Factorial design and response surface optimization of crude violacein for Chromobacterium violaceum production

Initially an eleven variable, sixteen assay 2^15–11 fractional factorial design, was used to determine the key factors in the production of violacein produced by Chromobacterium violaceum, CCT 3496. Subsequently five and three factor central composite designs were executed to determine response surfaces with the aim of optimizing cellular mass and crude violacein production. The 7.5 g l^–1 dry cell mass and 0.17 g l^–1 crude violacein productions obtained with our initial culture medium were increased to 21 g l^–1 and 0.43 g l^–1, respectively, for a medium investigated in the three factor design.     

Inhibition of quorum sensing in Chromobacterium violaceum by pigments extracted from Auricularia auricular

Aims: This study aimed to search for a novel quorum‐sensing inhibitor from some fungi and analyse its inhibitory activity. Methods and Results: Chromobacterium violaceum CV026, a double mini‐Tn5 mutant, was used as an indicator to monitor quorum‐sensing inhibition. Auricularia auricular pigments from fruiting bodies were extracted using hydrochloric acid as an infusion, dissolved in alkaline dimethylsulfoxide (DMSO), sterilized by filtration through a 0·22‐μm membrane filter and added to C. violaceum CV026 cultures. Inhibitory activity was measured by quantifying violacein production using a microplate reader. The results have revealed that the alkaline DMSO‐soluble pigments significantly reduced violacein production in a concentration‐dependent manner, a quorum‐sensing‐regulated behaviour in C. violaceum. Conclusions: Auricularia auricular pigments can inhibit bacterial quorum sensing. Significance and Impact of the Study: The results suggest the bioactive constituents from edible and medicinal fungi could interfere with bacterial quorum‐sensing system, regulate its associate functions and prevent bacterial pathogenesis. Further studies were in process in our laboratory to isolate specific compounds from A. auricular pigments, evaluate them as quorum‐sensing inhibitors and analyse the exact mechanism of action.     

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.     

Genotyping of Chromobacterium violaceum isolates by recA PCR-RFLP analysis

Intraspecies variation of Chromobacterium violaceum was examined by comparative sequence - and by restriction fragment length polymorphism analysis of the recombinase A gene (recA-PCR-RFLP). Primers deduced from the known recA gene sequence of the type strain C. violaceum ATCC 12472(T) allowed the specific amplification of a 1040bp recA fragment from each of the 13 C. violaceum strains investigated, whereas other closely related organisms tested negative. HindII-PstI-recA RFLP analysis generated from 13 representative C. violaceum strains enabled us to identify at least three different genospecies. In conclusion, analysis of the recA gene provides a rapid and robust nucleotide sequence-based approach to specifically identify and classify C. violaceum on genospecies level.     

生长素合成途径的研究进展

生长素是一类含有一个不饱和芳香族环和一个乙酸侧链的内源激素, 参与植物生长发育的许多过程。植物和一些侵染植物的病原微生物都可以通过改变生长素的合成来调节植株的生长。吲哚-3-乙酸(IAA)是天然植物生长素的主要活性成分。近年来, 随着IAA生物合成过程中一些关键调控基因的克隆和功能分析, 人们对IAA的生物合成途径有了更加深入的认识。IAA的生物合成有依赖色氨酸和非依赖色氨酸两条途径。依据IAA合成的中间产物不同, 依赖色氨酸的生物合成过程通常又划分成4条支路: 吲哚乙醛肟途径、吲哚丙酮酸途径、色胺途径和吲哚乙酰胺途径。该文综述了近几年在IAA生物合成方面取得的新进展。     

Diversity in antifungal activity of strains of <Emphasis Type="Italic">Chromobacterium violaceum</Emphasis> from the Brazilian Amazon

Chromobacterium violaceum is a free-living Gram-negative bacterium found in soil and aquatic habitats; abundantly present in the Brazilian Amazon, it is an important example of exploitable microbial diversity of the tropics. In this study, 24 strains from the Brazilian Amazon and ATCC 12472(T) were investigated for biocontrol potential of seven fungi pathogenic to soybean [Glycine max (L.) Merril] seed. Both cells and the supernatants of two Brazilian strains, 07-1 and 27-1, together with ATCC 12472(T) were strongly antagonistic to six out of the seven fungi. The antifungal activity of the Brazilian strains to Fusarium sp., Phomopsis sp. and Cercospora kikuchi was consistently stronger than that of ATCC 12472(T). In addition, the two Brazilian strains, but not ATCC 12472(T), were effective against Corynespora sp., and all three strains and their supernatants were equally effective against Aspergillus sp. and Colletotrichum sp. None of the strains had antifungal activity against Botroyodiplodia sp. Three potential mechanisms related to the antibiosis were investigated: violacein toxicity, cyanide production and chitinolytic activity; however, it was not possible to associate any of them with the antifungal activity. The results highlight the biotechnological potential still to be explored within the poorly characterized microbial biodiversity of the tropics.     

Conversion from Tryptophan Precursor into Violacein Pigments by a Cell-free System from Chromobacterium violaceum

A cell-free system for converting tryptophan precursor into violacein pigments is reported. Crucial factors were the requirements of the reduced nicotinamides as a cofactor and Zn²⁺ as a metal ion. Optimal pHs were in the range of 8.5–9.5. The effectiveness of NADH was thirty times lower than that of NADPH at a low concentration of 2mm. The oxygenation mechanism(s) is discussed by using oxygenase inhibitors such as amethopterin, metyrapone, ancymidol, and prohexadione. Metal-chelating agents strongly inhibited the biosynthesis, suggesting that metallo-enzymes are involved in the biosynthesis.     

Indole-3-butyric acid in Arabidopsis thaliana

Indole-3-butyric acid (IBA) was identified by HPLC and GC-MS as an endogenous compound in plantlets of the crucifer Arabidopsis thaliana (L.) Heynh. A. thaliana was cultivated under sterile conditions as shaking culture in different liquid media with and without supply of hormones. Free and total IBA and indole-3-acetic acid (IAA) were determined at different stages of development during the culture period as well as in culture media of different initial pH values. The results showed that IAA was present in higher concentrations than IBA, but both hormones seemed to show the same behaviour under the different experimental conditions. Differences were found in the mode of conjugation of the two hormones. While IAA was mostly conjugated via amide bonds, the main IBA conjugates were ester bound. The ethylene concentration derived from the seedlings, when they were grown in flasks of different size, seemed not to influence the auxin content in the same cultures.     

Peroxidase isoenzymes as markers for the rooting ability of easy-to-root and difficult-to-root <Emphasis Type="Italic">Grevillea</Emphasis> species and cultivars of <Emphasis Type="Italic">Protea obstusifolia</Emphasis> (Proteaceae)

Summary One easy-to-root and one difficult-to-root species of the ornamental plant Grevillea were investigated for rooting potential in relation to peroxidase activity. In vitro-grown shoot segments of both species started to root 30 d after transplanting to rooting medium containing indole-3-butyric acid (IBA), however, fewer roots were found on fewer segments of the difficult-to-root species G. petrophioides compared to the easy-to-root species G. rondeau. Total peroxidase (POX) activity was measured during the rooting process. G. petrophioides showed higher total POX activity at the time point of adventitious root formation than G. rondeau. Isoelectric focusing electrophoresis showed that G. rondeau contained more acidic isoforms than G. petrophioides, but the basic isoforms were more prominent in the difficult-to-root species, especially at the time point of lateral root emergence. In addition, the ability of different hormones to induced POX activity in upper and lower stem segments of both species was tested. Indole-3-acetic acid (IAA), IBA and α-naphthaleneacetic acid induced POX activity in the upper stem segments of G. rondeau, whereas the same hormones led to the induction of POX activity in the lower stem segments of G. petrophioides. Similar to the results obtained with Grevillea, the difficult-to-root variety of Protea showed higher POX activity, especially in the middle stem part and the leaves. Feeding of radiolabeled IAA to the Grevillea stem segments resulted in the synthesis of three different compounds in both species. After 1h incubation no differences were found in the uptake of IAA and the appearance of other labeled compounds. However, after 2 and 4 h incubation IAA uptake was faster in the easy-to-root species and IAA was also metabolized to a higher extent in G. rondeau. Three metabolites were found, tentatively identified as IAA-aspartate, IBA, and an IBA conjugate.     

Chlorophyll-protein levels and degree of thylakoid stacking in radish chloroplasts from high-light, low-light and bentazon-treated plants

Lemna gibba plants were incubated aseptically on medium containing labelled 10^-7 M indole-3-acetic acid (IAA-1-¹⁴C). Most of the radioactivity disappeared from the culture medium during a 24 h light period. A high percentage of the loss was due to photolysis and only a low percentage of the radioactivity was recovered in the plants. Uptake of ¹⁴C by the plants was strongly stimulated by light. The radioactivity taken up by the plants was the sum of photosynthetically taken up ¹⁴CO₂ and ¹⁴C taken up in IAA. Analyses with the indolo-α-pyrone fluorescence method revealed that the free IAA content was almost the same in plants grown in control and in IAA media for 5 h, whereas the amount of IAA which could be liberated by alkaline hydrolysis was doubled by the presence of IAA in the medium.     

Computational analysis suggests that virulence of Chromobacterium violaceum might be linked to biofilm formation and poly-NAG biosynthesis

Groups of genes that produce exopolysaccharide with a N-acetyl-D-glucosamine monomer are in the genome of several pathogenic bacteria. Chromobacterium violaceum, an opportunistic pathogen, has the operon hmsHFR-CV2940, whose proteins can synthesize such polysaccharide. In this work, multiple alignments among proteins from bacteria that synthesize such polysaccharide were used to verify the existence of amino acids that might be critical for pathogen activity. Three-dimensional models were generated for spatial visualization of these amino acid residues. The analysis carried out showed that the protein HmsR preserves the amino acids D135, D228, Q264 and R267, considered critical for the formation of biofilms and, furthermore, that these amino acids are close to each other. The protein HmsF of C. violaceum preserves the residues D86, D87, H156 and W115. It was also shown that these residues are also close to each other in their spatial arrangement. For the proteins HmsH and CV2940 there is evidence of conservation of the residues R104 and W94, respectively. Conservation and favorable spatial location of those critical amino acids that constitute the proteins of the operon indicates that they preserve the same enzymatic function in biofilm synthesis. This is an indicator that the operon hmsHFR-CV2940 is a possible target in C. violaceum pathogenicity.     

Molecular genetics of met 17 and met 25 mutants of Saccharomyces cerevisiae: intragenic complementation between mutations of a single structural gene

Summary A method for transposon mutagenesis in Azospirillum lipoferum 29708 is reported with transposon Tn5. The suicide plasmid pSUP2021 was used to deliver Tn5 in A. lipoferum using Escherichia coli SM10 as the donor. Neomycin-resistant transconjugants were detected at a frequency of 6x10^-6 per recipient. Different types of mutants were isolated, e.g. auxotrophic, coloured, IAA-negative, and IAA-overproducers. Among the auxotrophic mutants, cysteine and methionine requirers prevailed. Random Tn5-insertion with only one copy per mutant was demonstrated by Southern blotting and hybridization. Tn5-induced mutants are relatively stable, with reversion rates of 2–20×10^-8. A gene which is a part of the carotenoid pathway is closely linked to the histidine genes. The existence of two pathways for IAA production in A. lipoferum is discussed.     

解淀粉芽胞杆菌HZ-12中ysnE参与吲哚-3-乙酸合成的代谢途径研究

【目的】吲哚-3-乙酸是调控植物生长发育和生理活动的重要激素,吲哚-3-乙酸N-乙酰转移酶YsnE在吲哚-3-乙酸合成中发挥重要作用,本研究拟解析解淀粉芽胞杆菌中YsnE参与吲哚-3-乙酸合成的代谢途径。【方法】通过基因ysnE缺失和强化表达,分析ysnE对吲哚-3-乙酸合成影响,结合吲哚-3-乙酸合成中间物(吲哚丙酮酸、吲哚乙酰胺、色胺和吲哚乙腈)添加和体外酶转化实验,解析ysnE参与吲哚-3-乙酸合成的代谢途径。【结果】明确了YsnE在解淀粉芽胞杆菌HZ-12吲哚-3-乙酸合成中发挥重要作用。发现ysnE缺失菌株中的吲哚丙酮酸、吲哚乙酰胺和吲哚乙腈利用显著降低,揭示了YsnE主要发挥吲哚丙酮酸脱羧酶YclB和吲哚乙酰胺水解酶/腈水解酶/腈水合酶YhcX的功能,并通过参与吲哚丙酮酸、吲哚乙酰胺和吲哚乙腈途径来影响吲哚-3-乙酸合成。【结论】初步揭示了YsnE通过影响吲哚丙酮酸、吲哚乙酰胺和吲哚乙腈途径参与吲哚-3-乙酸合成的代谢机理,为吲哚-3-乙酸合成途径解析和代谢工程育种构建吲哚-3-乙酸高产菌株奠定了基础。     

Biosynthesis of novel polyhydroxyalkanoate containing 3-hydroxy-4-methylvalerate by Chromobacterium sp. USM2

Aims: Polyhydroxyalkanoate (PHA) with enhanced physicochemical properties will be ideal for a wide range of practical applications. The incorporation of 3‐hydroxy‐4‐methylvalerate (3H4MV) into the polymer backbone is known to improve the overall properties of the resulting polymer. However, the most suitable micro‐organism and PHA synthase that can synthesize this monomer efficiently still remain unknown at present. Therefore, we evaluated the abilities of a locally isolated Chromobacterium sp. USM2 to produce PHA containing 3H4MV. Methods and Results: The ability of Chromobacterium sp. USM2 to synthesize poly(3‐hydroxybutyrate‐co‐3‐hydroxy‐4‐methylvalerate) [P(3HB‐co‐3H4MV)] was evaluated under different culture conditions. It was found that Chromobacterium sp. USM2 can synthesize P(3HB‐co‐3H4MV) when glucose and isocaproic acid were fed as carbon source. However, the highest molar fraction of 3H4MV, 22 mol% was detected in Chromobacterium sp. USM2 when isocaproic acid was provided as the sole carbon source. In addition, aeration was identified as a crucial factor in initiating the accumulation of high 3H4MV molar fractions. Conclusions: Chromobacterium sp. USM2 was able to synthesize broad comonomer compositional distribution of P(3HB‐co‐3H4MV). Significance and Impact of the Study: Compared with Cupriavidus necator and Burkholderia sp., Chromobacterium sp. USM2 was found to have better ability to bioconvert isocaproic acid to form 3H4MV unit.     

Peroxidase assay in plants: Interference by ascorbic acid and endogenous inhibitors in Sedum and Pelargonium enzyme extracts

Sedum album and Pelargonium zonale extracts do not show any peroxidase activity. Both extracts provoke a lag phase in the horse-radish peroxidase-catalyzed oxidation of guaiacol by H₂O₂. Preincubation of Sedum album extract with ascorbate oxidase eliminated completely the lag phase. Ascorbic acid has been identified as the substance responsible for this lag phase by reacting with a coloured intermediary product of the analytical reaction. In the Pelargonium zonale extract, the lag phase seems to be due to competitive inhibitors of peroxidase, which are of a phenolic nature.     

Molecular confirmation of a Trichophyton violaceum isolate from human black-dot ringworm

A clinical isolate from a black-dot ringworm lesion of a 28-year-old female Japanese was investigated by morphological and biochemical analyses as well as molecular analyses. The isolate grew well onthiamine enriched agar and did not produce violetpigment, macroconidia or microconidia on Sabouraud's dextrose agar. Approximately 620-bp genomic DNA fragments of the CHS1 gene were amplified from Trichophyton mentagrophytes, T. rubrum, T. tonsurans and T. violaceum by polymerase chain reaction (PCR) and sequenced. The chitin synthase 1 (CHS1) nucleotide sequences of the clinical isolate showed more than 97% similarity to that of T. violaceum and less than 96% similarity to that of T. mentagrophytes, T. rubrum and T. tonsurans. The phylogenetic analysis of their sequences revealed that the clinical isolate was genetically close to T. violaceum and distinct from T. mentagrophytes, T. rubrum and T. tonsurans. Therefore, the isolate was confirmed as T. violaceum by mycological examination and molecular analyses. This revised version was published online in June 2006 with corrections to the Cover Date.