Biochemical basis for malate over production in Actinomycete spp.
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Transcript of Biochemical basis for malate over production in Actinomycete spp.
Biochemical Basis for Malate Overproduction in
Actinomycete spp. Isolated from Cotton Rhizosphere
Presented by:Anjali Joshi (11MMB005)
Nikul Parsana (11MMB011) Sakhmeet Patel (11MMB018)
Guided by:Dr. Shalini Rajkumar
Introduction
Actinobacteria
Fig.1. A typical Actinomycetes colony growing on agar•Gram positive, aerobic, filamentous, high G+C content (Williams et al.1989; Manfio et al. 1995).
Habitat of Actinomycetes
Habitat Actinomycetes Reference
Salt lake Streptomonospor amylolytica,Streptomonospora flavalba
Cai et al., 2009
Marine(pacific ocean)
Micromonospora, Rhodococcus, Streptomyces Maldonado et al.,2004
Tropical rain forest Streptosporangium, Nocardia, Micromonospora,Streptomyces, Actinomadura,
Wang et al., 1999
Stream Streptomyces,Micromonospora, Actinomadura, Pseudonocardia
Das et al., 2006
Marine sediment Salinispora tropica,Salinispora arenicola Jensen et al.,2006
Antarctica soil Streptomyces, Actinomycetales Moncheva et al.,2002
Mangrove soil Micromonospora, Streptomyces. Hong et al., 2009
Forest soil Micromonospora,Microbisporium,Actinosporium,Streptosporangium
Seong et al.,2001
Habitat for Actinomycetes
Habitat Actinomycetes Reference
Volcanic soil Streptomyces,Streptosporangium,Actinomadura Zenova et al.,2009
Fresh water Saccharopolyspora,Actinosynnema Sibanda et al.,2010
Agricultural soil Actinoallomurus,Actinopolyspora,Micromonospora Khanna et al.,2011
Mangrove ecosystem Actinomycetes Fredimose et al.,2011
Theobroma cacao Streptomyces Barreto et al., 2008
Lycopersicon esculentum(tomato)
Streptomyces species,Streptoverticillium,Nocardia
Cao et al.,2004
Pea (Pisum sativum) Streptomyces lydicus Tokala et al.,2002
Wheat Streptomyces Juhnke et al., 1987
Cotton S. purpureus, S. aurantiacus, S. microflavus. Hassanin et al., 2007
Actinomycetes in Plant Rhizosphere
Plant Rhizosphere Actinomycetes Reference
Medicinal plant Streptomyces,Actinomadura sp.,Microbispora sp., Micromonospora sp.,Nocardia sp,Nonomurea sp
Khamna et al., 2009
Mahuva (Madhuca induca),Karaj(Pongamia globra)
Streptomyces sp. Thangapandian et al., 2007
Maize (Zea mays) Actinomycetes Miller et al., 1989
Rice (Oryza sativa L.) Mycobacterium,Streptomyces,Micromonospora,Actinoplanes,Frankia,
Tian et al.,2007
Theobroma cacao Streptomyces Barreto et al., 2008
Lycopersicon esculentum (tomato)
Streptomyces species,Streptoverticillium,Nocardia
Cao et al.,2004
Wheat Streptomyces Juhnke et al., 1987
Cotton S. purpureus, S. aurantiacus, S. microflavus. Hassanin et al., 2007
PGP activity of Actinomycetes
Rhizospheric Actinomycetes
IAA Siderophore Phosphate Solubilization
Reference
Streptomyces viridis + + + Khamna et al., 2010, Gangwar et al., 2012.
Nocardia + + + Gangwar et al., 2012
Micromonospora + + + Gangwar et al., 2012
Saccharopolyspora + + + Gangwar et al., 2012
Actinopolyspora + + + Gangwar et al., 2012
Streptomyces rochei IDWR19, Streptomycescarpinensis IDWR53, Streptomyces thermolilacinus IDWR81
+ + + Jog et al., 2012
Fig.2. Mechanisms of P solubilization (Khan et al., 2010)
Mechanisms of P solubilization
Mineral Phosphate Solubilization by microorganisms in soil
Organic acid productionSOIL
Organic acid production by soil microbes
Soil Microbes Organic acid production Reference
Peniciilium bilaii Citric acid, oxalic acid Cunningham et al, 1992
Aspergillus candidus Oxalic acid, tartaric acid Banik et al, 1982
Bacillus firmus Oxalic acid, tartaric acid Banik et al, 1982
Streptomyces spp. Oxalic acid, tartaric acid Banik et al, 1982
Pseudomonas fluorescens 2-ketogluconic acid Duff et al, 1963
Enterobacter intermedium 2-ketogluconic acid Hwangdbo et al, 2003
Aspergillus niger Gluconic acid, fumaric acid, succinic acid, acetic acid, oxalic acid
Rashid et al, 2004
Rhizobium meliloti 2-ketogluconic acid Halder et al, 1993
Azospirillum Gluconic acid Hilda et al, 2004
Organic acid production by Actinomycetes
Actinomycetes Organic acid production Reference
Streptomyces spp.U121 Hydrocitric acid Hilda et al., 2006
Streptomyces lividans Pyruvic acid, 2-oxoglutaric acd
Madden et al., 1996
Acinetobacter rhizosphaere Gluconic acid, oxalic acid, lactic acid,malic acid, formic acid
Gulati et al., 2009
Micromonospora endolithica Unidentified organic acid Khaled et al., 2009
Actinomadura Unidentified organic acid Abdulla, 2009
Kitasatospora Unidentified organic acid Abdulla, 2009
Nocardioses Unidentified organic acid Abdulla, 2009
Origin of Study
• In previous studies, significantly high P-solubilizing actinomycete isolates from cotton rhizosphere were obtained. • The isolate (CR-16) over produced malate (as confirmed on TLC using malate standard) in minimal medium supplemented with 100 mM glucose. However, when grown on lower glucose concentration (50 mM), acid production was not observed.
• Literature lacks determined pathway for malate over production in actinomycetes
• Majority of actinomycetes are reported to follow glycolytic pathway for glucose metabolism . Glyoxylate shunt has also been reported in Streptomyces spp. (Han and Reynolds,1997).
TCP solubilization profile of CR-16
Fig.3. Phosphate solubilization on Tris buffered (50 mM) (pH- 8.0)Tricalcium Phosphate (TCP) Agar supplemented with 100 mM glucose by CR-16 isolate
Metabolic Pathway
Glucose(50 mM)
Pyruvateglycolysis
Fig.4. Metabolism of glucose
Metabolic Pathway at high concentration of glucose
Fig.5. Putative Pathway for production of malic acid
Glucose(100 mM)
Pyruvateglycolysis
Objectives
Confirmation of MPS phenotype of CR-16 and EC-11 in presence of rhizospheric carbon sources.
Elucidation of hypothesized pathway for malate over production in phosphate solubilising CR-16 isolate. Enzyme assays (Isocitrate lyase, Isocitrate dehydrogenase and Malate synthase). Gene expression (Isocitrate lyase, Isocitrate dehydrogenase and Malate synthase)
study by reverse transcriptase PCR (RT-PCR) Confirmation of gene expression by Real-time PCR (q-PCR)
In vivo studies for beneficial effect of isolates on chick pea plants. Pot experiment Hydroponic studies
▪ Rock phosphate containing MS medium▪ Coinoculation with commercially available biofertilizer (V Green)
▪ Biocontrol trait (chitinase production)▪ Organic phosphorous utilization (phytase production)▪ Potassium solubilization (Mica sol.)▪ Halotolerance of isolates
Materials and Methods
Selection of cultures to determine malate production pathway
• Test culture: Phosphate solubilizing actinomycete isolate (CR-16) showing over production of malic acid.
• Control: Phosphate solubilizing actinomycete isolate (EC-11) lacking malic acid over production served as a control.
Revival of cultures:
Organic acid production:
Minimal medium supplemented with 2% glucose was inoculated with CR-16 and incubated at 30o
C for 7 days.
Cells were centrifuged at 10,000 rpm for 10 min
Qualitative and quantitative organic acid profile of cell free supernatant by HPLC analysis (CSMCRI, Bhavnagar) using Supelcogel organic acid specific column (Sigma Aldirch)
CR-16 and EC-11 obtained in
previous studies were maintained
on ISP-3 and preserved as 20% glycerol
stock
Minimal medium
(Vellore, 2001) with glucose
unless mentioned otherwise
Incubated at 30 ◦C for 7 days
under static/shaking
condition
Sugar utilization profile and MPS phenotype of CR-16 and EC-11
Objective 1:
Sugar utilization profile and MPS phenotype of CR-16 and EC-11
CR-16 and EC-11 inoculated
in minimal medium
containing 1% sugar and
incubated at 30 oC for 10 days
pH of cell free supernatant
was measured and growth was visually
observed
Phosphate estimation by
phosphomolybdate method (Ames, 1964)
Gene Expression studies and Enzyme Assays of enzymes involved in malate over production
Isocitrate dehydrogenase Isocitrate lyase Malate synthase
Objective 2:
Enzyme Production
Cells grown in minimal medium with 50 mM and 100 mM glucose
seperately.
Incubated at 30 oC under shaking for
7 days
Cells were subjected to
freeze thaw and lysozyme
treatment for 1 h at 37 oC
Cells were then resuspended in
0.1 M phosphate buffer (pH- 6.8)
(Ball and McCarthy, 1988)
Cells were lysed by ultra
sonication for 10 sec five times with 1.5 min
interval at 700 WClear cell lysate obtained after
centrifugation at 8000 rpm for 10 min was used as enzyme source
Enzyme Assays: Isocitrate Dehydrogenase:
•Isocitrate dehydrogenase (IDH) was determined by continuous spectrophotometric rate determination depending on reduction of β-NADP ( Nicotinamide Adenine Dinuleotide Phosphate ) at 340 nm.
•The IDH activity was measured by decrease in the absorption of β-NADP at 340 nm. One unit IDH converts 1µmole of isocitrate to α-ketoglutarate per minute at pH 7.4 at 370 C (Bergmeyer, 1974).
Enzyme Assays: Isocitrate Lyase:
• Isocitrate lyase (ICL) was determined by continuous spectrophotometric rate determination depending on formation of phenylhydrazine glyoxylate and measuring absorption at 324 nm.
•The ICL activity was measured by decrease in absorption of phenylhydrazine glyoxylate at 324 nm. One unit ICL forms 1µmole of glyoxylate per minute at pH 6.8 at 300 C( Chell et al,1978).
Enzyme Assays: Malate Synthase:
•Malate synthase (MS) was determined by continuous spectrophotometric rate determination depending on reduction of 5,5’- Dithio bis 2- Nitrobenzoic acid (DTNB) to 5- Thio, 2- Nitrobenzoic acid (TNB) at 412 nm.
•The MS activity was measured by decrease in absorption of DTNB at 412 nm. One unit MS cleaves 1 µmole of acetyl CoA per minute at pH 8.0 at 300C in presence of glyoxylate (Silverstein, 1975).
Gene Expression (RT-PCR)
Primer designing RNA isolation Reverse Transcriptase PCR (RT-PCR) Data analysis
Primer Designing
Primer synthesis using Integrated DNA Technology (IDT)
Primer pairs fulfilling all criteria for primer selection were selected The primers were confirmed by Insilico PCR
Primers were reconfirmed by primer BLAST
Gene sequence of enzymes: Isocitrate dehydrogenase, Isocitrate lyase, Malate synthase of Streptomyces spp. from NCBI
Contig sequence from sequences of the same gene using codon aligner
Coding sequence of the gene using ORF Finder
RNA isolation Growth
Actinomycetes in minimal medium,
30 oC, 4-5 days
Cell LysisGTE solution, freeze thaw
bead beating, lysozyme
SDS-EDTA prolonged
incubation at 65 oC
Cell lysateProceeded
with High pure RNA isolation
kit (Roche)Steps
Silica Binding,DNase
treatment,Washing,Elution
RNA yield confirmed on 1.5% agarose
gel and quantified at
260 nm
Equal concentration of RNA (4 μg) was used for
gene amplification
of IDH, ICL and MS genes. DNA
gyrase gene was used as an internal control
Amplification of genes using Reverse Transcriptase-PCR reaction Kit (PrimeScript one tube reaction, Takara)
Reagents Volume
2x 1 step buffer (Reaction buffer, dNTP mixture, One step enhancer solution)
12.5 μl
Prime Script 1 step enzyme mix (PrimeScript RTase, DNA Polymerase- Ex Taq HS, RNase Inhibitor)
1 μl
Forward Primer 100 mmole
Reverse Primer 100 mmole
Template RNA 4 μg
Sterile Milli Q Make up volume upto 25 μl
Total Volume 25 μl
Specific for genes
PCR programme (IDH, ICL and MS)
c-DNA synthesis PCR
PCR programme (DNA gyrase)
c-DNA synthesis PCR
Data Analysis
RT-PCR products were resolved on 1.5 % and 1.2 % agarose gel respectively and viewed under gel documentation system.
The data was analyzed TotalLab quant (Ver. 10) as follows:
Detection of lanes, annotation of wells, band detection, removal of background signals
Different parameters of the bands were analyzed including area of band in pixels(for DNA concentration) and peak height (for purity)
Graph for area (in pixels) of each band was plotted
Confirmation of Gene Expression by Real Time PCR (q-PCR)
RNA isolation
c-DNA synthesis
q-PCR and data analysis
PCR conditions
Reagents Volume
SYBR Premix Ex Taq II (2X) 25 µl
Forward Primer (10 µM) 2 µl
Reverse Primer (10 µM) 2 µl
ROX Reference Dye (50X) 1 µl
c-DNA template 100 ng
sterile milliQ water Make upto 50 µl
Final Volume 50 µl
specific for genes
PCR Programme
Process Temperature Time
Initial denaturation 95 oC 30 sec
PCR (40 CYCLES) 95 oC 5 sec
60 oC 30 sec
Dissociation stage As recommended for ABI Real-time StepOnePLus PCR system
Prefixed
Data Analysis
Data were analysed in StepOne software (ver. 2.2.2), Applied BioSystems Life Technologies.
DNA gyrase was used as an internal control.
ΔCT values were calculated for each sample. Accordingly, RQ values were determined.
Graphs were plotted with RQ values for each samples.
In vivo study for beneficial effects of CR-16 and EC-11 inoculation on Cicer
arietinum (chick pea) growth and development
Objective 3:
Pot Experiment for in vivo study of PGP effects of CR-16 and EC-11 inoculation
Chick pea seeds surface sterilized by 0.1% HgCl2 and 70%
ethanol
Washed many times with sterile distilled
water and rolled over N-agar plate for
validation
Seeds germinated on 1% water agar plate were bacterized in
dense spore suspension of each isolate separately
(107-108/ml) for 1 h
Seeds were sown in sterile soil in equal
sized pits and grown at room temperature
using standard light-dark cycle
After Incubation for 40 days, different plant
parameters were measured.
Uninoculated plants served as control
Hydroponics for in vivo study of PGP effects of CR-16 and EC-11
Chick pea seeds surface sterilized by 0.1% HgCl2 and 70%
ethanol
Washed many times with sterile distilled
water and rolled over N-agar plate for confirmation of
sterilization
Seeds germinated on 1% water agar plate
Germinated seeds were placed in sterile partial MS medium
inoculated with CR-16 and EC-11
Plants were incubated in Plant
Growth Chamber at 28oC and 35%
relative humidity (Rh)
After 14 days of incubation different
plant parameters were measured
Hydroponics assay to study growth of inoculated plants in free phosphate deprived MS medium
Chick pea seeds surface sterilized by 0.1% HgCl2 and 70%
ethanol
Washed many times with sterile distilled
water and rolled over N-agar plate for confirmation of
sterilization
Seeds germinated on 1% water agar plate
Germinated seeds were placed in
sterile free phosphate deprived
MS medium containing rock phosphate and
inoculated with CR-16 and EC-11
Plants were incubated in Plant Growth
Chamber at 28oC and 35% relative humidity
(Rh)
After 30 days of incubation different
plant parameters were measured
Hydroponics - Coinoculation of commercial biofertilizer with CR-16 and EC-11
Chick pea seeds surface sterilized by 0.1% HgCl2 and 70%
ethanol
Washed many times with sterile distilled
water and rolled over N-agar plate for confirmation of
sterilization
Seeds germinated on 1% water agar plate
Germinated seeds were placed in
sterile partial MS medium
containing V-Green biofertilizer with and without CR-16 and EC-11
Plants were incubated in Plant Growth
Chamber at 28oC and 35% relative humidity
(Rh)
After 14 days of incubation different
plant parameters were measured
Chitinase production (Biocontrol)Phytase production (Organic P solubilization)
Potassium solubilizationHalotolerance of isolates
Objective 4:
Chitinase test
Cultures spotted on 2% colloidal chitin agar (Renwick et al.,1991)
Amount of NAG liberated was estimated by DNSA method (Miller, 1959)
Phytase test
Cultures spotted on 1% sodium phytate agar (Harland and Harland, 1980)
Amount of free soluble phosphate liberated was estimated (Ames, 1969)
Potassium solubilizationCultures spotted on 1% mica agar Amount of potassium solubilized was
estimated by flame photometry (Sugumaran and Janarthanam,2007)
Halotolerance of isolates
Cultures grown in minimal medium containing 2,4,6,8 and 10% NaCl and incubated and growth was visually observed
Results and Discussion
Revival of CR-16 and EC-11
Morphological and colonial characteristics of CR-16 and EC-11
Isolate Vegetative mycelium
Aerial mycelium
Pigmentation
Spore Size and Shape
Appearance
Substrate secretion
Additional feature
Microscopic arrangement
CR-16 Lightbrown
Cream Light brown
Light Grey (++)
Small, Even margin
Dry,Raised
None Nucleated colony
Point shapedSpores
EC-11 Lightbrown
White Black Light Grey(++)
Small,Evenmargin
Dry,Flat
Greenish None RegularSpores withHyphae
CR- 16 EC-11
Gram’s Staining
CR - 16ISP- 1 ISP - 4
ISP -3 ISP - 7
Colony morphology on ISP media
EC -11ISP-1 ISP -3
ISP - 4 ISP -7
Colony morphology on ISP media
MPS phenotype of CR-16 and EC-11 in presence of
different sugars
Acid production by CR-16 and EC-11 in presence of different carbon sources
Sugar
Suc
cina
te
Ace
tate
Glu
cona
te
Citr
ate
Oxa
late
Ara
bino
se
Rib
ose
Man
nito
l
Xyl
ose
Glu
cose
Lact
ose
Fruc
tose
Suc
rose
pH V
alue
0
2
4
6
8
10EC-11CR-16
(P ≤ 0.05, n=3)
• pH drop in glucose and fructose from 7.2 to 5.0 in both isolates
• In presence of organic acids succinate, acetate and oxalate CR-16 showed no significant change in pH; whereas increase in pH for same tubes was observed in EC-11
• CR-16 showed pH drop from 7.2 to 5.0 in sugars viz ribose, xylose , arabinose, sucrose, mannitol and lactose, indicating organic acid production
• Organic acid over production results in pH drop (Hilda et al 2006)
Growth in different rhizospheric sugars
Sugars
Suc
cina
te
Ace
tate
Ara
bino
se
Rib
ose
Oxa
late
Man
nito
l
Citr
ate
Fruc
tose
Xyl
ose
Suc
rose
Glu
cose
Lact
ose
Glu
cona
te
Gro
wth
0.0
0.2
0.4
0.6
0.8
EC-11 CR-16
• All 13 different rhizospheric sugars were utilized by both the isolates• Growth of EC-11 was faster compared to CR-16• However, CR-16 showed more pH drop compared to EC-11
Sugar utilization growth profile of CR-16 and EC-11
Qualitative assay of Phosphate Solubilization
Red halo zone surrounding EC-11
Fig.7. P solubilization by EC-11 on Tris (25 mM) (pH-8.0) buffered TCP agar
Phosphate Solubilization in different rhizospheric sugars
Sugar
Succ
inat
e
Ace
tate
Glu
cont
e
Oxa
late
Ara
bino
se
Rib
ose
Man
nito
l
Xyl
ose
Glu
cose
Lact
ose
Fruc
tose
Sucr
ose
Con
cent
ratio
n of
Pi (
mg
ml- )
0.0
0.2
0.4
0.6
0.8
1.0 EC-11CR-16
(P ≤ 0.05, n=3)
• Range of free phosphate liberated: 96.79 μg/ml to 267.14 μg/ml
• CR-16 solubilized maximum phosphate in presence of ribose followed by xylose and lactose
• EC-11 solubilized maximum phosphate in presence of fructose followed by succinate and glucose
• Phosphate solubilization is found in those sugars which showed organic acid production
• P solubilization by organic acid production is reported in actinomycetes( Gangwar et al., 2012)
Rock Phosphate Solubilization
1 2 3 4 5 6 7 8 9 10 110
0.1
0.2
0.3
0.4
0.5
0.6
f(x) = 0.0503727272727273 xR² = 0.99992219525961
Concentration of KH2PO4 (μg/ml)
O.D
. at 8
20 n
mStandard curve
Isolate Free phosphate concentration (μg/ml)
CR-16 95.24±0.034
EC-11 91.27±0.012
(P ≤ 0.05, n=3)
Biocontrol trait (chitinase production)Organic P utilization (phytase production)
Potassium solubilizationHalotolerance of isolates
Chitinase Production by EC-11
Clear zone of hydrolysis surrounding colonies indicate chitinase production
Fig.8.Chitinase production by EC-11 on 2% colloidal chitin agar
Quantitative assay for chitinase
0 0.2 0.4 0.6 0.8 1 1.20
0.1
0.2
0.3
0.4
0.5
0.6
0.7
0.8
f(x) = 0.677590909090911 xR² = 0.996071781343927
NAG conc. (mg ml-1)
O.D
. at 5
40 n
m
Isolate Chitinase production (units/ml)
EC-11 1.27±0.007
CR-16 -
(P ≤ 0.05, n=3)
Chitin is the major polymer present in fungal cell wall. Hence chitinase producing cultures are known to have antifungal activity. Chitinase production is common in actinomycetes (Gupta et al., 1995)
Actinomycete isolates producing chitinase serve as a natural plant protecting agent against phytopathogenic fungus (Shirokikh et al., 2007).
EC-11 produced 1.27 units/ml chitinase in 2% colloidal chitin medium which is within the known range reported for actinomycetes.
Quantitative assay for phytase
0 0.1 0.2 0.3 0.4 0.5 0.6 0.70
0.20.40.60.8
11.21.41.61.8
2f(x) = 3.29722222222222 xR² = 0.997507781354895
KH2PO4 conc. (mM ml-1)
O.D
. at 6
60 n
m
Isolates Phytase production (units/ml)
CR-16 0.68±0.007
EC-11 0.52±0.011
O.D. at 660 nm
(P ≤ 0.05, n=3)
Phytic acid, myo-inositolhexaphosphate, is a major storage form of phosphorus in cereals and legumes, representing 18–88% of total phosphorus. Phytic acid is also abundantly available in soil. However, plants cannot utilize it as phosphate is present in a bound form.
Phytate degrading enzymes (phytases) breakdown phytic acid and release inorganic phosphate (Pi) which can be taken up by plants (Reddy et al., 1982). The phenomena is called as organic phosphate solubilization.
The isolates under study (CR-16 and EC-11) produced 0.68 and 0.52 units/ml enzyme respectively. The values are higher than reported in Thermomonospora spp.RC7 (0.233 units/ml ) (Wittanalai et al., 2003).
Halotolerance of CR-16 and EC -11
Salinity (%) Growth of CR-16 Growth of EC-112 +++ +++4 +++ +++6 +++ +++8 +++ +++
10 +++ +++
• For the survival of cultures in saline conditions, salt tolerance is required. Saline soils usually have high NaCl concentration. Similarly, saline condition is created in fertile non-saline soils due to improper irrigation.
• Hence for a bio-inoculant, salt tolerance is a desirable trait. The cultures obtained showed tolerance to as high as 10% NaCl. In literature, halotolerant actinobacteria Haloactinospora alba (7-23% NaCl tolerance) are reported (Tang et al., 2008).
• Results indicate NaCl tolerant cultures, however tolerance to higher NaCl range needs to be examined.
Mica utilization
Isolate Free potassium concentration
CR-16 300 μg/ml
EC-11 400 μg/ml
• Crude powder of mica rocks was obtained. Mica contains potassium ions in bound form, thus making it unavailable. Mica solubilizing cultures liberate K+ ions from mica rocks which can be further utilized by plants.
• In the present study, CR-16 and EC-11 solubilized mica releasing K+ ions. The solubilization is relatively higher as compared to other mica solubilizing bacteria (MSc dissertation thesis, Archana D.S., 2007, University of Agricultural sciences, Dharwad) • To the best of our knowledge, there are no reports for mica solubilization for actinomycetes.
Enzyme Assays
HPLC Profile of culture supernatant
Fig.6.HPLC profile of isolate CR 16: Peak with RT 13.12 min corresponds to malate (RT 13.1 min)
Enzyme Assays: 1. Isocitrate dehydrogenase EC-11
• Significantly high activity of IDH in 50 mM glucose and low activity of IDH in 100 mM glucose.
Isocitrate dehydrogenase EC-11
Glucose concentration
0 1 2
Enz
yme
U m
l-1
0.00
0.01
0.02
0.03
0.04
0.05
0.06
EC-11 50 mM pH -2.7 EC-11 100 mM pH- 2.5EC-11 50 mM pH -7.0 EC-11 100 mM pH -7.0
(P ≤ 0.05, n=3)
• Significantly low activity of ICL in 50 mM glucose and high activity of ICL in 100 mM glucose
2. Isocitrate lyase EC-11
Isocitrate lyase EC-11
Glucose concentration
0 1 2
Enz
yme
U m
l-1
0.000
0.005
0.010
0.015
0.020
0.025
0.030
EC-11 50 mM pH -2.7 EC-11 100 mM pH -2.5 EC-11 50 mM pH -7.0 EC-11 100 mM pH -7.0
(P ≤ 0.05, n=3)
• Similar amount of malate synthase produced in 50 mM and 100 mM glucose.• We can conclude that EC-11 produces some unidentified organic acid other than malic acid.
3. Malate Synthase EC-11
Malate synthase EC-11
Glucose concentration
0 1 2
Enz
yme
U m
l-1
0.000
0.005
0.010
0.015
0.020
0.025
EC-11 50 mM pH -2.7 EC-11 100 mM pH -2.5EC-11 50 mM pH -7.0 EC-11 100 mM pH -7.0
4. Isocitrate dehydrogenase CR-16
Isocitrate dehyrogenase CR-16
Glucose concentration
0 1 2
Enz
yme
U m
l-1
0.00
0.01
0.02
0.03
0.04
0.05
0.06
CR-16 50 mM pH -2.8 CR-16 100 mM pH -2.1 CR-16 50 mM pH -7.0 CR-16 100 mM pH -7.0
• Significant low activity of IDH in 50 mM and high activity in 100 mM glucose
(P ≤ 0.05, n=3)
• Significant low activity of ICL in 50 mM and high activity in 100 mM glucose.
5. Isocitrate lyase CR-16
Isocitrate lyase CR-16
Glucose concentration
0 1 2
Enz
yme
U m
l-1
0.00
0.01
0.02
0.03
0.04
0.05
0.06
0.07
CR-16 50 mM pH -2.8 CR-16 100 mM pH -2.1 CR-16 50 mM pH -7.0CR-16 100 mM pH -7.0
(P ≤ 0.05, n=3)
• Significant low activity in 50 mM and high activity in 100 mM glucose
6. Malate synthase CR-16
Malate Synthase CR-16
Glucose concentration
0 1 2
Enz
yme
U m
l-1
0.00
0.02
0.04
0.06
0.08
0.10
CR-16 50 mM pH -2.8 CR-16 100 mM pH -2.1 CR-16 50 mM pH -7.0CR-16 100 mM pH -7.0
(P ≤ 0.05, n=3)
Summary of enzyme assays
Isolate IDH (U ml-1) ICL (U ml-1) MS (U ml-1)
After pH drop- 10 daysBefore pH drop- 3 days
After pH drop
Before pH drop
After pH drop
Before pH drop
After pH drop
Before pH drop
CR-16 (50 mM) 0.070 0.040 0.050 0.022 0.080 0.030
CR-16 (100 mM) 0.090 0.049 0.090 0.020 0.140 0.030
EC-11 (50 mM) 0.070 0.032 0.030 0.015 0.060 0.014
EC-11 (100 mM) 0.095 0.037 0.045 0.017 0.060 0.015
• CR-16 shows significant high activity of ICL and MS at high concentration of glucose • EC-11 shows similar malate synthase activity, no over production of malate in EC-11, organic acid produced by EC-11 is unidentified• Enzyme activity of all three enzymes almost similar in medium before pH drop• High activity of ICL and MS in CR-16 shows presence of glyoxylate shunt pathway
Gene Expression
Primers:
Enzymes Primer Sequence
Forward Primer Tm(◦c) Reverse Primer Tm(◦c)
Isocitrate dehyrogenase CCAACATCATCAAGCTGCCGAACA 60.1 AGACCTTCATCATCGTGGCCTTCA 60.2
Isocitrate lyase
TTCGAGCTGACCAAGGCGATGAT 60.7 CCAGGGTGATGAACTGGAACTTGT 59.1
Malate synthase
ACTTCGGCCTGTACTTCTTCCACA 60.0 TCGTAGAGGATCTCCTCCATCTCGAA 59.8
DNA gyrase
GAAGTCATCATGACCGTTCTGCA 59.2 AGCAGGGTACGGATGTGCGAGCC 65.6
RNA Isolation:
1 2 3 4
• Bands of RNA resolved on 1.5% agarose gel.• RNA concentration quantified at 260 nm • Same concentration of RNA template used for RT-PCR.
Isolate and glucose
concentrationO.D. at 260 nm Concentration (μg/μl)
CR-16 (50 mM) 0.270 4.32
CR-16 (100 mM) 0.266 4.26
EC-11 (50 mM) 0.284 4.54
EC-11( 100 mM) 0.282 4.51
• Lane 1: CR-16 (50 mM)• Lane 2: CR-16 (100 mM)• Lane 3: EC-11 (50 mM)• Lane 4: EC-11 (100
1. DNA gyrase:
• DNA gyrase, internal control• Band intensity not affected by glucose concentration
1.2 kb amplicon
2. Isocitrate dehydrogenase
200 bp amplicon
Isolate Band intensity
EC-11 (50 mM) high
EC-11 (100 mM) low
1 2
• Lane 1: EC-11 (50 mM)• Lane 2: EC-11 (100 mM)
3. Isocitrate lyase:
200 bp amplicon
Isolate Band intensity
CR-16 (50 mM) Low
CR-16 (100 mM) High
EC-11 (50 mM) Low
EC-11 (100 mM) High
1 2 3 4
• Lane 1: CR-16 (50 mM)• Lane 2: CR-16 (100 mM)• Lane 3: EC-11 (50 mM)• Lane 4: EC-11 (100 mM)
4. Malate synthase:
1 2 3 4
200 bp amplicon
Isolate Band intensity
CR-16 (50 mM) Low
CR-16 (100 mM) High
EC-11 (50 mM) Moderate
EC-11 (100 mM) Moderate
• Lane 1: CR-16 (100 mM)• Lane 2: CR-16 (50 mM)• Lane 3: EC-11 (100 mM)• Lane 4: EC-11 (50 mM)
TotalLab AnalysisIsocitrate dehydrogenase
EC-11 50 mM EC-11 100 mM0
500
1000
1500
2000
2500
3000
3500
4000
Area
(pixe
ls)
• Area covered under 50 mM is higher compared to 100 mM glucose concentration indicating higher IDH activity in 50 mM glucose for EC-11
Isocitrate lyase
CR-16 50 mM CR-16 100 mM EC-11 50 mM EC-11 100 mM0
1000
2000
3000
4000
5000
6000
7000
8000
Area
(pix
els)
•Area of band was higher for 100 mM glucose as compared to 50 mM glucose for both CR-16 and EC-11 indicating higher ICL activity at 100 mM glucose
Malate synthase
CR-16 50 mM CR-16 100 mM EC-11 50 mM EC-11 100 mM0
500
1000
1500
2000
2500
3000
3500
4000
4500
Are
a (p
ixel
s)
• Area covered under 50 mM is lesser compared to 100 mM glucose concentration in CR-16• EC-11 shows almost equal area covered under 50 mM and 100 mM glucose concentration
Real time PCR
EC11_100EC11_50
0
0.5
1
1.5
2
2.5
3
3.5
Isocitrate Dehydrogenase
Samples
RQ
• Expression in 50 mM is higher compared to 100 mM glucose concentration indicating higher IDH activity in 50 mM glucose for EC-11
EC11_100EC11_50
CR16_50CR16_100
012345678
Isocitrate Lyase
Samples
RQ
• Expression was higher for 100 mM glucose as compared to 50 mM glucose for both CR-16 and EC-11 indicating higher ICL activity at 100 mM glucose
CR16_100CR-16_50
EC-11_100EC-11_50
0
0.2
0.4
0.6
0.8
1
Malate Synthase
Samples
RQ
• Expression in 50 mM is lesser compared to 100 mM glucose concentration in CR-16• EC-11 shows almost equal expression in 50 mM and 100 mM glucose concentration
Summary of Gene Expression
Isolate IDH intensity ICL intensity MS intensity
CR-16 (50 mM) - Low Low
CR-16 (100 mM) - High High
EC-11 (50 mM) High Low Moderate
EC-11 (100 mM) Low High Moderate
• High intensity ICL and MS bands obtained in 100 mM glucose medium shows the presence of glyoxylate shunt in CR-16• Low intensity IDH band at 100 mM glucose concentration compared to 50 mM glucose concentration • EC-11 MS bands are of almost equal intensity indicating lack of malate over production• IDH gene amplification for CR-16 was not obtained due to inadequate initial m-RNA concentration
In vivo studies for effects of PGP activity of CR-16 and
EC-11 on chick pea growth
Plant growth experiment
CR-16 Control EC-11
Plant growth parameters
• Gopalkrishna et al., (2012) reported 39-65% increase in root length, shoot length and total dry weight by Streptomyces inoculation•Micromonospora endolithica promoted the growth of roots and shoots of bean plants (El-Tarabily et al.,2008)• Actinomycetes isolates CR-16 and EC-11 showed beneficial effects on chick pea development• Higher root length, shoot length, lateral roots, branches and plant biomass observed as compared to control
Plants Root length (cm) Shoot length (cm)
Shoot length increase (fold)
No. of lateral roots
No. of branches
Dry weight (mg)
Dry weight increase
(fold)
Control 1±0 2.5±0.70 - 1±0 4±0 265±0.04 -
CR-16 3.83±1.72 10.17±5.56 3 5±1.75 6±1.47 580±0.16 2.2
EC-11 5.00±1.41 16.00±1.41 5.4 10±0.70 10±0 550±0.01 2.0
Hydroponics- plant growth parameters
• The mechanisms by which PGPR promote plant growth are not fully understood but include : 1) ability to produce plant hormones (Mordukhova et al., 1991) 2) asymbiotic N2 fixation (Boddey and Dobereiner, 1995) 3) solubilization of mineral phosphate and other nutrients (De Freitas et al., 1997) •The production of hormones by PGPR in numerous reports indicate the importance of indolacetic acid (IAA) in the roots and shoots development (Aloni et al. 2006)• El tarabily (2008) reported PGP with S. filipinensis due to ability to produce IAA• CR-16 and EC-11 showed beneficial effects on chick pea growth in partial MS medium
Plants Root length (cm)
Shoot length (cm)
% increase with respect to
control
No. of lateral roots
No. of branches
Dry weight (mg)
% increase with respect
to control
Control 7.5±2.36 11.33±0.82 -- 10±1.18 7±1.89 120±0.001 --
CR-16 8.53±0.54 16.37±2.95 44% 15±1.93 11±0.61 120±0.024 --
EC-11 10.22±1.22 19.59±3.46 72% 15±2.25 10±0.92 130±0.006 8%
Hydroponics with rock phosphate
Plant growth parameters
• Saber et al., (2009) reported inoculation of mung bean with phosphate solubilizing fungi in presence of rock phosphate or calcium superphosphate increased significantly growth, seed yield and P-uptake • Plant promotion relies on the ability of the Actinomycetes to solubilize phosphate (El-Tarabily et al., 2008; Hamdali et al.,2008) •Root development in rock phosphate containing M.S. medium was low as compared to normal M.S. medium• EC-11 showed its beneficial effects on chick pea shoot development and CR-16 showed its beneficial effect on plant biomass
Root length (cm) Shoot length (cm)
Shoot length
% increase
No. of lateral roots
No. of branches
Dry weight (mg)
Dry weight % increase
Control 5.76±3.06 9.60±3.28 - 2±0.20 14±1.21 146±0.003 -
CR-16 4.08±0.26 8.57±0.28 - 1±0.80 13±0.91 169±0.018 15.75 %
EC-11 5.65±0.49 13.95±0.35 45 % 2±0.14 13±0.42 134±0.019 -
Hydroponics with commercial biofertilizer
Plant growth parameters
•Vessey (2003) defines biofertilizers as a substance which contains living microorganisms which, when applied to seed, plant surfaces, or soil colonizes the rhizosphere or the interior of the plant and promotes growth by increasing the supply or availability of primary nutrients the host plant.• Rhizobacteria, associated with rhizosphere, can fix nitrogen, and solubilizing phosphorus has been used as inoculum in nonleguminous species such as maize, rice, wheat, and sugar cane (Dobereiner 1997).• However, beneficial effects of isolates seen on shoot development and lateral roots development• Plant biomass in EC-11 inoculated plants was more as compared to control
Plants Root length (cm) Shoot length (cm)
Shoot length% increase
No. of lateral roots
No. of branches
Dry weight (mg)
Dry weight% increase/
decrease
Control 16.06±1.13 12.20±1.09 - 19±1.76 16±2.16 135±0.02 -
CR-16 15.10±1.33 14.33±1.21 17.3 % 26±2.23 12±3.78 117±0.06 -
EC-11 15.45±1.05 14.73±0.32 20.7 % 25±3.74 10±0.65 146±0.003 8.1 %
SUMMARY
CR-16 EC-11
MPS Ribose > Xylose > Lactose Fructose > Succinate > Glucose
Chitinase --- 1.27 units/ml
Phytase 0.68±0.007 units/ml 0.52±0.011 units/ml
Halotolerance Upto 10% NaCl Upto 10% NaCl
Mica 300 μg/ml 400 μg/ml
Pathway for malic acid Present: glyoxalate shunt present; malate synthase high
Absent: glyoxalate shunt present; malate synthase low
IAA + +
P Solubilization + +
Siderophore production + +
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