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.     

微生物CoQ合成途径及CoQ10生产菌株的分子生物学改造

CoQ10具有呼吸链电子传递者、抗氧化性、调控基因表达等多种生理生化功能,目前不仅用作药物也用作食品添加剂。微生物发酵法是目前生产CoQ10最有效的方法。本文就有关微生物CoQ合成途径及基于CoQ合成途径的CoQ10生产菌株分子生物学改造的策略与研究进展进行了综述和展望。     

Chorismate‐dependent transcriptional regulation of quinate/shikimate utilization genes by LysR‐type transcriptional regulator QsuR in Corynebacterium glutamicum: carbon flow control at metabolic branch point

The qsu operon of Corynebacterium glutamicum comprises four genes (qsuABCD) that underpin the microorganism's quinate/shikimate utilization pathways. The genes encode enzymes that catalyse reactions at the metabolic branch point between the biosynthesis route for synthesis of aromatic compounds and the catabolic route for degradation of quinate and shikimate for energy production. A qsuR gene located immediately upstream of qsuA encodes a protein (QsuR) which activates the operon in the presence of quinate or shikimate. Three observations support chorismate, an intermediate of the biosynthesis route, as a direct effector of QsuR: First, induction of qsuA mRNA in the presence of either quinate or shikimate disappears upon deletion of the gene encoding chorismate synthase. Second, chorismate accumulates when the operon is induced. Third, a DNase I‐protected segment by QsuR is shortened in the presence of chorismate. The QsuR tetramer senses the accumulation of chorismate and activates qsu genes that promote the quinate/shikimate catabolic instead of the aromatic compounds biosynthetic route. Such chorismate‐dependent control of carbon flow has not been previously described.     

Structure and function of a complex between chorismate mutase and DAHP synthase: efficiency boost for the junior partner

Chorismate mutase catalyzes a key step in the shikimate biosynthetic pathway towards phenylalanine and tyrosine. Curiously, the intracellular chorismate mutase of Mycobacterium tuberculosis (MtCM; Rv0948c) has poor activity and lacks prominent active‐site residues. However, its catalytic efficiency increases >100‐fold on addition of DAHP synthase (MtDS; Rv2178c), another shikimate‐pathway enzyme. The 2.35 Å crystal structure of the MtCM–MtDS complex bound to a transition‐state analogue shows a central core formed by four MtDS subunits sandwiched between two MtCM dimers. Structural comparisons imply catalytic activation to be a consequence of the repositioning of MtCM active‐site residues on binding to MtDS. The mutagenesis of the C‐terminal extrusion of MtCM establishes conserved residues as part of the activation machinery. The chorismate‐mutase activity of the complex, but not of MtCM alone, is inhibited synergistically by phenylalanine and tyrosine. The complex formation thus endows the shikimate pathway of M. tuberculosis with an important regulatory feature. Experimental evidence suggests that such non‐covalent enzyme complexes comprising an AroQ_δ subclass chorismate mutase like MtCM are abundant in the bacterial order Actinomycetales.     

A dual role for proline iminopeptidase in the regulation of bacterial motility and host immunity

During plant–pathogen interactions, pathogenic bacteria have evolved multiple strategies to cope with the sophisticated defence systems of host plants. Proline iminopeptidase (PIP) is essential to Xanthomonas campestris pv. campestris (Xcc) virulence, and is conserved in many plant‐associated bacteria, but its pathogenic mechanism remains unclear. In this study, we found that disruption of pip in Xcc enhanced its flagella‐mediated bacterial motility by decreasing intracellular bis‐(3′,5′)‐cyclic dimeric guanosine monophosphate (c‐di‐GMP) levels, whereas overexpression of pip in Xcc restricted its bacterial motility by elevating c‐di‐GMP levels. We also found that PIP is a type III secretion system‐dependent effector capable of eliciting a hypersensitive response in non‐host, but not host plants. When we transformed pip into the host plant Arabidopsis, higher bacterial titres were observed in pip‐overexpressing plants relative to wild‐type plants after Xcc inoculation. The repressive function of PIP on plant immunity was dependent on PIP's enzymatic activity and acted through interference with the salicylic acid (SA) biosynthetic and regulatory genes. Thus, PIP simultaneously regulates two distinct regulatory networks during plant–microbe interactions, i.e. it affects intracellular c‐di‐GMP levels to coordinate bacterial behaviour, such as motility, and functions as a type III effector translocated into plant cells to suppress plant immunity. Both processes provide bacteria with the regulatory potential to rapidly adapt to complex environments, to utilize limited resources for growth and survival in a cost‐efficient manner and to improve the chances of bacterial survival by helping pathogens to inhabit the internal tissues of host plants.     

Effect of oxidative stress on the biosynthesis of 2-C-methyl-d-erythritol-2,4-cyclopyrophosphate and isoprenoids by several bacterial strains

In this study, the gram-negative bacteria Xanthomonas campestris, Xanthomonas maltophilia, and Pseudomonas putida, facultative parasites of plants and animals, were shown to accumulate 2-C-methyl-d-erythritol-2,4-cyclopyrophosphate (MEC) in response to benzyl-viologen-induced oxidative stress. Corynebacterium ammoniagenes mutants capable of accumulating MEC in the absence of an exogenous oxidative stress inducer were obtained. Isoprenoid synthesis and MEC synthesis in these and other bacteria were shown to be alternative processes, while biosynthesis of brominated polyene xanthomonadin (an antioxidant pigment of X. campestris) increased concomitantly with the accumulation of MEC. Received: 30 March 1998 / Accepted: 16 October 1998     

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.     

Archaeal shikimate kinase, a new member of the GHMP-kinase family

Shikimate kinase (EC 2.7.1.71) is a committed enzyme in the seven-step biosynthesis of chorismate, a major precursor of aromatic amino acids and many other aromatic compounds. Genes for all enzymes of the chorismate pathway except shikimate kinase are found in archaeal genomes by sequence homology to their bacterial counterparts. In this study, a conserved archaeal gene (gi1500322 in Methanococcus jannaschii) was identified as the best candidate for the missing shikimate kinase gene by the analysis of chromosomal clustering of chorismate biosynthetic genes. The encoded hypothetical protein, with no sequence similarity to bacterial and eukaryotic shikimate kinases, is distantly related to homoserine kinases (EC 2.7.1.39) of the GHMP-kinase superfamily. The latter functionality in M. jannaschii is assigned to another gene (gi591748), in agreement with sequence similarity and chromosomal clustering analysis. Both archaeal proteins, overexpressed in Escherichia coli and purified to homogeneity, displayed activity of the predicted type, with steady-state kinetic parameters similar to those of the corresponding bacterial kinases: K(m,shikimate) = 414 +/- 33 microM, K(m,ATP) = 48 +/- 4 microM, and k(cat) = 57 +/- 2 s(-1) for the predicted shikimate kinase and K(m,homoserine) = 188 +/- 37 microM, K(m,ATP) = 101 +/- 7 microM, and k(cat) = 28 +/- 1 s(-1) for the homoserine kinase. No overlapping activity could be detected between shikimate kinase and homoserine kinase, both revealing a >1,000-fold preference for their own specific substrates. The case of archaeal shikimate kinase illustrates the efficacy of techniques based on reconstruction of metabolism from genomic data and analysis of gene clustering on chromosomes in finding missing genes.     

Reinvestigation of coenzyme Q10 isolation from Sporidiobolus johnsonii

There is considerable current interest in coenzyme Q10 (CoQ10) from a medical perspective. CoQ10 has been shown to alleviate the side effects of statin drugs, for instance, and so there is a push to find naturally high producers of the compound. Sporidiobolus johnsonii (S. johnsonii) has been reported to produce CoQ10 in studies that used only standards on thin‐layer chromatography (TLC) and also suggested the production of coenzyme Q9 (CoQ9). This work set out to verify CoQ9/CoQ10 production in S. johnsonii and quantify as appropriate. We show that S. johnsonii produces CoQ10 but found no evidence for CoQ9 biosynthesis. The specific production of CoQ10 was noted at 10 mg/g dry cell weight (DCW) in media supplemented with 4‐hydroxybenzoic acid (HBA). This makes S. johnsonii a naturally high CoQ10 producer. New methods for extraction and purification of CoQ10 are also discussed, and identification of a closely eluting side product under normal phase isolation is reported.     

Comparative and functional genomics reveals genetic diversity and determinants of host specificity among reference strains and a large collection of Chinese isolates of the phytopathogen Xanthomonas campestris pv. campestris

Background

Xanthomonas campestris pathovar campestris (Xcc) is the causal agent of black rot disease of crucifers worldwide. The molecular genetic diversity and host specificity of Xcc are poorly understood.

Results

We constructed a microarray based on the complete genome sequence of Xcc strain 8004 and investigated the genetic diversity and host specificity of Xcc by array-based comparative genome hybridization analyses of 18 virulent strains. The results demonstrate that a genetic core comprising 3,405 of the 4,186 coding sequences (CDSs) spotted on the array are conserved and a flexible gene pool with 730 CDSs is absent/highly divergent (AHD). The results also revealed that 258 of the 304 proved/presumed pathogenicity genes are conserved and 46 are AHD. The conserved pathogenicity genes include mainly the genes involved in type I, II and III secretion systems, the quorum sensing system, extracellular enzymes and polysaccharide production, as well as many other proved pathogenicity genes, while the AHD CDSs contain the genes encoding type IV secretion system (T4SS) and type III-effectors. A Xcc T4SS-deletion mutant displayed the same virulence as wild type. Furthermore, three avirulence genes (avrXccC, avrXccE1 and avrBs1) were identified. avrXccC and avrXccE1 conferred avirulence on the hosts mustard cultivar Guangtou and Chinese cabbage cultivar Zhongbai-83, respectively, and avrBs1 conferred hypersensitive response on the nonhost pepper ECW10R.

Conclusion

About 80% of the Xcc CDSs, including 258 proved/presumed pathogenicity genes, is conserved in different strains. Xcc T4SS is not involved in pathogenicity. An efficient strategy to identify avr genes determining host specificity from the AHD genes was developed.     

Biochemical insights into basal and induced resistance in cabbage to black rot

Black rot, caused by Xanthomonas campestris pv. campestris (Xcc), is the most devastating disease of brassica, but the mechanisms of basal or induced resistance in cabbage remain largely unknown. Here, we performed three experiments to investigate biochemical features associated with cabbage resistance to black rot. In the first experiment, biochemical changes were assessed in plants that were inoculated with a highly (UFPR 5) or a moderately (Xcc 10) aggressive Xcc isolate. In the second experiment, we examined the biochemical responses in two cultivars (Chato de Quintal [CQ] and Louco de Verão [LV], susceptible and moderately resistant to Xcc, respectively). Finally, we examined whether acibenzolar‐S‐methyl (ASM) could induce cabbage resistance to Xcc. Plants inoculated with the Xcc 10 isolate displayed higher activities of superoxide dismutase (SOD), peroxidase (POX) and ascorbate peroxidase (APX), whereas activities of chitinase (CHI), β‐1,3‐glucanase (GLU) and polyphenol oxidase (PPO) as well as the concentrations of hydrogen peroxide (H₂O₂) and malondialdehyde (MDA) were lower compared to plants inoculated with the UFPR 5 isolate. The resistance of the cultivar LV to Xcc was linked to increases in the activities of CHI, GLU, and PPO and decreases in the activities of SOD, POX and APX as well as in the concentrations of H₂O₂ and MDA relative to the cultivar CQ. In general, ASM‐sprayed plants displayed higher activities for the enzymes studied, which was associated with decreased disease symptoms and oxidative stress. Taken together, our results demonstrated that high activities of both defence and antioxidant enzymes played a major role in both basal and induced resistance of cabbage to black rot.     

The entry reaction of the plant shikimate pathway is subjected to highly complex metabolite-mediated regulation

The plant shikimate pathway directs bulk carbon flow toward biosynthesis of aromatic amino acids (AAAs, i.e. tyrosine, phenylalanine, and tryptophan) and numerous aromatic phytochemicals. The microbial shikimate pathway is feedback inhibited by AAAs at the first enzyme, 3-deoxy-d-arabino-heptulosonate 7-phosphate synthase (DHS). However, AAAs generally do not inhibit DHS activities from plant extracts and how plants regulate the shikimate pathway remains elusive. Here, we characterized recombinant Arabidopsis thaliana DHSs (AthDHSs) and found that tyrosine and tryptophan inhibit AthDHS2, but not AthDHS1 or AthDHS3. Mixing AthDHS2 with AthDHS1 or 3 attenuated its inhibition. The AAA and phenylpropanoid pathway intermediates chorismate and caffeate, respectively, strongly inhibited all AthDHSs, while the arogenate intermediate counteracted the AthDHS1 or 3 inhibition by chorismate. AAAs inhibited DHS activity in young seedlings, where AthDHS2 is highly expressed, but not in mature leaves, where AthDHS1 is predominantly expressed. Arabidopsis dhs1 and dhs3 knockout mutants were hypersensitive to tyrosine and tryptophan, respectively, while dhs2 was resistant to tyrosine-mediated growth inhibition. dhs1 and dhs3 also had reduced anthocyanin accumulation under high light stress. These findings reveal the highly complex regulation of the entry reaction of the plant shikimate pathway and lay the foundation for efforts to control the production of AAAs and diverse aromatic natural products in plants.

Characterization of Arabidopsis 3-deoxy-D-arabino-heptulosonate 7-phosphate synthase enzymes and mutants revealed highly complex metabolite-mediated feedback regulation of the plant shikimate pathway.