Presence or absence of lipopolysaccharide O antigens affects type III secretion by Pseudomonas aeruginosa

Pseudomonas aeruginosa is one of the major causative agents of mortality and morbidity in hospitalized patients due to a multiplicity of virulence factors associated with both chronic and acute infections. Acute P. aeruginosa infection is primarily mediated by planktonic bacteria expressing the type III secretion system (TTSS), a surface-attached needle-like complex that injects cytotoxins directly into eukaryotic cells, causing cellular damage. Lipopolysaccharide (LPS) is the principal surface-associated virulence factor of P. aeruginosa. This molecule is known to undergo structural modification (primarily alterations in the A- and B-band O antigen) in response to changes in the mode of life (e.g., from biofilm to planktonic). Given that LPS exhibits structural plasticity, we hypothesized that the presence of LPS lacking O antigen would facilitate eukaryotic intoxication and that a correlation between the LPS O-antigen serotype and TTSS-mediated cytotoxicity would exist. Therefore, strain PAO1 (A+ B+ O-antigen serotype) and isogenic mutants with specific O-antigen defects (A+ B-, A- B+, and A- B-) were examined for TTSS expression and cytotoxicity. A strong association existed in vitro between the absence of the large, structured B-band O antigen and increased cytotoxicity of these strains. In vivo, all three LPS mutant strains demonstrated significantly increased lung injury compared to PAO1. Clinical strains lacking the B-band O antigen also demonstrated increased TTSS secretion. These results suggest the existence of a cooperative association between LPS O-antigen structure and the TTSS in both laboratory and clinical isolates of P. aeruginosa.     

聚苹果酸生产菌出芽短梗霉CCTCC M2012223的全基因组测序及序列分析

【目的】解析出芽短梗霉CCTCC M2012223的基因组序列信息,分析其代谢产物聚苹果酸、黑色素、普鲁兰多糖合成相关基因,为深入研究遗传多样性和代谢工程改造提供序列背景信息。【方法】使用Illumina Hi Seq高通量测序平台对出芽短梗霉CCTCC M2012223菌株进行全基因组测序,并对测序数据进行序列拼接,基因预测与功能注释,COG/GO聚类分析,比较基因组学分析等。下载其他5株出芽短梗霉基因组序列,比较分析6株菌的种内同源基因、全基因组进化以及代谢产物合成相关基因。【结果】出芽短梗霉CCTCC M2012223基因组序列全长30756831 bp,GC含量47.49%,编码9452个基因。比较基因组分析表明出芽短梗霉CCTCC M2012223的基因组组装长度最长,6株菌的同源基因数达到7092个,普鲁兰多糖和聚苹果酸合成相关基因的蛋白序列有很高的保守性。出芽短梗霉CCTCC M2012223和Aureobasidium pullulans var.melanogenum亲缘关系最近,而这2株菌的黑色素合成相关基因的蛋白序列有一些插入和突变。【结论】本研究解析了出芽短梗霉CCTCC M2012223的基因组序列信息,获得黑色素、普鲁兰多糖和聚苹果酸合成相关基因,为后续的代谢机制解析和改造提供相关依据。     

菠菜BADH单抗细胞株及其应用初探

用菠菜甜菜碱醛脱氢酶 ( BADH)免疫巴比西 ( BALB/c)小鼠 ,将其脾细胞与骨髓瘤细胞 SP2 /O-Ag1 4融合 ,在 1 92孔中 ,有约 1 4 %孔生长的杂交瘤细胞 ,用间接酶联免疫方法 ( ELISA)检测表现为阳性。选择其中 2 G3和 2 D10 细胞系 ,用有限稀释法进行克隆化培养 ,约 2 0 %克隆化细胞为强阳性。选择其中 2 G3- H3细胞株注射到 BALB/c小鼠腹腔中诱导腹水 ,腹水的单抗效价为 1∶ 1 0 3。应用 BADH单抗检查了大麦、水稻、高粱、小麦幼苗的叶片和根的粗提物 ,均呈阳性反应 ,表明 BADH除在光合组织中存在外 ,在非光合组织中也可能存在。讨论了非光合组织 BADH的意义     

Functions of poly-gamma-glutamic acid (γ-PGA) degradation genes in γ-PGA synthesis and cell morphology maintenance

Poly-γ-glutamic acid (γ-PGA) is an important biopolymer with greatly potential in industrial and medical applications. In the present study, we constructed a metabolically engineered glutamate-independent Bacillus amyloliquefaciens LL3 strain with considerable γ-PGA production, which was carried out by single, double, and triple markerless deletions of three degradation genes pgdS, ggt, and cwlO. The highest γ-PGA production (7.12 g/L) was obtained from the pgdS and cwlO double-deletion strain NK-pc, which was 93 % higher than that of wild-type LL3 strain (3.69 g/L). The triple-gene-deletion strain NK-pgc showed a 28 % decrease in γ-PGA production, leading to a yield of 2.69 g/L. Furthermore, the cell morphologies of the mutant strains were also characterized. The cell length of cwlO deletion strains NK-c and NK-pc was shorter than that of the wild-type strain, while the ggt deletion strains NK-g, NK-pg, NK-gc, and NK-pgc showed longer cell lengths. This is the first report concerning the markerless deletion of γ-PGA degradation genes to improve γ-PGA production in a glutamate-independent strain and the first observation that γ-glutamyltranspeptidase (encoded by ggt) could be involved in the inhibition of cell elongation.     

应用纤维素结合域标签构建谷氨酸棒状杆菌表达系统

为优化谷氨酸棒状杆菌表达系统的纯化工艺,合成里氏木霉的CBD基因,将其与谷氨酸棒状杆菌分泌表达载体pXMJ19-sp连接,构建以CBD为纯化标签的重组载体pXMJ 19-sp-CBD.在该载体中插入GFP基因并转化至谷氨酸棒状杆菌,可获得分泌表达融合蛋白GFP-CBD的重组菌.该菌经IPTG诱导后的发酵液在紫外灯下显示强烈的绿色荧光,重组蛋白的分泌表达量达200 mg/L.利用CBD标签对纤维素柱的可逆性吸附,可直接对谷氨酸棒状杆菌分泌到培养基中的重组蛋白进行纯化,从而简化工艺和降低成本,为工业化大生产奠定基础.     

Study on fibroin heavy chain of the silkwormBombyx mori by fluorescencein situ hybridization (FISH)

The sericulture industry plays a very important role in our national economy. Silkworm (Bombyx mori) is always regarded as a model animal and biological reactor. There have been detailed studies on the structure, expression and control and molecular evolution of silk genes. However, few, if any, reports are available on the localization of structural genes in silkworm by molecular cytogenetics. The present experiment has tentatively localized theFib-H gene at the distal end of the 25th linkage group, namely at the 25-0.0 position, and verified thatFib-H has only one locus, thus providing a temporary solution to the problem about its localization.     

东亚中更新世古人类下颔第二臼齿釉质-齿质连接面三维形状和釉质厚度分布

东亚中更新世古人类在头骨、下颌骨、牙齿等解剖部位表现出不同程度的形态多样性,中期成员代表为直立人,而晚期成员的演化地位具有较大争议。为进一步了解东亚中更新世古人类内部的形态变异特点和为东亚中更新世晚期古人类分类提供依据,本文使用微分同胚的表面匹配（Diffeomorphic Surface Matching, DSM）和形态测量图（Morphometric map）对下颌第二臼齿（M₂）釉质-齿质连接面的形状和齿冠侧面釉质厚度分布模式进行了量化分析。结果显示：1）东亚中更新世古人类与晚期人属成员（尼安德特人和现代人）存在较明显的形态差别;2）该时段晚期的东亚古人类相对中期直立人在侧面釉质厚度分布规律上具有独特性,并在釉质-齿质连接面的三维形状上与晚期人属成员更加接近。本文在以往对东亚中更新世古人类牙齿内外结构单个性状研究的基础上,使用三维形态测量方法进一步量化了M₂釉质-齿质连接面三维形状和侧面釉质厚度分布模式两项重要特征的变异特点,这对未来该时段同类型牙齿的形态鉴定以及解决东亚中更新世晚期古人类的分类地位具有一定意义。     

高秆野生稻内生固氮细菌多样性

高秆野生稻(Oryza alta)是一种重要的种质资源, 其组织内也蕴藏着非常宝贵的功能微生物资源。本实验采用无氮培养基, 从高秆野生稻中分离到43株内生固氮菌, 结合乙炔还原法测定其固氮酶活性。经固氮酶基因(nifH)的PCR扩增检测, 43株内生固氮菌代表菌株均能扩增出固氮酶基因片段。利用IS-PCR DNA指纹图谱和SDS-PAGE全细胞蛋白电泳图谱将获得的菌株聚类为6个类群(I、II、III、IV、V、VI)。对各个类群的代表菌株(ZF3, ZF8, ZF13, ZF15, ZF24, ZF43)进行16S rRNA基因序列测定, 结果表明, 类群I属于土生拉乌尔菌(Raoultella terrigena), 类群II属于类肺炎克雷伯氏菌类肺炎亚种(Klebsiella quasipnmoniae subsp. quasipneumoniae), 类群III属于越南伯克氏菌(Burkholderia vietnamiensis), 类群IV的菌株代表肠秆菌属的一个类群(Enterobacter sp.), 类群V的菌株属于门多萨假单胞菌(Pseudomonas mendocina), 类群VI归属于固氮植物菌(Phytobacter diazotrophicus)。Biolog聚类结果与IS-PCR指纹图谱类型及SDS-PAGE全细胞蛋白聚类结果一致。Biolog板测定结果显示, 来自不同类群的代表菌株对碳源的利用差异显著, 说明野生稻内多样的内生固氮菌从环境中获取碳源和氮素的适应能力较强。     

中国西藏种子植物区系新资料

本文报道了采自西藏喜马拉雅南坡的8个中国种子植物新记录种以及1个西藏新记录属。前者分别是吉隆牛奶菜(Marsdenia roylei)、塔基棕榈(Trachycarpus takil)、喀西蜂斗草(Sonerila khasiana)、旋花锡生藤(Cissampelos convolvulacea)、吉隆角盘兰(Herminium edgeworthii)、尼泊尔西番莲(Passiflora napalensis)、椭穗姜花(Hedychium ellipticum)和藏南象牙参(Roscoea brandisii); 1个西藏新记录属为箭药藤属(Belostemma) (箭药藤 Belostemma hirsutum)。凭证标本存放于中国科学院西双版纳热带植物园标本馆(HITBC)和西藏自治区高原生物研究所标本室(XZ)。     

Evolution of an inducible penicillin-target protein in methicillin-resistant Staphylococcus aureus by gene fusion

A new beta-lactam-inducible penicillin-binding protein (PBP) that has extremely low affinity to penicillin and most other beta-lactam antibiotics has been widely found in highly beta-lactam(methicillin)-resistant Staphylococcus aureus (MRSA). The gene for this protein was sequenced and the nucleotide sequence in its promoter and close upstream area was found to show close similarity with that of staphylococcal penicillinase, while the amino acid sequence over a wide range of the molecule was found to be similar to those of two PBPs of Escherichia coli, the shape-determining protein (PBP 2) and septum-forming one (PBP 3). Probably the MRSA PBP (Mr 76462) evolved by recombination of two genes: an inducible type I penicillinase gene and a PBP gene of a bacterium, causing the formation of a beta-lactam-inducible MRSA PBP.