Molecular Identification of Methylorubrum extorquens using PCR-Amplified MxaF Gene Fragments as A Molecular Marker

: Methylotrophs bacteria are ubiquitous, and they have the ability to consume single carbon (C1) which makes them biological conversion machines. It is the first study to find facultative methylotrophic bacteria in contaminated soils in Iraq. Conventional PCR was employed to amplify MxaF that encodes methanol dehydrogenase enzyme. DNA templates were extracted from bacteria isolated from five contaminated sites in Basra. The gene specific PCR detected Methylorubrum extorquens as the most dominant species in these environments. The ability of M. extorquens to degrade aliphatic hydrocarbons compound was tested at the laboratory. Within 7 days, gas chromatographic (GC) studies of remaining utilized crude oil revealed that 61.14 % of the initial content had been degraded, and GC fingerprinting of the utilized aliphatic compounds revealed significant reductions in C 12 , C 13 , C 14 , and C 15 . Globally this is the first time found a new strain of M. extorquens has the ability to degrade aliphatic hydrocarbons compound. Conventional PCR and gene sequencing revealed the presence of the facilitative methylotrophic bacteria in polluted areas in Basra. M. extorquens was dominant and showed a substantial ability to degrade crude oil which makes them an important tool to be employed in bioremediation.


Introduction:
Various species of methylotrophic bacteria are distributed in nearly all natural environments 1 . Methylotrophs use reduced single-carbon (C1) molecules like methanol as carbon sources for growth, making them methanol biological convertors 2 . More than 50 methylotrophic taxa have been identified, including Alpha, Beta, and Gammaproteobacteria, Verrucomicrobia, Firmibacteria, Actinobacteria, and Flavobacteria 3 .
Pink-pigmented facultative methylotrophs (PPFMs), are present in the rhizosphere or exist in soil, air, or water. PPFMs belong to methyl bacteriaceae family, they are gram negative and utilize C1 molecules as their only source of energy and carbon, methanol, methylamine, formate, and formaldehyde are among the examples 4

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Methylobacterium has more species than any other within the family methylobacteriaceae, order rhizobiales, and class alphaproteobacteria. Methylobacterium includes facultative methylotrophs that may live on carbon and energy sources other than organic acids and sugars, such as methane or methanol 5 . All other previously PPFMs bacteria were classified within Methylobacterium in a taxonomic analysis 6 . Following that, according to 16S rRNA gene sequences, multi-locus sequence analysis (MLSA), phenotypic data, and genomics, eleven Methylobacterium species were reclassified as Methylorubrum, a new genus 7 .
Methanol dehydrogenase (MDH) converts the methanol to formaldehyde, which is the second crucial enzyme in methane metabolism. The MDH is a pyrroloquinoline quinone (PQQ)-containing soluble periplasmic enzyme with a α2β2 structure, consisting of two large subunits MxaF and two tiny subunits MxaI, the active site contains a Ca 2+ ion 8 .
MxaF encodes for the large alpha-subunit of MDH and other functional molecular marker genes are highly conserved across methylotrophs and have been utilized in environmental studies to identify methylotrophs in different environments 9 . One of the most significant environmental pollutants, with bad impacts on both people and the environment is hydrocarbons 10 . It is a crucial global environmental pollutant because it spills and leaks often throughout the exploration, transport, refining, and storage of petroleum and petroleum products. The fundamental approach for reducing biodegradable contaminants is biodegradation, which is cost effective alternative 11 . It is one of the most effective and promising methods for cleaning up soil that has been contaminated with diesel. This choice has the potential to remove harmful contaminants through biological activity 12 .
The aim of this study is to isolate methylotrophic bacteria from hydrocarbon-contaminated soils. Identification of these bacteria according to their morphological features and 16S rRNA gene sequencing. Determination of their capability to degrade aliphatic compounds in vitro.

Material and Methods: Sampling
A total of 10 g of oil-polluted soil was collected aseptically in sterile plastic bags from five oil sites in Basra city, southern Iraq in December 2020, Fig.  1 and Table 1.

Preparation of media for isolation and purification
The isolation basal salt medium (BSM) 13 has been modified according to Fujii et al 14 by increasing thiamine and biotin, Table 2. The medium components were dissolved in 1000 ml distilled water pH 6.8-7.0 and after sterilization, thiamine, biotin, and 20 ml of methanol was added aseptically to the medium in addition to that fluconazole was added as an antifungal. A 0.5 g of soil was placed in Erlenmeyer flasks containing 100 ml of BSM and incubated for 7 days at 30°C in a shaking incubator (Sartorius, Stidem, Germany) at 180 rpm. Methanol-utilizers were cultured for several replicates on a basal salt medium and were picked up after 5 -7 days of incubation at 30°C.  Methanol-salt medium (MSM) of 13 was modified also by increasing the weights of some ingredients and adding fluconazole as an antifungal, Table 3. The medium was dissolved in 1000 ml distilled water pH 6.8. Fluconazole and 10 ml of methanol were added after sterilization.

Morphological and Biochemical Tests
The morphological and biochemical tests were used to identify the isolates including to their cell shape, colony morphology, pigment production, Gram staining, catalase and oxidase 15 .

Extraction of Genomic DNA and Identification of bacteria using 16S rDNA
Bacterial genomic DNA was isolated using the Geneaid Presto TM Mini gDNA Kit (Korea) according to the manufacturer's instructions. A 0.5% agarose gel electrophoresis was used to determine the purity of the DNA. Eluted DNA concentrations were measured using Nano-Drop (Optizen/Korea). 16S rRNA gene sequencing was used to identify consuming methanol bacteria grown on MSM plates, using the following primers: 27F AGAGTTTGATCCTGGCTCAG, 1492R GGTTACCTTGTTACGACTT 16 . A master mix of 25 µl from Go Taq Green master mix (Promega, USA), was mixed with 19 µl of Nuclease Free water, 2 µl (10-20 ng) of DNA template, and 100 Pmol (2 µl) of each primer to a total volume of 50 µl PCR reaction. In order to perform the PCR reaction, the thermal cycler (Eppendorf, Germany) was programmed with the following parameters: 95 °C for 5 min, followed by 35 cycles of 95 °C for 30 Sec, 55 °C for 30 Sec, and 72 °C for 60 Sec, with a final extension at 72 °C for 5 min.

Gel Electrophoresis
Using a 100bp DNA ladder (Promega, USA) and a UV transilluminator (ATTA, Korea), agarose gel was prepared by dissolving 0.25 g agarose powder in 25 ml TBE buffer with 0.2 g of Ethidium bromide as visualizing dye was used to detect 16S rDNA bands. PCR amplicons were sent to Macrogen for further purification and sequencing. The National Center for Biotechnology Information's BLAST was employed to align sequences in order to identify the isolated

Identification of the isolates through MxaF gene
The methylotrophic isolates were identified using MxaF specific primers which encodes for gene methanol dehydrogenase enzyme, producing an amplicon of ~ 550bp. The primers sequences were: F1003degen 5-GGNCANACYTGGGGNTGGT-3, R1561degen 5-GGGARCCNTTYATGCTNCCN -3́ 17 . Each PCR reaction mixture included 12.5 µl of Go Taq Green master mix (Promega, USA), 7.5µl of Nuclease Free water, 3 µl (15-30 ng) of DNA template, and 1 µl (100 Pmol) of each primer make up a 25µl PCR reaction mixture. The thermal cycle was set (Eppendorf, Germany) as the following conditions were used for the PCR reaction: 94 °C for 45 Sec, 59 °C for 1 min, and 72 °C for 1.5 min, for 30 cycles, with a final extension at 72 °C for 10 min.

Biodegradation of crude oil
Pure cultures of the isolated bacteria were prepared by adding 1 ml of the liquid pure culture in a conical flask containing 100 ml of MSM and 0.5% (v/v) crude oil supplied from Al-Shua'aba Refinery-Basra city. The flasks were incubated in a shaking incubator for 7 days at 30 °C with 120 rpm 18 .

Extracting residual crude oil
A liquid-liquid extraction technique was used to extract the leftover crude oil by a separating funnel. The aqueous phase was discarded, and the remaining oil was dried in the oven at 40 °C to remove the chloroform. The aliphatic fraction was separated, and the residual oil was diluted in 25 ml of n-hexane. The aliphatic fraction was collected and sent to be analyzed by Gas Chromatography to estimate aliphatic compounds (Agilent Chem Station) 19 .

Isolation of Methanol utilizers
Little pink spherical colonies began to form after 3-5 days of incubation at 30 °C. gramnegative bacteria might be seen alone, in pairs, or in large numbers. There was no spore formation, and tests for catalase and oxidase were positive, Fig. 2.

Identification of the isolates through 16S rRNA gene
The sequencing of six nominated isolates revealed the presence of methylotrophic bacteria using16S rRNA amplicon size of 1500bp on a 1% agarose gel. Methylorubrum extorquens were identified at the species level based on a 99% similarity of 16S rRNA sequences to the intended type in GenBank Fig. 3.

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Identification of bacteria using MxaF gene
Using the MxaF-specific primers, MxaF was detected in four isolates at the conservative region of methylotrophic bacteria, the isolates were genetically identified. On a 2 % agarose gel, the MxaF amplicon names and affiliations of Methylorubrum genomic DNA were visualized at the expected size of 550bp, Fig. 4.

Biodegradation of crude oil
Significant increases in cell density were seen after 7 days of incubation when crude oil was used as the sole carbon and energy source, with simultaneous decreases in several components of the used crude oil, Fig. 5. During the incubation time, there were significant changes in numerous components of the used crude oil. After 7 days, most of the peaks had significantly shrunk. The short-chain alkanes nC12-nC15 were efficiently destroyed by presumptive M. extorquens which the best-growing isolate was used on crude oil, while long-chain alkanes nC16-nC37 degraded at a slower rate. The total degradation ratio was 61.14% (0.5% v/v) of crude oil, Fig. 6.  The study aimed to isolate and identify facultative methylotrophs from oil contaminated sites in Basra-Iraq and test their ability to naturally degrade the crude oil at the laboratory.
In this study, a new modified medium has been introduced according to the ability of bacteria to grow. The isolates grew very poorly on the methanol-salt medium and did not grow if the inoculum was small. Also, the isolates could not grow in the medium of Kouno et al 13 . This may be due to the harsh environment from which they were isolated. So, the increase of biotin and thiamine and the addition of antifungals led to the flourishing of these bacteria and increased their ability to consume methanol, which reached 6% (unpublished data) and reduced the incubation period.
The In the current study, we found that methanol can be consumed during colonization by Methylorubrum in addition to using carbon sources other than methanol, such as crude oil. This is consistent with the study conducted by Hu and Lidstrom 23 , which found that methylotrophs can consume single-carbon compounds or multiplecarbon compounds without a carbon-carbon bond.
The MxaF gene has been used to detect methylotrophs in the environment because it is highly conserved among the methylotrophs that have been studied 24 . A pair of specific primers was chosen from the common conserved area of the MxaF gene for the identification of methylotrophic bacteria, to validate the identification of the genus Methylorubrum and to detect the MxaF gene. The size was amplified by these primers, resulting in a 550-bp amplicon. The MxaF gene was employed by Lau et al 16 as a biomarker for methanotrophic proteobacteria found in the Methylocystaceae and Methylococcaceae families.
In contaminated environments, petroleum hydrocarbons are digested by bacteria and used as their sole source of carbon and energy. Genetics determines a microbe's ability to incorporate molecular oxygen into a hydrocarbon and generate intermediates that enter the cell's overall energyproducing metabolic pathway 25 .
A new strain of Methylorubrum extorquens has been isolated and with the ability to exploit the aliphatic hydrocarbon compounds as a source of energy in addition to methanol, in spite of carboncarbon bonds. This is a globally new finding. So, after 7 days of incubation, the rate of degradation was 61.14% and short-chain alkanes nC12-nC15 were broken down, whereas long-chain alkanes nC16-nC37 degraded at a slower rate. Moreover, these bacteria have the ability to degrade aromatic hydrocarbon compounds (unpublished data). Perhaps the reason behind that, is these bacteria have been isolated from harsh environments and exposed to many types of oil and chemical pollutants, in addition to high temperatures that may sometimes reach more than 65°C, forcing them to adapt to these conditions and exploit what is available to them from nutrients. This degradation rate is less than the 83.8 and 81.63% at 0.5 % crude oil reported for Vibrio vulnificus and Brevundimonas diminuta respectively and higher than 51.64 and 58.31 % at 7 days reported for Ochrobactrum anthropic and Sphingomonas paucimobilis isolated from contaminated soils in the Khor Al-Zubair channel, southern Iraq 18 .
As a source of carbon and energy, the bacteria used the hydrocarbon substrate, as evidenced by a considerable reduction in peaks between 0 to 7 days which coincided with the exponential development of bacteria. Salam et al. 26 also found that Methylobacterium mesophilicum strain RD1 destroyed 61.2 and 89.5 % of the starting concentration of the used motor oil during 12-21 days, respectively. They explain that this is because strain RD1 contains numerous degradative genes.

Conclusion:
Due to the high oil pollution at the locations of M. extorquens isolation, these sites are of significant interest for isolating novel hydrocarbondegrading bacteria with high catabolic abilities enhanced by living in a highly polluted site. Without no doubt, this elevated bioremediation ability is attributed to the high and continuous exposure to hydrocarbon chemicals over time. In the present study, conventional PCR was used to identify methylotrophic bacteria and the methanol dehydrogenase enzyme. The analysis of GC showed about 61.14% of crude oil was degraded, and the GC fingerprinting appears to show that C12, C13, C14, and C15 had decreased significantly. Our findings of the ability of M. extorquens to successfully metabolize aliphatic compounds can be employed and genetically upregulated to bioremediate aromatic compounds in other studies. M. extorquens' ability to break down crude oil is a good start for genetically modifying these strains to make them twice as good using advanced methods like CRISPR Cas9.