<Emphasis Type="Italic">Cupriavidus metallidurans</Emphasis>: evolution of a metal-resistant bacterium

Cupriavidus metallidurans CH34 has gained increasing interest as a model organism for heavy metal detoxification and for biotechnological purposes. Resistance of this bacterium to transition metal cations is predominantly based on metal resistance determinants that contain genes for RND (resistance, nodulation, and cell division protein family) proteins. These are part of transenvelope protein complexes, which seem to detoxify the periplasm by export of toxic metal cations from the periplasm to the outside. Strain CH34 contains 12 predicted RND proteins belonging to a protein family of heavy metal exporters. Together with many efflux systems that detoxify the cytoplasm, regulators and possible metal-binding proteins, RND proteins mediate an efficient defense against transition metal cations. To shed some light into the origin of genes encoding these proteins, the genomes of C. metallidurans CH34 and six related proteobacteria were investigated for occurrence of orthologous and paralogous proteins involved in metal resistance. Strain CH34 was not much different from the other six bacteria when the total content of transport proteins was compared but CH34 had significantly more putative transition metal transport systems than the other bacteria. The genes for these systems are located on its chromosome 2 but especially on plasmids pMOL28 and pMOL30. Cobalt–nickel and chromate resistance determinants located on plasmid pMOL28 evolved by gene duplication and horizontal gene transfer events, leading to a better adaptation of strain CH34 to serpentine-like soils. The czc cobalt–zinc–cadmium resistance determinant, located on plasmid pMOL30 in addition copper, lead and mercury resistance determinants, arose by duplication of a czcICAB core determinant on chromosome 2, plus addition of the czcN gene upstream and the genes czcD, czcRS, czcE downstream of czcICBA. C. metallidurans apparently evolved metal resistance by horizontal acquisition and by duplication of genes for transition metal efflux, mostly on the two plasmids, and decreased the number of uptake systems for those metals. This paper is dedicated to Dr. Max Mergeay for a long time of cooperation, constructive competition and friendship.     

The Chloroplast trnT–trnF Region in the Seed Plant Lineage Gnetales

The trnT–trnF region is located in the large single-copy region of the chloroplast genome. It consists of the trnL intron, a group I intron, and the trnT–trnL and trnL–trnF intergenic spacers. We analyzed the evolution of the region in the three genera of the gymnosperm lineage Gnetales (Gnetum, Welwitschia, and Ephedra), with especially dense sampling in Gnetum for which we sequenced 41 accessions, representing most of the 25–35 species. The trnL intron has a conserved secondary structure and contains elements that are homologous across land plants, while the spacers are so variable in length and composition that homology cannot be found even among the three genera. Palindromic sequences that form hairpin structures were detected in the trnL–trnF spacer, but neither spacer contained promoter elements for the tRNA genes. The absence of promoters, presence of hairpin structures in the trnL–trnF spacer, and high sequence variation in both spacers together suggest that trnT and trnF are independently transcribed. Our model for the expression and processing of the genes tRNA^Thr(UGU), tRNA^Leu(UAA), and tRNA^Phe (GAA) therefore attributes the seemingly neutral evolution of the two spacers to their escape from functional constraints. [Reviewing Editor: Debashish Bhattacharya]     

Transfer of megaplasmid pKB1 from the rubber-degrading bacterium <Emphasis Type="Italic">Gordonia westfalica</Emphasis> strain Kb1 to related bacteria and its modification

Because engineering of the 101.016-bp megaplasmid pKB1 of Gordonia westfalica Kb1 failed due to the absence of an effective transfer system, pKB1 was transferred by conjugation from G. westfalica Kb1 to a kanamycin-resistant mutant of Rhodococcus opacus PD630 at a frequency of about 6.2 × 10⁻⁸ events per recipient cell. Furthermore, pKB1 was transferred to G. polyisoprenivorans strains VH2 and Y2K and to Mycobacterium smegmatis by electroporation at frequencies of 5.5 × 10³, 1.9 × 10³, and 8.3 × 10² transformants per microgram plasmid DNA. The pKB1-encoded cadmium resistance gene cadA was used for selection in these experiments. Recombinant pKB1-containing G. polyisoprenivorans VH2 and M. smegmatis were then used to engineer pKB1. A kanamycin resistance cassette was inserted into the pKB1-encoded cadA gene, ligated to suicide plasmid pBBR1MCS-5, and the resulting plasmid was electroporated into plasmid-harboring strains. Homologous recombination between cadA on suicide plasmid and the respective sequence in pKB1 led to its integration into pKB1. Thus, two selection markers were accommodated in pKB1 to monitor plasmid transfer into Gordonia and related taxa for analysis of genes essential for rubber degradation and others. In this study, two transfer methods for large plasmids and strategies for engineering of pKB1 were successfully applied, thereby, extending the tool box for Gordonia.     

The ABC-transporter AtmA is involved in nickel and cobalt resistance of <Emphasis Type="Italic">Cupriavidus metallidurans</Emphasis> strain CH34

Cupriavidus metallidurans CH34 genome contains an ortholog of Atm1p named AtmA (Rmet_0391, YP_582546). In Saccharomyces cerevisiae, the ABC-type transport system Atm1p is involved in export of iron–sulfur clusters from mitochondria into the cytoplasm for assembly of cytoplasmic iron–sulfur containing proteins. An ∆atmA mutant of C. metallidurans was sensitive to nickel and cobalt but not iron cations. AtmA increased also resistance to these cations in Escherichia coli strains that carry deletions of the genes for other nickel and cobalt transport systems. In C. metallidurans, atmA expression was not significantly induced by nickel and cobalt, but repressed by zinc. AtmA was purified as a 70 kDa protein after expression in E. coli. ATPase activity of AtmA was stimulated by nickel and cobalt.     

Effect of chelating agents and respiratory inhibitors on regulation of the cadA gene in Escherichia coli

The cadA gene that encodes lysine decarboxylase in Escherichia coli is induced by low pH and – during anaerobic growth – by the substrate, lysine. We used operon fusions of cadA to lacZ to investigate the effects of aeration on cadA regulation. When an insertion mutation in osmZ (= hns) was introduced, a cadA-lacZ fusion was derepressed in the presence of air to approximately the same level as seen during anaerobic growth. However, the pH-dependent regulation of cadA was not affected by osmZ. Introduction of mutations in rpoS, fur, or fnr had no significant effect on cadA expression. However, defects in arcB or arcA largely abolished expression of cadA during anaerobic growth. Nonetheless, strains defective in both arcB and osmZ showed the same high cadA-lac expression in air as seen in the single osmZ derivatives. Blocking the respiratory chain with mutations or chemical inhibitors also caused derepression of a cadA-lacZ fusion in air, while agents affecting the proton gradient had no effect. Derepression of cadA in air was also mediated by several chelating agents, in particular by methoxyindole carboxylic acid. Addition of Fe²⁺ overcame this effect. Chelating agents also abolished the expression during aerobic growth of several genes known to be under arcAB control and which are normally repressed during anaerobic growth but induced in the presence of air. This implies that the effect of chelating agents on cadA expression is mediated via the arcAB regulatory system. Received: 16 August 1996 / Accepted: 12 November 1996     

Application of petG-trnP sequence to systematic study of Chinese Cupressus species

Chinese Cupressus L. includes five species. The molecular phylogenetic relationship of the Cupressus species and Chamaecyparis L. were determined by comparing 417–479 bp of chloroplast petG-trnP intergenic spacer sequence. In PAUP^* analysis, Platycladus orientalis was used as the functional out group. By using the maximum likelihood method 1 077 trees were examined and the result showed that one tree had a best score of -Ln=2 232.47. The phylogenetic tree clearly showed that Chamaecyparis nootkatensis was diverged from other Chamaecyparis species. Based on the results, together with evidences from other aspects, we consider that Cupressus funebris and Chamaecyparis nootkatensis should be placed in the genus Cupressus. The use of cpDNA intergenic spacer petG-trnP in Cupressus was also discussed. __________ Translated from Journal of Sichuan University (Natural Science Edition), 2005, 42(5): 1033–1037 [译自: 四川大学学报 (自然科学版) 2005, 42(5): 1033–1037]     

Purification and characterization of MerR, the regulator of the broad-spectrum mercury resistance genes in Streptomyces lividans 1326

Streptomyces lividans 1326 carries inducible mercury resistance genes on the chromosome, which are arranged in two divergently transcribed operons. Expression of the genes is negatively regulated by the repressor MerR, which binds in the intercistronic region between the two operons. The merR gene was expressed in E. coli using a T7 RNA polymerase/promoter expression system, and MerR was purified to around 95% homogeneity by ammonium sulfate precipitation, gel filtration and affinity chromatography. Gel filtration showed that the native MerR is a dimer with a molecular mass of 31 kDa. Two DNA binding sites were identified in the intercistronic mer promoter region by footprinting experiments. No evidence for cooperativity in the binding of MerR to the adjacent operator sequences was observed in gel mobility shift assays. The dissociation constants (K_D) for binding of MerR were: binding site I, 8.5 × 10⁻⁹ M; binding site II, 1.2 × 10⁻⁸ M; and for the complete promoter/operator region 1 × 10⁻⁸ M. The half-life of the MerR-DNA complex was 19.4 min and 18.8 min for binding site I and binding site II, respectively. The K_D value for binding of mercury(II)chloride to MerR, again determined by mobility shift assay, was 1.1 × 10⁻⁷ M. Received: 18 August 1998 / Accepted: 5 May 1999     

Rapid evolution of promoters for the plastome gene <Emphasis Type="Italic">ndhF</Emphasis> in flowering plants

Plastome is thought to be a very conservative part of plant genome but little is known about the evolution of plastome promoters. It was previously shown that one light-regulated promoter (LRPpsbD) is highly conserved in different flowering plant species and in black pine. We have undertaken search and demonstrated that gene ndhF is located in a plastome region that rarely underwent substantial rearrangements in terrestrial plants. However, alignment of sequences upstream ndhF suggests that promoters of this gene underwent comparatively rapid evolution in flowering plants. Probably, the ancestor of two basal Magnoliophyta branches (magnoliids and eudicotyledons) had the promoter P_A-ndhF, which was substituted with other promoters—P_B-ndhF and P_C-ndhF—in some phylogenetic lineages of dicots. We failed to reveal conservative sequences with potential promoters of −10/−35 type upstream ndhF genes of monocotyledonous plants, including nine representatives of the grass family (Poaceae). Multiple alignments of sequences from related taxa showed that the predicted ndhF promoters (A–C) underwent frequent mutations and these mutations are not only nucleotide substitutions but also small insertions and deletions. Thus, we can assume that at least some plastome promoters evolve rapidly.     

The Triticum aestivum non-specific lipid transfer protein (TaLtp) gene family: comparative promoter activity of six TaLtp genes in transgenic rice

Plant non-specific lipid transfer proteins (nsLTPs) are encoded by a multigene family and support physiological functions, which remain unclear. We adapted an efficient ligation-mediated polymerase chain reaction (LM-PCR) procedure that enabled isolation of 22 novel Triticum aestivum nsLtp (TaLtp) genes encoding types 1 and 2 nsLTPs. A phylogenetic tree clustered the wheat nsLTPs into ten subfamilies comprising 1–7 members. We also studied the activity of four type 1 and two type 2 TaLtp gene promoters in transgenic rice using the β-Glucuronidase reporter gene. The activities of the six promoters displayed both overlapping and distinct features in rice. In vegetative organs, these promoters were active in leaves and root vascular tissues while no β-Glucuronidase (GUS) activity was detected in stems. In flowers, the GUS activity driven by the TaLtp7.2a, TaLtp9.1a, TaLtp9.2d, and TaLtp9.3e gene promoters was associated with vascular tissues in glumes and in the extremities of anther filaments whereas only the TaLtp9.4a gene promoter was active in anther epidermal cells. In developing grains, GUS activity and GUS immunolocalization data evidenced complex patterns of activity of the TaLtp7.1a, TaLtp9.2d, and TaLtp9.4a gene promoters in embryo scutellum and in the grain epicarp cell layer. In contrast, GUS activity driven by TaLtp7.2a, TaLtp9.1a, and TaLtp9.3e promoters was restricted to the vascular bundle of the embryo scutellum. This diversity of TaLtp gene promoter activity supports the hypothesis that the encoded TaLTPs possess distinct functions in planta.     

Comparative study of promoters for the production of polyhydroxyalkanoates in recombinant strains of Wautersia eutropha

Recombinant strains of Wautersia eutropha expressing an artificial polyhydroxyalkanoate (PHA) biosynthesis operon under the control of different native promoters linked to polyhydroxybutyrate (PHB) (P_phb), acetoin (P_acoE, P_acoD, and P_acoX) or pyruvate (P_pdhE) metabolism were constructed and tested. The promoters were representative either of the enterobacterial σ⁷⁰ (P_phb, P_acoE, and P_pdhE)- or σ⁵⁴ (P_acoD and P_acoX)-dependent promoters. To obtain polymers consisting of C₄–C₁₂ monomer units, an artificial operon consisting of the PHA synthase gene from Pseudomonas sp. 61-3 (phaC1 _Ps) tandemly linked to the W. eutropha genes encoding β-ketothiolase (phbA _We) and nicotinamide adenine dinucleotide phosphate dependent acetoacetyl-coenzyme A (CoA) reductase (phbB _We) was constructed. All recombinant strains produced PHA, indicating that the PHA biosynthesis genes were expressed under the control of the different promoters. Cell growth and PHA synthesis on MS medium complemented with gluconate or octanoate, and different concentrations of acetoin (0, 0.15, and 0.3%) clearly differed among the recombinant strains. While the P_acoD and P_acoX promoters mediated only low PHA yields (<1%) in the presence of the inducer acetoin, the remaining promoters—independent of the addition of acetoin—resulted in the production of PHA polymers with high 3HB fractions (90–100 mol%) and with high 3HO contents (70–86 mol%) from gluconate and octanoate, respectively. Interestingly, on octanoate-MS medium with 0.15% acetoin, the P_acoE promoter mediated the synthesis of PHA with a relatively high 3HB fraction (48 mol%). While PHAs with high 3HB contents were obtained, the overall PHA product yields were low (<10%); thus, their potential application for further commercial exploitation appears limited.     

Heavy metal resistant <Emphasis Type="Italic">Distigma proteus</Emphasis> (Euglenophyta) isolated from industrial effluents and its possible role in bioremediation of contaminated wastewaters

Summary The alga, Distigma proteus, isolated from industrial wastewater showed tolerance against Cd²⁺ (8.0 μg/ml), Cr⁶⁺ (12 μg/ml), Pb²⁺ (15 μg/ml) and Cu²⁺ (10 μg/ml). The metal ions slowed down the growth of the organism after 4–5 days of exposure. The reduction in cell population was 90% for Cu²⁺, 84% for Cd²⁺, 71% for Cr⁶⁺, and 63% for Pb²⁺ after 8 days of metal stress. The order of resistance to heavy metal, in terms of reduction in the cellular population, was Cu²⁺ > Cd²⁺ > Cr⁶⁺ > Pb²⁺. Chromium- and cadmium-processing capabilities of the alga were worked out for its potential use as a bioremediator of wastewater. The reduction in the amount of Cr⁶⁺ after 2, 4, 6 and 8 days of algal culture containing 5.0 μg Cr⁶⁺ ml⁻¹ of culture medium was 77, 85, 92 and 97%, respectively. Distigma could also remove 48% Cd²⁺after 2 days, 68% after 4 days, 80% after 6 days and 90% after 8 days from the medium. The heavy metal uptake ability of Distigma can be exploited for metal detoxification and environmental clean-up operations.     

Characterization and role of a metalloprotease induced by chitin in <Emphasis Type="Italic">Serratia</Emphasis> sp. KCK

A metalloprotease induced by chitin in a new chitinolytic bacterium Serratia sp. Strain KCK was purified and characterized. Compared with other Serratia enzymes, it exhibited a rather broad pH activity range (pH 5.0–8.0), and thermostability. The cognate ORF, mpr, was cloned and expressed. Its deduced amino acid sequence showed high similarity to those of bacterial zinc-binding metalloproteases and a well-conserved serralysin family motif. Pretreatment of chitin with the Mpr protein promoted chitin degradation by chitinase A, which suggests that Mpr participates in, and facilitates, chitin degradation by this microorganism.     

The LRRK2 Gly2385Arg variant is associated with Parkinson’s disease: genetic and functional evidence

Evidence of LRRK2 haplotypes associated with Parkinson’s disease (PD) risk was recently found in the Chinese population from Singapore, and a common LRRK2 missense variant, Gly2385Arg, was independently detected as a putative risk factor for PD in the Chinese population from Taiwan. To test the association between the Gly2385Arg variant in a large case-control sample of Chinese ethnicity from Singapore, and to perform functional studies of the wild type and Gly2385Arg LRRK2 protein in human cell lines. In a case-control study involving 989 Chinese subjects, the frequency of the heterozygous Gly2385Arg genotype was higher in PD compared to controls (7.3 vs. 3.6%, odds ratio = 2.1, 95% CI: 1.1–3.9, P = 0.014); these values yield an estimated population attributable risk (PAR) of ∼4%. In a multivariate logistic regression analysis with the disease group (PD vs. controls) as the dependent variable and the genotype as an independent factor with adjustments made for the effect of age and gender, the heterozygous Gly2385Arg genotype remained associated with an increased risk of PD compared to wild type genotype (odds ratio = 2.67, 95% CI: 1.43–4.99, P = 0.002). The glycine at position 2385 is a candidate site for N-myristoylation, and the Gly2385Arg variant replaces the hydrophobic glycine with the hydrophilic arginine, and increases the net positive charge of the LRRK2 WD40 domain. In transfection studies, we demonstrated that both the wild type and Gly2385Arg variant LRRK2 protein localize to the cytoplasm and form aggregates. However, under condition of oxidative stress, the Gly2385Arg variant was more toxic and associated with a higher rate of apoptosis. Our study lends support to the contention that the Gly2385Arg is a common risk factor for PD in the Chinese population. Our bioinformatics and in-vitro studies also suggest that the Gly2385Arg variant is biologically relevant and it might act through pro-apoptotic mechanisms.     

Fluctuation in recoverable nickel and zinc resistant copiotrophic bacteria explained by the varying zinc ion content of Torsa River in different months

Heavy metal content analysis of River Torsa in India did not indicate any alarming level of toxicity for human consumption but revealed variation at the ppb level in different months. The variation in recoverable nickel and zinc resistant copiotrophic (or eutrophic) bacterial counts was explained by the variation of the zinc content (34.0–691.3 ppb) of the river water in different sampling months. Growth studies conducted with some purified nickel and/or zinc resistant strains revealed that pre-exposure of the cells to ppb levels of Zn²⁺, comparable to the indigenous zinc ion concentration of the river, could induce the nickel or zinc resistance. A minimum concentration of 5–10 μM Zn²⁺ (325–650 ppb) was found effective in inducing the Nickel resistance of the isolates. Zinc resistance of the isolates was tested by pre-exposing the cells to 4 μM Zn²⁺ (260 ppb). The lag phase was reduced by 6–8 h in all the cases. Biochemical characteristics and phylogenetic analysis based on 16S rDNA sequence indicated that some of the Torsa River isolates, having inducible nickel and zinc resistance, are members of the genus Pseudomonas, Acinetobacter, Bacillus, Enterobacter, Serratia and Moraxella.     

The Effect of Growth Regulators on Quality Parameters and Ginsenosides Accumulation in Panax quinquefolium L. Roots

American ginseng (Panax quinquefolium L.) is a perennial medicinal herb originally grown in Canada and USA, and recently also in China, Australia, Holland and Poland. Several commercial preparations are produced from ginseng roots, that are known for their antifatigue, antitumor, antistress and immune system stimulating functions. The medicinal properties are due mainly to the active components – ginsenosides. In this work, the results of field cultivation experiments are presented that examine the effects of foliar application of several growth regulators on quality parameters and ginsenoside content of P. quinuefolium roots. The growth regulators tested, i.e., kinetin, daminozide, mixture of gibberellic acid (GA₃) with potassium salt of α-naphthalene acetic acid (kNAA) and new preparation – IPO-1 – benzimidazole derivative (obtained from the Institute of Organic Industry in Warsaw – at present during the process of patent), were applied at a concentration of 100 or 200 mg l⁻¹ in the middle of June in the 2nd year of vegetation. After 4 years of cultivation, the roots were dug up and dried, and subsequently the quantitative analysis of individual saponins (R_b1, R_b2, R_c, R_d, R_e, R_g1) by HPLC was performed. Growth regulators significantly affected quality parameters, morphological features and accumulation of individual and total ginsenosides in ginseng roots. Regardless of doses, the plant roots treated with growth regulators had a higher content of total ginsenosides in comparison to the control. The growth regulators also affected individual ginsenosides level and narrowed the ratio of R_b:R_g group. The application of kinetin, daminozide and benzimidazole derivative for foliar spray during 2nd year of American ginseng vegetation caused a significant increase in air dry weight of roots and aboveground parts whereas the mixture of GA₃ and kNAA showed a decreasing effect. An increase of roots size was observed using higher doses (200 mg l⁻¹) of kinetin and daminozide while a decreasing tendency appeared with the application of the other preparations.     

Disruption of the copper efflux pump (CopA) of Serratia marcescens ATCC 274 pleiotropically affects copper sensitivity and production of the tripyrrole secondary metabolite, prodigiosin

The prodigiosin biosynthetic gene cluster (pig cluster) of Serratia marcescens ATCC 274 (Sma 274) is flanked by cueR/copA homologues. Inactivation of the copA homologue resulted in an increased sensitivity to copper, confirming that CopA is involved in copper homeostasis in Sma 274. The effect of copper on the biosynthesis of prodigiosin in Sma 274 and the copA mutant strain was investigated. Increased levels of copper were found to reduce prodigiosin production in the wild type Sma 274, but increase production in the copA mutant strain. The physiological implications for CopA mediated prodigiosin production are discussed. We also demonstrate that the gene products of pigB–pigE of Sma 274 are sufficient for the biosynthesis of 2-methyl-3-n-amyl-pyrrole and condensation with 4-methoxy-2,2′-bipyrrole-5-carboxyaldehyde to form prodigiosin, as we have shown for Serratia sp. ATCC 39006.