Quenching quorum-sensing-dependent bacterial infection by an N-acyl homoserine lactonase.

Bacterial cells sense their population density through a sophisticated cell-cell communication system and trigger expression of particular genes when the density reaches a threshold. This type of gene regulation, which controls diverse biological functions including virulence, is known as quorum sensing. Quorum-sensing signals, such as acyl-homoserine lactones (AHLs), are the essential components of the communication system. AHLs regulate virulence gene expression in a range of plant and animal (including human) bacterial pathogens. AHL-producing tobacco restored the pathogenicity of an AHL-negative mutant of Erwinia carotovora. Different bacterial species may produce different AHLs, which vary in the length and substitution of the acyl chain but contain the same homoserine lactone moiety. Here we show that the acyl-homoserine lactonase (AHL-lactonase), a new enzyme from Bacillus sp., inactivates AHL activity by hydrolysing the lactone bond of AHLs. Plants expressing AHL-lactonase quenched pathogen quorum-sensing signalling and showed significantly enhanced resistance to E. carotovora infection. Our results highlight a promising potential to use quorum-sensing signals as molecular targets for disease control, thereby broadening current approaches for prevention of bacterial infections.     

Retrovirus genomes methylated by mammalian but not bacterial methylase are non-infectious

The biological importance of DNA methylation for gene expression in eukaryotes is becoming increasingly evident, and a direct role of methylation in gene expression has been suggested by an analysis of the infectivity of integrated retroviral genomes in a transfection assay. These studies, however, did not address whether specific methylatable residues are involved in gene regulation. Methylation by sequence-specific bacterial DNA methylases has been shown to suppress the expression of some genes, but not others. To investigate the effect of methylation on gene expression without having to rely on sequence-specific methylases, a rat liver enzyme was used to methylate in vitro all C-G dinucleotides of a proviral genomic clone. This treatment reduced the biological activity of Moloney murine leukaemia virus (M-MuLV) proviral DNA by more than three orders of magnitude, whereas complete methylation of 35 HpaII sites in the same DNA had only a marginal effect. The rat methylase-induced inactivation was reversible, as treatment of recipient cells with 5-azacytidine rendered the non-infectious viral genomes biologically active. This suggests that methylation in other C-G dinucleotides than those detectable with restriction enzymes can be crucial for gene expression.     

Clusters of CpG dinucleotides implicated by nuclease hypersensitivity as control elements of housekeeping genes

DNA sequences of the X-chromosome-linked hypoxanthine phosphoribosyltransferase (HPRT) and glucose 6-phosphate dehydrogenase (G6PD) genes have revealed the presence of clusters of CpG dinucleotides, raising the possibility that such clusters are involved in the control of expression of these genes, which are expressed in all tissues. Although CpG clusters are not exclusive features of the X chromosome, the analysis of X-linked genes provides the means to determine whether CpG clusters are control elements; one of the two homologous X loci in female mammals is not expressed, so that active and inactive versions of the gene can be compared. In fact, it has been shown that these CpG clusters are undermethylated when the gene is active and extensively methylated when the gene is inactive. In addition to hypomethylation, chromatin hypersensitivity to endonuclease digestion is a known hallmark of regulatory sequences in eukaryotic genes. We report here that the CpG clusters of the active hprt and g6pd genes are not only undermethylated, but also hypersensitive to MspI, DNase I and S1 nuclease, further supporting the suggestion that they are involved in the control of expression of these genes.     

Preferential relaxation of supercoiled DNA containing a hexadecameric recognition sequence for topoisomerase I

In prokaryotes, the degree of supercoiling of DNA can profoundly influence the use of specific promoters. In eukaryotes, a variety of indirect observations suggest that DNA topology has a similar importance in proper gene expression. Much attention has therefore been focused on the cellular proteins that control DNA supercoiling, among which are the enzymes topoisomerase I and II. A hexadecameric sequence functions as a strong attraction site for topoisomerase I. Here we report that the interaction of topoisomerase I with this sequence motif is highly specific, because a single base-pair substitution prevents strand cleavage and thereby catalytic activity at the sequence. Thus, supercoiled DNA containing the recognition sequence is relaxed preferentially by topoisomerase I compared to a control, but no difference in the relaxation rate is observed for supercoiled DNA carrying the mutated sequence. The preference for the recognition sequence seems to be an intrinsic property of all eukaryotic type I topoisomerases, suggesting that the interaction might be important in a fundamental biological process.     

低盐刺激下副溶血弧菌基因转录表达分析

目的:应用全基因组DNA芯片技术分析低盐冷刺激作用下副溶血弧菌基因的转录表达变化.方法:分别采用"低盐持续刺激培养(continuous growth,CTG)"和"中间转入低盐环境培养(shift growth,STG)".CTG和STG下.分别采用含NaCl浓度为2%和0.66%的MV-5培养基孵育副溶血弧菌,收集菌体,提取RNA,应用全基因组DNA芯片分别比较两个不同的转录表达谱基因变化特点,分析其作用规律.同时,应用实时定量逆转录多聚酶联反应对芯片结果进行验证.结果:和对照组相比,STG实验中,共有205个基因的转录表达发生显著性变化,上调的基因占优势地位;CTG实验中,总计有298个基因的转录表达发生显著性变化,上、下涮的基因总体基本趋于平衡状态,没有明显差异.实时定量逆转录多聚酶联反应结果证实其和芯片数据结果有很强的相关性.结论:在低盐这一"胁迫环境"下,副溶血弧菌利用其存在的独特而精细的应对机制,能够顽强的生存下来并繁衍生殖,这一过程中,节能调节处于调控的核心地位.     

细菌群体感应机制与动植物病原菌的致病力

N 酰基高丝氨酸内酯 (AHLs)作为信号分子介导的细菌群体感应机制参与许多生物学功能的调节 ,当侵染动植物寄主组织的病原菌繁殖到一定量时 ,细菌本身产生的AHLs积累到临界浓度 ,AHLs与胞内特异受体结合 ,启动致病因子的表达。利用AHLs降解酶和AHLs类似物的特性 ,干扰和破坏病原菌的AHLs 群体感应机制 ,将为利用现代生物技术防治此类细菌病害开辟了一条全新的途径     

The major Vibrio cholerae autoinducer and its role in virulence factor production

Vibrio cholerae, the causative agent of the human disease cholera, uses cell-to-cell communication to control pathogenicity and biofilm formation. This process, known as quorum sensing, relies on the secretion and detection of signalling molecules called autoinducers. At low cell density V. cholerae activates the expression of virulence factors and forms biofilms. At high cell density the accumulation of two quorum-sensing autoinducers represses these traits. These two autoinducers, cholerae autoinducer-1 (CAI-1) and autoinducer-2 (AI-2), function synergistically to control gene regulation, although CAI-1 is the stronger of the two signals. V. cholerae AI-2 is the furanosyl borate diester (2S,4S)-2-methyl-2,3,3,4-tetrahydroxytetrahydrofuran borate. Here we describe the purification of CAI-1 and identify the molecule as (S)-3-hydroxytridecan-4-one, a new type of bacterial autoinducer. We provide a synthetic route to both the R and S isomers of CAI-1 as well as simple homologues, and we evaluate their relative activities. Synthetic (S)-3-hydroxytridecan-4-one functions as effectively as natural CAI-1 in repressing production of the canonical virulence factor TCP (toxin co-regulated pilus). These findings suggest that CAI-1 could be used as a therapy to prevent cholera infection and, furthermore, that strategies to manipulate bacterial quorum sensing hold promise in the clinical arena.     

Widespread distribution and fitness contribution of Xanthomonas campestris avirulence gene avrBs2

Disease-resistance genes introduced into cultivated plants are often rendered ineffective by the ability of pathogen populations to overcome host resistance. The bacterial pathogen Xanthomonas campestris pathovar vesicatoria causes bacterial spot disease of tomato and pepper, and this pathogen has been shown to overcome disease resistance in pepper (Capsicum annuum) by evading the recognition and defence response of the host plant. Numerous resistance genes to bacterial spot have been identified in pepper and its wild relatives, each providing resistance to specific races of X.c. vesicatoria. The resistance gene Bs1, for example, provides resistance to X.c. vesicatoria strains expressing the avirulence gene avrBs1; Bs2 provides resistance to stains expressing avrBs2 and so on. We now report that avr Bs2 is highly conserved among strains of X.c. vesicatoria, and among many other pathovars of X. campestris. Furthermore, we find that avrBs2 is in fact needed for full virulence of the pathogen on susceptible hosts. This implies that plants carrying Bs2 can recognize an essential gene of the bacterial pathogen, which may explain why Bs2 confers the only effective field resistance to X.c. vesicatoria in pepper.     

Specific expression of a foreign beta-globin gene in erythroid cells of transgenic mice

The globin gene family represents an attractive system for the study of gene regulation during mammalian development, as its expression is subject to both tissue-specific and temporal regulation. While many aspects of globin gene structure and expression have been described extensively, relatively little is known about the cis-acting DNA sequences involved in the developmental regulation of globin gene expression. To begin to experimentally define these regulatory sequences, we have taken the approach of introducing cloned globin genes into the mouse germ line and examining their expression in the resulting transgenic animals. Here we describe a series of transgenic mice carrying a hybrid mouse/human adult beta-globin gene, several of which express the gene exclusively or predominantly in erythroid tissues. These studies demonstrate that regulatory sequences closely linked to the beta-globin gene are sufficient to specify a correct pattern of tissue-specific expression in a developing mouse, when the gene is integrated at a subset of foreign chromosomal positions.     

A Study on the Molecular Switch of Gene Expression of the Mouse Heart Nuclear DNA Fragments

It is observed by in situ stain that LDH(1-5)…nNAD+ can probably enter the nucleopore and can be bound bound specifically with the genes that encode them. During the in vitro expression, the dilution of heart nuclear DNA fragments could enhance the expression activity of LDH/DNA and the amount of expressed LDH(1-5) is in proportion to the amount of dissociable LDH(1-5) on the LDH/DNA. With the integration of 14CLeu to the proteins, it is also observed that the addition of LDH(1-5)…nNAD+ can suppress the in vitro expression activity of LDH/DNA. AFM bservation shows that the regulation sequence at the both ends of active genes may be bound with such active factors as proteins encoded by the genes which probably is the main molecular switch of gene expression and regulation we have been always searching for. Our work shows the prospective application of the combination of AFM and isotope labeling in the research of biological reaction.