Authored by Moh’d Nizar Battikhi
Abstract
The aim of this study is to compare pattern of Isoprenoid quinine of unspecified bacterial isolates from dairy product to reference strains type species of the genus Microbacterium.Method: Twenty-eight strains of coryneform and related bacteria were studied for isoprenoid quinine content. Extraction of 100 mg of freezedried cells by chloroform: methanol (2:1v/v), extract was then run onto analytical TLC plates coated with o.5 mm Merck Silica Gel HF245.
Results: Out of the twenty-eight reference and unspecified strains M. lacticum and M. flavum are quite distinct and that the predominant of MK-8 in BL77/18. BL77/19 and BL77/20 support that these strains are related to M. flavum.
Coryneform bacteria classification in Bergy’s Manual of
Bacteriology [1] accommodate different genera as Cellumonas,
Arthrobacter, Corynebacterium and Microbacterium described
as incertea sedis. Other motile plant pathogens were added to
coryneform group [2,3]. Numerical phonetic studies improved
classification of coryneform [4,5]. Other markers were used to
improve taxonomic position of coryneform bacteria such as DNA
base composition determination [4,6-10], wall analysis [11-15]
and mycolic acid analysis [2,15,16]. The use of additional marker
(isoprenoid quinone) was recommended by [16,17] to improve
classification of coryneform and related taxa. The analysis of the
isoprenoid quinone of coryneform bacteria has been a useful aid
to establish the taxonomic relatedness of bacterial strains [2,
3,17-20]. These quinines can be divided into menaquinones and
ubiquinones abbreviation of their data is given in (Figure1a).
Substantial investigation amongst coryneform have been carried
out on the distribution of quinines bearing isoprenoid side
chain. Bacterial quinones of this type can be sub-divided into
menaquinones (2 methyl-3-polyisoprenyl 1-1,44 naphthoquinones
and ubiquinones (co-enzyme Q; 12,3-dimethoxy-5-methyl-1-6-
polyprenyl-1-1,4 benzoquinone (Figure1b). The menaquinones
(related to vitamin K) show structural variation at C-2 and ,more
especially, at C-3.Variation at C-3 include the length and degree of
un-saturation of the polyprenyl side-chain [22] and the presence of
hydroxyl [21] and epoxide groups [22]. Similarly, ubiquinones show
variation principally in the structure of polyprenyl sidechain [23].
Therefore, the structure of these compound might be of great value
in classification of coryneform and related bacteria [23]. Other
studies demonstrated that organisms can be clustered based on the
isoprenoid side chain of the menaquinones [2,3,20,24,25]. From
their analysis Minnikin et al [22] concluded that MK_8(H2) was
the major menaquinones of the animal associate corynebacterium.
Microbacterium flavum, with consistently shows affinities with the
animal associated corynebacteria in numerical phonetic studies,
also contains MK-8(H2). They also reported that Micro bacterium
ammoniaphilum contained MK-9(H2). Isoprenoid quinines are
membrane-bound compounds (lipid molecules) found in nearly
all living organisms. It marked structural variation depending
upon the microbial taxon [27-30]. Therefore, organisms can be
clustered based on isoprenoid side chain of their menaquinones.
and hence differentiation between various taxa could be improved
[3,17,18,27].
Twenty-eight strains of coryneform and related bacteria were
collected for the study. These were obtained from public and private
culture collections (Table 1). Each strain was given number for
ease handling. Several strains representing different taxa defined
in study [4,5] where numerical phonetic surveys of coryneform
and related bacteria were included as references. The unidentified
strains were received from the National Institute of Research in
Dairying, Sheffield, Reading and food Research institute, Norwich.
All strains were originally obtained in freeze dried cultures.
Strains were routinely subculture at three weeks intervals
onto nutrient agar (Difco) plates and stored at 4C. After 2-3 days
incubation at 37 °C. For broth culture Nutrient broth Media (Oxoid)
media was and culture were stored at lyophilized aliquots in glass
vials for future used were they resuscitated by transfer small
amount of nutrient onto nutrient agar plates and incubated for 7
days. Nutrient agar (Difco) and nutrient broth (Oxoid) were used
for growing all isolates. All media were sterilized by autoclaving
at 121 °C for 15min. Culture were incubated for 24-48 H at 37 °C.
Cultures were harvested by centrifugation at 8,000 rpm for 20min,
in an MSE high speed M-18 centrifuge, washed in distilled water and
re-harvested Organism were lyophilized and stored anhydrously
as a fine powder until required and freeze dried for future used.
Isoprenoid quinone analysis done by extraction of 100mg potions
freeze dried organisms by a method described by Collins et al.
[17] that proved to be satisfactory for the analysis of lipids. At the
preparation phase ,components with chromatograph with vitamin K
were the only isoprenoid quinines which were detected. Ultraviolet
spectroscopy of the eluted quinines showed absorption maxima
at 242,248,260,270 and 326nm (Figure 2), feature which are in
accordance with the published data for menaquinones [28] where
100mg of freeze dried cells were mixed with 20ml chloroform:
methanol (2:1v/v) in a 50ml conical flask and the suspension stirred
for16-18h in the dark (isoprenoid quinine are susceptible to strong
light). Organisms were removed by filtration and the extracts were
evaporated to dryness under reduced pressure and teem below 37
°C. Components were detected by observing chromatograms under
short-wave (254nm) ultra-violet light. The dried extracts were
prepared as described in above then were re-suspended in small
volume of chloroform: ethanol (2:1 v/v) and spotted onto analytical
TLC plates coated with 0.5mm Merck Silica Gel HF245 (prepared
by coating plates with slurry contains 40g gel HF245 per 100ml
distilled water and drying overnight at 65 °C).
Plates were developed in petroleum ether (BP 60-80): diethyl
ether (85:15 v/v). In this solvent system, the menaquinones
migrate further (Rf 0.7) than ubiquinones (Rf 0.4). Vitamin K
(sigma) an Ubiquinone’s -50 (BDH) were included as standard. The
isoprenoid quinines were also isolated by eluting with chloroform
from preparative TLC plates (1 mm, Merck silica Gel HF245) using
the same solvent system. The quinines were detected by brief
irradiation with ultraviolet light (245nm) when they appeared
as dark brown spots on a bright green fluorescent background.
Eluted samples were evaporated to dryness by a method based on
Collins et al [17] proved to be satisfactory for analysis of lipids at
the preparation phase, components with chromatographed with
vitamin K were the only isoprenoid quinines which were detected.
Ultraviolet spectroscopy of the eluted quinones showed maxima
at 242, 248, 269, 270 and 326nm (Figure 2) feature which are in
accordance with the published data from menaquinones [28].
The isoprenoid quinine was separated by reverse phase partition
chromatography using appropriate standards. Components were
detected by observing chromatogram under ultraviolet light
(254nm).
In describing the menaquinones of strains, the following
nomenclature was adopted. The symbol MK denoting menaquinone
is followed by a number (e.g. 8, 9 or 10) denoting the number of
isoprene units attached as a side chain (Table 2). A wide range
of manaquinones with varying numbers of isoprene units were
characterized within the group of the menaquinones although only
one type predominated of Corynebacterium, oortii (strain C617
and C618) was MK9, a result in agreement with other study [20]).
Likewise, the major quinine of Microbacterium flavum (C90) was
MK8, again in agreement with published data [17,29]. In this study
the menaquinones of 28 reference strains of Micro bacterium, Corynebacterium and unknown isolates were examined. The five
strains of M. lacticum (C88,C,89,C787,C788.C789) tested were all
contain eleven and twelve isoprene units as the predominant side
chain of their menaquinone in contrast M. flavum (C90) was shown
to contain MK-8 as the predominant menaquinones which is in
agreement with other study by [2]. These observations agree with
conclusion that M. lacticum and M. flavum are quite distinct, likewise
the major menaquinone (MK-7) of Brochothrix thermosphacta
(Microbacterium thermosphactum) support the other evidence
that this species is quite distinct from both M. lacticum and M.
flavum. The presence of MK-11 and MK12 in Corynebacterium
laevaniformans (C626) agrees with other study [20]. Strains
JP2/1/6 and strain JP2/1/5 were both contain MK-11 and MK12,
which is similar to M. lacticum that showed resemblances. M.
liquefaciens (C770, C771), Corynebacterium mediolanum (C69)
and Brevibacterium imperial (C25) all shown to contain MK-11 and
MK-12 a feature consistent with earlier study [19]. The unnamed
strains used in this study showed that the predominant of MK-8
in BL77/18. BL77/19 and BL77/20 support that these strains are
related to M. flavum. The similar menaquinones profiles of strains
JP2/1/1 and`JP2/1/21which contain MK-11 and MK-12 supports
their taxonomic relatedness but suggest that they may not closely
related to M.flavum. The predominant of MK-8 in strains.
The analysis of isoprenoid quinines of coryneform bacteria has
been a useful aid to establishing the taxonomic relatedness of strains
[2,18-21,26] These quinines can be divided into menaquinones
and ubiquinones. By far the majority of coryneform contains
menaquinones although different groups produce menaquinones
with different numbers of isoprene units and may also differ in their
degree of saturation. These differences appear to be taxonomically
significant [26]. Thus, organism regarded as closely related on other
evidence (numerical taxonomic data, cell-wall composition,) often
have similar menaquinones while unrelated organisms frequently
contain different components.
In this study the menaquinones of 28 strains were examined.
The five strains of M. lacticum (C88, C89, C787, C788, C789) used all contained eleven and twelve isoprene units as the predominant
side chain of their menaquinones. In contrast M. flavum (C90) was
shown to contain MK-8 as the predominant menaquinone which
agrees with other study [26]. These observations agree with the
conclusion that M. lacticum and M. flavum are quite distinct. Likewise,
the major menaquinone (ML-7) of Brochothrix thermosphata
(Microbacterium thermosphactum) supports the other evidence
that this species is quite distinct from both M. lacticum and M.
flavum. The presence of MK-11 and MK-12 in Corynebacterium lave
K-11 and MK-12 which showed their relatedness to each other and
similar to M. lacticum. The significant of manaquinone in taxonomy
was reported by Collins et al [18].
They showed that, amongst other criteria, the curtobacteria
can be divided into two groups based on their menaquinones.
The significant of menaquinone marker in taxonomy was clearly
appeared by showing relatedness of unknown isolates as BL77/18,
BL77/19. BL77/20 to each other and to M. flavum since they
showed MK-8. Likewise, the similar menaquinones profiles of
strain JP2/1/1 and JP2/1/21 which contain MK-12 supports their
similar taxonomic position but they are closely related to M. flavum.
Likewise, our result in this study showed the presence of MK-9 in
strains JP2/1/11, JP2/1/20, JP2/1/15 and JP2/1/3 which suggest
resemblance of these strains.
This study showed the significant of isoprenoid quinine
as marker of clustering different strains according to their
menaquinone content which may lay base line for future taxonomy.
However, other markers may be need for further support.
None.
No conflict of interest.
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