Identification and phylogenetic affiliation:
Microscopic identification
Strain RS1 formed ~ 1mm in size white to cream-coloured colonies on solid NMS media plates [1]. Live cells were 1.0–4.0 µm X 0.5-1 µm fat rod-elliptical pleomorphic bacilli and showed motility. Some cells were seen in the form of chains under phase-contrast microscopy [1].
Physiochemical characterization
Strain RS1 was a mesophilic methanotroph that grew in the 20–28ºC temperature range (Table 2). RS1 expressed growth of OD600 0.19 at 20°C, OD600 0.26 at 25°C and OD600 0.16 at 30°C. Beyond these temperatures, strain RS1 showed very low or no growth. This maximum growth was achieved after 10 days and the OD did not increase after that point. The doubling time of RS1 was very low ~ 0.0004 − 0.00008 per hour. At 20°C, 25°C, and 30°C, Methylomicrobium oryzae strain RS1 demonstrated growth of OD600 0.19, OD600 0.26, and OD600 0.16 respectively. Strain RS1 exhibited very little or no growth above these temperatures. At 20°C, 25°C, and 30°C, the specific growth rates were 0.0008, 0.001, and 0.0006 h− 1, respectively. Methylosoma difficile strain LC 2 is a methanotroph that grows slowly. The growth range of strain LC 2 was 16–30°C, with 25°C being the ideal growth temperature. According to M. difficile [16] the specific growth rates at 16, 25, and 30°C were 0.0024, 0.0041, and 0.0065 h− 1 [16]. Methylocucumis oryzae strain Sn10-6, another slow-growing methanotroph, demonstrated specific growth rates of 0.0006, 0.0023, 0.0038, and 0.0019 h− 1 at temperatures of 15℃, 20℃, 25℃, and 30℃ respectively [17; 18]. Methylomicrobium oryzae strain RS1 demonstrated relatively low specific growth rates at all tested temperatures, indicating that it is a slow-growing methanotroph.
Strain RS1 expressed growth under buffered and non-buffered conditions in the range between pH 5 to pH 10, with optimum growth around neutral pH 6.8-7 (Table 2). Strain RS1 was sensitive to the NaCl (salt) concentrations, as it was found to tolerate only up to 0.1% (w/v) NaCl (Table 2). Beyond this concentration, there was no growth observed.
Strain RS1 could tolerate and grow in methanol concentrations from 50 mM to 1000 mM with optimum growth between 200 mM to 300 mM methanol expressing a maximum OD of 0.2. RS1 did not utilize any other carbon source except methane and methanol. The strain did not utilize any of the multi-carbon (0.1%) such as glucose, fructose, sucrose, maltose, xylose, arabinose, and raffinose. Formate and formaldehyde were not utilized by RS1. The culture could use the following nitrogen sources: lysine, ammonium chloride, urea, valine, glutamate, peptone, and yeast extract. RS1 did not grow in the nitrogen-free medium under methane and air atmosphere. Strain RS1 could grow in 2.5mM and 25mM KNO3 but did not grow in 100mM KNO3. Successful growth was observed in all NH4Cl and urea concentrations tested. This tolerance to ammonium chloride and urea could have significance in the survival of the strain in rice field habitats where nitrogen-based fertilised are used in excess [4; 19; 20]. No growth of the strain RS1 was observed under hypo-oxic (1% oxygen) growth conditions and little amount of growth was seen under micro-oxic (5% oxygen) conditions. All three preservation techniques attempted for RS1 did not favour its revival thus, the only method currently employed is storing the liquid culture at 4°C in refrigeration for ~ 6 months, followed by its subculture for regular experimentations.
Phylogenetic and phylogenomic affiliation:
After the complete 16S rRNA gene (1523 bp) was obtained using the sequenced draft genome, the 16S rRNA strain RS1 expressed the closest similarity to 98.69% with Methylomocrobium agile ATCC 3508T. The complete 16S rRNA gene sequence has been submitted to the NCBI database with the accession number MH764455.2. The RS1 pmoA gene sequence expressed 94.01% similarity to its closest type species Methylomicrobium album ATCC 3303T. (ref- frontiers). The ANIb, AAI, and DDH values between RS1 and its closest members genomes are 69–91, 62–90, and 18–44% respectively, which are lesser than the thresholds for prokaryotic species description. (ref-frontiers) (Table 2). Based on this analysis, we propose a new name for the putative novel species Ca. Methylomicrobium oryzae strain RS1 sp. nov. [1].
Chemotaxonomic characterization: Strain RS1 contained maximum amounts of 25.06% of 16:0, followed by 24.73% of 16:0 3OH, and 23.57% of 16:1 ω7c/16:1 ω6c as its major cell wall fatty acids (Table 1). Methylomicrobium agile and Methylomicrobium album, the affiliate of strain RS1 possess 16:1 ω5t as its major fatty acid (Table 2). Polyphasic analysis of strain RS1 is shown schematically in Fig. 1.
Draft-genome features: The strain RS1 genome was sequenced using Illumina Hi-seq sequencing technology. A total of 432 scaffolds were constructed, with an N50 value of 233876 and 55 value of K-mer. The sequenced genome generated 69 contigs that were subjected to NCBI-blastx analysis, and it showed close similarity to the members of the genus Methylomicrobium. The contigs were uploaded in RAST (http://rast.nmpdr.org/) and NCBI-PGAP for genome annotation.
Annotated protein list of the genome RS1 using RAST v2.0 and NCBI is provided in Supplementary Table 1 which contains the name of the enzyme, and amino acid length, NCBI annotated accession number (wherever applicable), closest affiliate along with percent similarity, and the affiliate accession number.
Carbon metabolism
The genome of strain RS1 contained the complete set of proteins to oxidise methane gas into carbon dioxide. Two copies of particulate methane monooxygenase genes pmoCAB cluster were found to oxidise methane into methanol (Supplementary Table 1). One copy is identical to that of M. album. However, a second copy is detected which shows very less similarities to other pmoA genes. The dehydrogenation of methanol was done by three copies of PQQ-dependent dehydrogenase, Iron-containing alcohol dehydrogenase, zinc-dependent alcohol dehydrogenase family protein, and NAD(P)-dependent alcohol dehydrogenase (Supplementary Table 1). One of the copies of PQQ-dependent dehydrogenase was found to be a unique gene that showed ~ 70% similarity with that of Novimethylophilus kurashikiensis La2-4. This gene was also present in some species of the Methylocaldum genus (Supplementary Table 1). The H4MPT pathway for formaldehyde oxidation and formate dehydrogenations was also present in the genome of RS1. The assimilation of formaldehyde into the cell can be processed with RuMP or serine pathway by strain RS1; however, the RuMP pathway was used for formaldehyde assimilation by Type I methanotrophs. The genome also contains the complete glycolytic pathway, Entner-Doudoroff pathway, and citric acid.
Nitrogen metabolism
The strain RS1 draft genome contained the complete set of genes for environmental nitrogen fixation (Supplementary Table 1). Nitrogenase iron-molybdenum cofactor biosynthesis protein NifN of strain RS1 showed ~ 85% protein similarity with that of Proteobacteria bacterium ST_bin11. This protein was also present in other methanotrophs such as the genus Methylomonas, Methylobacter, and Methylomicrobium Cyanate hydratase genes were found in the draft genome of strain RS1 like other Methylomicrobium species (Supplementary Table 1). The draft genome also contains genes for the reduction of nitrate to nitrite to ammonia, as shown in Supplementary Table 1. NnrS protein was found in the genome, which is one of the important proteins in the nitrosative stress condition (overproduction of nitric oxide) [21].
Other critical proteins, such as proteins involved in hemerythrin metabolism, are also present and summarised in Supplementary Table 1. Hemerythrin genes are essential in oxygen-scavenging conditions and helpful in transporting oxygen to particulate methane monooxygenase [22; 23; 24].
Fermentation pathways
The genome of RS1 contains genes or enzymes for the fermentation process (Supplementary Table 1). Under oxygen limitation conditions, certain methanotrophs can switch to fermentation from methane respiration. It is concluded that oxygen is required only to activate the methane molecules but not for respiration [25; 26; 27].
Unique genes/enzymes present in the genome
The putative novel strain RS1 genome contains unique genes among methanotrophs. Some unique enzymes or genes are listed in Supplementary Table 1 along with their metabolic function that helps the survival of the methanotroph in its environment. The genome contained many unique genes and enzymes, including genes involved in methane and nitrogen metabolism pathways. The novel Methanol/ethanol family PQQ-dependent dehydrogenase, involved in the methanol oxidation reaction, showed the closest similarity to Novimethylophilus kurashikiensis La2-4. The novel nitrogenase iron-molybdenum cofactor biosynthesis protein NifN, an important nitrogen fixation gene, was most relative to Proteobacteria bacterium ST_bin11.
Taxonomic novelty of the strain RS1
Based on molecular analysis such as SIP-PLFA profiling, Methylomicrobium / Methylosarcina like methanotrophs were found to be dominant in rice paddies and forest soils [28; 29]. Strain RS1 shared 96–98% similarity with uncultured methanotrophs clades of paddy fields of the Italian Rice Research Institute in Vercelli, Italy (RT-U57-561; RT-PK57-526; RT-U57-577; RT- U57-550; RT-PK57-519; RT-PK57-486; RT-U57-554) as shown in phylogenetic tree (Supplementary Fig. 1) [30]. Strain RS1 like sequences were also present in Chinese rice fields (clone 50-50B-661r; 16-50B-661r; 14-50B-661r; 23-50B-661r) [31] and showed ~ 95% similarity, as shown in Supplementary Fig. 1. Similarly, RS1 like sequences were also detected in the KRF sample, Alleppey, Kerala, India [32]. The presence of similar sequences of strain RS1 in India, Italy, and China shows the diverse nature regardless of its geographical location.
Based on the culture-dependent studies, no Methylomicrobium sp. was cultivated so far from the rice fields worldwide. This strain RS1 was the first cultivated member of the Methylomicrobium genus isolated from the rice field sample.
The description of Methylomicrobium has been recently modified as per the new re-arrangements of the genus. It is as follows: Cells are short rods or coccobacilli. 0.5–1.0 µm × 1.0–2.5 µm in size. Cells may be motile using a single polar flagellum or nonmotile. Mostly mesophilic, with optimum temperature for growth at 25–30°C; range, 4–37°C. Optimum pH for growth from 5.5 to 7; range, pH 4–9. All species possess a pMMO but no sMMO. The most abundant fatty acids are C16:1 ω5t or C16:1 ω8c, with type depending on the species. DNA G + C content from 52 to 59.6 mol%. The current three species existing in the genus Methylomicrobium, Methylomicrobium album and Methylomicrobium agile were originally isolated from freshwater sediments and swampy soil [33; 34] and Methylomicrobium lacus (earlier named Methylosarcina lacus) from lake sediment [35; 36]. The newly added Ca. Methylomicrobium oryzae fits into the above description.
The earlier classified members of Methylomicrobium are all part of Methylotuvimicrobium: Methylomicrobium alcaliphilum, Methylomicrobium kenyense, and Methylomicrobium buryatense from soda lakes [37; 38]; Methylomicrobium japanense from marine mud [38]; Methylomicrobium pelagicum from upper mixing zone of oceans and, to a lesser extent, from estuarine waters [34; 39]. The morphological feature of the genus Methylomicrobium varies from rods as the major form to cocci and ellipsoids. It does not possess the ability to form cysts or other resting bodies [40].
Description of Ca. Methylomicrobium oryzae RS1:
Me.thy.lo.mi.cro’ bi.um. N.L. pref. methylo- refer to the methyl radical; Gr. adj. micros small; Gr. n. bios life; M.L. n. Methylomicrobium is a small organism able to utilise methyl units. Methylomicrobium oryzae o.ry'zae. L. fem. n. Oryza genus name of rice; L. gen. n. oryzae of rice, as the type strain was isolated from rice field soil. RS1 was a fat rod-elliptical pleomorphic bacillus with a moderate thickness (1.0–4.0µm x 0.5-1µm) and Gram-negative character. It showed motility and formed white to cream-colored ~ 1mm opaque colonies on solid NMS media plates under methane and air-rich environments. It can utilize methane and methanol as carbon sources; and lysine, ammonium chloride, urea, valine, glutamate, peptone, and yeast extract as nitrogen sources. Strain RS1 can grow under nitrogen-free conditions. The cell wall contained major fatty acids 16:0 (25.06%), 16:0 3OH (24.73%), and 16:1 ω7c/16:1 ω6c (23.57%).
The GC content of strain RS1 was 55 mol%. The whole-genome shotgun project was deposited at DDBJ/ENA/ GenBank, and the accession number is JAERVK000000000.1. The maintenance of RS1 was carried out in our house WDCM-approved culture collection, MACS collection of microorganisms, as MCMB-1473.
Ca. Methylomicrobium oryzae sp. nov. strain RS1T is a mesophilic methanotroph and grew in the 20–28ºC temperature range at a 3–10 pH range. The optimum temperature and pH were 25ºC and 6.8, respectively.