A cyclic GMP-dependent signalling pathway regulates bacterial phytopathogenesis

Cyclic guanosine 3′,5′‐monophosphate (cyclic GMP) is a second messenger whose role in bacterial signalling is poorly understood. A genetic screen in the plant pathogen Xanthomonas campestris (Xcc) identified that XC_0250, which encodes a protein with a class III nucleotidyl cyclase domain, is required for cyclic GMP synthesis. Purified XC_0250 was active in cyclic GMP synthesis in vitro. The linked gene XC_0249 encodes a protein with a cyclic mononucleotide‐binding (cNMP) domain and a GGDEF diguanylate cyclase domain. The activity of XC_0249 in cyclic di‐GMP synthesis was enhanced by addition of cyclic GMP. The isolated cNMP domain of XC_0249 bound cyclic GMP and a structure–function analysis, directed by determination of the crystal structure of the holo‐complex, demonstrated the site of cyclic GMP binding that modulates cyclic di‐GMP synthesis. Mutation of either XC_0250 or XC_0249 led to a reduced virulence to plants and reduced biofilm formation in vitro. These findings describe a regulatory pathway in which cyclic GMP regulates virulence and biofilm formation through interaction with a novel effector that directly links cyclic GMP and cyclic di‐GMP signalling.     

The role of PilZ domain proteins in the virulence of Xanthomonas campestris pv. campestris

Cyclic di‐GMP [(bis‐(3′–5′)‐cyclic di‐guanosine monophosphate)] is an almost ubiquitous second messenger in bacteria that is implicated in the regulation of a range of functions that include developmental transitions, aggregative behaviour, adhesion, biofilm formation and virulence. Comparatively little is known about the mechanism(s) by which cyclic di‐GMP exerts these various regulatory effects. PilZ has been identified as a cyclic di‐GMP binding protein domain; proteins with this domain are involved in regulation of specific cellular processes, including the virulence of animal pathogens. Here we have examined the role of PilZ domain proteins in virulence and the regulation of virulence factor synthesis in Xanthomonas campestris pv. campestris (Xcc), the causal agent of black rot of crucifers. The Xcc genome encodes four proteins (XC0965, XC2249, XC2317 and XC3221) that have a PilZ domain. Mutation of XC0965, XC2249 and XC3221 led to a significant reduction of virulence in Chinese radish. Mutation of XC2249 and XC3221 led to a reduction in motility whereas mutation of XC2249 and XC0965 affected extracellular enzyme production. All mutant strains were unaffected in biofilm formation in vitro. The reduction of virulence following mutation of XC3221 could not be wholly attributed to an effect on motility as mutation of pilA, which abolishes motility, has a lesser effect on virulence.     

Mutation of the cyclic di-GMP phosphodiesterase gene in <Emphasis Type="Italic">Burkholderia lata</Emphasis> SK875 attenuates virulence and enhances biofilm formation

Burkholderia sp. is a gram-negative bacterium that commonly exists in the environment, and can cause diseases in plants, animals, and humans. Here, a transposon mutant library of a Burkholderia lata isolate from a pig with swine respiratory disease in Korea was screened for strains showing attenuated virulence in Caenorhabditis elegans. One such mutant was obtained, and the Tn5 insertion junction was mapped to rpfR, a gene encoding a cyclic di-GMP phosphodiesterase that functions as a receptor. Mutation of rpfR caused a reduction in growth on CPG agar and swimming motility as well as a rough colony morphology on Congo red agar. TLC analysis showed reduced AHL secretion, which was in agreement with the results from plate-based and bioluminescence assays. The mutant strain produced significantly more biofilm detected by crystal violet staining than the parent strain. SEM of the mutant strain clearly showed that the overproduced biofilm contained a filamentous structure. These results suggest that the cyclic di-GMP phosphodiesterase RpfR plays an important role in quorum sensing modulation of the bacterial virulence and biofilm formation.     

The Host Plant Metabolite Glucose Is the Precursor of Diffusible Signal Factor (DSF) Family Signals in Xanthomonas campestris

Plant pathogen Xanthomonas campestris pv. campestris produces cis-11-methyl-2-dodecenoic acid (diffusible signal factor [DSF]) as a cell-cell communication signal to regulate biofilm dispersal and virulence factor production. Previous studies have demonstrated that DSF biosynthesis is dependent on the presence of RpfF, an enoyl-coenzyme A (CoA) hydratase, but the DSF synthetic mechanism and the influence of the host plant on DSF biosynthesis are still not clear. We show here that exogenous addition of host plant juice or ethanol extract to the growth medium of X. campestris pv. campestris could significantly boost DSF family signal production. It was subsequently revealed that X. campestris pv. campestris produces not only DSF but also BDSF (cis-2-dodecenoic acid) and another novel DSF family signal, which was designated DSF-II. BDSF was originally identified in Burkholderia cenocepacia to be involved in regulation of motility, biofilm formation, and virulence in B. cenocepacia. Functional analysis suggested that DSF-II plays a role equal to that of DSF in regulation of biofilm dispersion and virulence factor production in X. campestris pv. campestris. Furthermore, chromatographic separation led to identification of glucose as a specific molecule stimulating DSF family signal biosynthesis in X. campestris pv. campestris. ¹³C-labeling experiments demonstrated that glucose acts as a substrate to provide a carbon element for DSF biosynthesis. The results of this study indicate that X. campestris pv. campestris could utilize a common metabolite of the host plant to enhance DSF family signal synthesis and therefore promote virulence.     

Structure of the Legionella Effector,lpg1496, Suggests a Role in Nucleotide Metabolism

Pathogenic Gram-negative bacteria use specialized secretion systems that translocate bacterial proteins, termed effectors, directly into host cells where they interact with host proteins and biochemical processes for the benefit of the pathogen. lpg1496 is a previously uncharacterized effector of Legionella pneumophila, the causative agent of Legionnaires disease. Here, we crystallized three nucleotide binding domains from lpg1496. The C-terminal domain, which is conserved among the SidE family of effectors, is formed of two largely α-helical lobes with a nucleotide binding cleft. A structural homology search has shown similarity to phosphodiesterases involved in cleavage of cyclic nucleotides. We have also crystallized a novel domain that occurs twice in the N-terminal half of the protein that we term the KLAMP domain due to the presence of homologous domains in bacterial histidine kinase-like ATP binding region-containing proteins and S-adenosylmethionine-dependent methyltransferase proteins. Both KLAMP structures are very similar but selectively bind 3′,5′-cAMP and ADP. A co-crystal of the KLAMP1 domain with 3′,5′-cAMP reveals the contribution of Tyr-61 and Tyr-69 that produces π-stacking interactions with the adenine ring of the nucleotide. Our study provides the first structural insights into two novel nucleotide binding domains associated with bacterial virulence.     

野油菜黄单胞菌中烯脂酰ACP还原酶的功能鉴定

烯脂酰ACP还原酶是细菌脂肪酸合成的关键酶之一.本研究通过生物信息学分析发现,野油菜黄单胞菌Xanthomonas campestris(Xcc)8004基因组中XC_0119(Xccfab V)注释为反-2-烯脂酰Co A还原酶基因.但其编码产物与铜绿假单胞菌的烯脂酰ACP还原酶Fab V具有较高的同源性,并含有相同的催化活性中心Tyr-(Xaa)8-Lys序列.用携带Xccfab V的质粒载体互补大肠杆菌fab I温度敏感突变株JP1111,转化子能在42℃生长,表明Xccfab V能遗传互补大肠杆菌fab I突变.体外重建脂肪酸合成反应表明,Xcc Fab V能催化不同链长的烯脂酰ACP还原为脂酰ACP,且催化活性不受三氯森抑制.遗传学研究表明,Xccfab V是必需基因,不能获得Xccfab V基因敲除突变株.将携带大肠杆菌fab I的外源质粒导入野生菌后,可敲除染色体上的fab V基因,获得的替换突变株生长特性和脂肪酸组成未发生显著变化,但替换突变株对三氯森敏感.上述结果证实,野油菜黄单胞菌fab V是必需基因,编码烯脂酰ACP还原酶,参与脂肪酸从头合成反应,且Fab V是Xcc对三氯森耐受的根本原因.     

野油菜黄单胞菌中LipB-LipA是唯一的硫辛酰化途径

【目的】硫辛酸是细胞内重要的辅因子,参与多种基础代谢过程。野油菜黄单胞菌(Xcc)是十字花科植物黑腐病的病原菌,在全球范围内引起植物病害,引起重大经济损失。为此研究Xcc中硫辛酸的合成途径,为防治黑腐病提供新思路。【方法】利用大肠杆菌硫辛酸合成关键酶LipA和LipB序列,同源比对发现Xcc基因组中XC_0713 (XccLipA)和XC_0712 (XccLipB)具有较高的同源性。采用PCR方法分别扩增XccLipA和XccLipB基因,并连入表达载体pBAD24M后分别互补大肠杆菌突变株,并检测转化子生长表型。利用同源重组方法,获得替换突变株,分析其生长性状,并利用剪叶法检测替换突变株对寄主植物甘蓝的致病力。【结果】XcclipA和XcclipB能分别恢复大肠杆菌lipA和lipB突变株在基础培养上生长。XcclipA和XcclipB都是菌体生长的必需基因,不能直接被敲除。但导入pSRK-EclplA后,成功分别获得XcclipA和XcclipB敲除突变株。两种EclplA替换后的敲除突变株在基础培养上都不能生长,添加硫辛酸后能恢复生长表型。在丰富培养基上,XcclipB敲除突变株能正常生长,而XcclipA敲除突变株不能生长,添加硫辛酸后生长也能恢复。分别测定不同培养条件下生长曲线,也得到同样的结果。寄主植物侵染结果显示,与野生菌相比,XcclipA敲除突变株致病性几乎丧失,而XcclipB敲除突变株的致病性与野生菌无显著性差异。【结论】Xcc中lipA编码硫辛酸合成酶,lipB编码辛酰转移酶,两者都是必需基因。Xcc中LipB-LipA途径是唯一的硫辛酰化途径,而没有外源性的硫辛酸途径。lipA敲除后显著影响Xcc的致病性,可作为抗菌药物筛选的靶点。     

Characterization of the 2',3' cyclic phosphodiesterase activities of Clostridium thermocellum polynucleotide kinase-phosphatase and bacteriophage lambda phosphatase

Clostridium thermocellum polynucleotide kinase-phosphatase (CthPnkp) catalyzes 5′ and 3′ end-healing reactions that prepare broken RNA termini for sealing by RNA ligase. The central phosphatase domain of CthPnkp belongs to the dinuclear metallophosphoesterase superfamily exemplified by bacteriophage λ phosphatase (λ-Pase). CthPnkp is a Ni²⁺/Mn²⁺-dependent phosphodiesterase-monoesterase, active on nucleotide and non-nucleotide substrates, that can be transformed toward narrower metal and substrate specificities via mutations of the active site. Here we characterize the Mn²⁺-dependent 2′,3′ cyclic nucleotide phosphodiesterase activity of CthPnkp, the reaction most relevant to RNA repair pathways. We find that CthPnkp prefers a 2′,3′ cyclic phosphate to a 3′,5′ cyclic phosphate. A single H189D mutation imposes strict specificity for a 2′,3′ cyclic phosphate, which is cleaved to form a single 2′-NMP product. Analysis of the cyclic phosphodiesterase activities of mutated CthPnkp enzymes illuminates the active site and the structural features that affect substrate affinity and k_cat. We also characterize a previously unrecognized phosphodiesterase activity of λ-Pase, which catalyzes hydrolysis of bis-p-nitrophenyl phosphate. λ-Pase also has cyclic phosphodiesterase activity with nucleoside 2′,3′ cyclic phosphates, which it hydrolyzes to yield a mixture of 2′-NMP and 3′-NMP products. We discuss our results in light of available structural and functional data for other phosphodiesterase members of the binuclear metallophosphoesterase family and draw inferences about how differences in active site composition influence catalytic repertoire.     

A novel 3-oxoacyl-ACP reductase (FabG3) is involved in the xanthomonadin biosynthesis of Xanthomonas campestris pv. campestris

Xanthomonas campestris pv. campestris (Xcc), the causal agent of black rot in crucifers, produces a membrane-bound yellow pigment called xanthomonadin to protect against photobiological and peroxidative damage, and uses a quorum-sensing mechanism mediated by the diffusible signal factor (DSF) family signals to regulate virulence factors production. The Xcc gene XCC4003, annotated as Xcc fabG3, is located in the pig cluster, which may be responsible for xanthomonadin synthesis. We report that fabG3 expression restored the growth of the Escherichia coli fabG temperature-sensitive mutant CL104 under non-permissive conditions. In vitro assays demonstrated that FabG3 catalyses the reduction of 3-oxoacyl-acyl carrier protein (ACP) intermediates in fatty acid synthetic reactions, although FabG3 had a lower activity than FabG1. Moreover, the fabG3 deletion did not affect growth or fatty acid composition. These results indicate that Xcc fabG3 encodes a 3-oxoacyl-ACP reductase, but is not essential for growth or fatty acid synthesis. However, the Xcc fabG3 knock-out mutant abolished xanthomonadin production, which could be only restored by wild-type fabG3, but not by other 3-oxoacyl-ACP reductase-encoding genes, indicating that Xcc FabG3 is specifically involved in xanthomonadin biosynthesis. Additionally, our study also shows that the Xcc fabG3-disrupted mutant affects Xcc virulence in host plants.     

The HD-GYP Domain Protein RpfG of Xanthomonas oryzae pv. oryzicola Regulates Synthesis of Extracellular Polysaccharides that Contribute to Biofilm Formation and Virulence on Rice

Bacterial leaf streak caused by Xanthomonas oryzae pv. oryzicola (Xoc) is one of the most important diseases in rice. However, little is known about the pathogenicity mechanisms of Xoc. Here we have investigated the function of three HD-GYP domain regulatory proteins in biofilm formation, the synthesis of virulence factors and virulence of Xoc. Deletion of rpfG resulted in altered production of extracellular polysaccharides (EPS), abolished virulence on rice and enhanced biofilm formation, but had little effect on the secretion of proteases and motility. In contrast, mutational analysis showed that the other two HD-GYP domain proteins had no effect on virulence factor synthesis and tested phenotypes. Mutation of rpfG led to up-regulation of the type III secretion system and altered expression of three putative glycosyltransferase genes gumD, pgaC and xagB, which are part of operons directing the synthesis of different extracellular polysaccharides. The pgaABCD and xagABCD operons were greatly up-regulated in the Xoc ΔrpfG mutant, whereas the expression of the gum genes was unaltered or slightly enhanced. The elevated biofilm formation of the Xoc ΔrpfG mutant was dramatically reduced upon deletion of gumD, xagA and xagB, but not when pgaA and pgaC were deleted. Interestingly, only the ΔgumD mutant, among these single gene mutants, exhibits multiple phenotype alterations including reduced biofilm and EPS production and attenuated virulence on rice. These data indicate that RpfG is a global regulator that controls biofilm formation, EPS production and bacterial virulence in Xoc and that both gumD- and xagB-dependent EPS contribute to biofilm formation under different conditions.     

Structure–activity relationships in human RNA 3′-phosphate cyclase

RNA 3′-phosphate cyclase (Rtc) enzymes are a widely distributed family that catalyze the synthesis of RNA 2′,3′ cyclic phosphate ends via an ATP-dependent pathway comprising three nucleotidyl transfer steps: reaction of Rtc with ATP to form a covalent Rtc-(histidinyl-N)-AMP intermediate and release PP_i; transfer of AMP from Rtc1 to an RNA 3′-phosphate to form an RNA(3′)pp(5′)A intermediate; and attack by the terminal nucleoside O2′ on the 3′-phosphate to form an RNA 2′,3′ cyclic phosphate product and release AMP. Here we used the crystal structure of Escherichia coli RtcA to guide a mutational analysis of the human RNA cyclase Rtc1. An alanine scan defined seven conserved residues as essential for the Rtc1 RNA cyclization and autoadenylylation reactions. Structure–activity relationships were clarified by conservative substitutions. Our results are consistent with a mechanism of adenylate transfer in which attack of the Rtc1 His320 nucleophile on the ATP α phosphorus is facilitated by proper orientation of the PP_i leaving group via contacts to Arg21, Arg40, and Arg43. We invoke roles for Tyr294 in binding the adenine base and Glu14 in binding the divalent cation cofactor. We find that Rtc1 forms a stable binary complex with a 3′-phosphate terminated RNA, but not with an otherwise identical 3′-OH terminated RNA. Mutation of His320 had little impact on RNA 3′-phosphate binding, signifying that covalent adenylylation of Rtc1 is not a prerequisite for end recognition.