Assembly Dynamics and Stability of the Pneumococcal Epsilon Zeta Antitoxin Toxin (PezAT) System from Streptococcus pneumoniae

The pneumococcal epsilon zeta antitoxin toxin (PezAT) system is a chromosomally encoded, class II toxin antitoxin system from the human pathogen Streptococcus pneumnoniae. Neutralization of the bacteriotoxic protein PezT is carried out by complex formation with its cognate antitoxin PezA. Here we study the stability of the inhibitory complex in vivo and in vitro. We found that toxin release is impeded in Escherichia coli and Bacillus subtilis due to the proteolytic resistance of PezA once bound to PezT. These findings are supported by in vitro experiments demonstrating a strong thermodynamic stabilization of both proteins upon binding. A detailed kinetic analysis of PezAT assembly revealed that these particular features of PezAT are based on a strong, electrostatically guided binding mechanism leading to a stable toxin antitoxin complex with femtomolar affinity. Our data show that PezAT complex formation is distinct to all other conventional toxin antitoxin modules and a controlled mode of toxin release is required for activation.     

A novel mechanism of programmed cell death in bacteria by toxin-antitoxin systems corrupts peptidoglycan synthesis

Most genomes of bacteria contain toxin-antitoxin (TA) systems. These gene systems encode a toxic protein and its cognate antitoxin. Upon antitoxin degradation, the toxin induces cell stasis or death. TA systems have been linked with numerous functions, including growth modulation, genome maintenance, and stress response. Members of the epsilon/zeta TA family are found throughout the genomes of pathogenic bacteria and were shown not only to stabilize resistance plasmids but also to promote virulence. The broad distribution of epsilon/zeta systems implies that zeta toxins utilize a ubiquitous bacteriotoxic mechanism. However, whereas all other TA families known to date poison macromolecules involved in translation or replication, the target of zeta toxins remained inscrutable. We used in vivo techniques such as microscropy and permeability assays to show that pneumococcal zeta toxin PezT impairs cell wall synthesis and triggers autolysis in Escherichia coli. Subsequently, we demonstrated in vitro that zeta toxins in general phosphorylate the ubiquitous peptidoglycan precursor uridine diphosphate-N-acetylglucosamine (UNAG) and that this activity is counteracted by binding of antitoxin. After identification of the product we verified the kinase activity in vivo by analyzing metabolite extracts of cells poisoned by PezT using high pressure liquid chromatograpy (HPLC). We further show that phosphorylated UNAG inhibitis MurA, the enzyme catalyzing the initial step in bacterial peptidoglycan biosynthesis. Additionally, we provide what is to our knowledge the first crystal structure of a zeta toxin bound to its substrate. We show that zeta toxins are novel kinases that poison bacteria through global inhibition of peptidoglycan synthesis. This provides a fundamental understanding of how epsilon/zeta TA systems stabilize mobile genetic elements. Additionally, our results imply a mechanism that connects activity of zeta toxin PezT to virulence of pneumococcal infections. Finally, we discuss how phosphorylated UNAG likely poisons additional pathways of bacterial cell wall synthesis, making it an attractive lead compound for development of new antibiotics.     

Nucleotide sequence and DNA recognition elements of alc, the structural gene which encodes allantoicase, a purine catabolic enzyme of Neurospora crassa

The nitrogen regulatory circuit of Neurospora crassa contains structural genes that encode nitrogen catabolic enzymes which are subject to complex genetic and metabolic regulation. This set of genes is controlled by nitrogen limitation, by specific induction, and by the action of nit-2, a major positive-acting regulatory gene, and nmr, a negative-acting control gene. The complete nucleotide sequence of alc, the gene that encodes allantoicase, a purine catabolic enzyme, is presented. The alc gene contains a single intron, is transcribed from two initiation sites situated approximately 50 nb upstream of the translation start site, and encodes a protein comprised of 354 amino acids. Mobility shift and DNA footprint experiments identified a single binding site for the NIT2 regulatory protein in the alc promoter region. The binding site contains a 10 nucleotide base pair symmetrical sequence which is flanked by two possible core binding sequences, TATCT and TATCG. Mutant NIT2/beta-gal fusion proteins with amino acid substitutions in a putative zinc-finger motif were shown to be completely deficient in the ability to bind to the alc promoter DNA fragment.     

Cloning and sequencing of the HU-2 gene of Escherichia coli

Summary The Escherichia coli HU-2 gene was cloned using a DNA fragment from the HU-1 gene as a probe. The amino acid sequence of the HU-2 protein deduced from the nucleotide sequence is in good agreement with the published sequence. The nucleotide sequence has a possible promoter and a typical ribosomal binding site upstream of the translation initiation codon (AUG) and a possible rhoindependent terminater site downstream of the termination codon (UAA) of the gene.     

The toxin-antitoxin system of the streptococcal plasmid pSM19035

pSM19035 of the pathogenic bacterium Streptococcus pyogenes is a low-copy-number plasmid carrying erythromycin resistance, stably maintained in a broad range of gram-positive bacteria. We show here that the omega-epsilon-zeta operon of this plasmid constitutes a novel proteic plasmid addiction system in which the epsilon and zeta genes encode an antitoxin and toxin, respectively, while omega plays an autoregulatory function. Expression of toxin Zeta is bactericidal for the gram-positive Bacillus subtilis and bacteriostatic for the gram-negative Escherichia coli. The toxic effects of zeta gene expression in both bacterial species are counteracted by proper expression of epsilon. The epsilon-zeta toxin-antitoxin cassette stabilizes plasmids in E. coli less efficiently than in B. subtilis.     

Molecular cloning and nucleotide sequence of the HU-1 gene of Escherichia coli

Summary The Escherichia coli HU-1 was cloned by use of mixed synthetic oligonucleotides (17-mer) predicted from a portion of its amino acid sequence. The amino acid sequence of the HU-1 protein deduced from the nucleotide sequence is in good agreement with the published sequence. The nucleotide sequence has a possible promoter and a typical ribosomal binding site upstream from the translational initiation codon (GUG) of the HU-1 gene.     

CS3纤毛抗原表达调控机理的研究

CS3亚基结构基因的核苷酸序列分析表明,在其翻译起始位点的上游存在着rbs位点及原核启动子的—10区和—35区DNA序列。凝胶阻滞和启动报告基因表达的实验确证了CS3亚基结构基因具有自身的启动子(Ps),怛它的作用受其上游区域的抑制。核苷酸序列分析发现,在Ps—35区上游550bp和840bp处各存在一个富A-T簇。结合原核启动子的一般作用规律推知,CS3的表达可能受DNA结合蛋白型的正向调节因子的作用,互补实验结果表明cfaD基因不仅可消除上游区对Ps的抑制,而且可大幅度地提高Ps的启动能力。在分析表达调控的基础上获得CS3重组高效表达。同时提出了其表达调控模型。     

人晶体膜内蛋白MP19基因（LIM2）启动子上一个晶体特异顺式元件的鉴定

使用重叠和变异的寡核苷酸作为探针,凝胶迁移分析和竞争实验分析了LIM2转录起始位点上游-47至-32的区域,与其高度亲和结合的一个蛋白复合体看来仅仅结合到这个DNA双链区域的“敏感”位点。这个位点的序列由4个G核苷,接着7个其他核苷酸(AACCTAA）及连着另外4个G核苷组成,即GGGGAACCTAAGGGG; 我们称其为Hsu元件。使用含有这个元件或相应的变异元件所构建的LIM2基因启动子CAT质粒的活性分析表明Hsu元件是位于LIM2基因启动子之内,它是LIM2基因表达所必须的。结合到Hsu元件的反式因子存于晶体发育期间,看来是晶体特异性的。由于LIM2基因启动子并不包含一个经典的TATA盒,这个Hsu元件可能充当RNA复制酶复合体结合的位点。     

CS3纤毛抗原表达调控机理的研究

ＣＳ３是某些肠毒素大肠杆菌菌体表面上的多聚物,它能使病原菌粘附于宿主的小肠上皮细胞上,是致病的重要因素．为了探索ＣＳ３菌毛抗原基因的表达调控机制,根据ＣＳ３亚基结构基因的核苷酸序列分析表明,在其翻译起始位点的上游存在着ｒｂｓ位点及原核启动子的－１０区和－３５区ＤＮＡ序列．采用基因重组技术将ＣＳ３结构基因上游１２０ｂｐ的ＤＮＡ片段亚克隆进缺乏启动子而只含报告基因ｌａｃＺ的质粒ｐＣＢ２６７中．凝胶滞留和启动报告基因表达的实验证明了ＣＳ３亚基结构基因具有自身的启动子（Ｐｓ）．将该启动子上游区域不同长度的核苷酸片段克隆进ｐＣＢ２６７中,报告基因表达结果表明ＣＳ３结构基因的表达受其上游区域的抑制．核苷酸序列分析发现,在Ｐｓ－３５区上游５５０ｂｐ和８４０ｂｐ处各存在一个富Ａ－Ｔ簇．结合原核启动子的一般作用规律推知,ＣＳ３的表达可能受ＤＮＡ结合蛋白型的正向调节因子的作用．用ＣＦＡ／１菌毛抗原基因的正向调节基因ｃｆａＤ对ＣＳ３基因进行的互补表达试验表明ｃｆａＤ基因不仅可消除上游区对Ｐｓ的抑制,而且可大幅度地提高Ｐｓ的启动能力．在分析表达调控的基础上获得ＣＳ３重组高效表达．同时提出了其表达调控模型．