外源质粒电击转入Pseudomonas putida的条件研究

以自行筛选的恶臭假单胞菌（Pseudomonas putida）(命名为Rs198,Genbank登录号为FJ788425）为受体菌,将具有卡那霉素抗性标记的大肠杆菌假单胞菌穿梭质粒PDSK519通过电转化法导入到受体菌中,对细胞生长状态、电转化温度、质粒DNA及感受态细胞浓度、电击电压及电转化介质给予转化效率的影响进行研究。结果表明,在细胞生长至OD600为0.5左右时收集菌体,在低温条件下制备浓度为 4.6×1012/ml 的感受态细胞,以0.3mol/L的蔗糖为电转化介质,在13kV/cm的场强下电击能获得较高的转化效率,最高可达1.3×107个转化子/μ g DNA。为构建恶臭假单胞的遗传转化系统,利用基因工程手段为该菌的进一步研究奠定了理论基础。     

Mechanism of cyanide and thiocyanate decomposition by an association of Pseudomonas putida and Pseudomonas stutzeri strains

The intermediate and terminal products of cyanide and thiocyanate decomposition by individual strains of the genus Pseudomonas, P. putida strain 21 and P. stutzeri strain 18, and by their association were analyzed. The activity of the enzymes of nitrogen and sulfur metabolism in these strains was compared with that of the collection strains P. putida VKM B-2187^T and P. stutzeri VKM B-975^T. Upon the introduction of CN⁻ and SCN⁻ into cell suspensions of strains 18 and 21 in phosphate buffer (pH 8.8), the production of NH ₄ ⁺ was observed. Due to the high rate of their utilization, NH₃, NH ₄ ⁺ , and CNO⁻ were absent from the culture liquids of P. putida strain 21 and P. stutzeri strain 18 grown with CN⁻ or SCN⁻. Both Pseudomonas strains decomposed SCN⁻ via cyanate production. The cyanase activity was 0.75 μmol/(min mg protein) for P. putida strain 21 and 1.26 μmol/(min mg protein) for P. stutzeri strain 18. The cyanase activity was present in the cells grown with SCN⁻ but absent in cells grown with NH ₄ ⁺ . Strain 21 of P. putida was a more active CN⁻ decomposer than strain 18 of P. stutzeri. Ammonium and CO₂ were the terminal nitrogen and carbon products of CN⁻ and SCN⁻ decomposition. The terminal sulfur products of SCN⁻ decomposition by P. stutzeri strain 18 and P. putida strain 21 were thiosulfate and tetrathionate, respectively. The strains utilized the toxic compounds in the anabolism only, as sources of nitrogen (CN⁻ and SCN⁻) and sulfur (SCN⁻). The pathway of thiocyanate decomposition by the association of bacteria of the genus Pseudomonas is proposed based on the results obtained. Original Russian Text ? N.V. Grigor’eva, T.F. Kondrat’eva, E.N. Krasil’nikova, G.I. Karavaiko, 2006, published in Mikrobiologiya, 2006, Vol. 75, No. 3, pp. 320–328.     

趋化信号转导基因cheA突变对Pseudomonas putida DLL-1甲基对硫磷的趋化性及原位降解的影响

Pseudomonas putida DLL-1是一株甲基对硫磷(MP)高效降解菌株,同时对MP具有趋化性。cheA基因是菌株趋化信号转导过程中负责编码组氨酸激酶的基因,为了研究菌株趋化性在农药原位降解中的作用,通过基因打靶的方式使P.putida DLL-1染色体上单拷贝的cheA基因失活,成功地获得了MP的趋化突变株P.putida DAK,突变株与野生菌株生长能力没有显著差异。通过土壤盆钵试验(MP浓度为50mg/kg),发现在灭菌与未灭菌土壤中趋化突变株对MP的降解能力低于原始出发菌株DLL-1约20%~30%,说明菌株DLL-1趋化性的丧失会减慢其对农药的降解,趋化性在农药的原位降解过程中发挥重要作用。     

A novel pathway for the biodegradation of 3-nitrotoluene in Pseudomonas putida

Abstract: 3-Nitrotoluene was degraded when incubated with the resting cells of Pseudomonas putida OU83. Most of the 3-nitrotoluene (70%) was metabolized via reduction of the nitro group to form 3-aminotoluene (3-AT). A minor portion (30%) was degraded through a novel pathway involving oxidation of 3-NT to form 3-nitrophenol through a series of intermediary metabolites: 3-nitrobenzyl alcohol, 3-nitrobenzaldehyde and 3-nitrobenzoic acid. Degradation of 3-nitrophenol occurred with the formation of a transient intermediary metabolite, hydroxynitroquinone, which was further degraded with the near stoichiometric release of nitrite into the medium. 3-Nitrotoluene-induced cells showed increased oxygen consumption with 3-nitrotoluene, 3-nitrobenzaldehyde, 3-nitrobenzoate, and 3-nitrophenol as substrates in comparison to uninduced cells. Cell extracts prepared from strain OU83 contained benzylalcohol dehydrogenase and benzaldehyde dehydrogenase activities. The experimental evidence suggests a novel pathway for the degradation of 3-NT in which C-1 elimination is catalyzed by a cofactor-independent deformylase, rather than a decarboxylase or dioxygenase.     

基于蛋白质组学的假单胞菌耐溶剂机制研究

【目的】有机溶剂对微生物有强烈的毒害作用致使绝大多数微生物不能在较高的有机溶剂浓度下生长。为了探究微生物的耐溶剂性机制,由野生型假单胞菌Pseudomonas putida JUCS驯化获得一株能够在60%(V/V)的环己烷中生长的菌株P.putidaJUCT1。【方法】采用蛋白质二维电泳对P.putida JUCT1在不同溶剂条件下的蛋白组分表达量的差异进行分析比对。【结果】从总共22个表达量差异均超过50%的蛋白质中,选取了3个高丰度蛋白质,通过MALDI-TOF/TOF鉴定为:3-羟基异丁酸水解酶、蛋白质延伸因子EF-Ts、异分支酸水解酶超家族(编码基因分别为mmsB、tsf、PSEEN0851)。将这3个基因在大肠杆菌中重组表达,3个蛋白均能不同程度地提高E.coli JM109的耐溶剂性,其中3-羟基异丁酸脱氢酶(编码基因mmsB)对菌株的溶剂耐受性影响最为显著。【结论】证明了运用蛋白组学的方法研究微生物的耐溶剂性的可行性,并为构建适用于工业化应用的溶剂耐受性整体细胞生物催化剂提供理论依据。     

Solution structure of the ubiquitin-associated domain of human BMSC-UbP and its complex with ubiquitin

Ubiquitin is an important cellular signal that targets proteins for degradation or regulates their functions. The previously identified BMSC-UbP protein derived from bone marrow stromal cells contains a ubiquitin-associated (UBA) domain at the C terminus that has been implicated in linking cellular processes and the ubiquitin system. Here, we report the solution NMR structure of the UBA domain of human BMSC-UbP protein and its complex with ubiquitin. The structure determination was facilitated by using a solubility-enhancement tag (SET) GB1, immunoglobulin G binding domain 1 of Streptococcal protein G. The results show that BMSC-UbP UBA domain is primarily comprised of three alpha-helices with a hydrophobic patch defined by residues within the C terminus of helix-1, loop-1, and helix-3. The M-G-I motif is similar to the M/L-G-F/Y motifs conserved in most UBA domains. Chemical shift perturbation study revealed that the UBA domain binds with the conserved five-stranded beta-sheet of ubiquitin via hydrophobic interactions with the dissociation constant (KD) of approximately 17 microM. The structural model of BMSC-UbP UBA domain complexed with ubiquitin was constructed by chemical shift mapping combined with the program HADDOCK, which is in agreement with the result from mutagenesis studies. In the complex structure, three residues (Met76, Ile78, and Leu99) of BMSC-UbP UBA form a trident anchoring the domain to the hydrophobic concave surface of ubiquitin defined by residues Leu8, Ile44, His68, and Val70. This complex structure may provide clues for BMSC-UbP functions and structural insights into the UBA domains of other ubiquitin-associated proteins that share high sequence homology with BMSC-UbP UBA domain.     

假单胞菌海因酶基因在大肠杆菌中的高效表达(英文)

为实现利用生物酶转化法进行D 对羟基苯甘氨酸的工业化生产 ,构建了 3株海因酶基因工程菌 .利用PCR技术从恶臭假单胞菌 (Pseudomonasputida)CPU 980 1染色体DNA中扩增得到长约1.8kb的含编码区和自身启动子的海因酶全基因 .通过将海因酶全基因插入pMD18 T质粒、海因酶基因的编码区与pET 17 b质粒重组、海因酶基因编码区和T7强启动子一起插入pMD18 T质粒分别得到重组质粒pMD dht、pET dht和pMD T7 dht.将上述重组质粒分别转化大肠杆菌 (Escherichiacoli) ,通过地高辛标记菌落原位杂交和海因酶活力测定两种方法 ,筛选出具有海因酶活力的阳性转化子 .结果表明 ,大肠杆菌的RNA聚合酶能够识别和结合来自恶臭假单胞菌海因酶基因的自身启动子 ,该启动子在大肠杆菌中能够工作 .基因工程菌E .coliBL2 1 pMD dht、E .coliBL2 1 pET dht和E .coliBL2 1 pMD T7 dht的海因酶活力分别为 170 0U L、190 0U L和 2 5 0 0U L ,比野生菌P .putidaCPU 980 1的海因酶活力分别提高了 8倍、9倍和 12倍 .薄层扫描结果显示 ,这些工程菌的海因酶表达量分别约占菌体总可溶性蛋白质的 2 0 %、31%和 5 7%.SDS PAGE显示 ,海因酶的单体分子量约为 5 0kD .经工程菌E .coliBL2 1 pMD T7 dht催化 ,底物对羟基苯海因的转化率在 13h内可达到 9     

苯系物降解菌Pseudomonas putida SW-3的筛选及其降解苯的研究

【目的】以苯、甲苯和苯乙烯为唯一碳源,从工业石油废水中筛选苯系物降解菌,分析其降解特性,探讨底物间相互作用对降解情况的影响。【方法】经生理生化和16S r RNA基因分析进行菌种鉴定,采用顶空气相色谱法测定苯系物含量,通过细胞的疏水性、乳化能力、排油圈及透射电镜观察分析菌株降解特性。【结果】经鉴定该菌为Pseudomonas putida,命名为SW-3菌株。最适降解条件下,单位菌体对苯、甲苯和苯乙烯的最大降解速率分别为0.072、0.035和0.019 g/(L·h),苯系混合物的总降解率达79.99%。底物降解实验表明,苯可促进甲苯和苯乙烯的降解,而苯乙烯则能抑制甲苯的降解。菌株的吸附、摄取和降解特性的研究发现,菌株SW-3在自身分泌的表面活性剂的协助下以耗能的方式运输苯。【结论】菌株SW-3具有产生表面活性剂和降解苯系物的能力,且底物间的相互作用能够显著影响菌株对不同底物的降解。     

Involvement of the plasmid pPGH1 in the phenol degradation of Pseudomonas putida strain H

Pseudomonas putida strain H (wildtype) was shown to harbour two plasmids with a molecular mass of about 50 kb and 200–220 kb, respectively. Evidence is presented that the larger one, pPGH1, is involved in the phenol degradation via the meta-cleavage pathway.     

Identification and properties of an inducible phenylacyl-CoA dehydrogenase in Pseudomonas putida KT2440

A novel acyl-CoA dehydrogenase that initiates beta-oxidation of the side chains of phenylacyl-CoA compounds by Pseudomonas putida was induced by growth with phenylhexanoate as carbon source. It was identified as the product of gene PP_0368, which was cloned and overexpressed in Escherichia coli. This phenylacyl-CoA dehydrogenase was found to be dimeric with a subunit molecular mass of 66 kDa, to contain FAD and to be active with phenylacyl-CoA substrates having side chains from four to at least 11 carbon atoms. The same enzyme was induced by the aliphatic alkanoate octanoate. The optimal aliphatic substrates for the enzyme were palmitoyl-CoA and stearoyl-CoA, a property shared with mammalian very-long-chain acyl-CoA dehydrogenases. The FAD in the enzyme was reduced by aromatic and aliphatic substrates, with changes to the oxidation-reduction potential. Chemical reduction by dithionite ion and oxidation by ferricyanide ion showed that the enzyme can accept four electrons: two to reduce the flavin and two to slowly reduce an unknown acceptor, which in its reduced form interacts with the oxidized flavin in a charge-transfer complex. The experiments identify for the first time an acyl-CoA dehydrogenase that oxidizes the activated forms of aromatic acids similar to those used to first demonstrate the biological beta-oxidation of fatty acids.     

恶臭假单胞菌丙氨酸消旋酶基因的克隆、序列分析及表达

从恶臭假单胞菌(Pseudomonas putida)200的基因组出发,用PCR方法克隆到两个独立作用的丙氨酸消旋酶基因,称之为dadX和alr。DadX编码357个氨基酸长的多肽,计算分子量为38.82kDa,alr编码409个氨基酸长的多肽,计算分子量为44.182kDa。序列分析显示,DadX的氨基酸序列与Pseudomonas putidaKT2440,铜绿假单胞菌(Pseudomonas aeruginosa),鼠伤寒沙门氏菌(Salmonella typhimurium)和大肠杆菌(Escherichia coli)的DadX比较,相似性分别为96.64%、71.99%、44.88%和47.37%。Alr的氨基酸序列与Pseudomonas putidaKT2440比较,同源性为94.38%,而与铜绿假单胞菌(P.aeruginosa)、鼠伤寒沙门氏菌(S.typhimurium)和大肠杆菌(E.coli)的Alr比较,同源性均较低,分别为22.89%、25.72%和26.44%。在P.putida200的DadX和Alr氨基酸序列中部发现有对于酶活性至关重要的保守区域,如磷酸吡哆醛(PLP)结合位点。DadX和alr在大肠杆菌中得到表达,DadX丙氨酸消旋酶只对丙氨酸有消旋作用,而Alr丙氨酸消旋酶可以作用于丙氨酸和丝氨酸两种底物,且对丝氨酸特异性更高。Alr的表达不依赖于外源启动子,说明在其结构基因上游存在启动子结构。     

Solution structure of protein synthesis initiation factor 1 from Pseudomonas aeruginosa

Pseudomonas aeruginosa is an opportunistic bacterial pathogen and a primary cause of nosocomial infection in humans. The rate of antibiotic resistance in P. aeruginosa is increasing worldwide leading to an unmet need for discovery of new chemical compounds distinctly different from present antimicrobials. Protein synthesis is an essential metabolic process and a validated target for the development of new antibiotics. Initiation factor 1 from P. aeruginosa (Pa‐IF1) is the smallest of the three initiation factors that act to establish the 30S initiation complex during initiation of protein biosynthesis. Here we report the characterization and solution NMR structure of Pa‐IF1. Pa‐IF1 consists of a five‐stranded β‐sheet with an unusual extended β‐strand at the C‐terminus and one short α‐helix arranged in the sequential order β1‐β2‐β3‐α1‐β4‐β5. The structure adopts a typical β‐barrel fold and contains an oligomer‐binding motif. A cluster of basic residues (K39, R41, K42, K64, R66, R70, and R72) located on the surface of strands β4 and β5 near the short α‐helix may compose the binding interface with the 30S subunit.     

Use of bioluminescence for monitoring the viability of individual Pseudomonas putida KT2442 cells

Exponential phase cells of Pseudomonas putida KT2442 rapidly lost viability when incubated at 0°C without entering a viable but non-culturable state. The majority of dead cells retained their cellular integrity and contained DNA. However, their cellular rRNA content was substantially reduced. By employing a luciferase-marked derivative of P. putida KT2442 in combination with a highly sensitive low-light imaging system, live and dead cells could be distinguished.     

Engineering Pseudomonas putida KT2440 for the production of isobutanol

We engineered P. putida for the production of isobutanol from glucose by preventing product and precursor degradation, inactivation of the soluble transhydrogenase SthA, overexpression of the native ilvC and ilvD genes, and implementation of the feedback‐resistant acetolactate synthase AlsS from Bacillus subtilis, ketoacid decarboxylase KivD from Lactococcus lactis, and aldehyde dehydrogenase YqhD from Escherichia coli. The resulting strain P. putida Iso2 produced isobutanol with a substrate specific product yield (Y_Iso/S) of 22 ± 2 mg per gram of glucose under aerobic conditions. Furthermore, we identified the ketoacid decarboxylase from Carnobacterium maltaromaticum to be a suitable alternative for isobutanol production, since replacement of kivD from L. lactis in P. putida Iso2 by the variant from C. maltaromaticum yielded an identical Y_Iso/S. Although P. putida is regarded as obligate aerobic, we show that under oxygen deprivation conditions this bacterium does not grow, remains metabolically active, and that engineered producer strains secreted isobutanol also under the non‐growing conditions.     

Anti-Oomycete Activity and Pepper Root Colonization of Pseudomonas plecoglossicida YJR13 and Pseudomonas putida YJR92 against Phytophthora capsici

Previously, Pseudomonas plecoglossicida YJR13 and Pseudomonas putida YJR92 from a sequential screening procedure were proven to effectively control Phytophthora blight caused by Phytophthora capsici. In this study, we further investigated the anti-oomycete activities of these strains against mycelial growth, zoospore germination, and germ tube elongation of P. capsici. We also investigated root colonization ability of the bacterial strains in square dishes, including cell motility (swimming and swarming motilities) and biofilm formation. Both strains significantly inhibited mycelial growth in liquid and solid V8 juice media and M9 minimal media, zoospore germination, and germ tube elongation compared with Bacillus vallismortis EXTN-1 (positive biocontrol strain), Sphingomonas aquatilis KU408 (negative biocontrol strain), and MgSO₄ solution (untreated control). In diluted (nutrient-deficient) V₈ juice broth, the tested strain populations were maintained at >10⁸ cells/ml, simultaneously providing mycelial inhibitory activity. Additionally, these strains colonized pepper roots at a 10⁶ cells/ml concentration for 7 days. The root colonization of the strains was supported by strong swimming and swarming activities, biofilm formation, and chemotactic activity towards exudate components (amino acids, organic acids, and sugars) of pepper roots. Collectively, these results suggest that strains YJR13 and YJR92 can effectively suppress Phytophthora blight of pepper through direct anti-oomycete activities against mycelial growth, zoospore germination and germ tube elongation. Bacterial colonization of pepper roots may be mediated by cell motility and biofilm formation together with chemotaxis to root exudates.     

Broad spectrum and narrow spectrum mercury resistance encoded by different plasmids of a Pseudomonas putida strain

Abstract Pseudomonas putida strain H harbours two plasmids of different sizes. It was demonstrated that the large plasmid pPGH1 confers a broad spectrum resistance to mercurials, whereas the small plasmid pPGH2 confers a narrow spectrum one. Under the influence of the small plasmid the resistance of cells against poisoning with 2,4-di-chlorophenol or o -cresol increases in comparison to cells without this plasmid. Both plasmids proved to be not self-transmissible, but pPGH1 is transferable by mobilisation by means of the IncP-1 vectors R68.45 or RP1.