BTY 538

TERM PAPER

“ Methylibium petroleiphilum”

NAME : SHASHI PAUL

REG.NO. : 11006142

ROLL.NO : RP8003-B-15

COURSE : MSc.MICROBIOLOGY

COURSE CODE : 2403

SUBJECT : MICROBIAL PHYSIOLOGY

& METABOLISM

SUBJECT CODE : BTY538

SUBMITTED TO:-

DR. NEETA RAJ.

CONTENTS

INTRODUCTION

Classification o Higher order taxa

Classification

o Higher order taxa

o Species

Genome structure

Cell structure and metabolism

Ecology

Pathology

INDUSTRIAL IMPORTANCE

Importance Description and significance Application to Biotechnology

CURRENT RESEARCH

REFERENCES

REASON FOR SELECTION OF METHYLIBIUM PETROLIEPHILUM:

MEYHYLIBIUM PETROLIEPHIUM IS VERY KNOWN FOR ITS OIL DEGRADATION IN SEAS .ORGANIC COMPOUNDS LIKE PETROLEUM , KEROSENE , AND OTHER FUELS ARE DEGRADED IN LARGE AMOUNT S BY PETROLIEPHILUM . ITS VAST DEGRATION CAPABILTY HAS INCREASD ITS ROLE IN BIOREMEDATION. SO THAT IS WHY ABOVE BACTERIA IS CHOSEN .

“REVIEW OF TERM PAPER”

INTRODUCTION

ABSTRACT

A Gram-negative, rod-shaped, motile, non-pigmented, facultative

aerobe that grew optimally at pH 6.5 and 30 °C (strainPM1T) was isolated for its ability to completely degrade the gasoline additive methyl tert-butyl ether. Analysis of the 16S rRNA gene sequence indicated that this bacterium was a member of the class Betaproteobacteria in the Sphaerotilus–Leptothrix group. The 16S rRNA gene sequence identity to other genera in this group, Leptothrix, Aquabacterium, Roseateles, Sphaerotilus, Ideonella and Rubrivivax, ranged from 93 to 96 %. The chemotaxonomic data

including Q-8 as the major quinone, C16 : 1 7c and C16 : 0 as the major fatty acids and a DNA G+C content of 69 mol%, support the inclusion of strain PM1T in the class Betaproteobacteria. It differed from other members of the Sphaerotilus–Leptothrix group by being a facultative methylotroph that used methanol as a sole carbon source, and by also being able to grow heterotrophically in defined media containing ethanol, toluene, benzene, ethylbenzene and dihydroxybenzoates as sole carbon sources. On the basis of the morphological, physiological, biochemical and genetic information, a new genus and species, Methylibium petroleiphilum gen. nov., sp. nov., is proposed, with PM1T (=ATCC BAA-1232T=LMG 22953T) as the type strain.

Abbreviations: MTBE, methyl tert-butyl ether; PHB, poly- -hydroxybutyrate

CLASSIFICATION

Higher order taxa:

Kingdom: Bacteria Phylum: Proteobacteria Class: Betaproteobacteria Order: Burkholderiales Family: Comamonadaceae Genus: Methylibium Species: petroleiphilum Strain: PM1 (extensively studied and all available data on)

Species:

petroleiphilum Genus species

Methylibium petroliphilum

GENOME STRUCTURE

M. petroleiphilum PM1 has a 4,044,225 nucleotide (approximately 4Mb) circular chromosome and a 599,444 nucleotide (approximately 600kb) megaplasmid. The chromosome and megaplasmid contain 3,831 and 646 genes respectively. The G+C content of the chromosome and plasmid are 69.2% and 66% respectively; this suggests that the DNA has a relatively high melting temperature and is perhaps an adaptation to harsh environments that may cause irregular annealing.

CELL STRUCTURE AND METABOLISM

M. petroleiphilum PM1 is a Gram-negative, motile bacteria with a rod shape and is non-pigmented, aerobic bacteria. The rod structure of the bacteria can range in size from 0.5-2.0 μM. The rods also do not contain any sheaths.

ECOLOGY

M. petroleiphilum PM1 is found primarily in sites contaminated with MTBE as well as other aromatic hydrocarbon contaminated sites. The bacteria play a role in what appears to be a form of bioremediation by breaking down the hazardous compounds.

PATHOLOGY

M. petroleiphilum PM1 is not known to be pathogenic

INDUSTRIAL IMPORTANCE

IMPORTANCE

The contamination of groundwater by methyl tert-butyl ether (MTBE) is one of the most serious environmental problems around the world. MTBE degradation in a closed algal-bacterial symbiotic system, containing a mixed culture of Methylibium petroleiphilum PM1 and Chlorella ellipsoidea, was investigated. The algal-bacterial symbiotic system showed increased MTBE degradation.

DESCRIPTION AND SIGNIFICANCE

Methylibium petroleiphilum PM1 was first discovered in 1998 by Professor Kate Scow at UC Davis when purifying biofilters used for treating byproducts from oil refineries.

The discovery of M. petroleiphilum PM1 was significant in that it was determined to be the first and currently only known species of bacteria capable of using MTBE as a sole source of carbon.

APPLICATION TO BIOTECHNOLOGY

The identification of the key enzymes in M. petroleiphilum PM1 used for degrading MTBE and other organic compounds could be used to further aid in the progression of the field of bioremediation but using the enzymes alone to degrade organic toxins in vitro prior to release into the environment.

CURRENT RESEARCH

Most current research is focused on understanding the magnitude of novel metabolic pathways involved in MTBE and aromatic substrate degradation. At the present time it is not clearly understood how the bacteria successfully breakdown MTBE and other aromatic substrates in a energetically favorable manner.

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INTRODUCTION

Organisms that can use one-carbon compounds as energy sources are

called methylotrophs (Lidstrom, 2001 ). A subset of this group, the methanotrophs, can use methane as their sole carbon source. Methylotrophs have been extensively studied because of their potential use in biotechnology and bioremediation (Lidstrom & Stirling, 1990 ; Hanson & Hanson, 1996 ). The aerobic methylotrophs have representatives in the Proteobacteria, high-G+C and low-G+C Gram-positive bacteria that have been isolated from diverse environments. Within the Proteobacteria, the majority of the methylotrophs that have been isolated belong to either the Alphaproteobacteria or Gammaproteobacteria. Three genera,

Methylobacillus (Urakami & Komagata, 1986 ), Methylophilus (Jenkins et al., 1987 ) and Methylovorus (Govorukhina & Trotsenko, 1991 ), in the class Betaproteobacteria are considered to be restricted facultative methylotrophs because they can use methanol but not methane as a sole carbon source, and can use only a limited number of other carbon sources such as glucose and fructose. Phylogenetic analysis based on their 16S rRNA gene sequence resulted in all of them being grouped in the order Methylophilales (Bratina et al., 1992 ; Garrity & Holt, 2001 ). Currently,

none of the described methanotrophs belong to the class Betaproteobacteria. However, comparison of the 16S rRNA gene sequence indicated that isolate PM1T was most closely related to the class Betaproteobacteria in the Sphaerotilus–Leptothrix group (Bruns et al., 2001). In this study, morphological, physiological, biochemical and genetic

information is used to propose a new genus and species, Methylibium petroleiphilum gen. nov., sp. nov.

Strain PM1T was isolated from a mixed bacterial culture enriched with methyl tert-butyl ether (MTBE) using a bench-scale biofilter inoculated with material from a compost biofilter from the Los Angeles County Joint Water Pollution Control Plant (Carson, CA, USA) (Hanson et al., 1999 ). Isolates were obtained on minimal medium (Mu & Scow, 1994 ) with MTBE (25 mg HPLC grade, 99.9 % pure; Fisher Scientific) as the sole carbon source. MTBE utilization was confirmed by monitoring the disappearance of the substrate using gas chromatography (Shimadzu GC-14A, equipped with a photonionization detector). MTBE mineralization was determined by measuring 14CO2 production using uniformly labelled [14C]MTBE (NEN Life Science Products). Strain PM1T and its relatives have been found to completely mineralize this compound and can do so at rates that have made it an appealing choice for use in the bioremediation of contaminated sites (Hristova et al., 2001 ). MTBE is a gasoline additive that is not readily degraded in all environments and therefore has become a widespread

contaminant of groundwater in the USA (Squillace et al., 1996 ). The compound consists of four methyl groups surrounding a carbon monoxide

and is produced from chemically reacting methanol and isobutylene. Two pathways for the degradation of this compound have been described to date. The initial step for both pathways is the conversion of MTBE to hydroxymethyl tert-butyl ether; then, in the pathway described for propane-oxidizing bacteria, tert-butyl alcohol and formaldehyde are formed (Steffan et al., 1997 ). In the degradation pathway used by Mycobacterium species, MTBE is converted to tert-butyl ether and then hydrolysed to tert-butyl

alcohol and formate (François et al., 2002 ; Smith et al., 2003 ). Formaldehyde and formate both enter the C1 metabolic cycle, involved in the cycling of one-carbon compounds, where CO2 and NADH are generated (Ellis et al., 2001 ). Strain PM1T grows on tert-butyl alcohol, formaldehyde and formate (K. Hristova and K. M. Scow, unpublished results), suggesting that at least part of its MTBE biodegradation pathway is similar to that reported for cometabolizers.

Under all growth conditions tested, the cells grew singly as 0.5x1–2 µm rods, without a sheath , were motile by means of a single polar flagellum and reproduced by normal cell division. Strain PM1T was Gram-negative,

oxidase-positive and catalase-negative, and was capable of hydrolysing

urea but not starch, gelatin, aesculin, casein or DNA. Nitrate was reduced to nitrite, but nitrite was not reduced. API 20NE test results were negative for o-nitrophenyl -D-galactopyranoside, lysine decarboxylase, ornithine decarboxylase, citrate, indole, Voges–Proskauer test, glucose, rhamnose, sucrose, melibiose, arabinose and xylose. Centrally located intracellular granules were observed, which were considered to be poly- -hydroxybutyrate (PHB) granules . This reserve material is commonly found in members of the Sphaerotilus–Leptothrix group (Spring, 2002 ). The characteristic cell morphology of members of the Sphaerotilus–Leptothrix group of sheathed cells growing filamentously with oxidized manganese or iron deposits was not observed . Instead, the cell morphology was more similar to that of members of the genus Aquabacterium, which was also a member of this clade (Kalmbach et al., 1999 ), except that a surficial fibrillar matrix was not observed. Additionally, intracytoplasmic membrane structures of any type indicative of most methanotrophs were not observed (Bowman, 2000 ).

Fig. 1. Photomicrographs of Methylibium petroleiphilum strain PM1T. (a) Phase-contrast micrograph of unstained cells of strain PM1T after 2 days of growth on minimal medium plus a mixed carbon source. (b) Electron micrograph of negatively stained cells of strain PM1T after growth on minimal medium plus MTBE showing a single polar flagellum. (c). Electron micrograph of thin sections of negatively stained cells of strain PM1T after 2 days of growth on minimal medium plus MTBE. Bars, 5 µm (a) and 0.5 µm (b and c).

 

 Within 2–3 days on nutrient agar, strain PM1T formed cream-coloured, flat colonies with smooth margins, of 2–3 mm in diameter. Colonies were white in colour when the strain was grown on minimal media with MTBE as the sole carbon source. No pink or orange colony pigmentation was observed, which is often indicative of some methanotrophs (Bowman, 2000 ). Vitamins were not required for growth; subculture of strain PM1T in medium

without vitamins had no effect on growth. Trace metals required for the use of MTBE as a sole carbon source were Co, Cu, Mn, Zn, Mo, Ni and Fe. Strain PM1T could grow both aerobically and anaerobically. Other genera in the Leptothrix group whose members are also facultative aerobes are Rubrivivax, Ideonella and Aquabacterium (Spring, 2002 ). Facultatively anaerobic methylotroph representatives also occur (Lidstrom, 2001 ); therefore the occurrence of this phenotype is not unusual.

To gain insight into the novelty of the genes potentially involved in the utilization of some of the growth substrates tested, PCR was performed using a variety of primers that had been used previously to detect genes encoding oxygenase (Baldwin et al., 2003 ). PCR products were obtained with primers specific for genes encoding ring-hydroxylating toluene monooxygenase and phenol hydroxylase. The presence of these genes suggested that degradation of at least some of the aromatic hydrocarbons involved catabolic pathways that have been previously described in other

bacteria. No products were observed with the other oxygenase gene primers used. Also, no PCR amplicons were produced using primers for the

sMMO or pMMO genes that are typically found in methanotrophs. The 16S rRNA gene sequence of isolate L013.11 was found to closely match that of strain PM1T . This isolate has not been cultivated, but was found in peat soil after methane enrichment with 13CH4 (Morris et al., 2002 ). The authors of that study speculated that this strain was a novel representative of methanotrophs within the class Betaproteobacteria. The close phylogenetic relationship of isolate L013.11 to strain PM1T suggests that it is at least methylotrophic, but further research is needed to clarify the role played by this strain in methane-enriched communities.

 

Fig. 2. Phylogenetic position of strain PM1T among neighbouring species selected from the class Betaproteobacteria. Bar, 0.05 substitution per nucleotide position in 16S rRNA gene sequences. GenBank accession numbers and culture collection numbers (where available) used in the tree construction are included on the figure.

 Phylogenetic analysis using the 16S rRNA gene indicated that strain PM1T fell into the Sphaerotilus–Leptothrix subcluster within the class Betaproteobacteria (Spring, 2002 ). The sequence formed a separate branch from those of the described genera in this group, Leptothrix, Aquabacterium, Roseateles, Sphaerotilus, Ideonella and Rubrivivax . Using 16S rRNA gene sequence identity, the most closely related bacterium with a validly published name was Aquabacterium commune DSM 11901T (96 %), followed closely by others in the same clade,

Description of Methylibium gen. nov.Methylibium (Me.thy.li.bi'um. N.L. n. methyl the methyl radical, the methyl group; Gr. n. bios life; N.L. neut. n. Methylibium referring to methylotroph).

Cells are motile, Gram-negative straight rods. Oxidase-positive. Negative for gelatinase and catalase. Hydrolyse urea and reduce nitrate to nitrite. Cells possess PHB granules as a storage material and reproduce by binary fission. Growth occurs heterotrophically under aerobic conditions. Facultative methylotrophs able to use methanol as a sole carbon source in addition to a variety of other more complex carbon sources. The major quinone is Q-8. The major fatty acids are C16 : 1 7c and C16 : 0, and in lesser amounts C10 : 0 3-OH, C12 : 0, C12 : 0 2-OH, C12 : 0 3-OH, C14 : 0, C17 : 0 cyclo7–8c, C18 : 1 7c and C18 : 0. On the basis of the results of 16S rRNA gene sequence comparison, the bacteria belong to the class Betaproteobacteria. The DNA G+C content of the type species is 69 mol%. The type species is Methylibium petroleiphilum.

Description of Methylibium petroleiphilum sp. nov.Methylibium petroleiphilum (pe.tro.lei.phi'lum. Gr. n. petra stone, rock; L. n. oleum oil; Gr. adj. philos loving; N.L. neut. adj. petroleiphilum petrol loving).

Exhibits the following properties in addition to those given in the genus description. Colonies are cream in colour under conditions suitable for MTBE degradation. Grows well heterotrophically in media containing ethanol, methanol, toluene, benzene, ethylbenzene and dihydroxybenzoates as the sole carbon source. Vitamins are not required for growth. Optimum pH and temperature for growth are 6.5 and 30 °C, respectively. Does not grow at 37 °C. The genome size is 4.6 Mb. Inhabits subsurface environments highly contaminated with MTBE.

The type strain is PM1T (=ATCC BAA-1232T=LMG 22953T), which was isolated from a mixed bacterial culture enriched using a bench-scale biofilter inoculated with some solid support material from a compost biofilter located at the Los Angeles County Joint Water Pollution Control Plant (Carson, CA, USA).

CLASSIFICATION

Higher order taxa

Kingdom: Bacteria Phylum: Proteobacteria Class: Betaproteobacteria Order: Burkholderiales Family: Comamonadaceae Genus: Methylibium Species: petroleiphilum Strain: PM1 (extensively studied and all available data on) .

Species

petroleiphilum Genus species

Methylibium petroliphilum

GENOME STRUCTURE

M. petroleiphilum PM1 has a 4,044,225 nucleotide (approximately 4Mb) circular chromosome and a 599,444 nucleotide (approximately 600kb) megaplasmid. The chromosome and megaplasmid contain 3,831 and 646 genes respectively. The G+C content of the chromosome and plasmid are 69.2% and 66% respectively; this suggests that the DNA has a relatively high melting temperature and is perhaps an adaptation to harsh environments that may cause irregular annealing.

Genes of that encode enzymes that degrade and catabolize aromatic compounds and alkanes, pumps to uptake and expel metals (metal resistance), and methylotrophy are primarily found in the circular chromosome.

The megaplasmid also encodes genes for alkane degradation and MTBE degradation and metabolism. Also, noted was that the plasmid contains some anomalies unlike plasmids in other bacteria such as t-RNA islands (some of which do not have a valid anti-codon sequence), irregular insertions, and an unusually large number of repeated elements and genes such as a 40 kb region identical to the chromosome as well as a smaller 29 kb region identical to the chromosome. Comparative genome hybridization experiments provide compelling evidence that M. petroleiphilum PM1 acquired its plasmid recently and also that the megaplasmid is what may contain the most critical genes involved in MTBE degradation and metabolism. Another interesting attribute of M. petroleiphilum PM1 is that varying isolates of the identical strain show approximately 99% conservation of the megaplasmid but chromosomal similarities were of a much lower order of magnitude.

CELL STRUCTURE AND METABOLISM

M. petroleiphilum PM1 is a Gram-negative, motile bacteria with a rod shape and is non-pigmented, aerobic bacteria. The rod structure of the bacteria can range in size from 0.5-2.0 μM. The rods also do not contain any sheaths. Motility is achieved by the use of a polar flagellum. Reproduction occurs by generic binary fission. The fatty acids that make up the majority of the plasma membrane are composed of C16:1w7c and C16:0.

An attribute that M. petroleiphilum PM1 shares with members of the Sphaerotilus-Leptothrix group is the presence of intracellular granules of poly-β-hydroxybutyrate. In addition, M. petroleiphilum PM1 has a cell morphology characteristic of the genus Aquabacterium with the exception of lacking a fibrillar matrix.

M. petroleiphilum PM1is characterized as a methanotroph since it can use methane as a carbon source; however, M. petroleiphilum PM1 lacks all intracellular structures which normally characterize methanotrophs.

Under optimal growth conditions, the bacteria can form cream color flat colonies with smooth features ranging from 2-3 mm in diameter. When grown under very basic conditions, the colonies maintain the same size and shape however tend to be white. Lack of pigmentation (such as orange or pink) is another unusual attribute of M. petroleiphilum PM1 that other methanotrophs poses.

For proper metabolism, M. petroleiphilum PM1 does not require any vitamins or other organic nutrients and can derive all organic compounds from MTBE. The bacterium however does require the trace elements cobalt, copper, manganese, zinc, molybdenum, nickel, and iron. This strain also can metabolize and make use of other organic carbohydrates and amino acids as nutrient sources such as pyruvate, ethanol, L-aspargine, acetate, butanol, methanol, toluene, benzene, phenol, ethylbenzene, 3,4-dihydroxybenzoate, 2,5-dihydroxbenzoate, 3,5-dihydroxbenzoate, 2,6-dihydroxbenzoate and 2,3-dihydroxbenzoate. Unlike other methanotrophs, M. petroleiphilum PM1 has a wide variety of substrates to derive nutrients from.

M. petroleiphilum PM1 grows optimally at pH 6.5 and around 30 degrees Celsius.

ECOLOGY

M. petroleiphilum PM1 is found primarily in sites contaminated with MTBE as well as other aromatic hydrocarbon contaminated sites. The bacteria play a role in what appears to be a form of bioremediation by breaking down the hazardous compounds. It is intuitive to think that both the environment and bacteria benefit since the bacteria have an abundant food supply of hydrocarbons such as MTBE and they clean the environment at the same time. Grows well in aerobic, warm, and close to neutral pH conditions.

PATHOLOGY

M. petroleiphilum PM1 is not known to be pathogenic.

INDUSTRIAL IMPORTANCE

IMPORTANCE OF METHYLIBIUM PETROLIEPHILUM:

The contamination of groundwater by methyl tert-butyl ether (MTBE) is one

of the most serious environmental problems around the world. MTBE

degradation in a closed algal-bacterial symbiotic system, containing a

mixed culture of Methylibium petroleiphilum PM1 and Chlorella ellipsoidea,

was investigated. The algal-bacterial symbiotic system showed increased

MTBE degradation. The MTBE-degradation rate in the mixed culture

(8.808 ± 0.007 mg l−1 d−1) was higher than that in the pure bacterial culture

(5.664 ± 0.017 mg l−1 d−1). The level of dissolved oxygen was also higher in

the mixed culture than that in the pure bacterial culture. However, the

improved efficiency of MTBE degradation was not in proportional to the

biomass of the alga. The optimal ratio of initial cell population of bacteria to

algae was 100:1. An immobilized culture of mixed bacteria and algae also

showed higher MTBE degradation rate than the immobilized pure bacterial

culture. A mixed culture with algae and PM1 immobilized separately in

different gel beads showed higher degradation rate (8.496 ± 0.636 mg l−1

d−1) than that obtained with algae and PM1 immobilized in the same gel

beads (5.424 ± 0.010 mg l−1 d−1).

DESCRIPTION AND SIGNIFICANCE

(1)Methylibium petroleiphilum PM1 was first discovered in 1998 by Professor Kate Scow at UC Davis when purifying biofilters used for treating byproducts from oil refineries. The specific PM1 strain of the bacteria was isolated from a culture enriched with methyl tert-butyl

ether (MTBE) from a biofilter from the Los Angeles County Joint Water Pollution Control Plant.

(2)The discovery of M. petroleiphilum PM1 was significant in that it was determined to be the first and currently only known species of bacteria capable of using MTBE as a sole source of carbon. The fact that MTBE is the sole carbon source is a key property of M. petroleiphilum PM1 which makes is a great candidate for bioremediation.

(3) Starting in the last 15 years, oil refineries began using MTBE in oil and petroleum purification. MTBE however, is a carcinogen which on numerous occasions has entered water systems and caused massive contaminations. The detection of M. petroleiphilum PM1 in contaminated water supplies could be indicative that it plays a role in purifying the water supply.

(4) 16S rRNA sequence analysis revealed that M. petroleiphilum PM1 was a novel genus with 93-96% similarities to the genera Leptothrix, Aquabacterium, Roseateles, Sphaerotilus, Idenella, and Rubrivivax.

(5) To date, M. petroleiphilum PM1 has been discovered in a vast number of MTBE contaminated water supplies such as the US, Mexico, and Europe, and it is assumed that M. petroleiphilum PM1 is abundantly found in any other MTBE tainted water supply in other parts of the world.

(6) M. petroleiphilum PM1is not known to be pathogenic.

APPLICATION TO BIOTECHNOLOGY

M. petroleiphilum PM1 is viewed as being a novel tool used in bioremediation for MTBE and other aromatic contaminated sites. Because of M. petroleiphilum PM1’s ability to degrade a wide variety of normally toxic organic compounds, it can grow fairly well without needing any extra

nutrients. Superfund sites as well as contaminated aquifers in the United States are viewed as ideal candidates to see if perhaps M. petroleiphilum PM1 itself could be added directly into aquifers to reduce, if not completely eliminate, hazardous organic compounds. The largest concern of MTBE is that it readily enters aquifers and contaminates drinking water with a carcinogen. Experiments with gas chromatography have revealed that the presence of M. petroleiphilum PM1 in culture with MTBE has shown drastic decreases in MTBE levels and increase carbon dioxide levels and water.

In addition, understanding the novel genetic and metabolic pathways possessed by M. petroleiphilum PM1 could lead to possible ways for devising other strains of bacteria that could degrade a wider range of organic compounds that are carcinogen or toxic in other means to humans. The identification of the key enzymes in M. petroleiphilum PM1 used for degrading MTBE and other organic compounds could be used to further aid in the progression of the field of bioremediation but using the enzymes alone to degrade organic toxins in vitro prior to release into the environment.

CURRENT RESEACH

Most current research is focused on understanding the magnitude of novel metabolic pathways involved in MTBE and aromatic substrate degradation. At the present time it is not clearly understood how the bacteria successfully breakdown MTBE and other aromatic substrates in a energetically favorable manner.

In addition, isolating pure enzymes involved in these metabolic processes is another avenue of research. Pure enzymes could be used in industrial settings to prevent release of MTBE and other hydrocarbons that could be degraded by the appropriate enzymes.

How to directly use the bacteria in bioremediation such as the contaminated Superfund sites and aquifers is where most active research takes place. UC Davis Department of Land, Air and Water Resources is the main research center for the studies of M. petroleiphilum PM1. Water filtration using the bacteria to purify and breakdown MTBE.

Methylibium petroleiphilum strain PM1 is one of only a few pure culture isolates that can grow on and completely degrade the fuel additive MTBE (methyl tertiary butyl ether) . MTBE is a commonly used fuel-oxygenate in the last two decades that is far more recalcitrant to biodegradation than other gasoline components and has contaminated numerous subsurface drinking water supplies . Strain PM1 was isolated by

Dr. Scow’s lab in 1998 from a sewage treatment plant biofilter that was used for treating discharge from oil refineries . Strain PM1 is a methylotroph representing a new species within the Rubrivivax group (Comamonadaceae family) of the beta subclass of Proteobacteria .

Pilot and field studies have demonstrated the efficacy of aerobic bioremediation of MTBE by PM1 . Furthermore, PM1-like bacteria (98-99% similar based on 16S rDNA sequences) have been shown to be naturally occurring in a number of MTBE-contaminated aquifers in California . The presence of PM1-like bacteria has been correlated with MTBE degradation activity in numerous sites (S. Kane, K. Hristova, unpublished) using technologies such as real-time quantitative PCR . In field studies, increases

in PM1-like bacterial populations corresponded to MTBE removal. These results suggest that a PM1-like organism may play a major role in MTBE biodegradation under aerobic conditions in California aquifers.

To date, little to nothing is known concerning the biochemistry and genetics of aerobic MTBE metabolism, which involves a novel ether cleavage reaction described primarily for cometabolic MTBE-degrading organisms . In addition to MTBE catabolism, PM1 has a broad range of metabolic capabilities such as the ability to aerobically degrade aromatic hydrocarbons including benzene, toluene, xylenes and phenol. These unique metabolic capabilities point to a significant number of novel genes, providing fertile ground for genomics and proteomics studies. The genome sequence of PM1 provides a framework for characterizing the MTBE degradation pathway and other important metabolic pathways in this novel bacterium. Understanding PM1’s capacity to biodegrade petroleum constituents including relevant mixtures, and the genetic regulation of these catabolic processes will enhance our ability to protect and restore gasoline-impacted aquifers.

An in-depth analysis of the PM1 genome sequence was reported by Kane et al., describing genes and operons on a ~4-Mb circular chromosome and a ~600-kb megaplasmid. The genes for MTBE degradation were shown to be coded on the megaplasmid using plasmid curing experiments and

comparative genomic hybridization and resequencing analysis with PM1-like MTBE-degrading bacteria .

JOURNAL

A Gram-negative, rod-shaped, motile, non-pigmented, facultative aerobe that grew optimally at pH 6.5 and 30 °C (strain PM1T) was isolated for its ability to completely degrade the gasoline additive methyl tert-butyl ether. Analysis of the 16S rRNA gene sequence indicated that this bacterium was a member of the classBetaproteobacteria in the Sphaerotilus–

Leptothrix group. The 16S rRNA gene sequence identity to other genera in this group, Leptothrix, Aquabacterium,Roseateles, Sphaerotilus, Ideonella and Rubrivivax, ranged from 93 to 96 %. The chemotaxonomic data including Q-8 as the major quinone, C16 : 1 7c and C16 : 0 as the major fatty acids and a DNA G+C content of 69 mol%, support the inclusion of strain PM1T in the class Betaproteobacteria. It differed from other members of the Sphaerotilus–Leptothrix group by being a facultative methylotroph that used methanol as a sole carbon source, and by also being able to grow heterotrophically in defined media containing ethanol, toluene, benzene, ethylbenzene and dihydroxybenzoates as sole carbon sources. On the basis of the morphological, physiological, biochemical and genetic information, a new genus and species, Methylibium petroleiphilum gen. nov., sp. nov., is proposed, with PM1T (=ATCC BAA-1232T=LMG22953T) as the type strain.

Abbreviations: MTBE, methyl tert-butyl ether; PHB, poly- -hydroxybutyrate

RFERENCES

(1). NCBI website

(2). Hanson, J. R. et al. 1999. Biodegradation of methyl tert-butyl ether by a bacterial pure culture. Appl. Environ. Microbiol. 65:4788-4792.

(3). Kane, S. R. et al. 2001. Aerobic biodegradation of methyl tert-butyl ether by aquifer bacteria from leaking underground storage tank sites. Appl. Environ. Microbiol. 67:5824-5829.

(4). Kane, S. R. et al. 2003. Aerobic biodegradation of MTBE by aquifer bacteria from LUFT sites. E-12. In: V.S. Magar and M.E. Kelley (Eds.) Proceedings of the Seventh International In Situ and On-site Bioremediation Symposium. Battelle Press, Columbus, OH.

(5). Bruns et al. 2001. Isolate PM1 populations are dominant and novel methyl tert-butyl ether-degrading bacteria in compost biofilter enrichments. Environ Microbiol. 3: 220-225.

(6). Nakatsu, C. et al. 2006. Methylibium petroleiphilum gen. nov., sp. Nov., a novel methyl tert-butyl ether-degrading methylotroph of the Betaproteobacteria. International Journal of Systematic and Evolutionary Microbiology. 56: 983-989.

(7). Kane, S. R. et al. 2007. Whole-Genome Analysis of the Methyl tert-Butyl Ether-Degrading Beta-Proteobacterium Meythylibium petroleiphilum PM1. Journal of Bacteriology. 5: 1931-1945.

(8). Hristova K. B. et al. 2003. Naturally occurring bacteria similar to the methyl tert-butyl ether (MTBE)-degrading strain PM1 are present in MTBE-contaminated groundwater. Appl. Environ. Microbiol. 69(5):2616-2623.

(9). Hristova, K. R. et al. 2001. Detection and quantification of MTBE-degrading strain PM1 by real-time TaqMan PCR. Appl. Environ. Microbiol. 67: 5154-5160.