Chemical modification of intracellular cytosine deaminase fromChromobacterium violaceum YK 391

Cytosine deaminase (cytosine aminohydrolase, EC 3.5.4.1) stoichiometrically catalyzes the hydrolytic deamination of cytosine and 5-fluorocytosine to uracil and 5-fluorouracil, respectively. Amino acid residues located in or near the active sites of the intracellular cytosine deaminase fromChromobacterium violaceum YK 391 were identified by chemical modification studies. The enzymic activity was completely inhibited by chemical modifiers, such as 1 mM NBS, chloramine-T, ρ-CMB, ρ-HMB and iodine, and was strongly inhibited by 1 mM PMSF and pyridoxal 5′-phosphate. This chemical deactivation of the enzymic activity was reversed by a high concentration of cytosine. Furthermore, the deactivation of the enzymic activity by ρ-CMB was also reversed by 1 mM cysteine-HCl, DTT and 2-mercaptoethanol. These results suggested that cysteine, tryptophan and methionine residues might be located in or near the active sites of the enzyme, while serine and lysine were indirectly involved in the enzymic activity. The intracellular cytosine deaminase fromC. violaceum YK 391 was assumed to be a thiol enzyme.     

Seed fitness of hermaphrodites in areas with females and anther smut disease: Silene acaulis and Microbotryum violaceum

Sex-dependent infection rates could change the effective sex ratio of a population. Here, I tested whether females and hermaphrodites of Silene acaulis were equally likely to be infected by Microbotryum violaceum, a fungus that sterilizes the host, and whether sex allocation in hermaphrodites differed between low and high disease plots. Sex ratios of healthy and diseased plants were estimated in five natural plots. Fitness gained through seed production was estimated by measuring seed quantity and quality for each sex morph in eight plots for 2 yr; four plots had 1-5% disease frequency and four plots had 18-25% disease frequency. Sex ratios of healthy and diseased plants did not differ in five plots. The proportion of fitness hermaphrodites gained through ovules varied from 25 to 48%, indicating that this population is near the cosexual end of gynodioecy. Variation in functional gender of hermaphrodites was not explained by sex-dependent infection rates. Spatial heterogeneity in resources and microclimate seems to be important in explaining both disease frequency and variation in seed production by females and hermaphrodites.     

Natural selection on floral traits of female Silene dioica by a sexually transmitted disease

Floral traits endowing high reproductive fitness can also affect the probability of plants contracting sexually transmitted diseases. We explore how variations in floral traits influence the fitness of Silene dioica females in their interactions with pollinators carrying pollen or spores of the sterilizing anther-smut fungus Microbotryum violaceum. We collected healthy and infected plants in a highly diseased population and grew them under conditions that 'cure' infected individuals, and used standard regression methods to detect natural selection on floral traits. Narrow-sense heritabilities, coefficients of additive genetic variation (CV(A)) and genetic correlations among traits were estimated from paternal half-sib groups. Pollinator preferences imposed strong direct and directional selection on traits affecting female attractiveness and pollen-/spore-capturing abilities. Levels of additive genetic variance were high in these traits, suggesting that rapid responses to selection are possible. By considering our results in the light of spatial and temporal heterogeneity resulting from the colonization dynamics typical for this species, we suggest that the conflicting selective effects of pollen/spore loads lead to the maintenance of genetic variation in these traits.     

An In Vitro Inhibition Test That Predicts Toxicity of Bacterial Pathogen Combinations in the Colorado Potato Beetle

Many insect bacterial pathogens are not toxic enough for field control. Combinations of bacteria may increase toxicity. Bacteria toxic to Colorado potato beetle , Photorhabdus luminescens, Chromobacterium violaceum and Serratia marcescens , were tested in pair-wise combinations in an in vitro double streak test to determine bacterial compatibility. Only C. violaceum and S. marcescens grew to confluency. Their combined toxicity in vivo was additive. Other bacterial combinations had clear zones between bacterial streaks indicating inhibition. In the insect, the combined toxicity was less than the most toxic bacteria of the pair. For these strains, this in vitro test predicted compatibility in the insect.     

Within‐ and among‐population variation in infectivity,latency and spore production in a host–pathogen system

In spatially structured populations, host–parasite coevolutionary potential depends on the distribution of genetic variation within and among populations. Inoculation experiments using the plant, Silene latifolia, and its fungal pathogen, Microbotryum violaceum, revealed little overall differentiation in infectivity/resistance, latency or spore production among host or pathogen populations. Within populations, fungal strains had similar means, but varied in performance across plant populations. Variation in resistance among seed families indicates the potential for parasite‐mediated selection, whereas there was little evidence for local pathogen genotype × plant genotype interactions assumed by most theoretical coevolution models. Lower spore production on sympatric than allopatric hosts confirmed local fungal maladaptation already observed for infectivity. Correlations between infectivity and latency or spore production suggest a common mechanism for variation in these traits. Our results suggest low variation available to this pathogen for tracking its coevolving host. This may be caused by random drift, breeding system or migration characteristic of metapopulation dynamics.     

紫色色杆菌感染小熊猫引起肺炎的临床调查

本文报道了福州大熊猫研究中心小熊猫感染紫色色杆菌引起肺炎的群发病例。7 只小熊猫中,出现以呼吸衰竭为主要临床症状的3 只小熊猫在发病3 d内全部死亡,病死率100% 。死亡的小熊猫解剖病变为气管内有白色泡沫样分泌物,肺有化脓性坏死并严重淤血;胸腔,心包积液且胸水浑浊;肝脏呈弥漫性空泡变性,淤血和散在局灶性坏死。通过流行病学调查,病理解剖观察,细菌分离培养和鉴定及动物回归试验,诊断为紫色色杆菌无色变种感染。根据GenBank 数据库提供的紫色色杆菌16S RNA 基因序列,设计一对引物(5’GAG CAAACA GGA TTA GAT ACC 3’;5’TTA CGG TTA CCT TGT TAC GAC 3’),获得目的基因片段739 bp,将核苷酸序列测定的结果与GenBank 数据库提供的7 株紫色色杆菌进行同源性比较。分离的菌株(FJ08A) 与CV09 株、ESBV4400株同源性均为98.8% ,与AY117554 株、EAV2 株、AJ871127 株、LMG3953 株、JS1 株同源性分别为98. 2% 、98.0% 、94.9%、93.1% 、92.8% 。依据调查结果和药物敏感试验,对其余4 只未发病的小熊猫采取肌
肉注射头孢哌酮钠和口服复方新诺明进行预防性治疗,效果良好,未有新病发生。结果表明:(1)紫色色杆菌是革兰氏阴性菌,致病性强,致死率高,腹腔注射分离的紫色色杆菌菌液导致接种小白鼠在2 ～3 d 内全部死亡。小熊猫感染后,发病迅速,致死率高,应引起重视;(2)紫色色杆菌是一种条件致病菌,外伤感染是主要致病因素。     

Inhibition of bacterial quorum sensing by vanilla extract

AIMS: The purpose of this study was to search for a novel quorum sensing inhibitor and analyse its inhibitory activity. METHODS AND RESULTS: Quorum sensing inhibition was monitored using the Tn-5 mutant, Chromobacterium violaceum CV026. Vanilla beans (Vanilla planifolia Andrews) were extracted using 75% (v/v) aqueous methanol and added to C. violaceum CV026 cultures. Inhibitory activity was measured by quantifying violacein production using a spectrophotometer. The results have revealed that vanilla extract significantly reduced violacein production in a concentration-dependent manner, indicating inhibition of quorum sensing. CONCLUSIONS: Vanilla, a widely used spice and flavour, can inhibit bacterial quorum sensing. SIGNIFICANCE AND IMPACT OF THE STUDY: The results suggest that the intake of vanilla-containing food materials might promote human health by inhibiting quorum sensing and preventing bacterial pathogenesis. Further studies are required to isolate specific substances from vanilla extract acting as quorum sensing inhibitors.     

Evaluation of the effects of selected phytochemicals on quorum sensing inhibition and in vitro cytotoxicity

Quorum sensing (QS) is an important regulatory mechanism in biofilm formation and differentiation. Interference with QS can affect biofilm development and antimicrobial susceptibility. This study evaluates the potential of selected phytochemical products to inhibit QS. Three isothiocyanates (allylisothiocyanate – AITC, benzylisothiocyanate – BITC and 2-phenylethylisothiocyanate – PEITC) and six phenolic products (gallic acid – GA, ferulic acid – FA, caffeic acid – CA, phloridzin – PHL, (?) epicatechin – EPI and oleuropein glucoside – OG) were tested. A disc diffusion assay based on pigment inhibition in Chromobacterium violaceum CV12472 was performed. In addition, the mechanisms of QS inhibition (QSI) based on the modulation of N-acyl homoserine lactone (AHLs) activity and synthesis by the phytochemicals were investigated. The cytotoxicity of each product was tested on a cell line of mouse lung fibroblasts. AITC, BITC and PEITC demonstrated a capacity for QSI by modulation of AHL activity and synthesis, interfering the with QS systems of C. violaceum CviI/CviR homologs of LuxI/LuxR systems. The cytotoxic assays demonstrated low effects on the metabolic viability of the fibroblast cell line only for FA, PHL and EPI.