2013, 53(2):111-117.
Abstract:Abstract:In most Gram-negative bacteria,the Kdo2-lipid A moiety of lipopolysaccharide forms the outer monolayer of the outer membrane.Diverse covalent modifications of Kdo2-lipid A may occur in bacteria to survive different environmental conditions.Kdo2-lipid A is usually synthesized on the cytoplasmic surface of the inner membrane.After the core oligosaccharide is attached to Kdo2-lipid A,the molecule is flipped to the outer surface of the inner membrane,where the O-antigen repeats are attached to form lipopolysaccharide.Kdo2-lipid A could activate the innate immune system through TLR4.Therefore,researches on the structure modification of Kdo2-lipid A would be useful for developing new vaccines and adjuvants.
Jiucheng Liu , Wei Zhang , Xiaogang Wu , Liqun Zhang
2013, 53(2):118-126.
Abstract:Abstract:Regulator of exopolysaccharide and type Ⅲ secretion (RetS) is a hybrid sensor kinase/response regulator protein located on bacterial membrane,and essential for expression of numerous genes.In Pseudomonas aeruginosa,RetS modulates the phosphorylation state of another kinase GacS via a direct interaction.[Objective]The goal of this study is to study the effect of retS on the antibiotic 2,4-diacetyl-phloroglucinol (2,4-DAPG) production in P.fluorescens 2P24.[Methods]Production of 2,4-DAPG in strain 2P24 and its mutants was quantified by HPLC. To determine the effect of RetS on the Gac/Rsm pathway,the promoters of the small RNA genes rsmX/rsmY/rsmZ and regulatory genes rsmA/rsmE in the Rsm pathway were fused with a promoterless lacZ,and the promoter activities were measured in 2P24 and the retSdeficient mutants.[Results]Genetic inactivation of the retS in strain 2P24 increased the production of an uncharacterized red pigment and the antibiotic 2,4-DAPG.RetS negatively regulated the transcription of the small RNAs RsmX and RsmZ.In the retS and gacS double mutant or the retS and gacA double mutant,all the phenotypic changes caused by the retS deletion were reversed to the level of gacS or gacA single gene mutant.[Conclusion]In P.fluorescens 2P24,RetS negatively regulates the production of antibiotic 2,4-DAPG through the Gac/Rsm pathway.
Yuan Zhang , Qinna Cui , Zhe Zhao , Yongfei Ming , Xiaoyan Chi , Zhibin Feng , Shiwei Cheng , Weihai Xie , Yihe Ge
2013, 53(2):127-135.
Abstract:Abstract:Pseudomonas aeruginosa PAO1,an opportunistic pathogenic bacterium,produces phenazine and its derivatives which play a critical role in pathogen-host interaction during its infection.In a biological control strain P.chlororaphis PCL1391,Pip positively regulates PCN production.[Objective]Our aim is to identify the function and regulation of an ORF of PA0243 (the homolog of Pip) in Pseudomonas aeruginosa PAO1.[Methods]We first cloned the fragment of the pip gene from the chromosomal DNA of P.aeruginosa PAO1 and constructed the pip-defect mutant PA-PG with the insertion of gentamycin resistance cassette (aacC1).With construction and introduction of pME10P (containing the whole pip gene region),complementation of the pip was then carried out. With creation of the mutants PA-PD-Z1G and PA-PGZ2K,phenazine-1-carboxylic acid and pyocyanin were measured in GA medium in relative mutants,respectively.[Results]In GA medium,production of phenazine-1-carboxylic acid and pyocyanin in the mutant PA-PG decreased dramatically in comparison with that produced in the wild type strain PAO1.The amounts of phenazine-1-carboxylic acid and pyocyanin,however,were recovered with complementation of the derivative PA-PG bearing pME10P.The production of phenazine-1-carboxylic acid and pyocyanin in mutant PA-PG-Z2K were same to those in parental strain PA-Z2K.Phenazine-1-carboxylic acid and pyocyanin produced by the mutant PA-PD-Z1G were lower than those in the original strain PA-Z1G.[Conclusion]With these results,it is uggested that Pip exerts positively regulation in phenazine biosynthesis by specifically modulating expression of the phz2 operon,not by mediating expression of the phz1 operon in P.aeruginosa PAO1.
Li Zhang , Lujing Ren , Yuanwei Hu , Liang Qu , He Huang
2013, 53(2):136-144.
Abstract:Abstract:[Objective]The aim of our study was to find a novel method to evaluate the strain quality of Schizochytrium sp.HX-308.[Methods]An acclimatized strain and an original strain were cultivated under normal condition.Meanwhile,the same acclimatized strain was cultured under two different conditions,the optimum condition and the harsh condition.We detected the activity of superoxide dismutase and the contents of malondialdehyde and proline by WST-1 method,thiobarbituric acid test and colorimetry,respectively. The relationship between the three criteria and the fermentation performance of Schizochytrium sp.HX-308 was studied.[Results] The acclimatized strain cultured under the optimal fermentation condition had the lowest superoxide dismutase activity (0.025U/g protein),malondialdehyde (26.20 mmol/g·Fw) and proline contents(0.098 mg/g·Fw).In contrast,the superoxide dismutase activity of the original strain cultured in the harsh conditions was 5 times higher than that of the acclimatized strain,the malondialdehyde and proline contents were both 2 times as high as the acclimatized strain. [Conclusion]The three criteria were correlated negatively with the fermentation performance.Thus,they could be used to evaluate quality and fermentation performance of Schizochytrium sp.
2013, 53(2):145-153.
Abstract:Abstract:[Objective]To increase extracellular productivityof pullulanase,pullulanase gene from Bacillus naganoensis JNB-1 was expressed in recombinant Escherichia coli,followed by optimizing induction conditions and applying chemical additives.[Methods] We amplified pullulanase gene pul from B.naganoensis genome by PCR and constructed recombinant E.coli BL21/pET-20b-pul. Optimal induction conditions and additive parameters of glycine and Na + were determined by measuring the extracellular pullulanase activity. [Results]Pullulanase was expressed in E. coli with the molecular weight of 119 kDa. Under optimal induction conditions,i.e.induction was initiated with 0.4 mmol/L isopropyl β-D-1-Thiogalactopyranoside (IPTG) at 20℃ when OD600 of bacteria culture reached 1.2,total pullulanase activity including intracellular and extracellular enzyme reached 10.8 U/mL.Addition of glycine and Na+ enhanced the secretion of pullulanase.With the supplementation of 0.08 mol /L glycine and 0.2 mol/L Na+ ,extracellular pullulanase activity was increased up to 8.1 U/mL,10.3 times of that without additives. [Conclusion]A promising resource of pullulanase was achieved by construction of recombinant E.coli for industrial production of pullulanase,and additionally the efficient regulation method with chemical additives was developed for pullulanase secretion,which would also be useful for highlevel extracellular production of recombinant enzymes.
Chuanzhi Zhu , Yanlin Zhao , Xiangyu Huang , Yu Pang , Yazhen Zhao , Yuhui Zhuang , Xiuyun He
2013, 53(2):154-163.
Abstract:Abstract:[Objective]To identify specific antigens related to streptomycin resistant (SMr) Mycobacterium tuberculosis.[Methods]Cellular proteins were extracted from SMr clinical isolate 01108,SM-sensitive clinical isolate 01105 and H37Rv. Differential expression proteins were identified with isobaric tags for relative and absolute quantitation (iTRAQ) combined with Nano LC-MS/MS technology.[Results]Approximately 194 and 146 differential expression proteins were identified in 01108 strain compared with the proteomic profiles of 01105 strain and H37Rv,respectively,and 121 proteins were identified in 01108 strain compared with the proteomic profiles of both 01105 strain and H37Rv.Identified proteins showed a pI (isoelectric point) variation between 3.74-12.48 and a molecular mass (Mr) range between 7.63 and 326.2 kDa.Differential expression proteins were mainly associated with metabolism (involved in intermediary metabolism,respiration,and lipid metabolism) and took part in catalysis and binding function.Seven ribosomal proteins (Rv0056,Rv0641,Rv0652,Rv0701,Rv1630,Rv2442c and Rv2785c) and seven proteins (the ratios>1.20or <0.55) were commonly down-regulated in 01108 strain compared with both 01105 strain and H37Rv,i.e.the thiol peroxidase (Rv1932),acyl carrier protein dehydrogenase (Rv0824c),30S ribosomal protein S15 (Rv2785c),acetone acid dehydrogenase E2 part (Rv2215),two-component transcriptional regulatory protein (Rv3133c) and Hypothetical protein (Rv2466c and Rv2626c).[Conclusion]Differential expression proteins were found in SMr strain compared with both SM-sensitive strain and H37Rv. Further studies are needed to assess the role of these differential expression proteins in SM resistance.
Yongqing Ni , Yanling Gu , Xuewei Shi , Xiaoji Zheng , Liang Han , Hong Zhou , Guodong Cheng
2013, 53(2):164-172.
Abstract:Abstract:[Objective]We characterized proteolytic bacteria isolates from sediments of the bottom layer of the Glacier No.1 in the Tianshan Mountains,China.Physiological test and phylogenetic analysis were undertaken to expand our knowledge on diversity and ecological distribution of psycrotrophic and psycrophlic bacteria populations.[Methods]Using the screening media containing skim milk,we screened cold-adapted strains producing protease. Taxonomic identity and genetic variability of strains isolated was determined by partial 16S rRNA gene sequences and repetitive-element PCR fingerprint.[Results]Of the total 125 cold-adapted bacterial isolates,high levels of protease activity were observed from 27 isolates at optimal growth temperatures ranging from 15 to 24℃ in plate assay. Among 27 protease-producing strains, only 6 isolates were psychrophilic.The 16S rRNA gene phylogenetic analysis revealed that protease-producing isolates belonged to 5 phyum,namely α、,β and !of Proteobacteria,Actinobacteria and Cytophaga-Flexibacter-Bacteroides. They are affiliated to the genera Pseudomonas, Polaromonas,Brevundimonas, Rholococces,Cryobacterium, Kocuria,Arthrobacter,Chryseobacterium and Flavobacterium.The populations of the predominant cultivated protease-producing bacteria are the Pseudomonas spp.(40.7%).[Conclusion]The results enriched our knowledge on the phylogenetic and physiological diversity of cold-adapted strains producing protease in cold environments.
2013, 53(2):173-184.
Abstract:Abstract:[Objective]This study is aimed to establish an unbiased profiling strategy for investigating the microorganisms responsible for aerobic methane oxidation by pyrosequencing the total soil microbial communities at DNA and RNA levels,and to link aerobic methane oxidation activity with taxonomic identity of active microorganisms by DNA /RNA SIP in red paddy soils.[Methods]Three red paddy soils derived from quaternary red clay were collected from Gushi and Taoyuan cities of Hunan province and Leizhou city of Guangdong province,were incubated with the labeled 13 CH4 or 12 CH4 for determination of aerobic methane oxidation kinetics. Pyrosequencing of the 16S rRNA and16S rRNA gene at the whole microbial community levels were performed over the course of aerobic methane oxidation in soil microcosms. 13 C-DNA and 13 C-RNA were obtained through ultracentrifugation of the total soil DNA and RNA extracts,respectively. Clone library of pmoA genes in 13 C-DNA and 16S rRNA genes in 13 C-RNA were constructed.[Results] Pyrosequencing of the total microbial communities revealed significant increase in the relative abundance of aerobic methanotrophs in soil microcosms upon the completion of aerobic methane consumption. The proportional increase of aerobic methanotrophs was significantly higher at RNA than DNA levels. Type I and II aerobic methanotrophs significantly increased in Gushi soil,while the significant increase of type II aerobic methanotrophs was observed in Taoyuan soil. In the meantime,type I aerobic methanotrophs appeared to be stimulated exclusively in Leizhou soil. Sequencing analysis of the 13 C-labeled pmoA genes and 16S rRNA further demonstrate that phylogenetically distinct methanotrophs dominated aerobic methane oxidation activity in paddy soils of Gushi (Type I and II),Taoyuan (Type II) and Leizhou (Type I).[Conclusion] Highthroughput pyrosequencing at the whole community level of 16S rRNA genes provides an almost unbiased profiling stragety for measuring characteristic changes in relative proportions of aerobic methanotrophs responsible for aerobic methane oxidation activity in red paddy soils,and higher sensitivity was observed at RNA than DNA levels.DNA/RNA-SIP can accurately reveal the active microorganisms responsible for aerobic methane oxidation in read soil,being largely consistent to pyrosequencing-based fingerprinting analysis of the total microbial communities.
Jifei Ma , Zongjun Du , Wei Luo , Yong Yu , Yixin Zeng , Bo Chen , Huirong Li
2013, 53(2):185-196.
Abstract:Abstract:[Objective]In order to assess bacterial abundance and diversity within three different sections of summer seaice samples collected from the Prydz Bay,Antarctica.[Methods] Fluorescence in situ hybridization was applied to determine the proportions of Bacteria in sea-ice. Bacterial community composition within sea ice was analyzed by 16S rRNA gene clone library construction. Correlation analysis was performed between the physicochemical parameters and the bacterial diversity and abundance within sea ice.[Results]The result of fluorescence in situ hybridization shows that bacteria were abundant in the bottom section,and the concentration of total organic carbon,total organic nitrogen and phosphate may be the main factors for bacterial abundance.In bacterial 16S rRNA gene libraries of sea-ice,nearly complete 16S rRNA gene sequences were grouped into three distinct lineages of Bacteria (γ-Proteobacteria,α-Proteobacteria and Bacteroidetes).Most clone sequences were related to cultured bacterial isolates from the marine environment,arctic and Antarctic sea-ice with high similarity. The member of Bacteroidetes was not detected in the bottom section of sea-ice.The bacterial communities within sea-ice were little heterogeneous at the genus-level between different sections,and the concentration of NH4+ may cause this distribution.[Conclusion]The number of bacteria was abundant in the bottom section of sea-ice. Gamma-proteobacteria was the dominant bacterial lineage in sea-ice.
Wen Han , Yuzi Luo , Bibo Zhao , Yuan Sun , Su Li , Huaji Qiu
2013, 53(2):197-203.
Abstract:Abstract:Extreme varieties of viruses exist in the environment and animals,some of which are unknown.However,many unknown viruses are barely detected by means of conventional virus isolation and PCR assay.[Objective]To develop a technology platform for detecting unknown viruses. [Methods] We established the technology based on viral metagenomics in combination with novel molecular diagnostics. The technology is consisted of removal of host nucleic acid,random PCR amplification,large-scale sequencing,and bioinformatics.[Results]The technology was applied to detect classical swine fever virus (CSFV)-infected cells and a tissue sample of a pig infected with porcine circovirus type 2 (PCV2).We amplified 13.7% sequences of CSFV genome and 47.2% those of PCV2 genome,respectively.Moreover,we amplified 16. 4% sequences of the simian parainfluenza virus type 5 genome from an unknown virus cell culture using the developed method.In addition,using the developed method combined with the high-throughput sequencing,we detected 1. 1% virus sequences,including CSFV,PCV2,torque teno sus virus (TTSuV),porcine bocavirus (PBoV) and human adenovirus type 6 (Ad6) from 7 clinical swine samples of unknown causative agents.[Conclusion]The developed metagenomics-based method showed good sensitivity for detection of both DNA and RNA viruses from diverse swine samples,and has potential for universal detection of known and unknown viruses.It might facilitate the diagnosis of emerging viral diseases.
Xianhui An , Xiufen Zhou , Zhijun Wang , Zixin Deng , Jingdan Liang
2013, 53(2):204-209.
Abstract:Abstract:[Objective]DNA phosphorothioate modification (DNA sulfur modification,a non-bridging oxygen swapped with a sulfur) exists in diverse bacteria. Salmonella enterica serovar Cerro 87 is one of the bacteria that harbor the DNA sulfur modification. The modification is carried out by the products of a four-membered gene cluster,dptBCDE.Transformation of Escherichia coli DH10B with the dptBCDE gene cluster endows the strain with DNA sulfur modification capability. Deletion of dptC abolished the modification. Here,we studied the function of dptC in DNA sulfur modification.[Methods]Six cysteine residues in dptC were mutated individually within the dptBCDE gene cluster. Mutants were then tested for DNA sulfur modification.[Results]Among the 6 cysteine mutations (C39,C146,C262,C273,C280,and C283),5 abolished DNA modification except for C39,suggesting that C146,C262,C273,C280,and C283 are essential for DNA sulfur modification. Sequence alignment shows that these five cysteine residues are conserved among different strains.[Conclusion]Mutation at anyone of C146,C262,C273,C280 and C283 of dptC abolished DNA modification.Our results shed light on further study of DNA sulfur modification biochemical pathway.
Guojian Liao , Yanli Zhao , Kangli Xu , Dan Li , Ludan Dai , Changhua Hu
2013, 53(2):210-216.
Abstract:Abstract:[Objective]Daptomycin-resistance in environmental actinomycetes was studied to provide warning systems for emerging clinical resistance.[Methods]In total 49 soil and 10 endophytic acitnomycetes were used in this study.The daptomycin resistant strains were identified by measuring daptomycin resistance profile. Subsequently,daptomycin inactivating assay was preformed to distinguish resistance from other nondestructive mechanisms. Then,the strains of interest were determined by morphology and 16S rRNA gene sequence analysis.Finally,PCR analysis was used to detect the daptomycin acylase gene (dpa) encoding daptomycin acylase in those strains[Results]All strains tested in this study were resistant towards daptomycin. Of them 24 soil acitnomycetes and 4 endophytic actinomycetes had the ability to inactivate daptomycin,while the remaining strains used other measures to confer resistance.Sequence analysis demonstrated that strains inactivating daptomycin were Streptomyces,Micromonospora and Norcadia. PCR analysis shows that 17.9% strains contained the dpa.[Conclusion] There is very high percentage of resistance in environmental actinomycetes and inactivating daptomycin is one of the main resistant mechanisms.
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