Synthesis of chitinase in Streptomyces thermoviolaceus is regulated by a two-component sensor-regulator system

The chiS and chiR genes located upstream of the chitinase locus (chi40) on the chromosome of Streptomyces thermoviolaceus OPC-520 were cloned and sequenced. The deduced amino acid sequences revealed that ChiS (390 amino acids, 40.9 kDa) and ChiR (213 amino acids, 22 kDa) show significant sequence similarities to histidine kinases and response regulators, respectively, of typical prokaryotic two-component regulatory systems. The extracellular chitinase activity of Streptomyces lividans 66 (pTSR2 (bearing chiS, chiR and chi40)) was significantly enhanced by a high dosage of the chiS and chiR genes.     

Arabidopsis CaM Binding Protein CBP60g Contributes to MAMP-Induced SA Accumulation and Is Involved in Disease Resistance against Pseudomonas syringae

Salicylic acid (SA)-induced defense responses are important factors during effector triggered immunity and microbe-associated molecular pattern (MAMP)-induced immunity in plants. This article presents evidence that a member of the Arabidopsis CBP60 gene family, CBP60g, contributes to MAMP-triggered SA accumulation. CBP60g is inducible by both pathogen and MAMP treatments. Pseudomonas syringae growth is enhanced in cbp60g mutants. Expression profiles of a cbp60g mutant after MAMP treatment are similar to those of sid2 and pad4, suggesting a defect in SA signaling. Accordingly, cbp60g mutants accumulate less SA when treated with the MAMP flg22 or a P. syringae hrcC strain that activates MAMP signaling. MAMP-induced production of reactive oxygen species and callose deposition are unaffected in cbp60g mutants. CBP60g is a calmodulin-binding protein with a calmodulin-binding domain located near the N-terminus. Calmodulin binding is dependent on Ca²⁺. Mutations in CBP60g that abolish calmodulin binding prevent complementation of the SA production and bacterial growth defects of cbp60g mutants, indicating that calmodulin binding is essential for the function of CBP60g in defense signaling. These studies show that CBP60g constitutes a Ca²⁺ link between MAMP recognition and SA accumulation that is important for resistance to P. syringae.     

Assembly of the mitochondrial membrane system. Characterization of a yeast nuclear gene involved in the processing of the cytochrome b pre-mRNA

The cytochrome b gene of Saccharomyces cerevisiae D273-10B was previously shown to be composed of three exons and two introns (Nobrega, F.G., and Tzagoloff, A. (1980) J. Biol. Chem. 255, 9828-9837). In the present study nuclear respiratory deficient mutants of this strain have been screened for defects in processing of the cytochrome b pre-mRNA. Fifteen independently isolated mutants lacking cytochrome b have been assigned to a single genetic complementation group (G36). Members of this complementation group are blocked in the excision of the second intervening sequence of cytochrome b and consequently are unable to produce the mature mRNA. The wild type gene defined by this class of mutants has been named CBP2. A recombinant plasmid with the CBP2 gene has been selected from a library of wild type nuclear DNA and further subcloned by transformation of a cbp2 mutant to respiratory competency. The smallest plasmid (pG36/T5) capable of complementing cbp2 mutants and of restoring their ability to complete processing of the cytochrome b pre-mRNA has a nuclear DNA fragment of 2.6 kilobase pairs inserted at the BamHI site of the yeast vector YEp13. The sequence of the cloned DNA fragment has revealed an 1890-nucleotide-long reading frame encoding a basic protein with a molecular weight of 74,000. Deletion analysis confirms that the entire reading frame is required for complementation of cbp2 mutants. This reading frame is proposed to code for the CBP2 gene product.     

Generation of Temperature-Sensitive Cbp1 Strains of Saccharomyces Cerevisiae by Pcr Mutagenesis and in Vivo Recombination: Characteristics of the Mutant Strains Imply That Cbp1 Is Involved in Stabilization and Processing of Cytochrome B Pre-mRNA

Mitochondrial biogenesis is dependent on both nuclearly and mitochondrially encoded proteins. Study of the nuclearly encoded mitochondrial gene products and their effect on mitochondrial genome expression is essential to understanding mitochondrial function. Mutations in the nuclear gene CBP1 of Saccharomyces cerevisiae result in degradation of mitochondrially encoded cytochrome b (cob) RNA; thus, the cells are unable to respire. Putative roles for the CBP1 protein include processing of precursor RNA to yield the mature 5' end of cob mRNA and/or physical protection of the mRNA from degradation by nucleases. To examine the activity of CBP1, we generated temperature-sensitive cbp1 mutant strains by polymerase chain reaction (PCR) mutagenesis and in vivo recombination. These temperature-sensitive cbp1 strains lack cob mRNA only at the nonpermissive temperature. Quantitative primer extension analyses of RNA from these strains and from a cbp1 deletion strain demonstrated that CBP1 is required for the stability of precursor RNAs in addition to production of the stable mature mRNA. Thus, CBP1 is not involved solely in the protection of mature cob mRNA from nucleases. Moreover, we found that mature mRNAs are undetectable while precursor RNAs are reduced only slightly at the nonpermissive temperature. Collectively, these data lead us to favor a hypothesis whereby CBP1 protects cob precursor RNAs and promotes the processing event that generates the mature 5' end of the mRNA.     

Identification and characterization of a highly conserved calcineurin binding protein, CBP1/calcipressin, in Cryptococcus neoformans

Calcineurin is the conserved target of the immunosuppressants cyclosporin A and FK506. Using the yeast two-hybrid system, we identified a novel calcineurin binding protein, CBP1, from the pathogenic fungus Cryptococcus neoformans. We show that CBP1 binds to calcineurin in vitro and in vivo, and FKBP12-FK506 inhibits CBP1 binding to calcineurin. Cryptococcus neoformans cbp1 mutant strains exhibit modest defects in growth under stress conditions and virulence, similar to but less severe than the phenotypes of calcineurin mutants. Saccharomyces cerevisiae mutants lacking the CBP1 homolog RCN1 are, like calcineurin mutants, sensitive to lithium cation stress. CBP1 shares a central peptide sequence motif, SPPxSPP, with related proteins in S.CEREVISIAE:, Schizosaccharomyces pombe, Drosophila melanogaster, Caenorhabditis elegans and humans, and peptides containing this motif altered calcineurin activity in vitro. Interestingly, the human CBP1 homolog DSCR1 is encoded by the Down's syndrome candidate region interval on chromosome 21, is highly expressed in the heart and central nervous system, and may play a role in calcineurin functions in heart development, neurite extension and memory.     

家蚕不同茧色品种中cbp基因的结构和表达分析

类胡萝卜素结合蛋白(CBP)是唯一已被确认与家蚕黄色茧形成儡切相关的主要蛋白质。文章选择12个有色茧和白茧蚕品种, 调查了cbp基因结构、转录产物mRNA类型和丝腺类胡萝卜素紫外可见光吸收特征与其茧色的关系。结果表明: 黄色茧蚕品种含有2种或3种cbp基因结构, 同时转录具有CBP功能性的完整 mRNA和缺少第2外显子的mRNA; 绿茧品种间cbp基因结构存在差异, 转录缺少第2外显子的mRNA; 白茧蚕品种cbp基因只有1种结构, 转录缺少第2外显子的mRNA。文章在黄茧品种中新发现的cbp基因第1内含子序列可能具有茧色品种特异性, 家蚕黄茧品种丝腺的紫外可见吸收光谱特征显著区别于绿茧和白茧品种, cbp基因的结构和表达特征与家蚕茧色密切相关。     

Isolation of Citrobacter sp. mutants defective in decolorization of brilliant green by transposon mutagenesis

To identify genes involved in the decolorization of brilliant green, we isolated random mutants generated by transposon insertion in brilliant green-decolorizing bacterium, Citrobacter sp. The resulting mutant bank yielded 19 mutants with a complete defect in terms of the brilliant green color removing ability. Southern hybridization with a Tn5 fragment as a probe showed a single hybridized band in 7 mutants and these mutants appeared to have insertions at different sites of the chromosome. Tn5-inserted genes were isolated and the DNA sequence flanking Tn5 was determined. By comparing these with a sequence database, putative protein products encoded by bg genes were identified as follows: bg 3 as a LysR-type regulatory protein; bg 11 as a MalG protein in the maltose transport system; bg 14 as an oxidoreductase; and bg 17 as an ABC transporter. The sequences deduced from the three bg genes, bg 2, bg 7 and bg 16, showed no significant similarity to any protein with a known function, suggesting that these three bg genes may encode unidentified proteins responsible for the decolorization of brilliant green.     

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.     

CBP60g and SARD1 play partially redundant critical roles in salicylic acid signaling

Arabidopsis thaliana calmodulin binding protein 60g (CBP60g) contributes to production of salicylic acid (SA) in response to recognition of microbe‐associated molecular patterns (MAMPs) such as flg22, a fragment of bacterial flagellin. Calmodulin binding is required for the function of CBP60g in limiting growth of the bacterial pathogen Pseudomonas syringae pv. maculicola (Pma) ES4326 and activation of SA synthesis. Here, we describe a closely related protein, SARD1. Unlike CBP60g, SARD1 does not bind calmodulin. Growth of Pma ES4326 is enhanced in sard1 mutants. In cbp60g sard1 double mutants, growth of Pma ES4326 is greatly enhanced, and SA levels and expression of PR‐1 and SID2 are dramatically reduced. Expression profiling placed the CBP60g/SARD1 node between the PAD4/EDS1 and SA nodes in the defense signaling network, and indicated that CBP60g and SARD1 affect defense responses in addition to SA production. A DNA motif bound by CBP60g and SARD1, GAAATTT, was significantly over‐represented in promoters of CBP60g/SARD1‐dependent genes, suggesting that expression of these genes is modulated by CBP60g/SARD1 binding. Gene expression patterns showed a stronger effect of cbp60g mutations soon after activation of a defense response, and a stronger effect of sard1 mutations at later times. The results are consistent with a model in which CBP60g and SARD1 comprise a partially redundant protein pair that is required for activation of SA production as well as other defense responses, with CBP60g playing a more important role early during the defense response, and SARD1 to playing a more important role later.     

Cloning and characterization of a gene required for the secretion of extracellular enzymes across the outer membrane by Xanthomonas campestris pv. campestris.

Nonpathogenic mutants of Xanthomonas campestris pv. campestris, generated from transposon mutagenesis, accumulated extracellular polygalacturonate lyase, alpha-amylase, and endoglucanase in the periplasm. The transposon Tn5 was introduced by a mobilizable, suicidal plasmid, pSUP2021 or pEYDG1. Genomic banks of wild-type X. campestris pv. campestris, constructed on the broad-host-range, mobilizable cosmid pLAFR1 or pLAFR3, were conjugated with one of the mutants, designated XC1708. Recombinant plasmids isolated by their ability to complement XC1708 can be classified into two categories. One, represented by pLASC3, can complement some mutants, whereas the other, represented by a single plasmid, pLAHH2, can complement all of the other mutants. Restriction mapping showed that the two recombinant plasmids shared an EcoRI fragment of 8.9 kb. Results from subcloning, deletion mapping, and mini-Mu insertional mutation of the 8.9-kb EcoRI fragment suggested that a 4.2-kb fragment was sufficient to complement the mutant XC1708. Sequence analysis of this 4.2-kb fragment revealed three consecutive open reading frames (ORFs), ORF1, ORF2, and ORF3. Hybridization experiments showed that Tn5 in the genome of XC1708 and other mutants complemented by pLASC3 was located in ORF3, which could code for a protein of 83.5 kDa. A signal peptidase II processing site was identified at the N terminus of the predicted amino acid sequence. Sequence homology of 51% was observed between the amino acid sequences predicted from ORF3 and the pulD gene of Klebsiella species.