Ralfuranones contribute to mushroom‐type biofilm formation by Ralstonia solanacearum strain OE1‐1

After invasion into intercellular spaces of tomato plants, the soil‐borne, plant‐pathogenic Ralstonia solanacearum strain OE1‐1 forms mushroom‐shaped biofilms (mushroom‐type biofilms, mBFs) on tomato cells, leading to its virulence. The strain OE1‐1 produces aryl‐furanone secondary metabolites, ralfuranones (A, B, J, K and L), dependent on the quorum sensing (QS) system, with methyl 3‐hydroxymyristate (3‐OH MAME) synthesized by PhcB as a QS signal. Ralfuranones are associated with the feedback loop of the QS system. A ralfuranone productivity‐deficient mutant (ΔralA) exhibited significantly reduced growth in intercellular spaces compared with strain OE1‐1, losing its virulence. To analyse the function of ralfuranones in mBF formation by OE1‐1 cells, we observed cell aggregates of R. solanacearum strains statically incubated in tomato apoplast fluids on filters under a scanning electron microscope. The ΔralA strain formed significantly fewer microcolonies and mBFs than strain OE1‐1. Supplementation of ralfuranones A, B, J and K, but not L, significantly enhanced the development of mBF formation by ΔralA. Furthermore, a phcB‐ and ralA‐deleted mutant (ΔphcB/ralA) exhibited less formation of mBFs than OE1‐1, although a QS‐deficient, phcB‐deleted mutant formed mBFs similar to OE1‐1. Supplementation with 3‐OH MAME significantly reduced the formation of mBFs by ΔphcB/ralA. The application of each ralfuranone significantly increased the formation of mBFs by ΔphcB/ralA supplied with 3‐OH MAME. Together, our findings indicate that ralfuranones are implicated not only in the development of mBFs by strain OE1‐1, but also in the suppression of QS‐mediated negative regulation of mBF formation.     

Heavy metal-resistant bacteria as extremophiles: molecular physiology and biotechnological use of Ralstonia sp. CH34

In contrast to thermophilic or psychrophilic organisms, heavy metal-resistant bacteria do not supply enzymes that are active under harsh conditions, but are themselves tools for the evaluation and remediation of heavy metal-contaminated environments. Ralstonia sp. CH34 is a gram-negative bacterium with a remarkable set of resistance determinants, allowing this bacterium to live in extreme environments that are heavily contaminated with toxic metal ions. These heavy metal ions are mostly detoxified by inducible ion efflux systems that reduce the intracellular concentration of a given ion by active export. Because all metal resistance determinants in this bacterium are inducible, their regulatory systems can be used to develop biosensors that measure the biologically important concentrations of heavy metals in an environment. Resistance based on metal ion efflux detoxifies only the cytoplasm of the respective cell. Therefore, this resistance mechanism cannot be used directly to develop biotechnological procedures; however, metal ion efflux can protect a cell in a metal-contaminated environment. Thus, the cell can be enabled to mediate biochemical reactions such as precipitation of heavy metals with the carbon dioxide produced during growth or degradation of xenobiotics. Received: July 11, 1999 / Accepted: December 27, 1999     

Production of poly(3-hydroxybutyrate) by solid-state fermentation with <Emphasis Type="Italic">Ralstonia eutropha</Emphasis>

The use of solid-state fermentation is examined as a low-cost technology for the production of poly(hydroxyalkanoates) (PHAs) by Ralstonia eutropha. Two agroindustrial residues (babassu and soy cake) were evaluated as culture media. The maximum poly(hydroxybutyrate) (PHB) yield was 1.2 mg g^–1 medium on soy cake in 36 h, and 0.7 mg g^–1 medium on babassu cake in 84 h. Addition of 2.5% (w/w) sugar cane molasses to soy cake increased PHB production to 4.9 mg g^–1 medium in 60 h. Under these conditions, the PHB content of the dry biomass was 39% (w/w). The present results indicate that solid-state fermentation could be a promising alternative for producing biodegradable polymers at low cost.Revisions requested 31 August 2004; Revisions received 12 October 2004     

The role of the active site-coordinating cysteine residues in the maturation of the H2-sensing [NiFe] hydrogenase from Ralstonia eutropha H16

The H₂-splitting active site of [NiFe] hydrogenases is tightly bound to the protein matrix via four conserved cysteine residues. In this study, the nickel-binding cysteine residues of HoxC, the large subunit of the H₂-sensing regulatory hydrogenase (RH) from Ralstonia eutropha, were replaced by serine. All four mutant proteins, C60S, C63S, C479S, and C482S, were inactive both in H₂ sensing and H₂ oxidation and did not adopt the native oligomeric structure of the RH. Nickel was bound only to the C482S derivative. The assembly of the [NiFe] active site is a complex process that requires the function of at least six accessory proteins. Among these proteins, HypC has been shown to act as a chaperone for the large subunit during the maturation process. Immunoblot analysis revealed the presence of a strong RH-dependent HypC-specific complex in extracts containing the C60S, C63S, and C482S derivatives, pointing to a block in maturation for these mutant proteins. The lack of this complex in the extract containing C479S indicates that this specific cysteine residue might be crucial for the interaction between HoxC and HypC.This work is dedicated to Prof. H.G. Schlegel on the occasion of his 80th birthday.     

Biophysical characterization of the MerP-like amino-terminal extension of the mercuric reductase from<Emphasis Type="Italic"> Ralstonia metallidurans</Emphasis> CH34

The purified native mercuric reductase (MerA) from Ralstonia metallidurans CH34 contains an N-terminal sequence of 68 amino acids predicted to be homologous to MerP, the periplasmic mercury-binding protein. This MerP-like protein has now been expressed independently. The protein was named MerAa by homology with Ccc2a, the first soluble domain of the copper-transporting ATPase from yeast. a has been characterized using a set of biophysical techniques. The binding of mercury was followed using circular dichroism spectroscopy and electrospray mass spectrometry. The two cysteine residues contained in the consensus sequence GMTCXXC are involved in the binding of one mercury atom, with an apparent affinity comparable to that of MerP for the same metal. The metal-binding site is confirmed by NMR chemical shift changes observed between apo- and metal-bound MerAa in solution. NMR shift and NOE data also indicate that only minor structural changes occur upon metal binding. Further NMR investigation of the fold of MerAa using long-range methyl–methyl NOE and backbone residual dipolar coupling data confirm the expected close structural homology with MerP. ¹⁵N relaxation data show that MerAa is a globally rigid molecule. An increased backbone mobility was observed for the loop region connecting the first -strand and the first -helix and comprising the metal-binding domain. Although significantly reduced, this loop region keeps some conformational flexibility upon metal binding. Altogether, our data suggest a role of MerAa in mercury trafficking.Electronic Supplementary Material Supplementary material is available in the online version of this article at http://dx.doi.org/10.1007/s00775-003-0495-yAbbreviations CCA -cyano-4-hydroxy-trans-cinnamic acid - CSI chemical shift index - HSQC ¹H-detected heteronuclear single-quantum coherence - MerAa the 68 amino acid N-terminal extension of the mercuric reductase - NOE nuclear Overhauser effect - RDCs residual dipolar couplings - TCEP-HCl tris(2-carboxyethyl)phosphine hydrochloride     

The active component in the flax-retting system of the zygomycete Rhizopus oryzae sb is a family 28 polygalacturonase

The zygomycete Rhizopus oryzae sb is a very efficient organism for retting of flax, the initial microbiological step in the process of making linen. An extracellular polygalacturonase, when isolated could perform retting, and therefore probably is the key component in the retting system of R. oryzae. This was purified and characterized. The purified enzyme has a molecular mass of 37,436 Da from mass spectrometric determination, an isoelectric point of 8.4, and has non-methylated polygalacturonic acid as its preferred substrate. Peptide sequences indicate that the enzyme belongs to family 28, in similarity with other polygalacturonases (EC. 3.2.1.15). It contains, however an N-terminal sequence absent in other fungal pectinases, but present in an enzyme from the phytopathogenic bacterium Ralstonia solanacearum. The biochemical background for the superior retting efficiency of R. oryzae sb is discussed.     

Comparison of bioMérieux API 20NE and Remel RapID NF Plus, identification systems of type strains of Ralstonia pickettii

A : Comparison of two commercial miniaturized rapid systems for the identification of Ralstonia pickettii strains. METHODS AND RESULTS: Varying identification results were encountered using the bioMérieux API NE system and the Remel IDS RapID NF Plus commercial systems for R. pickettii. To compare these two systems, eight strains of R. pickettii were purchased from different commercial culture collections. Additionally, 32 industrial and eight clinical isolates, initially identified using the Vitek Junior (bioMérieux) were tested. Total number of isolates tested was 48. The API 20NE identified 29 isolates, as R. pickettii but was unsuccessful with 19 isolates. The Remel IDS RapID NF Plus identified 46 isolates as R. pickettii. One clinical and one industrial isolates was identified as non-R. pickettii with both systems. CONCLUSIONS: The above results indicate that the use of API 20NE system for examining the identification of R. pickettii strains is inconsistent. SIGNIFICANCE AND IMPACT OF THE STUDY: This study demonstrated that the RapID NF Plus is more accurate as an inexpensive identification system for the identification of R. pickettii, a potential emerging organism of medically and industrial importance.     

茄科劳尔氏菌复合体毒力基因及调控网络最新研究进展

植物青枯病是一种能造成巨大经济损失的土传病害,其病原茄科劳尔氏菌复合体(Ralstonia solanacearum species complex,RSSC)能通过复杂的毒力调控网络将毒力因子合成并分泌到植物细胞胞质间或细胞质内,从而引起寄主植物发病。本文详细分析了RSSC主要的毒力基因及调控网络,包括其运动性(鞭毛,菌毛)、细菌分泌系统(T2SS、T3SS以及T6SS)、毒力调控系统(Phc、Prh、Vsr、Peh、Sol)、毒力因子(CWDEs、T3Es、EPS)、群体信号因子AHL及植物激素,总结了近年来最新的研究进展并绘制了相关网络调控模式图,以期为进一步研究RSSC的致病机理及防控研究提供参考。     

丛枝菌根真菌与深色有隔内生真菌对姜瘟病的防效及抗病机理初探

【背景】青枯劳尔氏菌(Ralstonia solanacearum,R.S)引发的姜瘟病是生姜产业发展的瓶颈问题。丛枝菌根真菌(arbuscular mycorrhiza fungi, AMF)与深色有隔内生真菌(dark septate endophytes,DSE)是两类重要的共生微生物。【目的】前期研究发现,AMF与DSE可提高生姜对姜瘟病的抗性,但其抗病机制尚不清楚,极大地限制了利用这两类共生真菌对该病的防治。【方法】在温室条件下做盆栽试验,以生姜组培苗为材料,设立接种AMF、DSE和不接种AMF、DSE的对照(CK)处理,并在上述处理下的植物生长4周后淋入病原菌液,病原菌接种1周后,通过测定菌根侵染率、发病率、叶绿素含量、光合指标、磷(P)含量、防御性酶活性及丙二醛(malondialdehyde, MDA)含量,研究AMF和DSE互作对病原菌侵染后生姜生长和生理生化指标的影响。【结果】AMF和DSE分别使姜瘟病发病率降低了45.27%和52.04%(P<0.05)。AMF+DSE组合处理抑病效果更好,发病率较对照降低60.87%(P<0.05)。AMF、DSE及...     

丛枝菌根真菌响应桉树茄科雷尔氏菌侵染进程基因的筛选与鉴定

【背景】桉树(Eucalyptus)青枯病危害严重,丛枝菌根真菌(arbuscular mycorrhizal fungi,AMF)与桉树共生影响桉树对青枯病的抗性,而AMF响应桉树青枯菌侵染的机制仍不清楚。【目的】探索AMF响应桉树茄科雷尔氏菌(Ralstonia solanacearum)的侵染机制。【方法】以非菌根化和异形根孢囊霉(Rhizophagus irregularis)菌根化巨桉(Eucalyptus grandis)分别受茄科雷尔氏菌侵染0、24、48和96 h接种后(hour post-inoculated, hpi)的根系组织为研究对象,基于转录组测序筛选和鉴定菌根化巨桉根系中异形根孢囊霉响应茄科雷尔氏菌侵染的基因信息。【结果】与对应非菌根化桉树受茄科雷尔氏菌侵染的时间点相比,菌根化桉树中异形根孢囊霉响应青枯菌侵染显著差异表达基因为3 382–5 989个,随青枯侵染时间进程的增加,异形根孢囊霉特异性响应茄科雷尔氏菌侵染差异表达基因数量逐渐增多。茄科雷尔氏菌侵染24 hpi时,异形根孢囊霉显著富集共生体生长、孢子形成和凋亡信号通路、铁载体等相关基因;茄科雷尔氏菌侵...