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Research Article
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Molecular Characterization and Batch Fermentation of Bacillus subtilis as Biocontrol Agent, II |
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S. M. Matar,
S.A. El-Kazzaz,
E. E. Wagih,
A.I. El-Diwany,
M. A. El-Saadani,
E. E. Hafez,
H. E. Moustafa,
H.E. Abd-Elsalam,
G.A. Abo-Zaid
and
E. A. Serour
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ABSTRACT
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Fourteen Bacillus subtilis isolates (B1 to B14) obtained from different Egyptian sites had different antagonistic and inhibitory effect against six fungal isolates belonging to four different genera, Rhizoctonia solani, Helminthosporium sp., Alternaria sp. and Fusarium oxysporum. Random Amplified Polymorphic DNA (RAPD), differential display technique and partial sequencing of 16S rRNA gene were used for characterization of isolates. While RAPD technology, using 4 different random 20-mer primers, revealed different levels of molecular variation among twelve out of the fourteen isolates tested, the remaining two isolates B3 and B5, demonstrated identical profiles. Because of the high degree of homology (93%) between isolates B4 and B7 and the no variation observed between isolates B3 and B5, the differential display technique was used to test the reliability of these results. Isolate B7 was selected for batch cultivation in bioreactor, the biomass achieved was 3.2 g L-1. Inhibitory activity of supernatant was increased near the end of the stationary phase. The activity reached its highest value of 1.7 cm in death phase.
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How
to cite this article:
S. M. Matar, S.A. El-Kazzaz, E. E. Wagih, A.I. El-Diwany, M. A. El-Saadani, E. E. Hafez, H. E. Moustafa, H.E. Abd-Elsalam, G.A. Abo-Zaid and E. A. Serour, 2009. Molecular Characterization and Batch Fermentation of Bacillus subtilis as Biocontrol Agent, II. Biotechnology, 8: 35-43. DOI: 10.3923/biotech.2009.35.43 URL: https://scialert.net/abstract/?doi=biotech.2009.35.43
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INTRODUCTION
The Random Amplified Polymorphic DNA (RAPD) which is a Polymerase Chain Reaction
(PCR)-based technique in which amplification of random DNA segments with single
primers of arbitrary nucleotide sequence (Williams et
al., 1990; Hilton et al., 1997) is conducted
and used as powerful tool for genetic studies involving molecular genetic diversity
of microorganisms. A robust RAPD-PCR procedure for large-scale typing of B.
cereus was developed by Nilsson et al. (1998).
It is based on a simple DNA preparation, involving only freezing and boiling
of cells in water with active carbon. By using a computerized for data collection
and processing, an efficient system for handling RAPD patterns for large scale
investigation was achieved. The procedure was highly discriminatory for B.
cereus strains and was found to give reproducible classification of RAPD
fingerprints for five reference strains. Istock et al.
(2001) analyzed the geographical distribution and structure of B. subtilis
genomic lineages using RAPD fingerprints. Six major genomic lineages separating
at 9-18% similarity were found. One hundred and twenty-six strains of B.
thuringiensis representing 57 serovars were allocated to 58 genomic types
using random amplified polymorphic DNA (RAPD)-PCR patterns (Gaviria
and Priest, 2003), also Levy et al. (2006)
used the RAPD technique to distinguish between seven Bacillus species.
Differential display is a molecular biology technique that can distinguish between
genes that are up- or down regulated. Initially described by Liang
and Pardee (1992), differential display encompasses several reverse transcription
polymerase chain reaction (RT-PCR) techniques that can produce a large number
of cDNA fragments from cellular RNA. First, anchored 3` poly-T primers are used
to create cDNAs corresponding to all possible mRNA species. Next, specific gene
fragments are amplified by use of random 5` primers.
Fislage et al. (1997) developed a primer set
for a prokaryotic differential display of mRNA in the Enterobacteriaceae group.
The technique of mRNA differential display was used to identify simultaneously
two metabolic genes involved in the degradation of cyclohexanone
in a new halotolerant Brevibacterium isolate (Brzostowicz
et al., 2000). Chia et al. (2001)
analyzed differential gene expression of Streptococcus mutans
in response to environmental stress by RNA fingerprinting using
arbitrarily primed PCR with a panel of 11 mer primers designed for
differential display in Enterobacteriaceae. In a batch process, all substrates
are much available in the initial medium and none is supplied extra during the
process. The process is stopped after the substrate is converted and the whole
batch is harvested together (Sonnleitner, 1998; Wirtz,
2002). A batch growth is a non-steady state process; that is the content
of the bioreactor changes with time. The nutrients and the inoculum are added
to the bioreactor at the beginning of the batch operation and air is supplied
continuously. After a short lag time, B. thuringiensis cells begin
the exponential growth; the carbon source is oxidized and owing to organic acids
accumulation, the pH drops dramatically. The biomass concentration and the cell
count increases as the carbon source decreases. The oxygen demand increases
during the exponential stage and there are no spores present in the bioreactor
(Avignone-Rossa and Mignone, 1995). The production of
biocontrol agent of B. subtilis R14 was evaluated in a batch bioreactor
(Luna et al., 2002). Batch operation has been
used for comparison with other modes of growth as a reference. Liu
et al. (1994) for example compared the results obtained in a batch
growth with a fed-batch growth of B. thuringiensis subspecies
kurstaki. The maximum biomass concentration obtained in batch growth was
5.9 g dry weight mL-1 whereas the maximum biomass concentration was
53.7 g dry weight mL-1 in fed-batch growth.
The objective of this study was for molecular characterization of bacterial isolates which have an antagonistic and inhibitory effect against certain plant pathogenic fungi and study the behavior of B. subtilis isolate B7 during batch cultivation in bioreactor.
MATERIALS AND METHODS
Bacterial and fungal isolates: Fourteen B. subtilis isolates
were isolated in 2002-2003 from different locations in Egypt (El-Malaha, El-Amria,
El-Nobaria, Abo-Homos and El-Sharkia) and identified according to the morphological,
biochemical and physiological tests recommended by Sneath et
al. (1986) and Collee et al. (1996). Six
fungal isolates belonging to four different genera were isolated in 2002-2003
from different hosts and area in Egypt, R. solani, Helminthosporium
sp., Alternaria sp. and Fusarium oxysporum.
Antagonistic and inhibitory effect of B. subtilis isolates
(in vitro): Antagonistic effect of fourteen B. subtilis
isolates and inhibitory effect of supernatant fluid were performed against all
fungal isolates according to Toure et al. (2004)
and Chitarra et al. (2003). The data obtained
were statistically analyzed using the Statistical Analysis System (SAS).
Genotypic characterization
DNA extraction protocol: Total DNA was extracted from B. subtilis
isolates according to Istock et al. (2001).
Random Amplified Polymorphic DNA (RAPD): Four 20-mer arbitrary primers
were used for RAPD analysis (A1A13, A9B7, A1 and A7) (Abdel
Ghany and Zaki, 2003). Sequences of all primers are shown in Table
1. For RAPD analysis, PCR amplification was carried out in a total volume
25 μL containing 2.5 μL 10x buffer, 2 μL 25 mM MgCl2, 2 μL 2.5
mM dNTPs, 1 μL 10 pmol primer, 1 μL 50 ng of bacterial genomic DNA and 0.2
μL (5 U μL-1) Taq DNA polymerase (Promega, Germany). PCR amplification
was performed in a thermal cycler (Eppendorf, Germany) programmed for one cycle
at 95°C for 5 min followed by 34 cycles each with 30 sec at 95°C for denaturation,
1 min at 45°C for annealing and 2 min at 72°C for elongation. Reaction mixture
was then incubated at 72°C for 10 min for final extension (Istock
et al., 2001).
Differential display analysis: Total RNA was extracted from B.
subtilis isolates using TRI-reagent (Chin et al.,
2000). Reverse transcription reactions were performed using oligo dT primer
(5`-TTTTTTTTTTTTTTT-3`). Each 25 μL reaction mixture contained 2.5 μL 5x buffer
with MgCl2, 2.5 μL 2.5 mM dNTPs, 1 μL 10 pmol primer, 2.5 μL RNA
and 0.2 μL (5 U μL-1) reverse transcriptase (Promega, Germany).
PCR amplification was performed in a thermal cycler (Eppendorf, Germany) programmed
at 95°C for 5 min, 42°C for 1 h, 72°C for 10 min and a soak at 4°C (Chin
et al., 2000).
Table 1: |
Sequence of all primers used in this study |
 |
Three primers were used in the differential display analysis one of them is
specific for 16S rRNA gene (P0) but the other were arbitrary in sequence (P1
and P2). Sequence of primers is shown in Table 1. The reaction
mixture for differential display PCR was carried out in a total volume 25 μL
containing 2.5 μL 10x buffer with MgCl2, 2 μL 2.5 mM dNTPs, 1 μL
10 pmol primer, 1.5 μL cDNA and 0.2 μL (5 U μL-1) Taq DNA polymerase
(Promega, Germany). PCR amplification was performed in a thermal cycler (Eppendorf,
Germany) programmed for one cycle at 95°C for 5 min, then 34 cycles as follows:
30 sec at 95°C for denaturation, 1 min at 45°C for annealing and 2 min at
72°C for elongation. Reaction was then incubated at 72°C for 10 min for final
extension (Chin et al., 2000).
Amplification of 16S rRNA gene: The full length (1550 bp) of 16S rRNA
gene was amplified for all B. subtilis isolates by using two universal
primers (P0 and P6) (Sow et al., 2005). Sequence
of primers is illustrated in Table 1. PCR amplification was
carried out in a total volume 50 μL containing 5 μL 10x buffer, 4 μL 25 mM
MgCl2, 4 μL 2.5 mM dNTPs, 2 μL 10 pmol forward primer, 2 μL 10
pmol reverse primer, 2 μL 50 ng of bacterial genomic DNA and 0.4 μL (5 U μL-1)
Taq DNA polymerase (Promega, Germany). PCR amplification was performed in a
thermal cycler (Eppendorf, Germany) programmed for one cycle at 95°C for 5
min followed by 34 cycles each with 45 sec at 95°C for denaturation, 1 min
at 50°C for annealing and 2 min at 72°C for elongation. Reaction mixture was
then incubated at 72°C for 10 min for final extension (Sambrook
et al., 1989).
Sequencing of 16S rRNA gene and the accession number: QIAquick PCR purification kit (Qiagen, Germany) was used to purify the amplified products of 16S rRNA gene.
The amplified product (1550 bp) of 16S rRNA was sequenced by the use of a Big Dye terminator cycle sequencing kit. Sequencing products were purified using Centri-Sep spin columns and were resolved on an ABI PRISM model 310 automated DNA sequencer at the Agriculture Genetic Engineering Research Institute (AGERI) in Egypt.
Fermentation experiments
Bioreactor: Batch and fed-batch cultivations were performed in a
3 L bench-top bioreactor (Bioflow III, New Brunswick, N J, USA) equipped with
two 6-bladed disc-turbine impeller and four baffles and connected to a digital
control unit. The process was automated through a computer aided data bioprocessing
system AFS-BioCommand multi-process management program. The set points for temperature
and pH value were 30°C and 7, respectively and pH was controlled by automatic
feeding of 2N NaOH. Compressed air was supplied initially at 0.5-1.0 VVM (air
volume per broth volume per minute) through sterile filter. It was manually
controlled in parallel with agitation speed (150-900 rpm) to maintain the dissolved
oxygen level above 20%. Dissolved oxygen and pH values were determined on-line
using METTLER TOLEDO electrodes. Antifoam A (Sigma) was used for eliminating
foaming.
Batch fermentation: Batch cultivation was initiated in a bioreactor
using a volume of 1900 mL of fresh number 3 medium of which each 1 L contained
10 g peptone, 10 g glucose, 1 g KH2PO4 and 0.5 g MgSO4.
7H2O in distilled water and a pH of 6.8 (Asaka
and Shoda, 1996) which was then inoculated with 100 mL of shake flask pre-cultured
seeds which had previously been prepared as follows: a single colony of B.
subtilis B7 (Abo-Homos) which was selected from fourteen isolates had
antagonistic effect against all tested fungi, but showed more antagonistic effect
against Alternaria sp. (Matar et al., 2008(I))
was inoculated into a 500 mL Erlenmeyer flask containing 100 mL of No. 3 medium
and cultured overnight at 30°C with constant shaking at 200 rpm. During the
time of cultivation, several samples of culture were taken and cell number was
determined by measuring of optical density (OD) at 550 nm. Glucose level was
estimated by enzymatic (GOD-PAD) colorimetric kit (Diamond Diagnostic, Egypt)
and the inhibitory activity assay of supernatant fluid was performed against
Alternaria sp. according to Chitarra method (Chitarra
et al., 2003). Also, 10 mL of cultivated broth was separated overtime
to apply the dry test method.
RESULTS
Data of antagonistic and inhibitory effect of B. subtilis isolates
(in vitro) was explained by Matar et al. (2008(I)).
When Random Amplified Polymorphic DNA (RAPD) technique was used to molecularly
differentiate among the 14 B. subtilis isolates, using four 20-mer arbitrary
primers (Table 1), clear differences among twelve isolates
on the bases of amplified product band patterns observed with each primer (Fig.
1, 2). Notably, high similarity (93%) was observed between
B4 (El-Nobaria) and B7 (Abo-Homos) and also between B8 (El-Malaha) and B9 (El-Malaha).
Exceptionally, isolates B3 (El-Malaha) and B5 (El-Amria) gave identical band
patterns with all the four primers used. Results indicated that 256 amplicons
were produced by all primers. The number of amplified products produced by each
primer varied from 46 with primer A1A13 to 75 with primer A9B7. However, primer
A1A13 and A7 failed to show any amplified products with isolate B9 (El-Malaha).
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Fig. 1: |
RAPD-PCR patterns of Bacillus subtilis isolates with primer A9B7.
Lanes on both sides of the gel labeled M are loaded with PBR 328 Mix DNA
marker. B1 to B14 are the fourteen Bacillus subtilis isolates investigated.
Isolates B1, B3, B8 and B9 are those of El-Malaha; B5 and B6 are of El-Amria;
B4, B10, B11 and B12 are of El-Nobaria; B7, B13 and B14 are from Abo-Homos;
B2 is that isolated from El-Sharkia |
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Fig. 2: |
RAPD-PCR patterns of Bacillus subtilis isolates with primer A1.
Lanes on both sides of the gel labeled M are loaded with PBR 328 Mix DNA
marker. B1 to B14 are the fourteen Bacillus subtilis isolates investigated.
Isolates B1, B3, B8 and B9 are those of El-Malaha; B5 and B6 are of El-Amria;
B4, B10, B11 and B12 are of El-Nobaria; B7, B13 and B14 are from Abo-Homos;
B2 is that isolated from El-Sharkia |
The RAPD band patterns resulting from the use of the four primers were analyzed
using UPGMA method to construct a similarity matrix and to generate a dendrogram
indicating the relationship between the fourteen tested isolates of B.
subtilis.
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Fig. 3: |
Dendrogram obtained by clustering (UPGMA method) based on the band pattern
obtained by the RAPD-PCR analysis of fourteen Bacillus subtilis
isolates from different location according to Jaccard similarity |
The presence or absence of any particular DNA bands was the only parameter
considered in the computer analysis. The dendrogram generated to show linkage
distance (Fig. 3) indicated that the fourteen B. subtilis isolates
were classified into two main Clusters (Cluster I and Cluster II). Cluster I
represented only one group which included the two B. subtilis
isolates B3 (EL-Malaha) and B5 (El-Amria). Cluster II split into two Sub-clusters:
Sub-cluster 1 that divided further into two Groups: Group 1 which included isolate
B2 (El-Sharkia) and Group 2 which included B6 (El-Amria), whereas Sub-cluster
2 divided into two Groups: Group 1 included B1 (El-Malaha) and Group 2 divided
into two Sub-groups: Sub-group 1 contained B11 (El-Nobaria), B4 (El-Nobaria)
and B7 (Abo-Homos) and Sub-group 2 contained B10 (El-Nobaria), B12 (El-Nobaria),
B14 (Abo-Homos), B13 (Abo-Homos), B8 (El-Malaha) and B9 (El-Malaha), respectively.
The results obtained when the differential display technique was applied to
B. subtilis isolates B3 (El-Malaha) and B5 (El-Amria), which could
not be differentiated when analyzed by the RAPD-PCR, using the primer P0 of
the 16S rRNA gene revealed clear differences. While, three fragments differing
in molecular size, 637, 1083 and 1767 bp were shown with isolate B3 (El-Malaha)
but not shown with B5 (El-Amria). Two fragments of a size of 763 and 860 bp
were appeared with B5 (El-Amria) but not with B3 (El-Malaha). Similarity results
were obtained (Fig. 4) where isolate B4 (El-Nobaria) and B7 (Abo-Homos), which
showed high percentage of similarity when exposed to RAPD analysis, were tested
with the same primer (P0). While only one fragment of 860 bp was observed with
isolate B4 (El-Nobaria) but not with B7 (Abo-Homos), four fragments differing
in molecular size, 2361, 2457, 3398 and 3910 bp were detected with B7 but not
with B4.
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Fig. 4: |
Differential display RT-PCR for six Bacillus subtilis isolates
using primer P0. The lane labeled M at the left-hand side of the gel was
loaded with M25-3 k bp ladder; the other lanes were loaded with B1 and B3
(El-Malaha), B4 (El-Nobaria), B5 (El-Amria), B7 and B14 (Abo-Homos). Arrows
within the gel point out polymorphic bands |
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Fig. 5: |
Differential display RT-PCR for six Bacillus subtilis isolates
using primer P1. The lane labeled M at the left-hand side of the gel was
loaded with M25-3 k bp ladder; the other lanes were loaded with B1 and B3
(El-Malaha), B4 (El-Nobaria), B5 (El-Amria), B7 and B14 (Abo-Homos). Arrows
within the gel point out polymorphic bands |
Supportive results of polymorphism were observed (Fig. 5) when the arbitrary
primer (P1) was used as, one fragment of a size of 980 bp was shown with isolate
B3 (El-Malaha) but not with B5 (El-Amria). Three fragments of 295, 435 and 948
bp were appeared with B5 but not with B3. One fragment of 430 bp was evident
with B4 (El-Nobaria) but not with B7 (Abo-Homos).
Table 2: |
The accession No. and isolate code of 16S rRNA gene of Bacillus
subtilis isolates in the GenBank |
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Fig. 6: |
Differential display RT-PCR for six Bacillus subtilis isolates
using the arbitrary primer P2. The Lanes lablelled M at both sides of the
gel were loaded with M25-3 k bp ladder; the other lanes were loaded with
B1 and B3 (El-Malaha), B4 (El-Nobaria), B5 (El-Amria), B7 and B14 (Abo-Homos).
Arrows within the gel point out polymorphic bands |
When differential display analysis with the arbitrary primer P2 was conducted
(Fig. 6), two fragments of a size of 741 and 1186 bp were observed with B3 (El-Malaha),
but not with B5 (El-Amria) and only one fragment of 1199 bp was detected with
B5 with no corresponding band with B3. Likewise, the same primer gave six fragments
(286, 522, 596, 1856, 2094 and 2335 bp) with B7 but these fragments were not
generated with B4.
Approximately 1550 bp of the 16S rRNA gene was amplified for all the fourteen
B. subtilis isolates using universal primers (Table 2). Consequently,
the obtained amplicon was later subjected to sequencing analysis.
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Fig. 7: |
Biomass, glucose concentration and inhibitory activity of culture supernatant
as a function of time for batch cultivation of Bacillus subtilis
isolate B7 |
When the purified product (1550 bp) of the 16S rRNA gene was subjected to sequencing
using ABI PRISM model 310 automated DNA sequencer, the following partial sequence
was obtained for B. subtilis isolates B1 (El-Malaha), B4 (El-Nobaria),
B7 (Abo-Homos), B8 (El-Malaha), B10 (El-Nobaria), B11 (El-Nobaria) and B14 (Abo-Homos)
(Table 2).
Search in databases to identify the bacteria was achieved in BLAST search at
the National Center for Biotechnology Information site (http://www.ncbi.nlm.nih.gov).
The search revealed that these sequences were identical to that of B.
subtilis.
Bacterial isolates (B1, B4, B7, B8, B10, B11 and B14) were identified according to partial sequence of 16S rRNA gene as B. subtilis and scored in GenBank with accession No. (EF150883, EF150884, EF583053, EF583054, EF583055, EF583056 and EF150885) (Table 2).
Batch cultivation of B. subtilis isolate B7 was carried out in
3 L bench top bioreactor (Bioflow III, New Brunswich, NJ, USA) at a constant
temperature of 30°C. Figure 7 shows biomass, concentration
of consumed glucose and inhibitory activity of supernatant of isolate B7 plotted
against the time. Cells were grown directly after the lag phase which lasted
for about 2 h. Cell mass was then increased exponentially over time with a constant
specific growth rate, μ = 0.3 h-1 within the exponential phase.
The behavior in this phase allows to calculate the yield coefficient YX/S
(0.45 g cells g glucose). During this phase, the specific growth rate of 0.3
h-1 was determined from the logarithmic relationship shown in Fig.
8. In the Fig. 8, the values of the ln X (values of X
was obtained by using the correlation coefficient δ, 0.42 calculated from
the linear relationship between dry mass weight and optical density) was presented
by a straight line on a semi-logarithm plot versus time. The biomass achieved
was 3.2 g L-1. Inhibitory activity of supernatant was increased near
the end of the stationary phase. The activity reached its highest value of 1.7
cm in death phase.
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Fig. 8: |
ln biomass (X) in g L-1 as a function of time in the exponential
growth phase of batch cultivation of Bacillus subtilis isolate
B7 |
Modeling of batch fermentation: The most common culture system is the
batch culture, due to its simplicity and low cost. This is a closed system in
which there is no input or output of materials. The microbial population cell
density increases constantly until exhaustion of some limiting factor, while
other nutrient components of the culture medium decrease over time. Any products
produced by the cells during growth also increase in concentration in the culture
medium.
In batch culture, it was important to calculate growth yield for B.
subtilis during cultivation on glucose. Yield coefficient was calculated
based on the amount of glucose consumed (Shuler and Kargi,
1992). The following equation was used to calculate growth yield (YX/S).
where, X and X0 are biomass concentrations (g L-1)
at measuring time t and initial time t0, respectively. S and S0
are the consumed amounts of glucose (g L-1) at the same times mentioned
previously.
In logarithmic growth phase, cell mass density increases exponentially with
time t and specific growth rate μ (h-1) is independent of nutrient
concentration. According to Shuler and Kargi (1992), the
exponential growth rate is first order.
by integration of Eq. 2 yields
The exponential growth is characterized by a straight line on a semi logarithm plot of ln X versus time.
DISCUSSION
Molecular techniques, such as RAPD-PCR, are powerful tools for obtaining data
related to the genomic diversity that exists among the strains belonging to
a bacterial species (genetic diversity). Istock et al.
(2001) used RAPD-PCR for studying geographical diversity of genomic lineages
in B. subtilis and Drake et al. (1996)
used single primer G1, could distinguish 16 commercial strains of Lactobacillus
helveticus by RAPD-PCR profiling. One hundred and twenty-six strains
of B. thuringiensis representing 57 serovars were allocated to
58 genomic types using random amplified polymorphic DNA (RAPD)-PCR patterns
(Gaviria and Priest, 2003). The fourteen B. subtilis
isolates obtained in this study from different areas in Egypt could be differentiated
using random amplified polymorphic DNA technique and it was possible to categorize
them in two main Clusters, Cluster I which included one group with two B.
subtilis isolates, B3 and B5 (similarity 100%) and Cluster II which encompassed
four groups containing twelve B. subtilis isolates.
Differential display is a molecular biology technique that can distinguish between genes that are up- or down regulated. The failure of RAPD-PCR to show any variation between B. subtilis isolates B3 (El-Malaha) and B5 (El-Amria), acsside the high similarity percentage between B4 (El-Nobaria) and B7 (Abo-Homos) it demonstrated, differential display technique was performed for obtaining more confirmative results and to possibly differentiate between a members of each pair (B3, B5 and B4, B7). Differentiation was possible using a specific primer to the housekeeping gene, 16S rRNA. When differential display analysis with the arbitrary primer P2 was conducted. This was evident as the two fragment of a size of 741 and 1186 bp which were observed with B3 (El-Malaha) were not found with B5 (El-Amria) and the only one fragment of 1199 bp which was shown with B5 was not detectable with B3. Polymorphism reached its maximum when the same primer (P2) was used to analyze the pair of isolates B4 and B7. This was evident when the six fragments seizing 286, 522, 596, 1856, 2094 and 2335 bp which were obtained with B7 were not present with B4.
The sequence of the 16S rRNA gene has been widely used as a molecular clock
to estimate relationships among bacteria (phylogeny), but more recently it has
also become important as a means to identify an unknown bacterium to the genus
or species level (Sacchi et al., 2002). The sequencing
of the 16S rRNA and the alignment with sequences present in the GenBank database
resulted in the final identification of the studied isolates. The sequencing
of 16S rRNA has also successfully been used for identification of B.
subtilis, B. anthracis and B. cereus in other
studies (Ash et al., 1991; Ash
and Collins, 1992; Green et al., 1999; Siefert
et al., 2000).
In batch culture, the lag phase is characterized by a negligible increase in the biomass concentration which lasted for a bout 2 h. The exponential phase is characterized by a sharp exponential increase in the biomass concentration, cell mass was increased exponentially over time with a constant specific growth rate 0.3 h-1, the overall biomass yield was 0.45 g cell/g glucose. At the stationary phase, the growth rate was zero (no cell division), these due to the depletion of substrate, oxygen insufficiency, accumulation of inhibitory metabolites such as acetic acid and also due to the beginning of the sporulation process by B. subtilis. Finally during the death phase the cell growth had a negative growth. The growth process is divided into two main stages: vegetative stage and sporulative stage. A B. subtilis cell is in its vegetative stage if it is capable of dividing. On the other hand, a cell is in sporulative stage if the cell stops its division and uses the available energy to form a spore.
Unlike the growth phases, the metabolic stages cannot be identified by a change in the biomass concentration with time. The metabolic stages might, however, be identified by the cell morphology, the cell oxygen consumption and the change in the pH of the medium. Regarding the morphology of a vegetative B. subtilis cell it can be characterized on the basis of cell structure and the absence of a spore. In this respect, during the vegetative stage, the cells consume the substrates and require high amounts of oxygen and several acids such as acetic acid are produced. As a result, the pH decreases in the liquid medium.
Concerning sporulation, the shift from vegetative to sporulation stage occurs when the nutrients are depleted. In this case, the acids produced during the vegetative stage are already oxidized and the cells oxygen demand is very low. Therefore, the pH remains constant.
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REFERENCES |
1: Abel Ghany, A.A. and E.A. Zaki, 2003. DNA sequences of RAPD fragments in the Egyptian cotton Gossypium barbadense. Afr. J. Biotechnol., 2: 129-132. Direct Link |
2: Asaka, O. and M. Shoda, 1996. Biocontrol of Rhizoctonia solani damping-off of tomato with Bacillus subtilis RB14. Applied Environ. Microbiol., 62: 4081-4085. Direct Link |
3: Ash, C., J.A. Farrow, M. Dorsch, E. Stackebrandt and M.D. Collins, 1991. Comparative analysis of Bacillus anthracis, Bacillus cereus and related species on the basis of reverse transcriptase sequencing of 16S rRNA. Int. J. Syst. Bacteriol., 41: 343-346.
4: Ash, C. and M.D. Collins, 1992. Comparative analysis of 23S ribosomal RNA gene sequences of Bacillus anthracis and emetic Bacillus cereus determined by PCR-direct sequencing. FEMS Microbiol. Lett., 94: 75-80.
5: Avignone-Rossa, C. and C.F. Mignone, 1995. Bacillus thuringiensis growth and toxicity. Basic and applied considerations. Mol. Biotechnol., 4: 55-71.
6: Brzostowicz, P.C., K.L. Gibson, S.M. Thomas, M.S. Blasko and P.E. Rouviere, 2000. Simultaneous identification of two cyclohexanone oxidation genes from an environmental Brevibacterium isolate using mRNA differential display. J. Bacteriol., 182: 4241-4248. Direct Link |
7: Chia, J., Y. Lee, P. Huang and J. Chen, 2001. Identification of stress-responsive genes in Streptococcus mutans by differential display reverse transcription-PCR. Infection Immunity, 69: 2493-2501. Direct Link |
8: Chin, L.S., S.K. Singh, Q. Wang and S.F. Murray, 2000. Identification of Okadaic-acid-induced genes by mRNA differential display in glioma cells. J. Biomed. Sci., 7: 152-159. Direct Link |
9: Chitarra, G.S., P. Breeuwer, M.J. Nout, A.C. van Aelst , F.M. Rombouts and T. Abee, 2003. An antifungal compound produced by Bacillus subtilis YM 10-20 inhibits germination of Penicillium roqueforti conidiospores. J. Appl. Microbiol., 94: 159-166. Direct Link |
10: Collee, J.G., A.G. Fraser, B.P. Marmion and A. Simmons, 1996. Practical Medical Microbiology. 14th Edn., Churchill Livingstone, New York, pp: 131-149
11: Drake, M.A., C.L. Small, K.D. Spence and B.G. Swanson, 1996. Differentiation of Lactobacillus helveticus strains using molecular typing methods. Food Res. Int., 29: 451-455. Direct Link |
12: Fislage, R., M. Berceanu, Y. Humboldt, M. Wendt and H. Oberender, 1997. Primer design for a prokaryotic differential display RT-PCR. Nucl. Acids Res., 25: 1845-1850.
13: Gaviria, R.A.M. and F.G. Priest, 2003. Molecular typing of Bacillus thuringiensis serovars by RAPD-PCR. Syst. Applied Microbiol., 26: 254-261. Direct Link |
14: Green, D.H., P.R. Wakeley, A. Page, A. Barnes and L. Baccigalupi et al., 1999. Bacillus probiotics. Applied Environ. Microbiol., 65: 4288-4291.
15: Hilton, A.C., J.G. Banks and C.W. Penn, 1997. Optimization of RAPD for fingerprinting Salmonella. Lett. Applied Microbiol., 24: 243-284. CrossRef | PubMed | Direct Link |
16: Istock, C.A., N. Ferguson, N.L. Istock and K.E. Duncan, 2001. Geographical diversity of genomic lineages in Bacillus subtilis (Ehrenberg) Cohn sensu lato. Org. Divers. Evol., 1: 179-191. Direct Link |
17: Levy, H., M. Fisher, N. Ariel, Z. Altboum and D. Kobiler, 2006. Identification of strain specific markers in Bacillus anthracis by random amplification of polymorphic DNA. FEMS Microbiol. Lett., 244: 199-205. Direct Link |
18: Liang, P. and A.B. Pardee, 1992. Differential display of eukaryotic messenger RNA by means of the polymerase chain reaction. Science, 257: 967-971. CrossRef | PubMed | Direct Link |
19: Liu, W.M., R. Bajpai and V. Bihari, 1994. High-density cultivation of sporeformers. Ann. New York Acad. Sci., 721: 310-325.
20: Luna, C.L., R.L.R. Mariano and A.M. Souto-Major, 2002. Production of a biocontrol agent for crucifers black rot disease. Brazilian J. Chem. Eng., 19: 133-140. Direct Link |
21: Nilsson, J., B. Svensson, K. Ekelund and A. Christiansson, 1998. A RAPD-PCR method for large-scale typing of Bacillus cereus. Lett. Applied Microbiol., 27: 168-172. Direct Link |
22: Sacchi, C.T., A.M. Whitney, L.W. Mayer, R. Morey and A. Steigerwalt et al., 2002. Sequencing of 16S rRNA gene: A rapid tool for identification of Bacillus anthracis. Emerg. Infect. Dis., 8: 1117-1123. Direct Link |
23: Sambrook, J., E.F. Fritsch and T.A. Maniatis, 1989. Molecular Cloning: A Laboratory Manual. 2nd Edn., Cold Spring Harbor Laboratory Press, Cold Spring Harbor, New York, USA., ISBN-13: 9780879695774, Pages: 397 Direct Link |
24: Shuler, M.L. and F. Kargi, 1992. Bioprocess Engineering: Basic Concepts. 1st Edn., Prentice Hall, Englewood Cliffs, New Jersey, pp: 154-158
25: Siefert, J.L., M. Larios-Sanz, L.K. Nakamura, R.A. Slepecky and J.H. Paul et al., 2000. Phylogeny of marine Bacillus isolates from the gulf of Mexico. Curr. Microbiol., 41: 84-88. Direct Link |
26: Sneath, P.H.A., N.S. Mair, M.E. Sharpe and J.G. Holt, 1986. Bergey's Manual of Systematic Bacteriology. 1st Edn., Williams and Wilkins, Baltimore, USA., pp: 1104-1139
27: Sonnleitner, B., 1998. Dynamic adaptation of microbes. J. Biotechnol., 65: 47-60.
28: Sow, D.M., R.D. Dauphin, D. Roblain, A.T. Guiro and P. Thonart, 2005. Polyphasic identification of a new thermotolerant species of lactic acid bacteria isolated from chicken faeces. Afr. J. Biotechnol., 4: 409-421. Direct Link |
29: Toure, Y., M. Ongena, P. Jacques, A. Guiro and P. Thonart, 2004. Role of lipopeptides produced by Bacillus subtilis GA1 in the reduction of grey mould disease caused by Botrytis cinerea on apple. J. Applied Microbiol., 96: 1151-1160. Direct Link |
30: Williams, J.G.K., A.R. Kubelik, K.J. Livak, J.A. Rafalski and S.V. Tingey, 1990. DNA polymorphisms amplified by arbitrary primers are useful as genetic markers. Nucleic Acids Res., 18: 6531-6535. CrossRef | PubMed | Direct Link |
31: Wirtz, K.W., 2002. A generic model for changes in microbial kinetic coefficients. J. Biotechnol., 97: 147-162. Direct Link |
32: Matar, S.M., S.A. El-Kazzaz, E.E. Wagih, A.I. El-Diwany, M.A. El-Saadani, H.E. Abd-Elsalam, E.E. Hafez, H.E. Moustafa, G.A. Abo-Zaid and E.A. Serour, 2008. Antagonistic and inhibitory effect of Bacillus subtilis against certain plant pathogenic fungi, I. Biotechnology, (In Press).
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