Plant lectin-like bacteriocin from a rhizosphere-colonizing Pseudomonas isolate

Rhizosphere isolate Pseudomonas sp. strain BW11M1, which belongs to the Pseudomonas putida cluster, secretes a heat- and protease-sensitive bacteriocin which kills P. putida GR12-2R3. The production of this bacteriocin is enhanced by DNA-damaging treatment of producer cells. We isolated a TnMod mutant of strain BW11M1 that had lost the capacity to inhibit the growth of strain GR12-2R3. A wild-type genomic fragment encompassing the transposon insertion site was shown to confer the bacteriocin phenotype when it was introduced into Escherichia coli cells. The bacteriocin structural gene was identified by defining the minimal region required for expression in E. coli. This gene was designated llpA (lectin-like putidacin) on the basis of significant homology of its 276-amino-acid product with mannose-binding lectins from monocotyledonous plants. LlpA is composed of two monocot mannose-binding lectin (MMBL) domains. Several uncharacterized bacterial genes encoding diverse proteins containing one or two MMBL domains were identified. A phylogenetic analysis of the MMBL domains present in eukaryotic and prokaryotic proteins assigned the putidacin domains to a new bacterial clade within the MMBL-containing protein family. Heterologous expression of the llpA gene also conveyed bacteriocin production to several Pseudomonas fluorescens strains. In addition, we demonstrated that strain BW11M1 and heterologous hosts secrete LlpA into the growth medium without requiring a cleavable signal sequence. Most likely, the mode of action of this lectin-like bacteriocin is different from the modes of action of previously described Pseudomonas bacteriocins.     

Albusin B, a bacteriocin from the ruminal bacterium Ruminococcus albus 7 that inhibits growth of Ruminococcus flavefaciens

An approximately 32-kDa protein (albusin B) that inhibited growth of Ruminococcus flavefaciens FD-1 was isolated from culture supernatants of Ruminococcus albus 7. Traditional cloning and gene-walking PCR techniques revealed an open reading frame (albB) encoding a protein with a predicted molecular mass of 32,168 Da. A BLAST search revealed two homologs of AlbB from the unfinished genome of R. albus 8 and moderate similarity to LlpA, a recently described 30-kDa bacteriocin from Pseudomonas sp. strain BW11M1.     

Novel lectin-like bacteriocins of biocontrol strain Pseudomonas fluorescens Pf-5

Bacteriocin LlpA, produced by Pseudomonas sp. strain BW11M1, is a peculiar antibacterial protein due to its homology to mannose-binding lectins mostly found in monocots (A. H. A. Parret, G. Schoofs, P. Proost, and R. De Mot, J. Bacteriol. 185:897-908, 2003). Biocontrol strain Pseudomonas fluorescens Pf-5 contains two llpA-like genes, named llpA1(Pf-5) and llpA2(Pf-5). Recombinant Escherichia coli cells expressing llpA1(Pf-5) or llpA2(Pf-5) acquired bacteriocin activity and secreted a 31-kDa protein cross-reacting with LlpA(BW11M1) antibodies. Antibacterial activity of the recombinant proteins was evidenced by gel overlay assays. Analysis of the antimicrobial spectrum indicated that LlpA1(Pf-5) and LlpA2(Pf-5) are able to inhibit P. fluorescens strains, as well as the related mushroom pathogen Pseudomonas tolaasii. LlpA-type bacteriocins are characterized by a domain structure consisting of tandem monocot mannose-binding lectin (MMBL) domains. Molecular phylogeny of these MMBL domains suggests that the individual MMBL domains within an LlpA protein have evolved separately toward a specific, as yet unknown, function or, alternatively, were acquired from different ancestral sources. Our observations are consistent with earlier observations, which hinted that MMBL-like bacteriocins represent a new family of antibacterial proteins, probably with a novel mode of action.     

藤黄绿脓菌素的自诱导及假单胞菌M18抗生物质代谢相关性初步分析

假单胞菌M18的生防功能归功于其分泌吩嗪-1-羧酸和藤黄绿脓菌素。为了研究抗生物质合成代谢相关性及调控机制,分别构建了两种抗生物质合成基因簇插入突变株M18T和M18Z1。用翻译融合表达载体pMEAZ(pltA′-′lacZ)分别转化野生株和突变株M18T、发酵培养并测定β-半乳糖苷酶活性,结果显示,添加藤黄绿脓菌素使突变株M18T(pMEAZ)的β-半乳糖苷酶活性比野生株M18(pMEAZ)增加约6倍,表明藤黄绿脓菌素对自身基因簇具正向自诱导作用。抗生物质的测定结果显示,突变株M18T无藤黄绿脓菌素合成,而吩嗪-1-羧酸的合成量与野生株相同;突变株M18Z1与野生株相比,吩嗪-1-羧酸明显减少,藤黄绿脓菌素却显著提高。过量的吩嗪-1-羧酸又抑制藤黄绿脓菌素的合成。表明,假单胞菌M18中独有的代谢相关方式为:藤黄绿脓菌素不影响吩嗪-1-羧酸,但吩嗪-1-羧酸负调控藤黄绿脓菌素。     

RNA聚合酶σ^38亚基缺失对假单胞菌抗生物质合成代谢的影响

设计引物从假单胞菌M18基因组DNA中扩增并获得rpoS基因的378bp保守区段。以此为探针,从假单胞菌基因组文库中克隆了包括rpoS基因全序列及其相邻序列的3·1kbEcoRⅠ-XhoⅠ片段。通过抗性基因(抗庆大霉素基因)的定点插入构建了σ38亚基缺失突变株M18S。HPLC检测结果显示,σ38亚基缺失引起该菌株的抗生物质合成代谢的显著变化。与野生株相比,缺失突变株的吩嗪-1-羧酸在PPM和KMB中2种培养基中合成量由58μg/mL和10·2μg/mL分别减少到20·4μg/mL和0μg/mL;而缺失突变株的藤黄绿脓菌素则相反,在PPM和KMB两种培养基中合成量由0·5μg/mL和20·5μg/mL分别提高到75·4μg/mL和185·6μg/mL。表明σ38亚基可区别性调控假单胞菌M18的抗生物质合成代谢。rpoS基因的互补实验和两种抗生素基因与β-半乳糖苷酶基因的翻译融合表达实验进一步验证了上述的结果:σ38亚基正调控吩嗪-1-羧酸的表达,而负调控藤黄绿脓菌素的表达。     

假单胞菌M18调控因子GacA与RsmA的相关性及对抗生物质合成代谢调控机制的分析

假单胞菌M18是一株可同时合成并分泌吩嗪-1-羧酸(Phenazine-1-carboxylic acid,PCA)和藤黄绿脓菌素(Pyoluteorin,Plt)两种抗生物质的生防菌株。为了进一步研究假单胞菌M18抗生物质合成代谢的调控方式与机制,在分别构建gacAr、smA等单基因突变株基础上,又构建了gacArsmA双基因突变株M18GR以及gacA′-l′acZ和rsmA′-′lacZ等翻译融合表达载体(pMEGA和pMERA)。通过在PPM和KMB两种培养基中发酵培养和两种抗生物质PCA和Plt的HPLC定量测定显示,双突变株M18GR的PCA和Plt的合成量不论在PPM还是在KMB培养基中都介于单突变株M18G和M18R之间。由实验结果分析推测,两种调控因子对抗生物质合成的调控作用不是发生在转录水平,很可能发生在转录后水平。由β-半乳糖苷酶的定量分析表明,在假单胞菌M18中,两种调控因子不存在自诱导机制;虽然GacA未调控RsmA的合成,但RsmA可能部分正向调控GacA的表达。     

假单胞菌M18rsmA-突变株的构建及其对Plt和PCA合成的区别性调控作用

假单胞菌(Pseudomonas sp．)M18是促进植物生长的根际细菌,能产生吩嗪-1-羧酸(PCA)和藤黄绿菌素(Plt)两种不同的抗生素抑制植物病原菌,保护植物免受病害。运用PCR方法,从M18基因组中,扩增出rsmA基因部分片段,并以该片段为探针,从M18的基因组柯斯文库中筛出阳性克隆,切取带有rsmA基因及两侧序列的1．5kb片段,中间插入编码Km‘的DNA片段,获得rsmA^-体外突变体。运用同源重组剔除技术,构建了M18菌株的rsmA突变株M18R^-。突变株M18R^-生物合成Plt的能力比野生型M18提高4倍,但是,PCA产量仅为野生型的20％。研究结果表明,全局性调控基因rsmA可能通过不同的机制区别性地影响Plt和PCA的生物合成。     

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     

Phenazine-1-carboxylic acid is negatively regulated and pyoluteorin positively regulated by gacA in Pseudomonas sp. M18

The biosynthesis of antimicrobial metabolites is controlled by the GacS/GacA two-component regulatory system in Pseudomonas species. The production of phenazine-1-carboxylic acid and pyoluteorin is differentially regulated by GacA in Pseudomonas sp. M18. Pyoluteorin was reduced to nondetectable level in culture of the gacA insertional mutant strain M18G grown in King's medium B broth, whereas phenazine-1-carboxylic acid production was increased 30-fold over that of the wild-type strain. Production of both antibiotics was restored to wild-type levels after complementation in trans with the wild-type gacA gene. Expression of the translational fusions phzA'-'lacZ and pltA'-'lacZ confirmed the effect of GacA on both biosynthetic operons.     

假单胞菌M18调控基因ppbR的克隆及功能研究

假单胞菌(Pseudomonas sp.)M18是促进植物生长的根际细菌,能产生吩嗪-1-羧酸(PCA)和藤黄绿菌素(Plt)两种不同的抗生素.根据生物信息学分析,铜绿假单胞菌PA2572基因编码蛋白可能是一个双元调控系统的应答调节子.本研究从假单胞菌M18基因组中扩增出PA2572同源基因片段ppbR,利用体外定点插入突变和同源重组技术构建了M18的ppbR突变株M18P.研究结果表明,突变株M18P在泳动能力和群集运动能力上有显著的下降.突变株合成PCA的能力比野生型有显著的下降,在发酵液中PCA积累量仅为野生型的50%.在KMB培养基中,突变株Plt的积累量和野生型没有显著的差异.     

假单胞菌M18调控基因ppbR的克隆及功能研究

假单胞菌 (Pseudomonas sp.) M18 是促进植物生长的根际细菌, 能产生吩嗪-1-羧酸 (PCA) 和藤黄绿菌素 (Plt) 两种不同的抗生素。根据生物信息学分析, 铜绿假单胞菌PA2572基因编码蛋白可能是一个双元调控系统的应答调节子。本研究从假单胞菌M18基因组中扩增出PA2572同源基因片段ppbR, 利用体外定点插入突变和同源重组技术构建了M18 的ppbR突变株M18P。研究结果表明, 突变株M18P在泳动能力和群集运动能力上有显著的下降。突变株合成PCA 的能力比野生型有显著的下降, 在发酵液中PCA积累量仅为野生型的50%。在KMB培养基中, 突变株Plt的积累量和野生型没有显著的差异。     

Novel Lectin-Like Bacteriocins of Biocontrol Strain Pseudomonas fluorescens Pf-5

Bacteriocin LlpA, produced by Pseudomonas sp. strain BW11M1, is a peculiar antibacterial protein due to its homology to mannose-binding lectins mostly found in monocots (A. H. A. Parret, G. Schoofs, P. Proost, and R. De Mot, J. Bacteriol. 185:897-908, 2003). Biocontrol strain Pseudomonas fluorescens Pf-5 contains two llpA-like genes, named llpA1_Pf-5 and llpA2_Pf-5. Recombinant Escherichia coli cells expressing llpA1_Pf-5 or llpA2_Pf-5 acquired bacteriocin activity and secreted a 31-kDa protein cross-reacting with LlpA_BW11M1 antibodies. Antibacterial activity of the recombinant proteins was evidenced by gel overlay assays. Analysis of the antimicrobial spectrum indicated that LlpA1_Pf-5 and LlpA2_Pf-5 are able to inhibit P. fluorescens strains, as well as the related mushroom pathogen Pseudomonas tolaasii. LlpA-type bacteriocins are characterized by a domain structure consisting of tandem monocot mannose-binding lectin (MMBL) domains. Molecular phylogeny of these MMBL domains suggests that the individual MMBL domains within an LlpA protein have evolved separately toward a specific, as yet unknown, function or, alternatively, were acquired from different ancestral sources. Our observations are consistent with earlier observations, which hinted that MMBL-like bacteriocins represent a new family of antibacterial proteins, probably with a novel mode of action.     

An <Emphasis Type="Italic">Rhs</Emphasis>-like genetic element is involved in bacteriocin production by <Emphasis Type="Italic">Pseudomonas savastanoi</Emphasis> pv. <Emphasis Type="Italic">savastanoi</Emphasis>

The main aim of this work was the identification of genetic determinants involved in bacteriocin production by strain ITM317 of Pseudomonas savastanoi pv. savastanoi, besides bacteriocin characterization. The bacteriocin was observed to be a heat-sensitive, high molecular weight proteinaceous compound. We identified a transposon (Tn5)-induced mutant which had lost its ability to produce the bacteriocin. The Tn5 insertion’s responsibility for the above mutated phenotype was demonstrated by marker-exchange mutagenesis. An EcoRI DNA fragment, corresponding to the EcoRI Tn5-containing fragment of the mutant, was also cloned from the wild-type strain, and its introduction into the mutant complemented the mutation. Moreover, that fragment enabled bacteriocin production by P. s. pv. savastanoi ITM302, a strain not previously capable of doing so. DNA sequence analysis revealed that Tn5 insertion occurred in the mutant within a large ORF encoding a protein which showed similarity with proteins from the Rhs family. The DNA region including that ORF showed features which have been considered typical of the Rhs genetic elements previously identified in other bacteria but whose function is as yet unclear. The results of this study for the first time identify an Rhs-like element in P. s. pv. savastanoi, and for the first time indicate that an Rhs element is involved in bacteriocin production, also suggesting this possible function for Rhs genetic elements previously characterized in other bacteria.     

假单胞菌株M18 psrA突变株的构建及其对吩嗪-1-羧酸和藤黄绿菌素合成的调控

【目的】假单胞菌M18是一株能同时合成吩嗪-1-羧酸(PCA)和藤黄绿菌素(Plt)两种抗生素的植物根际促生细菌。PsrA为细菌TetR家族转录调控因子。为了研究PsrA对PCA与Plt生物合成的影响,从M18菌株基因组中扩增psrA基因。【方法】通过同源重组技术,构建庆大霉素抗性片段置换psrA的突变菌株M18psrA。利用基因互补、lacZ报告基因融合分析实验,验证PsrA对抗生素合成基因的调控作用。【结果】在PPM和KMB培养基中,分别比较野生型菌株M18和突变菌株M18psrA的PCA与Plt产量,突变菌株M18psrA的PCA产量显著下降;Plt产量显著升高,为野生型菌株的10-15倍。基因互补、lacZ报告基因融合分析,进一步证明了psrA正调控PCA的phz2合成基因簇,负调控Plt的合成基因簇。【结论】PsrA区别性调控抗生素PCA与Plt的生物合成。     

Identification and cloning of an Erwinia carotovora subsp. carotovora bacteriocin regulator gene by insertional mutagenesis

Avirulent Erwinia carotovora subsp. carotovora CGE234-M403 produces two types of bacteriocin. For the purpose of cloning the bacteriocin genes of strain CGE234M403, a spontaneous rifampin-resistant mutant of this strain, M-rif-11-2, was isolated. By Tn5 insertional mutagenesis using M-rif-11-2, a mutant, TM01A01, which produces the high-molecular-weight bacteriocin but not the low-molecular-weight bacteriocin was obtained. By thermal asymmetric interlaced PCR, the DNA sequence from the Tn5 insertion site and the DNA sequence of a contiguous 1,280-bp region were determined. One complete open reading frame (ORF), designated ORF2, was identified within the sequenced fragment. The 3' end of another ORF, ORF1, was located upstream of ORF2. A noncoding region and a putative promoter were located between ORF1 and ORF2. Downstream from ORF2, the 5' end of another ORF (ORF3) was found. Deduction from the nucleotide sequence indicated that ORF2 encodes a protein of 99 amino acids, which showed high homology with Yersinia enterocolitica Yrp, a regulator of enterotoxin (Y-ST) production; Escherichia coli host factor 1, required for Qbeta-replicase; and Azorhizobium caulinodans NrfA, required for the expression of nifA. ORF2 was designated brg, bacteriocin regulator gene. A fragment containing ORF2 and its promoter was amplified and cloned into pBR322 and pHSG415r, and the recombinant plasmids, pBYL1 and pHYL1, were transferred into E. coli DH5. Plasmid pBYL1 was reisolated and transferred into the insertion mutant TM01A01. Transformants carrying the plasmid, which was reisolated and designated pBYL1, re-produced the low-molecular-weight bacteriocin.     

Molecular cloning of a malyl coenzyme A lyase gene from Pseudomonas sp. strain AM1, a facultative methylotroph

A genomic library containing HindIII partial digests of Pseudomonas sp. strain AM1 DNA was constructed in the broad-host-range cosmid pVK100. PCT57, a Pseudomonas sp. strain AM1 methanol mutant deficient in malyl coenzyme A lyase activity, was complemented to a methanol-positive phenotype by mobilization of the pVK100 library into PCT57 recipients with the ColE1/RK2 mobilizing plasmid pRK2013. Six different complemented isolates all contained a recombinant plasmid carrying the same 19.6-kilobase-pair Pseudomonas sp. strain AM1 DNA insert. Subcloning and complementation analysis demonstrated that the gene deficient in PCT57 (mcl-1) was located in a 1.6-kilobase-pair region within a 7.4-kilobase-pair EcoRI-HindIII fragment.     

Genetic analysis of influenza virus NS1 gene: a temperature-sensitive mutant shows defective formation of virus particles

To perform a genetic analysis of the influenza A virus NS1 gene, a library of NS1 mutants was generated by PCR-mediated mutagenesis. A collection of mutant ribonucleic proteins containing the nonstructural genes was generated from the library that were rescued for an infectious virus mutant library by a novel RNP competition virus rescue procedure. Several temperature-sensitive (ts) mutant viruses were obtained by screening of the mutant library, and the sequences of their NS1 genes were determined. Most of the mutations identified led to amino acid exchanges and concentrated in the N-terminal region of the protein, but some of them occurred in the C-terminal region. Mutant 11C contained three mutations that led to amino acid exchanges, V18A, R44K, and S195P, all of which were required for the ts phenotype, and was characterized further. Several steps in the infection were slightly altered: (i) M1, M2, NS1, and neuraminidase (NA) accumulations were reduced and (ii) NS1 protein was retained in the nucleus in a temperature-independent manner, but these modifications could not justify the strong virus titer reduction at restrictive temperature. The most dramatic phenotype was the almost complete absence of virus particles in the culture medium, in spite of normal accumulation and nucleocytoplasmic export of virus RNPs. The function affected in the 11C mutant was required late in the infection, as documented by shift-up and shift-down experiments. The defect in virion production was not due to reduced NA expression, as virus yield could not be rescued by exogenous neuraminidase treatment. All together, the analysis of 11C mutant phenotype may indicate a role for NS1 protein in a late event in virus morphogenesis.     

Positive regulation of pyoluteorin biosynthesis in Pseudomonas sp. M18 by quorum-sensing regulator VqsR

The biocontrol rhizobacterium Pseudomonas sp. M18 can produce two kinds of antibiotics, namely pyoluteorin (Plt) and phenazine-1-carboxylic acid (PCA), and is antagonistic against a number of soilborne phytopathogens. In this study, a luxR-type quorum-sensing regulatory gene, vqsR, was identified and characterized immediately downstream of the Plt gene cluster in strain M18. A vqsR-inactivated mutant led to a significant decrease in the production of Plt and its biosynthetic gene expression. However, this was restored when introducing the vqsR gene by cloning into the plasmid pME6032 in trans. The vqsR mutation did not exert any obvious influence on the production of PCA and its biosynthetic gene expression and the production of Nacylhomoserine lactones (C4 and C8-HSLs) and their biosynthetic gene rhlI expression. Accordingly, these results introduce VqsR as a regulator of Plt production in Pseudomonas spp., and suggest that the regulatory mechanism of vqsR in strain M18 is distinct from that in P. aeruginosa. In addition, it was demonstrated that vqsR mutation did not have any obvious impact on the expression of Plt-specific ABC transporters and other secondary metabolic global regulators, including GacA, RpoS, and RsmA.