Biodegradation of trimethylamine by a halotolerant strain of Paracoccus sp. PS1 and in silico analysis of trimethylamine degrading enzymes

doi:10.21203/rs.3.rs-3135380/v1

Download PDF

Research Article

Biodegradation of trimethylamine by a halotolerant strain of Paracoccus sp. PS1 and in silico analysis of trimethylamine degrading enzymes

https://doi.org/10.21203/rs.3.rs-3135380/v1

This work is licensed under a CC BY 4.0 License

Version 1

posted

You are reading this older preprint version

Read the latest preprint version →

In recent times, trimethylamine N-oxide (TMAO) a gut metabolite generated by constitutive oxidation and reduction by gut microbial and host enzymes is gaining increased attention of scientists as it has been linked to the development of atherosclerosis and other ailments such as chronic kidney disease, type 2 diabetes mellitus, etc. TMAO which acts as a biomarker for health risk is generated by the oxidation of trimethylamine (TMA), produced by human gut microflora from proteinaceous food material. Microbial degradation of TMA can be a predicted approach towards the reduction of the effect of TMAO on human health. The isolated Paracoccus sp. strain PS1 could rapidly grow in mineral salt medium supplemented with TMA as the sole source of carbon and nitrogen. Its TMA degrading capacity was further confirmed through spectrophotometric, Electrospray Ionization Time-of-Flight Mass Spectrometry (ESI TOF-MS) and High performance liquid chromatography (HPLC) analysis. In silico analysis of the TMA/TMAO degrading enzymes were performed using bioinformatics tools.

TMA

TMAO

atherosclerosis

trimethylamine monooxygenase (Tmm)

trimethylamine-oxide aldolase (Tdm)

Paracoccus sp.

TMAO is known as a precursor of atherosclerosis (Zhu et al. 2020) which is generated inside the human body through a two-step process as colorless amine oxide that involves the participation of both gut microbiota and host liver enzyme flavin-containing monooxygenase (FMO3) (Velasquez et al. 2016). Initially, ingested L-carnitine, choline rich foods are metabolized by gut microbial choline TMA lyases (CutC/CutD complex) and carnitine oxygenases (CntA/B) (Rath et al. 2017) into TMA, producing y-butyrobetaine as an intermediate metabolite, which is then absorbed by the circulating blood of hepatic portal of the human body (Velasquez et al. 2016). Then the host hepatic FMO3 plays a crucial role in oxidation of TMA into TMAO, which enters into the main circulation. After entry, TMAO initiates atherosclerosis by activating macrophage scavenger receptors such as class A1 (SRA1) and CD36 for uptake of oxidized low-density lipoproteins (ox-LDL) (oxidized due to high TMAO levels) leading to upregulation and increase in transformation of macrophages into foam cells. TMAO also causes an increase in the expression of inflammatory cytokines IL-6 and TNFα, provoking an inflammatory response, resulting in the penetration of these modified macrophages through the endothelial barrier and accumulation in vessel intima forming atherosclerotic plaques (Zhu et al. 2020).

Elevated plasma TMAO level is also associated with numerous ailments such as chronic kidney disease (Tang et al. 2015), colorectal cancer (Xu et al. 2015), type 2 diabetes mellitus (Li et al. 2015), liver steatosis, hypertension (Coutinho-Wolino et al. 2021), ischaemic stroke (Schneider et al. 2020). High TMAO levels also acts as a biomarker in detecting neurodegenerative disorders such as Alzheimer’s disease (AD) (Xu and Wang 2016) and Autism Spectrum Disorder (ASD) (Quan et al. 2020). Increase in blood TMAO content also alters bile acid synthesis (Wilson et al. 2016), enhances thrombosis and causes hyperactivity of platelets leading to heart attack (Zhu et al. 2016a).

Presence of TMAO as an osmoprotectant (Yancey et al. 2014) in tissues of marine organisms helps them to withstand adverse sea environmental conditions. Consumption of sea food causes direct absorption of TMAO from the gut into the bloodstream (Velasquez et al. 2016). TMAO has also been detected in Arabidopsis thaliana as an osmolyte to combat adverse environmental conditions such as high soil salinity, temperatures and unfavorable natural calamities by preventing plant protein denaturation and upregulating stress-induced functional genes (Catalá et al. 2021).

TMA, the main precursor of TMAO in the human body, has a characteristic fishy odor and is generated in the gut in a number of ways. Members of human gut flora, such as Clostridium asparagiforme, Anaerococcus hydrogenalis, Escherichia fergusonii, etc. actively utilize choline and generates TMA with the help of choline TMA lyase (Velasquez et al. 2016) by introducing a cleavage in radical C-N bond of choline (Craciun and Balskus 2012). TMAO when reaches gut through food is reduced to TMA by TMAO degrading enzymes such as TMAO reductase (TorA) produced by gut bacteria such as Escherichia sp., Bacillus sp., Salmonella sp., etc (Fennema et al. 2016). Mutation of hepatic FMO3 gene which is involved in conversion of TMA to TMAO, leads to accumulation of TMA in the digestive system, causing a rare human disease known as Trimethylaminuria (TMAU) or fish odor syndrome (Ayesh et al. 1993) (Dolphin et al. 1997) resulting in rotten fish odor in urine, breath and sweat due to the release of TMA (Fennema et al. 2016).

TMA is also declared an air pollutant by The National Institute for Occupational Safety and Health (NIOSH) and its recommended exposure limit (REL) is 10 ppm (Wannomai et al. 2019). Long-term exposure to TMA has shown a visible rise in blood pressure levels and also kidney damage by upregulation of the markers of KIM-1 and proteinuria in rats (Maksymiuk et al. 2022).

So recently, scientists are trying to reduce human plasma TMAO levels using antibiotics, meldonium, 3,3-dimethyl-1-butanol, resveratrol, FMO3 inhibitors and by lowering the L-carnitine and choline consumption (Velasquez et al. 2016). Unfortunately, all these methods have considerable adverse effects on the human body. So degradation of TMAO or its precursor, TMA, using microbial enzymes in the gut may be a convenient way to reduce the detrimental effect of TMAO on human health with least side effects. The enzyme Tmm is the primary enzyme responsible for the oxidation of TMA to TMAO followed by the second enzyme, Tdm which carries out the oxygen independent demethylation of TMA into dimethylamine (DMA) and formaldehyde (Zhu et al. 2016b) leading to the release of ammonia as the final byproduct of the metabolism. This mechanism is carried out by some reported microbes such as Hyphomicrobium sp. (Meiberg 1979), Paracoccus sp. (Kim et al. 2001), Ruegeria pomeroyi DSS-3 (Lidbury et al. 2015), etc.

In present study, our primary aim was to isolate bacterial strains from naturally TMA/TMAO rich environment (marine fish) which can effectively degrade TMA/ TMAO and may be used as probiotic organism to reduce the adverse effect of these two harmful chemicals in human gut in the long run. For this purpose, we have screened 14 bacterial isolates from various marine fishes based on their effective utilization of TMA in mineral salt medium supplemented with TMA as sole carbon and nitrogen source. Finally, isolate PS1 screened from skin of Pomfret fish (Pampus argenteus), which was later identified as a strain of Paracoccus sp. was selected for further study on the basis of its higher growth rate on TMA. Study of antibiotic sensitivity profile and β-hemolysis test showed that Paracoccus sp. strain PS1 is not positive for such pathogenicity related trait. We have got positive results for PS1 when tested for qualitative and quantitative assays for TMA degradation, Nessler reaction, effective utilization of other amines for growth that are involved in TMA degradation pathway, spectrophotometric, ESI TOF-MS and HPLC assay methods. To get an insight into the genetic configuration of TMA/TMAO degrading enzymes and related genes we have performed the whole genome sequencing (WGS) of isolate PS1. From the functionally annotated WGS data of PS1, sequences of Tmm and Tdm enzymes, the main players behind the aerobic degradation of TMA are retrieved and their evolutionary relationship was established using MEGA7 software. In silico analysis for the determination of physicochemical properties of those two genes is done in detail with the help of the Expasy tool.

Chemicals used

The chemicals used in the experiments were of analytical grade and HPLC grade. Trimethylamine hydrochloride (TMA.HCl), Dimethylamine hydrochloride (DMA.HCl), Methylamine hydrochloride (MMA.HCl) were purchased from Himedia and Trimethylamine n-oxide dihydrate from Sigma Aldrich. The syringe-driven filters (MCE hydrophilic, 0.22µm pore size, 13mm diameter) were purchased from Himedia and Eppendorf tubes from Tarsons.

Isolation and characterization

Sample collection and isolation of TMA degrading bacteria

TMA degrading bacterial isolates used in this study were collected from five marine water fishes of the Bay of Bengal (Contai, East Midnapore, West Bengal, India). The samples were Pampus argenteus (Duremdez et al. 2004), Tenualosa ilisha (Sahoo et al. 2018), Nematalosa nasus (Habib and Islam 2020), Liza parsia (Guhathakurta and Kaviraj 2000) and Mystus gulio (Siddiqui et al. 2010) commonly known as Pomfret, Hilsa, Khoira, Parshe and Nuna Tengra fish respectively. A portion of skin and gut of each fish sample were cut with a sterilized blade and were inoculated into modified Davis Minimal broth (DMB) medium and incubated at 30^oC for 72 hrs. The modified DMB medium prepared consisted of 7g/L dipotassium phosphate, 2g/L monopotassium phosphate and 0.1g/L magnesium sulfate. After autoclaving and cooling the medium, filter sterilized TMA.HCl (supplemented as sole carbon and nitrogen source) of approximately 0.4M concentration was added and the final medium pH was adjusted to 7 ± 0.2. Fish samples were also inoculated in the same medium containing 3.5% sodium chloride (salinity of sea is 3.5% (Balebona et al. 1995)) to detect halotolerant nature of the bacterial isolates as they were isolated from sea fish. Bacterial growth observed in both types of broth mediums were serially diluted (tenfolds) and 100µl of each of the dilutions were spread on similar modified Davis Mingolis (DM) media plates containing 1.5% agar and incubated at 30^oC for 72 hrs.

Characterization of isolates and determination of optimum growth conditions:

Growth rate of the isolates were measured spectrophotometrically using a UV-VIS double beam Spectrophotometer of Shimadzu (Model – UV-1900) at a wavelength of 600nm. Based on faster growth rate in TMA broth medium PS1 was selected and its optimum growth pH, temperature and salinity were determined. PS1 was grown in modified DMB medium supplemented with TMA at temperatures of 10, 20, 30, 37, 40, 50 and 60^oC, pH values of 3, 4, 5, 6, 7, 8, 9, 10 and 11 and sodium chloride (NaCl) concentrations of 0, 0.3, 0.6, 0.9, 1.2, 1.5, 1.8, 2.1, and 2.4 molar respectively. The range of TMA utilization of isolate PS1 was also detected spectrophotometrically by observing its growth in varying molar concentrations of TMA (0–4). Isolate PS1 was tested for its Gram character, antibiotic sensitivity profile, biochemical characteristics such as carbon utilization using KB009 HiCarbohydrate™ kit (HIMEDIA) (35 types of carbon present in the kit), nitrate reduction, catalase, indole, Methyl red (MR), Voges–Proskauer (VP) and casein hydrolysis tests and for the presence of pathogenicity-related traits such as β-hemolysis using conventional techniques.

Ability to grow in various amines as a sole source of carbon and nitrogen.

Growth of isolate PS1 was monitored spectrophotometrically at 600 nm for 24, 48 and 72 hrs respectively when inoculated in both modified DM broth and agar mediums supplemented with TMA, DMA and MMA respectively as sole carbon and nitrogen source to detect its ability to utilize all three amines when incubated at 30^oC. PS1 was also grown in modified DM agar medium containing TMAO as the sole carbon and nitrogen source to observe its TMAO utilising capability for growth.

Qualitative and quantitative assay method for detection of TMA degrading property.

Isolate PS1 was inoculated in a modified DMB medium supplemented with TMA (0.4M) and incubated at 30^oC for 10 days in B.O.D. incubator shaker at 150 rpm. Both control (containing uninoculated TMA medium) and blank (medium with no added TMA) sets were maintained at the same incubation conditions. The TMA degradation assay was performed according to Miao-Kang Shen (Shen 1988) with modifications. The reagents used in this experiment were prepared as described by Miao-Kang Shen. Growth rate of isolated PS1 was evaluated for 10 days spectrophotometrically at 600nm. After 24hrs of growth, 1 ml of PS1 culture was taken in an Eppendorf tube and centrifuged at 10,000rpm for 2min. 1µl of the supernatant was taken in a fresh screw-capped tube and an equal volume of 25% HCl was added, and diluted to 4 ml with sterile distilled water. Appropriate volumes of formaldehyde solution, toluene and potassium carbonate solutions were added to the tube (Shen 1988), tightly screw capped and vortexed for a few seconds. 7 ml of the upper toluene layer was collected carefully to avoid any contamination of the lower aqueous layer and was transferred to a fresh screw-capped tube and was dried over 0.1g of anhydrous sodium sulfate salt. After the drying process, 5 ml of the toluene layer was collected and transferred into another screw-capped tube and an equal volume of picric acid solution was added and observed for the yellow color development of the solution. The intensity of the yellow color developed was measured spectrophotometrically at 410 nm. This was performed for both control and blank sets. A standard curve was prepared using known concentrations of TMA.HCl (0.05M, 0.1M, 0.15M, 0.2M, 0.25M, 0.3M, 0.35M, 0.4M, 0.45M and 0.5M) and absorbance values of the samples were plotted against the standard curve to quantitatively determine the rate of degradation of TMA by isolate PS1 with time as its growth increased compared to the control set.

Detection of TMA degradation using Nessler method

Ammonia produced as the final byproduct of aerobic TMA degradation pathway (Chen et al. 2011) by isolate PS1 was detected by Nessler method according to Bhushan Subhash Naphade et al. (Naphade and Hussain 2014) with modifications. Isolate PS1 was freshly inoculated in TMA containing modified DMB medium and incubated at 30^oC for upto 15 days in B.O.D. incubator shaker at 150 rpm. 1ml of the culture was taken in an Eppendorf tube after 24 hrs of incubation and centrifuged at 10,000rpm for 2 min. The supernatant was collected in a fresh Eppendorf tube and a few drops of Nessler’s reagent was added to it to observe the development of brown color precipitate due to ammonia production. This experiment was conducted for 15 days at an interval of 24 hours to detect the increase in ammonia production with the rate of utilization of TMA.

Easy method for qualitative and quantitative analysis of TMA by ESI TOF-MS.

For qualitative and quantitative ESI TOF-MS analysis of TMA degradation by isolate PS1, 100µl of overnight culture of PS1 isolate was inoculated into a modified DMB medium supplemented with TMA and incubated at 30^oC for 7 days in a B.O.D. incubator shaker at 150 rpm. Control sets of uninoculated TMA medium and blank sets of medium without TMA were also kept at the same incubation conditions. Supernatants of all the samples (test, control and blank) were collected by centrifugation at 10,000rpm for 2 min at 4^oC and filtered through a sterile 0.22µm syringe-driven filter. 1 mg/ml of TMA.HCl (dissolved in HPLC grade water) was taken as standard. Both the standard and the samples were diluted to 1000 folds using LC-MS grade water and were subjected to ESI TOF-MS. ESI TOF-MS (time-of-flight) TOF experiments were conducted (Mitra et al. 2022) using a Waters Xero-GS-XS-Q-Tof. All samples were ionized using electrospray ionization in a positive ion mode and were operated in a scanning range of m/z 50-1800 keeping a capillary voltage of 3 kV. Source and desolvation temperatures were set at 100^oC and 250^oC respectively. Nitrogen was used as nebulizing gas and the pressure was maintained at 300 L/hr. Argon was used as auxiliary gas and the pressure was maintained at 49 L/hr. After sterile filtration, all samples were infused into the ESI source maintaining a flow rate of 5µl/min and an acquisition time of 1min. Calibration was performed using sodium formate and leucine enkephalin as primary secondary calibrants respectively. The product of MS analysis was predicted using MassLynx software.

Qualitative and quantitative HPLC analysis of TMA

The HPLC method for qualitative and quantitative analysis of TMA degrading property of isolate PS1 was performed according to Consuelo Cháfer-Pericás et.al (2004) with modifications. PS1 isolate was grown in a modified DMB medium supplemented with TMA for 4 days at 30^oC in a B.O.D. incubator shaker at 150 rpm. An uninoculated TMA broth medium was kept as control and a blank was kept containing medium without TMA at same incubation conditions. TMA.HCl of 1 mg/ml was taken as standard which was dissolved in HPLC grade water (Merck). The culture was centrifuged at 10,000rpm for 2 min and the supernatant was collected. Borate buffer (200mM) of pH 10 and of 9-fluorenylmethyl chloroformate (FMOC) 15mM in ratio 1:1 was added to supernatant and was kept at room temperature for 5 min. Water to acetonitrile in a 1:1 ratio was added to the mixture and kept at rest for 1 min. The mixture was centrifuged at 10,000 rpm for 5–10 min. The reaction mixture was sterilized through a 0.22µm syringe-driven filter, diluted to 50 times and 100 µl of the sample was injected into the HPLC column using Hamilton Syringe. HPLC was carried on gradient mode using acetonitrile and phosphate buffer of pH 4.2 in the ratio 40:60. The instrument specifications were WATERS HPLC System, Model No. 2695, MDLC (Multi-Dimensional Liquid Chromatography), EMPOWER-2 Software from WATERS, Dual Binary Pump of Model No is 515 from WATERS. Dual lambda absorbance detector was used and the column was Hypersil Gold CI8 Reverse Phase Column from WATERS of dimensions 4.6 x 250mm and particle size of 5µm. Mobile phase A was 0.05M phosphate buffer (pH 4) and mobile phase B was 100% Acetonitrile. The flow rate was kept at 1 ml/min and the run time was 10 min at a wavelength of 262 nm.

Isolation of genomic DNA, whole genome sequencing (WGS) and phylogenetic analysis.

Genomic DNA isolation of PS1 was performed using a Genomic DNA kit (PureLink™ Genomic DNA Mini Kit Catalog number: K182001). The isolated DNA was subjected to whole genome sequence analysis at the National Institute of Biomedical Genomics (NIBMG), Kalyani, West Bengal, India. The whole genome of strain PS1 was sequenced using Illumina NovaSeq 6000 sequencing technology and the WGS is submitted to the NCBI database under the BioProject accession number of PRJNA858582. Primarily, raw sequences were visualized in an updated version of FastQC v0.11.9 (Andrews 2010), followed by trimming and size selection (> 200 bp) in Trim_Galore v0.6.5 (Andrews et al. 2015). High-quality reads (Phred score >20) obtained after trimming were then used for downstream analysis. Unicycler v0.4.8, a hybrid bacterial genome assembly pipeline (Wick et al. 2017), was used for de novo assembly. The assembly pipeline was optimized, which involved an error correction of sequenced reads with Bayes Hammer (Nikolenko et al. 2013) and assembly with SPAdes v3.15.3 (Bankevich et al. 2012), with a k-mer value (up to 99). Finally, the assembly quality was visualized in QUAST v5.2.0 (Gurevich et al. 2013) and annotated using Prokka v1.14 (Seemann 2014). Eventually, functional annotation of protein sequences was done using the PANNZER2 web server (Törönen et al. 2018). The full-length 16S rRNA sequence was retrieved from WGS of strain PS1 and a 16S rRNA similarity search was performed using BLAST (Johnson et al. 2008) of NCBI database. Similar reference sequences homologous to the 16S rRNA sequence of strain PS1 retrieved from the NCBI database and Multiple Sequence Alignment (MSA) was performed using the MUSCLE algorithm in the MEGA7 software (Hall 2013). Depending on the best fit model the maximum-likelihood tree (Kishino and Hasegawa 1989) was constructed using the MEGA7 program, bootstrapping with 1000 replications (Kumar et al. 1994). Bootstrap values are considered 1000 replicates, and branches lower than 50% of bootstrap values are not considered (Felsenstein 1985).

Retrieval of Tdm and Tmm sequences and in silico characterization of these enzymes

After functional annotation of the assembly file of WGS of Paracoccus sp strain PS1, FASTA sequences of TMA degrading enzymes such as Tmm and Tdm were retrieved and subjected to conserved domain (CD) search (domain responsible for the functioning of the proteins) using BLASTP tool of the NCBI database (Johnson et al. 2008) and KEGG BLASTP tool of the KEGG database (for functional characterization of the enzymes) (Kanehisa and Goto 2000). For comparative study, similar intragenus and intergenus reference sequences of Tmm and Tdm were retrieved from the NCBI protein database homologous to Tmm and Tdm sequences of PS1 and checked for the presence of their conserved domain (Marchler-Bauer et al. 2010) using BLASTP tool (Johnson et al. 2008) of NCBI database. The percentage similarity and identity between both the query (Tmm and Tdm of PS1) and reference sequences were calculated using the LALIGN tool (EMBOSS) (Huang and Miller 1991) following the Smith-Waterman algorithm (Farrar 2007). MSA of all the Tmm and Tdm sequences were performed using the MUSCLE algorithm (Edgar 2004) of the MEGA7 software (Kumar et al. 2016) and the phylogenetic trees were constructed using the MEGA7 software (Kumar et al. 2016) by maximum likelihood method (Kishino and Hasegawa 1989) based on the best-fit model. The tree’s branch length scale corresponds to the number of substitutions per site. Bootstrap values are considered 1000 replicates, and branches lower than 50% of bootstrap values are not considered (Felsenstein 1985).

Physicochemical characterization of Tmm and Tdm sequences of PS1

The physicochemical properties of the primary structure of Tmm and Tdm Paracoccus sp. strain PS1 were obtained using the ExPASy ProtParam tool (Gasteiger et al., 2005). The parameters such as pI value, molecular weight, instability and aliphatic index, and grand average of hydropathicity (GRAVY) of the Tmm and Tdm of PS1 sequences were calculated. Low pI values resembled the acidic nature of proteins and high pI values were predicted as basic (Moldoveanu and David, 2017). Proteins were considered unstable if their instability index values were above 40 (Guruprasad et al., 1990). High aliphatic index values represented high thermal stability of the proteins (Ikai, 1980). Negative GRAVY values predicted the hydrophilic nature of the proteins (Kyte and Doolittle, 1982). Using STRING (Szklarczyk et al. 2018) database, we estimated the protein-protein interactions between TMA degrading proteins.

Statistical analysis

All the experiments described above were run in triplicate. The standard curve of known TMA concentrations was performed in Microsoft Excel 2010. Statistical data analysis of the quantitative results obtained from the experiments such as mean and standard deviation were analysed using GraphPad Prism 7 software. T-test was employed to calculate P-values for the variations between the test compounds and the control. P value result was observed to be less than the alpha level of 0.05 (< 0.05) and so the results were considered statistically significant.

Selection of isolates

Morphologically different isolates were selected based on their faster growth rate on modified DM medium plates supplemented with TMA as carbon and nitrogen source. Five isolates (PS1, P4, PS, and PG) were obtained from Pomfret fish, two (KS1, KS2) from Khoira fish, two (NTS and NTG) from Nuna Tengra fish, three isolates (HS1, HS2 and HG) from Hilsa fish and three isolates (LS1, LS2 and LG) from Parshe fish. Out of all the 14 isolates, isolate PS1 was selected for further study based on its faster growth on medium containing TMA as both carbon and nitrogen source as spectrophotometrically measured at 600 nm.

Optimization of Growth conditions

Isolate PS1 was found to be Gram-positive, cocci-shaped bacteria. Antibiotic profiling of the isolate revealed that it was sensitive towards all conventional antibiotics tested, it could utilize various carbon compounds as substrate for growth and could effectively reduce nitrate for nitrate reduction test. PS1 showed positive for catalase activity, amylase activity and gave negative results for β hemolysis where no clear hemolytic zone surrounding the colonies in blood agar plates was observed (data not shown). It showed negative results for indole production, MR, VP and casein hydrolytic activity. The optimized growth pH and temperature for isolate PS1 were at neutral pH7 ± 0.2 and 30^oC respectively. The isolate could also grow up to 0.6M salt concentration when grown in a varying range of salt concentrations and could utilize trimethylamine up to 2M concentration (Fig. S1).

Growth of isolate PS1 in various amines

Isolate PS1 grew in both DM agar plates and broth medium containing TMA, DMA and MMA at 30^oC. Increase in the growth rate of PS1 in MMA, DMA and TMA broth mediums were recorded at 24, 48 and 72 hrs (Fig. S2a). PS1 was also able to grow in TMAO medium, however the growth rate was slow and required 72 hrs for full quadrant growth (Fig. S2b).

Result of TMA degradation assay of isolate PS1

The experiment showed that the reduction in TMA content in culture medium was directly proportional to the increase in growth compared to the control. Moreover, the highly basic pH of the medium (due to the presence of trimethylammonium ion) was reduced as TMA was taken up as both carbon and nitrogen by isolate PS1 during growth compared to the control (Fig. 1). The control set showed a negligible amount of TMA reduction/dissipation in the air with time due to its volatile nature whereas the supernatant of PS1 showed a huge reduction in TMA with time which is directly proportional to the increase in growth rate. The rate of TMA utilization (calculated from the standard curve (values in Table S1)), pH and growth of isolate PS1 to control were calculated. At 0 hr, TMA concentration of control was 46.11mg/ml and that of supernatant of PS1 was 46 mg/ml and at 240 hrs they were 25.11mg/ml and 2.67mg/ml respectively. The initial growth O.D. of PS1 at 600nm was 0.065 and at 240 hrs it was 1.572. At 0 hr, both the control and supernatant of PS1 showed pH values at 7.27 and at 240 hrs the pH values were 7.12 and 6.59 respectively. The result shows that the TMA content was reduced 9.4 times than the control and the pH was also reduced due to high uptake of TMA from the medium by isolate PS1 for its growth.

Nessler reaction result

According to the Nessler reaction, the medium color changes from transparent to brown due to the presence of ammonia after the addition of Nessler's reagent. Here, it was observed in Fig. S3 that the blank tube containing medium without TMA remains transparent due to absence of both ammonia and TMA whereas the tube containing TMA medium shows white precipitation. However, the TMA and ammonia medium-containing tube don’t give brown precipitate (ppt) despite the presence of ammonia (Vickery and Pucher 1929). So, it can be predicted that only when TMA is in high concentration it causes a white coloration which overpowers the brown ppt formed by ammonia, as a result when TMA is in elevated concentration in medium, negligible amount of ammonia produced cannot be detected. So at the initial stages of growth of isolate PS1 no brown ppt for ammonia release was detected but as the growth increased and TMA was reduced from the medium brown color precipitate of ammonia was observed with the increase in growth. This experiment proves that TMA has been utilized as both carbon and nitrogen source by PS1 releasing ammonia as the byproduct.

TMA degradation result by ESI TOF-MS

The ESI TOF-MS spectrum analysis of standard TMA detected an intense peak at m/z 61.0045 corresponding to the protonated (M + 1) form of TMA (the molar mass of TMA as obtained from literature is 59.11). The ESI TOF-MS spectra for the blank set (containing no TMA in the medium) showed no peak at m/z 61, however the sample peaks of PS1 showed lowering of the peak height at 61 compared to the control set kept at the same incubation conditions. The concentration of TMA degraded by the bacterium was evaluated by comparing the peak height at m/z 61 of control (61.0029) in respect to the sample PS1 (61.0061) is shown the rate of degradation of TMA by PS1 with control is shown in Fig. 2. The peak height of TOF MS ESI of standard was 9.48e3, control was 1.30e3 and cell-free culture filtrate of PS1 was 400. For quantitative analysis, TMA concentrations of the samples were calculated from the height of their respective peaks compared to the known standard. High TMA degradation was observed in the cell-free culture filtrate of PS1 of 0.042mg/ml compared to the control which was 0.14 mg/ml.

Confirmation result of TMA degradation by HPLC

The degradation of trimethylamine by isolate PS1 could be confirmed by the reduction of TMA content in the sterile cell-free culture filtrate of isolate PS1 compared to the uninoculated control set when kept under the same incubation conditions (Fig. 3). The retention time of standard TMA was 2.785 min, cell-free culture filtrate of PS1 was 2.786 min and control was 2.809 min. No peak of TMA was observed at standard TMA retention time for the blank set. The TMA concentration of control and cell-free culture filtrate of PS1 were calculated from the area of their respective chromatograms with respect to the known standard concentration. The TMA content of the control set was 1.3mg/ml whereas the TMA content of cell-free culture filtrate of isolate PS1 was reduced to 1.13mg/ml. Reduction in TMA content observed in the cell-free culture filtrate of PS1 then the control proves the utilisation of TMA by isolate PS1 for growth.

Whole genome assembly statistics and 16S rRNA phylogenetic analysis

The genomic DNA of PS1 was isolated and its band image is shown in the figure with ladder of 500 bp (O’Range Ruler of Fermentas brand) (Fig. S4). The assembly quality result obtained from QUAST analysis is represented in Table S2. 16S rRNA sequence retrieved from WGS of strain PS1 was aligned with reference 16S rRNA sequences available on the NCBI Database by using the BLAST tool. Phylogenetic tree constructed shows its close similarity with Paracoccus communis (Fig. 4). The 16S rRNA sequence is deposited in the GenBank database of NCBI and its accession number is OP070057. Rhodobacter capsulatus ATCC 11166 was used as an outgroup. Bar, 0.02 substitutions per nucleotide position is calculated (Pan et al., 2014).

KEGG BLASTP result, sequence identity and similarity result and phylogenetic tree construction of Tmm and Tdm sequences

The Tmm and Tdm sequences of PS1 showed functional identity with reported Tmm, i.e. trimethylamine monooxygenase and Tdm, i.e. trimethylamine-oxide aldolase sequences with an E-value of 0 in KEGG BLASTP software (Fig. S5a and Fig. S5b).

The Protein ID of gene sequence from WGS uploaded in NCBI database is given below:

Protein Name	Protein_ID	Product
Trimethylamine monooxygenase (Tmm)	PRJNA858582:NM680_20190	NAD(P)/FAD-dependent oxidoreductase
Trimethylamine-oxide aldolase (Tdm)	PRJNA858582:NM680_20185	DUF1989 domain-containing protein

The similarity and identity percentages of Tmm and Tdm sequences after alignment are provided (Tables S3 and S4). Sr. No. 1–9 of Table S3 contains NAD(P)/FAD-dependent oxidoreductase (containing the CD of Tmm) and reported Tmm sequences of 9 different species of Paracoccus genus for intragenus comparison and Sr. No. 10–11 contains Tmm sequences from various genus for intergenus comparison. Sr. No. 12 contains the outgroup (FAD-dependent oxidoreductase) sequence that doesn’t possess the CD for Tmm activity. Sr. No. 1–10 of Table S4 contains Tdm sequences reported as aminomethyltransferase family protein sequence of 10 different species of Paracoccus genus for intragenus comparison and Sr. No. 11–22 contains Tdm sequences of various other genus for intergenus comparison. Sr. No. 23 contains the outgroup (aminomethyltransferase) sequence that doesn't possess the CD for Tdm function. The Tmm sequence of Paracoccus sp. PS1 contains the FMO-like superfamily conserved domain which is essential for TMA degradation similar to the reported Tmm sequences of NCBI database (Fig. S6a and Fig. S6b). Intergenic and intragenic percentage similarity and identity of Tmm sequence of Paracoccus sp. strain PS1 showed Paracoccus sp. J39 is the closest relative of PS1 having 100% percentage similarity and identity, and highest intergenus similarity and identity with Ruegeria pomeroyi DSS-3 (Lidbury et al., 2015) of 94.4% and 82.6% respectively. The FAD oxidoreductase sequence (outgroup) of Paracoccus sanguinis does not contain the FMO family domain responsible for Tmm activity (Table S3). Maximum likelihood phylogenetic tree (Fig. 5a) was constructed based on the best-fit LG model with a gamma distribution (LG + G) (Nei and Zhang 2006. Bar, 5 substitutions per nucleotide position was considered (Pan et al. 2014).

Tdm protein sequences of genus Paracoccus, has not yet been reported by any researcher in NCBI. So, we have aligned reported Tdm sequences of other genus and the reported aminomethyltransferase family protein sequences of Paracoccus genus which showed homology with Tdm sequence of Paracoccus sp. strain PS1 using the BLASTP tool. In Table S4 the aminomethyltransferase family protein of Paracoccus dentrificans was considered an outgroup as it didn’t possess the conserved DUF1989 domain, which is responsible for Tdm activity (Zhu et al. 2016b). The aminomethyltransferase family protein (Tdm) of strain PS1 showed conserved domains of DUF1989 and GCV_T protein which is identical to reported TMAO aldolase (Tdm) sequences of Ruegeria pomeroyi (Lidbury et al. 2015) (Fig. S7a and S7b). The highest intergeneric and intrageneric similarity and identity percentages of strain PS1 was observed with Paracoccus sp. MA with 99.7% and 99.2% respectively and Ruegeria pomeroyi of 85.5% and 67.1% respectively. Maximum likelihood phylogenetic tree (Fig. 5b) was constructed using the LG + G best fit model. Bar, 2 substitutions per nucleotide position was considered (Pan et al. 2014).

Physicochemical properties of Tmm and Tdm sequences

The amino acid sequences of Tmm and Tdm are shown in Fig. S8 and their physicochemical properties are shown in Table S5. Both Tmm and Tdm of Paracoccus sp. PS1 are observed to be acidic in nature possessing the isoelectric point (pI) values of 5.65 and 5.86 respectively. The instability index of both Tmm and Tdm are lower than 40 with a value of 33.26 and 34.06, hence are considered to be stable in nature. The aliphatic index for PS1 Tmm is 67 and that of Tdm is 81.96, showing high thermal stability of the proteins. The GRAVY values of Tmm and Tdm were − 0.466 and − 0.115 which determines the hydrophilic nature of the proteins. STRING database predicted the proteins involved in the TMA degradation network in bacterial system such as Tmm, Tdm (belonging to aminomethyltransferase group), DmmA (dimethylamine monooxygenase A subunit), DmmB (dimethylamine monooxygenase B subunit) and DmmC (dimethylamine monooxygenase C subunit) of Paracoccus sp. strain PS1 on the basis of their similarity with the same proteins reported in Paracoccus aminovorans in the NCBI database (Fig. S9 and Table S6).

Only a few bacteria such as Hyphomcrobium sp (Meiberg 1979), Paracoccus sp. (Kim et al. 2001), Ruegeria pomeroyi DSS-3 (Lidbury et al. 2015) and Aminobacter aminovorans (Aguirre et al. 2018). are reported to possess TMA degrading enzymes. According to the reports, the growth medium required for culturing of those bacteria consists of mineral-salt medium enriched with TMA and ammonium salt (such as (NH₄)₂SO₄, NH₄Cl.) as carbon and nitrogen source. It is observed that PS1 has the ability to grow in a modified DM medium containing TMA as sole carbon and nitrogen source without any ammonium salt. This means PS1 possesses both trimethylamine monooxygenase (Tmm) and trimethylamine N-oxide aldolase (Tdm) activity to utilize TMA as carbon as well as nitrogen source. Additionally, the ability of Paracoccus sp. strain PS1 to grow on three different amines viz TMA, DMA and MMA, when used as sole carbon and nitrogen source, proved that it contains all the genes required for the breakdown of TMA to its respective amines releasing ammonia at the end product (Chen et al. 2011). As there is no direct assay method yet to quantify TMA concentration in the growth medium, we have tested some indirect quantitative and qualitative assays such as Nessler reaction, spectrophotometric, ESI TOF-MS, HPLC methods with modifications for that purpose. The Nessler method by Naphade et.al was carried out to detect ammonia production by agricultural isolate Pseudomonas putida but here we have performed the assay for ammonia detection as a by-product of TMA degradation by Paracoccus sp. strain PS1. Spectrophotometric assay method and HPLC analysis by Miao-Kang Shen (Shen 1988) and Cháfer-Pericás et al. respectively used for quantification of TMA, present in various environmental water samples whereas in this study those methods are modified for easy quantification of TMA concentration in the culture medium in different growth phase of the isolate PS1. ESI TOF-MS procedure has been earlier used for detection for TMA in urine and plasma samples (Jia et al. 2020). We have employed the ESI TOF-MS TOF method for quick determination of the rate of reduction in TMA content of the culture medium with increase in growth rate by PS1. Annotation of the whole genome of PS1 confirmed the presence of Tmm and Tdm (the two TMA degrading enzymes) enzymes. Physicochemical properties such as instability index and aliphatic index values of Tmm and Tdm are analysed by the ProtParam tool that recognizes them as thermally stable proteins. The degradation of gut generated TMA by PS1 like microbial strain may greatly reduce its availability for conversion into TMAO by host liver enzyme FMO3. In addition to that microbes like PS1 may reduce the chance of absorption of TMAO from sea food directly into the human circulation due to presence of TMAO degrading enzymes in them. In Trimethylaminuria patient intensity of the disease may be reduced by bacteria like PS1 which breaks down the TMA in the gut before absorption. So, according to the in vitro and in silico studies we can say PS1 has potential to degrade TMA and TMAO and can be developed in the long run into a suitable probiotic strain to reduce the adverse effect of TMA and TMAO in human gut.

Data availability

The 16S rRNA gene sequence of Paracoccus sp. strain PS1 has been submitted under the accession number OP070057. The whole Genome sequence of Paracoccus sp. strain PS1 has been deposited in GenBank of NCBI under the BioProject accession number PRJNA858582.

Declaration of Competing interest

The authors declare that they have no competing interests.

Funding

This work was supported by a grant of Department of Science and Technology Innovation in Science Pursuit for Inspired Research (DST INSPIRE) (Grant No. DST/INSPIRE Fellowship/2018/IF180258) Government of India, Ministry Of Science and Technology, Department of Science & Technology (DST), New Delhi, India. The funding body played a significant role in the data collection for the study, their analysis and finally data interpretation for writing the manuscript.

Consent for publication

Not applicable

Ethics approval

Not applicable

Acknowledgement

We thankfully acknowledge Dr. Wriddhiman Ghosh, Associate Professor of Microbiology, Bose Institute, Kolkata, for his valuable advice in conceptualization of the work and for providing instrumentation facilities of his laboratory for Genomic DNA extraction and ESI TOF-MS analysis. We are thankful to Dr. Ramdhan Majhi, Principal Technical Officer, HPLC, Incharge of Central Instrumentation Facility (CIF), IICB, Kolkata, India for HPLC analysis of the samples, Souvik Roy, Senior Laboratory Assistant, CIF, Bose Institute, Kolkata, India for ESI TOF-MS analysis of the samples. We also thankfully acknowledge the microbiology laboratory of Dr. Bomba Dam, Department of Botany, Institute of Science, Visva-Bharati (A Central University), Shantiniketan, India for providing the infrastructure for bioinformatics analysis and the Honourable Vice Chancellor, The University of Burdwan, for providing the necessary infrastructural facilities.

Author contributions

MS has designed, conducted the experiments, analysed the data and wrote the manuscript. PM and DG contributed in writing of Original draft preparation. RB conducted the bioinformatic analysis such as annotation of WGS data and phylogenetic analysis. SC helped in performing ESI TOF-MS and DNA isolation. SKM provided valuable contributions in project administration, conceptualization, review and editing of the manuscript, and supervision of this whole project.

Aguirre A, Bernal P, Maureira D, Ramos N, Vásquez J, Urrutia H, Gentina JC, Aroca G (2018) Biofiltration of trimethylamine in biotrickling filter inoculated with Aminobacter aminovorans. Electron J Biotechnol 33:63-67. https://doi.org/10.1016/j.ejbt.2018.04.004
Andrews S (2010) Babraham bioinformatics-FastQC a quality control tool for high throughput sequence data.
Andrews S, Krueger F, Segonds-Pichon A, Biggins L, Virk B, Dalle-Pezze P, Wingett S, Saadeh H, Ahlfors H (2015) Trim Galore.
Ayesh R, Mitchell S, Zhang A, Smith R (1993) The fish odour syndrome: biochemical, familial, and clinical aspects. Br Med J 307:655-657. https://doi.org/10.1136/bmj.307.6905.655
Balebona M, Morinigo M, Faris A, Krovacek K, Månsson I, Bordas M, Borrego J (1995) Influence of salinity and pH on the adhesion of pathogenic Vibrio strains to Sparus aurata skin mucus. Aquaculture 132:113-120. https://doi.org/10.1016/0044-8486(94)00376-Y
Bankevich A, Nurk S, Antipov D, Gurevich AA, Dvorkin M, Kulikov AS, Lesin VM, Nikolenko SI, Pham S, Prjibelski AD (2012) SPAdes: a new genome assembly algorithm and its applications to single-cell sequencing. J Comput Biol 19:455-477. https://doi.org/10.1089/cmb.2012.0021
Catalá R, López-Cobollo R, Berbís MÁ, Jiménez-Barbero J, Salinas J (2021) Trimethylamine N-oxide is a new plant molecule that promotes abiotic stress tolerance. Sci Adv 7:eabd9296. https://doi.org/10.1126/sciadv.abd9296
Cháfer-Pericás C, Herráez-Hernández R, Campı́ns-Falcó P (2004) Liquid chromatographic determination of trimethylamine in water. J Chromatogr A 1023:27-31. https://doi.org/10.1016/j.chroma.2003.10.003
Chen Y, Patel NA, Crombie A, Scrivens JH, Murrell JC (2011) Bacterial flavin-containing monooxygenase is trimethylamine monooxygenase. Proc Natl Acad Sci 108:17791-17796. https://doi.org/10.1073/pnas.1112928108
Coutinho-Wolino KS, de F. Cardozo LF, de Oliveira Leal V, Mafra D, Stockler-Pinto MB (2021) Can diet modulate trimethylamine N-oxide (TMAO) production? What do we know so far? Eur J Nutr 60: 3567–3584. https://doi.org/10.1007/s00394-021-02491-6
Craciun S, Balskus EP (2012) Microbial conversion of choline to trimethylamine requires a glycyl radical enzyme. Proc Natl Acad Sci 109:21307-21312. https://doi.org/10.1073/pnas.1215689109
Dolphin CT, Janmohamed A, Smith RL, Shephard EA, Phillips lR (1997) Missense mutation in flavin-containing mono-oxygenase 3 gene, FMO3, underlies fish-odour syndrome. Nat Genet 17(4):491-494. https://doi.org/10.1038/ng1297-491
Duremdez R, Al‐Marzouk A, Qasem J, Al‐Harbi A, Gharabally H (2004) Isolation of Streptococcus agalactiae from cultured silver pomfret, Pampus argenteus (Euphrasen), in Kuwait. J Fish Dis 27:307-310. https://doi.org/10.1111/j.1365-2761.2004.00538.x
Edgar RC (2004) MUSCLE: multiple sequence alignment with high accuracy and high throughput. Nucleic Acids Res 32:1792-1797. https://doi.org/10.1093/nar/gkh340
Farrar M (2007) Striped Smith-Waterman speeds database searches six times over other SIMD implementations. Bioinformatics 23:156-61. https://doi.org/10.1093/bioinformatics/btl582
Felsenstein J (1985) Confidence limits on phylogenies: an approach using the bootstrap. Evolution 39:783-791. https://doi.org/10.2307/2408678
Fennema D, Phillips IR, Shephard EA (2016) Trimethylamine and trimethylamine N-oxide, a flavin-containing monooxygenase 3 (FMO3)-mediated host-microbiome metabolic axis implicated in health and disease. Drug Metab Dispos 44:1839-1850. https://doi.org/10.1124/dmd.116.070615
Gasteiger E, Hoogland C, Gattiker A, Duvaud Se, Wilkins MR, Appel RD, Bairoch A (2005) Protein identification and analysis tools on the ExPASy server. The proteomics protocols handbook, pp 571-607. https://doi.org/10.1385/1-59259-890-0:571
Guhathakurta H, Kaviraj A (2000) Heavy metal concentration in water, sediment, shrimp (Penaeus monodon) and mullet (Liza parsia) in some brackish water ponds of Sunderban, India. Mar Pollut Bull 40:914-920. https://doi.org/10.1016/S0025-326X(00)00028-X
Gurevich A, Saveliev V, Vyahhi N, Tesler G (2013) QUAST: quality assessment tool for genome assemblies. Bioinformatics 29:1072-1075. https://doi.org/10.1093/bioinformatics/btt086
Guruprasad K, Reddy BB, Pandit MW (1990) Correlation between stability of a protein and its dipeptide composition: a novel approach for predicting in vivo stability of a protein from its primary sequence. Protein Eng Des Sel 4:155-161. https://doi.org/10.1093/protein/4.2.155
Habib KA, Islam MJ (2020) An updated checklist of Marine Fishes of Bangladesh. Bangladesh J Fish 32:357-367. https://doi.org/10.52168/bjf.2020.32.40
Hall BG (2013) Building phylogenetic trees from molecular data with MEGA. Mol Biol Evol 30:1229-1235. https://doi.org/10.1093/molbev/mst012
Huang X, Miller W (1991) Lalign-find the best local alignments between two sequences. Adv Appl Math 12:373-381.
Ikai A (1980) Thermostability and aliphatic index of globular proteins. J Biochem 88:1895-1898.
Jia X, Osborn LJ, Wang Z (2020) Simultaneous measurement of urinary trimethylamine (TMA) and trimethylamine N-oxide (TMAO) by liquid chromatography–mass spectrometry. Molecules 25:1862. https://doi.org/10.3390/molecules25081862
Johnson M, Zaretskaya I, Raytselis Y, Merezhuk Y, McGinnis S, Madden TL (2008) NCBI BLAST: a better web interface. Nucleic Acids Res 36:W5-W9. https://doi.org/10.1093/nar/gkn201
Kanehisa M, Goto S (2000) KEGG: kyoto encyclopedia of genes and genomes. Nucleic Acids Res 28:27-30. https://doi.org/10.1093/nar/28.1.27
Kim S-G, Bae H-S, Lee S-T (2001) A novel denitrifying bacterial isolate that degrades trimethylamine both aerobically and anaerobically via two different pathways. Arch Microbiol 176:271-277. https://doi.org/10.1007/s002030100319
Kishino H, Hasegawa M (1989) Evaluation of the maximum likelihood estimate of the evolutionary tree topologies from DNA sequence data, and the branching order in Hominoidea. J Mol Evol 29:170-179. https://doi.org/10.1007/BF02100115
Kumar S, Stecher G, Tamura K (2016) MEGA7: molecular evolutionary genetics analysis version 7.0 for bigger datasets. Mol Biol Evol 33:1870-1874. https://doi.org/10.1093/molbev/msw054
Kumar S, Tamura K, Nei M (1994) MEGA: molecular evolutionary genetics analysis software for microcomputers. Bioinformatics 10:189-191. https://doi.org/10.1093/bioinformatics/10.2.189
Kyte J, Doolittle RF (1982) A simple method for displaying the hydropathic character of a protein. J Mol Biol 157:105-132. https://doi.org/10.1016/0022-2836(82)90515-0
Li D, Kirsop J, Tang WW (2015) Listening to our gut: contribution of gut microbiota and cardiovascular risk in diabetes pathogenesis. Curr Diabetes Rep 15:1-9. https://doi.org/10.1007/s11892-015-0634-1
Lidbury ID, Murrell JC, Chen Y (2015) Trimethylamine and trimethylamine N-oxide are supplementary energy sources for a marine heterotrophic bacterium: implications for marine carbon and nitrogen cycling. The ISME J 9:760-769. https://doi.org/10.1038/ismej.2014.149
Maksymiuk KM, Szudzik M, Gawryś-Kopczyńska M, Onyszkiewicz M, Samborowska E, Mogilnicka I, Ufnal M (2022) Trimethylamine, a gut bacteria metabolite and air pollutant, increases blood pressure and markers of kidney damage including proteinuria and KIM-1 in rats. J Transl Med 20:470. https://doi.org/10.1186/s12967-022-03687-y
Marchler-Bauer A, Lu S, Anderson JB, Chitsaz F, Derbyshire MK, DeWeese-Scott C, Fong JH, Geer LY, Geer RC, Gonzales NR, Gwadz M, Hurwitz DI, Jackson JD, Ke Z, Lanczycki CJ, Lu F, Marchler GH, Mullokandov M, Omelchenko MV, Robertson CL, Song JS, Thanki N, Yamashita RA, Zhang D, Zhang N, Zheng C, Bryant SH (2010) CDD: a Conserved Domain Database for the functional annotation of proteins. Nucleic Acids Res 39:D225-D229. https://doi.org/10.1093/nar/gkq1189
Meiberg J (1978) Aerobic and anaerobic metabolism of trimethylamine, dimethylamine and methylamine in Hyphomicrobium X. J Gen Microbiol 106:265-276.
Mitra S, Naskar N, Ghosh K, Dutta A, Lahiri S, Chaudhuri P, Saha A (2022) Studies on radiation stability of natural caffeine. Appl Radiat and Isot 183:110148. https://doi.org/10.1016/j.apradiso.2022.110148
Moldoveanu SC, David V (2017) Properties of analytes and matrices determining HPLC selection. Sel HPLC Method Chem Anal pp 189-230. https://doi.org/10.1016/B978-0-12-803684-6.00005-6
Naphade BS, Hussain SS (2014) Isolation, characterization and evaluation of Pseudomonas putida as a plant growth promoting rhizobacteria. Int Sci J 1:30-33
Nei M, Zhang J (2006) Evolutionary distance: estimation. Encyclopedia of life sciences. 2005, John Wiley & Sons, Ltd, pp 1-4.
Nikolenko SI, Korobeynikov AI, Alekseyev MA (2013) BayesHammer: Bayesian clustering for error correction in single-cell sequencing. BMC Genom 14:1-11.
Pan J, Sun C, Zhang X-Q, Huo Y-Y, Zhu X-F, Wu M (2014) Paracoccus sediminis sp. nov., isolated from Pacific Ocean marine sediment. Int J Syst Evol 64:2512-2516. https://doi.org/10.1099/ijs.0.051318-0
Quan L, Yi J, Zhao Y, Zhang F, Shi X-T, Feng Z, Miller HL (2020) Plasma trimethylamine N-oxide, a gut microbe–generated phosphatidylcholine metabolite, is associated with autism spectrum disorders. Neurotoxicology 76:93-98. https://doi.org/10.1016/j.neuro.2019.10.012
Rath S, Heidrich B, Pieper DH, Vital M (2017) Uncovering the trimethylamine-producing bacteria of the human gut microbiota. Microbiome 5:1-14. https://doi.org/10.1186/s40168-017-0271-9
Sahoo AK, Wahab MA, Phillips M, Rahman A, Padiyar A, Puvanendran V, Bangera R, Belton B, De DK, Meena DK (2018) Breeding and culture status of Hilsa (Tenualosa ilisha, Ham. 1822) in South Asia: A review. Rev Aquac 10:96-110. https://doi.org/10.1111/raq.12149
Schneider C, Okun J, Schwarz K, Hauke J, Zorn M, Nürnberg C, Ungerer M, Ringleb P, Mundiyanapurath S (2020) Trimethylamine‐N‐oxide is elevated in the acute phase after ischaemic stroke and decreases within the first days. Eur J Neurol 27:1596-1603. https://doi.org/10.1111/ene.14253
Seemann T (2014) Prokka: rapid prokaryotic genome annotation. Bioinformatics 30:2068-2069. https://doi.org/10.1093/bioinformatics/btu153
Shen M-K (1988) Spectrophotometric determination of trimethylamine nitrogen in raw waters using picric acid. Analyst 113:1139-1140. https://doi.org/10.1039/AN9881301139
Siddiqui M, Biswas P, Ray S, Hasan M, Reza M (2010) Effect of Freezing Time on the Nutritional Value of Mystus gulio (Nuna tengra), Mystus tengra (Bazari tengra) and Mystus cavasius (Ghulsha tengra). J Sci Found 8:119-122. https://doi.org/10.3329/jsf.v8i1-2.14634
Szklarczyk D, Gable AL, Lyon D, Junge A, Wyder S, Huerta-Cepas J, Simonovic M, Doncheva NT, Morris JH, Bork P, Jensen LJ, Mering Christian v (2018) STRING v11: protein–protein association networks with increased coverage, supporting functional discovery in genome-wide experimental datasets. Nucleic Acids Res 47(D1):D607-D613 https://doi.org/10.1093/nar/gky1131
Tang WW, Wang Z, Kennedy DJ, Wu Y, Buffa JA, Agatisa-Boyle B, Li XS, Levison BS, Hazen SL (2015) Gut microbiota-dependent trimethylamine N-oxide (TMAO) pathway contributes to both development of renal insufficiency and mortality risk in chronic kidney disease. Circ Res 116:448-455. https://doi.org/10.1161/CIRCRESAHA.116.305360
Törönen P, Medlar A, Holm L (2018) PANNZER2: a rapid functional annotation web server. Nucleic Acids Res 46:W84-W88. https://doi.org/10.1093/nar/gky350
Velasquez MT, Ramezani A, Manal A, Raj DS (2016) Trimethylamine N-oxide: the good, the bad and the unknown. Toxins 8:326. https://doi.org/10.3390/toxins8110326
Vickery HB, Pucher GW (1929) The determination of ammonia and amide nitrogen in tobacco by the use of permutit. J Biol Chem 83:1-10. https://doi.org/10.1016/S0021-9258(20)70831-8
Wannomai T, Kemacheevakul P, Thiravetyan P (2019) Removal of trimethylamine from indoor air using potted plants under light and dark conditions. Aerosol Air Qual Res 19:1105-1113. https://doi.org/10.4209/aaqr.2018.09.0334
Wick RR, Judd LM, Gorrie CL, Holt KE (2017) Unicycler: resolving bacterial genome assemblies from short and long sequencing reads. PLoS Comput Biol 13:e1005595. https://doi.org/10.1371/journal.pcbi.1005595
Wilson A, McLean C, Kim RB (2016) Trimethylamine-N-oxide: a link between the gut microbiome, bile acid metabolism, and atherosclerosis. Curr Opin Lipidol 27:148-154. https://doi.org/10.1097/MOL.0000000000000274
Xu R, Wang Q (2016) Towards understanding brain-gut-microbiome connections in Alzheimer’s disease. BMC Syst Biol 10:277-285. https://doi.org/10.1186/s12918-016-0307-y
Xu R, Wang Q, Li L (2015) A genome-wide systems analysis reveals strong link between colorectal cancer and trimethylamine N-oxide (TMAO), a gut microbial metabolite of dietary meat and fat. BMC Genom 16:1-9. https://doi.org/10.1186/1471-2164-16-S7-S4
Yancey PH, Gerringer ME, Drazen JC, Rowden AA, Jamieson A (2014) Marine fish may be biochemically constrained from inhabiting the deepest ocean depths. Proc Natl Acad Sci 111(12):4461-4465. https://doi.org/10.1073/pnas.1322003111
Zhu W, Gregory JC, Org E, Buffa JA, Gupta N, Wang Z, Li L, Fu X, Wu Y, Mehrabian M (2016a) Gut microbial metabolite TMAO enhances platelet hyperreactivity and thrombosis risk. Cell 165:111-124. https://doi.org/10.1016/j.cell.2016.02.011
Zhu Y, Ksibe AZ, Schäfer H, Blindauer CA, Bugg TD, Chen Y (2016b) O2‐independent demethylation of trimethylamine N‐oxide by Tdm of Methylocella silvestris. The FEBS J 283:3979-3993. https://doi.org/10.1111/febs.13902
Zhu Y, Li Q, Jiang H (2020) Gut microbiota in atherosclerosis: focus on trimethylamine N‐oxide. Apmis 128:353-366. https://doi.org/10.1111/apm.13038.

No competing interests reported.

Download PDF

Version 1

posted

You are reading this older preprint version

Read the latest preprint version →

Biodegradation of trimethylamine by a halotolerant strain of Paracoccus sp. PS1 and in silico analysis of trimethylamine degrading enzymes

Status:

Version 1

Abstract

Figures

Introduction

Materials and Methods

Chemicals used

Isolation and characterization

Sample collection and isolation of TMA degrading bacteria

Characterization of isolates and determination of optimum growth conditions:

Detection of TMA degradation using Nessler method

Qualitative and quantitative HPLC analysis of TMA

Retrieval of Tdm and Tmm sequences and in silico characterization of these enzymes

Physicochemical characterization of Tmm and Tdm sequences of PS1

Statistical analysis

Results

Selection of isolates

Optimization of Growth conditions

Growth of isolate PS1 in various amines

Result of TMA degradation assay of isolate PS1

Nessler reaction result

TMA degradation result by ESI TOF-MS

Confirmation result of TMA degradation by HPLC

Whole genome assembly statistics and 16S rRNA phylogenetic analysis

Physicochemical properties of Tmm and Tdm sequences

Discussion

Declarations

References

Additional Declarations

Supplementary Files

Status:

Version 1