Comamonas resistens sp. nov. and Pseudomonas triclosanedens sp. nov., two members of the phylum Pseudomonadota isolated from the wastewater treatment system of a pharmaceutical factory.

Five strains of two novel species were isolated from the wastewater treatment systems of a pharmaceutical factory located in Zhejiang province, PR China. Strains ZM22T and Y6 were identified as belonging to a potential novel species of the genus Comamonas, whereas strains ZM23T, ZM24 and ZM25 were identified as belonging to a novel species of the genus Pseudomonas. These strains were characterized by polyphasic approaches including 16S rRNA gene analysis, multi-locus sequence analysis, average nucleotide identity (ANI), in silico DNA-DNA hybridization (isDDH), physiological and biochemical tests, as well as chemotaxonomic analysis. Genome-based phylogenetic analysis further confirmed that strains ZM22T and Y6 form a distinct clade closely related to Comamonas testosteroni ATCC 11996T and Comamonas thiooxydans DSM 17888T. Strains ZM23T, ZM24 and ZM25 were grouped as a separate clade closely related to Pseudomonas nitroreducens DSM 14399T and Pseudomonas nicosulfuronedens LAM1902T. The orthoANI and isDDH results indicated that strains ZM22T and Y6 belong to the same species. In addition, genomic DNA fingerprinting demonstrated that these strains do not originate from a single clone. The same results were observed for strains ZM23T, ZM24 and ZM25. Strains ZM22T and Y6 were resistant to multiple antibiotics, whereas strains ZM23T, ZM24 and ZM25 were able to degrade an emerging pollutant, triclosan. The phylogenetic, physiological and biochemical characteristics, as well as chemotaxonomy, allowed these strains to be distinguished from their genus, and we therefore propose the names Comamonas resistens sp. nov. (type strain ZM22=MCCC 1K08496T=KCTC 82561T) and Pseudomonas triclosanedens sp. nov. (type strain ZM23T=MCCC 1K08497T=JCM 36056T), respectively.

An Ultra-Sensitive Comamonas thiooxidans Biosensor for the Rapid Detection of Enzymatic Polyethylene Terephthalate (PET) Degradation.

Polyethylene terephthalate (PET) is a prevalent synthetic polymer that is known to contaminate marine and terrestrial environments. Currently, only a limited number of PET-active microorganisms and enzymes (PETases) are known. This is in part linked to the lack of highly sensitive function-based screening assays for PET-active enzymes. Here, we report on the construction of a fluorescent biosensor based on Comamonas thiooxidans strain S23. C. thiooxidans S23 transports and metabolizes TPA, one of the main breakdown products of PET, using a specific tripartite tricarboxylate transporter (TTT) and various mono- and dioxygenases encoded in its genome in a conserved operon ranging from tphC-tphA1. TphR, an IclR-type transcriptional regulator is found upstream of the tphC-tphA1 cluster where TPA induces transcription of tphC-tphA1 up to 88-fold in exponentially growing cells. In the present study, we show that the C. thiooxidans S23 wild-type strain, carrying the sfGFP gene fused to the tphC promoter, senses TPA at concentrations as low as 10 µM. Moreover, a deletion mutant lacking the catabolic genes involved in TPA degradation thphA2-A1 (ΔtphA2A3BA1) is up to 10,000-fold more sensitive and detects TPA concentrations in the nanomolar range. This is, to our knowledge, the most sensitive reporter strain for TPA and we demonstrate that it can be used for the detection of enzymatic PET breakdown products. IMPORTANCE Plastics and microplastics accumulate in all ecological niches. The construction of more sensitive biosensors allows to monitor and screen potential PET degradation in natural environments and industrial samples. These strains will also be a valuable tool for functional screenings of novel PETase candidates and variants or monitoring of PET recycling processes using biocatalysts. Thereby they help us to enrich the known biodiversity and efficiency of PET degrading organisms and enzymes and understand their contribution to environmental plastic degradation.

Quantitative proteomic analysis of the microbial degradation of 3-aminobenzoic acid by Comamonas sp. QT12.

A mab cluster associated with 3-aminobenzoic acid (3AB) degradation was identified in Comamonas sp. QT12. However, the cellular response of Comamonas sp. QT12 to 3-aminobenzoic acid remains unclear. In this study, label-free quantitative proteome analysis based on LC-MS/MS was used to study the protein expression difference of strain QT12 under the condition of using 3AB (3AB) and citric acid/ammonium chloride as substrates (3ABCon). A total of 2068 proteins were identified, of which 239 were significantly up-regulated in 3AB group, 124 were significantly down-regulated in 3AB group, 624 were expressed only in 3AB group, and 216 were expressed only in 3ABCon group in 3AB group. KEGG pathway analysis found that 83 pathways were up-regulated and 49 pathways were down-regulated, In GO analysis, 315 paths were up-regulated and 156 paths were down-regulated. There were 6 genes in the mab cluster that were only detected in the 3AB group.The mab cluster was found to be related to degradation of 3AB. By knockout, it was found that the growth rate of the mutant △orf7 and △orf9 were slowed down. HPLC results showed that the mutant △orf7 and △orf9 could still degrade 3AB, it was found that orf7, orf9 were not key genes about 3AB degradation and they could be replaced by other genes in strain QT12. These findings improve our understanding of the molecular mechanisms underlying the cellular response of 3AB degradation in Comamonas bacterium.

Comamonas fluminis sp. nov., isolated from the Han River, Republic of Korea.

A Gram-stain-negative, aerobic and motile bacterial strain, designated CJ34T, was isolated from Han River water in the Republic of Korea. Strain CJ34T grew optimally on tryptic soy agar at 30 °C and pH 7.0 in the absence of NaCl. Results of phylogenetic analysis based on 16S rRNA gene sequence showed that strain CJ34T belonged to the genus Comamonas within the family Comamonadaceae and was most closely related to Comamonas testosteroni ATCC 11996T and Comamonas thiooxydans DSM 17888T (both 98.63 % similarity). The average nucleotide identity values between strain CJ34T and two closely related type strains C. testosteroni ATCC 11996T and C. thiooxydans DSM 17888T were 82.77 and 82.73 %, respectively. The major isoprenoid quinone of strain CJ34T was ubiquinone Q-8. The major cellular fatty acids of strain CJ34T were C16 : 0, C16 : 1 ω6c and/or C16 : 1 ω7c and C18 : 1 ω6c and/or C18 : 1 ω7c. The predominant polar lipids of strain CJ34T were diphosphatidylglycerol, phosphatidylethanolamine, phosphatidylglycerol and an unidentified aminophospholipid. Whole genome sequencing revealed that strain CJ34T had a genome of 4.9 Mbp and the G+C content of the genomic DNA was 59.73 mol%. On the basis of the results of this polyphasic taxonomy study, strain CJ34T represents a novel species in the genus Comamonas, for which the name Comamonas fluminis sp. nov. is proposed. The type strain is CJ34T (=KACC 22237T=JCM 34454T).

Structural basis of terephthalate recognition by solute binding protein TphC.

Biological degradation of Polyethylene terephthalate (PET) plastic and assimilation of the corresponding monomers ethylene glycol and terephthalate (TPA) into central metabolism offers an attractive route for bio-based molecular recycling and bioremediation applications. A key step is the cellular uptake of the non-permeable TPA into bacterial cells which has been shown to be dependent upon the presence of the key tphC gene. However, little is known from a biochemical and structural perspective about the encoded solute binding protein, TphC. Here, we report the biochemical and structural characterisation of TphC in both open and TPA-bound closed conformations. This analysis demonstrates the narrow ligand specificity of TphC towards aromatic para-substituted dicarboxylates, such as TPA and closely related analogues. Further phylogenetic and genomic context analysis of the tph genes reveals homologous operons as a genetic resource for future biotechnological and metabolic engineering efforts towards circular plastic bio-economy solutions.

Genes Encoding Microbial Acyl Coenzyme A Binding Protein/Diazepam-Binding Inhibitor Orthologs Are Rare in the Human Gut Microbiome and Show No Links to Obesity.

Acyl coenzyme A (CoA) binding protein (ACBP), also called diazepam-binding inhibitor (DBI), is a phylogenetically conserved protein that is expressed by all eukaryotic species as well as by some bacteria. Since elevated ACBP/DBI levels play a major role in the inhibition of autophagy, increase in appetite, and enhanced lipid storage that accompany obesity, we wondered whether ACBP/DBI produced by the human microbiome might affect host weight. We found that the genomes of bacterial commensals rarely contain ACBP/DBI homologues, which are rather encoded by genomes of some pathogenic or environmental taxa that were not prevalent in human feces. Exhaustive bioinformatic analyses of 1,899 gut samples from healthy individuals refuted the hypothesis that bacterial ACBP/DBI might affect the body mass index (BMI) in a physiological context. Thus, the physiological regulation of BMI is unlikely to be affected by microbial ACBP/DBI-like proteins. However, at the speculative level, it remains possible that ACBP/DBI produced by potential pathogenic bacteria might enhance their virulence by inhibiting autophagy and hence subverting innate immune responses. IMPORTANCE Acyl coenzyme A (CoA) binding protein (ACBP) can be encoded by several organisms across the domains of life, including microbes, and has shown to play major roles in human metabolic processes. However, little is known about its presence in the human gut microbiome and whether its microbial counterpart could also play a role in human metabolism. In the present study, we found that microbial ACBP/DBI sequences were rarely present in the gut microbiome across multiple metagenomic data sets. Microbes that carried ACBP/DBI in the human gut microbiome included Saccharomyces cerevisiae, Lautropia mirabilis, and Comamonas kerstersii, but these microorganisms were not associated with body mass index, further indicating an unconvincing role for microbial ACBP/DBI in human metabolism.

First report of urinary tract infection caused by Comamonas kerstersii in a goat.

BACKGROUND: Comamonas kerstersii is rarely associated with infections in humans and has never been reported in animals until now. CASE PRESENTATION: Herein, we describe a case of urinary tract infection caused by C. kerstersii in a young goat. A seven-month-old male goat showed lethargy, generalised weakness and anorexia and in the last hours before its death, severe depression, slight abdominal distention, ruminal stasis, and sternal recumbency. Grossly, multifocal haemorrhages in different organs and tissues, subcutaneous oedema and hydrocele, serous fluid with scattered fibrin deposition on the serosa of the abdominal organs and severe pyelonephritis with multifocal renal infarction were detected. Histopathological examination confirmed severe chronic active pyelonephritis with renal infarcts, multi-organ vasculitis and thrombosis suggestive of an infectious diseases of bacterial origin. The bacterium was identified using routine methods, matrix assisted laser desorption/ionisation time-of-flight mass spectrometry (MALDI-TOF-MS), and sequencing of the gyrB gene. CONCLUSIONS: To the best of our knowledge, this is the first report of C. kerstersii infection in animals (goat). Our findings support the possibility of C. kerstersii isolation from extraintestinal sites and suggest this organism as a possible cause of urinary tract infection.

The Operon Encoding Hydrolytic Dehalogenation of 4-Chlorobenzoate Is Transcriptionally Regulated by the TetR-Type Repressor FcbR and Its Ligand 4-Chlorobenzoyl Coenzyme A.

The bacterial hydrolytic dehalogenation of 4-chlorobenzoate (4CBA) is a coenzyme A (CoA)-activation-type catabolic pathway that is usually a common part of the microbial mineralization of chlorinated aromatic compounds. Previous studies have shown that the transport and dehalogenation genes for 4CBA are typically clustered as an fcbBAT1T2T3C operon and inducibly expressed in response to 4CBA. However, the associated molecular mechanism remains unknown. In this study, a gene (fcbR) adjacent to the fcb operon was predicted to encode a TetR-type transcriptional regulator in Comamonas sediminis strain CD-2. The fcbR knockout strain exhibited constitutive expression of the fcb cluster. In the host Escherichia coli, the expression of the Pfcb -fused green fluorescent protein (gfp) reporter was repressed by the introduction of the fcbR gene, and genetic studies combining various catabolic genes suggest that the ligand for FcbR may be an intermediate metabolite. Purified FcbR could bind to the Pfcb DNA probe in vitro, and the metabolite 4-chlorobenzyl-CoA (4CBA-CoA) prevented FcbR binding to the P fcb DNA probe. Isothermal titration calorimetry (ITC) measurements showed that 4CBA-CoA could bind to FcbR at a 1:1 molar ratio. DNase I footprinting showed that FcbR protected a 42-bp DNA motif (5'-GGAAATCAATAGGTCCATAGAAAATCTATTGACTAATCGAAT-3') that consists of two sequence repeats containing four pseudopalindromic sequences (5'-TCNATNGA-3'). This binding motif overlaps with the -35 box of Pfcb and was proposed to prevent the binding of RNA polymerase. This study characterizes a transcriptional repressor of the fcb operon, together with its ligand, thus identifying halogenated benzoyl-CoA as belonging to the class of ligands of transcriptional regulators.IMPORTANCE The bacterial hydrolytic dehalogenation of 4CBA is a special CoA-activation-type catabolic pathway that plays an important role in the biodegradation of polychlorinated biphenyls and some herbicides. With genetic and biochemical approaches, the present study identified the transcriptional repressor and its cognate effector of a 4CBA hydrolytic dehalogenation operon. This work extends halogenated benzoyl-CoA as a new member of CoA-derived effector compounds that mediate allosteric regulation of transcriptional regulators.

Bacterial Community Analysis on the Skin of Odorrana grahami and Proposal of Comamonas aquatica subsp. aquatica subsp. nov. and Comamonas aquatica subsp. rana subsp. nov.

Several studies indicated that Odorrana grahami (O. grahami) skin contains abundant antimicrobial peptides, and the skin was recognized as hostile habitat for microorganisms. In this study, the microbial community was evaluated by 16S rRNA gene sequencing, and two associated bacterial isolates were obtained and characterized from the skin of O. grahami. Sixteen bacterial genera were identified from the O. grahami skin by uncultured clone sequences. The dominant groups were Comamonas, Bacillus and Morganella, and the genus Comamonas was the most abundant group (41.7% of the total) of the community. Fortunately, strains CW-25T and CW-518 belonging to genus Comamonas were isolated by plating dilutions. The polyphasic taxonomy results indicated that strain CW-25T was a member of Comamonas aquatica, it showed much higher antimicrobials resistance than the closest C. aquatica strains of LMG 2370T, LMG5937 and LMG 6112 isolated from freshwater. Based on the polyphasic taxonomic studies and antimicrobials resistance characteristics, two subspecies of Comamonas aquatica subsp. aquatica nov. and Comamonas aquatica subsp. rana nov. were proposed. The super-antimicrobial resistance endows the strains of Comamonas aquatica subsp. rana inhabit the O. grahami skin, and the primary defense of O. grahami might be composed by the antimicrobial peptides and the native bacteria.

Complete genome sequencing of Comamonas kerstersii 8943, a causative agent for peritonitis.

Because of poor differentiation among the members of genus Comamonas using phenotypic methods, human infections caused by C. kerstersii are sporadically reported in the literature. Here, we represent the first complete genome sequence of C. kerstersii 8943, which caused peritonitis in a patient with continuous ambulatory peritoneal dialysis (CAPD). The complete genome with no gaps was obtained using third-generation Pacific Biosciences (PacBio) RSII sequencing system with single-molecule real-time (SMRT) analysis. Protein-coding genes, rRNAs and tRNAs were predicted. Functional annotations of the genome using different databases revealed several genes related to pathogenicity including antibiotic resistance genes and prophages. Our work demonstrates that whole genome sequencing can enhance the resolution of clinical investigations and our data can be used as a reference genome during the rapid diagnosis of C. kerstersii infections in the future.

A novel gene, encoding 3-aminobenzoate 6-monooxygenase, involved in 3-aminobenzoate degradation in Comamonas sp. strain QT12.

The biodegradation pathway of 3-aminobenzoate has been documented, but little is known about the sequence and biochemical properties of the proteins involved. In the present study, a 10,083-bp DNA fragment involved in 3-aminobenzoate degradation was identified in 3-aminobenzoate-degrading Comamonas sp. strain QT12. The mabA gene, whose encoded protein shares 39% amino acid sequence identity with 3-hydroxybenzoate 6-hydroxylase of Polaromonas naphthalenivorans CJ2, was identified on this DNA fragment, and the mabA-disrupted mutant was unable to grow on and convert 3-aminobenzoate. MabA was heterologously expressed in Escherichia coli and purified to homogeneity as an approximately ~ 48-kDa His-tagged protein. It was characterized as 3-aminobenzoate 6-hydroxylase capable of catalyzing the conversion of 3-aminobenzoate to 5-aminosalicylate, incorporating one oxygen atom from dioxygen into the product. It contains a non-covalent but tightly bound FAD as the prosthetic group and NADH as an external electron donor. 5-Aminosalicylate was produced with equimolar consumption of NADH. The apparent Km and kcat values of the purified enzyme for 3-aminobenzoate were 158.51 ± 4.74 µM and 6.49 ± 0.17 s-1, respectively, and those for NADH were 189.85 ± 55.70 µM and 7.41 ± 1.39 s-1, respectively. The results suggest that mabA is essential for 3-aminobenzoate degradation in strain QT12, and that 3-aminobenzoate is the primary and physiological substrate of MabA.

Massive over-representation of solute-binding proteins (SBPs) from the tripartite tricarboxylate transporter (TTT) family in the genome of the α-proteobacterium Rhodoplanes sp. Z2-YC6860.

Lineage-specific expansion (LSE) of protein families is a widespread phenomenon in many eukaryotic genomes, but is generally more limited in bacterial genomes. Here, we report the presence of 434 genes encoding solute-binding proteins (SBPs) from the tripartite tricarboxylate transporter (TTT) family, within the 8.2 Mb genome of the α-proteobacterium Rhodoplanes sp. Z2-YC6860, a gene family over-representation of unprecedented abundance in prokaryotes. Representing over 6 % of the total number of coding sequences, the SBP genes are distributed across the whole genome but are found rarely in low-GC islands, where the gene density for this family is much lower. This observation, and the much higher sequence identity between the 434 Rhodoplanes TTT SBPs compared with the average identity between homologues from different species, is indicative of a key role for LSE in the expansion. The TTT SBP genes were found in the vicinity of genes encoding membrane components of transport systems from different families, as well as regulatory proteins such as histidine-kinases and transcription factors, indicating a broad range of functions around the sensing, response and transport of organic compounds. A smaller expansion of TTT SBPs is known in some species of the ß-proteobacteria Bordetella and we observed similar expansions in other ß-proteobacterial lineages, including members of the genus Comamonas and the industrial biotechnology organism Cupriavidus necator, indicating that strong environmental selection can drive SBP duplication and specialisation from multiple evolutionary starting points.

Comamonas aquatilis sp. nov., isolated from a garden pond.

A beige-pigmented bacterial strain, SB30-Chr27-3T, isolated from a garden pond, was studied for its taxonomic position. Cells of the isolate were rod-shaped and stained Gram-negative. A comparison of the 16S rRNA gene sequence with the sequences of the type strains of the most closely related species showed that the strain belongs to the genus Comamonas and showed highest sequence similarities to the type strains of Comamonas jiangduensis (97.5 %), Comamonas aquatica (97.4 %) and Comamonas phosphati (97.3 %). The 16S rRNA gene sequence similarities to all other Comamonas species were below 97.0 %. The fatty acid profile of strain SB30-Chr27-3T consisted of the major fatty acids C16 : 0, C15 : 0iso 2-OH/ C16 : 1ω7c, C18 : 1ω7c/C18 : 1ω9c and, in a minor amount, C10 : 0 3-OH. Major compounds in the polar lipid profile were phosphatidylethanolamine, phosphatidylglycerol, phosphatidylserine and diphosphatidylglycerol. The quinone system was exclusively composed of ubiquinone Q-8. The polyamine pattern contained the major compounds putrescine, cadaverine and 2-hydroxyputrescine. These data and the differentiating biochemical properties indicated that isolate SB30-CHR27-3T represents a novel species of the genus Comamonas, for which we propose the name >Comamonas aquatilis sp. nov. with the type strain SB30-Chr27-3T (=CIP 111491T=CCM 8815T).

Novel Gene Encoding 5-Aminosalicylate 1,2-Dioxygenase from Comamonas sp. Strain QT12 and Catalytic Properties of the Purified Enzyme.

The 1,125-bp mabB gene encoding 5-aminosalicylate (5ASA) 1,2-dioxygenase, a nonheme iron dioxygenase in the bicupin family that catalyzes the cleavage of the 5ASA aromatic ring to form cis-4-amino-6-carboxy-2-oxohexa-3,5-dienoate in the biodegradation of 3-aminobenzoate, was cloned from Comamonas sp. strain QT12 and characterized. The deduced amino acid sequence of the enzyme has low sequence identity with that of other reported ring-cleaving dioxygenases. MabB was heterologously expressed in Escherichia coli cells and purified as a His-tagged enzyme. The optimum pH and temperature for MabB are 8.0 and 10°C, respectively. FeII is required for the catalytic activity of the purified enzyme. The apparent Km and Vmax values of MabB for 5ASA are 52.0 ± 5.6 µM and 850 ± 33.2 U/mg, respectively. The two oxygen atoms incorporated into the product of the MabB-catalyzed reaction are both from the dioxygen molecule. Both 5ASA and gentisate could be converted by MabB; however, the catalytic efficiency of MabB for 5ASA was much higher (∼70-fold) than that for gentisate. The mabB-disrupted mutant lost the ability to grow on 3-aminobenzoate, and mabB expression was higher when strain QT12 was cultivated in the presence of 3-aminobenzoate. Thus, 5ASA is the physiological substrate of MabB.IMPORTANCE For several decades, 5-aminosalicylate (5ASA) has been advocated as the drug mesalazine to treat human inflammatory bowel disease and considered the key intermediate in the xenobiotic degradation of many aromatic organic pollutants. 5ASA biotransformation research will help us elucidate the microbial degradation of these pollutants. Most studies have reported that gentisate 1,2-dioxygenases (GDOs) can convert 5ASA with significantly high activity; however, the catalytic efficiency of these enzymes for gentisate is much higher than that for 5ASA. This study showed that MabB can convert 5ASA to cis-4-amino-6-carboxy-2-oxohexa-3,5-dienoate, incorporating two oxygen atoms from the dioxygen molecule into the product. Unlike GDOs, MabB uses 5ASA instead of gentisate as the primary substrate. mabB is the first reported 5-aminosalicylate 1,2-dioxygenase gene.

The O-antigen structure of bacterium Comamonas aquatica CJG.

Genus Comamonas is a group of bacteria that are able to degrade a variety of environmental waste. Comamonas aquatica CJG (C. aquatica) in this genus is able to absorb low-density lipoprotein but not high-density lipoprotein of human serum. Using 1H and 13C NMR spectroscopy, we found that the O-polysaccharide (O-antigen) of this bacterium is comprised of a disaccharide repeat (O-unit) of d-glucose and 2-O-acetyl-l-rhamnose, which is shared by Serratia marcescens O6. The O-antigen gene cluster of C. aquatica, which is located between coaX and tnp4 genes, contains rhamnose synthesis genes, glycosyl and acetyl transferase genes, and ATP-binding cassette transporter genes, and therefore is consistent with the O-antigen structure determined here.

Bacterial Metabolism Affects the C. elegans Response to Cancer Chemotherapeutics.

The human microbiota greatly affects physiology and disease; however, the contribution of bacteria to the response to chemotherapeutic drugs remains poorly understood. Caenorhabditis elegans and its bacterial diet provide a powerful system to study host-bacteria interactions. Here, we use this system to study how bacteria affect the C. elegans response to chemotherapeutics. We find that different bacterial species can increase the response to one drug yet decrease the effect of another. We perform genetic screens in two bacterial species using three chemotherapeutic drugs: 5-fluorouracil (5-FU), 5-fluoro-2'-deoxyuridine (FUDR), and camptothecin (CPT). We find numerous bacterial nucleotide metabolism genes that affect drug efficacy in C. elegans. Surprisingly, we find that 5-FU and FUDR act through bacterial ribonucleotide metabolism to elicit their cytotoxic effects in C. elegans rather than by thymineless death or DNA damage. Our study provides a blueprint for characterizing the role of bacteria in the host response to chemotherapeutics.

Mn accumulation in a submerged plant Egeria densa (Hydrocharitaceae) is mediated by epiphytic bacteria.

Many aquatic plants act as biosorbents, removing and recovering metals from the environment. To assess the biosorbent activity of Egeria densa, a submerged freshwater macrophyte, plants were collected monthly from a circular drainage area in Lake Biwa basin and the Mn concentrations of the plants were analysed. Mn concentrations in these plants were generally above those of terrestrial hyperaccumulators, and were markedly higher in spring and summer than in autumn. Mn concentrations were much lower in plants incubated in hydroponic medium at various pH levels with and without Mn supplementation than in field-collected plants. The precipitation of Mn oxides on the leaves was determined by variable pressure scanning electron microscopy-energy dispersive X-ray analysis and Leucoberbelin blue staining. Several strains of epiphytic bacteria were isolated from the field-collected E. densa plants, with many of these strains, including those of the genera Acidovorax, Comamonas, Pseudomonas and Rhizobium, found to have Mn-oxidizing activity. High Mn concentrations in E. densa were mediated by the production of biogenic Mn oxide in biofilms on leaf surfaces. These findings provide new insights into plant epidermal bacterial flora that affect metal accumulation in plants and suggest that these aquatic plants may have use in Mn phytomining.

Description of Comamonas sediminis sp. nov., isolated from lagoon sediments.

Strain S3T was isolated from lagoon sediments, and appeared as transparent colonies on agar plates, with cells staining Gram-negative. Catalase and oxidase were positive. S3T hydrolyzed starch, casein and tween-20, while urea, chitin, gelatin and tween-80 were not hydrolysed. C18 : 1ω6c/C18 : 1ω7c, C16 : 1ω6c/C16 : 1ω7c,C17 : 0 cyclo and C16 : 0 were the predominant fatty acids with minor amounts of C10 : 0 3-OH, C12 : 0, C14 : 0 and C16 : 0 2-OH. S3T contained diphosphatidylglycerol (DPG), phosphatidylglycerol (PG), and phosphatidylethanolamine (PE) as major polar lipids with minor amounts of unidentified phospholipid (PL) and unidentified lipids (L1-2). Genomic DNA G+C content was 68.3 mol%. 16S rRNA gene sequence comparisons indicated that S3T represents a member of the genus Comamonas in family Comamonadaceae of the class Betaproteobacteria. S3T has a sequence similarity of 98.96 % with Comamonas koreensis YH12T, 97.93 % with Comamonas guangdongensis CY01T and <96.97 % with other members of the genus Comamonas. DNA-DNA hybridization values between S3T and the type strains of the most closely related species were clearly below the 70 % threshold. On the basis of phenotypic, genotypic and phylogenetic analysis, it is proposed that S3T represents a novel species of the genus Comamonas, for which the name Comamonas sediminis sp. nov. is proposed. The type strain is S3T (=KEMB 563-466T =JCM 31169T).

Improved efficiency of asymmetric hydrolysis of 3-substituted glutaric acid diamides with an engineered amidase.

AIMS: To improve the efficiency of asymmetric hydrolysis of 3-(4-chlorophenyl) glutaric acid diamide (CGD) using a recombinant Comamonas sp. KNK3-7 amidase (CoAM) produced in Escherichia coli. METHODS AND RESULTS: The CoAM gene was cloned, sequenced and found to comprise 1512 bp, encoding a polypeptide of 54 054 Da. CoAM-transformed E. coli were able to perform R-selective hydrolysis of CGD; however, complete conversion of 166·2 mmol l(-1) CGD in 28 h could not be obtained. We attempted to optimize the reactivity of CoAM by mutating single amino acids in the substrate-binding domain. Notably, the methionine-substituted L146M mutant enzyme showed increased reactivity, completing the conversion of 166·2 mmol l(-1) CGD in just 4 h. The Km value for L146M was lower than that of CoAM. CONCLUSIONS: We succeeded in creating the L146M mutant of CoAM with increased substrate affinity and found that this was the best mutant for the hydrolysis of CGD. SIGNIFICANCE AND IMPACT OF THE STUDY: Increasing the efficiency of hydrolysis of 3-substituted glutaric acid diamides is useful to improve the synthesis of optically active 3-substituted gamma-aminobutyric acid. This is the first report of efficient hydrolysis of CGD using amidase mutant-producing E. coli cells.