Lectins and polysaccharide EPS I have flow-responsive roles in the attachment and biofilm mechanics of plant pathogenic Ralstonia.

Bacterial biofilm formation and attachment to hosts are mediated by carbohydrate-binding lectins, exopolysaccharides, and their interactions in the extracellular matrix (ECM). During tomato infection Ralstonia pseudosolanacearum (Rps) GMI1000 highly expresses three lectins: LecM, LecF, and LecX. The latter two are uncharacterized. We evaluated the roles in bacterial wilt disease of LecF, a fucose-binding lectin, LecX, a xylose-binding lectin, and the Rps exopolysaccharide EPS I. Interestingly, single and double lectin mutants attached to tomato roots better and formed more biofilm under static conditions in vitro. Consistent with this finding, static bacterial aggregation was suppressed by heterologous expression of lecFGMI1000 and lecXGMI1000 in other Ralstonia strains that naturally lack these lectins. Crude ECM from a ΔlecF/X double mutant was more adhesive than the wild-type ECM, and LecF and LecX increased Rps attachment to ECM. The enhanced adhesiveness of the ΔlecF/X ECM could explain the double mutant's hyper-attachment in static conditions. Unexpectedly, mutating lectins decreased Rps attachment and biofilm viscosity under shear stress, which this pathogen experiences in plant xylem. LecF, LecX, and EPS I were all essential for biofilm development in xylem fluid flowing through cellulose-coated microfluidic channels. These results suggest that under shear stress, LecF and LecX increase Rps attachment by interacting with the ECM and plant cell wall components like cellulose. In static conditions such as on root surfaces and in clogged xylem vessels, the same lectins suppress attachment to facilitate pathogen dispersal. Thus, Rps lectins have a dual biological function that depends on the physical environment.

Engineering a nicotinamide mononucleotide redox cofactor system for biocatalysis.

Biological production of chemicals often requires the use of cellular cofactors, such as nicotinamide adenine dinucleotide phosphate (NADP+). These cofactors are expensive to use in vitro and difficult to control in vivo. We demonstrate the development of a noncanonical redox cofactor system based on nicotinamide mononucleotide (NMN+). The key enzyme in the system is a computationally designed glucose dehydrogenase with a 107-fold cofactor specificity switch toward NMN+ over NADP+ based on apparent enzymatic activity. We demonstrate that this system can be used to support diverse redox chemistries in vitro with high total turnover number (~39,000), to channel reducing power in Escherichia coli whole cells specifically from glucose to a pharmaceutical intermediate, levodione, and to sustain the high metabolic flux required for the central carbon metabolism to support growth. Overall, this work demonstrates efficient use of a noncanonical cofactor in biocatalysis and metabolic pathway design.

Recent advances in constructing artificial microbial consortia for the production of medium-chain-length polyhydroxyalkanoates.

Polyhydroxyalkanoates (PHAs) are a class of high-molecular-weight polyesters made from hydroxy fatty acid monomers. PHAs produced by microorganisms have diverse structures, variable physical properties, and good biodegradability. They exhibit similar physical properties to petroleum-based plastics but are much more environmentally friendly. Medium-chain-length polyhydroxyalkanoates (mcl-PHAs), in particular, have attracted much interest because of their low crystallinity, low glass transition temperature, low tensile strength, high elongation at break, and customizable structure. Nevertheless, high production costs have hindered their practical application. The use of genetically modified organisms can reduce production costs by expanding the scope of substrate utilization, improving the conversion efficiency of substrate to product, and increasing the yield of mcl-PHAs. The yield of mcl-PHAs produced by a pure culture of an engineered microorganism was not high enough because of the limitations of the metabolic capacity of a single microorganism. The construction of artificial microbial consortia and the optimization of microbial co-cultivation have been studied. This type of approach avoids the addition of precursor substances and helps synthesize mcl-PHAs more efficiently. In this paper, we reviewed the design and construction principles and optimized control strategies for artificial microbial consortia that produce mcl-PHAs. We described the metabolic advantages of co-cultivating artificial microbial consortia using low-value substrates and discussed future perspectives on the production of mcl-PHAs using artificial microbial consortia.

Stable-Isotope Probing-Enabled Cultivation of the Indigenous Bacterium Ralstonia sp. Strain M1, Capable of Degrading Phenanthrene and Biphenyl in Industrial Wastewater.

To identify and obtain the indigenous degraders metabolizing phenanthrene (PHE) and biphenyl (BP) from the complex microbial community within industrial wastewater, DNA-based stable-isotope probing (DNA-SIP) and cultivation-based methods were applied in the present study. DNA-SIP results showed that two bacterial taxa (Vogesella and Alicyclobacillus) were considered the key biodegraders responsible for PHE biodegradation only, whereas Bacillus and Cupriavidus were involved in BP degradation. Vogesella and Alicyclobacillus have not been linked with PHE degradation previously. Additionally, DNA-SIP helped reveal the taxonomic identity of Ralstonia-like degraders involved in both PHE and BP degradation. To target the separation of functional Ralstonia-like degraders from the wastewater, we modified the traditional cultivation medium and culture conditions. Finally, an indigenous PHE- and BP-degrading strain, Ralstonia pickettii M1, was isolated via a cultivation-dependent method, and its role in PHE and BP degradation was confirmed by enrichment of the 16S rRNA gene and distinctive dioxygenase genes in the DNA-SIP experiment. Our study has successfully established a program for the application of DNA-SIP in the isolation of the active functional degraders from an environment. It also deepens our insight into the diversity of indigenous PHE- and BP-degrading communities.IMPORTANCE The comprehensive treatment of wastewater in industrial parks suffers from the presence of multiple persistent organic pollutants (POPs), such as polycyclic aromatic hydrocarbons (PAHs) and polychlorinated biphenyls (PCBs), which reduce the activity of activated sludge and are difficult to eliminate. Characterizing and applying active bacterial degraders metabolizing multiple POPs therefore helps to reveal the mechanisms of synergistic metabolism and to improve wastewater treatment efficiency in industrial parks. To date, SIP studies have successfully investigated the biodegradation of PAHs or PCBs in real-world habitats. DNA-SIP facilitates the isolation of target microorganisms that pose environmental concerns. Here, an indigenous phenanthrene (PHE)- and biphenyl (BP)-degrading strain in wastewater, Ralstonia pickettii M1, was isolated via a cultivation-dependent method, and its role in PHE and BP degradation was confirmed by DNA-SIP. Our study provides a routine protocol for the application of DNA-SIP in the isolation of the active functional degraders from an environment.

Identification and characterization of chemosensors for d-malate, unnatural enantiomer of malate, in Ralstonia pseudosolanacearum.

Ralstonia pseudosolanacearum Ps29 is attracted by nonmetabolizable d-malate, an unnatural enantiomer. Screening of a complete collection of single-mcp-gene deletion mutants of Ps29 revealed that the RSc1156 homologue is a chemosensor for d-malate. An RSc1156 homologue deletion mutant of Ps29 showed decreased but significant responses to d-malate, suggesting the existence of another d-malate chemosensor. McpM previously had been identified as a chemosensor for l-malate. We constructed an RSc1156 homologue mcpM double deletion mutant and noted that this mutant failed to respond to d-malate; thus, the RSc1156 homologue and McpM are the major chemosensors for d-malate in this organism. To further characterize the ligand specificities of the RSc1156 homologue and McpM, we constructed a Ps29 derivative (designated K18) harbouring deletions in 18 individual mcp genes, including mcpM and RSc1156. K18 harbouring the RSc1156 homologue responded strongly to l-tartrate and d-malate and moderately to d-tartrate, but not to l-malate or succinate. K18 harbouring mcpM responded strongly to l-malate and d-tartrate and moderately to succinate, fumarate and d-malate. Ps29 utilizes l-malate and l-tartrate, but not d-malate. We therefore concluded that l-tartrate and l-malate are natural ligands of the RSc1156 homologue and McpM, respectively, and that chemotaxis toward d-malate is a fortuitous response by the RSc1156 homologue and McpM in Ps29. We propose re-designation of the RSc1156 homologue as McpT. In tomato plant infection assays, the mcpT deletion mutant of highly virulent R. pseudosolanacearum MAFF106611 was as infectious as wild-type MAFF106611, suggesting that McpT-mediated chemotaxis does not play an important role in tomato plant infection.

Analysis of stable 1,2-dichlorobenzene-degrading enrichments and two newly isolated degrading strains, Acidovorax sp. sk40 and Ralstonia sp. sk41.

Stable degrading 1,2-dichlorobenzene (1,2-DCB) enrichments were generated from original contaminated soil and groundwater via enrichment procedures using a mineral salt medium containing 1,2-DCB as the sole carbon and energy source. Four transferred enrichments showed stable 1,2-DCB-degrading ability and completely degraded 1,2-DCB within 32 h. PCR-denaturing gradient gel electrophoresis (DGGE) and 16S rRNA gene clone library analyses indicated that two bacterial strains, belonging to Acidovorax spp. and Ralstonia spp., respectively, were the predominant organisms in each enrichment. Moreover, these strains maintained a stable coexistence in the four transferred enrichments. These two bacteria were subsequently identified as Acidovorax sp. strain sk40 and Ralstonia sp. strain sk41. Strain sk40 was more tolerant to higher concentrations of 1,2-DCB than strain sk41, while strain sk41 maintained a shorter degradation time under lower concentrations of 1,2-DCB. Notably, however, both strains exhibited similar growth rates and degradation rates in media containing 40 mg/l 1,2-DCB, as well as complete degradation of the 1,2-DCB (40 mg/l) within 32 h. It is expected that these two strains will be used in future applications of bioremediation of 1,2-DCB contamination.

EliA is required for inducing the stearyl alcohol-mediated expression of secretory proteins and production of polyester in Ralstonia sp. NT80.

Addition of stearyl alcohol to the culture medium of Ralstonia sp. NT80 induced expression of a significant amount of secretory lipase. Comparative proteomic analysis of extracellular proteins from NT80 cells grown in the presence or absence of stearyl alcohol revealed that stearyl alcohol induced expression of several secretory proteins including lipase, haemolysin-coregulated protein and nucleoside diphosphate kinase. Expression of these secreted proteins was upregulated at the transcriptional level. Stearyl alcohol also induced the synthesis of polyhydroxyalkanoate. Secretory protein EliA was required for all these responses of NT80 cells to stearyl alcohol. Accordingly, the effects of stearyl alcohol were significantly reduced in the eliA deletion mutant cells of NT80 (ΔeliA). The remaining concentration of stearyl alcohol in the culture supernatant of the wild-type cells, but not that in the culture supernatant of the ΔeliA cells, clearly decreased during the course of growth. These observed phenotypes of the ΔeliA mutant were rescued by gene complementation. The results suggested that EliA is essential for these cells to respond to stearyl alcohol, and that it plays an important role in the recognition and assimilation of stearyl alcohol by NT80 cells.

Metabolic engineering in chemolithoautotrophic hosts for the production of fuels and chemicals.

The ability of autotrophic organisms to fix CO2 presents an opportunity to utilize this 'greenhouse gas' as an inexpensive substrate for biochemical production. Unlike conventional heterotrophic microorganisms that consume carbohydrates and amino acids, prokaryotic chemolithoautotrophs have evolved the capacity to utilize reduced chemical compounds to fix CO2 and drive metabolic processes. The use of chemolithoautotrophic hosts as production platforms has been renewed by the prospect of metabolically engineered commodity chemicals and fuels. Efforts such as the ARPA-E electrofuels program highlight both the potential and obstacles that chemolithoautotrophic biosynthetic platforms provide. This review surveys the numerous advances that have been made in chemolithoautotrophic metabolic engineering with a focus on hydrogen oxidizing bacteria such as the model chemolithoautotrophic organism (Ralstonia), the purple photosynthetic bacteria (Rhodobacter), and anaerobic acetogens. Two alternative strategies of microbial chassis development are considered: (1) introducing or enhancing autotrophic capabilities (carbon fixation, hydrogen utilization) in model heterotrophic organisms, or (2) improving tools for pathway engineering (transformation methods, promoters, vectors etc.) in native autotrophic organisms. Unique characteristics of autotrophic growth as they relate to bioreactor design and process development are also discussed in the context of challenges and opportunities for genetic manipulation of organisms as production platforms.

Characterization of Ralstonia insidiosa C1 isolated from Alpine regions: Capability in polyhydroxyalkanoates degradation and production.

This study ventures into the exploration of potential poly-3-hydroxybutyrate (PHB) degradation in alpine environments. PHB-degrading bacteria were identified in both campus soil, representing a residential area, and Mt. Kurodake soil, an alpine region in Hokkaido, Japan. Next-generation sequencing analysis indicated that the campus soil exhibited higher microbial diversity, while Ralstonia insidiosa C1, isolated from Mt. Kurodake soil, displayed the highest proficiency in PHB degradation. R. insidiosa C1 efficiently degraded up to 3% (w/v) of PHB and various films composed of other biopolymers at 14 °C. This bacterium synthesized homopolymers using substrates such as 3-hydroxybutyric acid, sugars, and acetic acid, while also produced copolymers using a mixture of fatty acids. The analysis results confirmed that the biopolymer synthesized by strain C1 using glucose was PHB, with physical properties comparable to commercial products. The unique capabilities of R. insidiosa C1, encompassing both the production and degradation of bioplastics, highlight its potential to establish a novel material circulation model.

Evolutionary analysis and lateral gene transfer of two-component regulatory systems associated with heavy-metal tolerance in bacteria.

Microorganisms have adapted intricate signal transduction mechanisms to coordinate tolerance to toxic levels of metals, including two-component regulatory systems (TCRS). In particular, both cop and czc operons are regulated by TCRS; the cop operon plays a key role in bacterial tolerance to copper, whereas the czc operon is involved in the efflux of cadmium, zinc, and cobalt from the cell. Although the molecular physiology of heavy metal tolerance genes has been extensively studied, their evolutionary relationships are not well-understood. Phylogenetic relationships among heavy-metal efflux proteins and their corresponding two-component regulatory proteins revealed orthologous and paralogous relationships from species divergences and ancient gene duplications. The presence of heavy metal tolerance genes on bacterial plasmids suggests these genes may be prone to spread through horizontal gene transfer. Phylogenetic inferences revealed nine potential examples of lateral gene transfer associated with metal efflux proteins and two examples for regulatory proteins. Notably, four of the examples suggest lateral transfer across major evolutionary domains. In most cases, differences in GC content in metal tolerance genes and their corresponding host genomes confirmed lateral gene transfer events. Three-dimensional protein structures predicted for the response regulators encoded by cop and czc operons showed a high degree of structural similarity with other known proteins involved in TCRS signal transduction, which suggests common evolutionary origins of functional phenotypes and similar mechanisms of action for these response regulators.

Novel gene clusters and metabolic pathway involved in 3,5,6-trichloro-2-pyridinol degradation by Ralstonia sp. strain T6.

3,5,6-Trichloro-2-pyridinol (TCP) is a widespread pollutant. Some bacteria and fungi have been reported to degrade TCP, but the gene clusters responsible for TCP biodegradation have not been characterized. In this study, a fragment of the reduced flavin adenine dinucleotide (FADH2)-dependent monooxygenase gene tcpA was amplified from the genomic DNA of Ralstonia sp. strain T6 with degenerate primers. The tcpA disruption mutant strain T6-ΔtcpA could not degrade TCP but could degrade the green intermediate metabolite 3,6-dihydroxypyridine-2,5-dione (DHPD), which was generated during TCP biodegradation by strain T6. The flanking sequences of tcpA were obtained by self-formed adaptor PCR. tcpRXA genes constitute a gene cluster. TcpR and TcpX are closely related to the LysR family transcriptional regulator and flavin reductase, respectively. T6-ΔtcpA-com, the complementation strain for the mutant strain T6-ΔtcpA, recovered the ability to degrade TCP, and the strain Escherichia coli DH10B-tcpRXA, which expressed the tcpRXA gene cluster, had the ability to transform TCP to DHPD, indicating that tcpA is a key gene in the initial step of TCP degradation and that TcpA dechlorinates TCP to DHPD. A library of DHPD degradation-deficient mutants of strain T6 was obtained by random transposon mutagenesis. The fragments flanking the Mariner transposon were amplified and sequenced, and the dhpRIJK gene cluster was cloned. DhpJ could transform DHPD to yield an intermediate product, 5-amino-2,4,5-trioxopentanoic acid (ATOPA), which was further degraded by DhpI. DhpR and DhpK are closely related to the AraC family transcriptional regulator and the MFS family transporter, respectively.

Genome sequence of Ralstonia sp. strain PBA, a bacterium involved in the biodegradation of 4-aminobenzenesulfonate.

Ralstonia sp. strain PBA was isolated from textile wastewater in a coculture with Hydrogenophaga sp. strain PBC. Here we present the assembly and annotation of its genome, which may provide further insights into the mechanism of its interaction with strain PBC during 4-aminobenzenesulfonate degradation.

Molecular detection of plant pathogenic bacteria using polymerase chain reaction single-strand conformation polymorphism.

The application of polymerase chain reaction (PCR) technology to molecular diagnostics holds great promise for the early identification of agriculturally important plant pathogens. Ralstonia solanacearum, Xanthomoans axonopodis pv. vesicatoria, and Xanthomonas oryzae pv. oryzae are phytopathogenic bacteria, which can infect vegetables, cause severe yield loss. PCR-single-strand conformation polymorphism (PCR-SSCP) is a simple and powerful technique for identifying sequence changes in amplified DNA. The technique of PCR-SSCP is being exploited so far, only to detect and diagnose human bacterial pathogens in addition to plant pathogenic fungi. Selective media and serology are the commonly used methods for the detection of plant pathogens in infected plant materials. In this study, we developed PCR-SSCP technique to identify phytopathogenic bacteria. The PCR product was denatured and separated on a non-denaturing polyacrylamide gel. SSCP banding patterns were detected by silver staining of nucleic acids. We tested over 56 isolates of R. solanacearum, 44 isolates of X. axonopodis pv. vesicatoria, and 20 isolates of X. oryzae pv. oryzae. With the use of universal primer 16S rRNA, we could discriminate such species at the genus and species levels. Species-specific patterns were obtained for bacteria R. solanacearum, X. axonopodis pv. vesicatoria, and X. oryzae pv. oryzae. The potential use of PCR-SSCP technique for the detection and diagnosis of phytobacterial pathogens is discussed in the present paper.

Biodegradation kinetics of 4-fluorocinnamic acid by a consortium of Arthrobacter and Ralstonia strains.

Arthrobacter sp. strain G1 is able to grow on 4-fluorocinnamic acid (4-FCA) as sole carbon source. The organism converts 4-FCA into 4-fluorobenzoic acid (4-FBA) and utilizes the two-carbon side-chain for growth with some formation of 4-fluoroacetophenone as a dead-end side product. We also have isolated Ralstonia sp. strain H1, an organism that degrades 4-FBA. A consortium of strains G1 and H1 degraded 4-FCA with Monod kinetics during growth in batch and continuous cultures. Specific growth rates of strain G1 and specific degradation rates of 4-FCA were observed to follow substrate inhibition kinetics, which could be modeled using the kinetic models of Haldane-Andrew and Luong-Levenspiel. The mixed culture showed complete mineralization of 4-FCA with quantitative release of fluoride, both in batch and continuous cultures. Steady-state chemostat cultures that were exposed to shock loadings of substrate responded with rapid degradation and returned to steady-state in 10-15 h, indicating that the mixed culture provided a robust system for continuous 4-FCA degradation.

Intermittent trickling bed filter for the removal of methyl ethyl ketone and methyl isobutyl ketone.

Biodegradations of methyl ethyl ketone and methyl isobutyl ketone were performed in intermittent biotrickling filter beds (ITBF) operated at two different trickling periods: 12 h/day (ITBF-12) and 30 min/day (ITBF-0.5). Ralstonia sp. MG1 was able to degrade both ketones as evidenced by growth kinetic experiments. Results show that trickling period is an important parameter to achieve high removal performance and to maintain the robustness of Ralstonia sp. MG1. Overall, ITBF-12 outperformed ITBF-0.5 regardless of the target compound. ITBF-12 had high performance recovery at various inlet gas concentrations. The higher carbon dioxide production rates in ITBF-12 suggest higher microbial activity than in ITBF-0.5. Additionally, lower concentrations of absorbed volatile organic compound (VOC) in trickling solutions of ITBF-12 systems also indicate VOC removal through biodegradation. Pressure drop levels in ITBF-12 were relatively higher than in ITBF-0.5 systems, which can be attributed to the decrease in packed bed porosity as Ralstonia sp. MG1 grew well in ITBF-12. Nonetheless, the obtained pressure drop levels did not have any adverse effect on the performance of ITBF-12. Biokinetic constants were also obtained which indicated that ITBF-12 performed better than ITBF-0.5 and other conventional biotrickling filter systems.

Complete biodegradation of 4-fluorocinnamic acid by a consortium comprising Arthrobacter sp. strain G1 and Ralstonia sp. strain H1.

A consortium of the newly isolated bacterial strains Arthrobacter sp. strain G1 and Ralstonia sp. strain H1 utilized 4-fluorocinnamic acid for growth under aerobic conditions. Strain G1 converted 4-fluorocinnamic acid into 4-fluorobenzoic acid and used the two-carbon side chain for growth, with some formation of 4-fluoroacetophenone as a dead-end side product. In the presence of strain H1, complete mineralization of 4-fluorocinnamic acid and release of fluoride were obtained. Degradation of 4-fluorocinnamic acid by strain G1 occurred through a ß-oxidation mechanism and started with the formation of 4-fluorocinnamoyl-coenzyme A (CoA), as indicated by the presence of 4-fluorocinnamoyl-CoA ligase. Enzymes for further transformation were detected in cell extract, i.e., 4-fluorocinnamoyl-CoA hydratase, 4-fluorophenyl-ß-hydroxy propionyl-CoA dehydrogenase, and 4-fluorophenyl-ß-keto propionyl-CoA thiolase. Degradation of 4-fluorobenzoic acid by strain H1 proceeded via 4-fluorocatechol, which was converted by an ortho-cleavage pathway.

Differentiating the growing nosocomial infectious threats Ralstonia pickettii and Ralstonia insidiosa.

Differentiation of the growing nosocomial infectious threats, Ralstonia pickettii and Ralstonia insidiosa, based on nitrate reduction, desferrioxamine susceptibility, arabinose, N-acetyl-glucosamine and phenylacetate assimilation is described. These tests can be used for preliminary identification of Ralstonia pickettii and Ralstonia insidiosa resulting in more accurate identification of these species.

Characterization and quantification of the nickel resistant microbial community in activated sludge using 16S rDNA and nickel resistance genes.

The effect of nickel on the microbial community in the activated sludge of a sequencing batch reactor (SBR) reactor was investigated by continuously dosing nickel from 60 to 240 mg Ni(II) L(-1). The diversity of the microbial community was investigated by polymerase chain reaction (PCR) and denaturing gradient gel electrophoresis of the variable V3 region of the bacterial 16S rDNA. The experimental results showed that the community structure changed significantly after dosing with nickel with a shift in dominant species, the disappearance of some original species and the emergence of some new species. The existence of a nickel resistant gene was also investigated using PCR. The obtained nickel resistance gene had a maximum homology with the plasmid pMOL30 of Ralstonia metallidurans CH34. The quantitative real-time PCR results indicated that the quantity of the nickel resistance gene was related to the nickel concentration loaded to the reactor.

PhcQ mainly contributes to the regulation of quorum sensing-dependent genes, in which PhcR is partially involved, in Ralstonia pseudosolanacearum strain OE1-1.

The gram-negative plant-pathogenic ß-proteobacterium Ralstonia pseudosolanacearum strain OE1-1 produces methyl 3-hydroxymyristate as a quorum sensing (QS) signal via the methyltransferase PhcB and senses the chemical through the sensor histidine kinase PhcS. This leads to functionalization of the LysR family transcriptional regulator PhcA, regulating QS-dependent genes responsible for the QS-dependent phenotypes including virulence. The phc operon consists of phcB, phcS, phcR, and phcQ, with the latter two encoding regulator proteins with a receiver domain and a histidine kinase domain and with a receiver domain, respectively. To elucidate the function of PhcR and PhcQ in the regulation of QS-dependent genes, we generated phcR-deletion and phcQ-deletion mutants. Though the QS-dependent phenotypes of the phcR-deletion mutant were largely unchanged, deletion of phcQ led to a significant change in the QS-dependent phenotypes. Transcriptome analysis coupled with quantitative reverse transcription-PCR and RNA-sequencing revealed that phcB, phcK, and phcA in the phcR-deletion and phcQ-deletion mutants were expressed at similar levels as in strain OE1-1. Compared with strain OE1-1, expression of 22.9% and 26.4% of positively and negatively QS-dependent genes, respectively, was significantly altered in the phcR-deletion mutant. However, expression of 96.8% and 66.9% of positively and negatively QS-dependent genes, respectively, was significantly altered in the phcQ-deletion mutant. Furthermore, a strong positive correlation of expression of these QS-dependent genes was observed between the phcQ-deletion and phcA-deletion mutants. Our results indicate that PhcQ mainly contributes to the regulation of QS-dependent genes, in which PhcR is partially involved.

Modulating the active site lid of an alcohol dehydrogenase from Ralstonia sp. enabled efficient stereospecific synthesis of 17ß-hydroxysteroids.

Enzymatic stereospecific reduction of 17-oxosteroids offers an attractive approach to access 17ß-hydroxysteroids of pharmaceutical importance. In this study, by adjusting the flexibility of α6-helix at the substrate entrance of the alcohol dehydrogenase from Ralstonia sp. (RasADH), the catalytic activity toward the stereospecific 17ß-reduction of androstenedione was improved without sacrifice of the enantioselectivity. Among the mutants, F205I and F205A exhibited up to 623- and 523-fold improvement in catalytic efficiency, respectively, towards a range of different 17-oxosteroids compared to the wild-type enzyme. The corresponding 17ß-hydroxysteroids were prepared in optically pure form with high space-time productivity and isolated yields using F205I as the biocatalyst, indicating that these mutants are promising biocatalysts for this useful transformation. These results suggest that modulating the flexibility of the active site lid offers an effective approach to engineer alcohol dehydrogenase for accommodating bulky steroidal substrates.