Comamonas sp. 3ah48 is a dibenz[a,h]anthracene-degrading bacterium that is tolerant to heavy metals.

Industrialization often causes polycyclic aromatic hydrocarbon (PAH) and heavy metal contamination of soil and water. In this study, we isolated a bacterium from bottom mud water around a park of Kawasaki Port, Japan, that degrades the 5-ring PAH dibenz[a,h]anthracene (DBA). The strain, Comamonas sp. 3ah48, degraded 29% of DBA (30 µg ml-1 ) in 7 days, and the degradation level increased drastically, to 59%, by the addition of glutamate to the medium. The strain also degraded 40, 14, 15 and 19% of pyrene (Pyr), benzo[b]fluoranthene (BbF), benzo[k]fluoranthene (BkF) and benzo[g,h,i]perylene (BghiP) respectively. Benzo[a]pyrene (BaP) was degraded only when glutamate was added to the medium. Strain 3ah48 retained its degradation levels in the presence of 2 mmol l-1 Co2+ , Zn2+ or Cr2+ , at almost the same level as that without metal, and increased the DBA degradation level to 57% in the presence of 2 mmol l-1 Cu2+ , suggesting the possibility of the presence of laccase. SIGNIFICANCE AND IMPACT OF THE STUDY: Sixteen polycyclic aromatic hydrocarbons (PAHs) are listed as priority pollutants by the United States Environmental Protection Agency (USEPA). Information about the biodegradation of one of those PAHs, dibenz[a,h]anthracene (DBA), is limited. The present study focuses on DBA degradation by Comamonas sp. 3ah48 strain isolated around Kawasaki Port, Japan. Comamonas sp. 3ah48, cultured with the addition of glutamate to the medium, was found to increase the degradation level of DBA and to degrade DBA even in the presence of high concentrations of heavy metals.

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.

Isolation, characterization, and identification of potential Diuron-degrading bacteria from surface sediments of Port Klang, Malaysia.

Diuron is an alternative biocide suggested to replace organotin in formulating antifouling paints to be applied on water-going vessels hull. However, it is potentially harmful to various non-targeted marine organisms due to its toxic properties. Present study aimed to isolate, screen and identify the potential of Diuron-degrading bacteria collected from the marine sediments of Port Klang, Malaysia. Preliminary screening was conducted by exposing isolated bacteria to 430ng/L (background level), followed by 600ng/L and 1000ng/L of Diuron concentrations. Nine bacteria colonies survived the exposure of the above concentrations. However, only two strains can tolerate to survive up to 1000µg/L, which were then characterised and identified using phenotypic tests and the standard 16S rRNA molecular identification. The strains were identified as Comamonas jiangduensis SZZ 10 and Bacillus aerius SZZ 19 (GenBank accession numbers: KU942479 and KU942480, respectively). Both strains have the potential of Diuron biodegradation for future use.

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.

Single cell growth rate and morphological dynamics revealing an "opportunistic" persistence.

Bacteria persistence is a well-known phenomenon, where a small fraction of cells in an isogenic population are able to survive high doses of antibiotic treatment. Since the persistence is often associated with single cell behaviour, the ability to study the dynamic response of individual cells to antibiotics is critical. In this work, we developed a gradient microfluidic system that enables long-term tracking of single cell morphology under a wide range of inhibitor concentrations. From time-lapse images, we calculated bacterial growth rates based on the variations in cell mass and in cell number. Using E. coli and Comamonas denitrificans to amoxicillin inhibition as model systems, we found the IC50 determined via both methods are in a good agreement. Importantly, the growth rates together with morphological dynamics of individual cells has led to the discovery of a new form of persistence to amoxicillin. Normal cells that are sensitive to amoxicillin gain persistence or recover from the killing process, if they have had an opportunity to utilise the cytoplasm released from lysed cells close-by. We term this acquired persistence in normal growing cells "opportunistic persistence". This finding might shed new insights into biofilm resistance and the effect of antibiotics on environmental microbes.

The effect of ammonium chloride and urea application on soil bacterial communities closely related to the reductive transformation of pentachlorophenol.

Pentachlorophenol (PCP) is widely distributed in the soil, and nitrogen fertilizer is extensively used in agricultural production. However, studies on the fate of organic contaminants as affected by nitrogen fertilizer application have been rare and superficial. The present study aimed to examine the effect of ammonium chloride (NH4Cl) and urea (CO(NH2)2) application on the reductive transformation of PCP in a paddy soil. The study showed that the addition of low concentrations of NH4Cl/CO(NH2)2 enhanced the transformation of PCP, while the addition of high concentrations of NH4Cl/CO(NH2)2 had the opposite effect. The variations in the abundance of soil microbes in response to NH4Cl/CO(NH2)2 addition showed that both NH4Cl and CO(NH2)2 had inhibitory effects on the growth of dissimilatory iron-reducing bacteria (DIRB) of the genus Comamonas. In contrast, for the genus Shewanella, low concentrations of NH4Cl inhibited growth, and high concentrations of NH4Cl enhanced growth, whereas all concentrations of CO(NH2)2 showed enhancement effects. In addition, consistent patterns of variation were found between the abundances of dechlorinating bacteria in the genus Dehalobacter and PCP transformation rates under NH4Cl/CO(NH2)2 addition. In conclusion, nitrogen application produced variations in the structure of the soil microbial community, especially in the abundance of dissimilatory iron-reducing bacteria and dechlorinating bacteria, which, in turn, affected PCP dechlorination.

In vitro studies on degradation of synthetic dye mixture by Comamonas sp. VS-MH2 and evaluation of its efficacy using simulated microcosm.

Reactive azo dyes are considered as one of the most detrimental pollutants from industrial effluents and therefore their biodegradation is receiving constant scientific consideration. A bacterial isolate VS-MH2, originating from dye contaminated sites of Gujarat, India, was exploited for its ability to degrade a synthetic dye mixture (SDM) (comprising of four azo reactive dyes) under static conditions. The identification of the isolate by 16S rRNA gene sequencing revealed it to be Comamonas sp. The biodegradation of the SDM was analyzed by UV-vis spectroscopy, IR spectroscopy and GC-MS analysis. The isolate showed high metabolic activity towards SDM and degraded it completely (100 mg L(-1)) within 30 h at pH 7 and 35 °C. Simulated microcosm studies in the presence and absence of indigenous microflora confirmed the ability of Comamonas sp. VS-MH2 for dye degradation and to colonize the soil. This is the first investigation reporting the degradation of SDM by Comamonas sp. under simulated soil microcosms.

Isolates of Comamonas spp. exhibiting catalase and peroxidase activities and diversity of their responses to oxidative stress.

For survival isolates of Comamonas testosteroni CCM 1931, C. testosteroni K3, C. terrigena N3H or N1C and C. terrigena CCM 2409, selected largely from polluted environments, the production of catalase and dianisidine-peroxidase activity was important. Electrophoretic resolution of cell-free extracts of aerobically grown strains in Luria-Bertani medium during the exponential phase revealed distinctive expression of catalatic and peroxidatic activities detected with 3,3'-diaminobenzidine tetrahydrochloride (DAB). The protection of isolates from 20 or 40 mM H(2)O(2) stress was characterized with a considerable diversity in catalase and peroxidase responses that resulted from hydroperoxidase's variant of original isolates, indicating also a selective pressure of environment. Results indicate catalase to be important for adaptation of cultures to high concentration of 60mM H(2)O(2). The greatest appreciable differences in sensitivity to toxic effect of H(2)O(2) (20 or 40 mM) treatment between individual isolates and their adapted variants during the growth were observed until the middle of exponential phase. Isolates exhibited diversity in catalases responses to possible contaminants o-or p-phenylenediamine (PDA) as well. Only positional isomer p-PDA (1 or 2mM) stimulated catalase activity unlike from isomer o-PDA in C. terrigena N3H cells. The study can contribute to understanding of bacterial antioxidative enzymatic responses in the presence of possible physiological stress resulting mainly from environmental pollutants.

Fe(III)-enhanced anaerobic transformation of 2,4-dichlorophenoxyacetic acid by an iron-reducing bacterium Comamonas koreensis CY01.

This work studied the ability of Comamonas koreensis CY01 to reduce Fe(III) (hydr)oxides by coupling the oxidation of electron donors and the enhanced biodegradation of 2,4-dichlorophenoxyacetic acid (2,4-D) by the presence of Fe(III) (hydr)oxides. The experimental results suggested that strain CY01 can utilize ferrihydrite, goethite, lepidocrocite or hematite as the terminal electron acceptor and citrate, glycerol, glucose or sucrose as the electron donor. Strain CY01 could transform 2,4-D to 4-chlorophenol through reductive side-chain removal and dechlorination. Under the anaerobic conditions, Fe(III) reduction and 2,4-D biodegradation by strain CY01 occurred simultaneously. The presence of Fe(III) (hydr)oxides would significantly enhance 2,4-D biodegradation, probably due to the fact that the reactive mineral-bound Fe(II) species generated from Fe(III) reduction can abiotically reduce 2,4-D. This is the first report of a strain of C. koreensis capable of reducing Fe(III) (hydr)oxides and 2,4-D, which extends the diversity of iron-reducing bacteria associated with dechlorination.

Responses to arsenate stress by Comamonas sp. strain CNB-1 at genetic and proteomic levels.

Comamonas sp. strain CNB-1, a chloronitrobenzene-degrading bacterium, was demonstrated to possess higher arsenate tolerance as compared with the mutant strain CNB-2. pCNB1, a plasmid harboured by CNB-1 but not CNB-2, contained the genetic cluster ars(RPBC)Com, which putatively encodes arsenate-resistance regulator, family II arsenate reductase, arsenite efflux pump and family I arsenate reductase, respectively, in Comamonas strain CNB-1. The arsC-negative Escherichia coli could gain arsenate resistance by transformation with arsPCom or arsCCom, indicating that these two genes might express functional forms of arsenate reductases. Intriguingly, when CNB-1 cells were exposed to arsenate, the transcription of arsPCom and arsCCom was measurable by RT-PCR, but only ArsPCom was detectable at protein level. To explore the proteins responding to arsenate stress, CNB-1 cells were cultured with and without arsenate and differential proteomics was carried out by two-dimensional PAGE (2-DE) and MALDI-TOF MS. A total of 31 differential 2-DE spots were defined upon image analysis and 23 proteins were identified to be responsive specifically to arsenate. Of these spots, 18 were unique proteins. These proteins were identified to be phosphate transporters, heat-shock proteins involved in protein refolding, and enzymes participating in carbon and energy metabolism.

Production of catalases by Comamonas spp. and resistance to oxidative stress.

Bacterial isolates Comamonas terrigena N3H (from soil contaminated with crude oil) and C. testosteroni (isolated from the sludge of a wastewater treatment plant), exhibit much higher total catalase activity than the same species from laboratory collection cultures. Electrophoretic resolution of catalases revealed only one corresponding band in cell-free extracts of both C. testosteroni cultures. Isolates of C. terrigena N3H exhibited catalase-1 and catalase-2 activity, whereas in the collection culture C. terrigena ATCC 8461 only catalase-1 was detected. The environmental isolates exhibited much higher resistance to exogenous H2O2 (20, 40 mmol/L) than collection cultures, mainly in the middle and late exponential growth phases. The stepwise H2O2-adapted culture of C. terrigena N3H, which was more resistant to oxidative stress than the original isolate, exhibited an increase of catalase and peroxidase activity represented by catalase-1. Pretreatment of cells with 0.5 mmol/L H2O2 followed by an application of the oxidative agent in toxic concentrations (up to 40 mmol/L) increased the rate of cell survival in the original isolate, but not in the H2O2-adapted variant. The protection of bacteria caused by such pretreatment corresponded with stimulation of catalase activity in pretreated culture.

Novel alpha-glucosidase inhibitors, CKD-711 and CKD-711a produced by Streptomyces sp. CK-4416. II. Biological properties.

CKD-711 and CKD-711a are aminooligosaccharide alpha-glucosidase inhibitors discovered during the bioactive material screening for antibacterial agent. Their inhibitory activities were studied and compared with those of acarbose in vitro and in vivo with animals. In in vitro study, CKD-711 showed similar effects to acarbose on porcine intestinal maltase and sucrase, IC50s of 2.5 and 0.5 microg/ml, respectively, whereas it had about 2 fold lower alpha-amylase inhibitory activity (IC50, 78.0 microg/ml) than acarbose (IC50, 36 microg/ml). CKD-711a showed less inhibitory activity than CKD-711 against all the enzymes tested. In rat fed on starch and sucrose meals, the dose of CKD-711 which reduced the postprandial blood glucose increment by 50 percent in comparison to control rats (ED50) were 3.07 and 1.15 mg/kg, respectively, and acarbose had ED50s of 1.94 and 1.15 mg/kg, respectively. CKD-711 and CKD-711a also showed antibacterial activity against Comamonas terrigena.

Comamonas denitrificans sp. nov., an efficient denitrifying bacterium isolated from activated sludge.

To find a biomarker for denitrification in activated sludge, five denitrifying strains isolated from three wastewater treatment plants were studied. These strains were selected from among 1,500 isolates for their excellent denitrifying properties. They denitrify quickly and have no lag phase when switching from aerobic to anoxic conditions. All strains have the cd1-type of nitrite reductase. The strains are Gram-negative rods and they all grow as filamentous chains when cultivated in liquid solution. The strains differ in colony morphology when grown on nutrient agar. Almost full-length 16S rDNA sequences were determined and phylogenetic analysis revealed that these strains are positioned among members of the genus Comamonas in the beta-subclass of the Proteobacteria. Signature nucleotides and bootstrap percentages were also analysed to verify this position. Strains 110, 123T, 2.99g, 5.38g and P17 were < or = 96.7% similar to known strains, but > or = 99.7% similar to each other, as judged from their 16S rDNA sequences, and grouped tightly together in the phylogenetic tree. Sequence motifs in the 16S rRNA gene were also found, suggesting the monophyletic origin of these strains. Nevertheless, some strains differed from the others, for example strain 110 branches early from the other strains and 5.38g is phenotypically more inert. Therefore, it is proposed that strains 110, 123T, 2.99g and P17 are classified into a new species, Comamonas denitrificans sp. nov., while the taxonomic status of strain 5.38g will have to await the outcome of further studies. The type strain of Comamonas denitrificans is 123T (ATCC 700936T).

Effect of starvation and chloramphenicol on acceleration of bacterial dihexyl sulfosuccinate biotransformation.

Starvation for carbon and energy sources accelerated the biotransformation of the anion-active surfactant dihexyl sulfosuccinate (DHS) by Comamonas terrigena cells. Chloramphenicol (Cm) added at different time intervals to non-starved cells inhibited the DHS transformation. The largest difference between cells treated and non-treated by Cm was observed for a 16-h-starvation period. Protein synthesis de novo during starvation enhanced the DHS biotransformation efficiency. A partial transformation of DHS in the presence of Cm indicated the constitutive character of enzymes involved in primary DHS biodegradation.