Intracellular degradation of two structurally different polyhydroxyalkanoic acids accumulated in Pseudomonas putida and Pseudomonas citronellolis from mixtures of octanoic acid and 5-phenylvaleric acid

From a set of mixed carbon sources, 5-phenylvaleric acid (PV) and octanoic acid (OA), polyhydroxyalkanoic acid (PHA) was separately accumulated in the two pseudomonads Pseudomonas putida BM01 and Pseudomonas citronellolis (ATCC 13674) to investigate any structural difference between the two PHA accumulated under a similar culture condition using one-step culture technique. The resulting polymers were isolated by chloroform solvent extraction and characterized by fractional precipitation and differential scanning calorimetry. The solvent fractionation analysis showed that the PHA synthesized by P. putida was separated into two fractions, 3-hydroxy-5-phenylvalerate (3HPV))-rich PHA fraction in the precipitate phase and 3-hydroxyoctanoate (3HO)-rich PHA fraction in the solution phase whereas the PHA produced by P. citronellolis exhibited a rather little compositional separation into the two phases. According to the thermal analysis, the P. putida PHA exhibited two glass transitions indicative of the PHA not being homogeneous whereas the P. citronellolis PHA exhibited only one glass transition. It was found that the structural heterogeneity of the P. putida PHA was caused by a significant difference in the assimilation rate between PV and OA. The structural heterogeneity present in the P. putida PHA was also confirmed by a first order degradation kinetics analysis of the PHA in the cells. The two different first-order degradation rate constants (k₁), 0.087 and 0.015/h for 3HO- and 3HPV-unit, respectively, were observed in a polymer system over the first 20 h of degradation. In the later degradation period, the disappearance rate of 3HO-unit was calculated to be 0.020 h. The k₁ value of 0.083/h, almost the same as for the 3HO-unit in the P. putida PHA, was obtained for the P(3HO) accumulated in P. putida BM01 grown on OA as the only carbon source. In addition, the k₁ value of 0.015/h for the 3HPV-unit in the P. putida PHA, was also close to 0.019/h for the P(3HPV) homopolymer accumulated in P. putida BM01 grown on PV plus butyric acid. On the contrary, the k₁ values for the P. citronellolis PHA were determined to be 0.035 and 0.029/h for 3HO- and 3HPV-unit, respectively, thus these two relatively close values implying a random copolymer nature of the P. citronellolis PHA. In addition, the faster degradation of P(3HO) than P(3HPV) by the intracellular P. putida PHA depolymerase indicates that the enzyme is more specific against the aliphatic PHA than the aromatic PHA.     

Isolation and Characterization of Group II Introns from Pseudomonas alcaligenes and Pseudomonas putida

Group II introns isolated from Pseudomonas alcaligenes NCIB 9867, Pseudomonas putida NCIB 9869, and P. putida KT2440 were closely related with nucleotide sequence identities of between 87 and 96%. The genome of P. alcaligenes also harbored a truncated group II intron of 682 bp that lacks the gene for the intron-encoded protein (IEP). Unlike most bacterial group II introns, the Pseudomonas introns were found to lack the Zn domains in their IEPs, did not appear to interrupt any genes, and were located downstream of open reading frames which were adjacent to hairpin loop structures that resemble rho-independent terminators. These structures also contain the intron binding sites 1 and 2 (IBS1 and IBS2 sequences) that were required for intron target site recognition in transposition. One of the group II introns found in P. alcaligenes, Xln3, was shown to have transposed from the chromosome to the endogenous pRA2 plasmid at a site adjacent to IBS1- and IBS2-like sequences.     

铜绿假单胞菌锌离子摄取系统的研究进展

锌作为一种结构、催化和信号的成分,在许多生理过程中起着关键的作用.它也是病原微生物生长所必需的,不但参与病原微生物代谢和各种毒力因子的调控,而且是病原微生物在宿主中感染和定殖所必需的.铜绿假单胞菌侵染宿主发挥毒力时,宿主会采取营养免疫的策略来限制体内环境中游离的锌离子浓度而抑制该病原菌的感染和定殖.反过来,铜绿假单胞菌...     

Association of the biocide 5-chloro-2-methyl-isothiazol-3-one with Pseudomonas aeruginosa and Pseudomonas fluorescens

The biocide 5-chloro-2-methyl-isothiazol-3-one (CMI) associated rapidly with cells of Pseudomonas aeruginosa and Pseudomonas fluorescens with association being nearly complete within 10–15 min. The cells serve as a sink for CMI, concentrating it up to 400-fold. Kinetics of association are very similar amongst the strains examined. Examination of the relation of CMI concentration to the rate of association indicates that there are two kinetically distinguishable processes, with the breakpoint occurring around the transition from inhibitory to suprainhibitory levels of CMI. This suggests the rapid onset of toxic effects at suprainhibitory CMI concentrations affects the associative process. Discharging the proton motive force by treatment with uncoupling agents or selectively depleting it by treatment with inhibitors reduces the amount of CMI which associates with the cells. Selective depletion of the ATP pool has no effect. These results suggest that either the proton motive force (pmf) is involved directly in CMI association in an active transport process, or that an intact pmf is required for some facet of the cells metabolism which maintains the cells as a sink for CMI. The nonchlorinated analogue 2-methyl-isothiazol-3-one is a poor competitor for CMI association.     

转录调节因子σ~(38)介导铜绿假单胞菌绿脓菌素合成代谢调控

【目的】为了进一步鉴定铜绿假单胞菌转录调控因子σ~(38)对2个拷贝吩嗪合成基因簇(phz A1-G1和phz A2-G2)的具体调控方式并推定介导绿脓菌素合成代谢的可能调控机制。【方法】根据铜绿假单胞菌基因组信息,利用同源重组原理构建rpo S基因缺失突变株Δrpo S以及克隆全长rpo S基因作互补分析;再以单一吩嗪基因簇缺失突变株Δphz1和Δphz2为出发菌株,分别构建rpo S缺失突变株Δrpo Sphz1和rpo S插入突变株Δrpo Sphz2,测定并比较野生株及相关突变株的绿脓菌素合成量,初步推定σ~(38)因子对2个不同吩嗪基因簇表达的调控方式。【结果】在GA培养基中,突变株Δrpo S的绿脓菌素合成量比野生株显著增加;互补分析证实,σ~(38)可使突变株Δrpo S的绿脓菌素降低并接近野生株PAO1水平;与对照株Δphz1相比,突变株Δrpo Sphz1的绿脓菌素合成量因σ~(38)因子缺失而显著减少;而与对照株Δphz2相比,突变株Δrpo Sphz2的绿脓菌素合成量因σ~(38)因子缺失显著增加。【结论】转录调控因子σ~(38)对铜绿假单胞菌绿脓菌素的合成代谢的确具一定的负调控作用;结合已报道的研究结果,初步推定:σ~(38)因子通过负调控吩嗪基因簇phz1,正调控吩嗪基因簇phz2的表达实现对绿脓菌素合成代谢的调控。     

假单胞菌属双组分信号转导系统调控吩嗪生物合成研究进展

吩嗪是由假单胞菌或链霉菌产生的一类具有抗菌、抗肿瘤和抗寄生虫活性的含氮杂环代谢物,在农业和医疗领域具有广泛的应用.但吩嗪的合成受到复杂的级联网络调控.本文总结了假单胞菌属中双组分信号转导系统(two-component signal transduction system,TCS)对吩嗪生物合成的调控机制,阐明在双组分...     

Directed Evolution of Toluene Dioxygenase from Pseudomonas putida for Improved Selectivity Toward cis-Indandiol during Indene Bioconversion

Toluene dioxygenase (TDO) from Pseudomonas putida F1 converts indene to a mixture of cis-indandiol (racemic), 1-indenol, and 1-indanone. The desired product, cis-(1S, 2R)-indandiol, is a potential key intermediate in the chemical synthesis of indinavir sulfate (Crixivan), Merck's HIV-1 protease inhibitor for the treatment of AIDS. To reduce the undesirable byproducts 1-indenol and 1-indanone formed during indene bioconversion, the recombinant TDO expressed in Escherichia coli was evolved by directed evolution using the error-prone polymerase chain reaction (epPCR) method. High-throughput fluorometric and spectrophotometric assays were developed for rapid screening of the mutant libraries in a 96-well format. Mutants with reduced 1-indenol by-product formation were identified, and the individual indene bioconversion product profiles of the selected mutants were confirmed by HPLC. Changes in the amino acid sequence of the mutant enzymes were identified by analyzing the nucleotide sequence of the genes. A mutant with the most desirable product profile from each library, defined as the most reduced 1-indenol concentration and with the highest cis-(1S, 2R)-indandiol enantiomeric excess, was used to perform each subsequent round of mutagenesis. After three rounds of mutagenesis and screening, mutant 1C4-3G was identified to have a threefold reduction in 1-indenol formation over the wild type (20% vs 60% of total products) and a 40% increase of product (cis-indandiol) yield.     

A novel Pseudomonas putida strain with high levels of hydantoin-converting activity, producing -amino acids

Optically pure chiral amino acids and their derivatives can be efficiently synthesised by the biocatalytic conversion of 5-substituted hydantoins in reactions catalysed by stereo-selective microbial enzymes: initially a hydantoinase catalyses the cleavage of the hydantoin producing an N-carbamyl amino acid. In certain bacteria where an N-carbamyl amino acid amidohydrolase (NCAAH) is present, the N-carbamyl amino acid intermediate is further converted to amino acid, ammonia and CO₂. In this study we report on a novel Pseudomonas putida strain which exhibits high levels of hydantoin-converting activity, yielding -amino acid products including alanine, valine, and norleucine, with bioconversion yields between 60% and 100%. The preferred substrates are generally aliphatic, but not necessarily short chain, 5-alkylhydantoins. In characterizing the enzymes from this microorganism, we have found that the NCAAH has -selectivity, while the hydantoinase is non-stereoselective. In addition, resting cell reactions under varying conditions showed that the hydantoinase is highly active, and is not subject to substrate inhibition, or product inhibition by ammonia. The rate-limiting reaction appears to be the NCAAH-catalysed conversion of the intermediate. Metal-dependence studies suggest that the hydantoinase is dependent on the presence of magnesium and cobalt ions, and is strongly inhibited by the presence of copper ions. The relative paucity of -selective hydantoin-hydrolysing enzyme systems, together with the high level of hydantoinase activity and the unusual substrate selectivity of this P. putida isolate, suggest that is has significant potential in industrial applications.     

铜绿假单胞菌cntRLMN操纵子介导锌离子摄取的功能鉴定

【目的】本研究以铜绿假单胞菌PAO1 (Pseudomonas aeruginosa PAO1,菌种编号ATCC15692)为对象,研究cntRLMN在锌离子摄取中的功能。【方法】在ΔznuBC的基础上,以同源重组的方法构建了cntRLMN的各种突变菌株,通过质粒接合转移的方法构建其互补菌株及lacZ转录融合报告菌株,运用β-半乳糖苷酶酶活检测研究了Zur蛋白对cntRLMN的转录调控,凝胶阻滞实验(EMSA)检验Zur蛋白与cnt启动子及cnt启动子的突变片段的体外结合,并进一步通过生长曲线分析对cntRLMN中cntR、cntL、cntN等基因的锌离子摄取功能进行了分析和鉴定。最终,通过构建大蜡螟幼虫的侵染模型来研究cntRLMN对铜绿假单胞菌毒力发挥的影响。【结果】lacZ转录融合的酶活分析显示cntRLMN受Zur蛋白的负调控,其表达以Zur蛋白依赖的方式受锌离子饥饿的诱导;EMSA实验的结果显示cntRLMN的启动子可以与His-Zur结合形成DNA-蛋白质复合体,结合位点为GCGTTATAGTATATCAT;生长曲线和大蜡螟幼虫侵染实验的分析结果显示ZnuBC和CntRLMN的功能存在互补性,仅znuBC和cntRLMN双缺失突变时菌株在限锌培养条件下的生长和对大蜡螟幼虫的毒性才受到显著抑制,说明CntRLMN代表另一种独立的锌离子摄取系统。【结论】cntRLMN是受Zur直接负调控的另一种独立的铜绿假单胞菌锌离子摄取系统,对铜绿假单胞菌毒力的发挥起重要作用。     

Algicidal activity of rhamnolipid biosurfactants produced by Pseudomonas aeruginosa

The algicidal activity of the rhamnolipid biosurfactants (the mixture of Rha-Rha-C₁₀-C₁₀ and Rha-C₁₀-C₁₀) produced by Pseudomonas aeruginosa was investigated in the present paper. The results indicated that the biosurfactants had potential algicidal effects on the harmful algal bloom (HAB) species, Heterosigma akashiwo. The growth of H. akashiwo was strongly inhibited in medium containing rhamnolipids (0.4–3.0 mg L⁻¹); moreover, the rhamnolipids showed strong lytic activity toward H. akashiwo at higher concentrations (≥4.0 mg L⁻¹). In addition, the effects of the rhamnolipids on the growth of Gymnodinium sp. and Prorocentrum dentatum, another two kinds of HAB species, were also studied. Compared with the dramatic algicidal effect on H. akashiwo, the cells of P. dentatum were inhibited or lysed at higher concentrations (1.0–10.0 mg L⁻¹), while the cells of Gymnodinium sp. were not suppressed with the same treatment, indicating the rhamnolipids had the potential for the selective control of HABs.Morphometric analysis at ultrastructural level by transmission electron micrographs indicated that the extent of ultrastructural damage of the alga was severe at high concentrations of rhamnolipids and during extended periods of contact. The first response occurred in the plasma membrane which partly disintegrated. The lack of membrane facilitated the rhamnolipid biosurfactants into the cells and allowed damage to other organelles, which resulted in the injury of chloroplast, vacuolization of mitochondria and deformation of the cristae, disruption of nuclear membrane and condensation of chromatin in nucleus, suggesting that the lytic activity of rhamnolipids was mainly due to their powerful surfactivity and their tendency to cohere on the surface of phospholipids bimolecular layer of the cells and further destroyed the layers, and then the structure of quasi-membrane configurations inside the cells was disintegrated, following by the irreversible damage to the ultrastructure and the loss of the function of organelles, consequently leading the cells to lyse.     

鳖源铜绿假单胞菌的分离鉴定及多位点序列与全基因组分析

【目的】查明引起湖北仙桃某中华鳖养殖场中华鳖发病死亡的病原及其特征。【方法】本研究分离患病中华鳖的病原,并结合形态特征、生理生化试验、16SrRNA基因鉴定,鉴定分离菌株;通过人工回归感染试验、药敏试验、全基因组测序与分析对分离菌株的致病性和耐药性进行研究;通过多位点序列分型(multilocus sequence type,MLST)分析,对分离菌株的流行情况进行探究。【结果】从患病中华鳖肝脏等部分分离到3株优势菌株HX8、FG10和GC20,16SrRNA基因同源性和生化特征分析鉴定为铜绿假单胞菌(Pseudomonas aeruginosa)。回归感染试验证实该菌株是引起本次中华鳖患病的病原菌。3株分离株的药敏实验结果基本一致,均对诺氟沙星、恩诺沙星等8种抗生素敏感,对氟苯尼考、多西环素、磺胺异恶唑等6种抗生素耐药。MLST鉴定表明,3株分离株均属于序列型(sequencetype,ST)252型,eBURST分析进一步显示ST252型与一些ST共同构成了克隆复合体(clonal complexes,CC) CC252,且ST252是CC252的原始序列型(founder ST)...     

Localization of functional domains in the Escherichia coli coprogen receptor FhuE and the Pseudomonas putida ferric-pseudobactin 358 receptor PupA

Transport of ferric-siderophores across the outer membrane of gram-negative bacteria is mediated by specific outer membrane receptors. To localize the substrate-binding domain of the ferric-pseudobactin 358 receptor, PupA, of Pseudomonas putida WCS358, we constructed chimeric receptors in which different domains of PupA were replaced by the corresponding domains of the related ferric-pseudobactin receptors PupB and PupX, or the coprogen receptor FhuE of Escherichia coli. None of the chimeric proteins composed of pseudobactin receptor domains facilitated growth on any of the original substrates, or they showed only an extremely low efficiency. However, these receptors enabled cells of Pseudomonas BN8 to grow on media supplemented with uncharacterized siderophore preparations. These siderophore preparations were isolated from the culture supernatant of WCS358 cells carrying plasmids that contain genes of Pseudomonas B10 required for the biosynthesis of pseudobactin B10. Hybrid proteins that contained at least the amino-terminal 516 amino acids of mature FhuE were active as a receptor for coprogen and interacted with the E. coli TonB protein. A chimeric PupA-FhuE protein, containing the amino-terminal 94 amino acids of mature PupA, was also active as a coprogen receptor, but only in the presence of Pseudomonas TonB. It is concluded that the carboxy-terminal domain of ferric-pseudobactin receptors is important, but not sufficient, for ligand interaction, whereas binding of coprogen by the FhuE receptor is not dependent on this domain. Apparently, the ligand-binding sites of different receptors are located in different regions of the proteins. Furthermore, species-specific TonB binding by the PupA receptor is dependent on the amino-terminal domain of the receptor.     

铜绿假单胞菌生物被膜组成及其受群体感应系统和c-di-GMP调控的研究进展

作为人类条件性感染的前三大病原菌之一的铜绿假单胞菌,是一种革兰氏阴性细菌,对免疫功能低下和囊性纤维化患者可以造成严重和持续性感染。造成这种持续感染的原因主要是由于细菌接收外界信号后,在自身调控网络的协同作用下,会依附于固体表面,并产生胞外多糖、基质蛋白和胞外DNA等大分子物质形成高度结构化的膜状复合物将自身包裹形成生物被膜群体结构。生物被膜可以有效帮助细菌定殖、提高细菌对抗菌物质和宿主免疫反应的抵抗能力、促进群落细菌的细胞-细胞之间的信号交流等,是临床治疗中病原菌慢性感染和反复感染最重要的原因之一。本篇综述重点介绍了铜绿假单胞菌生物被膜的各组成成分及其在生物被膜形成中的重要功能,并进一步阐述了群体感应系统(las、rhl、pqs与iqs)和c-di-GMP对铜绿假单胞菌生物被膜形成的调控作用。通过本篇综述可以更清晰地了解细菌生物被膜形成和调控的过程,为开发新的治疗生物被膜感染策略提供帮助。     

维拉帕米对铜绿假单胞菌群体感应抑制作用的体外研究

考察维拉帕米对铜绿假单胞菌（Pseudomonas aeruginosa, PA）群体感应（Quorum sensing, QS）的抑制作用。测定维拉帕米最小抑菌浓度(Minimal inhibit concentration,MIC) ;构建培养环境,考察维拉帕米对PA生长的影响,绘制生长曲线;毒力因子表达实验中,分别考察维拉帕米对PA弹性蛋白酶表达、蛋白水解酶表达和绿脓毒素表达的影响。结果表明,维拉帕米MIC₅₀为128 μg/mL,MIC₉₀为512 μg/mL,最低抑菌质量浓度范围为128~512 μg/mL时,具有较好抑菌活性;生长曲线表明,维拉帕米质量浓度为16 μg/mL时,开始抑制PA的生长,随着浓度的增加,抑制作用逐渐增大;当维拉帕米质量浓度为512、256、128、64、32、16 μg/mL时,明显抑制弹性蛋白酶表达（P<0.01）,质量浓度为8 μg/mL时,对弹性蛋白酶有一定抑制作用（P<0.05）,维拉帕米质量浓度为512、256、128、64、32 μg/mL时,明显抑制蛋白水解酶的活性和绿脓毒素的表达（P<0.01）,当质量浓度为16 μg/mL时,对蛋白水解酶活性和绿脓毒素的表达有一定抑制作用（P<0.05）;维拉帕米对QS的抑制有浓度依赖。维拉帕米对PA的QS有明显抑制作用,体外可明显抑制PA生长,可作为抗菌增效的潜在开发药物。     

恶臭假单胞菌ND6菌株catA基因的克隆和表达及其儿茶酚裂解途径探讨

恶臭假单胞菌ND6菌株的萘降解质粒pND6-1中编码儿茶酚1,2-双加氧酶的catA基因在大肠杆菌中进行了克隆和表达,并研究表达产物的酶学性质。结果表明:酶的K_m为0.019μmol/L,V_max为1.434μmol/(min.mg);具有很好的耐热性,在50℃保温45min后仍能够保留酶活力的93.7%;Fe²⁺对酶活性有显著的促进作用,其比活力是对照反应的292%;酶对4-氯儿茶酚的催化活性非常低,属于Ⅰ型儿茶酚1,2-双加氧酶。以萘为底物生长时,ND6菌株的细胞提取液中既存在催化邻位裂解途径的儿茶酚1,2-双加氧酶活性,也存在催化间位裂解途径的儿茶酚2,3-双加氧酶活性。以苯甲酸、对羟基苯甲酸和苯乙酸为唯一碳源生长时,ND6菌株细胞提取液的儿茶酚1,2-双加氧酶活性远远大于儿茶酚2,3-双加氧酶活性。表明ND6菌株既能通过儿茶酚间位裂解途径降解萘,也能通过儿茶酚邻位裂解途径降解萘,而以苯甲酸、对羟基苯甲酸和苯乙酸为诱导物时只利用儿茶酚邻位裂解途径。     

抗微生物肽CRAMP联合抗生素分散铜绿假单胞菌生物被膜的增效作用研究

【目的】研究修饰后的鼠源抗微生物肽CRAMP联用抗生素对铜绿假单胞菌PAO1成熟生物被膜的分散作用,为临床联合应用抗生物被膜药物提供理论依据。【方法】采用微量肉汤稀释法测定CRAMP修饰肽和抗生素对PAO1的最低抑菌浓度(MIC)、最低杀菌浓度(MBC)和生物被膜最小根除浓度(MBEC);采用时间杀菌曲线(time-kill curve,TKC)法测定CRAMP修饰肽及抗生素单用和联用对PAO1成熟生物被膜的杀菌活性;采用菌落计数法和激光共聚焦扫描显微镜(CLSM)评估CRAMP修饰肽联用抗生素对PAO1成熟生物被膜的分散作用。【结果】与单用抗生素组相比,除2种碳青霉烯类药物和4种β-内酰胺类药物,其他抗生素联合CRAMP修饰肽后的MBEC值均有不同程度的下降,万古霉素、罗红霉素和阿奇霉素下降倍数最明显(4倍)。TKC试验结果表明,CRAMP修饰肽分别联用万古霉素、罗红霉素和阿奇霉素均具有比单用药更快且更强的杀菌作用,尤其是与万古霉素联用仅在3h时杀灭了全部(100%)的生物被膜细菌。随后,通过CLSM观察发现生物被膜数量、体积、面积和单位面积荧光强度均有明显的变化。【结论】CRAMP...     

Pseudomonas mosselii E1铁载体合成相关基因cysI的克隆与功能初步分析

从棉花根际分离的铁载体产生菌E1,其16SrDNA与Pseudomonas mosselii ATCCBAA-99的同源性为100%。采用三亲本杂交方法将携带转座子Tn5-1063的质粒pRL1063a导入E1中进行转座子插入诱变。利用CAS法,从1000个突变株中,筛选到一株铁载体合成缺失突变株E1-185。利用TAIL-PCR方法,扩增位于Tn5-1063两端的侧翼序列。测序结果表明,转座子插入到E1的cysI基因内。该基因与Pseudomonas entomophila L48的cysI同源性为96%,其CysI氨基酸序列相似性为97%。该基因与半胱氨酸的合成密切相关,而在加有半胱氨酸的CAS平板上,突变株恢复了铁载体产生能力,证明cysI在E1铁载体合成过程中具有重要作用。据推测,cysI可能与铁载体合成途径中关键蛋白acyl-S-PCPs的形成有关。     

Use of resazurin to detect mefloquine as an efflux-pump inhibitor in Pseudomonas aeruginosa and Escherichia coli

Multi-drug-resistant bacteria can cause serious infections that are extremely difficult to treat. Bacterial efflux pumps are known to contribute to multi-drug resistance and, thus, constitute a promising target for novel antibacterial agents. Resazurin is widely used to monitor bacterial growth because resazurin is reduced to the fluorescent resorufin by live cells. We have shown by flow cytometric analysis and by accumulation studies with wild type and efflux deficient strains that resazurin is a substrate of efflux pumps in Escherichia coli and Pseudomonas aeruginosa. Our investigations showed that the conversion rate of resazurin to resorufin is affected by efflux pumps. This finding was used to design an assay useful to detect efflux pump activity and to find potential efflux-pump inhibitors in a microtiter plate format. Mefloquine was detected as efflux-pump inhibitor when a panel of selected chemical compounds was tested for assay validation purposes.