Download results and discussion discussion

RESULTS
RESULTS AND
DISCUSSION
Results and discussion
4.0 RESULTS AND DISCUSSION
4.1 ISOLATION OF METAGENOMIC DNA
Water from a small pond in University of Delhi campus was collected. The surface of the
water was covered with algal mass, which was avoided during collection of the water
sample. The water appeared greenish-brown in colour and had a pH of 8.5. Microscopic
observation revealed presence of bacterial, cyanobacterial, filamentous algal cells, and few
possible members of invertebrate groups. The pond water metagenomic DNA was isolated
by the in situ lysis method as described earlier (Zhou et al., 1996), with some minor
modifications. The metagenomic DNA obtained after initial procedure was resistant to
digestion with restriction enzymes. It was further purified by CTAB and readily digestible
pure DNA was obtained. CTAB is known to remove polysaccharides and humic acids
(Zhou et al., 1996; Ausubel et al., 2001). This indicated that the sample contained high
amount of these impurities, which were not removed completely by the CTAB present in
extraction buffer used during the initial lysis steps.
Fig. 4. 1: Isolation of metagenomic DNA from pond microbial community.
Lane 1: λ mix-19 marker, Lane 2: Pond metagenomic DNA
57
Results and discussion
The size of the recovered DNA from pond microbial community sample was more then 48
kb (Fig.4.1). The DNA could be digested with restriction enzymes to carry out partial
digestion and construction of metagenomic library.
4.2 PREPARATION OF METAGENOMIC LIBRARY
Metagenomes are virtually inexhaustible reservoirs for the recovery of novel and
potentially economically valuable genes. Various technologies have been developed to
target specific genes within environmental samples. One of the earliest and still most
popular approaches is activity based screening of metagenomic DNA libraries. Most
reports describe the construction of small insert DNA libraries (2-10 kbp) and the
subsequent recovery of various novel genes (Cottrell et al., 1999; Henne et al., 1999;
Henne et al., 2000). Other workers constructed large insert libraries that could be used to
identify entire metabolic pathways and other biomolecules (Brady et al., 2001; Gillespie et
al., 2002; Rondon et al., 2000).
The recovered metagenomic DNA from pond microbial community sample was partially
digested by using Sau3AӀ and fragments in range from 2-12 kb were excised and purified
(Fig. 4.2). The partially digested DNA was cloned into cloning vector pUC19 and the
library was maintained in E. coli DH10B. Multiple ligations and transformations were
done to increase the number of clones in the metagenomic library.
Pond water metagenomic library contained
more then 1, 40,000 recombinant colonies.
More than 90% of the colonies in the library were recombinant. Analysis of the insert
fragments generated by EcoRI and HindIII restriction digestion of 40 recombinant
plasmids was used to estimate an average insert size of ~3.8 kb. The metagenomic library
represented about 532 Mb of the pond water microbial community DNA.
58
Results and discussion
Fig. 4.2: Partial digestion of pond metagenomic DNA.
Lane1: Undigested pond metagenomic DNA, Lane 2-7: Different stages of
partial digestion after 10, 20, 30, 40, 50, and 60 min respectively, Lane 8: λ
BamHI/ EcoRI marker.
4.3 SCREENING OF METAGENOMIC LIBRARY FOR SALT TOLERANT
CLONES
Gene enrichment strategies have been used to target the active salt tolerance clones from
pond water microbial community. Enriched in LB ampicillin broth and 4%, 5% or 6%
NaCl, the amplified pond water metagenomic library clones were selected on agar plates
containing the same solid medium. Randomly, ten colonies were picked from each plate
after 72 h incubation at 37 °C. These selected colonies were re-streaked with the help of
sterile tooth pick into LB containing ampicillin agar plates. The plasmids of the colonies
there by obtained were isolated, which were further screened for unique insert carrying
plasmids, through restriction digestion analysis.
59
Results and discussion
4.4 IDENTIFICATION OF UNIQUE SALT TOLERANCE CLONES
All the selected salt tolerance clones plasmids were digested with restriction enzymes
EcoRI and HindIII. Those showing unique digestion pattern were one from 5% NaCl
containing flask designated as p5B2 and two from 6% NaCl containing flask designated
as, p6S2 and p6B4. Restriction analysis of p5B2, p6S2 and p6B4 revealed that they
contained insert of ~2.6 kb, ~4.0 kb and ~2.3 kb, respectively (Fig. 4.4.1). These unique
digestion pattern plasmids having different insert size and were retransformed into E. coli
DH10B host for further study. The selected plasmids clones growth pattern were checked
into LB ampicillin and 750 mM NaCl/KCl and 400 mM LiCl containing LB ampicillin
medium. All the colonies grew in this medium indicating plasmid borne nature of salt
tolerance, the control strain with pUC19 failed to grow under same conditions (Fig. 4.4.2,
4.4.3, 4.4.4, and 4.4.5).
Fig. 4.4.1: Restriction digestion of plasmids of unique salt tolerant clones
from pond water metagenomic library.
Lane 1: 1kb marker, Lane 2: p5B2, Lane 3: p6S2, Lane 4: p6B4.
60
Results and discussion
Fig. 4.4.2: Growth pattern of salt tolerant clones in LB ampicillin medium.
(●) E. coli DH10B/ (pUC19), (○) E. coli DH10B/p5B2, (▼) E. coli DH10B/p6S2, (∇) E.
coliDH10B/p6B4. Experiments were performed in triplicates, values are averages and
standard deviations are shown as error bars.
61
Results and discussion
Fig. 4.4.3: Growth pattern of salt tolerant clones in LB ampicillin medium
containing 750 mM NaCl.
(●) E. coli DH10B/ (pUC19), (○) E. coli DH10B/p5B2, (▼) E. coli DH10B /p6S2, (∇) E. coli
DH10B/p6B4. Experiments were performed in triplicates, values are averages and standard
deviations are shown as error bars.
62
Results and discussion
Fig. 4.4.4: Growth pattern of salt tolerant clones in LB ampicillin medium
containing 750 mM KCl.
(●) E. coli DH10B /(pUC19), (○) E. coli DH10B/p5B2, (▼) E. coli DH10B/p6S2, (∇) E. coli
DH10B/p6B4. Experiments were performed in triplicates, values are averages and standard
deviations are shown as error bars.
63
Results and discussion
Fig. 4.4.5: Growth pattern of salt tolerance clones in LB ampicillin medium
containing 400 mM LiCl.
(●) E. coli DH10B/(pUC19), (○) E. coli DH10B/p5B2, (▼) E. coli DH10B/p6S2, (∇) E. coli
DH10B/p6B4, Experiments were performed in triplicates, values are averages and standard
deviations are shown as error bars.
64
Results and discussion
The growth rate of clones and control in LB containing ampicillin medium was different
i.e. clone 5B2 and 6S2 growth rate was double as compared to the clone 6B4 and control
E. coli/pUC19 (Fig. 4.4.2). Similarly, growth rate of the clones and control with the
different salt stress condition such as NaCl, KCl and LiCl showed different pattern. In case
of 750 mM NaCl/KCl clone 5B2 growth rate was slightly more as compare to the clone
6S2 and 6B4, where as the control does not support growth in high concentration of salt
(Fig. 4.4.3 and 4.4.4). Since, all the clones in presence of NaCl/KCl salt medium grew
normally, where as in case of 400 mM LiCl, 5B2 and 6S2 clones showed normal growth
as similar to NaCl/KCl salt medium, but the clone 6B4 growth rate was similar to the
control (Fig. 4.4.5).
The physicochemical basis for this striking salt stress tolerance is not fully understood.
When bacteria are subjected to a sudden shift in one or several parameters affecting their
growth or survival, a program of gene expression is initiated, which is manifested as an
increased or decreased amount of set of proteins synthesized in response to stress. For
example, in Bacillus subtilis, salt stress strongly stimulates the expression of a set of
proteins that probably allow the bacterium to survive in the rapidly changing environment.
Salt also down regulates the expression of other proteins which do not appears to be
necessary for survival (Kunst and Rapoport 1995; Gerth et al., 1998). In case of
Lysteria
monocytogenes, it has been shown that the microorganism responds to elevated osmolarity
in the environment by the intracellular accumulation of compatible solutes, called
osmolytes, through osmotic activation of their transport from the medium rather than
through de novo synthesis. The osmolytes act in the cytosole by counter balancing the
external osmolarity, thus preventing water loss from the cell and plasmolysis without
adversely affecting macromolecular structure and function (Csonka and Hanson, 1991).
65
Results and discussion
4.5 SEQUENCING OF THE GENES CONFERRING SALT TOLERANCE
4.5.1 Transposon mutagenesis and mapping
DNA transposition is an important biological phenomenon that mediates genome rearrangements, inheritance of antibiotic resistance determinants, and integration of
retroviral DNA. Transposition has also become a powerful tool in genetic analysis, with
applications in creating insertional knockout mutations, generating gene, operon fusions to
reporter functions, providing physical or genetic landmarks for the cloning of adjacent
DNAs, and locating primer binding sites for DNA sequence analysis.
Plasmids of three unique salt tolerance clones were mutated by using an in-vitro Tranposon
mutagenesis kit (Template Generation System (TGS) Finnzyme, Finland). Mutated
plasmids were electroporated in E. coli DH10B and transformants were plated on LB
ampicillin/kanamycin agar plates. Resulting clones were patched on LB ampicillin
/kanamycin and replica of LB ampicillin/kanamycin containing 750 mM NaCl agar plates,
incubated at 37 °C. After overnight incubation, some of the mutants were not showing
growth on LB ampicillin/kanamycin containing 750 mM NaCl agar plates. This clearly
indicates the disruption in the salt tolerance gene or regulatory region. Six salt sensitive
insertional mutants were obtained for plasmid p5B2 out of the 400 mutants screened,
where nine mutants from p6S2 out of 900 and approximately 1000 screened for p6B4 but
didn’t find any significant result, but consider five positive and nine negative mutants
according to growth (Table 4.5.1). Plasmids of the negative as well as positive salt
tolerance mutants were isolated, to determine the site of transposon insertion by PCR
amplification. On the basis of PCR insertion map distance of 400-500 bps of insertion of
mutants were sequenced and double stranded sequence was obtained by designing the
primers manually.
66
Results and discussion
Table: 4.5.1 Screened transposon mutants of salt tolerant clones.
Clones
Screened
5B2
6S2
6B4
400
900
1000
Negative
Mutants
6
9
9
Negative
Sequenced
Mutants
6
9
9
Positive
Sequenced
Mutants
14
5
5
Total
Sequenced
Mutants
20
14
14
Fig. 4.5.1: Growth analysis of transposon mutants of p5B2 in LB and LB containing
750 mM NaCl.
The transposon mutants of 5B2 and control 6S2 were grown in LB ampicillin (kanamycin
for mtants) medium containing 750 mM of NaCl. The growth was monitored after 24 h of
incubation at 37 °C, 200 rpm by measuring absorbance at 600 nm. The mutants were
grown in standard LB ampicillin/kanamycin medium as a control. Experiments were
performed in triplicates; values are shown in average.
67
Results and discussion
Fig. 4.5.2: Growth analysis of transposon mutants of p6S2 in LB and LB containing
750 mM NaCl.
The transposon mutants of 6S2 and control 6S2 were grown in LB ampicillin (kanamycin
for mutants) medium containing 750 mM of NaCl. The growth was monitored after 24 h of
incubation at 37 °C, 200 rpm by measuring absorbance at 600 nm. The mutants were
grown in standard LB ampicillin/kanamycin medium as a control. Experiments were
performed in triplicates; values are shown in average.
68
Results and discussion
Fig. 4.5.3: Growth analysis of transposon mutants of p6B4 in LB and LB containing
750 mM NaCl.
The transposon mutants of 6B4 and control 6B4 were grown in LB ampicillin (kanamycin
for mutants) medium containing 750 mM of NaCl. The growth was monitored after 24 h of
incubation at 37°C, 200 rpm by measuring absorbance at 600 nm. The mutants were grown
in standard LB ampicillin/kanamycin medium as a control. Experiments were performed in
triplicates; values are shown in average.
69
Results and discussion
The selected negative as well as positive mutants were checked into 750 mM NaCl
containing LB ampicillin/kanamycin medium shown as bar graphs (Fig 4.5.1, 4.5.2 and
4.5.3). The negative mutants were not showed any growth as compare to the positive
mutants of 5B2 and 6S2. Which are indicating insertions of transposon into regulatory
region of the salt tolerance gene (Fig. 4.5.4). Where, the transposon mutant of 6B4 clone
didn’t show growth as compare to the original clone. Therefore transposon mutants of 6B4
clones in 750 mM NaCl containing LB ampicillin/kanamycin medium shown growth
nearer to or above to 0.5 optical density at 600nm consider as positive and the mutants
having similar growth as control E. coli DH10B/pUC19 consider as negative.
4.6 BIOINFORMATIC ANALYSIS
4.6.1 Sequence analysis of the clones
A 2593 bp inserts of p5B2 DNA sequence with G+C composition of 59.58% was obtained.
The search using nucleotide BLAST did not result in any significant match for the DNA
sequence obtained from p5B2 (Table 4.6.1). Mapping and sequencing of the mutants
revealed that the transposons were located in four different ORFs i.e. Hypothetical protein
Msil_1796, Two-component response regulator, and Hypothetical protein and ECF
subfamily RNA polymerase sigma-24 factor. Only hypothetical protein Msil_1796 region
showed negative mutants and rest of the protein gene regions showed positive mutants.
Two-component response regulator encoding 274 amino acids with 64% identity with
Methylocella silvestris BL2. It had two complete domains of REC signal receiver
(cd00156), and RpoE DNA directed RNA polymerase specialized sigma subunit, sigma24
homolog (COG1595). many truncated domains AtoC, response regulator containing CheYlike receiver (COG2204), CitB, response regulator of citrate/malate (COG4565), response
regulator containing CheY-like receiver (COG4753),
70
Results and discussion
p5B2
EcoRI
Hypothetical protein
(GspM)
SalI HindIII
SalI
SalI
Hypothetical
Protein
Response regulator receiver protein
RNA polymerase
sigma-70 factor
pRK1
p6S2
PstI
Acetyl-CoA acetyltransferase
AMP-dependent synthetase and ligase
pRK3
71
PstI
Enoyl-CoA hydratase/isomerase
(EchM)
Results and discussion
p6B4
HindIII
EcoRI
Poly-3-hydroxybutyrate
synthase
ATP-dependent Clp
Permease YjgP/YjgQ
Protease adaptor protein (ClpS)
Phasin protein
……… LacZ
Fig. 4.5.4: Schematic representation of the genetic organization of the salt tolerant clones p5B2 p6S2 and p6B4, with respect to pUC19
lacZ promoter.
Vertical arrows with round head represent the positive transposon mutant and with triangular head represent negative transposon mutant
(numbers indicate the mutant number). A bold arrow with pointed head represents a complete ORF and with diamond head represents a
truncated.
72
Results and discussion
LytT, response regulator of the LytR/AlgR (COG3279), response regulator containing
CheY-like receiver and SARP (COG3947), CheB, chemotaxis response regulator
containing a CheY-like receiver and a methylesterase ( COG2201), CitB, response
regulator containing a CheY-like receiver and an HTH DNA-binding (COG2197) protein
domain, OmpR, response regulators consisting of a CheY-like receiver domain and a
winged-helix DNA-binding (COG0745), AmiR, response regulator with putative anti
terminator output (COG3707), response regulator containing a CheY-like receiver domain
and an HD-GYP (COG3437). Another ORF is hypothetical protein encoding 149 amino
acid with 69% identity with Beijerinckia indica subsp. indica ATCC 9039. The last
positive mutant insertion at the position of ECF subfamily RNA polymerase sigma-24
factor protein encoding 190 amino acids and 75% identity with Beijerinckia indica subsp.
indica ATCC 9039. It had three complete domains Sigma-70; region 2 (pfam04542),
Sigma-70; region 4 (pfam04545), RpoE DNA-directed RNA polymerase specialized sigma
subunit, sigma 24 homolog (COG1595) and a truncated domain of RNA polymerase sigma
factor (COG4941).
Mapping and sequencing of negative mutants revealed that the transposons were located in
an ORF encoding for 94 amino acids long protein in five of them indicated that disruption
of structural gene of this ORF was responsible for the salt sensitive phenotype of the
mutants (Fig. 4.5.4). The encoded protein of about 10 kDa showed best match with a
hypothetical protein of Methylocella silvestris BL2 with 64% amino acid identity. It had a
complete domain of general stress protein family GsiB (COG3729). Many of the other
matches were annotated as general stress proteins in the database. We designated the gene
gspM because of its similarity with genes encoding for putative general stress proteins and
metagenomic origin.
73
Results and discussion
Inter Pro Scan search revealed similarity of GspM with members of Family PD027049
(ProDom release 2005.1). The most well characterized member of this family is glucose
starvation induced protein (GsiB) of Bacillus subtilis, which is a hydrophilic protein of 123
amino acids and is composed almost entirely of five repeating motifs (Stacy and Aalen,
1998). Sequence analysis of GspM by radar repeat finder revealed that it was not formed of
perfect repeats similar to GsiB but a motif R(K/T)GG was found repeated three time in
GspM (Fig.4.6.2). Sequence alignment of GspM and its homologues with proteins from
GsiB and PD027049 families revealed significant difference between these proteins and
GspM (Fig.4.6.3). Phylogenetic analysis revealed that GspM and its homologues formed a
distinct clade separate from the clades formed by GsiB, YciG or LEA homologues (Fig.
4.6.1). GspM grouped with homologous proteins from alpha-proteobacteria indicating its
possible origin from an alpha-proteobacteria of pond water sample.
The DNA sequences of p6S2 as obtained by transposon mutagenesis and primer walking.
The p6S2 of insert having DNA sequence of 4019 bp with a G+C composition of 63.97%
was obtained. The search using nucleotide BLAST showed some significant match with
the DNA sequence. Mapping and sequencing of the mutants revealed that the transposons
were located into three different ORFs (Fig. 4.5.4). The DNA sequence from p6S2 resulted
in significant match in the coding regions with Rhodoferax ferrireducens DSM15236 with
nucleotide identity ranging from 81-92% and with Polaromonas sp. JS666 with nucleotide
identity ranging from 81-90%. ORFs encoding enoyl-CoA hydratase/isomerase, feruloylCoA synthase and acetyl-CoA acetyltransferase with amino acid identity ranging from
77% to 84% were identified from p6S2.
The Tn-mutant of feruloyl-CoA synthase and acetyl-CoA acetyltransferase grew normal,
but Tn insertion in enoyl-CoA hydratase resulted in negative phenotype.
74
Results and discussion
GsiB
Lsa
LEA
Ghi
Hvu
Bcl
Bli
Bsu
Ath
Pae2
YciG
Pae1
Eco2
Afu
Ani
Eco1
Aor
Cgl
Con-10
Mma
Ncr
Ccr
Afu1
GspM
AvaNos
Xca
Rru2
Nmu
Xau
Rru1
Psp
GspM
Fig. 4.6.1: Phylogenetic analysis of GspM and related proteins.
GspM: GspM from p5B2 (EF611421)), Ava: Anabaena variabilis ATCC 29413:
YP_320833, Nos: Nostoc sp. PCC 7120: NP_485758, Xca: Xanthomonas campestris pv.
campestris ATCC 33913: NP_638171, Nmu: Nitrosospira multiformis ATCC 25196:
YP_412183, Psp: Polaromonas sp. JS666: YP_548573, Rru1: Rhodospirillum rubrum
ATCC 11170: YP_428101, Rru2: Rhodospirillum rubrum ATCC 11170: YP_428100,
Xau: Xanthobacter autotrophicus Py2: ZP_01200453, Ccr: Caulobacter crescentus CB15:
NP_419994, Mma: Methanoculleus marisnigri JR1: ZP_01392511, Eco1: Escherichia coli
K12: NP_415525, Eco2: Escherichia coli K12: NP_415775, Pae1: Pseudomonas
aeruginosa C3719: ZP_00969497, Pae2: Pseudomonas aeruginosa PAO1: AAG05578,
Bsu: Bacillus subtilis: P26907, Bli: Bacillus licheniformis ATCC 14580: YP_077772, Bcl:
Bacillus clausii KSM-K16: YP_177001, Lsa: Lactobacillus sakei: BAC99042, Hvu:
Hordeum vulgare: Q05191, Ghi: Gossypium hirsutum: AAB00728, Ath: Arabidopsis
thaliana: AAO63884, Afu: Aspergillus fumigatus Af293: XP_747756, Ani: Aspergillus
nidulans FGSC A4: XP_662619, Aor: Aspergillus oryzae: BAE65121, Cgl: Chaetomium
globosum CBS 148.51: EAQ87406, Ncr: Neurospora crassa: P10713, Afu1: Aspergillus
fumigatus Af293: XP_747961.
75
Results and discussion
Bacillus subtilis: GsiB: 123 aa
MADNNKMSREEAGR
KGGETTSKNHDKEFYQEIG
KGGEATSKNHDKEFYQEIG
KGGEATSKNHDKEFYQEIG
KGGEATSENHDKEFYQEIG
KGGEATSKNHDKEFYQEIG
Q
E
E
R
S
KGGNARNND
5B2: GspM: 93 aa
MTEQKKSKRGFASMDPEKRREIA
RKGGLSVPDEKRIFSKNPDLAARAG
RTGGKNVKPANRSFARDPALAAAAG
RKGGQASPRVERVQVIDAAE
Fig. 4.6.2: Repeat analysis of GsiB and GspM using radar repeat finder.
GspM
YciG
Con-10
GsiB
LEA
--------------------------------------MTEQ-KKSKRGFASMDPEKRRE
--------------------------------------MAEH-RGGSGNFAE-DREKASD
--------------------------------------MAGTGNDNPGNFANRPKEEVQA
MADNNKMSREE----------------------------AGR-KGGETTSKNHDKEFYQE
MAS-KQLSREELDEKAKQGETVVPGGTGGHSLEAQEHLAEGRSKGGQTRKEQLGHEGYQE
. .
.
*
21
20
22
31
59
GspM
YciG
Con-10
GsiB
LEA
IARKGGLSV---------------PDEKRIFSKNPDLAARAGRTGGKNVKPANRSFARDP
AGRKGGQHS---------------GGN---FKNDPQRASEAGKKGGQ------------IASKGGQAS---------------HSG-GFASMDPEKQREIASKGGK---ASSGSFEPGS
IGQKGGEATSKNHDKEFYQEIGEKGGEATSKNHDKEFYQEIGEKGGE-----ATSENHDK
IGHKGGEARKEQLGHEGYQEMGHKGGEARKEQLGHEGYQEMGHKGGE-----ARKEQLGH
. ***
.
. . :
. . .**:
66
49
63
86
114
GspM
YciG
Con-10
GsiB
LEA
ALAAAAGRKGGQASPRVERVQVIDAAE-------------QSGGNKSGKS------------------------EKAREAGRKGGKASGGTG----ADDDE----------EFYQEIGRKGGEATSKNHDKEFYQEIGSKGGNARNNDEGYKEMGRKGGLSTMEKSGGERAEEEGIEIDESKFTNK
*.*.* :
93
59
86
123
152
Fig. 4.6.3: Sequence alignment of general stress tolerance protein.
(Accession numbers given in parenthesis): GspM: GspM from p5B2 (EF611421), YciG:
Escherichia coli K12 (NP_415775), Con-10: Neurospora crassa (P10713), GsiB: Bacillus
subtilis (P26907), LEA: Arabidopsis thaliana (AAO63884).
76
Results and discussion
It proves that the enoyl CoA hydratase was responsible for salt stress tolerance in
metagenome clone. The feruloyl-CoA synthase (YP_981036) encoding 618 amino acids
having 77% amino acid identity with Polaromonas naphthalenivorans CJ2. It had a
complete domain of superfamily acyl-protein synthetase LuxE (cl10450) LuxE is an acylprotein synthetase found in bioluminescent bacteria. LuxE catalyses, formation of an acylprotein thioester from a fatty acid and protein. Other domains CaiC acyl-CoA synthetases
(AMP-forming)/AMP-acid ligases II (COG0318), AMP binding enzymes (pfam00501),
Acs acyl-coenzyme A synthetases (COG0365) , EntF non-ribosomal peptide synthetase
modules and related proteins (COG1020). Where as acetyl-CoA acetyltransferase
(YP_981037) encoding 416 amino acid with the 84% identities with Polaromonas
naphthalenivorans CJ2.
It had one complete domain Thiolase, N-terminal (pfam00108) and then truncated domain
Thiolase (cd00751), Non decarboxylating condensing enzyme (cd00826), SCP- X_thiolase
(cd00829), Condensing enzymes (cd00327), Ketoacyl-acyl carrier protein synthase III
(cd00830), Initial condensing enzymes (cd00827), Beta-ketoacyl-acyl carrier protein
(ACP) synthase (KAS), type I and II (cd00834).
The transposon was located in a gene encoding for a protein of 294 amino acids in nine of
these mutants and 230 bp upstream of this gene in one mutant. The encoded protein of
about 31kDa showed best match to a putative enoyl-CoA hydratase/isomerase from
Polaromonas sp. JS666 with 77% amino acid identity. The gene was designated as echM
because of its similarity with enoyl-CoA hydratase and metagenomic origin.
EchM
contained conserved domains for enoyl-CoA hydratase/isomerase (pfam00378, ECH),
enoyl-CoA hydratase/carnithine racemase (COG1024, CaiD) and dihydroxynapthoic acid
synthase
(COG0447,
menB).
Prosite search
77
revealed
presence
of enoyl-CoA
Results and discussion
hydratase/isomerase signature pattern [LIVM]-[STAG]-X-[LIVM]-[DENQRHSTA]-GX(3)-[AG](3)-X(4)-[LIVMST]-X-[CSTA]-[DQHP]-[LIVMFYA] between position 134 to
154 amino acid of the EchM as IAaLHGavvGGGlelaSaSHI. Alignment of the EchM with
enoyl-CoA hydratase from rat mitochondria (Hofstein el al., 1999; Bell et al, 2001)
showed presence of conserved residues G144, E147 and E167 in EchM corresponding to rat
ECH catalytic residues G141, E144 and E164 (Fig. 4.6.5).
Sequence similarity analysis with BLAST two sequences between EchM and characterized
and putative enoyl-CoA hydratases from E. coli, YgfG, YfcX/FadJ, PaaG, PaaF, MenB,
CaiD, FadB (Yang et.al,1991) and MaoC (Park and lee 2003) revealed low sequence
identity of EchM with E. coli proteins. Amino acid identity between EchM and the E. coli
proteins ranged from 24-35% with maximum of 35% between EchM and FadJ.
Phylogenetic analysis was carried out for EchM, various E. coli enoyl-CoA hydratase
proteins and their homologues. EchM with its homologues formed a distinct clade separate
from the clades containing E. coli proteins and their homologs (Fig. 4.6.4).
The p6B4 insert of 2254 bp had a G+C composition of 64.06%. The search using
nucleotide BLAST did not result in any significant match for the DNA sequence obtained
from p6B4. The encoded proteins with similarity amino acid identity ranging between 57%
and 81% were identifying from p6B4. Mapping and sequencing of the mutants revealed
that the transposons were located in a four different ORFs i.e. poly-3-hydroxybutyrate
synthase (Erythrobacter sp. SD-21), ATP-dependent Clp protease adaptor protein ClpS
(Erythrobacter sp. NAP1), permease YjgP/YjgQ (Novosphingobium aromaticivorans
DSM 12444) and Phasin (Novosphingobium aromaticivorans DSM 12444).
Transposon mutation analysis did not reveal any clear negative mutants hence, it was not
possible to identify gene responsible for salt tolerance from p6B4 (Fig. 4.5.4).
78
Results and discussion
MenB
Msu.c
MenB.c
Asu.c
YgfG
YgfG.g
Mma.g
CaiD
Psp.b
CaiD.b
Gur.g
Rsp.b
Osp.d
Ksp.e
PaaG
PaaF
PaaG.e
PaaF.d
Psp.e
Pin.d
MaoC.h
Bli
EchM
Rfe
MaoC
Osp.h
Psp
Pna
Rpa
Ppu.h
Bsp
EchM
Bja
Eca.f
Oba
Plu.f
Atu
YcfX.f
YfcX
Ecl.a
Rba
FadB.a Eca .a
FadB
Fig. 4.6.4: Phylogenetic affiliation of EchM with other enoyl-CoA hydratases.
EchM: EchM from p6S2 (EF611422), Atu: Agrobacterium tumefaciens str. C58:
NP_354425, Rba: Rhodobacterales bacterium HTCC2654: ZP_01012481, Oba:
Oceanicola batsensis HTCC2597: ZP_00998324, Bja: Bradyrhizobium japonicum USDA
110: NP_774484, Bsp: Bradyrhizobium sp. BTAi1: ZP_00861107, Rpa:
Rhodopseudomonas palustris BisB5: YP_569022, Pna: Polaromonas naphthalenivorans
CJ2: EAQ19525, Psp: Polaromonas sp. JS666: ABE45025, Rfe: Rhodoferax ferrireducens
DSM 15236: ABD68031, Bli: Brevibacterium linens BL2: ZP_00378475, Pin.d:
Psychromonas ingrahamii 37: EAT18296, PaaF.d: Escherichia coli: P76082, Osp.d:
Oceanospirillum sp. MED92: EAR60003, Gur.g: Geobacter uraniumreducens Rf4:
EAR38053, Mma.g: Magnetospirillum magnetotacticum MS-1: ZP_00208575, YgfG.g:
Escherichia coli: P52045, Asu.c: Actinobacillus succinogenes 130Z: EAO50532, Msu.c:
Mannheimia succiniciproducens MBEL55E: YP_088984, MenB.c: Escherichia coli:
P0ABU1, Psp.b: Proteus sp. LE138: Q8GB17, CaiD.b: Escherichia coli: P31551, Rsp.b:
Roseovarius sp. 217: EAQ26258, Ksp.e: Klebsiella sp. PAMU-1.2: BAE02692, PaaG.e:
Escherichia coli: P77467, Psp.e: Pseudomonas sp. Y2: CAD76917, MaoC.h: Escherichia
coli K12: ZP_01167918, Osp.h: Oceanospirillum sp. MED92: NP_415905, Ppu.h:
Pseudomonas putida AAC24340, Eca.f: Erwinia carotovora subsp. atroseptica SCRI1043:
79
Results and discussion
Y P_051168, Plu.f: Photorhabdus luminescens subsp. laumondii TTO1: NP_930429,
YcfX.f: Escherichia coli: P77399, Ecl.a: Enterobacter cloacae: Q9F0Y7, FadB.a:
Escherichia coli: P21177, Eca.a: Erwinia carotovora subsp. atroseptica SCRI1043:
YP_048335.
EchR
EchM
MAALRALLPRACNSLLSPVRCPEFRRFASGANFQYIITE--KKGKNSSVGLIQLNRPKA 57
MQTASSPDNMVIVHNFIDPRTFPGQGPHMSHPLLERAAQNGVTLEMRGAVAVVTLRRPGK 60
:* .
:.::.*
*
. * . ::
: .
..:*.:: *.**
EchR
EchM
LNALCNGLIEELNQALETFEEDPAVGAIVLTGGEKAFAAGADIKEMQNRTFQD--CYSGK 115
RNALSDALIEAIRDTFQNLP--AEARAAVIDGEGEHFCAGLDLSELKERDAGEGVHHSRG 118
***.:.*** :.::::.:
. . * *: * : *.** *:.*:::*
:
:*
EchR
EchM
FLSHWDHITRIKKPVIAAVNGYALGGGCELAMMCDIIYAGEKAQFGQPEILLGTIPGAGG 175
WHVALDAVQFGRVPVIAALHGAVVGGGLELASASHIRVADDSTFYALPEGTRGIFVGGGG 178
:
* :
: *****::* .:*** *** ..* *.:.: :. **
* : *.**
EchR
EchM
TQRLTRAVGKSLAMEMVLTGDRISAQDAKQAGLVSKIFPVETLVEEAIQCAEKIANNSKI 235
SVRIPKLIGVARMTDMMLTGRVYNAVEGERLGFAQYLVPQGTALDKAVELATRIATNAPL 238
: *:.: :* :
:*:***
.* :.:: *:.. :.* * :::*:: * :**.*: :
EchR
EchM
IVAMAKESVNAAFEMTLTEGNKLEKKLFYSTFATDDRREGMSAFVEKRKANFKDH- 290
TNYALMHALPRIAEQPADHGFLTEALMASIAQSAPEAKARVRAFLEGKAAKVKKAD 294
.::
* . .*
* :
: :: : : : **:* : *:.*.
Fig. 4.6.5: Sequence alignment of Enoyl-CoA hydratase.
EchM: EchM from p6S2 # (EF611422), EchR: Rattus norvegicus # (P14604).
Poly-3-hydroxybutyrate synthase encoding 626 amino acids with 63% identity with
Erythrobacter sp. SD-21 and permease YjgP/YjgQ encoding 414 amino acids with 78%
identity with Novosphingobium aromaticivorans DSM 12444. Both having truncated
domains PhaC Poly(3-hydroxyalkanoate) synthetase (COG3243), PHA_synth_I, poly(R)hydroxyalkanoic acid synthase, class I (TIGR01838), and PHA_synth_II, poly(R)hydroxyalkanoic acid synthase, class II (TIGR1839) were from poly-3-hydroxybutyrate
synthase, where as predicted permease YjgP/YjgQ family (pfam03739) and predicted
permeases (COG0795) from permease YjgP/YjgQ. ATP-dependent Clp protease adaptor
protein ClpS and phasin having a complete domain of ClpS (pfam02617) and Phasin_2
(pfam09361) respectively. ATP-dependent Clp protease adaptor protein ClpS encoded 140
amino acid with 81% identity with Erythrobacter sp. NAP1 and phasin encode 288 amino
acids with 57% identity with Novosphingobium aromaticivorans DSM 12444.
80
Results and discussion
Table 4.6.1: Bioinformatics analysis of salt tolerance clones.
S.No
Clone
1
5B2
Double
Strand
2593 bp
GC
(%)
59.58
ORF
(aa)
93
263
97
159
2
6S2
4019 bp
63.97
294
6B4
2254
64.06
Hypothetical protein Msil_1796
(YP_002362103)
Two component response regulator
(YP_002360627)
Hypothetical protein Bind_ 3240
(YP_001831149)
ECF subfamily RNA polymerase sigma24 factor (YC_001834290)
ORF
(aa)
94
Organism
Identity
Methyllocella silvestris BL2
54/79 (68%)
Complete
274
Methyllocella silvestris BL2
170/265 (64%)
Complete
146
Beijerinckia indica subsp.
Indica ATCC 9039
Beijerinckia indica subsp.
Indica ATCC 9039
23/33 (69%)
Complete
108/143 (75%)
Truncated
190
Domain
Enoyl-CoA hydratase/isomerase
(YP_549923)
Feruloyl-CoA synthase (YP_981036)
264
Polaromonas sp. Js666
197/255 (77%)
Complete
618
477/618 (77%)
Complete
279
Acetyl-CoA acetyltransferase
(YP_981037)
416
Polaromonas
naphthalenivorans cj2
Polaromonas
naphthalenivorans cj2
258/306 (84%)
Truncated
133
ATP-dependent Clp protease adaptor
protein ClpS ZP_01040407
Phasin ZP_01864749
140
Erythrobacter sp. NAP
100/122 (81%)
Complete
267
86/149 (57%)
Complete
Poly-3-hydroxybutyrate synthase
ZP_01864750
Permease YjgP/YjgQ
YP_495778
626
Novosphingobium
aromaticivorans DSM 12444
Erythrobacter sp. SD-21
617
3
Match
235
99
121
441
81
Novosphingobium
aromaticivorans DSM 12444
61/96 (63%)
Truncated
84/107 (78%)
Truncated
Results and discussion
4.7 CHARACTERIZATION OF SALT TOLERANT CLONES
Restriction digestion of pRK1 showing fragment of salt tolerant gspM gene (Fig.4.8.1).
The E. coli/pRK1 growth pattern showed similar growth to E. coli/p5B2 in LB ampicillin
and LB ampicillin containing 750 mM NaCl medium (Fig. 4.7.4) confirming that the salt
tolerance was due to gspM. The mapping and sequencing of negative transposon mutants
obtained for plasmid p5B2 results clearly indicate that disruption of structural gene solely
responsible for salt sensitive phenotype. It was also seen that GspM (10 kDa ) showed best
match with a hypothetical protein of Methylocella silvestris BL2 along with 68% amino
acid identities and other matches such as general stress protein present in database. GspM
had a complete domain of general stress protein family GsiB (COG3729) of Bacillus
subtillis. The characteristic of these families are based on its transcription under glucose
starvation (Muller et al., 1992) and later it was found that gsiB transcribed exclusively
from a SigB-dependent promoter in many starvation and stress conditions including salt
stress (Maul et al., 1995). Family PD027049 contain two members from E.coli K12 named
YciG and YmdF. In Salmonella enterica serotype Typhimurium, expression of YciG and
its homologue genes has been shown to be regulated by the stress-induced sigma factor
RpoS (Ibanez-Ruiz et al., 2000; Robbe-Saule at el., 2001). In E. coli, the expression of
yciG has also been found to be regulated by RpoS (Van Dyk et al., 1998). YciG is assigned
to a cluster of orthologous proteins (COG3279), which includes the general stress protein
GsiB from Bacillus subtilis (E = 0.007) (Tatusov et al., 2001). GsiB is involved in an
adaptive non-sporulation response to nutrient deprivations (Mueller et al., 1992) and its
expression is induced by starvation for glucose or phosphate and by heat shock, salt stress,
and oxidative stress (Volker et al., 1994). Late embryogenesis abundant (LEA) proteins
are hydrophilic and vary mainly in the numbers of an extremely hydrophilic internal 20
82
Results and discussion
amino-acid motif. This motif was not only present in numerous plant species but also
stress-related protein (GsiB) from Bacillus subtilis and their role similar to general stress
protein in adverse environmental conditions. However, experimental and bioinformatics
studies revealed GspM protein belongs to stress tolerance protein family, which is induced
under various environmental stress conditions.
Where as, 1277 bp long PstӀ fragment carrying echM gene from 6S2 clone plasmid recloned in pUC19. Due to the single restriction site cloning, orientation of the gene in
pUC19 possibly change, so the plasmids were digested with BamHӀ to obtained small
fragment approximately 500bp and big 900bp, designated as pRK3 and pRK4 respectively
(Fig.4.7.2). The phenotypic and restriction observation of 6S2 sub-clones RK3 indicate
echM fragment having same orientation as in clone 6S2 plasmid i.e upstream of lacZ
promoter of pUC19 and RK4 having opposite orientation of clone 6S2 plasmid i.e.
opposite to lacZ promoter of pUC19 (Fig. 4.7.1). These results conclude echM regulate
with the lacZ promoter of pUC19. Therefore RK3 and RK4 clones growth was checked
into LB ampicillin and LB ampicillin containing 750 mM NaCl medium. RK3 clone
showed normal growth in salt stress condition, where as RK4 didn’t show growth
(Fig.4.7.3). When, E. coli/ pRK3 grown in LB ampicilin medium containing 750 mM NaCl
showed similar growth pattern to E. coli/p6S2 indicate echM gene was responsible for salt
stress tolerance (Fig. 4.7.4). The evolutionary lineage of EchM in comparison to E. coli
enoyl-CoA hydratases indicated that EchM may have different substrate specificity and
may be performing a unique function in as yet uncharacterized pond water bacterium. It
has been also observed that both bacteria and plant can modulate the composition of lipids
as well as fatty acids in their membrane in response of environmental stresses, which
includes temperature, osmotic and salt stress (Rodriguez-Vargas et al., 2007). In these
83
Results and discussion
cases, salt tolerance requires removal and metabolism of unnecessary lipids and fatty acids.
Enoyl-CoA hydratases play important role in β-oxidation pathway of fatty acid
metabolism. Where as, EchM may play a role at a rate limiting step in lipid/fatty acid
degradation during its adaptation towards salt stress in E. coli. EchM in salt stress tolerant
may play another role by carrying out synthesis of a compatible solute such as carnitine.
CaiD, a crotonobetainyl-CoA hydratase was shown to be involved in carnitine synthesis
(Elssner et al., 2001). Therefore, EchM posses complete conserved domain for CaiD and
might be involved in shifting the metabolic flux towards carnitine synthesis.
Fig. 4.7.1: Orientation of echM gene fragment
in pUC19 vector.
Fig. 4.7.2: Restriction analysis showing
orientation of pRK3 and pRK4 clones.
Lane 1- 1 kb Ladder, Lane 2- pRK3,
Lane 3- pRK4
84
Results and discussion
Fig. 4.7.3: Growth of RK 1 - 4 clones in salt medium.
The RK1– 4, clones and a control (E. coli DH10B/pUC19) were grown in LB
ampicillin medium containing 750 mM of NaCl. The growth was monitored after 24
h of incubation at 37 °C, 200 rpm by measuring absorbance at 600nm. The clones
were also grown in standard LB ampicillin medium as a control. Experiments were
performed in duplicate; values are shown in average.
85
Results and discussion
Fig. 4.7.4: Growth pattern of RK clones in LB ampicillin medium containing 750
mM NaCl.
(●) E. coli DH10B/(pUC19), (○) E. coli DH10B/p5B2, (▼) E. coli DH10B /pRK1, (∇) E.
coli DH10B /p6S2, (■) E. coli DH10B /pRK3. Experiments were performed in triplicates,
values are averages and standard deviations are shown as error bars.
86
Results and discussion
4.7.1 GspM and EchM together in response to salt stress
Clone pRK5 and pRK6 inserts orientation have been confirmed with reference to promoter
(Fig. 4.7.5 and 4.7.6). E. coli/pRK6 was expected to express both the proteins, GspM with
native promoter and EchM with lacZ promoter, whereas E. coli/pRK5 was expected to
express only GspM and not EchM. E. coli/pRK6 formed smaller colonies on LB agar
plates and also grew at a slower rate than the other clones and control on LB medium (Fig.
4.7.7). Growth curve analysis of these clones in LB medium with 750 mM NaCl revealed
that E. coli/pRK5 grew similar to E. coli/pRK1 but unexpectedly E. coli/pRK6 grew at a
slower rate than the individual clones, E. coli/pRK1 and E. coli/pRK3 (Fig. 4.7.8). These
results indicated that expression of both the proteins together from high copy number
plasmid caused stress to the E. coli culture. Optimization of appropriate expression of both
the proteins will be required to obtain better salt tolerance from the recombinant clone
expressing both the proteins.
Fig. 4.7.5: Orientation of double salt
tolerance genes containing plasmid.
Fig. 4.7.6: Restriction analysis of
pRK5 and pRK6 by HindIII/XhoI.
Lane1- 1 kb Ladder, Lane 2-pRK5,
Lane 3- pRK6,
87
Results and discussion
Fig. 4.7.7: Growth pattern of RK clones in LB ampicillin medium.
(●) E. coli DH10B/pUC19, (○) E. coli DH10B/pRK1, (▼) E. coli DH10B/pRK3,
(■) E. coli DH10B/pRK5, (∇) E. coli DH10B/pRK6. Experiments were performed in
triplicates, values are averages and standard deviations are shown as error bars.
88
Results and discussion
Fig. 4.7.8: Growth pattern of RK clones in LB ampicillin medium containing
750 mM NaCl.
(●) E. coli DH10B/pUC19, (○) E. coli DH10B/pRK1, (▼) E. coli DH10B/pRK3, (■)
E.coli DH10B/pRK5, (∇) E. coli DH10B/pRK6. Experiments were performed in
triplicates, values are averages and standard deviations are shown as error bars.
89
Results and discussion
4.8 PROMOTER ANALYSIS
Fusion of promoter with a reporter gene has become an important tool for the analysis of
gene regulation in both prokaryotic and eukaryotic systems. In general, the attachment of
the regulatory sites of a given gene upstream of a reporter gene that encodes an easily
assayed enzyme facilitates the analysis of expression of the affixed gene. The reporter gene
may be used in either transcriptional (operon) fusions, where it retains its own translational
start site but is dependent on the attached DNA for transcription, or in translational
(protein) fusions, where both its transcription and translation are dependent on signals in
the attached upstream DNA.
Process such as, mapping and sequencing of p5B2 (gspM) negative mutants indicate that
the transposons were located in an ORF encoding for 94 amino acids long protein and
three in the upstream region of this gene within 60 bp of the start site (Fig. 4.5.4). Which
shows disruption of structural gene or
putative
promoter
region of this ORF was
responsible for the salt sensitive phenotype of the mutants. The gspM gene having its own
promoter was confirmed by cloning into pUC19 and pUC18 vector, this contained multiple
cloning sites (MCS) which were arranged in opposite orientations. These are identical but
the arrangements of multiple cloning sites are opposite, due to this reason orientation of the
gene was reversed automatically, designated as RK1 and RK2 respectively and resultant
plasmids were confirmed by EcoRI and SalI digestion (Fig. 4.8.1). A 24 h bar graph of
clones RK1 and RK2 in LB ampicillin containing 750 mM NaCl medium was constructed,
which confirmed gspM has its own promoter that helped in the expression of gspM in salt
stress condition (Fig.4.7.3). For functional characterization of gspM promoter was cloned
into a low-copy-number, promoter less lacZ fusion vector pTL61T and resultant plasmid
90
Results and discussion
was confirmed by restriction digestion with EcoRI/BamHI having approximately 300 bp of
promoter DNA, designated as pT13 (Fig. 4.8.2).
Fig. 4.8.1: Restriction analysis of pRK1 and pRK2 clone.
Lane 1- 1 kb Ladder, Lane 2- pRK1, Lane 3- pRK2,
This gspM promoter of pT13 construct and promoter less control plasmid pTL61T basal
level study of β -galactosidase activity was done in normal LB ampicillin medium as
well as stress condition of salt and
temperature. However, in general, log and
exponential phase of growth of E. coli/pT13 construct in LB and stress (temperature and
salt) medium, lacZ expression of the construct was gradually increased as the cells growing
but the control has constant background β-galactosidase activity in all conditions. In
stationary phase of LB ampicillin medium clone showed 30,000 ± 5000 more βgalactosidase unit in 9 to 15 h of observation but, the control had only background activity
91
Results and discussion
(Fig 4.8.3). E. coli/pT13 in normal LB ampicillin and LB ampicillin with 200 mM and 400
mM of salt, stress showed similar β-galactosidase activity in exponential phase of growth.
It showed change in lacZ expression i.e, maximum expression achieved by the construct in
limited period of stress condition than the non stress (Fig. 4.8.4). Now, at 42 °C of
temperature stress clone as well as control growth was suddenly discontinued due to heat
shock, but it continues after an hour. It has been observed that β-galactosidase unit of clone
at normal and after heat shock remains same. Finally, it was observed that in both the stress
conditions lacZ gene expression induction by gspM promoter showed maximum βgalactosidase units in exponential period of time but, in simple medium it has been
observed at stationary phase of growth and the control background activity was same as the
normal in all condition (Fig. 4.8.5).
Fig. 4.8.2: Restriction analysis of pT13 clone.
Lane 1- 1 kb Ladder, Lane 2- pT13
92
Results and discussion
Fig. 4.8.3: Regulation of expression of lacZ with the fusion of gspM gene promoter.
Clone E. coli/pT13 and control E. coli/pTL61T cell were grown in LB ampicillin medium
(line graph) and β-galactosidase unit was monitored at various time points along with bar
graph. Experiments were performed in triplicates, values are averages and standard
deviations are shown as error bars.
93
Results and discussion
Fig. 4.8.4: Regulation of gspM gene promoter by salt stress.
Clone E. coli/pT13 and control E. coli/pTL61T cell were grown in LB ampicillin and LB
ampicillin medium containing 200 mM and 400 mM of NaCl. β-galactosidase unit was
monitored after stress at various time points along with growth curve. Experiments were
performed in triplicates values are averages and standard deviations are shown as error
bars.
94
Results and discussion
Fig. 4.8.5: Regulation of gspM gene promoter by heat shock.
Clone E. coli/pT13 and control E. coli/pTL61T cell were grown in LB ampicillin at 42 °C
and β-galactosidase unit was monitored after stress at various time points along with
growth curve. Experiments were performed in triplicates, values are averages and standard
deviations are shown as error bars.
95
Results and discussion
4.9 CLONING, OVEREXPRESSION AND OPTIMIZATION OF gspM and echM
4.9.1 Cloning
The putative salt tolerance gspM from clone 5B2 was amplified by PCR and ligated into
the pET21d expression vector of an NheI and XhoI site, and transformed into strain E. coli
BL21 (DE3) by electroporation. Inserts of resulting plasmid was verified by DNA
sequencing and restriction digestion. The obtained clone was designated as RK16 (Fig:
4.9.1). Putative salt tolerance gene EchM from clone 6S2 have been amplified by PCR and
ligated into pET21d and electroporated into host of E. coli BL21 (DE3). The constructed
plasmid was verified by DNA sequencing and restriction digestion. The obtained clone
was designated a RK17 (Fig. 4.9.1).
Fig. 4.9.1: Cloning of gspM and echM gene in expression vector pET21d.
Lane 1: 1kb Ladder, Lane 2:pRK-16,Lane 3: pRK-17
96
Results and discussion
4.9.2 Overxpression and optimization of protein
Both the clones RK16 (GspM, protein size approx 10 kDa) and RK17 (EchM, protein size
approx 31kDa) were used for protein over expression. The expression of clones RK16 and
RK17 have been optimized by using different concentration of IPTG as inducer, with
respect to time along with growth condition of 37 °C at 200 rpm. GspM over-expression
and optimization was carried out by addition of 5.0 µM or 50 µM of IPTG (isopropyl-Dthiogalactopyranoside) and incubated for 2 h or 4 h in order to obtain the highest amount of
soluble protein. It has been observed that RK16 yields highest amount of soluble GspM at
5.0 µM of IPTG after 2 h of incubation post induction (Fig. 4.9.2 and 4.9.3). When EchM
was induced at 5.0, 50 and 100 µM of IPTG with the time of 2, 4, and 6 h of incubation
after post induction, it was observed that, at 5.0 µM of IPTG and 2 h after induction was
the best optimization for EchM production (Fig. 4.9.4, 4.9.5 and 4.9.6).
Fig. 4.9.2: Expression of GspM (E. coli BL21 (DE3)/pRK16) after 2 h or 4 h or 6 h
induction with 5 µM IPTG.
Lane1: LMW, Lane 2: Debris (2 h), Lane 3: Lysate (2 h), Lane 4: Debris (4 h), Lane 5::
Lysate (4 h), Lane 6: Debris (6 h), Lane 7: : Lysate (6 h).
97
Results and discussion
Fig. 4.9.3: Expression of GspM (E. coli BL21 (DE3)/pRK16) after 2 h or 4 h or 6 h
induction with 50 µM IPTG.
Lane1: LMW, Lane 2: Debris (2 h), Lane 3: Lysate (2 h), Lane 4: Debris (4 h), Lane 5:
Lysate (4 h), Lane 6: Debris (6 h), Lane 7: : Lysate (6 h)
Fig. 4.9.4: Expression of GspM (E. coli BL21(DE3) /pRK17) after 2 h or 4 h or 6 h
induction with 5 µM IPTG.
Lane1: LMW, Lane 2: Debris (2 h), Lane 3: Lysate (2 h), Lane 4: Debris (4 h), Lane 5:
Lysate (4 h), Lane 6: Debris (6 h), Lane 7: Lysate (6 h).
98
Results and discussion
Fig. 4.9.5: Expression of GspM (E. coli BL21 (DE3)/pRK17) after 2 h or 4 h or 6 h
induction with 50 µM IPTG.
Lane1: LMW, Lane 2: Debris (2 h), Lane 3: Lysate (2 h), Lane 4: Debris (4 h), Lane 5:
Lysate (4 h), Lane 6: Debris (6 h), Lane 7: : Lysate (6 h)
Fig. 4.9.6: Expression of GspM (E. coli BL21 (DE3)/pRK16) after 2 h or 4 h or 6 h
induction with 100 µM IPTG.
Lane1: LMW, Lane 2: Debris (2 h), Lane 3: Lysate (2 h), Lane 4: Debris (4 h), Lane 5:
Lysate (4 h), Lane 6: Debris (6 h), Lane 7: Lysate (6 h)
99
Results and discussion
4.10 PURIFICATION AND OPTIMIZATION OF GspM and EchM PROTEINS
4.10.1 Purification of GspM and EchM
The over expressed GspM and EchM with a carboxy-terminal hexahistidyl tag under T7
promoter were purified by Ni2+-NTA affinity at room temperature using 5 mM of DTT
(Di- thiotritole). After single step affinity purification, GspM and EchM were analyzed by
sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) where GspM
purity approximately 90% and size about 10 kDa and the EchM had 95% purity and
approximate size of 31 kDa (Fig. 4.10.1 and 4.10.2).
Fig. 4.10.1: Purification of GspM from E. coli BL21 (DE3)/pRK16.
Lanes1 LMW, Lane 2: Debris, Lane 3: Lysate, Lane 4: Unbound GspM, Lane 5: Pass
through of native wash, Lane 6: Pass through of 25 mM Imidazole wash, Lane 7: Purified
GspM.
100
Results and discussion
Fig. 4.10.2: Purification of GspM from E. coli BL21 (DE3)/pRK17.
Lanes: 1 LMW, Lane 2: Debris, Lane 3: Lysate, Lane 4: Unbound EchM, Lane 5: Pass
through of native wash, Lane 6: Pass through of 50 mM Imidazole wash, Lane 7: Purified
EchM. Lane 8: LMW.
4.10.2 pH Optimization
The purified enoyl-CoA hydratase (EchM) having reducing activity with crotonyl-CoA
substrate was found to be 100% activity in slightly alkaline condition with a pH range of
8.0 to 9.0. The enzyme retained more then 90% of the activity at pH 8.0 after 10 min at 30
°C (Fig. 4.10.3).
101
Results and discussion
Fig. 4.10.3: Effect of pH on the activity of EchM.
The pH profile of the enzyme was evaluated by assay of the enzyme for 10 min at
30 °C in following buffers: 50 mM citrate buffer pH-5.0 to 6.0, 50 mM phosphate
buffer pH-6.0 to 8.0 and 50 mM Tris-HCl buffer pH-8.0 to 9.0. Values ploted are
average of assays performed in triplicate.
4.10.3 Temperature optimization
The temperature optimum for enoyl-CoA hydratase (EchM) was at 30 °C and it retained
more then 100% of its activity, below this temperature (at 25°C) the activity was
appxomately 70% at 35 °C, it was approximately 85%. Therefore we have selected the
temperature of 30 °C is more suitable for the activity assay (4.10.4).
102
Results and discussion
Fig. 4.10.4: Effect of temperature on the activity of EchM.
The optimum temperature for EchM was determined by incubating enzyme for 10 min
at different temperature ranging from 15 to 55 °C at the optimum buffer 50 mM TrisHCl (pH 8.0). Values plotted are average of assays performed in triplicate.
4.10.4 EchM activity
The single step purified EchM enzyme showed reducing activity with crotonyl-CoA
substrate of specific activity of 5.6 U/mg with 350 µg crotonyl-CoA at optimized pH 8.0
and temperature 30 °C (Fig. 4.10.5).
This activity (EchM) has potential biotechnological interest which may be further explored
for producing higher quantities of “crotonyl-CoA”, an important intermediate for “1103
Results and discussion
Fig. 4.10.5: EchM showed enoyl-CoA hydratase activity with crotonyl-CoA.
Experiments were performed in triplicates, values are averages and standard
deviations are shown as error bars.
butanol” production (Zheng et al., 2009). The expression of a non-clostridial butanol
producing pathway in E. coli is probably the most promising strategy for butanol
biosynthesis. Butanol has been regarded as a biofuel (less hygroscopic and less volatile
versus ethanol) which is suitable as a chemical feedstock in the plastic & flavor industries.
Thus, metagenomic library of pond water genome has resulted in the expression of an
enzyme that has manifold application in biotechnology. Further, elaborate characterization
104
Results and discussion
in terms of converting “crotonyl-CoA” to butanol couldn’t be investigated for want of time
and resources. We have plans to investigate this aspect as a post-doctoral assignment.
4.11 TRANSPOSON CASSETTE WITH gspM AND echM FOR ENGINEERING
RHIZOSPHERIC BACTERIA
One area of applied research that is of global interest is the enhancement of the beneficial
associations between microorganisms and plants, particularly in the rhizosphere (Brown,
1974). Nonsymbiotic nitrogen-fixing bacteria have been successfully used in field
inoculation studies. For example, soil and/or plant inoculation with members of the genera
Azotobacter, Azospirillum and Klebsiella have led to significant increases in both the yield
and nitrogen content of a number of forage and grain crops (Baldani et al., 1986; Imhoff,
1986; Kapulnik et al., 1981; Madkour et al., 1987) However, nitrogen fixation is impaired
by unfavourable environmental conditions, such as salt stress.
Azospirillum is a plant growth-promoting rhizobacterium associated with roots of
monocots, including important crops, such as wheat, corn, and rice. Both in greenhouse
and in field trials, Azospirillum was shown to exert beneficial effects on plant growth and
crop yields (Dobbelaere et al., 2001). The actual benefit from biological nitrogen fixation
has been questioned, and plant growth promotion by Azospirillum seems to be mainly due
to production of phytohormones, allowing an increase in the number of lateral roots and
root hairs; this results in a higher absorption of water and minerals from the soil (Bashan et
al., 2004; Dobbelaere et al., 2001). Our strategy is to enhance the growth of free living
nitrogen fixing bacteria, such as Azotobacter and Azospirillum to obtained biologically
fixed nitrogen for improved plant growth in saline soil.
To achieve this objective we selected a transposon construction vectors pMOD3<R6Kγori/MCS>, which is a pUC-based vector. They consists of Mosaic Ends (ME)
105
Results and discussion
sequences that flank an MCS in a vector with a colE1 origin of replication, and also
contains an R6Kγori within the ME sequences. A multiple cloning site (MCS) flanked by
the hyperactive 19-bp Mosaic Ends (ME) that are specifically and uniquely recognized by
EZ-Tn5 Transposase and the MCS is useful for a variety of cloning applications. In the
MCS a selectable marker kanamycin gene cassette was cloned (Fig. 4.11.1) and then the
constructed vector was followed by cloning of gspM and echM gene to generate as clone
RK9 and pRK10, respectively (Fig. 4.11.1).
Fig. 4.11.1: Cloning of Kanamycine cassette, gspM and echM genes into pMOD
vector.
Lane1: 1 kb ladder,Lane2:pMOD + Kan cassette ,Lane 3: pMOD + Kan cassette, Lane 4
& 5: pRK9, Lane 6 & 7: pRK10.
4.11.1 Growth pattern of E. coli DH10B and Azospirillum brasilense (MTCC125)
strain with Tn integrated salt tolerance genes
The growth pattern of transposon integrated E .coli DH10B and A. brasilense (MTCC125
106
Results and discussion
strain) were checked into LB kanamycin medium containing 750 mM and 1.0 M NaCl at
37 °C and 30 °C, 200 rpm for 48 h respectively. The obtained results showed no change
in mutants growth in comparision to the control E. coli DH10B and A. brasilense wild type
strain (Fig.4.11.2 and 4.11.3).
Fig. 4.11.2: Growth pattern of E. coli / Tn-integrated E. coli mutants with gspM
and echM gene in LB/LB kanamycin medium containing 750 mM NaCl.
(●) E . coli DH10B strain, (○) E . coli DH10B strain, (▼) E . coli DH10B/gspM, (∇) E
. coli DH10B/gspM. (■) E. coli DH10B/echM, (□) E . coli DH10B/echM . Experiments
were performed in triplicates, values are averages and standard deviations are shown as
error bars.
107
Results and discussion
Fig. 4.11.3: Growth pattern of A. brasilense (MTCC125)/Tn-integrated A.
brasilense (MTCC125) mutants with gspM and echM gene in LB/LB
kanamycin medium containing 1.0 M NaCl at 30 °C.
(●) A. brasilense (MTCC125) strain, (○) A. brasilense (MTCC125) strain, (▼)
A. brasilense (MTCC125)/gspM, (∇) A. brasilense (MTCC125)/gspM, (■) A.
brasilense (MTCC125)/echM, (□) A. brasilense (MTCC125)/echM .Experiments
were performed in triplicates, values are averages and standard deviations are
shown as error bars.
4.11.2 Integration of genes into E. coli DH10B and Azospirillum brasilense
(MTCC125) strain
The prepared transposon clone of salt stress tolerance gene, generate the transposon by
PvuII restriction enzyme digestion of pRK9 and pRK10. Both the digested fragments
108
Results and discussion
containing gene with transposon were transformed by electroporated into the high
efficiency cells (107) of E. coli DH10B. The transforments were plated on the LB
kanamycin plates and incubated at 37 °C. The LB kanamycin plate having approximately
300 colonies as well as the colonies appearing on LB kanamycin agar plates have
phenotypically proved that, integration of Pvu11 digested fragment of gspM and echM into
the genome of E. coli DH10B. The same fragments of salt stress tolerance genes were
electroporated into the Azospirillum brasilense (MTCC125) of efficiency 104 cells/ ml. The
transforments were plated into LB kanamycin agar plate and incubated at 30 °C. It has
been observed that LB kanamycin plate showed 30 to 40 colonies on plates indicated
integration of Pvu11 digested fragment (containing kanamycin marker with gspM/echM)
into the genome of Azospirillum brasilense (MTCC125).
The integration of genes into the genome was confirmed by isolating genomic DNA of
obtained mutants as well as their control wild type strains were digested with the Pvu11
restriction enzyme and southern hybridization was performed. The integration of Tncassete with gspM and echM into the genome of E. coli DH10B and A. brasilense has been
confirmed (Fig. 4.11.4 and 4.11.5).
It has been observed that both the genes were integrated into the genome of the A.
bracilense, but it didn’t produce significant result, because strain already had tolerant
ability. Perhaps integration of these genes into other rhizospheric bacteria will give
significant salt tolerance.
109
Results and discussion
(a)
(b)
Fig. 4.11.4: Southern hybridization analysis of E. coli for integration of Tn-cassete.
The genomic DNA from E. coli and Tn-integrated E. coli were isolated and digested with
Pvu11. Blot was probed with purified PCR product of gspM and echM. (a) Lane 1: 2 Log
DNA ladder, Lane 2: E. coli digested genomic DNA, Lane 3 & 4: Tn-integrated E.
coli/gspM digested genomic DNA, Lane 5: Pvu11 digested gspM fragment. (b) Lane 1: 2
Log DNA ladder, Lane 2: E. coli digested genomic DNA, Lane 3 & 4: Tn-integrated E.
coli/echM digested genomic DNA, Lane 5: Pvu11 digested echM fragment.
110
Results and discussion
(a)
(b)
Fig. 4.11.5: Southern hybridization analysis of A. brasilense for integration of Tncassette.
The genomic DNA from A. brasilense and Tn-integrated A. brasilense were isolated and
digested with Pvu11. Blot was probed with purified PCR product of gspM and echM. (a)
Lane 1: 2 Log DNA ladder, Lane 2: A. brasilense digested genomic DNA, Lane 3 & 4: Tnintegrated A. brasilense/gspM digested genomic DNA, Lane 5: Pvu11 digested gspM
fragment. (b) Lane 1: 2 Log DNA ladder, Lane 2: A. brasilense digested genomic DNA,
Lane 3 & 4: Tn-integrated A. brasilense/echM digested genomic DNA, Lane 5: Pvu11
digested echM fragment.
111