Succinate Mediated Repression of Gluconic Acid Dependent Phosphate Solubilization in

Rhizobium sp. Isolated From Nodules of Cajanus cajan and Sesbania rostrata

Abstract:

Two rhizobial strains which could solubilize TCP and RP under 25mM buffering conditions were
isolated. HPLC analysis indicated production of 19.2mM and 9.4mM Gluconic acid in the presence
of Glucose and Xylose respectively by Td3 and 29.6mM and 32.4mM Gluconic acid in the presence
of Glucose and Xylose respectively by SN1. Periplasmic Gluconic acid production indicates
presence of functional direct glucose oxidation pathway in these rhizobia. However, this P
solubilizing ability was repressed in the presence of succinate. Even though 28-40mM Gluconic
acid production was seen in media with glucose/ xylose and succinate, no detectable P release was
obtained. This implies that succinate represses P solubilization but not Gluconic acid production in
these isolates as shown by the HPLC and enzyme activity studies. This succinate mediated
repression of P solubilization may be one of the reasons of failure of PSBs in field conditions.

Key words:

Rhizobium, Phosphate Solubilization, Succinate, Catabolite Repression, Gluconic acid.

Abbreviations:

Introduction:

Rhizosphere, region of soil influenced by root secretion, supports multitude of active microbial
population that exerts beneficial, neutral as well as detrimental effects on plant
growthRhizobacteria, a class of root colonizing bacteria exerts various plant growth promoting
effects by direct and indirect mechanisms (Glick, 1995) and Mineral Phosphate Solubilization
(MPS) phenotype is one of the most important direct mechanism. Phosphorous (P) the second major
macronutrient next to Nitrogen (N) (Ezawa et al., 2002) is highly critical for plant growth and
development. However, bioavailability of P in soil is very limited because of its adsorption to
charged metal ions forming complexes which are unavailable for plant uptake. Even in fertile soil,
at pH 6.5 where P is most soluble, bioavailable P is not higher than 10µM (Gyaneshwar et al.,
2002). This makes employment of P solubilizing microbes for plant growth promotion an important
practice for enhancing plant nutrient uptake. Bacteria are effective P solubilizers than fungi and P
solubilizing bacteria and fungi constitute 1 to 50% and 0.1 to 0.5% of the soil microbial population
respectively. Major classes of Phosphate solubilizing bacteria (PSBs) include Pseudomonas (Patel
et al., 2011), Bacillus (Maheshwar and Sathiyavani, 2012), Arthrobacter, Phyllobacterium,
Rhodococcus, Serratia, Chryseobacterium, Delftia sp. (Chen et al. 2006), Enterobacter, Pantoea,
and Klebsiella (Rajput et al., 2013),etc. PSBs are known for solubilizing insoluble P compounds
like tricalcium phosphate, dicalcium phosphate, hydroxyapatite and rock phosphate (Goldstein,
1986) through organic acid secretion.

Rhizobia, class of gram negative diazotrophic N2 fixing bacteria living in symbiotic association with
legumes supplies plant with fixed N and gets energy from the plants in form of organic acids,
principally dicarboxylic acids. Rhizobia can invade the plant roots of legumes inducing nodule
formation in which the bacteria reduce atmospheric N 2 to ammonia and supply the plant with
nitrogenous compounds (Young, 1989) thereby promoting plant growth. Rhizobia can serve as
excellent PGPR for non-legumes as well (Antoun, 1998). In 1990, Halder et al., reported the ability
of Rhizobium sp. to solubilize inorganic P. Earlier reports of P solubilizing rhizobia include PQQ
dependent Gluconic acid production by Rhizobium sp, Agrobacterium sp. (Van Schie et al., 1987),
2-ketogluconic acid mediated P solubilization in Rhizobium leguminosarum biovar viciae (Halder et
al, 1990), hydroxyapatite solubilization by Rhizobium sp. and Bradyrhizobium sp. (Abd Alla, 1993),
TCP and inositol hexaphosphate solubilization by rhizobia (Sridevi and Mallaiah, 2009), TCP
solubilization by Sinorhizobium sp., Rhizobium sp., and Agrobacterium sp. isolated from Vigna
trilobata (Kranthi Kumar and Raghuram, 2014), etc. Enhancement of common bean growth by
PQQ dependent production of Gluconic and 2-ketogluconic acid has been shown in Rhizobium
tropici CIAT 899 (Young et al., 2003).

Like Pseudomonas sp., Rhizobium sp. also prefer organic acids over glucose and other sugars
(Ucker and Singer, 1978) and these dicarboxylic acids influence several metabolic functions. In E.
coli and B. subtilis, glucose represses utilization of other carbon sources by a mechanism termed as
Carbon catabolite repression (CCR). This CCR is cAMP dependent in E.coli whereas cAMP
independent in B. subtilis (Gorke and Stulke 2008). But for Pseudomonas sp. and Rhizobium sp.,
glucose is only a secondary carbon source and so mechanism reverse to what operates in E.coli
exists in these organisms, termed as Reverse CCR. The process is cAMP independent in
Pseudomonas sp. (Collier et al 1996) and in S. meliloti (Ucker and Signer 1978). Several reports
have shown the existence of catabolite repression in rhizobia. Presence of succinate reduces α-
glucosidase, β –glucosidase and β-galactosidase activity in R. meliloti (Ucker and Singer, 1978).
Saroso et al.1986, showed low activities of enzymes invertase, fructokinase, glucose-6 phosphate
dehydrogenase and ED pathway enzymes in the presence of succinate in Rhizobium sp. NGR234.
High activities of key enzymes of ED and PP pathway have been shown in sucrose, glucose and
fructose grown free living R. tropici CFN299 whereas when grown in C4 acids, activities of ED
enzymes were only about 30-38% of that of sucrose grown cells (Romanov et al, 1994). 40-50%
decreased activity of ED enzymes were obtained when cells were grown on succinate as compared
to glucose in R. meliloti (Irigoyen et al, 1990). Activities of key enzymes of PP and ED pathways
had 3-18 folds high activity in glucose as compared to succinate in Rhizobium sp. NGR234 (Saroso
et al, 1986) and Rhizobium meliloti (Finan et al, 1991). Hence, it can be said that presence of C 4
acids does not favour sugar uptake and metabolism in rhizobia. So, sugar utilization dependent
phenotypes like P solubilization may not be expressed in the presence of C 4 acids. C4 acid mediated
repression of P solubilization has been reported in very few bacterial species. Succinate repressed
oxalic acid mediated mps phenotype in Klebsiella pneumoniae (Rajput et al., 2013) whereas both
succinate and malate repressed Gluconic acid mediated mps phenotype in Pseudomonas aeruginosa
(Patel et al. 2011). The present study has been carried out with an aim to isolate and characterize P
solubilizing Rhizobium sp. for succinate mediated repression of their mps phenotype which is an
important reason for the failure of P solubilizers in field conditions where presence of multiple C
sources affect bacterial phenotypes.

Materials and Methods

Isolation and Characterization of rhizobia

Intact root nodules of Cajanus cajan and Sesbania rostrata were collected, surface sterilized using
0.1% HgCl2, crushed in a drop of sterile distilled water and plated on sterile yeast extract mannitol
agar with Congo red and incubated at 28°C for 48-72 hours. Opaque, white and mucoid colonies
were characterized biochemically as per Bergey’s Manual of Systematic Bacteriology and
molecularly through 16S rRNA sequencing using F27 (5’-AGAGTTTGATCATGGCTCAG-3’)
and R1492 (5’-TACGGTTACCTTGTTACGACTT-3’) by both end sequencing (forward and
reverse) and the sequences were submitted to NCBI database. Amplification of nodC and nifH
genes was carried out as described by Laguerre et al., 2001.

Determining the Phosphate solubilization ability of rhizobia

All the isolates were spot inoculated on Pikovskya’s agar plates (Pikovskaya, 1948) and 25mM Tris
buffered rock phosphate agar (TRP) with pH indicator methyl red (Gyaneshwar et al., 1998) to
check tricalcium phosphate (TCP) and rock phosphate (RP) solubilization respectively. P
solubilization was quantitatively estimated in 50mL Pikovskaya’s broth and 25mM Tris buffered
RP broth, incubated at 28°C for 4 days. Total carbon source supplemented was 100mM in TRP agar
and broth. Samples were withdrawn at 24 hr intervals, centrifuged at 9000 rpm for 10 mins and the
culture supernatant was used to determine the pH drop, soluble P (Ames 1966) and residual glucose
concentration using Glucose-SLR reagent (Labcare Diagnostics, India).

Determining repression of Phosphate solubilization

All the isolates were spot inoculated on Pikovskaya’s agar plates (Pikovskaya, 1948) supplemented
with equimolar concentration of succinate and 25mM Tris buffered rock phosphate agar (TRP) agar
with pH indicator methyl red (Gyaneshwar et al., 1998) with 100mM Glucose or Xylose and 50mM
Succinate to check repression of TCP and RP solubilization respectively. Repression of P
solubilization was quantitatively estimated in 50mL Pikovskaya’s broth with equimolar
concentration of Succinate and 25mM Tris buffered RP broth with 100mM Glucose and 50mM
Succinate, incubated at 28°C for 4 days. Samples were withdrawn at 24 hr intervals, centrifuged at
9000 rpm for 10 mins and the culture supernatant was used to determine pH drop, soluble P (Ames
1966) and residual glucose concentration using Glucose-SLR reagent (Labcare Diagnostics, India).

HPLC analysis of Organic acids produced

Cell were grown in TRP broth (Gyaneshwar et al., 1998) with 100mM Glucose, 100mM Xylose,
100mM Glucose with 50mM Succinate and 100mM Xylose with 50mM succinate, cells were
pelleted and the supernatant was filter sterilized using 0.22-μm Nylon filter and subjected to HPLC
analysis in Perkin Elmer and series 200 with Hypersil C18-column. 25mM KH 2PO4 with pH 2.5 set
with orthophosphoric acid was used as a mobile phase at a flow rate of 1mL/min and the organic
acids were detected using UV/VIS detector at 214nm. Gluconate, malate, citrate, acetate and
succinate were taken as standards. Retention time of the organic acid and area under peak were
compared with the standards to identify the type and concentration of the organic acid produced.

Effect of pH of Gluconic acid on TCP Solubilization

To determine the effect of pH of Gluconic acid on solubilization of TCP, Pikovskaya’s broth was
supplemented with 20mM Gluconic acid with pH set to 3.0, 4.0, 5.0, 6.0, 7.0 and 8.0 with HCl or
NaOH. pH of the media and P release (Ames, 1966) was checked before and after adding acid.

Physiological experiments
Isolates were grown in minimal medium with appropriate C sources which were autoclaved
separately. Overnight grown cells were grown in 100mL M9 medium with 10mM Glucose or
Succinate for studying monoauxic growth pattern and 5mM Glucose and 5mM Succinate each for
studying diauxic growth pattern. Flasks were incubated at 28°C on shaker incubator at 150 rpm,
cultures were taken at specific intervals to determine cell density and residual glucose
concentration. Increase in cell density was checked by measuring absorbance at 600nm and residual
glucose concentration was detected using Glucose-SLR reagent (Labcare Diagnostics, India).

Glucose Dehydrogenase enzyme activity

Cells grown in M9 minimal media, Pikovskaya’s and TRP broth were used as enzyme source for
determining gcd activity. Cells were washed and resuspended in same volume of 0.8% NaCl prior
to estimating enzyme activity to remove the residual media components. Cells were grown in M9
minimal media with 50mM Glucose, 50mM Succinate, 50mM Glucose with 50mM Succinate and
50mM succinate was added to cells growing on 50mM Glucose when OD at 600nm reaches 0.7. All
the four samples were allowed to grow till OD 0.7 and samples were withdrawn followed by sample
withdrawl every 1.5 hours upto 6 hours. For cells grown in Pikovskaya’s and TRP broth, sample
showing minimum pH was taken for determining gcd activity. Glucose dehydrogenase (gcd) assay
was performed by checking spectrophotometric reduction in absorbance of 2,6-
dichlorophenolindophenol (DCIP) at 600 nm (Eisenberg et al. 1972). Assay mixture included the
following components: 16.66 mM Tris-Cl buffer (pH 8.75), 66mM D-glucose, 0.05mM DCIP,
0.66mM phenazine methosulfate, 4mM sodium azide, whole cells and distilled water to final
volume 3 mL. Molar extinction coefficient of DCIP was taken as 15.1 mM-1cm-1 at pH 8.75. The
units of gcd activity was defined as μmol of DPIP reduced per minute .

Results and Discussion:

Isolation and Characterization of rhizobia

Nodule sap of Cajanus cajan and Sesbania rostrata was spread onto yeast extract mannitol agar
plates with congo red and a total of 6 isolates with opaque, white and mucoid colonies were taken
for biochemical characterization. All of the isolates were gram negative, lactose non fermenters,
starch hydrolysis negative, grew on nitrogen free media, showed phenylalanine deaminase activity,
were ketolactose negative, urease positive with no or very poor growth on peptone agar and could
tolerate 2% NaCl. All of them were characterized molecularly through complete 16S rRNA
sequencing and were found to be belonging to Rhizobium sp and was validated by nodC and nifH
amplification.
Determining the Phosphate Solubilization ability of rhizobia
All the 6 isolates were spot inoculated on Pikovskaya’s and TRP agar with Glucose and Xylose of
which two could solubilize TCP and RP after two days of incubation. TCP solubilization was seen
as a clear halo zone around the colony whereas RP solubilization was seen as red coloration around
the colony indicating acid production and pH reduction. Minimum pH of 4.1, 4.2 and 3.7 for
Rhizobium sp. Td3 and 3.3, 3.7 and 3.6 for Rhizobium sp. SN1 was obtained in Pikovskaya’s broth
and TRP broth with glucose and xylose respectively. Maximum P release from TCP was found to
be 423μg/mL and 428 μg/mL, P release from RP was found to be 23ug/mL and 15 μg/mL in the
presence of Glucose and 103ug/mL and 30μg/mL in the presence of Xylose for Rhizobium sp. Td3
and Rhizobium sp. SN1 respectively. Increased P release in the presence of Xylose showed that
Xylose may be a better Carbon source for P solubilization for these isolates. Maximum P release
was found after 2 days of incubation for Pikovskaya’s and TRP broth with glucose whereas it
increased for 4 days in TRP broth with Xylose. Several reports have shown TCP solubilization in
different bacteria. pH of ~6.0 with P release of 150-600ug/mL for Rhizobium sp. (Sridevi and
Mallaiah, 2009), pH 3.86 and P release of 130 ug/mL in Klebsiella sp.(Rajput et al., 2013), pH 5.3
and maximum P release of 510 ug/mL in S. kositense (Kranthi kumar and Raghu Ram, 2014) have
been reported. RP solubilization upto 77 ug/mL in Enterobacter asburiae PSI3 (Gyaneshwar et al.,
1998), 76.12mg/mL in P. aeruginosa M3 and 63.45mg/mL in P. aeruginosa SP1 (Patel et al., 2011)
have also been reported.

Td3 Td3 Td3

SN1 SN1
SN1
(a) (b) (c)
Figure 1: TCP and RP solubilization in (a) Pikovskaya’s agar and 25mM Tirs buffered RP
agar with (b) Glucose and (c) Xylose.

Determining repression of Phosphate solubilization

Both the isolates were spotted on Pikovskaya’s agar with equimolar concentration of Glucose and
Succinate and TRP agar with 100mM Glucose or Xylose with 50mM Succinate. However, no halo
zone or red coloration around the colonies was seen even after prolonged incubation. Instead, the
colony proximity turned dark indicating nutrient stress. This showed that succinate repressed P
solubilization as seen by no lowering of pH and no P release even after 4 days of incubation.
Succinate mediated repression of P solubilization has been reported earlier for Pseudomonas
aeruginosa (Patel et al., 2011) and Klebsiella pneumoniae (Rajput et al, 2013). Reports have shown
organic acid mediated repression of glucose utilization (Mandal and Chakrabartty, 1993) but
organic acid mediated repression of P solubilization has not been shown for Rhizobium sp.

Td3 Td3
Td3
SN1 SN1 SN1
(a) (b) (c)
Figure 2: Repression of TCP and RP solubilization in (a) Pikovskaya’s agar and 25mM Tirs
buffered RP agar with (b) Glucose and (c) Xylose.

pH and Residual Glucose (*10mM)

Piko G and GS Comparitive-Td3
P release-G P release-GS Piko G and GS Comparitive-SN1
500 pH-G pH-GS 12 P release-G P release-GS
pH and Residual Glucose (*10mM)

450 Glucose-GS Glucose-G pH-G Glucose-G

10 450 pH-GS Glucose-GS 12
400
350 400
P release (ug/ml)

8 10
350
300
P release (μg/ml)

300 8
250 6
250
200 6
4 200
150
150 4
100 2 100
50 2
50
0 0
0 0
0 1 2 3 4 0 1Time (In
2 days) 3 4
Time (In days)

(a) (d)
pH and Residual Glucose (*10mM)
pH and Residual Glucose (*10mM)

TRP G and GS Comparitive-Td3 TRP G and GS Comparitive-SN1

P release-G P release-GS P release-G P release-GS
pH-G pH-GS pH-G Glucose-G
25 Glucose-GS Glucose-G 12 16 pH-GS Glucose-GS 12

10 14
20 10
P release (ug/ml)

12
P release (μg/ml)

8 8
15 10
6 8 6
10
4 6
4
5 4
2 2
2
0 0 0 0
0 1 2 3 4 0 1 Time (In
2 days)3 4
Time (In days)

(b) (e)
 P release and pH change in TRP X P release and pH change in TRP-X
and XS-Td3 and XS-SN1
P release-X P release-XS P release -X P release-XS
35 9
pH-X pH-GS pH-X pH-XS
120 9 8
30
8
100 7
7 25

P release (μg/ml)
6
P release (ug/ml)

80 6
20 5
5
60

pH
4

pH
4 15
40 3 3
10
2 2
20
1 5 1
0 0
0 0
0 1Time (In2 days) 3 4 Time (In days)
1 2 3 4 5

(c) (f)
Figure 3: pH change, P release and residual Glucose concentration in (a, d) Pikovskaya’s
broth (b, e) TRP broth with Glucose and (c,f) TRP broth with Xylose of Rhizobium sp. Td3
and Rhizobium sp. SN1 respectively. Values are mean ± SD of 3 independent observations.

No significant decrease in pH and P release was obtained in Pikovskaya’s and TRP broth
supplemented with succinate as compared to media with Glucose and Xylose as sole C sources even
after 4 days of incubation. Complete repression of P solubilization in Td3and partial repression in
SN1 was observed from the quantitative studies. Rate of Glucose utilization was high and rapid in
media supplemented with Glucose and Succinate as compared to media with Glucose alone. In
media with Succinate and Glucose, the cell density at the start of Glucose utilization phase will be
higher than that in media with Glucose alone because of the increase in growth during succinate
utilization phase, which is a favoured C source for rhizobia. This may be one of the probable
reasons which may explain the increased rate of Glucose utilization in media supplemented with
Succinate.

HPLC analysis of Organic acids produced

TRP broth samples showing lowest pH and maximum P release in the presence of Glucose/Xylose
and the same day samples of TRP broth supplemented with Succinate in addition to Glucose/Xylose
were taken for HPLC analysis. HPLC profile of cell free supernatants of Rhizobium sp. Td3 and
Rhizobium sp. SN1 grown in TRP broth showed Gluconic acid as major acid responsible for P
solubilization. Trace amounts of malic acid, citric acid and succinic acid were also identified for
some samples. Gluconic acid produced in gram negative bacteria through periplasmic glucose
oxidation by quinoprotein glucose dehydrogenase is shown to result in most efficient P
solubilization (Hilda and Fraga, 1999). Gluconic acid production in these Rhizobium sp. indicates
the presence of holoenzyme quinoprotein glucose dehydrogenase and the direct glucose oxidation
pathway as reported earlier for S.meliloti (Boiardi et al., 1996) and in contrast to other existing
reports where Gluconic acid production by Rhizobium sp. was obtained only when PQQ was
externally supplemented (Van Schie et al., 1987; Boiardi et al., 1996; Young et al. 2003). Up to 4
fold increased Gluconic acid production was obtained in media supplemented with Succinate in
addition to Glucose/ Xylose as compared to Glucose/ Xylose alone. In addition, intact membrane
bound glucose is a prerequisite for effective symbiosis as gcd mutants of S. meliloti were found to
show delayed and reduced nodulation in addition to reduced competitiveness (Bernardelli et al,
2008).

Isolate Carbon source added pH of the Organic acid Organic acid

medium produced concentration
Rhizobium sp. Td3 100mM Glucose 4.2 Gluconic acid 19.2 mM
Malic acid 0.42mM
Rhizobium sp. Td3 100mM Glucose 7.1 Gluconic acid 40.6mM
50mM Succinate Malic acid 0.54mM
Citric acid 3.15mM
Succinic acid 0.54mM
Rhizobium sp. Td3 100mM Xylose 3.7 Gluconic acid 9.4mM
Malic acid 5.6mM
Citric acid 0.5mM
Rhizobium sp. Td3 100mM Xylose 7.0 Gluconic acid 40mM
50mM Succinate Malic acid 7.6mM
Citric acid 7.5mM
Succinic acid 0.66mM
Rhizobium sp. SN1 100mM Glucose 3.7 Gluconic acid 29.6mM
Malic acid 2.3mM
Citric acid 0.9mM
Rhizobium sp. SN1 100mM Glucose 4.7 Gluconic acid 28.2mM
50mM Succinate Malic acid 2.8mM
Citric acid 2.5mM
Rhizobium sp. SN1 100mM Xylose 3.6 Gluconic acid 32.4mM
Malic acid 8.6mM
Citric acid 1.1mM
 Rhizobium sp. SN1 100mM Xylose 4.6 Gluconic acid 36mM
50mM Succinate Citric acid 9.5mM
Succinic acid 1.8mM
Table 1: HPLC detection of organic acid production of both the isolates

Effect of pH of Gluconic acid on TCP Solubilization

pH of 1M Gluconic acid was set to 3.0-8.0 with HCl or NaOH and added to Pikovskaya’s broth to a
final concentration of 20mM. Media pH and P release was estimated before and after adding
Gluconic acid. P release was seen when pH of Gluconic acid was set to pH 3.0 and 4.0 which
altered the pH of the medium to 4.3 and 5.0 respectively from 7.1 This indicates that Gluconic acid
can solubilize TCP only when the pH of the medium is less than 5.0 which would explain why no P
solubilization was seen in Pikovskaya’s and TRP broth with Glucose and Xylose along with
succinate. Chelation property of Gluconic acid and its protons (Lin et al., 2006) are important
factors for its P solubilizing ability.
pH of 1M Media pH before P release (ug/mL) Media pH after P release (ug/mL)
Gluconic acid adding Gluconic before adding adding Gluconic after adding
acid Gluconic acid acid Gluconic acid
3.0 7.1 102.1 4.3 333
4.0 7.1 96.7 5.0 163
5.0 7.1 99.1 5.8 92
6.0 7.1 108.2 6.1 101
7.0 7.1 103 6.2 95
8.0 7.1 116 6.2 98

Table 2: Effect of pH on P solubilization ability of Gluconic acid

Physiological experiments
Isolates when grown on 10mM Glucose or 10mM Succinate showed monauxic growth pattern
whereas diauxic growth pattern was obtained when 5mM Glucose and 5mM Succinate each was
added to the medium. Samples were withdrawn every 1-2 hour to determine growth increase by
measuring the optical density at 600nm and residual glucose concentration. Isolate Rhizobium sp.
Td3 reached a maximum O.D of 2.4 and 0.96 in the presence of Glucose (10mM) and Succinate
(10mM) respectively and 1.6 when both of them were present in equimolar concentration (5mM +
5mM). In monoauxie, glucose utilization started after 6 hrs of inoculation and at the end of 26 hours
Glucose and was completely utilized whereas in diauxie, Glucose utilization started after 10 hours
and was completely utilized after 21 hours of inoculation. Isolate Rhizobium sp. SN1 reached a
maximum O.D of 1.74 and 0.72 in the presence of Glucose (10mM) and Succinate (10mM)
respectively and 1.02 when both of them were present in equimolar concentration (5mM + 5mM).
In monoauxie, glucose utilization started after 8 hrs of inoculation and at the end of 21 hours
Glucose and was completely utilized whereas in diauxie, Glucose utilization started after 11 hours
and was completely utilized after 17 hours of inoculation. Though several reports say that organic
acids are preferred over sugar for rhizobia (Ucker and Singer, 1978) growth rate was more in the
presence of Glucose as compared to Succinate.

Monoauxic growth curve in Glucose and Residual Glucose concentration (mM) Monoauxic growth curve in Glucose and
Succinate of Td3 Succinate of SN1
Growth in Succinate
Growth in Glucose
Growth in Glucose
2.5 Growth in Succinate 12
1.8 Glucose concentration 12

Residul Glucose Concentration (mM)

Glucose Concentration
10 1.6
2 10
1.4
8
OD at 600nm

1.5 1.2 8
OD at 600nm

6 1
6
1 0.8
4 0.6 4
0.5 0.4
2 2
0.2
0 0 0 0
0 2 4 6 8 9 10 12 14 16 18 20 22 24 0 4 8 10 12 14 16 18 20
Time (In hours) Time (In hours)

(a) (c)
Residual Glucose concentration (mM)

Residual Glucose Concnetration (mM)

Glucose Succinate Diauxie of
Glucose Succinate Diauxie of SN1
1.8 Td3 6 Growth
Growth Glucose concentration 1.20 6
Glucose Concentration
1.5 5
1.00 5
1.2 4
OD at 600 nm

0.80 4
OD at 600nm

0.9 3
0.60 3
0.6 2
0.40 2
0.3 1
0.20 1
0 0
0.00 0
0 2 4 6 8 10 12 13 14 15 16 17 18 20 21 22
1 2 3 4 5 6 7 8 9 10 11 12 13 14
Time (In hours) Time (In hours)

(b) (d)
Figure 4: Monoauxic (a, c) and Diauxic (b, d) growth profiles of Rhizobium sp. Td3 and
Rhizobium sp. SN1 respectively. Values are mean ± SD of three independent observations.
Glucose dehydrogenase enzyme activity
Both the isolates were grown on minimal media as described above and samples were withdrawn
every 1.5 hour to check the gcd activity. Gcd activity increased with time even in the presence of
succinate indicating that succinate which is known to repress intracellular glucose metabolizing
enzymes (Mandal and Chakrabartty, 1993) is not repressing periplasmic glucose dehydrogenase
activity. Increase in Gcd activity was higher for SN1 as compared to Td3. Maximum specific
activity of 13.2 and 20μmol/min/mg protein was obtained for Td3 and SN1 respectively.

25
Specific activity (μmol/min/mg protein)

Effect of Succinate on gcd activity in Effect of Succinate on gcd activity in

14 Rhizobium sp. Td3 13.2 Rhizobium sp. SN1

Specific activity (μmol/min/mg protein)

20
G-Gcd GS-Gcd G(S)-Gcd 20
12 G-Gcd GS-Gcd G(S)-Gcd
17
10.2
10
8.68.7 15

8
6.6 10.2
5.6 5.9 10 8.3
6 5.5 5.4
4.2 4.5
3.7 5.6
4 5 3.8
3.7
1.92.2
2 1 1.21.4
0
0
0 1.5 3 4.5
0 1.5 3 4.5
Time (In hours) Time (In hours)

(a) (b)
Figure 5: Effect of succinate on periplasmic gcd activity

Conclusion:
The study shows that Succinate which is known to repress expression of glucose
metabolism enzymes does not repress the expression of membrane bound
quinoprotein glucose dehydrogenase. However, Gluconic acid mediated P
solubilization phenotype of rhizobia is repressed in the presence of succinate because
of the preferential utilization of C4 acids by the rhizobia in the rhizosphere which
affects the field performance of most of the isolates.