INTERNATIONAL
JOURNAL
OF SYSTEMATIC
BACTERIOLOGY,
Oct. 1987, p. 446448
0020-7713/87/040446-03$02.00/0
Copyright 0 1987, International Union of Microbiological Societies
Vol. 37, No. 4
Methylophilus: a New Genus of Methanol-Utilizing Bacteria
OWEN JENKINS,l DAVID BYROM,2 AND DOROTHY JONES1*
Department of Microbiology, University of Leicester, Leicester LEI 7RH,l and Biochemical Group, Research
Department, ICI Agricultural Division, Billingham, Cleveland TS23 1LB,2 United Kingdom
A new genus, Methyluphilus, and species of restricted facultative methanol-utilizing bacteria are described.
These bacteria are aerobic gram-negative rods that occur singly and in pairs. In addition to methanol and
glucose, a limited range of other carbon compounds including fructose and methylamines may be used as the
sole carbon and energy source. The fatty acid composition is primarily of the nonhydroxylated straight-chain
saturated and monounsaturatedtypes. The major isoprenoid quinone components are ubiquinones with eight
isoprene units. The major polar lipid components are phosphatidylglycerol and phosphatidylethanolamine.
The deoxyribonucleic acid base composition is 50 to 53 mol% guanine plus cytosine. The name of the genus
proposed for these bacteria is Methyluphilus gen. nov. The name of the type species is Methyluphilus
methylutruphus sp. nov. The type strain is AS1 (= NCIB 10515).
utilizing gram-negative bacteria be classified in one species,
Methylobacillus glycogenes, and emended the genus
Methylobacillus (Yordy and Weaver 1977) (19) to accommodate the restricted facultative methanol utilizers. Urakami
and Komagata (16) argued that although the species Methylobacillus glycogenes as circumscribed by them was heterogeneous in DNA base composition, DNA-DNA homologies, electrophoretic patterns of enzymes, and polar lipid
composition, the bacteria all exhibited very similar morphological and physiological characters and possessed the same
fatty acid and ubiquinone composition, and consequently,
“for practical purposes,” they included them all in one
species.
On the basis of the results of numerical taxonomic (11,12),
polar lipid (11, 12), DNA base composition (11, 12), DNADNA homology (2; Jenkins, Ph.D. thesis), and polyacrylamide gel electrophoresis whole-cell protein (2; P. Prave, D.
Sukatsch, and U. Faust, U.S. patent 4,166,004, May 1979)
studies, we consider that the restricted facultative methanolutilizing bacteria exemplified by “Methylophilus methylotrophus” (NCIB 10515) and “Methylomonas Clara” (ATCC
31226) are sufficiently distinct from the type strain of
Methylobacillus glycogenes (ATCC 29475) to merit separate
genus status. The restricted facultative methanol utilizers
differ from Methylobacillus glycogenes (ATCC 29475T) in
utilizing glucose as the sole carbon and energy source and in
polar lipid composition. Methylobacillus glycogenes (ATCC
29475T) contains diphosphatidylglycerol, phosphatidylglycerol, phosphatidylethanolamine, and one unidentified glycolipid, while “Methylophilus methylotrophus” (NCIB
10515) contains phosphatidylglycerol, phosphatidylethanolamine, and two unidentified glycolipids, one of which may
be identical with that of Methylobacillus glycogenes (11, 12).
Further, DNA-DNA homology studies (2; Jenkins, Ph.D.
thesis) indicate a low level of homology between Methylobacillus glycogenes (ATCC 29475T) and both “Methylophilus methylotrophus” (NCIB 10515) and “Methylomonas
Clara” (ATCC 31226). Byrom (2) using the filter membrane
technique and 32P-labeled DNA from “Methylophilus
methylotrophus” (NCIB 10515)detected approximately 22%
homology with DNA from Methylobacillus glycogenes
(ATCC 29475T).A later study (Jenkins, Ph.D. thesis), using
essentially the same technique but with the addition of S1
nuclease treatment, showed only about 5% DNA-DNA
homology between the same strains. These results are in
The ability to utilize methanol and some other singlecarbon compounds, but not methane, as the sole carbon and
energy source is found among a variety of gram-positive and
gram-negative bacteria (17). On the basis of the range of
carbon compounds utilized, the bacteria can be divided into
three groups: the obligate methanol utilizers that can use
only single-carbon compounds such as methanol, methylamine, and formate; the restricted facultative methanol
utilizers that also grow on a limited range of more complex
organic compounds; and the less restricted facultative methanol utilizers that grow on a wider range of more complex
carbon compounds as the sole carbon and energy source (4,
12). The majority of the last group and all members of the
first two groups so far studied are gram-negative, aerobic,
rod-shaped bacteria that superficially resemble pseudomonads. Until recently the taxonomy and nomenclature of
these bacteria were unclear (17, 18). Consequently, they
were not treated in Bergey’s Manual of Systematic Bacteriology (18). Subsequent studies of the pink-pigmented, less
restricted facultative methanol utilizers (7) have resulted in
their classification in the genus Methylobacterium (1,8). The
results of recent numerical taxonomic and chemical studies
on a number of obligate, restricted, and less restricted
methanol-utilizing, gram-negative bacteria and some
pseudomonads confirmed the distinctness of the genus
Methylobacterium and indicated that the obligate and restricted facultative forms represented two distinct taxa of
generic status (11, 12; 0. Jenkins, Ph.D. thesis, University
of Leicester, Leicester, United Kingdom, 1984). The obligate methanol utilizers studied could be equated with the
genus Methylobacillus (Yordy and Weaver 1977). The restricted facultative methanol utilizers, many of which were
cultures used for the production of single-cell protein, could
not be placed in any validly named taxon (11, 12; Jenkins,
Ph.D. thesis) but did include the organisms labeled
“Methylophilus methylotrophus’’ (NCIB 10515) and “Methylomonas Clara” (ATCC 31226). These results are in agreement with the two groups detected by Byrom (2) on the basis
of polyacrylamide gel electrophoresis of whole-cell proteins
and deoxyribonucleic acid (DNA)-DNA homology studies of
a limited number of strains.
Very recently, Urakami and Komagata (16) proposed that
both the obligate and the restricted facultative methanol-
* Corresponding author
446
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NOTES
VOL. 37, 1987
accord with the clustering of the strains in the numerical
taxonomic study (12) and with the two groups noted on the
basis of polyacrylamide gel electrophoretic studies of wholecell proteins (2).
Consideration of all these data including the recent work
of Urakami and Komagata (16) leads us to propose that
“Methylophilus methylotrophus” (NCIB 10515) and
“Methylomonas Clara” (ATCC 31226) be removed from the
species Methylobacillus glycogenes (Yordy and Weaver
1977 emend. Urakami and Komagata 1986) and be reclassified, together with some industrial isolates (12), in a new
genus named Methylophilus (Me.thyl.o.phil’us. M. L. n.
methyl, the methyl radical; Gr. adj. philos, loving; M . L.
masc. n. Methylophilus, methyl radical loving). The type
species is Methylophilus methylotrophus sp. nov. This name
is chosen because, although never validly published, it was
suggested as a name for NCIB 10515 (2) and subsequently
has been so much used in the biochemical and genetic
literature for the strain, AS1 (= NCIB 10515) that we
propose it as the type strain (see reference 2, 3, 5 , 6, 9, 13).
Description of the new genus Methylophilus. The salient
characters of the genus, based on the literature descriptions
(2, W 1 5 ; Jenkins, Ph.D. thesis; D. G. MacLennan, J. C.
Ousby, T. R. Owen, and D. C. Steer, British patent 1370892,
October, 1974) and on our own observations, are as follows.
When grown on methanol-mineral salts agar or in methanolmineral salts liquid medium, straight or slightly curved rods
usually 0.3 to 0.6 pm in diameter and 0.8 to 1.5 pm in length
occur singly or in pairs. Gram negative, but the stain is often
not taken up well. Motile by polar flagella or nonmotile.
Endospores are absent. No cellular inclusions. No sheath or
prosthecae detected. No capsules formed, but slime may be
produced by some strains. Colonies on methanol-mineral
salts agar plates incubated for 2 days at 30 or 37°C are
circular, 1 to 2 mm in diameter, with entire edge, convex,
translucent to opaque. Pyocyanin and fluorescein are not
produced. No, or extremely poor, growth on nutrient agar
and in nutrient broth incubated at 30 or 37°C for 2 days. No,
or extremely poor, growth on blood agar; no hemolysis.
Optimum temperature, 30 to 37°C; no growth occurs at 4 or
45°C.Optimum pH for growth, 6.5 to 7.2 Aerobic. Metabolism respiratory; very little or no acid is produced from
glucose. Methanol is utilized as the sole carbon and energy
source by all strains. In addition, a limited range of other
carbon compounds such as methylamines, formate, glucose,
and fructose may be utilized as the sole carbon and energy
source. Nutritionally nonexacting; nitrate and ammonium
salts serve as nitrogen sources. Catalase positive and oxidase positive.
The fatty acid composition is primarily of the nonhydroxylated straight-chain saturated and monounsaturated types
with Clbz0and C16:1predominating (2, 10, 14, 16). The major
isoprenoid quinone components are ubiquinones with eight
isoprene units (Q-8)(11, 12, 14, 16; Jenkins, Ph.D. thesis).
DNA base composition is 50 to 53 mol% guanine plus
cytosine as determined by estimation of the melting point
(11, 12; Jenkins, Ph.D. thesis).
Bacteria of this kind have been isolated from activated
sludge, mud, and river and pond water (MacLennan et al.,
British patent 1370892).
The type species is Methylophilus methylotrophus.
Description of Methylophilus methylotrophus. Methylophilus methylotrophus (me.thyl.o.tro‘phus. M. L. n. methyl,
the methyl radical; Gr. adj. troph-o, pertaining to nutrition;
M. L. adj. methylotrophus, methyl radical consuming).
This description is based on studies of the type strain AS1
447
(= NCIB 10515) and strain ATCC 31226 (2, 10,-16; Jenkins,
Ph.D. thesis: MacLennan et al., British patent 1370892;
Prave et al., U. S. patent 4,166,004) and our own observations.
Morphology and general characteristics are as given for
the genus. Colonies on methanol-mineral salts agar are
greyish white. The cells are motile by single flagella. In
addition to growth on methanol as the sole carbon and
energy source, good growth occurs on glucose and may or
may not occur on methylamines as the sole carbon and
energy source (12; Jenkins, Ph.D. thesis). Variable results
are obtained with fructose as the sole carbon and energy
source (12; Jenkins, Ph.D. thesis). Poor, variable growth
may occur on lactose, sucrose, D-ribose, D-xylose, ethanol,
propanol, butanol, acetate, and formate. Acid is not produced from glucose. Acetoin, tested by the Voges-Proskauer
method, may or may not be produced. Tween 20,40, and 60
are hydrolyzed; Tween 80 is not hydrolyzed. Urease is
produced. Leucine arylamidase is produced. Phosphatase
production weak and variable. Sulfatase is not produced.
Hydrogen sulfide is not produced. Gelatin is not liquefied.
Extracellular deoxyribonuclease and ribonuclease are not
produced. 2,3,5-Triphenyltetrazoliumchloride (0.01%, wt/vol)
is reduced. No growth occurs in the presence of 0.01%
(wthol) potassium tellurite, or with 5% (wthol) NaC1.
Resistant to penicillin, oleandomycin; sensitive to nalidixic
acid, streptomycin, and a number of other antibiotics. The
guanine-plus-cytosine content of the DNA is about 50 mol%;
strain NCIB 10515T, 50.3 mol% (T,,J; strain ATCC 31226T,
49.8 mol% (TnJ (11, 12). DNA-DNA homology values of
88% (2) and 83% (Jenkins, Ph.D. thesis) have been reported
between strain NCIB 10515T and strain ATCC 31226T.
The type strain is AS1 (= NCIB 10515). This strain was
isolated from activated sludge.
Description of the type strain. In most respects the description of the type strain is as for the species. The type strain
grows well on dimethylamine and weakly on monomethylamine, trimethylamine, fructose, lactose, sucrose, D-ribose,
D-xylose, formate, acetate, citrate, ethanol, propanol, and
butanol as the sole carbon and energy source. Acetoin is not
produced. Alkaline phosphatase and esterase are produced.
One of us (O.J.) thanks the SERC and ICI plc for support.
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