Subterranean Depth Dependent Protein Constitutions of the Micrococcus sp., Isolated from the Kotumsar Cave, India
The subterranean caves represent one of the most suitable examples of extremities in ecosystem and the microbes abiding in such environments represent truly extremophiles in nature. In the present study, Micrococcus bacterial strains were isolated from various depths of the Kotumsar cave (India) and further 12 strains of 3 specific species (M. luteus, M. radiodurans and M. agilis) have been selected for further studies. The protein profiles by SDS-PAGE technique were estimated for each strain and the effect of subterranean depth on the characterization of protein profiles have been tried to establish by the linear regression method. The strains isolated from the deeper zones of the cave exhibits more number of protein bands, carrying higher molecular weights. Statistical analysis also support the same i.e., the strains isolated from the deeper zones of the cave revealed much protein as compared to the strains isolated from the anterior zones. The chances of developing extremozymes (biocatalysts) in the strains, isolated from the deeper zones could not be denied. Further, the result obtained from this study also suggests redrawing the evolutionary tree of studied bacterial strains.
November 03, 2011; Accepted: December 15, 2011;
Published: March 08, 2012
The subterranean caves represent one of the most suitable examples of extremities in ecosystem for several particularities of biotic and abiotic factors. The high humidity and CO2 concentration along with the constancy of other geophysical factors make the caves one of the most vulnerable environments in the Earth. Thus, the microbes living in such environments could be safely referred as extremophiles and the study of such microbes are always interesting for discovering potential microorganisms, important for several biotechnological tools.
Subterranean caves could be divided into various zones depending upon the characteristics
of constancy of their immediate geophysical parameters: (1) The entrance zone
i.e., the space around the cave entrance from where the inner zones of the cave
remain in direct contact with the external environmental condition. (2) The
twilight zone; nearer to the entrance, where light intensity, humidity and temperature
varies according to its ambient environment. (3) The transition zone; almost
complete dark with variable humidity and temperature. (4) The deep zone; complete
dark with almost 100% humidity, constant temperature and almost complete detachment
from the external environmental conditions (Aden, 2005;
Biswas, 2009, 2010). Further,
in addition to isolated environment, accumulation of various other internal
factors viz., guano deposition, fungal colonies, biofilm/foval soil (Camassa,
1997) etc. have also been known to cause gradual alteration of cave habitat.
Kotumsar cave of India is the most biologically explored limestone cave of
India (18°5209 N; 81°5605 E). Time to time several
troglobiotic as well as troglophilic species have been reported from this cave
(Biswas, 1992, 2010; Biswas
et al., 2011). Till date the total passage mapped in the cave is
nearly 660 m, out of which nearly 400 m is easily approachable. The roofs and
walls of the different chambers are lined with colorful dripstone formations
resulting from the precipitation of calcite-dissolved carbonate lime. The chambers
of the cave are floored with either rocks or pebbles of various dimensions or
by surface-derived soil/clay deposits. The cave is subject to frequent flooding
during the monsoon season which usually begins in the middle of June and continues
till the mid of October.
Screening of the bacterial populations inhabiting Kotumsar cave and updating
the list is a continuous study being performed by us. Till date we have succeeded
to isolate total 146 bacterial colonies from the soil samples collected from
seven different depth and habitats dependent sites of Kotumsar cave. On the
basis of PIB-win (probabilistic identification of bacteria) identification system,
twenty one selected strains have been identified and confirmed with the help
of ARDRA techniques (Thakur, 2009).
Microorganisms occupying heterogeneous habitats usually reveal apparent diversity
in their biochemical characteristics. The characterizations of any available
collection of microorganisms generally yield novel information regarding the
nature of the existing gene pool and which could be varied in any parameter
right from the morphological, biochemical, physiological to the molecular level
during identification. Thus, the current conception suggests that the conventional
tests based on the phenotypic characteristics may confuse the classification
of some bacterial taxa. As per Murray et al. (1990),
the electrophoresis technique, as a practical method, is necessary for integrated
use of phenotypic characters in identification of bacterial genera at all level.
In the present study protein profiles of twelve selected strains belonging to three different species of Micrococcus were studied by using SDS-Polyacrylamide Gel Electrophoresis (PAGE) technique. Further, a linear correlation between the total number of protein bands exhibited by each strain and the average of molecular weight (kDa) revealed by each strain have been tried to establish separately with their respective subterranean depth of occurrences.
MATERIALS AND METHODS
Strains selection: Total 12 PIB win and ARDRA based strains belonging to 3 different species of Micrococcus sp. (viz., M. luteus; M. radiodurans and M. agilis) were selected from isolated cultures for the study of protein profile. The strains of only those species were selected which were isolated at least from 4 different depth dependant habitats of the cave amounting to a total 12 strains (3 species X 4 habitats = 12) (Table 1).
Protein extraction: Bacterial cultures were grown overnight in LB broth
(Luria-Bertani medium) pH 7, tryptone, 10 g; yeast extract, 5 g; NaCl, 10 g;
distilled water, 1000 mL. 1.0 mL of bacterial broth was centrifuged at 5,000
rpm for 15 min in a microtube. The pellet was vertexed in sterilized normal
saline and sonicated (Vibra Cell, USA) for 20 min at 50 kHz. Sonicated samples
were again centrifuged at 15,000 rpm at 4°C for 30 min. The supernatant
were passed through the syringe filter (Axiva, dia 2.5 cm, rating, 0.2 μm).
Protein estimation was done by following Lowry et al.
(1951) method. The purified protein solutions were mixed with sample buffer
in the ratio of 1:2. The samples were taken in the Eppendorf tube and kept in
boiling water bath for 5 min, centrifuged to eliminate the cell debris and used
directly for electrophoresis.
||Isolated Micrococcus strains following PIB win scores
and ARDRA support with respect to their habitat types, depth of occurrences
|KCB: Kotumsar cave bacteria, 1: Micrococcus luteus 4-KCB11,
2: Micrococcus radiodurans KCB21, 3: Micrococcus luteus
3-KCB38, 4: Micrococcus radiodurans KCB39, 5: Micrococcus radiodurans
KCB82, 6: Micrococcus agilis KCB63, 7: Micrococcus luteus
1-KCB90, 8: Micrococcus radiodurans KCB93, 9: Micrococcus
agilis KCB111, 10: Micrococcus luteus 2-KCB113, 11: Micrococcus
agilis KCB140, 12: Micrococcus agilis KCB125
Electrophoresis analysis of proteins: The protein profile of all the
bacterial isolates were determined by SDS-polyacrylamide gel electrophoresis
using 12.5% acrylamide gel and the marker following Laemmlis protocol
(Laemmli, 1970). Ready-to-use protein molecular weight
marker (Banglore Genei) was used for following the procedure and which was a
mixture of purified protein as follows: myosin rabbit muscle, 205,00 Da; phosphorylase
b, 97,400 Da, bovine serum albumin 66,000 Da; ovalbumin, 43,000 Da; carbonic
anhydrase, 29,000 Da; soybean trypsin inhibitor, 20,100 Da; lysozyme, 14,300
Da; aprotinin, 6,500 Da; insulin, 3000 Da (2300-3400 Da).
Statistical analysis: To employ the statistical analysis first of all the observed results were quantified. The total number of protein bands (n) exhibited by each bacterial isolates taken as a parameter to verify the linear correlation between the obtained protein bands by each isolates and the subterranean depth (d) of its occurrences. The subterranean depth of each sample collection site were calculated (in meter) from the available map by graph measuring method. Though, the distances (in meter) of data collection sites were not equally distributed, to minimize the variances an equally distributed numerical code were assigned against each site i.e., 5, 10, 15, 20, 25, 30 and 35 (Table 1).
Further, linear correlation was also verified between the sum of total molecular weights (kDa) of the obtained proteins against each strain and their respective subterranean depth of collection site. To calculate it, the average molecular weights i.e., for each isolates the sum of molecular weight revealed by total number of exhibited protein bands were divided by number of total segaments of occurrence to quantify the average molecular weight of each strain revealed by SDS-PAGE analysis.
Cell filtrates protein profiles obtained by SDS-PAGE for 12 bacterial strains belongs to 3 specific species of Kotumsar cave are presented in Fig. 1 and Table 2.
Irrespective to the subterranean depth dependent sites of occurrences and the specific Micrococcus sp., the total numbers of clear protein bands were found to vary in between 8-21.
|| Protein profiles of bacterial strains, isolated from different
depth dependent habitats of Kotumsar cave, India
|Molecular weight (kDa) of bacterial protein *Distance from
cave entrance. #Average n/∑z (total numbers of segments = 34) Bacterial
isolates were as follows; 1: Micrococcus luteus 4-KCB11, 2: Micrococcus
radiodurans KCB21, 3: Micrococcus luteus 3-KCB38, 4: Micrococcus
radiodurans KCB39, 5: Micrococcus radiodurans KCB82, 6: Micrococcus
agilis KCB63, 7: Micrococcus luteus 1-KCB90, 8: Micrococcus
radiodurans KCB93, 9: Micrococcus agilis KCB111, 10: Micrococcus
luteus 2-KCB113, 11: Micrococcus agilis KCB140, 12: Micrococcus
Strains of Micrococcus agilis, isolated from the deepest zones of the cave exhibited highest number of bands, revealed high molecular weights; M. agilis KCB140 (bands-18, sum of mol. wts.-1037.55) M. agilis KCB125 (bands-21, sum of mol. wts.-1383.1).
||A comparative perspective among marker and Protein bands exhibited
by the isolated depth dependent strains of three Micrococcus sps.
Script on specific bands represents its respective molecular
weight (kDa). (a) Micrococcus radiodurans (*-205 kDa; a-81.7-82 kDa;
b-80 kDa; c-24.5 kDa) and (b) Micrococcus agilis (*-99 kDa; a-97.4
kDa; b-24.5-25 kDa)
|| (a) A positive linear correlation between the number
of protein bands exhibited by each strain of Micrococcus sp. during
SDS-PAGE electrophoresis and their respective subterranean depth of occurrences
and (b) A positive linear correlation between the average of total molecular
weight (kDa) of the protein bands exhibited by each strain of Micrococcus
sp. during SDS-PAGE electrophoresis and their respective subterranean depth
Comparative chart clearly indicate that protein band variations were mostly
restricted to the molecular proteins having lower molecular weights. However,
excluding the strain 3-KCB38 of Micrococcus luteus, isolated from the
twilight zone, rest 3 other strains of the same species isolated from three
different subterranean depth dependent habitats neither exhibited any difference
in their total sum of obtained molecular weight of the protein bands nor in
the total numbers of protein bands revealed from SDS-PAGE electrophoresis. Nevertheless,
the remaining 8 strains belonging to 2 different species (viz., Micrococcus
radiodurans; Micrococcus agilis) revealed apparent depth dependant variations
(Table 2; Fig. 1, 2).
Further, the total number of protein bands obtained against each strain evidenced positive correlation with their respective subterranean depth of occurrences (r = 0.633; p<0.027). More or less the same result was seen when the linear correlation was employed between the average value of total sum of molecular weight and the subterranean depth of their respective occurrences (r = 0.582; p<0.046). The result simply revealed that the strains which were isolated from the deeper zones i.e., more extreme conditions were having more protein and with higher molecular weights whereas the strains isolated from such zones which more or less remain under the influence of external environmental conditions were with low protein profiles.
In extremophiles a structural adaptations at the molecular level have been
proposed to cope up with the ambient exacting conditions. Further, some extremozymes
(biocatalysts) which are produced by these microorganisms have been shown to
play the key role to withstand such harsh conditions (Kuddus
et al., 2011). In our study, the strains which were isolated from
the deeper zones revealed few additional specific protein bands which were altogether
absent in their remaining counterparts; isolated from the anterior (nearer to
the entrance) zones of cave. The strain; KCB21 of Micrococcus radiodurans,
isolated from the soil sample of entrance zone did not show any specific band
in its 1st segment of protein profiles, whereas the remaining 3 strains of the
same species isolated from the deeper zones of the cave exhibited specific protein
bands (mol. wts. 205 kDa). The same patterns were repeated in further
9th, 10th as well as in 28th segments. Not a single band was revealed in those
strains of M. radiodurans which were isolated from the entrance zones
whereas the other remaining strains, isolated from the deeper zones revealed
specific protein bands (segment 9:-81.7-82 kDa; segment 10:-82 kDa and segment
While analyzing the protein profiles of various strains of Micrococcus agilis, isolated from different depth dependent habitats, we found some interesting results, revealing almost similar prototype. During our study we failed to isolate any strain of M. agilis from the entrance and/or other anterior zones of the cave. The strain; KCB63 of M. agilis which was isolated from the anterior most (transient) zone of the cave was also found to deviate enough in the prototype of protein profiles from its counterparts, isolated from the deeper zones of the cave. The strain KCB 111, KCB140 and KCB125 of M. agilis, isolated from the innermost zones of the cave revealed some extra bands at the protein profile segments-4 (kDa-99), 6 (kDa-97.4) and 28 (kDa-24.5 to 25) which were altogether lacking in the strain KCB63 of Micrococcus agilis. Absence of this protein bands in organisms living in transient zone possibly attributable to the external environmental conditions of the cave as the transient zone of the cave is under the influence of the external environmental conditions to a limited extent.
Total 4 Micrococcus luteus strains named; Micrococcus luteus 4, from external zone, Micrococcus luteus 3, from twilight zone and Micrococcus luteus 1 and 2 from two different habitats of the deeper zone were isolated, identified by PIB win and ARDRA methods. Surprisingly, while analyzing the protein profiles of all these strain we found the strains isolated from the entrance and the deeper zones are almost alike, where as the strain isolated from the twilight zone revealed dissimilarities in positions and numbers in the occurrences of protein bands. Although, the specific reason for such differences among all the strains of this particular species revealed by PIB Win identification method is yet to be elucidated, the subterranean depth was found to exert almost nil effect in the spectrum of protein profiles of the same.
Bacterial community which was discovered from the depths of more than a kilometer
was found to play a key role in geochemical processes and thus provoked the
cave-microbiologists to search for those microbes which might play a major geochemical
role as well (Culver, 2005). In the present study, recurrence
of some additional protein bands in the strains isolated from the deeper zones
of the cave possibly offering paramount importance for understanding the effects
of extreme environment on living organism as this part of the cave always represent
an extreme condition. Characterization of these extreme conditions proteins
produced by the respective resident microbes for their survivability can be
exploited for several biotechnological means. Nevertheless, studies of extremophiles
have also helped to redraw the evolutionary tree of life (Madigan
and Marrs, 1997) Conclusively, a further study of these extremophile proteins
produced by microbes of the deeper zones of Kotumsar cave will certainly not
be restricted to the beneficial of its utilities in several biotechnological
tools but also throw sufficient light for reconstruction of evolutionary tree
We are highly grateful to the Director, Kanger Valley National Park for allowing
us to collect the samples. 1st author (YR) is highly grateful to the Head, School
of Life Sciences, Pt. Ravishankar shukla University, Raipur for allowing her
to conduct the laboratory work. Thanks are also due to Prof. R.K. Pradhan, School
of Life Sciences, Pt. Ravishankar Shukla University, Raipur, India for his valuable
suggestions and critics for the earlier version of the current manuscript.
Aden, E., 2005. Adaptation to Darkness. In: Encyclopedia of Caves, Culver, D.C. and W.B. White (Eds.). 1st Edn. Elsevier Academic Press, Burlington, CA., USA., ISBN-13: 978-0121986513, pp: 1-3.
Biswas, J., 1992. Kotumsar cave ecosystem: An interaction between geophysical, chemical and biological characteristics. NSS Bull., 139: 7-10.
Direct Link |
Biswas, J., 2009. The biodiversity of Krem Mawkhyrdop of Meghalaya, India, on the verge of extinction. Curr. Sci., 96: 904-910.
Direct Link |
Biswas, J., 2010. Kotumsar Cave biodiversity: A review of cavernicoles and their troglobiotic traits. Biodivers. Conser., 19: 275-289.
CrossRef | Direct Link |
Biswas, J., S. Shrotriya, Y. Rajput and S. Sasmal, 2011. Impacts of ecotourism on bat habitats in caves of Kanger Valley National Park, India. Res. J. Environ. Sci., 5: 752-762.
CrossRef | Direct Link |
Camassa, M., 1997. Biological observations in an artificial cave of Ginosa (Southern Italy): Role of the foval in cave ecosystems. Thalassia Salentina, 23: 189-191.
Culver, D.C., 2005. Microbes. In: Encyclopedia of Caves, Culver, D.C. and W.B. White (Eds.). 1st Edn. Elsevier Academic Press, Burlington, CA., USA., ISBN-13: 978-0121986513, pp: 369-371.
Kuddus, M., Roohi, J.M. Arif and P.W. Ramteke, 2011. An overview of cold-active microbial α-amylase: Adaptation strategies and biotechnological potentials. Biotechnology, 10: 246-258.
CrossRef | Direct Link |
Laemmli, U.K., 1970. Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature, 227: 680-685.
Direct Link |
Lowry, O.H., N.J. Rosebrough, A.L. Farr and R.J. Randall, 1951. Protein measurement with the folin phenol reagent. J. Biol. Chem., 193: 265-275.
PubMed | Direct Link |
Madigan, M.T. and B.L. Marrs, 1997. Extremophiles. Sci. Am., 276: 82-87.
Murray, R.G.E., D.J. Brenner, R.R. Colwell, P. Devos and M. Goodfellow et al., 1990. Report of the ad hoc committee on approaches to taxonomy within the proteobacteria. Int. J. Syst. Bacteriol., 40: 213-215.
Direct Link |
Thakur, Y., 2009. Isolation and characterization of bacteria from kotumsar cave of chhattisgarh. Ph.D. Thesis, Pandit Ravishankar Shukla University, Raipur, India.