Karishma et al.
Carbon source preference in Pseudomonas sp.
S1
FEMSLE-15-04-0276
Revised Manuscript
Supplementary data
Analysis of preference for carbon source utilization among three strains of
aromatic compounds degrading Pseudomonas
Karishma M+, Vikas D. Trivedi+, Alpa Choudhary, Akanksha Mhatre,
Pranita Kambli, Jinal Desai and Prashant S. Phale
Department of Biosciences and Bioengineering, Indian Institute of Technology-Bombay,
Powai, Mumbai, 400 076, India.
Pseudomonas sp. display the carbon source preference in the order, organic
acids>glucose>aromatic compounds. Whereas, Pseudomonas putida CSV86 prefers aromatic
compounds over glucose and co-metabolizes aromatics and organic acids (Basu et al., 2006,
Shrivastava et al., 2011). In the current study, the unique property of strain CSV86 was
investigated and compared with other soil isolates.
Aromatic compound adapted strain CSV86 showed diauxic growth profile in
aromatics plus glucose. Additionally metabolic studies suggest the utilization of aromatics
over glucose. Conversely results obtained in strains PP4, C5pp suggested the preference for
glucose over aromatics. Thus, to avoid bias in the results, growth profiles of three strains
were studied using glucose adapted culture as inoculum. Studies performed with cultures
grown on respective aromatic compounds as inoculum are presented in the main manuscript.
‘Supplementary data’, contains experiments pertaining to cultures grown on glucose as
inoculum.
Soil isolates Pseudomonas putida CSV86 (Mahajan et al., 1994), Pseudomonas
aeruginosa strain PP4 (Vamsee-Krishna et al., 2006) and Pseudomonas sp. strain C5pp
(Swetha and Phale, 2005) were grown on MSM (150 ml in baffled 500 ml Erlenmeyer flask,
Basu et al., 2006) containing aromatic compounds and glucose at 30°C at 200 rpm. The
glucose adapted cells were used as inoculum. The growth was monitored
spectrophotometrically at 540 nm. Concentration of reducing sugar in the medium was
measured using DNSA reagent with glucose as the standard (Miller et al, 1959). The enzymes
were assayed as described in the ‘Material and Methods’.
Glucose adapted strain CSV86 showed diauxic growth pattern on phenylpropanoids
plus glucose (Figure S1). The observed first lag-phase duration was significantly longer
(ranging from 2h for vanillic acid to 26h for vanillin) than that observed for aromatic grown
inoculum. During the growth on double carbon source, cells were assayed for glucose 6phosphate dehydrogenase (zwf) and protocatechuate 3,4-dioxygenase (PDO) activity. When
Karishma et al.
Carbon source preference in Pseudomonas sp.
S2
grown on veratraldehyde plus glucose, in the first log-phase the glucose concentration
remained constant and cells showed significantly less zwf (22.1 nmol min-1 mg-1) activity and
maximum PDO (541 nmol min-1 mg-1) activity. However, cells from second log-phase
showed gradual decrease in the glucose concentration and PDO activity with concomitant
increase in the zwf activity (102 nmol min-1 mg-1) (Figure S1A). Thus, irrespective of the
inoculum (aromatic or glucose grown), diauxic growth profile with preference of aromatics
over glucose was observed. The glucose adapted cells showed a longer duration for first lagphase of 8h in contrast to 2h when cells adapted on veratraldehyde was used as inoculum.
Similar diauxic growth profile with longer duration for the first lag-phase and enzyme
activity pattern were observed for other phenylpropanoid compounds plus glucose (Figure
S1 B-D). On phenylpropanoid compounds (vanillin or ferulic acid) plus succinate, strain
CSV86 showed a single log-phase (Figure S2). Thus, confirming the co-metabolism of
organic acids and aromatics as reported earlier.
The glucose adapted cells of strain PP4 showed a diauxic pattern with the maximum
zwf activity (267 nmol min-1 mg-1, 10h, Figure S3) in the first log-phase in tandem with
gradual decrease in the glucose concentration. The activity for PDO (314 min-1 mg-1, 26h)
was maximum in the second log-phase indicating the metabolism of terephthalate.
Interestingly, the duration of the second lag-phase observed to be 10h when the glucose
adapted culture was used as an inoculum as compared to terephthalate adapted culture (2h).
Overall, these results validate the utilization of glucose over terephthalate in strain PP4.
Similar results were observed for strain C5pp with glucose grown inoculum which gave a
diauxic growth profile with glucose (maximum zwf activity in the first log-phase) preferred
over carbaryl (maximum GDO activity in the second log-phase) (Figure S4).
Strains CSV86, PP4 and C5pp utilize benzoate as carbon source and follow same
degradation pathway via catechol 1,2-dioxygenase (C12DO). The glucose adapted cells of
strains CSV86, PP4 and C5pp were used as inoculums on benzoate plus glucose. Strain
CSV86 showed a distinct diauxic growth profile with utilization of benzoate over glucose
(Figure S5A). The maximum C12DO activity (271 nmol min-1 mg-1, 6h) was observed in the
first log-phase followed by increase in zwf activity (16 nmol min-1 mg-1, 17h) in the second
log-phase. Whereas, in strains PP4 and C5pp cultures showed a growth profile with glucose
being utilized in the early half of log-phase (Figure S5B & C). Though distinct diauxic
growth pattern was not observed, the glucose was completely consumed in the initial stages
of growth along with increase in zwf activity. The C12DO activity was maximum in the late
log-phase, indicating the preference of glucose over benzoate in these two strains.
1.5
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Enzyme activity, nmole mg -1min-1
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Glucose, mg ml-1
Growth, O.D. at 540 nm
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Growth, O.D. at 540 nm
A
Carbon source preference in Pseudomonas sp.
Enzyme activity, nmole mg-1min-1
Karishma et al.
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Time (h)
Figure S1. Growth and enzyme activity profile of P. putida CSV86 using glucose adapted culture as inoculum.
Solid circle represents the growth profile on double carbon sources: veratraldehyde (0.1%) plus glucose (0.25%) (A); ferulic acid (0.1%) plus
glucose (0.5%) (B); vanillin (0.1%) plus glucose (0.25%) (C); and vanillic acid (0.1%) plus glucose (0.25%) (D). Open circle represents the
glucose concentration in the medium. The specific activity of protocatechuate 3,4-dioxygenase (PDO, grey bar) and zwf (solid bar) is depicted
in panels (A-D).
Karishma et al.
Carbon source preference in Pseudomonas sp.
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Growth, O.D. at 540 nm
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Time (h)
Figure S2. Growth profile of P. putida CSV86 on phenylpropanoid plus succinate. Open circle
represents growth profile of CSV86 in ferulic acid (0.1%) plus succinate (0.25%) and solid
inverted triangle represents vanillin (0.1%) plus succinate (0.25%). Culture grown on respective
aromatics was used as inoculum.
Carbon source preference in Pseudomonas sp.
PDO
ZWF
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Time (h)
GDO
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Enzyme activity, nmole mg-1min-1
Figure S3. Growth and enzyme activity profile of P. aeruginosa strain PP4 on terephthalate
(0.1%) plus glucose (0.05%) using glucose adapted culture as inoculum. Solid circle
represents the growth profile, open circle represents the glucose concentration in the medium,
grey bar represents protocatechuate 3,4-dioxygenase (PDO) and solid bar represents the
specific activity of zwf.
Time (h)
Figure S4. Growth and enzyme activity profile of Pseudomonas sp. C5pp on carbaryl (0.1%)
plus glucose (0.025%) using glucose adapted culture as inoculum. Solid circles represents
growth profile, open circle represents the glucose concentration in the medium. The specific
activities of enzyme involved in carbaryl (GDO, open bar) and glucose (zwf, solid bar)
degradation are depicted.
Carbon source preference in Pseudomonas sp.
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A
C12DO
ZWF
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Karishma et al.
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Time (h)
Figure S5. Growth and enzyme activity profile of P. putida CSV86, P. aeruginosa strain PP4
and Pseudomonas sp. C5pp. Solid circles represent growth profile of (A) strain CSV86 on
benzoate (0.1%) plus glucose (0.25%), (B) strain PP4 on benzoate (0.1%) plus glucose
(0.05%) and (C) strain C5pp on benzoate (0.1%) plus glucose (0.025%). The inoculum used
was prepared by growing respective strains on glucose. Open circle represents the glucose
concentration in the medium. The specific activities of C1,2DO (grey bar) and zwf (solid bar)
are depicted in panel (A-C).