Unconventional membrane lipid biosynthesis in Xanthomonas campestris

All bacteria are surrounded by at least one bilayer membrane mainly composed of phospholipids (PLs). Biosynthesis of the most abundant PLs phosphatidylethanolamine (PE), phosphatidylglycerol (PG) and cardiolipin (CL) is well understood in model bacteria such as Escherichia coli. It recently emerged, however, that the diversity of bacterial membrane lipids is huge and that not yet explored biosynthesis pathways exist, even for the common PLs. A good example is the plant pathogen Xanthomonas campestris pv. campestris. It contains PE, PG and CL as major lipids and small amounts of the N‐methylated PE derivatives monomethyl PE and phosphatidylcholine (PC = trimethylated PE). Xanthomonas campestris uses a repertoire of canonical and non‐canonical enzymes for the synthesis of its membrane lipids. In this minireview, we briefly recapitulate standard pathways and integrate three recently discovered pathways into the overall picture of bacterial membrane biosynthesis.     

XC1028 from Xanthomonas campestris adopts a PilZ domain‐like structure without a c‐di‐GMP switch

The crystal structure of XC1028 from Xanthomonas campestris has been determined to a resolution of 2.15 Å using the multiple anomalous dispersion approach. It bears significant sequence identity and similarity values of 64.10% and 70.09%, respectively, with PA2960, a protein indispensable for type IV pilus‐mediated twitching motility, after which the PilZ motif was first named. However, both XC1028 and PA2960 lack detectable c‐di‐GMP binding capability. Although XC1028 adopts a structure comprising a five‐stranded β‐barrel core similar to other canonical PilZ domains with robust c‐di‐GMP binding ability, considerable differences are observed in the N‐terminal motif; XC1028 assumes a compact five‐stranded β‐barrel without an extra long N‐terminal motif, whereas other canonical PilZ domains contain a long N‐terminal sequence embedded with an essential “c‐di‐GMP switch” motif. In addition, a β‐strand (β1) in the N‐terminal motif, running in exactly opposite polarity to that of XC1028, is found inserted into the parallel β3/β1′ strands, forming a completely antiparallel β4↓β3↑β1↓β1′↑ sheet in the canonical PilZ domains. Such dramatic structural differences at the N‐terminus may account for the diminished c‐di‐GMP binding capability of XC1028, and suggest that interactions with additional proteins are necessary to bind c‐di‐GMP for type IV fimbriae assembly. Proteins 2009. © 2008 Wiley‐Liss, Inc.     

Xanthomonas campestris pv. campestris possesses a single gluconeogenic pathway that is required for virulence

Disruption of ppsA, a key gene in gluconeogenesis, of Xanthomonas campestris pv. campestris resulted in the failure of the pathogen to grow in medium with pyruvate or C4-dicarboxylates as the sole carbon source and a significant reduction in virulence, indicating that X. campestris pv. campestris possesses only the malic enzyme-PpsA route in gluconeogenesis, which is required for virulence.     

Benefits and potential trade‐offs associated with yeast‐like symbionts during virulence adaptation in a phloem‐feeding planthopper

Insect herbivores form symbioses with a diversity of prokaryotic and eukaryotic microorganisms. A role for endosymbionts during host feeding on nutrient‐poor diets – including phloem – is now supported by a large body of evidence. Furthermore, symbiont‐herbivore associations have been implicated in feeding preferences by host races (mainly aphids) on multiple plant species. However, the role of symbionts in mediating herbivore preferences between varieties of the same plant species has received little research attention despite the implications for virulence adaptation to resistant crops. This study investigates the role of yeast‐like symbionts (YLS) in virulence adaptation and host plant switching among populations of the brown planthopper, Nilaparvata lugens (Stål) (Hemiptera: Delphacidae), that were selected on various rice [Oryza sativa L. (Poaceae)] lines differing in their resistance against herbivores. Planthopper fitness (nymph weight) declined where YLS densities were depleted through heat treatment. However, compared to normal symbiotic planthoppers, the depletion of symbionts did not generally change the relative fitness of planthoppers (each ‘adapted’ to a single natal host) when switched to feed on a range of rice lines (exposed hosts). In some cases, this occurred despite differences in YLS density responses to the various hosts. Furthermore, we detected no fitness costs associated with YLS in adapted populations. Therefore, the results of this study suggest that, whereas YLS are essential for planthopper nutrition, changes in YLS density play little role during virulence adaptation and host plant switching by the brown planthopper.     

Lipopolysaccharide biosynthesis in Xanthomonas campestris pv. campestris: a cluster of 15 genes is involved in the biosynthesis of the LPS O-antigen and the LPS core

As a result of mutational and DNA sequence analysis, a wxc gene cluster involved in the synthesis of the surface lipopolysaccharide (LPS) was identified in Xanthomonas campestris pv. campestris. This gene cluster comprises 15 genes. It was located on a cloned 35-kb fragment of chromosomal DNA, close, but not directly adjacent, to previously characterized genes for LPS biosynthesis. The G + C content of all but one of the wxc genes was atypically low for X. campestris pv. campestris, while the G + C distribution was uniform throughout the cluster. An SDS-PAGE analysis of mutant strains defective in various wxc genes confirmed that genes from this cluster were involved in LPS biosynthesis. The mutant phenotypes allowed the differentiation of three regions within the wxc cluster. Genes from wxc region 1 are necessary for the biosynthesis of the water-soluble LPS O-antigen. Analysis of DNA and deduced amino acid sequences led to the identification of two glycosyltransferases, two components of an ABC transport system, and a possible kinase among the seven putative proteins encoded by genes constituting wxc region 1. The two genes in wxc region 2 were similar to gmd and rmd, which direct the synthesis of the sugar nucleotide GDP-D-rhamnose. Mutations affecting wxc region 2 demonstrated its involvement in the formation of the LPS core. Genes from wxc region 3 showed similarities to genes that code for enzymes that modify nucleotide sugars, and to components of sugar translocation systems that have so far been rarely described in bacteria.     

Direct biosynthesis of ascorbic acid from glucose by Xanthomonas campestris through induced free-radicals

Ascorbic acid (20.4 g l^-1 in 50 h) was synthesized directly from glucose by Xanthomonas campestris as an adaptive response to induced free-radicals through HOCl treatment. Identity of ascorbic acid was confirmed through IR and NMR spectroscopy.     

Clues from Xanthomonas campestris about the evolution of aromatic biosynthesis and its regulation

The recent placement of major Gram-negative prokaryotes (Superfamily B) on a phylogenetic tree (including, e.g., lineages leading to Escherichia coli, Pseudomonas aeruginosa, and Acinetobacter calcoaceticus) has allowed initial insights into the evolution of the biochemical pathway for aromatic amino acid biosynthesis and its regulation to be obtained. Within this prokaryote grouping, Xanthomonas campestris ATCC 12612 (a representative of the Group V pseudomonads) has played a key role in facilitating deductions about the major evolutionary events that shaped the character of aromatic biosynthesis within this grouping. X. campestris is like P. aeruginosa (and unlike E. coli) in its possession of dual flow routes to both L-phenylalanine and L-tyrosine from prephenate. Like all other members of Superfamily B, X. campestris possesses a bifunctional P-protein bearing the activities of both chorismate mutase and prephenate dehydratase. We have found an unregulated arogenate dehydratase similar to that of P. aeruginosa in X. campestris. We separated the two tyrosine-branch dehydrogenase activities (prephenate dehydrogenase and arogenate dehydrogenase); this marks the first time this has been accomplished in an organism in which these two activities coexist. Superfamily B organisms possess 3-deoxy-D-arabino-heptulosonate 7-P (DAHP) synthase as three isozymes (e.g., in E. coli), as two isozymes (e.g., in P. aeruginosa), or as one enzyme (in X. campestris). The two-isozyme system has been deduced to correspond to the ancestral state of Superfamily B. Thus, E. coli has gained an isozyme, whereas X. campestris has lost one. We conclude that the single, chorismate-sensitive DAHP synthase enzyme of X. campestris is evolutionarily related to the tryptophan-sensitive DAHP synthase present throughout the rest of Superfamily B. In X. campestris, arogenate dehydrogenase, prephenate dehydrogenase, the P-protein, chorismate mutase-F, anthranilate synthase, and DAHP synthase are all allosteric proteins; we compared their regulatory properties with those of enzymes of other Superfamily B members with respect to the evolution of regulatory properties. The network of sequentially operating circuits of allosteric control that exists for feedback regulation of overall carbon flow through the aromatic pathway in X. campestris is thus far unique in nature.     

Modelling Xanthomonas campestris batch fermentations in a bubble column

Rate and yield expressions relating to biomass and xanthan formation and to nitrogen, glucose, and oxygen consumption were established for Xanthomonas campestris batch fermentations in a bubble column. Microbial growth was described by the logistic rate equation, characterized by a maximum specific growth rate mu(M) = 0.5 h(-1) and a maximum attainable cell concentration provided by nitrogenous compounds. With regard to carbon metabolism, the decrease with time in experimental yields and in the experimental specific rates of xanthan production and glucose assimilation demonstrated the inadequacy of the Luedeking-Piret model. These decreases were connected to the simultaneous drop in dissolved-oxygen tension observed during xanthan synthesis. The knowledge of metabolic pathways and energetic balance were used to establish the relationships between substrate utilization, ATP generation, and xanthan production. The model was structured by assuming the oxygen limitation of both the respiration rate and the efficiency of the oxidative phosphorylation mechanism (P/O ratio). Consequently, the specific rates and yield expressions became dependent on the dissolved-oxygen tension, i.e., of the volumetric oxygen transfer in the fermentor.     

Glucagon‐like peptide‐1 ameliorates cardiac lipotoxicity in diabetic cardiomyopathy via the PPARα pathway

Lipotoxicity cardiomyopathy is the result of excessive accumulation and oxidation of toxic lipids in the heart. It is a major threat to patients with diabetes. Glucagon‐like peptide‐1 (GLP‐1) has aroused considerable interest as a novel therapeutic target for diabetes mellitus because it stimulates insulin secretion. Here, we investigated the effects and mechanisms of the GLP‐1 analog exendin‐4 and the dipeptidyl peptidase‐4 inhibitor saxagliptin on cardiac lipid metabolism in diabetic mice (DM). The increased myocardial lipid accumulation, oxidative stress, apoptosis, and cardiac remodeling and dysfunction induced in DM by low streptozotocin doses and high‐fat diets were significantly reversed by exendin‐4 and saxagliptin treatments for 8 weeks. We found that exendin‐4 inhibited abnormal activation of the (PPARα)‐CD36 pathway by stimulating protein kinase A (PKA) but suppressing the Rho‐associated protein kinase (ROCK) pathway in DM hearts, palmitic acid (PA)‐treated rat h9c2 cardiomyocytes (CMs), and isolated adult mouse CMs. Cardioprotection in DM mediated by exendin‐4 was abolished by combination therapy with the PPARα agonist wy‐14643 but mimicked by PPARα gene deficiency. Therefore, the PPARα pathway accounted for the effects of exendin‐4. This conclusion was confirmed in cardiac‐restricted overexpression of PPARα mediated by adeno‐associated virus serotype‐9 containing a cardiac troponin T promoter. Our results provide the first direct evidence that GLP‐1 protects cardiac function by inhibiting the ROCK/PPARα pathway, thereby ameliorating lipotoxicity in diabetic cardiomyopathy.     

十字花科黑腐病菌中一个与抗逆有关的小RNA的鉴定

十字花科黑腐病菌(Xanthomonas campestris pathovar campestris,Xcc),是引起十字花科植物黑腐病的病原菌。Xcc要经历寄生、腐生等多种环境变化,为适应这些环境变化,需要调控相应基因的表达。除蛋白外,小RNA在基因表达调控中也起到关键作用。本实验室前期实验从Xcc 8004中鉴定出数百个小RNA,但是绝大多数小RNA的功能仍然未知。本研究通过构建一个小RNA(sRNA3843)的过量表达株来研究其生物学功能。确定该小RNA过量表达后,对其过量表达株OE3843进行了一系列的表型检测。结果发现,s RNA3843过量表达株OE3843对金属离子Cu^2+、Zn^2+、Cd^2+及蛋白变性剂SDS的耐受能力明显下降,表明s RNA3843与Xcc的抗逆有关。本研究的实验结果为深入研究小RNA在Xcc抗逆中所起的作用及其作用机理打下基础。     

Genetic and physical analyses of a cluster of genes essential for xanthan gum biosynthesis in Xanthomonas campestris.

Xanthomonas campestris produces copious amounts of a complex exopolysaccharide, xanthan gum. Nonmucoid mutants, defective in synthesis of xanthan polysaccharide, were isolated after nitrosoguanidine mutagenesis. To isolate genes essential for xanthan polysaccharide synthesis (xps), a genomic library of X. campestris DNA, partially digested with SalI and ligated into the broad-host-range cloning vector pRK293, was constructed in Escherichia coli. The pooled clone bank was conjugated en masse from E. coli into three nonmucoid mutants by using pRK2013, which provides plasmid transfer functions. Kanamycin-resistant exconjugants were then screened for the ability to form mucoid colonies. Analysis of plasmids from several mucoid exconjugants indicated that overlapping segments of DNA had been cloned. These plasmids were tested for complementation of eight additional nonmucoid mutants. A 22-kilobase (kb) region of DNA was defined physically by restriction enzyme analysis and genetically by ability to restore mucoid phenotype to 10 of the 11 nonmucoid mutants tested. This region was further defined by subcloning and by transposon mutagenesis with mini-Mu(Tetr), with subsequent analysis of genetic complementation of nonmucoid mutants. A region of 13.5 kb of DNA was determined to contain at least five complementation groups. The effect of plasmids containing cloned xps genes on xanthan gum synthesis was evaluated. One plasmid, pCHC3, containing a 12.4-kb insert and at least four linked xanthan biosynthetic genes, increased the production of xanthan gum by 10% and increased the extent of pyruvylation of the xanthan side chains by about 45%. This indicates that a gene affecting pyruvylation of xanthan gum is linked to this cluster of xps genes.     

Phosphatidylcholine biosynthesis and function in bacteria

Phosphatidylcholine (PC) is the major membrane-forming phospholipid in eukaryotes and is estimated to be present in about 15% of the domain Bacteria. Usually, PC can be synthesized in bacteria by either of two pathways, the phospholipid N-methylation (Pmt) pathway or the phosphatidylcholine synthase (Pcs) pathway. The three subsequent enzymatic methylations of phosphatidylethanolamine are performed by a single phospholipid N-methyltransferase in some bacteria whereas other bacteria possess multiple phospholipid N-methyltransferases each one performing one or several distinct methylation steps. Phosphatidylcholine synthase condenses choline directly with CDP-diacylglycerol to form CMP and PC. Like in eukaryotes, bacterial PC also functions as a biosynthetic intermediate during the formation of other biomolecules such as choline, diacylglycerol, or diacylglycerol-based phosphorus-free membrane lipids. Bacterial PC may serve as a specific recognition molecule but it affects the physicochemical properties of bacterial membranes as well. This article is part of a Special Issue entitled Phospholipids and Phospholipid Metabolism.     

Expression of the gum operon directing xanthan biosynthesis in Xanthomonas campestris and its regulation in planta

The gum gene cluster of Xanthomonas campestris pv. campestris comprises 12 genes whose products are involved in the biosynthesis of the extracellular polysaccharide xanthan. These genes are expressed primarily as an operon from a promoter upstream of the first gene, gumB. Although the regulation of xanthan synthesis in vitro has been well studied, nothing is known of its regulation in planta. A reporter plasmid was constructed in which the promoter region of the gum operon was fused to gusA. In liquid cultures, the expression of the gumgusA reporter was correlated closely with the production of xanthan, although a low basal level of beta-glucuronidase activity was seen in the absence of added carbon sources when xanthan production was very low. The expression of the gumgusA fusion also was subject to positive regulation by rpfF, which is responsible for the synthesis of the diffusible signal factor (DSF). The expression of the gumgusA fusion in bacteria recovered from inoculated turnip leaves was maximal at the later phases of growth and was subject to regulation by rpfF. These results provide indirect support for the operation of the DSF regulatory system in bacteria in planta.     

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.