Isotope discrimination by form IC RubisCO from Ralstonia eutropha and Rhodobacter sphaeroides, metabolically versatile members of 'Proteobacteria' from aquatic and soil habitats.

RubisCO, the CO2 fixing enzyme of the Calvin-Benson-Bassham (CBB) cycle, is responsible for the majority of carbon fixation on Earth. RubisCO fixes 12 CO2 faster than 13 CO2 resulting in 13 C-depleted biomass, enabling the use of δ13 C values to trace CBB activity in contemporary and ancient environments. Enzymatic fractionation is expressed as an ε value, and is routinely used in modelling, for example, the global carbon cycle and climate change, and for interpreting trophic interactions. Although values for spinach RubisCO (ε = ~29‰) have routinely been used in such efforts, there are five different forms of RubisCO utilized by diverse photolithoautotrophs and chemolithoautotrophs and ε values, now known for four forms (IA, B, D and II), vary substantially with ε = 11‰ to 27‰. Given the importance of ε values in δ13 C evaluation, we measured enzymatic fractionation of the fifth form, form IC RubisCO, which is found widely in aquatic and terrestrial environments. Values were determined for two model organisms, the 'Proteobacteria' Ralstonia eutropha (ε = 19.0‰) and Rhodobacter sphaeroides (ε = 22.4‰). It is apparent from these measurements that all RubisCO forms measured to date discriminate less than commonly assumed based on spinach, and that enzyme ε values must be considered when interpreting and modelling variability of δ13 C values in nature.

Paleomicrobiology to investigate copper resistance in bacteria: isolation and description of Cupriavidus necator B9 in the soil of a medieval foundry.

Remains of a medieval foundry were excavated by archaeologists in 2013 in Verdun (France). Ancient workshops specialized in brass and copper alloys were found with an activity between 13th to 16th c. Levels of Cu, Zn and Pb reached 20000, 7000 and 6000 mg kg-1 (dw), respectively, in several soil horizons. The objective of the present work was to examine the microbial community in this contaminated site. A total of 8-22 106 reads were obtained by shotgun metagenomics in four soil horizons. Bioinformatic analyses suggest the presence of complex bacterial communities dominated by Proteobacteria. The structure of the community was not affected by metals, contrary to the set of metal-resistance genes. Using selective media, a novel strain of Cupriavidus necator (eutrophus), strain B9, was isolated. Its genome was sequenced and a novel metal resistance gene cluster with Hg resistance genes (merRTPCA) followed by 24 copper-resistance genes (actP, cusCBAF, silP, copK1, copH4QLOFGJH3IDCBARS, copH2H1, copK2) was found. This cluster is partly homologous to the cop genes of Cupriavidus gilardii CR3 and C. metallidurans CH34. Proteomics indicated that the four copH genes were differentially expressed: CopH1 and CopH2 were mostly induced by Cd while CopH4 was highly expressed by Cu.

Inoculating Helianthus annuus (sunflower) grown in zinc and cadmium contaminated soils with plant growth promoting bacteria--effects on phytoremediation strategies.

Plant growth promoting bacteria (PGPR) may help reducing the toxicity of heavy metals to plants in polluted environments. In this work the effects of inoculating metal resistant and plant growth promoting bacterial strains on the growth of Helianthus annuus grown in Zn and Cd spiked soils were assessed. The PGPR strains Ralstonia eutropha (B1) and Chrysiobacterium humi (B2) reduced losses of weight in metal exposed plants and induced changes in metal bioaccumulation and bioconcentration - with strain B2 decreasing up to 67% Zn accumulation and by 20% Zn bioconcentration factor (BCF) in the shoots, up to 64% Zn uptake and 38% Zn BCF in the roots, and up to 27% Cd uptake and 27% Cd BCF in plant roots. The impact of inoculation on the bacterial communities in the rhizosphere of the plant was also assessed. Bacterial community diversity decreased with increasing levels of metal contamination in the soil, but in rhizosphere soil of plants inoculated with the PGPR strains, a higher bacterial diversity was kept throughout the experimental period. Inoculation of sunflower, particularly with C. humi (B2), appears to be an effective way of enhancing the short term stabilization potential of the plant in metal contaminated land, lowering losses in plant biomass and decreasing aboveground tissue contamination.

Complete genome sequence of the type strain Cupriavidus necator N-1.

Here we announce the complete genome sequence of the copper-resistant bacterium Cupriavidus necator N-1, the type strain of the genus Cupriavidus. The genome consists of two chromosomes and two circular plasmids. Based on genome comparison, the chromosomes of C. necator N-1 share a high degree of similarity with the two chromosomal replicons of the bioplastic-producing hydrogen bacterium Ralstonia eutropha H16. The two strains differ in their plasmids and the presence of hydrogenase genes, which are absent in strain N-1.

Characterization of Acrylamidase isolated from a newly isolated acrylamide-utilizing bacterium, Ralstonia eutropha AUM-01.

A mesophilic bacterium capable of utilizing acrylamide was isolated, AUM-01, from soil collected from leaf litter at Picnic Point on the UW-Madison campus. In minimal medium with acrylamide as the sole carbon and nitrogen source, a batch culture of AUM-01 completely converted 28.0 mM acrylamide to acrylic acid in 8 h and reached a cell density of 0.3 (A600)). Afterward all the acrylic acid was degraded by 20 h with the cell density increasing to 1.9 (A600). The acrylamide-utilizing bacterium was identified as Ralstonia eutropha based on morphological observations, the BiOLOG GN2 MicroPlate™ identification system for Gram-negative bacteria, and additional physiological tests. An acrylamidase that hydrolyzes acrylamide to acrylic acid was purified from the strain AUM-01. The molecular weight of the enzyme from AUM-01 was determined to be 38 kDa by SDS-PAGE. The enzyme had pH and temperature optima of 6.3 and 55°C, and the influence of different metals and amino acids on the ability of the purified protein to transform acrylamide to acrylic acid was evaluated. The enzyme from AUM-01 was totally inhibited by ZnSO4 and AgNO3.

Isolation and characterization of a phorate degrading bacterium.

AIMS: To study the degradation of phorate by a bacterium isolated from phorate-contaminated sites. METHODS AND RESULTS: Ralstonia eutropha strain AAJ1 isolated from soil was found to degrade phorate (supplied as sole carbon source) upto 85% in 10 days in liquid medium. Half-life (t((1/2))) of phorate in the liquid medium in control (uninoculated) and in experimental (inoculated with R. eutropha, strain AAJ1) samples was recorded as 36.49 and 6.29 days, respectively. Kinetics revealed that phorate degradation depends on time and the reaction follows the first order kinetics. Diethyl dithiophosphate was one of the degradation products, which is markedly less toxic than the parent compound; other degradation products included phorate sulfoxide and phorate sulfone. Release of inorganic phosphates and sulfates indicated the potential of the isolate to further degrade the above-mentioned metabolites to simpler forms. The strain was also found to poses phosphomonoesterase and phosphodiesterase enzymatic activity, which are involved in biodegradation of organophosphorus compounds. CONCLUSIONS: Ralstonia eutropha AAJ1 could degrade and detoxify phorate upto 85% in 10 days in laboratory conditions. SIGNIFICANCE AND IMPACT OF THE STUDY: The isolate has the potential to be utilized for remediation of phorate-contaminated water and soil.

Characterization of new isolated Ralstonia eutropha strain A-04 and kinetic study of biodegradable copolyester poly(3-hydroxybutyrate-co-4-hydroxybutyrate) production.

A new isolated bacterial strain A-04 capable of producing high content of polyhydroxyalkanoates (PHAs) was morphologically and taxonomically identified based on biochemical tests and 16S rRNA gene analysis. The isolate is a member of the genus Ralstonia and close to Ralstonia eutropha. Hence, this study has led to the finding of a new and unexplored R. eutropha strain A-04 capable of producing PHAs with reasonable yield. The kinetic study of poly(3-hydroxybutyrate-co-4-hydroxybutyrate) [P(3HB-co-4HB)] production by the R. eutropha strain A-04 was examined using butyric acid and gamma-hydroxybutyric acid as carbon sources. Effects of substrate ratio and mole ratio of carbon to nitrogen (C/N) on kinetic parameters were investigated in shake flask fed-batch cultivation. When C/N was 200, that is, nitrogen deficient condition, the specific production rate of 3-hydroxybutyrate (3HB) showed the highest value, whereas when C/N was in the range between 4 and 20, the maximum specific production rate of 4-hydroxybutyrate (4HB) was obtained. Thus, the synthesis of 3HB was growth-limited production under nitrogen-deficient condition, whereas the synthesis of 4HB was growth-associated production under nitrogen-sufficient condition. The mole fraction of 4HB units increased proportionally as the ratio of gamma-hydroxybutyric acid in the feed medium increased at any value of C/N ratio. Based on these kinetic studies, a simple strategy to improve P(3HB-co-4HB) production in shake flask fed-batch cultivation was investigated using C/N and substrate feeding ratio as manipulating variable, and was successfully proved by the experiments.

Isolation of the acrylamide denitrifying bacteria from a wastewater treatment system manufactured with polyacrylonitrile fiber.

Acrylamide has carcinogenicity and toxicity, so its discharge to natural water and soil systems might have an adverse impact on water quality, endangering public health and welfare. The investigation attempts to isolate acrylamide denitrifying bacteria from a wastewater treatment system manufactured with polyacrylonitrile (PAN) fiber. The goal is to elucidate the effectiveness of isolated pure strain and PAN mixed strains in treating acrylamide from synthetic wastewater. The results reveal that Ralstonia eutropha TDM-3 was isolated from the wastewater treatment system manufactured with PAN fiber. The PAN mixed strains and R. eutropha TDM-3 can consume up to 1446 mg/L acrylamide to denitrify from synthetic wastewater. Complete acrylamide removal depended on the supply of sufficient electron acceptors (nitrate). Strain R. eutropha TDM-3, Azoarcus sp. pF6, Azoarcus sp. T, and Herbaspirillum sp. G8A1 are related closely, according to the phylogenetic analyses of 16S rDNA sequences.

Heterodimeric nitrate reductase (NapAB) from Cupriavidus necator H16: purification, crystallization and preliminary X-ray analysis.

The periplasmic nitrate reductase from Cupriavidus necator (also known as Ralstonia eutropha) is a heterodimer that is able to reduce nitrate to nitrite. It comprises a 91 kDa catalytic subunit (NapA) and a 17 kDa subunit (NapB) that is involved in electron transfer. The larger subunit contains a molybdenum active site with a bis-molybdopterin guanine dinucleotide cofactor as well as one [4Fe-4S] cluster, while the small subunit is a di-haem c-type cytochrome. Crystals of the oxidized form of this enzyme were obtained using polyethylene glycol 3350 as precipitant. A single crystal grown at the High Throughput Crystallization Laboratory of the EMBL in Grenoble diffracted to beyond 1.5 A at the ESRF (ID14-1), which is the highest resolution reported to date for a nitrate reductase. The unit-cell parameters are a = 142.2, b = 82.4, c = 96.8 A, beta = 100.7 degrees, space group C2, and one heterodimer is present per asymmetric unit.

Recovery of poly(3-hydroxybutyrate) from high cell density culture of Ralstonia eutropha by direct addition of sodium dodecyl sulfate.

A simple and effective method for the recovery poly(3-hydroxybutyrate) [P(3HB)] directly from high cell density culture broth with no pretreatment steps has been developed. This method consists of direct addition of sodium dodecyl sulfate (SDS) to the culture broth, shaking, heat treatment, and washing steps. When the SDS/biomass ratio was higher than 0.4, the purity of recovered P(3HB) was over 95% for various cell concentrations of 50-300 g dry cell l(-1), with the highest value of 97%. The recovery of P(3HB) was over 90% regardless of cell concentration and SDS dosage (SDS/biomass ratios, 0.1-0.7). One g SDS digests 0.72 g non-P(3HB) cell materials. The reduction in molecular weight, due to degradation of P(3HB) by SDS, was negligible.

Microbial contamination of dialysate and its prevention in haemodialysis units.

At the haemodialysis centres of nine hospitals in Japan, microbial contamination of treated water (reverse osmosis method), acid and bicarbonate concentrates, and dialysate was investigated. Among these fluids used in haemodialysis, the dialysate was most frequently contaminated and had the highest concentration of bacteria. Of 40 dialysate samples analysed, 42.5% showed a bacterial count of more than 2000cfu/mL, which was above the Association for the Advancement of Medical Instrumentation (AAMI) standard. However, among the 40 samples from 20 dialysis machines, all six dialysate samples from three dialysis machines that used an ultrafiltration membrane in the circuit before the entrance of the dialysate into the dialyser, showed a bacterial count of < or =10 cfu/mL. In addition, when an ultrafiltration membrane was used in the circuit before the entrance of the dialysate into the dialyser for four dialysis machines showing dialysate samples contaminated with 10(4)-10(5)cfu/mL the bacterial count in dialysate samples from these machines became zero. Because dialysis machines are susceptible to microbial contamination, it is necessary to take measures such as placing an ultrafiltration membrane into the circuit before the entrance of dialysate into the dialyser.

Characterization of polychlorinated biphenyl-degrading bacteria isolated from contaminated sites in Czechia.

Biphenyl-utilizing polychlorinated biphenyls (PCB)-degrading bacteria were isolated from sites highly contaminated by PCBs, and their degradation abilities were determined using GC for typical commercial PCB mixtures (Delor 103 and Delor 106). Out of twelve strains which utilized biphenyl as a sole source of carbon and energy, strains Pseudomonas alcaligenes KP2 and P. fluorescens KP12, characterized by the BIOLOG identification system and the NEFERM test, were shown to significantly co-metabolize the PCB mixture Delor 103. DNA-DNA hybridization was used to compare both strains with well-known PCB-degraders Burkholderia cepacia strain LB400 and Ralstonia eutropha strain H850. The strain KP12 employs the same meta-fission route for degradation of chlorobenzoates as a chlorobiphenyl degrader Pseudomonas cepacia P166. Both isolates KP2 and KP12 belong to different phylogenetic groups, which indicates that the same geographical location does not ensure the same ancestor of degradative enzymes. We confirmed that also highly chlorinated and the most toxic congeners, which are contained in commercial PCB mixtures, can be biotransformed by members of indigenous bacterial-soil community under aerobic conditions.

Monitoring of Ralstonia eutropha KT1 in groundwater in an experimental bioaugmentation field by in situ PCR.

Ralstonia eutropha KT1, which degrades trichloroethylene, was injected into the aquifer after activation with toluene, and then the number of bacteria was monitored by in situ PCR targeting the phenol hydroxylase gene and by fluorescent in situ hybridization (FISH) targeting 16S rRNA. Before injection of the bacterial suspension, the total concentration of bacteria in the groundwater was approximately 3 x 10(5) cells/ml and the amount of Ralstonia and bacteria carrying the phenol hydroxylase gene as a percentage of total bacterial cells was less than 0.1%. The concentration of bacteria carrying the phenol hydroxylase gene detected by in situ PCR was approximately 3 x 10(7) cells/ml 1 h after injection, and the concentration of Ralstonia detected by FISH was similar. The number of bacteria detected by in situ PCR was similar to that detected by FISH 4 days after the start of the extraction of groundwater. On and after day 7, however, the number of bacterial cells detected by FISH was less than that detected by in situ PCR.

Isolation and characterization of a thermotolerant bacterium Ralstonia sp. strain PHS1 that degrades benzene, toluene, ethylbenzene, and o-xylene.

A thermotolerant bacterium, designated as PHS1, was isolated from a hot spring in Pohang, Korea, on the basis of its ability to grow on benzene, toluene, ethylbenzene, and xylenes (BTEX) as a sole carbon source. Strain PHS1 is a gram-negative, rod-shaped aerobe and grows optimally at 42 degrees C and pH 7.2. According to 16 S rDNA analysis, strain PHS1 showed highest similarity to Ralstonia eutropha (previously named Alcaligenes eutrophus). Unlike its closest known Ralstonia species, however, strain PHS1 was able to utilize toluene, ethylbenzene, o-xylene, and both m- and o-cresol. The degradation of o-xylene by strain PHS1 is particularly important, since o-xylene is a compound of considerable environmental interest, owing to its recalcitrance; and very few microorganisms have been reported to utilize o-xylene as a sole carbon source. It was found that strain PHS1 transformed o-xylene to 2,3-dimethylphenol through direct oxygenation of the aromatic ring. The unique properties of strain PHS1, such as thermotolerance and the ability to degrade o-xylene, may have important implications for the treatment of BTEX-contaminated industrial effluents.

Rapid identification of Ralstonia eutropha strain CH34 using monoclonal antibodies.

Ralstonia eutropha strain CH34 (formerly Alcaligenes eutrophus CH34) is an aerobic Gram-negative and facultative chemolithotrophic bacteria with plasmid-bound resistance to heavy metals. The presence of Ralstonia eutropha strain CH34 is an indication of environmental heavy metals pollution. The major purpose of this work was to produce monoclonal antibodies (MAbs) against the metal transport outer membrane proteins. In this way, bacteria outer membranes, grown with or without iron, were purified. The electrophoretic pattern of the outer membrane revealed that, in iron starvation conditions, at least four proteins were overexpressed. These outer membranes were used to immunize mice to produce MAbs. About 200 hybridomas were tested by enzyme-linked immunoadsorbent assay (ELISA). Most of these hybridomas exhibited cross reactions with Escherichia coli and Klebsiella aerogenes. Two hybridomas, AE5/7 and AE5/9, produced MAbs that detected specifically Ralstonia eutropha strain CH34. Analysis by Western blotting showed that these MAbs recognized a protein with a molecular weight of about 41 kDa. Moreover, the presence of the two megaplasmids was required for the full expression of the 41-kDa protein, as demonstrated by screening of the derivatives strains by ELISA. These MAbs could be used for a specific and rapid detection of Ralstonia eutropha strain CH34, using direct immunological methods.

Simultaneous detection of gfp-marked Moraxella sp. G21r and lux-marked Ralstonia eutrophas H850Lr using most-probable-number method.

The green fluorescent protein encoded by gfp gene and the luminescent protein encoded by luxAB genes were used as markers to detect p-nitrophenol (PNP)-degrading Moraxella sp. G21r and polychlorinated biphenyl (PCB)-degrading Ralstonia eutrophas H850Lr cells, respectively, in mixed liquid cultures and in soil samples using a most-probable-number (MPN) assay. Population estimates for both gfp-marked G21r and lux-marked H850Lr by using MPN assays were similar to viable colony counts. The MPN assay with microtiter plates permitted the simultaneous detection of fluorescent and luminescent bacteria in soil samples faster than conventional plate counting.

Modification of the aggregation behaviour of the environmental Ralstonia eutropha-like strain AE815 is reflected by both surface hydrophobicity and amplified fragment length polymorphism (AFLP) patterns.

After inoculation of the plasmid-free non-aggregative Ralstonia eutropha-like strain AE815 in activated sludge, followed by reisolation on a selective medium, a mutant strain A3 was obtained, which was characterized by an autoaggregative behaviour. Strain A3 had also acquired an IncP1 plasmid, pLME1, co-aggregated with yeast cells when co-cultured, and stained better with Congo red than did the AE815 strain. Contact angle measurements showed that the mutant strain was considerably more hydrophobic than the parent strain AE815, and scanning electron microscopy (SEM) revealed the production of an extracellular substance. A similar hydrophobic mutant (AE176R) could be isolated from the AE815-isogenic R. eutropha-like strain AE176. With the DNA fingerprinting technique repetitive extragenic palindromic-polymerase chain reaction (REP-PCR), no differences between these four strains, AE815, A3, AE176 and AE176R, could be revealed. However, using the amplified fragment length polymorphism (AFLP) DNA fingerprinting technique with three different primer combinations, small but clear reproducible differences between the banding patterns of the autoaggregative mutants and their non-autoaggregative parent strains were observed for each primer set. These studies demonstrate that, upon introduction of a strain in an activated sludge microbial community, minor genetic changes readily occur, which can nevertheless have major consequences for the phenotype of the strain and its aggregation behaviour.