共查询到20条相似文献,搜索用时 31 毫秒
1.
M. Veselova V. Lipasova M. A. Protsenko N. Buza I. A. Khmel 《Folia microbiologica》2009,54(5):401-408
Pseudomonas chlororaphis strain 449 isolated from the rhizosphere of maize suppresses numerous plant pathogens in vitro. The strain produces phenazine antibiotics and synthesizes at least three types of quorum sensing signaling molecules, N-acylhomoserine lactones. Here we have shown that the rhizospheric P. chlororaphis strains 449, well known strain 30–84 as well as two other P. chlororaphis strains exhibit polygalacturonase activity. Using mini-Tn5 transposon mutagenesis, four independent mutants of strain P. chlororaphis 449 with insertion of mini-Tn5 Km2 in gene gacS of two-component GacA-GacS system of global regulation were selected. All these mutant strains were deficient in production
of extracellular proteinase(s), phenazines, N-acylhomoserine lactones synthesis, and did not inhibit the growth of G+ bacteria in comparison with the wild type strain. The P. chlororaphis 449-06 gacS
− mutant studied in greater detail was deficient in polygalacturonase, pectin methylesterase activities, swarming motility
and antifungal activity. It is the first time the involvement of GacA-GacS system in the regulation of enzymes of pectin metabolism,
polygalacturonase and pectin methylesterase, was demonstrated in fluorescent pseudomonads. 相似文献
2.
V. A. Lipasova E. E. Atamova M. A. Veselova N. N. Tarasova I. A. Khmel 《Russian Journal of Genetics》2009,45(1):30-34
The introduction into strain Pseudomonas chlororaphis 449 of plasmid pME6863 that contains the cloned gene for N-acyl-homoserine lactonase, AiiA, leads to the degradation of all three types of N-acylhomoserine lactones produced by this strain (N-butanoyl-homoserine lactone, N-hexanoyl-homoserine lactone, and N-3-oxo-hexanoyl-homoserine lactone). This causes a drastic reduction in the synthesis of phenazine pigment and decreases the ability of cells to migrate on the surface of nutrient medium. However, the antagonistic activity of P. chlororaphis 449 toward phytopathogenic fungi Sclerotinia sclerotiorum and Rhizoctonia solani is not only decreased, but is even slightly increased; no essential changes in the exoprotease activity were observed. It is assumed that one of the QS systems of P. chlororaphis 449 may exert the repression effect on the expression of genes, which determine the two latter cell activities. 相似文献
3.
Ye Ni Zhenwei Li Zhihao Sun Pu Zheng Yongmei Liu Leilei Zhu Ulrich Schwaneberg 《Current microbiology》2009,58(6):593-598
Arginine deiminase (ADI), an arginine-degrading enzyme, has been studied as a potential anti-cancer agent in clinical trials
for the treatment of arginine-auxotrophic tumors, such as hepatocellular carcinomas (HCCs) and melanomas. The arcA gene encoding ADI was cloned from a recently isolated strain Pseudomonas plecoglossicida CGMCC2039. The nucleotide sequence of ADI comprises an ORF of 1,254 bp encoding 417 amino acids. The deduced ADI protein
sequence has a calculated molecular weight of 46.5 kDa and shows 97% and 85% identity to ADIs from P. putida and P. aeruginosa, respectively. The arcA from P. plecoglossicida CGMCC2039 was expressed in Escherichia coli BL21 with a N-terminal His6-tag, and purified to homogeneity. A molecular mass of approximate 49 kDa was confirmed by SDS-PAGE analysis and specific
activity was determined to be 4.76 U/mg (pH 6.0 and 37°C). In vivo activity study showed that the rADI could effectively inhibit
H22 tumor growth at a total dose of 5 U/mouse over a 2-week dosing period. 相似文献
4.
Ogino H Hiroshima S Hirose S Yasuda M Ishimi K Ishikawa H 《Molecular genetics and genomics : MGG》2004,271(2):189-196
A lipase gene (lip3) was cloned from the Pseudomonas aeruginosa strain LST-03 (which tolerates organic solvents) and expressed in Escherichia coli. The cloned sequence includes an ORF consisting of 945 nucleotides, encoding a protein of 315 amino acids (Lip3 lipase, 34.8 kDa). The predicted Lip3 lipase belongs to the class of serine hydrolases; the catalytic triad consists of the residues Ser-137, Asp-258, and His-286. The gene cloned in the present study does not encode the LST-03 lipase, a previously isolated solvent-stable lipase secreted by P. aeruginosa LST-03, because the N-terminal amino acid sequence of the Lip3 lipase differs from that of the LST-03 lipase. Although the effects of pH on the activity and stability of the Lip3 lipase, and the temperature optimum of the enzyme, were similar to those of the LST-03 lipase, the relative activity of the Lip3 lipase at lower temperatures (0–35°C) was higher than that of the LST-03 lipase. In the absence of organic solvents, the half-life of the Lip3 lipase was similar to that of the LST-03 lipase. However, in the presence of most of the organic solvents tested in this study (the exceptions were ethylene glycol and glycerol), the stability of the Lip3 lipase was lower than that of the LST-03 lipase.Communicated by H. Ikeda 相似文献
5.
We investigated the expression of (R)-specific enoyl coenzyme A hydratase (PhaJ) in Pseudomonas putida KT2440 accumulating polyhydroxyalkanoate (PHA) from sodium octanoate in order to identify biosynthesis pathways of PHAs from
fatty acids in pseudomonads. From a database search through the P. putida KT2440 genome, an additional phaJ gene homologous to phaJ4
Pa from Pseudomonas aeruginosa, termed phaJ4
Pp, was identified. The gene products of phaJ1
Pp, which was identified previously, and phaJ4
Pp were confirmed to be functional in recombinant Escherichia coli on PHA synthesis from sodium dodecanoate. Cytosolic proteins from P. putida grown on sodium octanoate were subjected to anion exchange chromatography and one of the eluted fractions with hydratase
activity included PhaJ4Pp, as revealed by western blot analysis. These results strongly suggest that PhaJ4Pp forms a channeling route from β-oxidation to PHA biosynthesis in P. putida. Moreover, the substrate specificity of PhaJ1Pp was suggested to be different from that of PhaJ1Pa from P. aeruginosa although these two proteins share 67% amino acid sequence identity. 相似文献
6.
7.
Yang TH Jung YK Kang HO Kim TW Park SJ Lee SY 《Applied microbiology and biotechnology》2011,90(2):603-614
Previously, we have developed metabolically engineered Escherichia coli strains capable of producing polylactic acid (PLA) and poly(3-hydroxybutyrate-co-lactate) [P(3HB-co-LA)] by employing evolved Clostridium propionicum propionate CoA transferase (Pct
Cp
) and Pseudomonas sp. MBEL 6-19 polyhydroxyalkanoate (PHA) synthase 1 (PhaC1
Ps6-19). Introduction of mutations four sites (E130, S325, S477, and Q481) of PhaC1
Ps6-19 have been found to affect the polymer content, lactate mole fraction, and molecular weight of P(3HB-co-LA). In this study, we have further engineered type II Pseudomonas PHA synthases 1 (PhaC1s) from Pseudomonas chlororaphis, Pseudomonas sp. 61-3, Pseudomonas putida KT2440, Pseudomonas resinovorans, and Pseudomonas aeruginosa PAO1 to accept short-chain-length hydroxyacyl-CoAs including lactyl-CoA and 3-hydroxybutyryl-CoA as substrates by site-directed
mutagenesis of four sites (E130, S325, S477, and Q481). All PhaC1s having mutations in these four sites were able to accept
lactyl-CoA as a substrate and supported the synthesis of P(3HB-co-LA) in recombinant E. coli, whereas the wild-type PhaC1s could not accumulate polymers in detectable levels. The contents, lactate mole fractions, and
the molecular weights of P(3HB-co-LA) synthesized by recombinant E. coli varied depending upon the source of the PHA synthase and the mutants used. PLA homopolymer could also be produced at ca.
7 wt.% by employing the several PhaC1 variants containing E130D/S325T/S477G/Q481K quadruple mutations in wild-type E. coli XL1-Blue. 相似文献
8.
V. N. Verbenko L. V. Kuznetsova E. P. Krupyan V. I. Shalguev 《Russian Journal of Genetics》2009,45(10):1192-1199
Plasmids pKS5 and pKSrec30 carrying normal and mutant alleles of the Deinococcus recA gene controlled by the lactose promoter slightly increase radioresistance of Escherichia coli cells with mutations in genes recA and ssb. The RecA protein of D. radiodurans is expressed in E. coli cells, and its synthesis can be supplementary induced. The radioprotective effect of the xenologic protein does not exceed
1.5 fold and yields essentially to the contribution of plasmid pUC19-recA1.1 harboring the E. coli recA
+ gene in the recovery of resistance of the ΔrecA deletion mutant. These data suggest that the expression of D. radiodurans recA gene in E. coli cells does not complement mutations at gene recA in the chromosome possibly due to structural and functional peculiarities of the D. radiodurans RecA protein. 相似文献
9.
Kateryna Zelena Holger Zorn Manfred Nimtz Ralf Günter Berger 《Archives of microbiology》2009,191(5):397-402
For the heterologous expression of the msp2 gene from the edible mushroom Marasmius scorodonius in Escherichia coli the cDNA encoding the extracellular Msp2 peroxidase was cloned into the pBAD III expression plasmid. Expression of the protein
with or without signal peptide was investigated in E. coli strains TOP10 and LMG194. Different PCR products were amplified for expression of the native target protein or a protein
with a signal peptide. Omitting the native stop codon and adding six His-residues resulted in a fusion protein amenable to
immune detection and purification by immobilised metal affinity chromatography. In E. coli the recombinant protein was produced in high yield as insoluble inclusion bodies. The influence of different parameters on
MsP2 refolding was investigated. Active enzyme was obtained by glutathione-mediated oxidation in a medium containing urea,
Ca2+, and hemin. 相似文献
10.
Sanath Kumar Ammini Parvathi Ricardo L. Hernandez Kathleen M. Cadle Manuel F. Varela 《Archives of microbiology》2009,191(5):425-429
MurA [UDP-N-acetylglucosamine (UDP-NAG) enolpyruvyl transferase] is a key enzyme involved in bacterial cell wall peptidoglycan synthesis
and a target for the antimicrobial agent fosfomycin, a structural analog of the MurA substrate phosphoenol pyruvate. In this
study, we identified, cloned and sequenced a novel murA gene from an environmental isolate of Vibrio fischeri that is naturally resistant to fosfomycin. The fosfomycin resistance gene was isolated from a genomic DNA library of V. fischeri. An antimicrobial agent hypersensitive strain of Escherichia coli harboring murA from V. fischeri exhibited a high fosfomycin resistance phenotype, with minimum inhibitory concentration of 3,000 μg/ml. The cloned murA gene was 1,269 bp long encoding a 422 amino acid polypeptide with an estimated pI of 5.0. The deduced amino acid sequence
of the putative protein was identified as UDP-NAG enolpyruvyl transferase by homology comparison. The MurA protein with an
estimated molecular weight of 44.7 kDa was expressed in E. coli and purified by affinity chromatography. MurA of V. fischeri will be a useful target to identify potential inhibitors of fosfomycin resistance in pharmacological studies. 相似文献
11.
Smita Dube Kamna Nanda Reema Rani Namrata Jit Kaur Jatin Kumar Nagpal Dilip J. Upadhyay Ian A. Cliffe Kulvinder Singh Saini Kedar P. Purnapatre 《World journal of microbiology & biotechnology》2010,26(9):1623-1629
Multi-drug resistant Pseudomonas aeruginosa (MDRPA) are emerging as a major threat in the hospitals as they have become resistant to current antibiotics. There is an
immediate requirement of drugs with novel mechanisms as the pipeline of investigational drugs against these organisms is lean.
UDP-N-acetylglucosamine enolpyruvyl transferase (MurA) enzyme that catalyzes the first committed step of bacterial cell wall
biosynthesis is an ideal target for the discovery of novel antibiotics against Gram negative pathogens as they have only one
copy of murA gene in its genome. We have performed biochemical characterization and comparative kinetic analysis of MurA from E. coli and P. aeruginosa. Both enzymes were active at broad range of pH with temperature optima of 37°C. Metal ions did not enhance the activity of
both enzymes. These enzymes had an apparent affinity constant (K
m
) for its substrate UDP-N-acetylglucosamine 36 ± 5.2 and 17.8 ± 2.5 μM and for phosphoenolpyruvate 0.84 ± 0.13 μM and 0.45 ± 0.07 μM
for E. coli and P. aeruginosa enzymes respectively. Both the enzymes showed 5–7 fold shift in IC50 for the known inhibitor fosfomycin upon pre-incubation with the substrate UDP-N-acetylglucosamine. This observation was used
to develop a novel rapid sensitive high throughput assay for the screening of MurA inhibitors. 相似文献
12.
Previous work from our laboratory has shown that most of Bacillus thuringiensis strains possess the ability to produce melanin in the presence of l-tyrosine at elevated temperatures (42 °C). Furthermore, it was shown that the melanin produced by B. thuringiensis was synthesized by the action of tyrosinase, which catalyzed the conversion of l-tyrosine, via l-DOPA, to melanin. In this study, the tyrosinase-encoding gene (mel) from B. thuringiensis 4D11 was cloned using PCR techniques and expressed in Escherichia coli DH5 . A DNA fragment with 1179 bp which contained the intact mel gene in the recombinant plasmid pGEM1179 imparted the ability to synthesize melanin to the E. coli recipient strain. The nucleotide sequence of this DNA fragment revealed an open reading frame of 744 bp, encoding a protein of 248 amino acids. The novel mel gene from B.thuringiensis expressed in E. coli DH5 conferred UV protection on the recipient strain. 相似文献
13.
K. Murugan K. Selvanayaki Saleh Al-Sohaibani 《World journal of microbiology & biotechnology》2011,27(7):1661-1668
Respiratory tract and device associated infections caused by biofilm forming Pseudomonas aeruginosa play a primary role in the pathogenesis and prognosis of cystic fibrosis (CF) diseases. The biofilm formed by these pathogens
attributes to the antibiotic resistance and protection from host immune response. Once established, the pathogens respond
poorly to therapeutic agents. Recently medicinal plants are largely explored as potential source of bioactive agents. In this
context the present study reports the antibiofilm activity of the folkloric medicinal plant Andrographis paniculata against biofilm forming CF causative Pseudomonas aeruginosa isolated from CF sputum. P. aeruginosa was also assessed for their growth and development of the biofilm, phylogenetic relationship and antibiotic susceptibility.
Antibiogram of the strains indicated that they were resistant to more than one antibiotic. Six extracts of A. paniculata showed significant antibiofilm activity. P. aeruginosa strains, KMS P03 and KMS P05, were found to be maximally inhibited by the methanol extract to an extent of 88.6 and 87.5%
respectively. This is the first report on antibiofilm activity of A. paniculata extracts, and our results indicate scope for development of complementary medicine for biofilm associated infections. 相似文献
14.
Effects of quorum sensing autoinducer degradation gene on virulence and biofilm formation of <Emphasis Type="Italic">Pseudomonas aeruginosa</Emphasis>
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The aiiA gene from Bacillus thuringiensis was cloned into the Pseudomonas/E. coli shuttle vector and transformed into Pseudomonas aeruginosa strain PAO1. Western blotting showed that the AiiA protein was expressed in PAO1. After induction by IPTG for 6 h and 18
h, expression of the aiiA gene in PAO1 completely degraded the quorum sensing autoinducers N-acylhomoserine lactones (AHLs): N-oxododecanoyl-L-homoserine lactone (OdDHL) and N-butyryl-L-homoserine lactone (BHL). The reduced amount of AHLs in PAO1 was also correlated with decreased expression and
production of several virulence factors such as elastase and pyocyanin. AiiA expression also influenced bacterial swarming
motility. Most importantly, our studies indicated that aiiA played significant roles in P. aeruginosa biofilm formation and dispersion, as observed by the differences of the biofilm formation on liquid and solid surfaces, and
biofilm structures under a scanning electron microscope. 相似文献
15.
Susana Patrícia Lopes Idalina Machado Maria Olívia Pereira 《Journal of industrial microbiology & biotechnology》2011,38(1):133-140
Bacterial species are found primarily as residents of complex surface-associated communities, known as biofilms. Although
these structures prevail in nature, bacteria still exist in planktonic lifestyle and differ from those in morphology, physiology,
and metabolism. This study aimed to investigate the influence of physiological states of Pseudomonas aeruginosa and Escherichia coli in cell-to-cell interactions. Filtered supernatants obtained under planktonic and biofilm cultures of each single species
were supplemented with tryptic soy broth (TSB) and used as the growth media (conditioned media) to planktonic and sessile
growth of both single- and two-species cultures. Planktonic bacterial growth was examined through OD640 measurement. One-day-old biofilms were evaluated in terms of biofilm biomass (CV), respiratory activity (XTT), and CFU number.
Conditioned media obtained either in biofilm or in planktonic mode of life triggered a synergistic effect on planktonic growth,
mainly for E. coli single cultures growing in P. aeruginosa supernatants. Biofilms grown in the presence of P. aeruginosa biofilms-derived metabolites presented less mass and activity. These events highlight that, when developed in biofilm, P. aeruginosa release signals or metabolites able to prejudice single and binary biofilm growth of others species and of their own species.
However, products released by their planktonic counterparts did not impair biofilm growth or activity. E. coli, living as planktonic or sessile cultures, released signals and metabolites or removed un-beneficial compounds which promoted
the growth and activity of all the species. Our findings revealed that inter and intraspecies behaviors depend on the involved
bacteria and their adopted mode of life. 相似文献
16.
Prema K. Latha Ravindra Soni Mahejibin Khan Soma S. Marla Reeta Goel 《Current microbiology》2009,58(4):343-348
The metagenomic Csp library was constructed from the temperate and glacier soils of central Himalaya, India followed by polymerase
chain reaction (PCR) amplification. The library was further screened for low-temperature adaptation, and the positive recombinants
were sorted out by determining changes in the melting temperature (Tm). A homology search of cloned sequence showed their identity with the Csp genes of Pseudomonas fluorescens, Psychrobacter cryohalolentis K5, and Shewanella spp MR-4. Amino acid sequence analysis annotated the presence of conserved aromatic and basic amino acids as well as RNA
binding motifs from the cold shock domain. Furthermore, a PROSITE scan showed a moderate identity of less than 60% with the
known cold shock-inducible proteins (ribosomal proteins, rbfA, DEAD-box helicases), cold acclimation protein, and temperature-induced
protein (SRP1/TIP1). This study highlighted the prevalence of Csp genes from cold Himalayan environments that can be explored
for tailor-made crop constructions in future. 相似文献
17.
Twitching motility allows Pseudomonas aeruginosa to respond to stimuli by extending and retracting its type IV pili (TFP). PilJ is a protein necessary for this surface-associated
twitching motility and bears high sequence identity with Escherichia coli methyl-accepting chemotaxis proteins (MCP). Here, we report that whereas wild-type P. aeruginosa PAO1 cells have extended pili at a single pole, pilJ mutant cells have shortened pili often at both poles despite normal levels of pilin accumulation, suggesting that PilJ is
required for full TFP assembly/extension. Using yellow fluorescent protein fusions (pilJ-yfp), both plasmid born and in-frame chromosomal constructs, we determined that PilJ localizes to both poles of the cell. Overexpression
of pilJ-yfp resulted in the protein accumulating between the poles.
Paul DeLange and Tracy Collins contributed equally to this work. 相似文献
18.
In order to produce (S) 10-monohydroxy-8E-octadecenoic acid (MHOD) from oleic acid, a full-length probable lipoxygenase cDNA from Pseudomonas aeruginosa 42A2 was cloned and expressed in Escherichia coli BL21(DE3). The recombinant protein was purified by affinity chromatography to electrophoretic homogeneity and specifically stained. Its molecular mass was 70 kDa. The activity of the rec-LOX with oleic acid was about 30% of that of the prefered substrate, linoleic acid (100%). Bacterial LOX forms a new subfamily in the lipoxygenase phylogenetic tree. 相似文献
19.
Attacin, a 20 kDa antibacterial peptide, plays an important role in immunity. To understand this gene better, gene cloning,
expression and biological activity detection of Attacin A was carried out in present study. The full-length open reading frame
(ORF) coding for Attacin A gene was generated using RT-PCR which takes total RNA extracted from Drosophila as the template. The gene was inserted directionally into the prokaryotic expression vector pET-32a (+). The resulting recombinant
plasmid was transformed into E. coli
Rosetta. SDS–PAGE was carried out to detect the expression product which was induced by IPTG. The antimicrobial activity and hemolysis
activity were tested in vitro after purification. Agarose gel electrophoresis indicated that the complete ORF of Attacin A
gene has been cloned successfully from Drosophila stimulated by E. coli which includes 666 bp and encodes 221 AA. The gene encoding mature Attacin A protein was amplified by PCR from the recombinant
plasmid containing Attacin A, which includes 570 bp in all. SDS–PAGE analysis demonstrated that the fusion protein expressed
was approximately 39.2 kDa. Biological activities detection showed that this peptide exhibited certain antibacterial activity
to several G− bacteria, as well as minor hemolysis activity for porcine red blood cells. In conclusion, Attacin A gene was
cloned and expressed successfully. It was the basis for further study of Attacin. 相似文献
20.
Xylella fastidiosa was the first phytopathogen to be completely sequenced, and its genome revealed several interesting features to be used in functional studies. In the present work, the htpX gene, which encodes a protein involved in the heat shock response in other bacteria, was analyzed by RT-PCR by using cells derived from different cultural conditions. This gene was induced after a temperature upshift to 37°C after growth in minimal medium, XDM, but showed constitutive expression in rich medium or in XDM plus plant extracts. Sequences upstream to the htpX gene, containing a putative regulatory region, were also transferred to E. coli, and the thermoregulation was maintained in the new host, since it was constitutively transcribed at 37°C or 45°C in all culture media tested, but not at 28°C in minimal culture medium. The gene was also cloned into the expression vector pET32Xa/LIC, and the expression of the corresponding protein was confirmed by Western blotting. 相似文献