| Vibrios are widely distributed in lakes,marine environmrents,high salt soil and other natural environments.Some species are probiotics of aquaculture animals,and some species can cause human and animal diseases,which are closely related to human life.Many bacteria are able to enter into the viable but non-culturable(VBNC)state in low temperature,radiation,oligo-nutritional and other harsh environmental conditions.The VBNC cells could not be detected by conventional methods,but still have some metabolic activity,and able to grow under favourable conditions.A resuscitation promoting factor of gram-positive bacteria can promote the growth of bacterial cells in different states and resuscitate the VBNC cells.The Rpf-like proteins were found in some gram-negative bacteria,which could promote the cell growth significantly.It is very important to study the mechanism for the recovering of the VBNC bacteria,which help us understand surviving abilities of the bacteria in extrame environments and to isolate more valuable new species.The genes of resuscitation-promoting factor YeaZ were cloned from genomic DNA of Vibrio harveyi SF-1 and Vibrio alginolyticus HW283.The nucleotide sequences of the yeaZ genes consist of 702 bp,which encoded polypeptides of 233 amino acids.They showed 59%and 55%of similarities with the Rpf of Escherichia coli and the M22 peptidase YeaZ of Yersinia,respectively.The yeaZ genes are widespread in vibrio groups and the sequence similarity is 67%-94%by detecting the yeaZ genes of different vibrios.The yeaZ genes were expressed in prokaryotic cells and the purified recombinant proteins showed specific band of 30 kDa on SDS-PAGE.The YeaZ was added to the VBNC cell suspension of Vibrio harveyi.Addition of the YeaZ significantly promoted the recovery of VBNC cells.The culturable cells could be detected when the VBNC cells were incubated at 28°C for 8 h with addition YeaZ with the cell counts of 2.88×105 cfu/ml.The culturability of the VBNC cells was found to decrease gradually during the development of the VBNC state.No resuscitation of the culturable cells could be detected 120 d after they entered into the VBNC state,however,addition of the YeaZ effectively promoted the resuscitation with the culturable cell counts of 1.13×103 cfu/ml.The YeaZ showed low muralytic activity when measured with 4-methylumbelliferyl-?-D-N,N',N,"-triacetylchitotrioside(4-MUF-3-NAG)as substrate.The specific lysozyme activity of YeaZ was 7.05 U/mg.The YeaZ showed maximal activity at pH 5.0 and was stable at pH 4.0-7.0 and 20-50?.When the temperature exceeded 50?,the enzyme activity was lost.The enzyme activities were inhibited by Ca2+,Mg2+ and Zn2+.One mmol/1 of Co2+ had a weak positive effect on the enzyme activity.The catalytic aspartic acid residues(Asp-112)and the catalytic threonine residues(Thr-71)of the YeaZ were mutated by PCR site-directed mutagenesis.It was found that the single point mutations at the conservative residues reduced the enzyme activity to a certain degree,with the specific activity of 4.75 and 2.50 U/mg.The mutanted YeaZ proteins promoted the resuscitation of VBNC cells.The protease activity of the purified YeaZ was detected with azocasein,BAPNA,ATEE and BTEE as substrates.It was found that the YeaZ could hydrolyze these protein substrates to some degrees.The specific activity of proteolytic and amidase with azocasein and BAPNA as substrate were 30 and 1190 U/mg.The specific activity of esterase with ATEE and BTEE as substrate were 365 and 197.5 U/mg,respectively.The possible protease active sites(Asp88,Ser185 and Trp169)of the YeaZ were mutated by PCR sit-directed mutagenesis.All mutants were expressed in E.coli BL21.It was found that all of the single point mutations at the conservative residues reduced or lost the protease activity.The proteolytic activity of mutated purified proteins of Asp88-Ala,Ser185-Leu and Trp169-Glu decreased by 66.67%,0.000%and 50.00%.The trypsin activity of mutants of Asp88-Ala and Trp169-Glu decreased by 84.93%and 32.88%,respectively.The protease activity of Asp88-Ala and Ser185-Leu mutations was completely lost.The results showed that these amino acids are protease activity centers.The effect of the YeaZ for the culturable bacteria in the soil samples of extreme environment were studied by using plate count and denaturing gradient gel electrophoresis(DGGE)analysis methods.The study found that the populations and diversities of culturable bacteria in the experimental group with addition YeaZ were improved.Addition of the YeaZ increased the culturable populations from 0.17×103 and 2.03x103 cfu/g to 1.00×103 and 5.55×103 cfu/g,respectively.The multiplications of the total number of bacteria were 2-5 times in soil samples.DGGE fingerprint showed that the fingerprint bands in the treatment group(with addition YeaZ)are more than control group(without YeaZ).More culturable bacteria genus were isolated in the treatment groups with addition of the YeaZ including of Micrococcus antarcticus,Kocuria ros',Salinibacterium xinjiangense,Planococcus antarcticus,Bacillus oceanisediminis,Brevibacillus brevis,Paenibacillys xylanilyticus,Microbacteriu,maritypicum,Bacillus subtilis,Bacillus alcalophilus,Bacillus niabensis,Oceanzimonas doudoroffii and Zobellella taiwanensis.The culturable bacterial community deversities and their correlation with physicochemical parameters of desert steppe soils in northwestern China were investigated.The results showed that the culturable bacterial populations in different seasons varied in the desert steppe soils and the vertical distribution of the bacterial quantity was remarkable.The number of culturable bacteria is associated with annual rainfall and temperature.The culturable bacterial counts in January,April,July and October were 0.13x107,4.09x107,5.33x107 and 1.80x107 cfu/g respectively.Seventy two bacterial strains were isolated in four seasons and belonged to four phylogenetic groups of Firmicutes.Actinobacteria,Proteobacteria and Bacteroidates.Among which Bacillus was the dominant species in the desert steppe soil systems in all seasons and accounts for 5.560%,28.57%,50.00%and 20.83%,respectively.The actinomycetes strains were belong to four phylogenetic groups:Atreptomyces,Micromonspora,Streptovrticillium and Intrasporangium which isolated from the desert steppe soil.Atreptomyces was the dominant community in desert steppe and accounts for 50.00%.The fungi strains from the desert steppe were identified and designated as Alternaria and Cladosporium.Our results indicated that soil moisture content and temperature exerted marked influence on bacterial quantities and diversities.In addition,the soil physicochemical parameters(N,P,K)are other critical factors of culturable bacterial populations and diversity in the desert steppe. |
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