假单胞菌株M18中pqsA突变株的构建及其对plt的调控

【目的】根际铜绿假单胞菌M18能产生藤黄绿菌素(Plt)和吩嗪-1-羧酸(PCA)两种主要的抗生素。其PqsR/PQS群体感应系统由应答调控蛋白PqsR与信号分子PQS组成。前期研究已经表明pqsR负调控Plt生物合成及基因簇表达。本论文旨在研究PQS分子对Plt合成及基因表达的调控作用。【方法】从M18基因组中扩增PQS合成基因pqsA,通过同源重组技术构建假单胞菌M18的pqsA突变菌株M18pqsA。利用lacZ报告基因分析、信号分子添加实验等,研究PQS对Plt合成及基因表达的调控作用。【结果】在KMB培养基中,分别比较野生型菌株M18和突变菌株M18pqsA的Plt产量,突变菌株的Plt产量存在较小幅度的升高,约为野生型菌株的1.53倍。添加PQS对plt表达存在一定程度但不是很显著的负调控作用。【结论】PQS分子对Plt生物合成及基因表达存在部分负调控作用。     

Characterization of a Genomic Region Required for Production of the Antibiotic Pyoluteorin by the Biological Control Agent Pseudomonas fluorescens Pf-5

A 21-kb region required for the biosynthesis of the polyketide antibiotic pyoluteorin by the biological control agent Pseudomonas fluorescens Pf-5 was identified and cloned. Seven previously isolated mutants deficient in pyoluteorin production (Plt(sup-)) had Tn5 insertions spanning the 21-kb region. Sequences flanking Tn5 inserts were cloned from genomic DNA of three Plt(sup-) mutants and used as probes to identify wild-type alleles of the plt loci from a genomic library of Pf-5. Five cosmids containing overlapping regions of genomic DNA hybridized to one or more of the probes. One cosmid, pJEL1938, contained the entire 21-kb region and, when introduced into a Plt(sup-) mutant, partially restored pyoluteorin production. To study the expression of the genes required for pyoluteorin biosynthesis, the transposon Tn3-nice, which contains a promoterless ice nucleation gene (inaZ) and a type I neomycin phosphotransferase gene, was introduced into the genomic plt region of Pf-5. Carbon sources that influenced pyoluteorin production by Pf-5 had parallel effects on ice nucleation activity of Pf-5 containing a genomic plt::Tn3-nice fusion, indicating that inaZ was transcribed from a promoter of the plt region. Cells of Pf-5 containing a genomic plt::Tn3-nice fusion expressed ice nucleation activity on cotton and cucumber seeds planted in field soil. The expression of plt genes by Pf-5 in the cucumber spermosphere was delayed in comparison with expression in the cotton spermosphere. This study demonstrates that genes required for pyoluteorin production were expressed in situ by the biological control bacterium.     

假单胞菌gacA插入突变对藤黄绿脓菌素和吩嗪-1-羧酸合成代谢的差异性调控

假单胞菌株M18分泌藤黄绿脓菌素 (Pyoluteorin ,Plt )和吩嗪 1 羧酸 (Phenazine 1 carboxylicacid ,PCA)并抑制多种植物病菌的生长。从M18中克隆双基因调控系统gacS gacA的组成基因gacA ,并构建了该基因抗性插入突变株M18G。在KMB培养基中 ,M18G合成Plt的能力受到完全抑制 ,而PCA的积累约比野生型提高 31倍左右。Plt合成基因簇突变株M18T和在M18G基础上构建的PCA合成基因簇突变株M18GA的Plt和PCA合成的动力学变化表明 ,在M18G菌株中 ,Plt合成的抑制并不引起PCA的过量积累 ,PCA的过量积累也不引起Plt合成的抑制。由此推测 ,gacA在基因表达的水平上全局性地执行着调控功能     

Cellular prion proteins in human platelets show a phenotype different to those in brain tissues

Prion diseases are characterized by high accumulation of infectious prion proteins (PrP(Sc)) in brains. PrP(Sc) are propagated by the conversion of host-encoded cellular prion proteins (PrP(C)) which are essential for developing the disease but are heterogeneously expressed in brains. The disease can be transmitted to humans and animals through blood and blood products, however, little attention has been given to molecular characterization of PrP(C) in blood cells. In this presented study, we characterized phenotypically PrP(C) of platelets (plt) and characterized the proteins regarding their glycobanding profiles by quantitative immunoblotting using a panel of monoclonal antibodies. The glycosylation patterns of plt and brain PrP(C) were compared using the ratios of di-, mono-, and non-glycosylated prions. The detergent solubility of plt and brain PrP(C) was also analyzed. The distinct banding patterns and detergent solubility of plt PrP(C) differed clearly from the glycosylation profiles and solubility characteristics of brain PrP(C). Plt PrP(C) exhibited single or only few prion protein types, whereas brain PrP(C) showed more extensive banding patterns and lower detergent solubility. Plt PrP(C) are post-translational modified differently from PrP(C) in brain. These findings suggest other or less physiological functions of plt PrP(C) than in brain.     

Molecular characterization of lactococcal bacteriophage Tuc2009 and identification and analysis of genes encoding lysin, a putative holin, and two structural proteins.

Bacteriophage Tuc2009 is a temperate bacteriophage with a small isometric head and is isolated from Lactococcus lactis subsp. cremoris UC509. The phage genome is packaged by a headful mechanism, giving rise to circularly permuted molecules with terminal redundancy. The unit genome size is approximately 39 kb. A map of the phage genome on which several determinants could be localized was constructed: pac, the site of initiation of DNA packaging; lys (1,287 bp), specifying the phage lysin; S (267 bp), specifying a putative holin; and mp1 (522 bp) and mp2 (498 bp), each specifying one of the phage's structural proteins. lys, S, mp1, and mp2 were further characterized. lys and S are partially overlapping and appear to be part of one operon. The lysin shows homology to the lysins of the Streptococcus pneumoniae phages Cp-9, Cp-1, and Cp-7. The putative holin, which is thought to be involved in the release of lysin from the cytoplasm, contains two strongly hydrophobic presumptive transmembrane domains and a highly charged C-terminal domain.     

Characterization of the 2-ketogluconate utilization operon in Pseudomonas aeruginosa PAO1

The Pseudomonas aeruginosa protein PtxS negatively regulates its own synthesis by binding to the upstream region of its gene. We have recently identified a 14 bp palindromic sequence within the ptxS upstream region as the PtxS operator site (OP1). In this study, we searched the P. aeruginosa genomic sequence to determine whether this 14 bp sequence exists in other regions of the P. aeruginosa chromosome. Another PtxS operator site (OP2) was located 47 bp downstream of ptxS. DNA gel shift experiments confirmed that PtxS specifically binds to a 520 bp fragment that carries OP2. The DNA segment 3' of OP2 contains four open reading frames (ORF1-ORF4), which code for 29, 32, 48 and 35 kDa proteins respectively. The molecular weight of the products of ORFs 2 and 3 were confirmed by T7 expression experiments. Computer analyses suggest that ORF2 encodes an ATP-dependent kinase; ORF3, a transporter; and ORF4, a dehydrogenase. The predicted product of ORF1 showed no homology to previously identified proteins and contains all the conserved amino acids within the aldose 1-epimerase protein motif. Examination of the ptxs-ORF1 intergenic region (using promoter fusion experiments) showed that no potential promoter exists. An isogenic mutant defective in ORF1 was constructed in the P. aeruginosa strain PAO1. In contrast to its parent strain, the mutant failed to grow on a minimal medium in which 2-ketogluconate was the sole carbon source. Similarly, a previously constructed ptxS isogenic mutant of PAO1 did not grow in a minimal medium containing 2-ketogluconate as the sole carbon source. Furthermore, a plasmid carrying a fragment that contains ptxS and ORFs 1-4 complemented the defect of the previously described P. aeruginosa 2-ketogluconate-negative mutant. In the presence of 10 mM 2-ketogluconate, the in vitro binding of PtxS to a DNA fragment that carries either OP1 or OP2 was inhibited. These results suggest that: (i) ptxS together with the other four ORFs constitute the 2-ketogluconate utilization operon (kgu) in P. aeruginosa. Therefore, ORFs 1-4 were designated kguE, kguK, kguT and kguD respectively. (ii) PtxS regulates the expression of the kgu operon by binding to two operators (OP1 and OP2) within the operon; and (iii) 2-ketogluconate is the molecular inducer of the kgu operon or the molecular effector of PtxS.     

Binding of the TorR regulator to cis-acting direct repeats activates tor operon expression

The expression of the Escherichia coli torCAD operon, which encodes the anaerobically expressed trimethylamine N-oxide (TMAO) reductase respiratory system, requires the presence of TMAO in the medium. The response regulator, TorR, has recently been identified as the regulatory protein that controls the expression of the torCAD operon in response to TMAO. The torC regulatory region contains four direct repeats of a decameric consensus motif designated the tor boxes. Alteration by base substitutions of any of the four tor boxes in a plasmid containing a torC'-lacZ fusion dramatically reduces TorR-dependent torC expression. In addition, deletion of the distal tor box (box1) abolishes torC induction whereas the presence of a DNA fragment starting three bases upstream from box1 suffices for normal torC expression. Footprinting and gel-retardation experiments unambiguously demonstrated that TorR binds to the torC regulatory region. Three distinct regions are protected by TorR binding. One of approximately 24 nucleotides covers the first two tor boxes (box1 and box2); the second is located upstream from the −35 promoter sequence and includes the third tor box (box3); the last is found downstream from the −35 sequence and corresponds to the fourth tor box (box4). Binding to the upstream tor boxes (box1 and box2) appears to be stronger than binding to the downstream tor boxes (box3 and box4) since only the upstream region is protected at the lower concentration of TorR used in the footprinting experiments.
We propose a model in which multiple binding sites (i.e. the tor boxes) contribute to the formation of a nucleoprotein complex, but only one particular proximal site positions TorR properly so that it interacts with RNA polymerase.