The pigment ofXanthomonas populi is a nonbrominated aryl-heptaene belonging to xanthomonadin pigment group 11

Based on mass spectrometry and electronic absorption spectroscopy, the main pigment fromXanthomonas populi (synonym:Aplanobacter populi) was identified as a nonbrominated aryl-heptaene. TheX. populi pigment was indistinguishable—on the basis of cochromatography and electronic absorption spectroscopy—from an authentic sample of a xanthomonadin belonging to Pigment Group 11, previously found as minor components in strains belonging to theXanthomonas campestris taxospecies (specifically in theXanthomonas pruni andXanthomonas vesicatoria nomenspecies). Possession of a xanthomonadin pigment confirms the placement ofX. populi in the genusXanthomonas and this particular pigment serves to distinguishX. populi from the five other taxospecies presently assigned to that genus. TheXanthomonas sp. isolated fromSalix, which purportedly shows affinities toX. populi, forms a monobrominated aryl-polyene pigment and —on that basis—is unlikeX. populi.     

Biological Role of Xanthomonadin Pigments in Xanthomonas campestris pv. Campestris

Previous studies have indicated that the yellow pigments (xanthomonadins) produced by phytopathogenic Xanthomonas bacteria are unimportant during pathogenesis but may be important for protection against photobiological damage. We used a Xanthomonas campestris pv. campestris parent strain, single-site transposon insertion mutant strains, and chromosomally restored mutant strains to define the biological role of xanthomonadins. Although xanthomonadin mutant strains were comparable to the parent strain for survival when exposed to UV light; after their exposure to the photosensitizer toluidine blue and visible light, survival was greatly reduced. Chromosomally restored mutant strains were completely restored for survival in these conditions. Likewise, epiphytic survival of a xanthomonadin mutant strain was greatly reduced in conditions of high light intensity, whereas a chromosomally restored mutant strain was comparable to the parent strain for epiphytic survival. These results are discussed with respect to previous results, and a model for epiphytic survival of X. campestris pv. campestris is presented.     

Biological role of xanthomonadin pigments in Xanthomonas campestris pv. campestris

Previous studies have indicated that the yellow pigments (xanthomonadins) produced by phytopathogenic Xanthomonas bacteria are unimportant during pathogenesis but may be important for protection against photobiological damage. We used a Xanthomonas campestris pv. campestris parent strain, single-site transposon insertion mutant strains, and chromosomally restored mutant strains to define the biological role of xanthomonadins. Although xanthomonadin mutant strains were comparable to the parent strain for survival when exposed to UV light; after their exposure to the photosensitizer toluidine blue and visible light, survival was greatly reduced. Chromosomally restored mutant strains were completely restored for survival in these conditions. Likewise, epiphytic survival of a xanthomonadin mutant strain was greatly reduced in conditions of high light intensity, whereas a chromosomally restored mutant strain was comparable to the parent strain for epiphytic survival. These results are discussed with respect to previous results, and a model for epiphytic survival of X. campestris pv. campestris is presented.     

The brominated aryl-polyene (xanthomonadin) pigments ofXanthomonas juglandis protect against photobiological damage

The brominated aryl-polyene, (xanthomonadin) pigments-characteristic of the nonphotosynthetic and phytopathogenic bacteria belonging to the genusXanthomonas-were examined for their role in protecting these bacteria against photobiological destruction of some as-yet unknown vital cellular component(s). Survival of a pigmented wild-type strain (ICPB XJ103) ofXanthomonas juglandis and a colorless mutant strain (ICPB XJ103-31) derived from it were compared after exposure to visible light in the presence of an exogenous photosensitizer, toluidine blue. Under these conditions, the survival of the pigmented wild-type strain was approximately two order of magnitude greater than the survival of the colorless mutant strain. Both strains were fully protected in the dark in the presence of toluidine blue. Although the oxygen-free controls were inconclusive-and the mechanisms(s) and target(s) remain unknown-the results do indicate that xanthomonadin pigments protect against photokilling.     

<Emphasis Type="Italic">Xanthomonas gardneri</Emphasis> exoenzymatic activity towards plant tissue

Bacterial spot caused by Xanthomonas spp. is an important tomato and pepper disease worldwide. Recent outbreaks of bacterial spot disease in Central Brazil and Canada have been attributed to Xanthomonas gardneri, which is also recognized as group D of Xanthomonas campestris pv. vesicatoria. Carotenoid-like pigments called xanthomonadins, which are diagnostic for yellow Xanthomonas spp., were extracted from X. gardneri. It was shown that the model plant Arabidopsis thaliana, member of the Brassicaceae family, can develop disease symptoms in response to different isolates of X. gardneri. Secretion of enzymes has been shown to play an important role in pathogenicity for different pathogens, and to begin to understand the interaction of X. gardneri and A. thaliana, a biochemical analysis of secreted proteins in the presence of A. thaliana leaves was performed. Different enzymatic activities such as for cellulase, α-arabinofuranosidase, pectinase, invertase and xylanase were assayed. In the presence of leaves, cellulase activity was highest after 60 and 72 h of growth and α-arabinofuranosidase activity was detected between 12 and 72 h of growth. Pectinase, invertase and xylanase activities were not detected. Cellulase and α-arabinofuranosidase activities may be important for X. gardneri acquisition of plant nutrients through degradation of cellulose fibers and hemicellulose of the cell wall, respectively, to the invasion of the host tissue and/or may generate signal molecules that are recognized by the plant. This is the first study to address how X. gardneri responds to host plant tissue.     

Cibacron blue 3G-A inhibits cell separation of gram-positive bacteria

A triazine dye, Cibacron blue 3G-A (CB), is an inhibitor of cell separation of staphylococcal spp. therefore, we examined the effect of CB on growth of grampositive bacteria other than Staphylococcus. CB added to the medium of growing cultures of strains of genus Micrococcus, Streptococcus, Lactobacillus and Bacillus caused inhibition of cell separation. Moreover, in case of Bacillus and Lactobacillus, individual cells were elongated as filament. Strains of the genus Micrococcus were as sensitive to CB as genus Staphylococcus in which the minimum concentrations of CB needed for inhibition of cell separation ranged from 15 to 100 M. Other strains belong to genus Streptococcus, Bacillus and Lactobacillus were less sensitive; the minimum concentrations were 100 M–25 mM.     

The diffusible factor synthase XanB2 is a bifunctional chorismatase that links the shikimate pathway to ubiquinone and xanthomonadins biosynthetic pathways

The diffusible factor synthase XanB2, originally identified in Xanthomonas campestris pv. campestris (Xcc), is highly conserved across a wide range of bacterial species, but its substrate and catalytic mechanism have not yet been investigated. Here, we show that XanB2 is a unique bifunctional chorismatase that hydrolyses chorismate, the end‐product of the shikimate pathway, to produce 3‐hydroxybenzoic acid (3‐HBA) and 4‐HBA. 3‐HBA and 4‐HBA are respectively associated with the yellow pigment xanthomonadin biosynthesis and antioxidant activity in Xcc. We further demonstrate that XanB2 is a structurally novel enzyme with three putative domains. It catalyses 3‐HBA and 4‐HBA biosynthesis via a unique mechanism with the C‐terminal YjgF‐like domain conferring activity for 3‐HBA biosynthesis and the N‐terminal FGFG motif‐containing domain responsible for 4‐HBA biosynthesis. Furthermore, we show that Xcc produces coenzyme Q8 (CoQ8) via a new biosynthetic pathway independent of the key chorismate‐pyruvate lyase UbiC. XanB2 is the alternative source of 4‐HBA for CoQ8 biosynthesis. The similar CoQ8 biosynthetic pathway, xanthomonadin biosynthetic gene cluster and XanB2 homologues are well conserved in the bacterial species within Xanthomonas, Xylella, Xylophilus, Pseudoxanthomonas, Rhodanobacter, Frateuria, Herminiimonas and Variovorax, suggesting that XanB2 may be a conserved metabolic link between the shikimate pathway, ubiquinone and xanthomonadin biosynthetic pathways in diverse bacteria.     

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     

Characterization and identification of bacteria isolated from micropropagated mint plants

Summary Bacterial isolates from contaminated mint shoot cultures were characterized and identified as a preliminary step in determining an elimination treatment. The 22 bacteria were characterized using biochemical and morphological tests and subjected to sensitivity tests with four antibiotics. The isolates were compared with known organisms and assigned to genera according to similarities in characteristics. Seven isolates were analyzed by fatty acid analysis carried out by a commercial laboratory. Six were classified asAgrobacterium radiobacter; eight asXanthomonas; one each asPseudomonas fluorescens, Micrococcus spp.,Corynebacterium spp., andCurtobacterium spp.; four could not be assigned to genera. Inhibition of growth of the bacteria by most antibiotics was best at pH 7.5. Minimal inhibitory concentration and minimal bactericidal concentrations of gentamicin, rifampicin, streptomycin sulfate, and Timentin varied with genotype.     

Pair-dependent co-aggregation behavior of non-flocculating sludge bacteria

Two strains of non-flocculating sewage sludge bacteria (Xanthomonassp. S53 and Microbacterium esteraromaticum S51) showed 91% and 77% co-aggregation, respectively, with Acinetobacter johnsonii S35 using a spectrophometric assay. The co-aggregates in case of Xanthomonas sp. S53 and A. johnsonii S35 were above 100 m and stable against EDTA (2 mM) and a commercial protease (0.2 mg ml^–1). Protease/periodate pretreatment of the partners did not affect this co-aggregation. On the other hand, co-aggregates of M. esteraromaticum S51 and A. johnsonii S35 (50–70 m) were deflocculated by EDTA or protease. Protease pretreatment of M. esteraromaticum S51 and periodate pretreatment of A. johnsonii S35 prevented their co-aggregation with respective untreated partners. The potential co-aggregation mechanisms of A. johnsonii S35 varied depending upon the other partner involved.     

一株拮抗黄单胞菌的贝莱斯芽孢杆菌的分离和鉴定

【目的】为了筛选防治水稻条斑病(bacterial leaf streak,BLS)的生防细菌。【方法】以水稻条斑病菌(Xanthomonas oryzae pv. oryzicola,Xoc)的模式菌株RS105为靶标菌,采用平板稀释和抑菌圈法,从空心菜根际土壤中筛选到一株对RS105具有拮抗作用的细菌菌株504。通过形态学、生理生化特征以及16SrDNA和gyrA序列分析对菌株504进行了鉴定。利用牛津杯法测定504对植物病原黄单胞菌的拮抗活性及其无菌发酵液拮抗活性的稳定性。通过PCR扩增预测504编码合成脂肽类和聚酮类化合物的合成相关基因。采用苗期水稻注射接菌法来评价水稻组织中504对Xoc的拮抗活性。【结果】菌株鉴定结果表明504为贝莱斯芽孢杆菌,命名为Bacillusvelezensis504。抑菌实验显示,B.velezensis504对黄单胞菌属的细菌具有较好的抑菌活性,对水稻白叶枯病菌(X. oryzae pv. oryzae,Xoo)的拮抗效果最显著。基因预测结果显示,B. velezensis 504含有fenA、dhbA、sfrA、bmyA、beaS、dfnA及bacA等编码脂肽类和聚酮糖类抑菌化合物的基因簇。其无菌发酵液的活性物质耐高温和蛋白酶降解,但不耐强酸、强碱,在pH值为5.5–8.9时仍具有稳定的拮抗活性。在高感水稻品种原丰早上,B. velezensis 504对Xoc在水稻叶片中引起的水渍症状具有显著的抑制作用。【结论】B. velezensis 504能够特异性拮抗黄单胞菌,在黄单胞菌引起的细菌性病害的生物防治中将具有较大的应用潜力。     

Genetic locus encoding functions involved in biosynthesis and outer membrane localization of xanthomonadin in Xanthomonas oryzae pv. oryzae

Xanthomonadins are membrane-bound, brominated, aryl-polyene pigments specific to the genus Xanthomonas. We have characterized a genetic locus (pig) from Xanthomonas oryzae pv. oryzae which contains four open reading frames (ORFs) that are essential for xanthomonadin production. Three of these ORFs are homologous to acyl carrier proteins, dehydratases, and acyl transferases, suggesting a type II polyketide synthase pathway for xanthomonadin biosynthesis. The fourth ORF has no homologue in the database. For the first time, we report that a putative cytoplasmic membrane protein encoded in the pig locus is required for outer membrane localization of xanthomonadin in X. oryzae pv. oryzae. We also report the identification of a novel 145-bp palindromic Xanthomonas repetitive intergenic consensus element that is present in two places in the pig locus. We estimate that more than 100 copies of this element might be present in the genome of X. oryzae pv. oryzae and other xanthomonads.     

Pigment and Virulence Deficiencies Associated with Mutations in the aroE Gene of Xanthomonas oryzae pv. oryzae

Xanthomonadins are yellow, membrane-bound pigments produced by members of the genus Xanthomonas. We identified an ethyl methanesulfonate-induced Xanthomonas oryzae pv. oryzae mutant (BXO65) that is deficient for xanthomonadin production and virulence on rice, as well as auxotrophic for aromatic amino acids (Pig⁻ Vir⁻ Aro⁻). Reversion analysis indicated that these multiple phenotypes are due to a single mutation. A genomic library of the wild-type strain was used to isolate a 7.0-kb clone that complements BXO65. By transposon mutagenesis, marker exchange, sequence analysis, and subcloning, the complementing activity was localized to a 849-bp open reading frame (ORF). This ORF is homologous to the aroE gene, which encodes shikimate dehydrogenase in various bacterial species. Shikimate dehydrogenase activity was present in the wild-type strain and the mutant with the complementing clone, whereas no activity was found in BXO65. This clone also complemented an Escherichia coli aroE mutant for prototrophy, indicating that aroE is functionally conserved in X. oryzae pv. oryzae and E. coli. The nucleotide sequence of the 2.9-kb region containing aroE revealed that a putative DNA helicase gene is located adjacent to aroE. Our results indicate that aroE is required for normal levels of virulence and xanthomonadin production in X. oryzae pv. oryzae.     

Detection of virulence and pathogenicity genes in selected phytopathovars

Plant pathogenic organisms are known to infect host cell using various range of secretory proteins. Amongst all other secretion systems, type III secretion system (T3SS) is a key mechanism for bacterial pathogenesis for establishing and maintaining infection into the host. Expression levels of seven genes viz. avrXacE1, avrXacE2, hpaA and hrpG along with bacterial endogenous control lrp (leucine-responsive protein) were studied. The pathogenic organisms selected for the present study includes Enterobacter cloacae, Enterobacter spp., Pantoea ananatis, Xanthomonas campestris pv. Citri, Pantoea agglomerans, Ochrobactrum anthropi and Erwinia chrysanthemi. P. agglomerans and Enterobacter spp. gave high expression of above-mentioned virulence genes compared to Xanthomonas, while E. cloacae and P. ananatis showed similar expression with that of Xanthomonas. The detailed relationship of the expression profiles with respect to the selected organisms is discussed.     

Reinstatement of the genus Paralona Sramek-Husek et al., 1962, and assessment of morphological variability in the Paralona pigra-group (Anomopoda: Chydoridae)

We studied the morphology of some members of the Chydorus piger-group, using populations from Karelia (Russia), France, Germany, the Azores, Magadan (East Siberia), Newfoundland (Canada), Venezuela, Zambia, and Bolivia, and move Chydorus piger Sars, 1861 to the genus Paralona Sramek-Husek et al., 1962, which we redefine using traditional characters (first antenna with subapical and apical esthetascs, second antenna with only six setae, ventral valve rim fully setulated with setae in marginal position except between anterior bend and well-developed anterior flange), as well as structural features of the trunk limbs, especially 1 and 2. Chydorus and Pleuroxus are the nearest relatives of Paralona.The genus presently contains only the species P. pigra (Sars, 1861). This, however, shows considerable morphological variation and might therefore be composed of several related taxa, in need of a closer definition.     

Draft genome sequence of Xanthomonas fragariae reveals reductive evolution and distinct virulence-related gene content

Background

Xanthomonas fragariae (Xf) is a bacterial strawberry pathogen and an A2 quarantine organism on strawberry planting stock in the EU. It is taxonomically and metabolically distinct within the genus Xanthomonas, and known for its host specificity. As part of a broader pathogenicity study, the genome of a Belgian, virulent Xf strain (LMG 25863) was assembled to draft status and examined for its pathogenicity related gene content.

Results

The Xf draft genome (4.2 Mb) was considerably smaller than most known Xanthomonas genomes (~5 Mb). Only half of the genes coding for TonB-dependent transporters and cell-wall degrading enzymes that are typically present in other Xanthomonas genomes, were found in Xf. Other missing genes/regions with a possible impact on its plant-host interaction were: i) the three loci for xylan degradation and metabolism, ii) a locus coding for a ß-ketoadipate phenolics catabolism pathway, iii) xcs, one of two Type II Secretion System coding regions in Xanthomonas, and iv) the genes coding for the glyoxylate shunt pathway. Conversely, the Xf genome revealed a high content of externally derived DNA and several uncommon, possibly virulence-related features: a Type VI Secretion System, a second Type IV Secretion System and a distinct Type III Secretion System effector repertoire comprised of multiple rare effectors and several putative new ones.

Conclusions

The draft genome sequence of LMG 25863 confirms the distinct phylogenetic position of Xf within the genus Xanthomonas and reveals a patchwork of both lost and newly acquired genomic features. These features may help explain the specific, mostly endophytic association of Xf with the strawberry plant.

Electronic supplementary material

The online version of this article (doi:10.1186/1471-2164-14-829) contains supplementary material, which is available to authorized users.     

Some applications of deoxyribonucleic acid base composition in bacterial taxonomy

The melting pointT _m, the mean molar (guanine+cytosine) composition and the compositional distribution of purified DNA from several strains ofXanthomonas, Chromobacterium and yellow-pigmented marine bacteria have been determined. These groups were selected because they had been analyzed adansonially. Ten strains ofXanthomonas had an average molar (guanine+cytosine) composition within the range 66.0–68.2%, which was very close to that ofPseudomonas (60–68%), as expected. All strains ofChromobacterium (six of theviolaceum biotype and three of thelividum biotype) had mean molar (guanine+cytosine) compositions within the range 63.4–71.4%. The yellow-pigmented marine flavobacteria had mean molar (guanine+cytosine) compositions of 35.6–40.6%. This suggests that they would not be genetically related to the yellow-pigmentedXanthomonas, nor to facultative aerobic organisms, such asAeromonas and theEnterobacteriaceae. The yellow-pigmented marine swarming bacteria, which resembleCytophaga, fell into two separate groups: some had a mean molar (guanine+cytosine) composition of about 34%, others were around 63%. This suggests genetic heterogeneity. The compositional distribution of DNA molecules was on the whole more narrow in polarly flagellated than in peritrichous organisms.     

Isolation and growth of a bacterium able to degrade nitrilotriacetic acid under denitrifying conditions

A Gram-negative bacterium was isolated from river sediment which was able to grow with nitrilotriacetic acid as a combined carbon, nitrogen and energy source in the absence of molecular oxygen using nitrate as the terminal electron acceptor. Batch growth parameters and mass balances are reported for growth under both aerobic and denitrifying conditions.The strain was characterized with respect to its substrate spectrum and other physiological properties. This denitrifying isolate is serologically unrelated to the comprehensively described Gram-negative obligately aerobic NTA-degrading bacteria all of which belong to the -subclass of Proteobacteria. Chemotaxonomic characterization, which revealed the presence of spermidine as the main polyamine and ubiquinone Q-8, excludes the new isolate from the phylogenetically redefined genus Pseudomonas and indicates a possible location within the -subclass of Proteobacteria close to, but separate from the genus Xanthomonas.