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1.
West TP 《Antonie van Leeuwenhoek》2005,88(2):181-186
Control of pyrimidine biosynthesis was examined in Pseudomonas mucidolens ATCC 4685 and the five de novo pyrimidine biosynthetic enzyme activities unique to this pathway were influenced by pyrimidine supplementation in cells grown on glucose or succinate as a carbon source. When uracil was supplemented to glucose-grown ATCC 4685 cells, activities of four de novo enzymes were depressed which indicated possible repression of enzyme synthesis. To learn whether the pathway was repressible, pyrimidine limitation experiments were conducted using an orotate phosphoribosyltransferase (pyrE) mutant strain identified in this study. Compared to excess uracil growth conditions for the glucose-grown mutant strain cells, pyrimidine limitation of this strain caused aspartate transcarbamoylase, dihydroorotase and dihydroorotate dehydrogenase activities to increase by more than 3-fold while OMP decarboxylase activity increased by 2.7-fold. The syntheses of the de novo enzymes appeared to be regulated by pyrimidines. At the level of enzyme activity, aspartate transcarbamoylase activity in P. mucidolens ATCC 4685 was subject to inhibition at saturating substrate concentrations. Transcarbamoylase activity was strongly inhibited by UTP, ADP, ATP, GTP and pyrophosphate. 相似文献
2.
Pyrimidine salvage pathways are vital for all bacteria in that they share in the synthesis of RNA with the biosynthetic pathway
in pyrimidine prototrophs, while supplying all pyrimidine requirements in pyrimidine auxotrophs. Salvage enzymes that constitute
the pyrimidine salvage pathways were studied in 13 members of Pseudomonas and former pseudomonads. Because it has been established that all Pseudomonas lack the enzyme uridine/cytidine kinase (Udk) and all contain uracil phosphoribosyl transferase (Upp), these two enzymes
were not included in this experimental work. The enzymes assayed were: cytosine deaminase [Cod: cytosine + H2O → uracil + NH3], cytidine deaminase [Cdd: cytidine + H2O → uridine + NH3], uridine phosphorylase [Udp: uridine + Pi ↔ uracil + ribose – 1 - P], nucleoside hydrolase [Nuh: purine/pyrimidine nucleoside + H2O → purine/pyrimidine base + ribose], uridine hydrolase [Udh: uridine/cytidine + H2O → uracil/cytosine + ribose]. The assay work generated five different Pyrimidine Salvage Groups (PSG) designated PSG1 – PSG5
based on the presence or absence of the five enzymes. These enzymes were assayed using reverse phase high-performance liquid
chromatography techniques routinely carried out in our laboratory. Escherichia coli was included as a standard, which contains all seven of the above enzymes. 相似文献
3.
Wenjuan Yao Xiaozhao Deng Hui Zhong Miao Liu Pu Zheng Zhihao Sun Yun Zhang 《Journal of industrial microbiology & biotechnology》2009,36(7):911-921
Corynebacterium glutamicum strains are used for the fermentative production of l-glutamate. Five C. glutamicum deletion mutants were isolated by two rounds of selection for homologous recombination and identified by Southern blot analysis.
The growth, glucose consumption and glutamate production of the mutants were analyzed and compared with the wild-type ATCC
13032 strain. Double disruption of dtsR1 (encoding a subunit of acetyl-CoA carboxylase complex) and pyc (encoding pyruvate carboxylase) caused efficient overproduction of l-glutamate in C. glutamicum; production was much higher than that of the wild-type strain and ΔdtsR1 strain under glutamate-inducing conditions. In the absence of any inducing conditions, the amount of glutamate produced by
the double-deletion strain ΔdtsR1Δpyc was more than that of the mutant ΔdtsR1. The activity of phosphoenolpyruvate carboxylase (PEPC) was found to be higher in the ΔdtsR1Δpyc strain than in the ΔdtsR1 strain and the wild-type strain. Therefore, PEPC appears to be an important anaplerotic enzyme for glutamate synthesis in
ΔdtsR1 derivatives. Moreover, this conclusion was confirmed by overexpression of ppc and pyc in the two double-deletion strains (ΔdtsR1Δppc and ΔdtsR1Δpyc), respectively. Based on the data generated in this investigation, we suggest a new method that will improve glutamate production
strains and provide a better understanding of the interaction(s) between the anaplerotic pathway and fatty acid synthesis. 相似文献
4.
Pyrimidine biosynthesis was investigated in Pseudomonas cepacia ATCC 17759. The presence of the de novo pyrimidine biosynthetic pathway enzyme activities was confirmed in this strain. Following transposon mutagenesis of the wild-type cells, a mutant strain deficient for orotidine 5-monophosphate decarboxylase activity (pyrF) was isolated. Uracil, cytosine or uridine supported the growth of this mutant. Uracil addition to minimal medium cultures of the wild-type strain diminished the levels of the de novo pyrimidine biosynthetic enzyme activities, while pyrimidine limitation of the mutant cells increased those de novo enzyme activities measured. It was concluded that regulation of pyrimidine biosynthesis at the lelel of enzyme synthesis in P. cepacia was present. Aspartate transcarbamoylase activity was found to be regulated in the wild-type cells. Its activity was shown to be controlled in vitro by inorganic pyrophosphate, adenosine 5-triphosphate and uridine 5-phosphate. 相似文献
5.
In studying the pyrimidine synthesising pathway in Deinococcus radiophilus two instances of anomalous behaviour were observed. One was the strikingly different results obtained for two types of assay
for carbamoyl phosphate synthetase. Both depend on the fixation of 14C from the substrate bicarbonate to give radioactive products. In the coupled assay the carbamoyl phosphate product of the
enzyme is converted to carbamoyl aspartate in the presence of aspartate and aspartate transcarbamoylase. In the direct assay
aspartate is omitted from the reaction mixture and the carbamoyl phosphate is converted to urea. It was found that the radioactive
counts in the direct assay were about 5% of those measured in the coupled assay. The second anomaly was that omission of glutamine
from both assay mixtures had no significant effect on the fixation of radioactive carbon. These results suggested that aspartate
amino-N could be the source of nitrogen for glutamine synthesis by a substrate-channelled pathway which delivered glutamine
to carbamoyl phosphate synthetase, and that externally added glutamine could not access its binding site on the enzyme. 相似文献
6.
The foodborne pathogen Bacillus cereus can form biofilms on various food contact surfaces, leading to contamination of food products. To study the mechanisms of
biofilm formation by B. cereus, a Tn5401 library was generated from strain UW101C. Eight thousand mutants were screened in EPS, a low nutrient medium. One mutant
(M124), with a disruption in codY, developed fourfold less biofilm than the wild-type, and its defective biofilm phenotype was rescued by complementation.
Addition of 0.1% casamino acids to EPS prolonged the duration of biofilms in the wild-type but not codY mutant. When decoyinine, a GTP synthesis inhibitor, was added to EPS, biofilm formation was decreased in the wild-type but
not the mutant. The codY mutant produced three times higher protease activity than the wild-type. Zymogram and SDS-PAGE data showed that production
of the protease (∼130 kDa) was repressed by CodY. Addition of proteinase K to EPS decreased biofilm formation by the wild-type.
Using a dpp-lacZ fusion reporter system, it was shown that that the B. cereus CodY can sense amino acids and GTP levels. These data suggest that by responding to amino acids and intracellular GTP levels
CodY represses production of an unknown protease and is involved in biofilm formation. 相似文献
7.
We investigated chilling-induced changes in ethylene levels in Arabidopsis to find plants with distinct patterns of ethylene
production in the cold-related biosynthetic pathway. The sensitive mutants identified here includedchs1-2,chs4-2, andchs6-2. Among these, plants of thechs4-2 mutant produced more ethylene than did the wild type after both were transferred from 4°C or 10°C to 22°C. This mutant also
showed less freezing tolerance and more electrolyte leakage than the wild-type plants. Our results suggest a relationship
between ethylene biosynthesis and chilling sensitivity in the mutant To determine which of the enzymes involved in ethylene
biosynthesis were induced by chilling, we tested the activities of ACC synthase and ACC oxidase in both mutant and wild-type
plants, and found greater activity by ACC synthase as well as a higher ACC content in the mutants after all the plants were
transferred from 10°C to 22°C. However, ACC oxidase activity did not differ between mutant and wild-type plants in response
to chilling treatment Therefore, we conclude thatchs4-2 mutants produce more ethylene than do other mutants or the wild type during their recovery from chilling conditions. Furthermore,
we believe that ACC synthase is the key enzyme involved in this response. 相似文献
8.
9.
Corynebacterium ammoniagenes N424 was metabolically modified to isolate overproducers of deoxycytidine. Inosine auxotrophy (ino) was initially introduced
to prevent the flow of PRPP (phosphoribosyl pyrophosphate) into the purine biosynthetic pathway by random mutagenesis using
N-methyl-N′-nitro-N-nitrosoguanidine. Following that, mutants possessing hydroxyurea resistance (HUr) were isolated to increase the activity of ribonucleoside diphosphate reductase, which catalyzes the reduction of ribonucleoside
diphosphate to deoxyribonucleoside diphosphate. Then, in order to block the flow of dCTP into the TMP biosynthetic pathway
via dUTP, thymine auxotrophy (thy−) was introduced into the mutant IH30 with ino− and Hlf. The resulting mutant IM7, possessing the characteristics of ino−, HUr, and thy−, was deficient in dCTP deaminase and produced significantly higher amounts of deoxycytidine (81.3 mg/L) compared to its mother
strain IH30 (6.2 mg/L). Deoxycytidine productivity was further enhanced by isolating the mutant IU19, which was resistant
to 5-fluorouracil, an inhibitor of carbamoyl phosphate synthase. This enzyme catalyzed the synthesis of carbamoyl phosphate
from glutamine, HCO3−, and ATP. 5-Fluorouracil also inhibited aspartate trans-carbamoylase, catalyzeing the condensation of carbamoyl phosphate
and aspartate. Finally, 5-fluorocytosine resistance (FCr) was introduced into the mutant strain IU19 to relieve the repression caused by accumulation of pyrimidine nucleosides. The
mutant strain IC14-C6 possessing all the five characteristics described above produced 226.3 mg/L of deoxycytidine, which
was at least 2,000 fold higher compared to the wild type, and accumulated only a negligible amount of other pyrimidines under
shake flask fermentation. 相似文献
10.
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
11.
Thomas P. West 《Archives of microbiology》1994,162(1-2):75-79
The five de novo enzyme activities unique to the pyrimidine biosynthetic pathway were found to be present in Pseudomonas pseudoalcaligenes ATCC 17440. A mutant strain with 31-fold reduced orotate phosphoribosyltransferase (encoded by pyrE) activity was isolated that exhibited a pyrimidine requirement for uracil or cytosine. Uptake of the nucleosides uridine or cytidine by wild-type or mutant cells was not detectable; explaining the inability of the mutant strain to utilize either nucleoside to satisfy its pyrimidine requirement. When the wildtype strain was grown in the presence of uracil, the activities of the five de novo enzymes were depressed. Pyrimidine limitation of the mutant strain led to the increase in aspartate transcarbamoylase and dihydroorotate dehydrogenase activities by more than 3-fold, and dihydroorotase and orotidine 5-monophosphate decarboxylase activities about 1.5-fold, as compared to growth with excess uracil. It appeared that the syntheses of the de novo enzymes were regulated by pyrimidines. In vitro regulation of aspartate transcarbamoylase activity in P. pseudoalcaligenes ATCC 17440 was investigated using saturating substrate concentrations; transcarbamoylase activity was inhibited by Pi, PPi, uridine ribonucleotides, ADP, ATP, GDP, GTP, CDP, and CTP. 相似文献
12.
Soon-Yong Choi Hee Yun Park Aron Paek Gil Seob Kim Seong Eun Jeong 《Molecules and cells》2009,28(6):575-581
Ornithine decarboxylase (ODC) is a rate-limiting enzyme in the biosynthesis of polyamines, which are essential for cell growth,
differentiation, and proliferation. This report presents the characterization of an ODC-encoding cDNA (SlitODC) isolated from
a moth species, the tobacco cutworm, Spodoptera litura (Lepidoptera); its expression in a polyamine-deficient strain of yeast, S. cerevisiae; and the recovery in polyamine levels and proliferation rate with the introduction of the insect enzyme. SlitODC encodes 448 amino acid residues, 4 amino acids longer than B. Mori ODC that has 71% identity, and has a longer C-terminus, consistent with B. mori ODC, than the reported dipteran enzymes. The null mutant yeast strain in the ODC gene, SPE1, showed remarkably depleted polyamine levels; in putrescine, spermidine, and spermine, the levels were > 7, > 1, and > 4%,
respectively, of the levels in the wild-type strain. This consequently caused a significant arrest in cell proliferation of
> 4% of the wild-type strain in polyaminefree media. The transformed strain, with the substituted SlitODC for the deleted endogenous ODC, grew and proliferated rapidly at even a higher rate than the wild-type strain. Furthermore,
its polyamine content was significantly higher than even that in the wild-type strain as well as the spe1-null mutant, particularly with a very continuously enhanced putrescine level, reflecting no inhibition mechanism operating
in the putrescine synthesis step by any corresponding insect ODC antizymes to SlitODC in this yeast system. 相似文献
13.
Cardoso PG Ribeiro JB Teixeira JA de Queiroz MV de Araújo EF 《Journal of industrial microbiology & biotechnology》2008,35(3):159-166
The pectin lyase (PL) is an industrially important enzyme since it is used for maceration and clarification in the process
of fruit juice production in food industries. In order to increase the yields of pectin lyase we cloned the plg1 (pectin lyase 1) from Penicillium griseoroseum gene under the control of the strong constitutive promoter of the glyceraldehyde-3-phosphate dehydrogenase gene (gpdA) and the terminator region of the tryptophan synthetase (trpC) gene from Aspergillus nidulans (plasmid pAN52-Plg1) and transformed this construct into the P. griseoroseum strain PG63. One of the pAN52-Plg1 multi-copy transformants (strain 105) grown in culture medium containing glucose or sugar
cane juice showed PL activities of 4,804 or 5,202 U ml−1 respectively, which represented 57- and 132-fold increases. In addition, the apparent specific activity of PL produced by
this strain was much higher than the one observed for a commercial pectinase preparation. Evaluation of the extracellular
proteins in the culture supernatant of strain 105 by SDS-PAGE showed the presence of a clear and strong band of approximately
40 kDa that probably corresponds to PL. The enzyme yields reported here demonstrate that the system we developed is able to
express pectin lyase at levels comparable to, or exceeding, previously reported data. 相似文献
14.
Shockey J Chapital D Gidda S Mason C Davis G Klasson KT Cao H Mullen R Dyer J 《Applied microbiology and biotechnology》2011,92(6):1207-1217
Saccharomyces cerevisiae is frequently used as a bioreactor for conversion of exogenously acquired metabolites into value-added products, but has
not been utilized for bioconversion of low-cost lipids such as triacylglycerols (TAGs) because the cells are typically unable
to acquire these lipid substrates from the growth media. To help circumvent this limitation, the Yarrowia lipolytica lipase 2 (LIP2) gene was cloned into S. cerevisiae expression vectors and used to generate S. cerevisiae strains that secrete active Lip2 lipase (Lip2p) enzyme into the growth media. Specifically, LIP2 expression was driven by the S. cerevisiae PEX11 promoter, which maintains basal transgene expression levels in the presence of sugars in the culture medium but is rapidly
upregulated by fatty acids. Northern blotting, lipase enzyme activity assays, and gas chromatographic measurements of cellular
fatty acid composition after lipid feeding all confirmed that cells transformed with the PEX11 promoter–LIP2 construct were responsive to lipids in the media, i.e., cells expressing LIP2 responded rapidly to either free fatty acids or TAGs and accumulated high levels of the corresponding fatty acids in intracellular
lipids. These data provided evidence of the creation of a self-regulating positive control feedback loop that allows the cells
to upregulate Lip2p production only when lipids are present in the media. Regulated, autonomous production of extracellular
lipase activity is a necessary step towards the generation of yeast strains that can serve as biocatalysts for conversion
of low-value lipids to value-added TAGs and other novel lipid products. 相似文献
15.
Exposure of cells to ultraviolet radiation (UVR) is one of the best studied and most used model system for the examination
of the biological effects of DNA damage, its repair and tolerance. The major product after UVR treatment is cyclobutane pyrimidine
dimer (TT, TC, CC). Pyrimidine dimers are repaired by a direct reversal called photoreactivation or by excision of damage
in a process of nucleotide excision repair. Several methods have been developed for the detection and quantification of pyrimidine
dimers in DNA. The technique of Small and Greimann, in which DNA is incubated with the pyrimidine dimer-specific endonuclease,
was used for the analysis of mutant strains with impaired excision repair system of the unicellular green alga Chlamydomonas reinhardtii. Another method is based on the binding of specific monoclonal antibodies to pyrimidine dimers. The aim of our work was to
compare these two techniques with the use of mutant strains of C. reinhardtii — uvsX1 and uvsX2 which are assumed to be deficient in DNA damage recognition. One of their traits was sensitivity to UVR which could be caused
by breakdown of the excision repair pathway. The results suggest that the immuno-approach is suitable for the detection of
DNA damage induced by UVR.
Presented at the International Symposium Biology and Taxonomy of Green Algae V, Smolenice, June 26–29, 2007, Slovakia. 相似文献
16.
The operon of the anabolic pyruvate oxidoreductase (POR) of Methanococcus maripaludis encodes two genes (porEF) whose functions are unknown. Because these genes possess sequence similarity to polyferredoxins, they may be electron carriers to the POR. To elucidate whether the methanococcal POR requires PorEF for activity, a deletion mutant, strain JJ150, lacking porEF was constructed. Compared to the wild-type strain JJ1, the mutant grew more slowly in minimal medium and minimal plus acetate medium, and pyruvate-dependent methanogenesis was inhibited. In contrast, the methyl-viologen-dependent pyruvate-oxidation activity of POR, carbon monoxide dehydrogenase, and hydrogenase activities of the mutant were similar to those of the wild-type. Upon genetic complementation of the mutant with porEF in the methanococcal shuttle vector pMEV2+porEF, growth in minimal medium and pyruvate-dependent methanogenesis were restored to wild-type levels. Complementation with porE alone restored methanogenesis from pyruvate but not growth in minimal medium. Complementation with porF alone partially restored growth but not methanogenesis from pyruvate. Although the specific roles of porE and porF have not been determined, these results suggest that PorEF play important roles in the anabolic POR in vivo even though they are not required for the dye-dependent activity.Abbreviations
CODH/ACS
Carbon monoxide dehydrogenase/acetyl-CoA synthase
-
POR
Pyruvate oxidoreductase 相似文献
17.
A major limitation on the expression of some foreign proteins in transgenic plants is the toxic effect of such proteins on
the host plant resulting in inhibition of normal growth and development. A solution to this problem is to control the expression
of genes for such proteins by means of inducible promoters, as is frequently done in microbial systems. A cDNA clone was obtained
from subtractive hybridization of non-harvested and harvested alfalfa leaf tissue, named hi12. The hi12 cDNA was identified as part of the S-adenosyl-l-methionine: trans-caffeoyl-CoA3-O-methyltransferase gene of alfalfa, a gene encoding an essential key enzyme in lignin synthesis. The hi12 gene was strongly induced by harvesting and wounding but not by heat shock. The promoter of the hi12 gene, isolated by genomic walking, contained several stress response cis-elements. Transgenic plants of tobacco and Medicago truncatula containing the GUS gene driven by the promoter showed GUS expression following harvesting, demonstrating the activity of
these regulatory regions in other plant species. 相似文献
18.
Shahin Negari Jeff Sulpher Francesca Pacello Keely Ingrey Andrea Battistoni B. Craig Lee 《Biometals》2008,21(3):249-258
The Cu,Zn superoxide dismutase (Cu,ZnSOD) from Haemophilus
ducreyi is the only enzyme of this class which binds a heme molecule at its dimer interface. To explore the role of the enzyme in
this heme-obligate bacterium, a sodC mutant was created by insertional inactivation. No difference in growth rate was observed during heme limitation. In contrast,
under heme rich conditions growth of the sodC mutant was impaired compared to the wild type strain. This growth defect was abolished by supplementation of exogenous catalase.
Genetic complementation of the sodC mutant in trans demonstrated that the enzymatic property or the heme-binding activity of the protein could repair the growth
defect of the sodC mutant. These results indicate that Cu,ZnSOD protects Haemophilus ducreyi from heme toxicity. 相似文献
19.
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
20.
Biotransformation of flavonoids using Escherichia coli harboring nucleotide sugar-dependent uridine diphosphate-dependent glycosyltransferases (UGTs) commonly results in the production
of a glucose conjugate because most UGTs are specific for UDP-glucose. The Arabidopsis enzyme AtUGT78D2 prefers UDP-glucose as a sugar donor and quercetin as a sugar acceptor. However, in vitro, AtUGT78D2 could
use UDP-N-acetylglucosamine as a sugar donor, and whole cell biotransformation of quercetin using E. coli harboring AtUGT78D2 produced quercetin 3-O-N-acetylglucosamine. In order to increase the production of quercetin 3-O-N-acetylglucosamine via biotransformation, two E. coli mutant strains deleted in phosphoglucomutase (pgm) or glucose-1-phosphate uridylyltransferase (galU) were created. The galU mutant produced up to threefold more quercetin 3-O-N-acetylglucosamine than wild type, resulting in the production of 380-mg/l quercetin 3-O-N-acetylglucosamine and a negligible amount of quercetin 3-O-glucoside. These results show that construction of bacterial strains for the synthesis of unnatural flavonoid glycosides
is possible through rational selection of the nucleotide sugar-dependent glycosyltransferase and engineering of the nucleotide
sugar metabolic pathway in the host strain. 相似文献