The cDNA encoding long-chain fatty-acid alcohol oxidase(FAO) was isolated by using RT-PCR method from Candida cloacae grown in a medium containing oleic acid. The cDNA of fatty-acid alcohol oxidase was cloned into exp...The cDNA encoding long-chain fatty-acid alcohol oxidase(FAO) was isolated by using RT-PCR method from Candida cloacae grown in a medium containing oleic acid. The cDNA of fatty-acid alcohol oxidase was cloned into expression plasmid pET17b under T7 promoter and then transformed into E.coli BL21/DE3. The molecular weight of the expressed protein was estimated to be approximately 64 kD by SDS-PAGE. The expressed protein had a specific catalytic activity of fatty-acid alcohol oxidase and its activity was 1090±116 U/ml medium. Northern blotting revealed that there was high expression of fao mRNA in Candida cloacae yeast grown in a medium containing oleic acid.展开更多
Objective To establish a domestic database of Enterobacteria cloacae （E. cloacae）, and improve the identification efficiency using peptide mass fingerprinting. Methods Peptide mass fingerprinting was used for the id...Objective To establish a domestic database of Enterobacteria cloacae （E. cloacae）, and improve the identification efficiency using peptide mass fingerprinting. Methods Peptide mass fingerprinting was used for the identification and subtyping of E. cloacae. Eighty-seven strains, identified based on hsp60 genotyping, were used to construct and evaluate a new reference database. Results Compared with the original reference database, the identification efficiency and accuracy of the new reference database was greatly improved at the species level. The first super reference database for E. cloacae identification was also constructed and evaluated. Based on the super reference database and the main spectra projection dendrogram, E. cloacae strains were divided into two clades. Conclusion Peptide mass fingerprinting is a powerful method to identify and subtype E. cloacae, and the use of this method will allow us to obtain more information to understand the heterogeneous organism E. cloacae.展开更多
Introduction: Enterobacter cloacae strains have been isolated from Eastern Cape hospitalised patients. Methodology: We have molecularly characterised blaOXA-48-, blaIMP- and blaVIM-expressing E. cloacae isolates demon...Introduction: Enterobacter cloacae strains have been isolated from Eastern Cape hospitalised patients. Methodology: We have molecularly characterised blaOXA-48-, blaIMP- and blaVIM-expressing E. cloacae isolates demonstrating resistance to carbapenems from five hospitals by multilocus sequence typing. Organism identification and antimicrobial susceptibility testing was done using automated systems and the isolates were screened for carbapenemases using either conventional or real-time PCR and then typed using multilocus sequence typing. Further characterisation of IMP-type-producing E. cloacae isolates, an unusual occurrence in South Africa, was performed by pulsed-field gel electrophoresis. Results and Conclusion: Twenty-five E. cloacae isolates from 24 patients were investigated. Eighteen (72%) isolates harboured either one of the following genes: blaIMP, blaVIM or blaOXA-48. Multilocus sequence typing data and pulsed-field gel electrophoresis showed that several strains from the same geographical region and hospitals were genetically related.展开更多
Enterobacter cloacae CYS-25 strain was isolated from a chromate plant. This bacterium was capable of resisting high hexavalent chromium concentration and reducing Cr（VI） under aerobic condition. CrO4^2- stimulated t...Enterobacter cloacae CYS-25 strain was isolated from a chromate plant. This bacterium was capable of resisting high hexavalent chromium concentration and reducing Cr（VI） under aerobic condition. CrO4^2- stimulated the increase of bacterial size and production of compact convex paths containing chromium on the bacterial surface. The increase of bacterial size was caused by integrative growth but not extracellular polymeric substance hyperplasia. IR and SDS-PAGE analyses showed the extracellular polymeric substance （EPS） components were mainly proteins and had no obvious changes whether the strains were induced by Cr（VI） or not. The EPS was amorphous and contained trivalent chromium. Under CrO4^2- growth condition, the extracellular substance of Enterobacter cloacae CYS-25 strains and Cr（VI） had redox reaction. The products were Cr^3＊-protein complexes which formed a piece of compact convex paths on the surface of bacteria and prevented Cr（VI） from entering into cells.展开更多
Using the minitransposon pMini-Tn5 and the ice-nucleation active (INA) gene of iceA, a suicide recombinant plasmid pTnice1 was constructed, which has the ability of broad-host-range conjugal mobilization and integrati...Using the minitransposon pMini-Tn5 and the ice-nucleation active (INA) gene of iceA, a suicide recombinant plasmid pTnice1 was constructed, which has the ability of broad-host-range conjugal mobilization and integration of iceA into chromosomal DNA of many gram-negative bacteria by Tn5 transposition. We used this plasmid to integrate the iceA into chromosomal DNA of Ent. cloacae and obtained the transgentic strain Enc1.2022 ina. In this transgenic Ent. cloacae, iceA would never be transferred elsewhere through transposition, and constantly expressed high ice nucleation activity even in the absence of antibiotic pressure. The transgenic strain was ingested by corn borer larvae. Over the 7 d after ingestion, the mean supercooling points (SCPs) of the larvae was about 10℃ higher than those of larvae treated with distilled water (control). The maintenance of these high SCPs was related to the stable gut colonization of transgenic strain. At 6th day post ingestion, the larva was exposed at 5 or 7℃ for 12 h, the percentages of larvae frozen to death were 85 and 100%, respectively. In contrast, none or a small proportion of control larvae was frozen to death under the same conditions. Further studies demonstrated that this transgenic strain bore weak epiphytic ability. Therefore, this genetically engineered strain may be a promising candidate for control of insect pests in agricultural fields.展开更多
Fourier Transform Infrared Spectroscopy (FTIR) was used to quantify total monosaccharide content in the bacterium Enterobacter cloacae and several of its biofilm mutants. Bacterial biofilm samples were grown on trypti...Fourier Transform Infrared Spectroscopy (FTIR) was used to quantify total monosaccharide content in the bacterium Enterobacter cloacae and several of its biofilm mutants. Bacterial biofilm samples were grown on trypticase soy agar, and 30 μL aliquots of aqueous sample bacterial plus biofilm were deposited into the center of barium fluoride crystals and dried at 50°C for 1-hour before being scanned by FTIR. The total amounts of monosaccharides were estimated using the absorbance of the mono-saccharide peak, 1192 - 958 cm–1, and normalized using the amide II peak, 1585 - 1483 cm–1. This method provided a linear correlation between the absorbance of the monosaccharide peak and concentration of monosaccharide in standard monosaccharides, fructose, glucose, mannose, and rhamnose, over a concentration range of 0.5 - 2.0 mg/mL.展开更多
A bacteria strain for the degradation of hydrolyzed polyacrylamide (HPAM) was isolated from a curing pot in HPAM distribution station of Daqing Oilfield using Hungate anaerobic technique.The isolate was investigated f...A bacteria strain for the degradation of hydrolyzed polyacrylamide (HPAM) was isolated from a curing pot in HPAM distribution station of Daqing Oilfield using Hungate anaerobic technique.The isolate was investigated from morphological,physiological,biochemical and molecular characterization.It is a Gram-negative,short-bacillus,non-spore-forming anaerobic bacteria with an optimum growth at 8.0 pH at 40℃.It can reduce sulfate to H2S.Alignment of 16S ribosomal DNA and 16S-23S ribosomal DNA intergenic spacer sequences suggests that this isolate is closely related to the Enterobacter cloacae.The isolate is identified as a new strain belonging to Enterobacter genus,temporarily named as Enterobacter cloacae I7.Analysis results of infrared spectroscopy (IR) show that the bacteria can use HPAM as the only carbon source,change the structure of HPAM polymer surface,and realize the hydrolysis of amide to carboxyl group by hydrolysis mechanism.It can degrade the side chain and change some functional groups,which obviously decreases the viscosity.GC-MS analysis indicates that the determined low-molecular weight degradation products of HPAM are polyacrylamide fragments with duplet bond,epoxy as well as carbonyl group,but most of them are acrylamide oligomer derivatives.展开更多
Expression of the nitrogen fixation (nif ) genes is tightly regulated by two proteins NifA and NifL in the (-subdivision of the proteobacteria. NifA is a transcriptional activator, which can be inactivated by NifL in ...Expression of the nitrogen fixation (nif ) genes is tightly regulated by two proteins NifA and NifL in the (-subdivision of the proteobacteria. NifA is a transcriptional activator, which can be inactivated by NifL in the presence of oxygen or excess fixed nitrogen. A direct interaction between E. cloacae NifL and NifA was detected using the bacterial two-hybrid system. This interaction was accelerated in the presence of fixed nitrogen, while oxygen had no effect. NifL proteins, with their C-terminus being deleted, completely lost the ability to interact with NifA. The data suggest that the C-terminal domain of NifL acts as a sensor of the nitrogen status of the cell and mediates interaction with NifA.展开更多
文摘The cDNA encoding long-chain fatty-acid alcohol oxidase(FAO) was isolated by using RT-PCR method from Candida cloacae grown in a medium containing oleic acid. The cDNA of fatty-acid alcohol oxidase was cloned into expression plasmid pET17b under T7 promoter and then transformed into E.coli BL21/DE3. The molecular weight of the expressed protein was estimated to be approximately 64 kD by SDS-PAGE. The expressed protein had a specific catalytic activity of fatty-acid alcohol oxidase and its activity was 1090±116 U/ml medium. Northern blotting revealed that there was high expression of fao mRNA in Candida cloacae yeast grown in a medium containing oleic acid.
基金supported by the Mega Project of Research on the Prevention and Control of HIV/AIDS,Viral Hepatitis Infectious Diseases 2011ZX10004-001,2013ZX10004-101 to YE Chang Yun
文摘Objective To establish a domestic database of Enterobacteria cloacae （E. cloacae）, and improve the identification efficiency using peptide mass fingerprinting. Methods Peptide mass fingerprinting was used for the identification and subtyping of E. cloacae. Eighty-seven strains, identified based on hsp60 genotyping, were used to construct and evaluate a new reference database. Results Compared with the original reference database, the identification efficiency and accuracy of the new reference database was greatly improved at the species level. The first super reference database for E. cloacae identification was also constructed and evaluated. Based on the super reference database and the main spectra projection dendrogram, E. cloacae strains were divided into two clades. Conclusion Peptide mass fingerprinting is a powerful method to identify and subtype E. cloacae, and the use of this method will allow us to obtain more information to understand the heterogeneous organism E. cloacae.
文摘Introduction: Enterobacter cloacae strains have been isolated from Eastern Cape hospitalised patients. Methodology: We have molecularly characterised blaOXA-48-, blaIMP- and blaVIM-expressing E. cloacae isolates demonstrating resistance to carbapenems from five hospitals by multilocus sequence typing. Organism identification and antimicrobial susceptibility testing was done using automated systems and the isolates were screened for carbapenemases using either conventional or real-time PCR and then typed using multilocus sequence typing. Further characterisation of IMP-type-producing E. cloacae isolates, an unusual occurrence in South Africa, was performed by pulsed-field gel electrophoresis. Results and Conclusion: Twenty-five E. cloacae isolates from 24 patients were investigated. Eighteen (72%) isolates harboured either one of the following genes: blaIMP, blaVIM or blaOXA-48. Multilocus sequence typing data and pulsed-field gel electrophoresis showed that several strains from the same geographical region and hospitals were genetically related.
基金This work was supported by the National Natural Science Foundation of China (20501020, 40772034) Nanoscience Foundation of China (90406024)+2 种基金Natural Science Foundation of Fujian Province (No. X0650094/2006J0383)973 program (2007CB815601) the Special Project on Science and Technology of Fujian Province (2005YZ1026)
文摘Enterobacter cloacae CYS-25 strain was isolated from a chromate plant. This bacterium was capable of resisting high hexavalent chromium concentration and reducing Cr（VI） under aerobic condition. CrO4^2- stimulated the increase of bacterial size and production of compact convex paths containing chromium on the bacterial surface. The increase of bacterial size was caused by integrative growth but not extracellular polymeric substance hyperplasia. IR and SDS-PAGE analyses showed the extracellular polymeric substance （EPS） components were mainly proteins and had no obvious changes whether the strains were induced by Cr（VI） or not. The EPS was amorphous and contained trivalent chromium. Under CrO4^2- growth condition, the extracellular substance of Enterobacter cloacae CYS-25 strains and Cr（VI） had redox reaction. The products were Cr^3＊-protein complexes which formed a piece of compact convex paths on the surface of bacteria and prevented Cr（VI） from entering into cells.
基金This work was supported by the National Natural Science Foundation of China(30170624).
文摘Using the minitransposon pMini-Tn5 and the ice-nucleation active (INA) gene of iceA, a suicide recombinant plasmid pTnice1 was constructed, which has the ability of broad-host-range conjugal mobilization and integration of iceA into chromosomal DNA of many gram-negative bacteria by Tn5 transposition. We used this plasmid to integrate the iceA into chromosomal DNA of Ent. cloacae and obtained the transgentic strain Enc1.2022 ina. In this transgenic Ent. cloacae, iceA would never be transferred elsewhere through transposition, and constantly expressed high ice nucleation activity even in the absence of antibiotic pressure. The transgenic strain was ingested by corn borer larvae. Over the 7 d after ingestion, the mean supercooling points (SCPs) of the larvae was about 10℃ higher than those of larvae treated with distilled water (control). The maintenance of these high SCPs was related to the stable gut colonization of transgenic strain. At 6th day post ingestion, the larva was exposed at 5 or 7℃ for 12 h, the percentages of larvae frozen to death were 85 and 100%, respectively. In contrast, none or a small proportion of control larvae was frozen to death under the same conditions. Further studies demonstrated that this transgenic strain bore weak epiphytic ability. Therefore, this genetically engineered strain may be a promising candidate for control of insect pests in agricultural fields.
文摘Fourier Transform Infrared Spectroscopy (FTIR) was used to quantify total monosaccharide content in the bacterium Enterobacter cloacae and several of its biofilm mutants. Bacterial biofilm samples were grown on trypticase soy agar, and 30 μL aliquots of aqueous sample bacterial plus biofilm were deposited into the center of barium fluoride crystals and dried at 50°C for 1-hour before being scanned by FTIR. The total amounts of monosaccharides were estimated using the absorbance of the mono-saccharide peak, 1192 - 958 cm–1, and normalized using the amide II peak, 1585 - 1483 cm–1. This method provided a linear correlation between the absorbance of the monosaccharide peak and concentration of monosaccharide in standard monosaccharides, fructose, glucose, mannose, and rhamnose, over a concentration range of 0.5 - 2.0 mg/mL.
基金Sponsored by the Country from Branch Fund Significant International Cooperation Item(Grant No.50521140075)
文摘A bacteria strain for the degradation of hydrolyzed polyacrylamide (HPAM) was isolated from a curing pot in HPAM distribution station of Daqing Oilfield using Hungate anaerobic technique.The isolate was investigated from morphological,physiological,biochemical and molecular characterization.It is a Gram-negative,short-bacillus,non-spore-forming anaerobic bacteria with an optimum growth at 8.0 pH at 40℃.It can reduce sulfate to H2S.Alignment of 16S ribosomal DNA and 16S-23S ribosomal DNA intergenic spacer sequences suggests that this isolate is closely related to the Enterobacter cloacae.The isolate is identified as a new strain belonging to Enterobacter genus,temporarily named as Enterobacter cloacae I7.Analysis results of infrared spectroscopy (IR) show that the bacteria can use HPAM as the only carbon source,change the structure of HPAM polymer surface,and realize the hydrolysis of amide to carboxyl group by hydrolysis mechanism.It can degrade the side chain and change some functional groups,which obviously decreases the viscosity.GC-MS analysis indicates that the determined low-molecular weight degradation products of HPAM are polyacrylamide fragments with duplet bond,epoxy as well as carbonyl group,but most of them are acrylamide oligomer derivatives.
基金This work was supported by a grant in aid of the Shanghai Institutes of Biological Sciences, the Chinese Academy of Sciences "973" Project of the Ministry of Science and Technology, China (Grant No. 2001CB108901)
文摘Expression of the nitrogen fixation (nif ) genes is tightly regulated by two proteins NifA and NifL in the (-subdivision of the proteobacteria. NifA is a transcriptional activator, which can be inactivated by NifL in the presence of oxygen or excess fixed nitrogen. A direct interaction between E. cloacae NifL and NifA was detected using the bacterial two-hybrid system. This interaction was accelerated in the presence of fixed nitrogen, while oxygen had no effect. NifL proteins, with their C-terminus being deleted, completely lost the ability to interact with NifA. The data suggest that the C-terminal domain of NifL acts as a sensor of the nitrogen status of the cell and mediates interaction with NifA.
