Potential of predominant activated sludge bacteria as recipients in conjugative plasmid transfer

We investigated the possibility of conjugative plasmid transfer to the predominant bacteria in activated sludge and the factors influencing the transfer frequency in the activated sludge process. We performed conjugative transfers of a self-transmissible, broad-host-range plasmid RP4 from Escherichia coli C600 to activated sludge bacteria by broth mating. Most of the activated sludge bacteria tested could acquire plasmid RP4, although the transfer frequencies varied from 8.8 x 10(-7) to 1.3 x 10(-2) transconjugants per recipient. The transfer frequencies in several strains were similar to, or higher than, that in intraspecific transfer to E. coli HB101. Matings under various environmental conditions showed that factors relevant to physiological activity, such as temperature and nutrient conditions, seemed to affect the transfer frequency. In addition, conjugative transfer was detected even in filtered raw and treated wastewaters. Thus, the predominant activated sludge bacteria seem to have sufficient potential as recipients in conjugative plasmid transfer under the conditions likely to occur in the activated sludge process. Transfer frequency was reduced by agitation in the presence of suspended solid. This may suggest that conjugative plasmid transfer is physically inhibited in aeration tanks.     

Simultaneous overexpression of enzymes of the lower part of glycolysis can enhance the fermentative capacity of Saccharomyces cerevisiae

Recombinant S. cerevisiae strains, with elevated levels of the enzymes of lower glycolysis (glyceraldehyde-3-phosphate dehydrogenase, phosphoglycerate mutase, phosphoglycerate kinase, enolase, pyruvate kinase, pyruvate decarboxylase and alcohol dehydrogenase) were physiologically characterized. During growth on glucose the enzyme levels in the recombinant strains (YHM4 and YHM7) were 1.1-3.4-fold higher than in the host strain (CEN.PK.K45). The recombinant strains were grown in aerobic or anaerobic batch cultures on glucose or a mixture of glucose and galactose. The specific ethanol production rates in the recombinant strains were the same as for the host strain and the physiological behaviour of the recombinant strains and the host strain was similar. When the cellular demand for ATP was increased by means of glucose pulses (final concentrations of 3.9 g/l or 2.0 g/l, respectively) to aerobic chemostat cultures maintained at a dilution rate of 0.08/h, the specific carbon dioxide production rate (qCO(2)) of CEN.PK.K45 accelerated at 6x10(-3) mmol/g/min(2) during the first 15 min, whereas during the same time period the qCO(2) of YHM7 accelerated twice as fast at 12x10(-3) mmol/g/min(2), indicating a higher fermentative capacity in the recombinant strain.     

Transfer of antibiotic multiresistant plasmid RP4 from escherichia coli to activated sludge bacteria

In situ transfer of a self-transmissible, antibiotic-multiresistant plasmid RP4 from a laboratory Escherichia coli strain C600 to indigenous activated sludge bacteria was investigated using filter mating. The transfer frequency of RP4 from the donor E. coli to the bacteria that was sampled from two wastewater treatment plants was 5.1x10(-2) to 7.5x10(-1) and 4.6x10(-3) to 1.3x10(-2)/potential recipient. The isolated transconjugants showed resistance to Ap, Km, and Tc and the presence of a plasmid with a similar size to RP4. The traG gene on RP4 was also detected from all transconjugants. Reverse-transfer experiments from the transconjugants to E. coli HB101 indicated that RP4 maintained self-transmissibility in the transconjugants. The transconjugant strains were dominant bacteria in activated sludge including Pseudomonas fluorescens, P. putida, and Ochrobactrum anthropi and minor populations of enteric bacterial strains including Citrobacter freundii, E. coli, Enterobacter cloacae, E. asburiae, and Klebsiella pneumoniae ssp. pneumoniae. The transconjugant strains K. pneumoniae ssp. pneumonia, E. cloacae, and E. asburiae had several naturally occurring plasmids. These results suggest that in situ transfer of plasmids and the exchange of antibiotic-resistant genes can occur between released and indigenous bacteria in activated sludge.     

ARO10基因在Saccharomyces cerevisiae中的克隆表达及其对3-甲硫基丙醇合成代谢的影响

研究了脱羧酶ARO10基因克隆与过量表达对酿酒酵母INVSc1 3-甲硫基丙醇合成途径的代谢流量影响。将脱羧酶基因ARO10与穿梭质粒pYES2连接,构建其酿酒酵母表达质粒(载体)pYES2-ARO10,LiAc/SSD-NA/PEG方法转化酿酒酵母菌株INVSc1中进行表达,验证ARO10基因过量表达对发酵产物3-甲硫基丙醇影响。结果表明,构建的酿酒酵母转化子SC10-1发酵120 h时,3-甲硫基丙醇生成量达到0.90 g/L,与未导入脱羧酶ARO10基因的对照菌株相比,3-甲硫基丙醇产量提高55.2%。因此,S.cerevisiae s288c中脱羧酶(EC 4.1.1.72)是3-甲硫基丙醇生物合成途径的关键限速酶,其增强脱羧酶基因ARO10的克隆及基因表达,有利于提高3-甲硫基丙醇的合成代谢流量。     

Development of a Rhodococcus recombinant strain for degradation of products from anaerobic dechlorination of PCBs

The Gram-positive bacterium Rhodococcus sp. strain RHA1, naturally containing the biphenyl pathway, was electroporated with a broad host range plasmid containing the 4-chlorobenzoate (4-CBA) degradation operon (fcb) isolated from Arthrobacter globiformis strain KZT1. The recombinant strain grew in medium containing 4-CBA and 4-chlorobiphenyl (4-CB) as the only source of carbon, with stoichiometric release of chloride and a molar growth yield on 4-CB that suggested utilization of both biphenyl rings. In resting cell assays, similar rates of degradation were observed for wild-type and recombinant strains for the most common eight congeners from the anaerobic dechlorination of Aroclor 1242, but the recombinant strain accumulated lower amounts of chlorinated meta-cleavage products and no 4-CBA. Recombinant cells inoculated at 10(4) cells/g into nonsterile soil amended with 4-CB grew to 6-10(5) cells/g, a density consistent with the 4-CB consumed. 4-CB was removed only in the inoculated soil, and the recombinant strain did not grow in the same soil when it was not amended with 4-CB. The fcb operon remained stable in the recombinant strain reisolated from soil after 60 days. This work provides proof of concept that a Rhodococcus strain constructed to grow on a PCB would grow in nonsterile soil if the appropriate chlorobiphenyl is available.     

碱性芽孢杆菌木聚糖酶基因的克隆及表达

本实验从广西红树林土壤中筛选出一株产木聚糖酶的耐碱菌HSL38,经过16S rDNA鉴定,其与碱性芽孢杆菌Bacillus halodurans C-125(GenBank：NC_002570.2)的同源性达到99%。参考Bacillus halodurans C-125的β-1,4-木聚糖酶基因xynA设计引物,扩增出HSL38中的β-1,4-木聚糖酶基因xyl-BH-G10,其与xynA的同源性达到99%,编码396个氨基酸残基(45.27kD),属于糖基水解酶GH10家族。将xyl-BH-G10基因与表达载体pET-30a-c(+)连接,构建重组载体,转化大肠杆菌E.coli BL21(DE3),IPTG诱导表达重组蛋白。结果表明,在最佳诱导条件下,木聚糖酶基因xyl-BH-G10在大肠杆菌细胞内高效表达,酶活力达到55.28U/mL,是原菌株HSL38发酵液酶活力的4倍。     

基于低拷贝质粒的高产紫色杆菌素谷氨酸棒杆菌的构建和发酵

该研究以公认安全(Generally Recognized as Safe,GRAS)的谷氨酸棒杆菌(Corynebacterium glutamicum)为宿主,构建高产紫色杆菌素的重组菌株。利用谷氨酸棒杆菌天然大质粒pTET3的复制与分配元件,构建了低拷贝质粒pOK12CG1,该质粒在谷氨酸棒杆菌中的拷贝数约为6拷贝/基因组,且与谷氨酸棒杆菌常用质粒pEC-XK99E和pXMJ19兼容。以低拷贝质粒pOK12CG1为骨架构建了携带紫色杆菌素合成操纵子(vioABCDE)的质粒pCGvio,并分别以谷氨酸棒杆菌标准株ATCC 13032和插入序列(Insertion Sequence,IS)元件删除株为宿主,构建了7株合成紫色杆菌素的重组菌株。通过初步筛选,发现基于低拷贝质粒的重组菌株ATCC13032/p CGvio,其紫色杆菌素产量(508.24 mg/L)高于基于中高拷贝质粒的重组菌株ATCC 13032/pECvio(376.16 mg/L),而基于低拷贝质粒的IS元件删除重组菌株ISDM023/pCGvio紫色杆菌素产量达到了610.13mg/L。进一步采用正交实验设计对重...     

Anammox bacterial abundance, biodiversity and activity in a constructed wetland

An integrated approach to document high anammox activity and biodiversity in a constructed wetland (CW) was performed and showed that substantial anammox activity could mitigate undesirable N(2)O emission. The enhanced anammox bacterial abundance, biodiversity and activity were achieved by supplementing activated sludge to the CW. Up to 3.38 × 10(7) gene copies g(-1) dry soil of anammox bacteria were enriched in the CW. The activity measured by isotope pairing technique increased from 1.6 nmol N g(-1) sludge h(-1) in the original activated sludge to 18 nmol N g(-1) soil h(-1) in the CW, with the specific cellular activity increased from 5.1 to 12.8 fmol cell(-1) d(-1). Up to 33% of produced N(2) could be attributed to anammox process in the CW, with the remainder being due to denitrification. Phylogenetic analysis of anammox bacterial 16S rRNA genes indicated a shift of community from single Candidatus "Brocadia fulgida" in sludge to multiple "Jettenia", "Brocadia", and "Anammoxoglobus" species in the CW. With static chambers and control experiments, the CW with supplemented sludge had a 30% reduced N(2)O emission flux compared with the tests without adding biomass during an 8 month testing period.     

Three-dimensional dual-morphotype species modeling of activated sludge flocs

An individual-based model, originally developed for a biofilm system, was adapted to simulate three-dimensional formation of activated sludge flocs. The model was extended to two different bacterial morphotypes (floc-forming and filamentous bacteria), allowing spatial development of the floc according to the bacterial morphology, diffusion, reaction, and growth processes. The model needed also extension with a process for attachment of individual cells. Despite being in an early stage of development, the model is already a tool that enables us to obtain useful information about the microfloc environment. The model indicates that filamentous bacterial morphology and substrate microgradients are important aspects in the formation of bacterial structures. In mass transport-limited regimes filamentous bacterial structures prevail, whereas in growth-limited regimes irregular shaped flocs with fingerlike structures are dominant. These modeling results suggest that activated sludge flocs and biofilms might be different manifestations of the same phenomena. The model results support the hypothesis that floc-macrogradients can be the most important parameter for development of bulking sludge. The model suggests that attachment has a very strong effect on floc structure, leading to enhancement of the effect of substrate microgradients.     

重组海藻糖合酶工程菌高密度发酵条件的研究

目的:通过对前期构建的海藻糖合酶基因工程菌进行高密度发酵的研究,获得了其高密度工艺条件。方法:采用摇瓶发酵和10L自控罐高密度发酵研究了培养基、pH、发酵方式对工程菌生长及目的蛋白表达的影响,并考察了工程菌中重组质粒的遗传稳定性。结果:海藻糖合酶基因工程菌高密度发酵的培养基为2YT+0.2%葡萄糖,最适pH为7.0,发酵方式为分批补料,通过10L自控罐高密度发酵最终得到的工程菌细胞密度达到了50.78g/L,酶活达到了3.197U/mL。所构建的重组质粒在宿主中得到了稳定遗传。结论:优化了海藻糖合酶基因工程菌高密度发酵的条件,为海藻糖规模化生产奠定了基础。     

米曲霉giF-10内切葡聚糖酶基因的克隆表达及酶学性质分析

根据NCBI(GenBank Accession No.:D83732.1)中注册的米曲霉内切葡聚糖酶CelB基因序列设计引物,以本实验室自行筛选的天然米曲霉基因组DNA为模板,PCR高保真扩增出内切葡聚糖酶基因eg,将其定向插入到酵母表达载体pPICZαA上,转化酵母宿主菌X33,刚果红水解圈筛选结果表明已成功表达。SDS-PAGE分析表明,表达产物分子量约为65 ku。对酵母表达工程菌X33-eg进行发酵条件优化,结果显示最适甲醇诱导浓度为0.75%,用1 L三角瓶诱导培养5天达到最高酶活120 U/mL。对重组酶的酶学性质分析表明,其最适反应pH值和温度分别为pH4.0和45℃,在30～45℃和pH3.4～pH6.9范围内可保持内切葡聚糖酶最高酶活力70%以上。     

生物合成γ-氨基丁酸酿酒酵母谷氨酸脱羧酶基因的克隆与表达

为了提高酵母菌的γ-氨基丁酸产量,本研究以十六烷基三甲基溴化铵法(hexadecyl trimethy ammonium bromide,CTAB)提取的酿酒酵母28基因组DNA为模板,扩增得到序列长度为1 758 bp的GAD1基因,经比对与S.cerevisiae S288c的一致性达到98.58%,并设计引物扩增包括GAD1基因上游启动子及调控序列,下游终止子在内的全长基因序列,长度为3 490 bp。将全长基因序列克隆至高拷贝质粒p UG6,构建重组质粒p28。以菌株28作为出发菌株进行转化,经G418抗性筛选得到转化子,进一步经过聚合酶链式反应(polymerase chain reaction,PCR)实验验证,质粒提取及酶切验证,确证得到重组子DL28。经重组质粒p28的特性研究得知,重组质粒p28具有良好的遗传稳定性,无抗性传代培养10次重组质粒不丢失。转化后进行酶活力测定,重组子DL28的谷氨酸脱羧酶(glutamic acid decarboxylase,GAD)酶活力比菌株28提高73.8%。通过以上实验结果得出结论,GAD基因高表达菌株DL28具有更高的G418抗性和GAD酶活性。     

Oxygen tolerance of sulfate-reducing bacteria in activated sludge

The oxygen tolerance of sulfate-reducing bacteria (SRB) present in activated sludge was studied in batch incubations using radiolabeled [35S]sulfate and a most probable number (MPN) technique employing activated sludge medium. Sulfate reduction (SR) could not be detected in activated sludge during oxic incubation or in the presence of nitrate. However, upon anoxic incubation of both freshly sampled activated sludge and activated sludge preaerated for 40 min, SR resumed immediately at an initial rate of 2 microM h(-1). During long-term aeration of activated sludge, the number of viable and culturable SRB remained constant at around 10(6) SRB mL(-1) throughout a 121 h aeration period. During the first 9 h of the 121 h aeration period, the anaerobic SR activitywas unaffected, as compared to that of an unaerated control sample, and recommenced instantaneously upon anoxic incubation. Even after 121 h of continuous aeration, SR took place within 1.5 h after anoxic incubation albeit at a rate less than 20% that of the unaerated control. As suggested by MPN estimates and the observed kinetics of SR, oxygen exposure resulted in temporary metabolic inactivation of SRB but did not cause cell death. Consequently, SRB have the potential for quick proliferation during anoxic storage of activated sludge.     

一株好氧反硝化细菌的分离与鉴定

为研究活性污泥中的反硝化细菌，从实验室驯化培养的活性污泥反应器中，分离获得1株反硝化细菌P7，进行了菌株形态观察及生理生化测定，并在此基础上，经PCR扩增后测定该菌株的16SrRNA基因序列，由16SrRNA基因序列比较可知，菌株P7与睾丸酮假单胞菌（Comamonas testosteroni）和稻草假单胞菌（Pseudomonas straminea）亲缘关系最为接近，16SrRNA基因相似性达到99．65％。可初步判定其为丛毛单胞菌属（Comamonas sp．）。结合生理生化实验结果，综合分析后，将其鉴定为丛毛单胞菌属（Comamonas sp．）的睾丸酮假单胞菌（Comamonastes tosteroni）。     

Hyaluronic acid production by recombinant Streptococcus thermophilus

Generally recognized as safe, Streptococcus thermophilus was transformed using a plasmid expressing endogenous hyaluronic acid (HA) synthase genes. A single expression of hyaluronic acid synthase (hasA), uridine diphosphate-glucose dehydrogenase gene (hasB), or pyrophosphorylase gene (glmU) and double expression of hasA and hasB were attempted. A streptococcus-Escherichia coli shuttle vector, pBE31, was successfully transfected in S. thermophilus. The single expression of hasA or hasB allowed S. thermophilus to produce about 0.5-1.0 g/l HA. The strains coexpressing of hasA and hasB showed a markedly increased HA production (1.2g/l) which was six-fold increase compared with the wild-type strain. The maximum cell concentration and specific growth rate of each recombinant strain were lower than those of the wild-type strain; however, the specific production rate was more than 100-fold higher. Galactose concentration decreased in the coexpressing strain after depletion of lactose. The bacterial metabolism would be altered in order to achieve a higher production by changing the intracellular metabolism. The average molecular weight of HA (1.0 × 10(6) Da) was not affected by the expression of hasA and hasB. HA produced from recombinant strain could be an alternative material for medical, cosmetic and food utilization instead of HA from conventional pathogenic streptococci.     

酿酒酵母表面展示β-淀粉酶及其酶学性质研究

通过PCR技术扩增来源于大麦的β-淀粉酶基因,将其与酿酒酵母细胞壁蛋白α凝集素基因在读框内融合,构建得到表面展示载体pBA-AG,进一步将该重组质粒通过遗传转化,整合到酿酒酵母W303-1A的染色体中,获得了β-淀粉酶经过α凝集素锚定信号结合到细胞壁上的重组酵母。重组酵母表面展示的β-淀粉酶活力为131U/g干细胞。对展示的β-淀粉酶酶学性质研究表明,其最适反应温度为50℃,最适作用pH为5.0,与游离酶相比,其温度稳定性和pH稳定性均得到提高。本研究利用α凝集素系统首次将β-淀粉酶成功展示在酿酒酵母表面,为以酿酒酵母为基础的全细胞催化剂研究与应用打下了一定基础。     

Cloning and characterization of baker's yeast alpha-glucosidase: over-expression in a yeast strain devoid of vacuolar proteinases

Two alpha-glucosidase (maltase) genes, designated GLUCPI and GLUCPII, have been cloned from an industrial strain of baker's yeast (Saccharomyces cerevisiae) by complementation of a maltase-negative mutant strain. The different genes were identified according to their alternatively expressed isoenzymes PI and PII in transformants after isoelectric focusing and activity staining in separated cell lysates. The gene encoding alpha-glucosidase PI (GLUCPI), which was not present in laboratory strains of S. carlsbergensis with a defined MAL1, 2, 3, 4 or 6 locus, was sequenced and compared with the recently published MAL6S gene. This comparison revealed single amino acid deviations at three positions in the predicted polypeptide sequence. In addition, the divergent promoter region of GLUCPI differed from MAL6S by a triple repeated 147-bp DNA segment. Maltose induction and glucose repression of alpha-glucosidase PI were not affected by the deletion of the repeated DNA segment. However, the absolute expression of alpha-glucosidase PI increased two- to four-fold. In addition, a two-fold increase in the maltase synthesis occurred when the cloned positive regulator gene MAL2-8ep was on the same plasmid. Furthermore, stability of the alpha-glucosidase in cultures in the stationary growth phase was greatly enhanced using a host strain lacking the proteinases A and B and the carboxypeptidases Y and S. Promoter trimming, MAL2-8cp stimulation and the use of a host strain deficient in four vacuolar proteinases resulted in alpha-glucosidase PI expression of about 13% of the soluble protein.     

New industrial brewing yeast strains with ILV2 disruption and LSD1 expression

New industrial brewing yeast strains, free of vector sequences and drug-resistance genes, were constructed by disrupting alpha-acetohydroxyacid synthase (AHAS) gene (ILV2) and introducing Lipomyces starkeyi dextranase (DEX) gene (LSD1) as a selective marker. The resulting recombinant strains can survive on YNB minimal medium plate with dextran T-70 as sole carbon source and showed lower AHAS activity. Fermentation test with recombinant strains in 500 ml conical flask confirmed DEX activity and lower AHAS activity compared with their host strain. Moreover, the fermentative performance of recombinant strains T1 and Q9 was better than their host, and the residual sugar content was reduced by 20-25% in fermented wort with recombinant strains compared to their host, too.