Association between gentamicin resistance and stress tolerance in water isolates of Ralstonia pickettii and R. mannitolilytica.

Members of the species Ralstonia pickettii and R. mannitolilytica, although ubiquitous and lacking major virulence factors, have been associated with nosocomial outbreaks. Tolerance to metals, antibiotics, and disinfectants may represent an advantage for their ubiquity and opportunistic pathogenic potential. In this study, we compared five strains that differed on the origin (hospital effluent, tap water, mineral water) and in the susceptibility to aminoglycosides, regarding their tolerance to metals and disinfection. The growth kinetics and biofilm formation capacity were tested in four R. pickettii strains and one R. mannitolilytica at sub-inhibitory concentrations of aminoglycosides or arsenite. The survival to UV radiation, chlorine, or hydrogen peroxide was also compared in aminoglycoside resistant and susceptible strains. Aminoglycoside-resistant strains presented a higher tolerance to arsenite than the susceptible ones and either aminoglycosides or arsenite was observed to stimulate the biofilm formation. Sub-inhibitory concentrations of the aminoglycoside gentamicin or arsenite significantly decreased the growth rate and yield, but only arsenite caused a significant increase of the lag phase. Hydrogen peroxide presented higher disinfection effectiveness against aminoglycoside susceptible than against resistant strains, an effect that was not observed for UV or chlorine. Although this conclusion needs validation based on a larger number of isolates, including clinical, the results suggest that aminoglycoside resistance may be associated with traits that influence Ralstonia spp. fitness in the environment.

An innovative nutritional slow-release packing material with functional microorganisms for biofiltration: Characterization and performance evaluation.

The type of packing material for biofiltration has a great impact on microbial growth and pollutant removal. This study evaluated the feasibility of a nutritional slow-release packing material with functional microorganisms (NSRP-FM) in a biofilter for the removal of gaseous n-butyl acetate. Through the emulsification-cross linked process and microbial immobilization, an innovative packing material was obtained, with a specific surface area of 2.45 m2 g-1 and a bulk density of 40.75 kg m-3. The cumulative release rates of total phosphorus and total nitrogen were 90.6% and 75.6%, respectively, as measured while continuously spraying deionized water. To evaluate the performance of biofiltration, NSRP-FM was compared with the commercial polyurethane foam (PU-foam), in two identical biotrickling filters (BTFs). The BTF packed with the prepared NSRP-FM maintained a consistent removal efficiency (over 95%) without nutrients addition and pH adjustment. The other BTF had poor removal performance, and the removal efficiency declined to 65% when there was no pH adjustment. Energy dispersive X-ray spectroscopy (EDS) analysis of NSRP-FM showed that inorganic elements were released during the operation of BTF. The abundance of functional microorganisms suggested that the prepared NSRP-FM provided a better environment for microbial growth, despite changes in the operating conditions.

Kinetic Mechanism of the Dechlorinating Flavin-dependent Monooxygenase HadA.

The accumulation of chlorophenols (CPs) in the environment, due to their wide use as agrochemicals, has become a serious environmental problem. These organic halides can be degraded by aerobic microorganisms, where the initial steps of various biodegradation pathways include an oxidative dechlorinating process in which chloride is replaced by a hydroxyl substituent. Harnessing these dechlorinating processes could provide an opportunity for environmental remediation, but detailed catalytic mechanisms for these enzymes are not yet known. To close this gap, we now report transient kinetics and product analysis of the dechlorinating flavin-dependent monooxygenase, HadA, from the aerobic organism Ralstonia pickettii DTP0602, identifying several mechanistic properties that differ from other enzymes in the same class. We first overexpressed and purified HadA to homogeneity. Analyses of the products from single and multiple turnover reactions demonstrated that HadA prefers 4-CP and 2-CP over CPs with multiple substituents. Stopped-flow and rapid-quench flow experiments of HadA with 4-CP show the involvement of specific intermediates (C4a-hydroperoxy-FAD and C4a-hydroxy-FAD) in the reaction, define rate constants and the order of substrate binding, and demonstrate that the hydroxylation step occurs prior to chloride elimination. The data also identify the non-productive and productive paths of the HadA reactions and demonstrate that product formation is the rate-limiting step. This is the first elucidation of the kinetic mechanism of a two-component flavin-dependent monooxygenase that can catalyze oxidative dechlorination of various CPs, and as such it will serve as the basis for future investigation of enzyme variants that will be useful for applications in detoxifying chemicals hazardous to human health.

The regulatory mechanism of 2,4,6-trichlorophenol catabolic operon expression by HadR in Ralstonia pickettii DTP0602.

Ralstonia pickettii DTP0602 utilizes 2,4,6-trichlorophenol (2,4,6-TCP) as its sole source of carbon. The expression of catabolic pathway genes (hadA, hadB and hadC) for 2,4,6-TCP has been reported to be regulated by the LysR-type transcriptional regulator (LTTR) HadR. Generally, coinducers are recognized as being important for the function of LTTRs, and alteration of the LTTR-protection sequence and the degree of DNA bending are characteristic of LTTRs with or without a recognized coinducer. In this study, we describe the mechanism by which HadR regulates the expression of 2,4,6-TCP catabolic genes. The 2,4,6-TCP catabolic pathway genes in DTP0602 consist of two transcriptional units: hadX-hadA-hadB-hadC and monocistronic hadR. Purified HadR binds to the hadX promoter and HadR-DNA complex formation was induced in the presence of 16 types of substituted phenols, including chloro- and nitro-phenols and tribromo-phenol. In contrast with observations of other well-characterized LTTRs, the tested phenols showed no diversity of the bending angle of the HadR binding fragment. The expression of 2,4,6-TCP catabolic pathway genes, which are regulated by HadR, was not influenced by the DNA bending angle of HadR. Moreover, the transcription of hadX, hadA and hadB was induced in the presence of seven types of substituted phenols, whereas the other substituted phenols, which induced formation of the HadR-DNA complex, did not induce the transcription of hadX, hadA or hadB in vivo.

Surveillance of single-cell behavior in different subpopulations of Ralstonia pickettii AR1 during growth and polyhydroxybutyrate production phases by flow cytometry.

Most bacterial strains accumulate intracellular polyhydroxybutyrate (PHB) granules as an energy reservoir, in response to fluctuations in their microenvironment. Flow cytometry was applied for the analysis of single cells of Ralstonia pickettii AR1 in response to changes in the culture conditions. Two parameters, the PHB production-related FL2 and side scatter (SSC) parameters, were used to monitor, distinguish and characterize different subpopulations in the growth and PHB production phases. A high SSC level was observed in the mid-log exponential growth phase. When the nitrogen source reached a limiting level, the SSC started to decrease, in contrast to the intracellular PHB granules-related FL2 parameter. The results show that ammonium limitation is a critical and important factor for the accumulation of reserve compounds. Four subpopulations were observed and distinguished upon entrance of the cells into the exponential growth phase. When the cells entered the late exponential growth or early stationary phase, two subpopulations had disappeared and only two, different subpopulations were dominant. One of the subpopulations with changed SSC and PHB production activity switched to another subpopulation that was only active in PHB production in the stationary phase. The whole cells of R. pickettii AR1 tended to form a homogeneous population at the end of the stationary phase. In fact, the changes in the subpopulations of a single strain are related to different physiological states of the cells. The observation of different subpopulations suggests that each subpopulation shows a specific response to changes in the surrounding microenvironment, nutrients and limiting factors.

Degradation of chlorobenzene by strain Ralstonia pickettii L2 isolated from a biotrickling filter treating a chlorobenzene-contaminated gas stream.

A Ralstonia pickettii species able to degrade chlorobenzene (CB) as the sole source of carbon and energy was isolated from a biotrickling filter used for the removal of CB from waste gases. This organism, strain L2, could degrade CB as high as 220 mg/L completely. Following CB consumption, stoichiometric amounts of chloride were released, and CO2 production rate up to 80.2% proved that the loss of CB was mainly via mineralization and incorporation into cell material. The Haldane modification of the Monod equation adequately described the relationship between the specific growth rate and substrate concentration. The maximum specific growth rate and yield coefficient were 0.26 h⁻¹ and 0.26 mg of biomass produced/mg of CB consumed, respectively. The pathways for CB degradation were proposed by the identification of metabolites and assay of ring cleavage enzymes in cell extracts. CB was degraded predominantly via 2-chlorophenol to 3-chlorocatechol and also partially via phenol to catechol with subsequent ortho ring cleavage, suggesting partially new pathways for CB-utilizing bacteria.

Biosequestration via cooperative binding of copper by Ralstonia pickettii.

Ralstonia pickettii isolated from copper-contaminated lake sediment are adapted to high levels of copper after 100 years of selective pressure. Two R. pickettii strains (12D and 12J) were selected for the studies reported herein due to their distinct differences in genomic structure, different metal resistance patterns and carriage of a filamentous phage. Copper sequestration studies revealed that these strains could bind up to 27.44 (12D) and 38.19 (12J) mg copper per g dry weight of cells and that viable cells sequestered more copper than heat-killed cells. Viable cells and heat-killed cells had significantly different saturation binding curves, indicating that one or more unique copper sequestration mechanism(s) was involved in binding by viable cells. Electron microscopy showed alteration of cell outer envelope after cells were grown in the presence of copper, suggesting that the accumulation of copper was membrane associated. X-ray Absorption Near Edge Structure and Extended X-ray Absorption Fine Structure revealed that the copper sequestered was present as Cu(II) and bound to oxygen and/or nitrogen. Recent completion of the genome sequence revealed that an approximately 220 kb region was enriched with metal resistance and transporter genes found in multiple copies. Comparative sequence analysis revealed that several genes may have been derived from horizontal transfer. Hence, rapid adaptation of R. pickettii to high concentrations of metal appears due to robust gene duplication and importation of several types of resistance determinants.

Characterization of regulatory element Rp3 of regulation of beta-lactamases from Ralstonia pickettii.

The two chromosomally encoded beta-lactamases, OXA-22 and OXA-60, from Ralstonia pickettii are inducible by beta-lactam molecules. Disruption of RP3 abolished induction of both beta-lactamases and the resistance to pH, osmolarity and survival in the stationary phase, suggesting that RP3 might be a global regulator. Interactions between RP3, OXA-22 and OXA-60 were investigated at a transcript and protein level using 5'-rapid amplification of cDNA ends experiments, real-time reverse transcription (RT)-PCR and footprinting assays. The rp3 gene was actively transcribed and the promoter sequences corresponded to a nontypical sigma(70)-type promoter. RT-PCR analysis showed that rp3 expression as well as that of the bla(OXA) genes was positively regulated: the level of transcripts of rp3, bla(OXA-22) and bla(OXA-60) genes were, respectively, increased 20-, 100- and 2000-fold upon imipenem induction. DNAse I footprinting showed that RP3 specifically bound to tandem repeats centered at positions -55.5 and -73.5 upstream from the bla(OXA-22) and bla(OXA-60) transcriptional start sites. Interestingly, the binding site at bla(OXA-60) overlapped the -35 region of the rp3 promoter, although the region essential for induction lies at the beginning of the orf-rp3. This result indicates that RP3 is most probably only one component of a novel regulatory system involved in the expression of beta-lactamases in R. pickettii.

Benzene degradation by Ralstonia pickettii PKO1 in the presence of the alternative substrate succinate.

The regulation of benzene degradation by Ralstonia pickettii PKO1 in the presence of the alternative substrate succinate was investigated in batch and continuous culture. In batch culture, R. pickettii PKO1 achieved a maximum specific growth rate with benzene of 0.18 h(-1), while succinate allowed much faster growth (mu(max) = 0.5 h(-1)). Under carbon excess conditions succinate repressed benzene consumption resulting in diauxic growth whereas under carbon-limited conditions in the chemostat both substrates were used simultaneously. Moreover, the effect of succinate on the adaptation towards growth with benzene was investigated in carbon-limited continuous culture at a dilution rate of 0.1 h(-1) by changing the inflowing carbon substrate from succinate to different mixtures of benzene and succinate. The adaptation process towards utilisation of benzene was rather complex. Three to seven hours after the medium shift biomass production from benzene started. Higher proportions of succinate in the mixture had a positive effect on both the onset of biomass production and on the time required for induction of benzene utilisation. Strikingly, after the initial increase in biomass and benzene-catabolising activities, the culture collapsed regularly and wash-out of biomass was observed. After a transient phase of low biomass concentrations growth on benzene resumed so that finally rather stable and high biomass concentrations were reached. The decrease in biomass and degradative activities cannot be explained so far, but the possibilities of either intoxication of the cells by benzene itself, or of inhibition by degradation intermediates were ruled out.

Efficient biocatalytic production of D-4-hydroxyphenylglycine by whole cells of recombinant Ralstonia pickettii.

The first establishment of a homologous expression system in the host Ralstonia pickettii CGMCC1596 using the compatible broad-host-range plasmid pWB5 is described. When whole cells of the recombinant strain R. pickettii MMYY01 (CGMCC1596/pYY05) were used as the biocatalyst to transform DL-4-hydroxyphenylhydantoin (DL-HPH) to D-4-hydroxyphenylglycine (D-HPG), the conversion rate reached 94 % in first 9 h, at a production rate of 2.8 g L(-1) h(-1), with the rapid reduction of the intermediate [N-carbamoyl-2-(4-hydroxyphenyl)glycine], compared with 80 % in >50 h at a rate of 0.5 g L(-1) h(-1) for the CGMCC1596. The stability of the recombinant plasmid pYY05 is sufficient for its application in industrial batch fermentation. An alternative strategy for the conversion of DL-HPH to D-HPG by resting CGMCC1596 cells and heterologous DCase expressed by E. coli is discussed.

Outer-membrane transport of aromatic hydrocarbons as a first step in biodegradation.

Bacterial biodegradation of hydrocarbons, an important process for environmental remediation, requires the passage of hydrophobic substrates across the cell membrane. Here, we report crystal structures of two outer membrane proteins, Pseudomonas putida TodX and Ralstonia pickettii TbuX, which have been implicated in hydrocarbon transport and are part of a subfamily of the FadL fatty acid transporter family. The structures of TodX and TbuX show significant differences with those previously determined for Escherichia coli FadL, which may provide an explanation for the substrate-specific transport of TodX and TbuX observed with in vivo transport assays. The TodX and TbuX structures revealed 14-stranded beta-barrels with an N-terminal hatch domain blocking the barrel interior. A hydrophobic channel with bound detergent molecules extends from the extracellular surface and is contiguous with a passageway through the hatch domain, lined by both hydrophobic and polar or charged residues. The TodX and TbuX structures support a mechanism for transport of hydrophobic substrates from the extracellular environment to the periplasm via a channel through the hatch domain.

Streptomyces griseus enhances denitrification by Ralstonia pickettii K50, which is possibly mediated by histidine produced during co-culture.

Ralstonia pickettii K50 (strain K50) is a denitrifying bacterium that produces low levels of N(2)O under aerobic conditions. In this study, we found that co-culturing of strain K50 with Streptomyces griseus significantly enhanced the denitrification activity of strain K50 in an artificial wastewater (AWW) system. Most factors that enhance denitrification activity were in the high molecular weight fraction of the cell-free broth of S. griseus, and were suggested to be extracellular proteases. Further investigation revealed that the cultivation of strain K50 in protease-treated AWW medium fully enhanced denitrification, and that a shortage of amino acids in the medium limited it. Among the 20 standard amino acids tested, only histidine had a significant effect in inducing denitrification by strain K50. Our results indicate that histidine is a novel inducer of bacterial denitrification.

Metabolism of thioamides by Ralstonia pickettii TA.

Information on bacterial thioamide metabolism has focused on transformation of the antituberculosis drug ethionamide and related compounds by Mycobacterium tuberculosis. To study this metabolism more generally, a bacterium that grew using thioacetamide as the sole nitrogen source was isolated via enrichment culture. The bacterium was identified as Ralstonia pickettii and designated strain TA. Cells grown on thioacetamide also transformed other thioamide compounds. Transformation of the thioamides tested was dependent on oxygen. During thioamide degradation, sulfur was detected in the medium at the oxidation level of sulfite, further suggesting an oxygenase mechanism. R. pickettii TA did not grow on thiobenzamide as a nitrogen source, but resting cells converted thiobenzamide to benzamide, with thiobenzamide S-oxide and benzonitrile detected as intermediates. Thioacetamide S-oxide was detected as an intermediate during thioacetamide degradation, but the only accumulating metabolite of thioacetamide was identified as 3,5-dimethyl-1,2,4-thiadiazole, a compound shown to derive from spontaneous reaction of thioacetamide and oxygenated thioacetamide species. This dead-end metabolite accounted for only ca. 12% of the metabolized thioacetamide. Neither acetonitrile nor acetamide was detected during thioacetamide degradation, but R. pickettii grew on both compounds as nitrogen and carbon sources. It is proposed that R. pickettii TA degrades thioamides via a mechanism involving consecutive oxygenations of the thioamide sulfur atom.

Effects of mutations in the substrate-binding domain of poly[(R)-3-hydroxybutyrate] (PHB) depolymerase from Ralstonia pickettii T1 on PHB degradation.

Poly[(R)-3-hydroxybutyrate] (PHB) depolymerase from Ralstonia pickettii T1 (PhaZ(RpiT1)) adsorbs to denatured PHB (dPHB) via its substrate-binding domain (SBD) to enhance dPHB degradation. To evaluate the amino acid residues participating in dPHB adsorption, PhaZ(RpiT1) was subjected to a high-throughput screening system consisting of PCR-mediated random mutagenesis targeted to the SBD gene and a plate assay to estimate the effects of mutations in the SBD on dPHB degradation by PhaZ(RpiT1). Genetic analysis of the isolated mutants with lowered activity showed that Ser, Tyr, Val, Ala, and Leu residues in the SBD were replaced by other residues at high frequency. Some of the mutant enzymes, which contained the residues replaced at high frequency, were applied to assays of dPHB degradation and adsorption, revealing that those residues are essential for full activity of both dPHB degradation and adsorption. These results suggested that PhaZ(RpiT1) adsorbs on the surface of dPHB not only via hydrogen bonds between hydroxyl groups of Ser in the enzyme and carbonyl groups in the PHB polymer but also via hydrophobic interaction between hydrophobic residues in the enzyme and methyl groups in the PHB polymer. The L441H enzyme, which displayed lower dPHB degradation and adsorption abilities, was purified and applied to a dPHB degradation assay to compare it with the wild-type enzyme. The kinetic analysis of the dPHB degradation suggested that lowering the affinity of the SBD towards dPHB causes a decrease in the dPHB degradation rate without the loss of its hydrolytic activity for the polymer chain.

Comparison of bioMérieux API 20NE and Remel RapID NF Plus, identification systems of type strains of Ralstonia pickettii.

UNLABELLED: A : Comparison of two commercial miniaturized rapid systems for the identification of Ralstonia pickettii strains. METHODS AND RESULTS: Varying identification results were encountered using the bioMérieux API NE system and the Remel IDS RapID NF Plus commercial systems for R. pickettii. To compare these two systems, eight strains of R. pickettii were purchased from different commercial culture collections. Additionally, 32 industrial and eight clinical isolates, initially identified using the Vitek Junior (bioMérieux) were tested. Total number of isolates tested was 48. The API 20NE identified 29 isolates, as R. pickettii but was unsuccessful with 19 isolates. The Remel IDS RapID NF Plus identified 46 isolates as R. pickettii. One clinical and one industrial isolates was identified as non-R. pickettii with both systems. CONCLUSIONS: The above results indicate that the use of API 20NE system for examining the identification of R. pickettii strains is inconsistent. SIGNIFICANCE AND IMPACT OF THE STUDY: This study demonstrated that the RapID NF Plus is more accurate as an inexpensive identification system for the identification of R. pickettii, a potential emerging organism of medically and industrial importance.