Eicosapentaenoic acid facilitates the folding of an outer membrane protein of the psychrotrophic bacterium, Shewanella livingstonensis Ac10.

Polyunsaturated fatty acids, such as eicosapentaenoic acid (EPA), are found in various cold-adapted microorganisms. We previously demonstrated that EPA-containing phospholipids (EPA-PLs) synthesized by the psychrotrophic bacterium Shewanella livingstonensis Ac10 support cell division, membrane biogenesis, and the production of membrane proteins at low temperatures. In this article, we demonstrate the effects of EPA-PLs on the folding and conformational transition of Omp74, a major outer membrane cold-inducible protein in this bacterium. Omp74 from an EPA-less mutant migrated differently from that of the parent strain on SDS-polyacrylamide gel, suggesting that EPA-PLs affect the conformation of Omp74 in vivo. To examine the effects of EPA-PLs on Omp74 protein folding, in vitro refolding of recombinant Omp74 was carried out with liposomes composed of 1,2-dipalmitoleoyl-sn-glycero-3-phosphoglycerol and 1,2-dipalmitoleoyl-sn-glycero-3-phosphoethanolamine (1:1 molar ratio) with or without EPA-PLs as guest lipids. SDS-PAGE analysis of liposome-reconstituted Omp74 revealed more rapid folding in the presence of EPA-PLs. CD spectroscopy of Omp74 folding kinetics at 4 °C showed that EPA-PLs accelerated ß-sheet formation. These results suggest that EPA-PLs act as chemical chaperones, accelerating membrane insertion and secondary structure formation of Omp74 at low temperatures.

[Isolation and characterization of an atrazine-degrading bacterium strain SA1].

Atrazine (AT), a kind of herbicide for the pre and post-emergence control of annual and broad leaved weeds and perennial grasses, had been widely used in the world. However, the extensive use of atrazine had led to widespread environmental pollution. A bacterium strain SA1, which could degrade AT completely, was isolated from an atrazine-degrading consortium by long-time repeated alternative cultivation and plate striking. Combining cultural and physiobiochemical characteristics with 16S rDNA sequence analysis, SA1 was identified as Pseudomonas sp.. SAl could use atrazine as the sole carbon, nitrogen and energy sources for growth, and the main product of AT biodegradation was cyanuric acid. AT degrading activity of SA1 was not affected by the addition of nitrogen resources. However, cyanuric acid could be degraded quickly to an undetectable level when glucose was added. The optimal temperature and pH value for SAl growth was 37 degrees C and pH7, respectively. Atrazine could be degraded efficiently by the resting cells of SAl under the conditions of 10 degrees C - 40 degrees C or pH value 4-11, and SA1 had a wide range of temperature and pH value for AT degradation when compared with ADP. atzABCD and conserved sequence of tnpA gene of IS1071 could be amplified from SA1, and these genes could be lost during subculture.

[Horizontal transfer of environmental pollutant-degrading gene and application in bioremediation].

Horizontal gene transfer, unlike vertical gene transfer, is a means of genetic communication in bacteria. In the special polluted environment, horizontal transfer of polluted-degrading gene has significant functions. Study on horizontal transfer of degrading gene in polluted environment may deepen our understanding of the mechanism of bacterial adaptation to the organic-polluted environment. In the practical application in bioremediation, horizontal transfer of degrading gene can be regulated to promote degrading ability of microorganisms. In this article, we will review the advances in the study on mechanisms of genetic interactions among bacteria, the effect of degrading gene transfer in contaminated environment on microorganisms'adaptation to contaminated environment and the degradation of the pollutants.

[Study on the atrazine-degrading genes in Arthrobacter sp. AG1].

Atrazine could be used as the sole carbon, nitrogen and energy sources for growth by strain Arthrobacter sp. AG1, and the atrazine-degrading genes of AG1 were found to be the combination of trzN, atzB and atzC. The atrazine chloride hydrolysase gene trzN was cloned by PCR amplification,whose sequence shared 99% identity with that of Norcardioides sp. C190. Two large plasmids were found in AG1,and trzN and atzB were confirmed to be localized on the larger plasmid pAG1 by the method of southern hybridization. Subculture of AG1 in liquid LB for three generations, 34% of the subsequent cells were found to lose degrading activity, however, neither plasmid was lost. PCR amplification results showed that the mutants had only lost the trzN gene instead of atzB and atzC. It was deduced that mutation might be due to the trzN gene deletion from the plasmid. This study provided new evidence that atrazine metabolic genotypes were resulted from horizontal gene transfer between different bacteria under environmental selective pressure.

[Construction of multifunctional genetically engineered pesticides-degrading bacteria by homologous recombination].

Construction of multifunctional pesticides-degrading genetically engineered microorganisms (GEMs) is increasing important in the bioremediation of various pesticides contaminants in environment. However, construction of genetically stable GEMs without any exogenous antibiotic resistance is thought to be one of the bottlenecks in GEMs construction. In this article, homologous recombination vectors with the recipient's 16S rDNA as homologous recombination directing sequence (HRDS) and sacB gene as double crossover recombinants positive selective marker were firstly constructed. The methyl parathion hydroalse gene (mpd) was inserted into the 16S rDNA site of the carbofuran degrading strain Sphingomonas sp. CDS-1 by homologous recombination single crossover in the level of about 3.7 x 10-(7) - 6.8 x 10(-7). Multifunctional pesticides-degrading GEMs with one or two mpd genes inserted into the chromosome without any antibiotic marker were successfully constructed. The homologous recombination events were confirmed by PCR and southern blot methods. The obtained GEMs were genetically stable and could degrade methyl parathion and carbofuran simultaneously. The insertion of mpd gene into rrn site did not have any significant effect on recipient' s physiological and original degrading characteristics. The methyl parathion hydrolase (MPH) was expressed at a relatively high level in the recombinants and the recombinant MPH specific activity in cell lysate was higher than that of original bacterium (DLL-1) in every growth phase tested. The highest recombinant MPH specific activity was 6.22 mu/tg. In this article, we describe a first attempt to use rRNA-encoding regions of Sphingomonas strains as target site for expression of exogenous MPH, and constructed multifunctional pesticides degrading GEMs, which are genetically stable and promising for developing bioremediation strategies for the decontamination of pesticides polluted soils.