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1.
We assess the microbial assay-dependent effect of AgNP on gram-negative Escherichia coli and gram-positive Bacillus subtilis. The experiment was conducted via three different assays: a growth inhibition assay, a colony forming unit assay, and a liquid-to-plate
assay. AgNP were exposed either as liquid suspensions or in an agar state. Bacterial sensitivity to AgNP was found to be dependent
on the microbial assay employed. E. coli was more sensitive than B. subtilis in the growth inhibition and CFU assays, but B. subtilis was more vulnerable than E. coli in the liquid-to-plate assay, ostensibly owing to the food stress mechanisms of B. subtilis in exposure medium. The dissolution of silver from AgNP could not explain the observed toxicity of AgNP. We detected clear
evidence of AgNP uptake by cells. The results of this study showed that the microbial toxicity of AgNP and the effects of
dissolved silver ions were influenced profoundly by the microbial test method employed. 相似文献
2.
Nattokinase producing bacterium, B. subtilis YF38, was isolated from douchi, using the fibrin plate method. The gene encoding this enzyme was cloned by polymerase chain reaction (PCR). Cytoplasmic
expression of this enzyme in E. coli resulted in inactive inclusion bodies. But with the help of two different signal peptides, the native signal peptide of nattokinase
and the signal peptide of PelB, active nattokinase was successfully expressed in E. coli with periplasmic secretion, and the nattokinase in culture medium displayed high fibrinolytic activity. The fibrinolytic
activity of the expressed enzyme in the culture was determined to reach 260 urokinase units per micro-liter when the recombinant
strain was induced by 0.7 mmol l−1 isopropyl-β-D- thiogalactopyranoside (IPTG) at 20°C for 20 h, resulting 49.3 mg active enzyme per liter culture. The characteristic
of this recombinant nattokinase is comparable to the native nattokinase from B. subtilis YF38. Secretory expression of nattokinase in E. coli would facilitate the development of this enzyme into a therapeutic product for the control and prevention of thrombosis diseases. 相似文献
3.
Joong-Chul Lee Jeong-Heon Cha Dennis B. Zerbv George C. Stewart 《Current microbiology》2003,47(2):0146-0152
The divIVB operon of Bacillus subtilis includes the cell shape-associated mre genes, including the membrane-associated proteins MreC and MreD. TnphoA mutagenesis was utilized to analyze a topological model for MreC. MreC has a short cytoplasmic amino terminus, a single membrane-spanning domain, and a large carboxy terminal domain which lies externally to the outer leaflet of the cell membrane. Expression of the B. subtilis MreB protein, or the Mre C and D proteins, results in a morphological conversion of the Escherichia coli host cells from a rod to a roughly spherical cell, morphologically similar to mre-negative mutants of E. coli. Immunolocalization of the MreC protein in B. subtilis revealed that this protein is found at the midcell division site of the bacterial cells, consistent with the postulated role of the Mre proteins in the regulation of septum-specific peptidoglycan synthesis. RID= ID= <E5>Correspondence to: </E5>G.C. Stewart; <E5>email:</E5> stewart@vet.ksu.edu Received: 5 August 2002 / Accepted: 7 October 2002 相似文献
4.
5.
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
6.
Lee YK Yoon BD Yoon JH Lee SG Song JJ Kim JG Oh HM Kim HS 《Applied microbiology and biotechnology》2007,75(3):567-572
The srfA operon is required for the nonribosomal biosynthesis of the cyclic lipopeptide, surfactin. The srfA operon is composed of the four genes, srfAA, srfAB, srfAC, and srfAD, encoding the surfactin synthetase subunits, plus the sfp gene that encodes phosphopantetheinyl transferase. In the present study, 32 kb of the srfA operon was amplified from Bacillus subtilis C9 using a long and accurate PCR (LA-PCR), and ligated into a pIndigoBAC536 vector. The ligated plasmid was then transformed
into Escherichia coli DH10B. The transformant ET2 showed positive signals to all the probes for each open reading frame (ORF) region of the srfA operon in southern hybridization, and a reduced surface tension in a culture broth. Even though the surface-active compound
extracted from the E. coli transformant exhibited a different R
f value of 0.52 from B. subtilis C9 or authentic surfactin (R
f = 0.63) in a thin layer chromatography (TLC) analysis, the transformant exhibited a much higher surface-tension-reducing
activity than the wild-type strain E. coli DH10B. Thus, it would appear that an intermediate metabolite of surfactin was expressed in the E. coli transformant harboring the srfA operon. 相似文献
7.
Background
Phospholipid biosynthesis commences with the acylation of glycerol-3-phosphate (G3P) to form 1-acyl-G3P. This step is catalyzed by the PlsB protein in Escherichia coli. The gene encoding this protein has not been identified, however, in the majority of bacterial genome sequences, including that of Bacillus subtilis. Recently, a new two-step pathway catalyzed by PlsX and PlsY proteins for the initiation of phospholipid formation in Streptococcus pneumoniae has been reported. 相似文献8.
D. V. Karelov R. A. Kreneva L. Errais Lopes D. A. Perumov A. S. Mironov 《Russian Journal of Genetics》2011,47(6):757-761
The nucleotide sequence of the ribC gene encoding the synthesis of bifunctional flavokinase/flavine adenine nucleotide (FAD) synthetase in Bacillus subtilis have been determined in a family of riboflavinconstitutive mutants. Two mutations have been found in the proximal region
of the gene, which controls the transferase (FAD synthase) activity. Three point mutations and one double mutation have been
found (in addition to the two mutations that were detected earlier) in the distal region of the gene, which controls the flavokinase
(flavin mononucleotide (FMN) synthase) activity. On the basis of all data known to date, it has been concluded that the identified
mutations affect riboflavin and ATP binding sites. No mutations have been found in the PTAN conserved sequence, which forms
the magnesium and ATP common binding site and is identical for organisms of all organizational levels, from bacteria too humans. 相似文献
9.
Xiaoyan Liu Donghai Peng Yi Luo Lifang Ruan Ziniu Yu Ming Sun 《Applied microbiology and biotechnology》2009,82(4):765-772
Shuttle vectors for Bacillus thuringiensis or Bacillus cereus usually cannot hold fragments larger than 20 kb. With the development of genome research, shuttle vectors with higher loading
capacity are necessary. We constructed an Escherichia coli to B. thuringiensis shuttle vector, pEMB0557, with a large loading capacity. This vector incorporated the ori60 replicon from B. thuringiensis subsp. kurstaki YBT-1520, erythromycin resistance (B. thuringiensis), and chloromycetin resistance (E. coli) genes. A bacterial artificial chromosome library of B. thuringiensis strain CT-43 was constructed and pEMB0557 was able to accommodate at least a 70-kb DNA fragment. Simultaneously, the cry1B gene on a 40-kb fragment could express a 140-kDa protein in plasmid-cured B. thuringiensis BMB171. Due to its high capacity and utility in expressing exogenous genes, pEMB0557 will be useful in cloning (especially
silencing genes) and expressing large DNA fragments (e.g., gene clusters) in B. thuringiensis. Plasmid pEMB0557 provides a new tool for B. thuringiensis genome or B. cereus group research. 相似文献
10.
The effects of cell wall mutation on the oxygenation of linoleic acid (M.W. 280) by recombinant Escherichia coli expressing the CYP102A2 gene encoding self-sufficient P450 monooxygenase of Bacillus subtilis was investigated. After the CYP102A2 gene was heterologously expressed in E. coli W3110 and its isogenic lipopolysaccharide (LPS) structural mutant strains, their whole-cell biotransformation activities
were compared. The mutants used in this study had previously been designated as MLK53, MLK1067, and MLK986. These strains
carry one or two defined mutations in the secondary acyl fatty acids of the LPS lipid A constituent. The CYP102A2 gene was
overexpressed in both wild type E. coli W3110 and its mutant strains, with the specific activity ranging from 1.7 to 2.1 U/mg protein. Interestingly, the whole-cell
biotransformation activity of those recombinant biocatalysts differed significantly. Indeed, MLK986 possessing the tetraacylated
LPS showed a higher oxygenation activity of linoleic acid than those in wild type or other mutant strains having hexa- or
penta-acylated LPSs. These results suggest that the biotransformation efficiency of E. coli-based biocatalysts, especially for medium- to large-sized lipophilic organic substrates, can be enhanced via engineering their LPS, which is known to function as a formidable barrier for hydrophobic molecules. 相似文献
11.
In the Tat protein export pathway of Gram-negative bacteria, TatA and TatB are homologous proteins that carry out distinct
and essential functions in separate sub-complexes. In contrast, Gram-positive Tat systems usually lack TatB and the TatA protein
is bifunctional. We have used a mutagenesis approach to delineate TatA/B-type domains in the bifunctional TatAd protein from
Bacillus subtilis. This involved expression of mutated TatAd variants in Escherichia coli and tests to determine whether the variants could function as TatA or TatB by complementing E. coli
tatA and/or tatB mutants. We show that mutations in the C-terminal half of the transmembrane span and the subsequent FGP ‘hinge’ motif are
critical for TatAd function with its partner TatCd subunit, and the same determinants are required for complementation of
either tatA or tatB mutants in Escherichia coli. This is thus a critical domain in both TatA and TatB proteins. In contrast, substitution of a series of residues at the
N-terminus specifically blocks the ability of TatAd to substitute for E. coli TatB. The results point to the presence of a universally conserved domain in the TatA/B-family, together with a separate
N-terminal domain that is linked to the TatB-type function in Gram-negative bacteria. 相似文献
12.
Xin Zhou Zhengping Zhang Xiaohe Jia Yifan Wu Lan Luo Zhimin Yin 《World journal of microbiology & biotechnology》2008,24(8):1267-1272
Bacillus subtilis glutamine synthetase (GS) was highly expressed (about 86% of total protein) as soluble protein in Escherichia coli BL21(DE3) containing pET28a-glnA, which was induced by 0.4 mM IPTG in LB medium, and maximal theanine-forming activity of
the recombinant GS induced in LB is 6.4 U/mg at a series concentration (0–100 mM) of Mn2+ at optimal pH 7.5. In order to get GS with high theanine-forming activity, safety, and low cost for food and pharmaceutics
industry, M9-A (details are described in “Materials and methods”) and 0.1% (w/v) lactose were selected as culture medium and
inducer respectively. Recombinant GS was also highly expressed (84% of total protein) and totally soluble in M9-A and the
specific activity of the recombinant GS is 6.2 U/mg which is approximate to that (6.4 U/mg) induced in LB in the presence
of 10 mM Mn2+ at optimal pH 7.5. The activity is markedly higher activated by Mn2+ than that by other nine bivalent cations. Furthermore, M9-B (5 μM Mn2+ was added into M9-A) was used to culture the recombinant strain and theanine-forming activity of the recombinant GS induced
in M9-B was improved 20% (up to 7.6 U/mg). Finally, theanine production experiment coupled with yeast fermentation system
was carried out in a 1.0 ml reaction system with 0.1 mg crude GS from M9-B or M9-A, and the yield of theanine were 15.3 and
13.1 g/L by paper chromatography and HPLC, respectively. 相似文献
13.
Function of the <Emphasis Type="Italic">rel</Emphasis> Gene in <Emphasis Type="Italic">Escherichia coli</Emphasis> 总被引:6,自引:0,他引:6
Sokawa et al. suggest that rel- strains of Escherichia coli possess abnormal protein synthesizing machinery, which cannot carry out normal protein synthesis when the supply of amino-acids is limited. 相似文献
14.
15.
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
16.
Pazlarova J 《Journal of industrial microbiology & biotechnology》2010,37(12):1257-1261
Anomalous forms of Bacillus subtilis A32 produced by prolonged cultivation in a chemostat under nitrogen limitation are described. A change in the cultivation
conditions brought about a transformation of these forms to bacillar rods. The transformation was gradual and lasted for several
generations. 相似文献
17.
To elucidate the physiological adaptation of Escherichia coli due to cra gene knockout, a total of 3,911 gene expressions were investigated by DNA microarray for continuous culture. About 50 genes
were differentially regulated for the cra mutant. TCA cycle and glyoxylate shunt were down-regulated, while pentose phosphate (PP) pathway and Entner Doudoroff (ED)
pathway were up-regulated in the cra mutant. The glucose uptake rate and the acetate production rate were increased with less acetate consumption for the cra mutant. To identify the genes controlled by Cra protein, the Cra recognition weight matrix from foot-printing data was developed
and used to scan the whole genome. Several new Cra-binding sites were found, and some of the result was consistent with the
DNA microarray data. The ED pathway was active in the cra mutant; we constructed cra.edd double genes knockout mutant to block this pathway, where the acetate overflowed due to the down-regulation of aceA,B and icd gene expressions. Then we further constructed cra.edd.iclR triple genes knockout mutant to direct the carbon flow through the glyoxylate pathway. The cra.edd.iclR mutant showed the least acetate production, resulting in the highest cell yield together with the activation of the glycolysis
pathway, but the glucose consumption rate could not be improved.
Dayanidhi Sarkar and Khandaker Al Zaid Siddiquee have contributed equally. 相似文献
18.
COLICINOGENIC factor E1 (Col E1) is a small bacterial plasmid (4.2×106 daltons) present in colicinogenic strains of Escherichia coli1 to the extent of about twenty-four copies per cell (Clewell and Helinski, unpublished results), which continues to replicate in the presence of high levels of chloramphenicol, a specific inhibitor of protein synthesis, although the chromosome only completes current rounds of replication and ceases (Clewell and Helinski, unpublished results). The average rate of Col E1 semiconservative replication in the absence of protein synthesis is, in certain conditions, faster than (as much as eight times) the normal rate of synthesis (Clewell, unpublished results). Replication continues for 10–15 h after the addition of chloramphenicol, resulting in nearly 3,000 copies of Col E1 DNA per cell. We are taking advantage of this system to study the effects of a number of antibiotics on DNA replication and now report evidence that rifampicin (an active semisynthetic derivative of rifamycin B)2, an antibiotic known specifically to inhibit bacterial DNA dependent RNA polymerase3–6, has a dramatic inhibitory effect on Col E1 DNA replication. 相似文献
19.
Natural wild-type strains of Bacillus subtilis spore is regarded as a non-pathogenic for both human and animal, and has been classified as a novel food which is currently
being used as probiotics added in the consumption. To identify B. subtilis spore proteins, we have accomplished a preliminary proteomic analysis of B. subtilis spore, with a combination of two-dimensional electrophoretic separations and matrix-assisted laser desorption ionization
tandem time of flight mass spectrometry (MALDI–TOF–MS). In this article, we presented a reference map of 158 B. subtilis spore proteins with an isoelectric point (pI) between 4 and 7. Followed by mass spectrometry (MS) analysis, we identified
71 B. subtilis spore proteins with high level of confidence. Database searches, combined with hydropathy analysis and GO analysis revealed
that most of the B. subtilis spore proteins were hydrophilic proteins related to catalytic function. These results should accelerate efforts to understand
the resistance of spore to harsh conditions. 相似文献
20.
Cytochrome bd from Escherichia coli is able to oxidize such substrates as guaiacol, ferrocene, benzohydroquinone, and potassium ferrocyanide through the peroxidase
mechanism, while none of these donors is oxidized in the oxidase reaction (i.e. in the reaction that involves molecular oxygen
as the electron acceptor). Peroxidation of guaiacol has been studied in detail. The dependence of the rate of the reaction
on the concentration of the enzyme and substrates as well as the effect of various inhibitors of the oxidase reaction on the
peroxidase activity have been tested. The dependence of the guaiacol-peroxidase activity on the H2O2 concentration is linear up to the concentration of 8 mM. At higher concentrations of H2O2, inactivation of the enzyme is observed. Guaiacol markedly protects the enzyme from inactivation induced by peroxide. The
peroxidase activity of cytochrome bd increases with increasing guaiacol concentration, reaching saturation in the range from 0.5 to 2.5 mM, but then starts falling.
Such inhibitors of the ubiquinol-oxidase activity of cytochrome bd as cyanide, pentachlorophenol, and 2-n-heptyl 4-hydroxyquinoline-N-oxide also suppress its guaiacol-peroxidase activity; in contrast, zinc ions have no influence
on the enzyme-catalyzed peroxidation of guaiacol. These data suggest that guaiacol interacts with the enzyme in the center
of ubiquinol binding and donates electrons into the di-heme center of oxygen reduction via heme b
558, and H2O2 is reduced by heme d. Although the peroxidase activity of cytochrome bd from E. coli is low compared to peroxidases, it might be of physiological significance for the bacterium itself and plays a pathophysiological
role for humans and animals. 相似文献