The natural transformation of the soil bacteria Pseudomonas stutzeri and Acinetobacter sp. by transgenic plant DNA strictly depends on homologous sequences in the recipient cells

The nptII(+) gene present in the genome of transgenic potato plants transforms naturally competent cells of the soil bacteria Pseudomonas stutzeri and Acinetobacter BD413 (both harboring a plasmid with an nptII gene containing a small deletion) with the same high efficiency as nptII(+) genes on plasmid DNA (3x10(-5)-1x10(-4) transformants per nptII(+)) despite the presence of a more than 10(6)-fold excess of plant DNA. However, in the absence of homologous sequences in the recipient cells the transformation by nptII(+) dropped by at least about 10(8)-fold in P. stutzeri and 10(9)-fold in Acinetobacter resulting in the latter strain in < or =1x10(-13) transformants per nptII(+). This indicated a very low probability of non-homologous DNA fragments to be integrated by illegitimate recombination events during transformation.     

固氮施氏假单胞菌亚硝酸盐还原酶基因nirS转录特性及功能鉴定

【目的】研究固氮施氏假单胞菌(Pseudomonas stutzeri)A1501亚硝酸盐还原酶结构基因nir S的转录调控机制及其在反硝化过程中的功能。【方法】构建nir S-lac Z融合载体,利用三亲本结合法将其导入野生型A1501,通过β-半乳糖苷酶活性的测定,分析不同供氧状况、不同浓度的硝酸盐、亚硝酸盐对nir S基因表达的影响;同时将该载体导入rpo N突变株中,研究氮代谢调控因子Rpo N对nir S基因转录影响。通过同源重组方法构建nir S突变株,通过生化表型测定明确nir S在反硝化过程中的功能。【结果】启动子活性测定表明,nir S基因厌氧条件下高水平表达,是好氧条件下表达水平的4倍;nir S的表达受硝酸盐诱导,但不受亚硝酸盐的诱导;Rpo N突变株中,nir S的表达活性为野生型的1/4,nir S启动子未发现Rpo N的保守结合位点,表明nir S的表达受Rpo N间接调控。表型测定显示以硝酸盐为电子受体时Δnir S的反硝化能力降低了约20%;以亚硝酸盐为电子受体时Δnir S仅有微弱的反硝化能力,并且nir S的突变使得菌体在反硝化条件下利用亚硝酸盐的能力显著减弱。nir S突变提高了菌体在亚硝酸为电子受体的反硝化条件下的固氮酶活。【结论】A1501中nir S基因的转录受外界氧及硝酸盐的影响,同时受氮代谢Sigma因子Rpo N的调控。nir S在A1501菌反硝化过程中起关键作用,参与了亚硝酸盐的转化。     

Role of IncP-1 plasmid primase in conjugation between Pseudomonas species

Abstract A Tn7 insertion in the DNA primase gene of the promiscuous IncP-1 plasmid R18 specifically reduced plasmid conjugational transfer from Pseudomanas aeruginosa donors to Pseudomonas stutzeri recipients. The cloned primase gene was found to efficiently complement the mutation in both the donor and in the recipient suggesting that the primase is required for priming single-stranded plasmid DNA in the donor prior to its transit to the recipient where it is converted to the double- stranded form.     

施氏假单胞菌氮代谢调控蛋白GlnK与碳信号分子α-酮戊二酸的体外互作研究

细菌中PⅡ蛋白是一类氮代谢调控因子,可通过感知胞内碳氮信号变化调整自身与靶蛋白的相互作用,从而实现对下游基因的级联调控。α-酮戊二酸是胞内碳状态的重要信号分子,前期研究发现PⅡ蛋白可结合α-酮戊二酸感应细胞内的碳状态,但不同菌中二者的结合存在差异。施氏假单胞菌A1501（Pseudomononas stutzeri A1501）只含有1种PⅡ蛋白GlnK,其在A1501固氮调控中发挥重要作用,但具体碳信号感知与转导机制有待进一步探索。基于此,通过大肠杆菌外源表达GlnK蛋白,并通过微量热泳动（microscale thermophoresis,MST）方法研究GlnK蛋白与碳信号分子α-酮戊二酸的体外互作,发现二者可以体外结合,且进一步证实GlnK蛋白的第89位甘氨酸（G89,Gly89）可能与互作相关。研究结果为进一步解析α-酮戊二酸在A1501中的信号转导奠定了基础,也为深入解析固氮菌的碳氮代谢偶联机制提供了理论支持。     

Isolation and Identification of Polycyclic Aromatic Hydrocarbon–Degrading Bacteria from Crude Oil Exploration Bore Well Sludge

Two bacterial strains capable of degrading polycyclic aromatic hydrocarbons were isolated from the crude oil exploration bore well sludge and identified by 16s rRNA gene sequencing as Pseudomonas stutzeri and Bacillus subtilis. The bacterial strains Pseudomonas stutzeri and Bacillus subtilis were able to degrade 95.1% and 99.4% of naphthalene (100 mg L^?1) and 99.5% and 94.6% of anthracene (50 mg L^?1), respectively, as a sole carbon and energy source in the liquid phase within a period of 6 days. The specific growth rate was determined for both the species and found to be 0.169 and 0.124 day^?1.     

Isolation and Characterization of Pseudomonas stutzeri Capable of Reducing Fe(III) and Nitrate from Skarn-type Copper Mine Tailings

Nitrate and Fe(III) are two terminal electron acceptors in anaerobic respiration by microorganisms growing in the anoxic soil or sediment environment. In the current paper a facultative anaerobic dissimilatory Fe(III)- and nitrate-reducing bacterium was isolated from the Linchong tailings, a skarn-type copper mine tailings, located in Anhui. Skarn often formed at the contact zone between intrusions of granitic magma bodies into contact with carbonate sedimentary rocks. This made the tailings possessed strong acid neutralizing capacity and pH of the pore water was 6.8–8.6. The isolate, which was designated strain CW (CCTCC AB 2013114), was a gram-negative rod bacterium and belonged to the gamma subgroup p of the proteobacteria, closely (99.0%) related to Pseudomonas stutzeri. In defined medium, strain CW was shown to grow anaerobically with the acetate using the ferric iron or nitrate as the electron acceptors. Results also showed that strain CW could not grow in the presence of ferrous iron and nitrite.     

Bioconversion of starches into maltotetraose using Pseudomonas stutzeri maltotetraohydrolase in a membrane recycle bioreactor: Effect of multiple enzyme systems and mass balance study

A simplified single-step method involving simultaneous production and purification of maltotetraose (G₄) by employing ultrafiltration (UF) membranes was previously proposed. The addition of a pretreatment step using pullulanase and then the G₄-amylase was expected to increase the yield of G₄. The single-enzyme system, however, showed 0.42 g higher total product output than the successive dual-enzyme system throughout 6 h reaction. The G₄ yield using the successive dual-enzyme system could be improved after removing the unwanted side product with UF. Experiments were conducted with membranes of larger pore size, but this did not significantly increase the total product output. The membrane unit with a molecular weight cutoff of 1,000 was the most appropriate membrane pore size for the G₄-exo--amylase membrane recycle bioreactor system. The total amount of substrate fouled in the membrane during a 6-h reaction was estimated as 69 mg glucose equivalent when substrate concentration was 0.25% (w/v). The mass balance equation indicated that the percent conversion of soluble starch to G₄ at steady state was 65%.