBSII SK+ vector.     

Hydrolytic action of phospholipases on bacterial membranes.

Substrate specificities of phospholipases C[EC 3.1.4.3] from Clostridium novyi, Clostridium perfringens, Bacillus cereus, and Pseudomonas aureofaciens were studied under the same conditions. Phospholipases C from Clostridium novyi and Bacillus cereus show wide substrate specificities while those of Clostridium perfringens and Pseudomonas aureofaciens show relatively narrow specificities. On the basis of these results, the hydrolytic actions of these phospholipases on membrane lipids of Escherichia coli, Bacillus cereus, and Clostridium novyi were examined under the same conditions. The enzymes of Clostridium novyi and Bacillus cereus attacked all the membranes and their lipid extracts, hydrolyzing phosphatidylethanolamine, phosphatidylglycerol, lyso-phosphatidylethanolamine, and o-aminoacylphosphatidylglycerol. Phospholipase C from Pseudomonas aureofaciens attacked these three membranes and their lipid extracts, hydrolyzing phosphatidylethanolamine. Phospholipase C from Clostridium perfringens hardly attacked the phospholipids of these bacterial membranes. However, phospholipase C from Clostridium perfringens hydrolyzed phosphatidylethanolamine in a mixture containing lipid extract from Escherichia coli membrane and purified phosphatidylcholine from egg yolk.     

Cloning and characterization of a new polyketide synthase gene cluster in Streptomyces aureofaciens CCM 3239.

We cloned a new polyketide gene cluster, aur2, in Streptomyces aureofaciens CCM3239. Sequence analysis of the 9531-bp DNA fragment revealed 10 open reading frames, majority of which showed high similarity to the previously characterized type II polyketide synthase (PKS) genes. An unusual feature of the aur2 cluster is a disconnected organization of minimal PKS genes; ACP is located apart from the genes for ketosynthases KSalpha and KSbeta. The aur2 gene cluster was disrupted in S. aureofaciens CCM3239 by a homologous recombination, replacing the four genes (aur2A, E, F, G) including ketosynthase KSalpha, with antibiotic resistance marker gene. The disruption did not affect growth and differentiation, and disrupted strain produced spores with wild-type grey-pink pigmentation. The biochromatographic analysis of the culture extracts from S. aureofaciens wild type and aur2-disrupted strains did not reveal any difference in the pattern of antibacterial compounds.     

Effect of a genetically modified Pseudomonas aureofaciens on indigenous microbial populations of wheat

Abstract An isolate of Pseudomonas aureofaciens from the phylloplane of sugar beet which was chromosomally modified for monitors purposes by the insertion of two gene cassettes (km^r- xyl E and lac ZY) was introduced to the phytosphere of spring wheat in a number of experiments and the resulting microbial perturbations quantified. Such studies involving innocuous bacterial isolates can serve as a guide in the assessment of risk associated with the release of functionally modified microorganisms. Introductions of P. aureofaciens on seeds caused large microbial perturbations (up to 2 log units) at the seedling stage on seeds and roots. As the inoculated plants matured (tillering, flowering and ripening), perturbations of total microbial populations were found to be non-significant. Microbial perturbation on maturing wheat roots as a result of seed inoculations with P. aureofaciens could only be detected using more sensitive monitoring procedures describing the Pseudomonas community in terms of colony appearance rate on a selective Pseudomonas medium. Spray applications of the marked P. aureofaciens isolate onto the leaf surface of wheat caused no significant perturbations of the indigenous microbial present on the phylloplane.     

Characteristics of NADPH-dependent thymidylate synthetase purified from Streptomyces aureofaciens.

NADPH-dependent thymidylate synthetase from Streptomyces aureofaciens has been purified to homogenity by a two-step chromatographic procedure including anion-exchange chromatography and affinity chromatography on methotrexate-Sepharose 4B. The enzyme was purified 1025-fold with a 34% yield. Basic characteristics of the enzyme were determined: molecular weight of the enzyme subunit (28,000), pH and temperature optimum, effect of cations, dependency on reducing agents, Km values for dUMP, mTHF, and NADPH (3.78, 21.1, and 38.9 microM, respectively), and inhibition effect of 5-FdUMP. Binding studies revealed the enzyme mechanism to be ordered sequential: dUMP bound before mTHF. S. aureofaciens thymidylate synthetase exhibits an absolute requirement for NADPH for the enzyme activity--a unique feature not displayed by any of the thymidylate synthetases isolated so far. NADPH is not consumed during enzyme reaction, indicating its regulatory role. The properties of S. aureofaciens thymidylate synthetase show that it is a monofunctional bacterial enzyme.     

Molecular cloning of chlortetracycline resistance gene from chlortetracycline producer Streptomyces aureofaciens

The chlortetracycline (CT) resistance gene ctr was cloned from S. aureofaciens 633, a strain producing the antibiotic. The 6.6-kb DNA Bam HI fragment containing the resistance gene was cloned with the plasmid vector pIJ699. Comparison of the restriction maps of the cloned gene and the oxytetracycline (OT) resistance gene otrA from S. rimosus revealed their similarity which enabled identification of the cloned resistance gene as otrA. Investigation of the resistance determinants in S. aureofaciens 633 made it possible to identify a mtr gene(s). It was demonstrated that introduction of a ctrA gene into S. lividance provided a simultaneous increase in the resistance of the recipient strain to CT and a number of macrolide antibiotics. The CT resistance determinants in S. lividans TK64 showed properties of exogenous induction by CT and the macrolide antibiotics similar to the properties of the mtr gene(s) of S. aureofaciens. Possible adaptation properties of mtr genes are discussed.