A 1.7 kb fragment of lat was obtained from Streptomyces clavuligerus NRRL 3585, and recombinant plasmid pKC1139-lat, which was used to disrupt the lat gene was constructed. pKC1139-lat was introduced into S. clavuligerus by bi-parental conjugation from Escherichia coli ET12567 to S. clavuligerus. The apramcin-resistant transformants were obtained and through homogeneous single-crossover between recombinant plasmid pKC1139-lat and the S. clavuligerus chromosome lat disrupted mutant strains were obtained. The genome of S. clavuligerus NRRL 3585 and the lat disrupted mutants were analyzed by PCR technique, the bioactivity of cephamycin C in the two kinds of strains were also tested. Both results proved that lat was disrupted by the insertion of pKC1139 in the lat disrupted mutants. And the production of clavulanic acid of these two kinds of strains were analyzed by HPLC with different incubation time interval (96 and 120 h), and the yield in the lat mutants was approximately 2.6 fold higher at their highest production point.

细胞膜的通透性与细菌多重耐药性的关系非常密切，我们通过对实验室使用的铜绿假单胞菌菌株进行筛选，得到一株对氨基糖苷类抗生素产生耐药性的自发突变株PAK1-3，采用互补实验从铜绿假单胞菌的基因文库中筛选到一组与铜绿假单胞菌耐药性相关的基因pprA和pprB，经在大肠埃希菌M15中表达、纯化后，进行体外磷酸化实验，证明其表达产物具有磷酸化作用，是一组肯有应答调节作用的双分子调节系统，并首次通过膜通透性实验证明PAK1-3的通透性降低，抗生素的耐药性增强。

The rapid and sensitive determination of pathogenic bacteria is extremely important in biotechnology, medical diagnosis, and the current fight against bioterrorism. Current methods either lack ultrasensitivity or take a long time for analysis. Here, we report a bioconjugated nanoparticle-based bioassay for in situ pathogen quantification down to single bacterium within 20 min. The bioconjugated nanoparticle provides an extremely high fluorescent signal for bioanalysis and can be easily incorporated with biorecognition molecules, such as antibody. The antibody-conjugated nanoparticles can readily and specifically identify a variety of bacterium, such as Escherichia coli O157:H7, through antibody–antigen interaction and recognition. The single-bacterium-detection capability within 20 min has been confirmed by the plate-counting method and realized by using two independent optical techniques. The two detection methods correlated extremely well. Furthermore, we were able to detect multiple bacterial samples with high throughput by using a 384-well microplate format. To show the usefulness of this assay, we have accurately detected 1–400 E. coli O157 bacterial cells in spiked ground beef samples. Our results demonstrate the potential for a broad application of bioconjugated nanoparticles in practical biotechnological and medical applications in various biodetection systems. The ultimate power of integrating bionanotechnology into complex biological systems will emerge as a revolutionary tool for ultrasensitive detection of disease markers and infectious agents.

采用反相高效液相色谱法对发酵液中的风味强化肽进行定量分析。色谱条件：色谱柱为日本shimadzu公司的Shimadzu VP-ODS C18（5um，150mm×4.6mm i.d.）不锈钢柱，流动相为乙腈水（含体积分数为0.1%的三氟乙酸）溶液，采用线性梯度洗脱方式，流动相B（含0.1%三氟乙酸的乙腈溶液）全程分数在30min内由5%线性升至50%，流速为1.0mL/min，检测波长为230nm，室温测定。结果：RP-HPLC法测定浓度在0.125-2.000mg/min，检测波长为230nm，回收率为86.2%-103.4%，最低检测限量为125ng，该方法精密度高，相对标准偏差（RSD）为1.4%。此方法准确、灵敏、重现性好，适合于发酵液这种复杂体系中风味强化肽的定量分析。

为实现牛奶成分的快速检测，研究了近红外光谱法在牛奶主要成分分析研究中的应用，重点对比了不同近红外区域的检测结果。研究中利用偏最小二乘法（PLS）建立校正模型，探讨了不同光谱区域和数据预处理对模型准确性的影响。模型结果表明在长波段（1700mm-2500mm）检测牛奶脂肪及蛋白质含量的准确性最高。

DsbA is a periplasmic thiol:disulfide oxidoreductase which contributes to the process of protein folding by catalyzing the formation of disulfide bonds. In this study, we demonstrate that the dsbA gene is required for the expression of the type III secretion system under low-calcium inducing conditions, intracellular survival of P. aeruginosa upon infection of HeLa cells, and twitching motility. The diverse phenotypes of the dsbA mutant are likely due to its defect in the folding of proteins that are involved in various biological processes, such as signal sensing, protein secretion, and defense against host clearing. In light of its effect on various virulence factors, DsbA could be an important target for the control of P. aeruginosa infections.

Membrane impermeability is the major contributing factor to multidrug resistance in clinical isolates of Pseudomonas aeruginosa. By using laboratory strain PAK, a spontaneous P. aeruginosa mutant (mutant PAK1-3) whose membrane had reduced permeability and which displayed increased levels of resistance to various antibiotics, especially aminoglycosides, was isolated. By complementation of the mutant with a genomic clone library derived from wild-type strain PAK, a novel two-component regulatory system (PprA and PprB) was identified and was found to be able to increase the permeability of the bacterial membrane and render PAK1-3 sensitive to antibiotics. Furthermore, specific phosphorylation of the response regulator (PprB) by histidine kinase (PprA) was observed in vitro, demonstrating that they are cognate two-component regulatory genes. Introduction of a plasmid expressing the pprB gene into randomly chosen clinical isolates (n 17) resulted in increased sensitivity to aminoglycosides in the majority of isolates (n 13) tested. This is the first demonstration that P. aeruginosa membrane permeability can be regulated, providing an important clue in the understanding of the mechanism of membrane impermeability-mediated multidrug resistance in P. aeruginosa.