Phylotype classification of Ralstonia solanacearum biovar 1 strains isolated from banana (Musa spp) in Malaysia

During 2011–2012, 15 bacterial isolates were obtained from wilting banana plants from seven locations in Malaysia. Characterisation of the Malaysian isolates was determined by biovar determination, pathogenicity test, phylotype-specific multiplex PCR (Pmx-PCR) and endoglucanase (egl) gene amplification. Based on the genotype, phenotype and pathogenic characteristics, all isolates were identified as Ralstonia solanacearum. Pmx- and egl-PCRs indicated that all isolates belong to phylotype II of Ralstonia species complex hierarchical classification. The neighbour joining phylogenetic tree of egl sequences also verified the results where the isolates were all clustered into phylotype II, together with the reference sequences strains, UW070 and UW162. Therefore, the results of our study may provide a better understanding on the taxonomy of R. solanacearum species occupying banana plantations in Malaysia. This study is indeed the first report of phylotype II classification of R. solanacearum biovar 1 strains isolated from banana plants in Malaysia.     

Involvement of a PadR regulator PrhP on virulence of Ralstonia solanacearum by controlling detoxification of phenolic acids and type III secretion system

Ralstonia solanacearum can metabolize ferulic acid (FA) and salicylic acid (SA), two representative phenolic acids, to protect it from toxicity of phenolic acids. Here, we genetically demonstrated a novel phenolic acid decarboxylase regulator (PadR)-like regulator PrhP as a positive regulator on detoxification of SA and FA in R. solanacearum. Although the ability to degrade SA and FA enhances the infection process of R. solanacearum toward host plants, PrhP greatly contributes to the infection process besides degradation of SA and FA. Our results from the growth assay, promoter activity assay, RNA-seq and qRT-PCR revealed that PrhP plays multiple roles in the virulence of R. solanacearum: (1) positively regulates expression of genes for degradation of SA and FA; (2) positively regulates expression of genes encoding type III secretion system (T3SS) and type III effectors both in vitro and in planta; (3) positively regulates expression of many virulence-related genes, such as the flagella, type IV pili and cell wall degradation enzymes; and (4) is important for the extensive proliferation in planta. The T3SS is one of the essential pathogenicity determinants in many pathogenic bacteria, and PrhP positively regulates its expression mediated with the key regulator HrpB but through some novel pathway to HrpB in R. solanacearum. This is the first report on PadR regulators to regulate the T3SS and it could improve our understanding of the various biological functions of PadR regulators and the complex regulatory pathway on T3SS in R. solanacearum.     

Control of the Ralstonia solanacearum Type III secretion system (Hrp) genes by the global virulence regulator PhcA

Expression of several virulence factors in the plant pathogen bacterium Ralstonia solanacearum is controlled by a complex regulatory network, at the center of which is PhcA. We provide genetic evidence that PhcA also represses the expression of hrp genes that code for the Type III protein secretion system, a major pathogenicity determinant in this bacterium. The repression of hrp genes in complete medium is relieved in a phcA mutant and two distinct signals, a quorum-sensing signal and complex nitrogen sources, appear to trigger this PhcA-dependent repression. This control of hrp gene expression by PhcA is realized at the level of the HrpG regulatory protein.     

Isolation of poly(beta-L-malic acid)-degrading bacteria and purification and characterization of the PMA hydrolase from Comamonas acidovorans strain 7789

Several bacteria were isolated which were able to utilize poly(beta-L-malic acid) as sole carbon source for growth. The poly(beta-L-malic acid) hydrolyzing enzyme of Comamonas acidovorans strain 7789 was detected in the membrane fraction. The enzyme was purified by isolation of crude cell membranes by ultracentrifugation of disrupted cells, solubilization of the membrane fraction with octylglucoside, selective precipitation with 50% saturated ammonium sulfate and preparative isolectric focusing. SDS-PAGE analysis revealed a M(r) of 43,000. The pH optimum was 8.1 and the Km was 0.13 microM (in terms of monomeric units) and 0.0021 microM poly(beta-L-malic acid) at pH 8.1 (100 mM glycylglycine buffer). Addition of NaCl, KCl, CaCl2 or MgCl2 (from 25 to 100 mM) decreased the hydrolase activity, whereas EDTA or polymethane sulfonic acid fluoride had no influence on the enzyme. The depolymerization of poly(beta-L-malic acid) proceeded from the ends of the polyester resulting in the formation of L-malate. Esterase activity was not detectable with p-nitrophenyl acetate or p-nitrophenyl butyrate, which is used to determine for example poly(3-hydroxybutyric acid) depolymerase activity.     

Structural and functional characterization of a promiscuous feruloyl esterase (Est1E) from the rumen bacterium Butyrivibrio proteoclasticus

The release of polysaccharide from the plant cell wall is a key process to release the stored energy from plant biomass. Within the ruminant digestive system, a host of commensal microorganisms speed the breakdown of plant cell matter releasing fermentable sugars. The presence of phenolic compounds, most notably ferulic acid (FA), esterified within the cell wall is thought to pose a significant impediment to the degradation of the plant cell wall. The structure of a FA esterase from the ruminant bacterium Butyrivibrio proteoclasticus has been determined in two different space groups, in both the apo‐form, and the ligand bound form with FA located in the active site. The structure reveals a new lid domain that has no structural homologues in the PDB. The flexibility of the lid domain is evident by the presence of three different conformations adopted by different molecules in the crystals. In the FA‐bound structures, these conformations show sequential binding and closing of the lid domain over the substrate. Enzymatic activity assays demonstrate a broad activity against plant‐derived hemicellulose, releasing at least four aromatic compounds including FA, coumaric acid, coumarin‐3‐carboxylic acid, and cinnamic acid. The rumen is a complex ecosystem that efficiently degrades plant biomass and the genome of B. proteoclasticus contains greater than 130 enzymes, which are potentially involved in this process of which Est1E is the first to be well characterized. Proteins 2010. © 2009 Wiley‐Liss, Inc.     

一种不对称水解（R,S）-2,6-二甲基苯基氨基丙酸甲酯的新酯酶基因的克隆表达和酶学性质研究

本文将来自反硝化无色杆菌Achromobacterdenitrificans1104的酯酶基因EHest,转化大肠杆菌中,成功表达了具有不对称水解农药甲霜灵的中间体(R,S)-2,6-二甲基苯基氨基丙酸甲酯( MAP )活性的酯酶EHesterase。用重组酯酶EHesterase催化MAP 的水解,底物浓度50 g/L,反应1h的转化率29.5%,产物( R-酸)的eep 是85.1%。该酶的最适反应pH和温度分别为9.0和50℃,在50℃以下和pH5~9之间具有较好的稳定性。该酶水解MAP 的米氏动力学参数Vm、Km 分别是0.733 g/(L·min)和7.49 g/L。加入10%DMSO对酶EHesterase的立体选择性和催化速度有一定的促进作用。 Cu2+、Fe3+对酶活有明显抑制作用。该酶水解MAP 的活性与水解p-对硝基苯乙酸酯的活性数量级相当,是水解橄榄油活性的333倍。     

A novel dienelactone hydrolase from the thermoacidophilic archaeon Sulfolobus solfataricus P1: Purification,characterization, and expression

Background

Dienelactone hydrolases catalyze the hydrolysis of dienelactone to maleylacetate, which play a key role for the microbial degradation of chloroaromatics via chlorocatechols. Here, a thermostable dienelactone hydrolase from thermoacidophilic archaeon Sulfolobus solfataricus P1 was the first purified and characterized and then expressed in Escherichia coli.

Methods

The enzyme was purified by using several column chromatographys and characterized by determining the enzyme activity using p-nitrophenyl caprylate and dienelactones. In addition, the amino acids related to the catalytic mechanism were examined by site-directed mutagenesis using the identified gene.

Results

The enzyme, approximately 29 kDa monomeric, showed the maximal activity at 74 °C and pH 5.0, respectively. The enzyme displayed remarkable thermostability: it retained approximately 50% of its activity after 50 h of incubation at 90 °C, and showed high stability against denaturing agents, including various detergents, urea, and organic solvents. The enzyme displayed substrate specificities toward trans-dienelactone, not cis-isomer, and also carboxylesterase activity toward p-nitrophenyl esters ranging from butyrate (C₄) to laurate (C₁₂). The k_cat/K_m ratios for trans-dienelactone and p-nitrophenyl caprylate (C₈), the best substrate, were 92.5 and 54.7 s⁻¹ μM⁻¹, respectively.

Conclusions

The enzyme is a typical dienelactone hydrolase belonging to α/β hydrolase family and containing a catalytic triad composed of Cys151, Asp198, and His229 in the active site.

General significance

The enzyme is the first characterized archaeal dienelactone hydrolase.