Preferential Utilization of Aromatic Compounds over Glucose by Pseudomonas putida CSV86

Pseudomonas putida CSV86, a naphthalene-degrading organism,exhibited diauxic growth on aromatic compounds plus glucose,with utilization of aromatics in the first log phase and ofglucose in the second log phase. Glucose supplementation didnot suppress the activity of degrading enzymes, which were inducedupon addition of aromatic compounds. The induction was inhibitedby chloramphenicol, suggesting that de novo protein synthesiswas essential. Cells showed cometabolism of aromatic compoundsand organic acids; however, organic acids suppressed glucoseutilization.

INTRODUCTION

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Introduction

The most effective and economical way to remove aromatic pollutantsis by means of microbial degradation (1). However, microorganismsin nature show a preference for a simple carbon source suchas glucose, and unless it is completely depleted, the complexcarbon sources such as aromatic compounds are not degraded (3).Therefore, the growth response on two carbon sources is diauxic,reflecting their sequential utilization. During the first growthphase, the simple carbon source is utilized and the enzymesrequired for the utilization of the second carbon source arerepressed. This phenomenon of carbon catabolite repression (CCR)has been shown to occur in several microorganisms. A well-studiedexample is the repression of lactose utilization by glucosein Escherichia coli (30). Here CCR is mediated through cyclicAMP (cAMP) (7, 18). CCR in nonenteric bacteria such as pseudomonadsis not clearly understood. Irrespective of the carbon source,the intracellular cAMP levels and adenylate cyclase remain constant,and external addition of cAMP does not alter the repression(20, 28). In pseudomonads, organic acids are found to suppressglucose uptake and its catabolizing enzymes (6, 16, 17, 23,32). Enzymes necessary for the utilization of amide (29), histidine(20), protocatechuate (35), and xylene (4, 34) are suppressedin the presence of organic acids. Catabolite repression at thetranscriptional level by glucose, gluconate, and organic acidshas been reported for the enzymes involved in catechol and chlorocatecholdegradation (13) and for those involved in methyl phenol degradation(15). Glucose is known to repress the enzymes responsible forbenzyl alcohol degradation in Pseudomonas putida (5) and todelay induction of the phenylacetic acid transport system inP. putida U (26). Recently, repression of phenanthrene degradationin P. putida by a plant root extract and exudates containingglucose, acetate, and amino acids has been reported (22). Thus,preferential utilization of a simple carbon source repressesthe degradation of complex compounds, leading to their accumulationin nature, thereby locking off the carbon and aggravating pollution.Attempts have been made to engineer organisms for efficientutilization of aromatics in the presence of glucose (24). However,the stability and viability of such strains in nature posesa challenge.

We report that Pseudomonas putida CSV86 shows an unusual preferencefor aromatics when grown on an aromatic compound plus glucose.The strain cometabolizes aromatic compounds plus organic acids,and organic acids suppress glucose utilization.

Growth conditions, chemical estimations, and enzyme assays.

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Growth conditions, chemical...

Pseudomonas putida CSV86 (12) was grown on 150 ml mineral saltmedium (MSM) (2) at 30°C on a rotary shaker. Aromatic compounds(0.1%), glucose (0.25%), or organic acids (0.25% succinate orcitrate, or 0.1% pyruvate) were added aseptically as carbonsources either alone or in combination. Growth was monitoredat 540 nm. Reducing sugar concentrations were estimated as describedby Miller (14) using glucose as a standard. Salicylate was estimatedby the ferric nitrate-HCl reagent (33) using salicylic acidas a standard. Cell extracts were prepared as described earlier(2). Protein was estimated as described by Lowry et al. (11)using bovine serum albumin as a standard. 1,2-Dihydroxynaphthalenedioxygenase (12DHNO) (31), benzyl alcohol dehydrogenase (BADH)(27), catechol 1,2-dioxygenase (C12O) (8), catechol 2,3-dioxygenase(C23O) (19), and glucose 6-phosphate dehydrogenase (ZWF) (10)were monitored. Specific activities are expressed as nanomolesper minute per milligram of protein.

Growth profiles.

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Growth profiles.

P. putida CSV86 utilized naphthalene, methylnaphthalenes, benzylalcohol, salicylate, and benzoate as the sole source of carbonand energy (2, 12). It also utilized glucose, glycerol, pyruvate,succinate, and citrate but failed to grow on gluconate, 2-ketogluconate,mannitol, or fructose. Pseudomonads sequester glucose as gluconate,and under carbon-limiting conditions the sequestered gluconateis used as a carbon source (9, 25). The inability of CSV86 togrow and respire on gluconate and 2-ketogluconate, the absenceof gluconate oxidase activity (data not shown), and the presenceof ZWF activity suggest that the organism utilizes glucose bythe phosphorylative pathway and that the direct oxidative pathwayis absent. Figure 1 depicts the growth profile on naphthaleneplus glucose by using naphthalene- or glucose-grown cells (Fig.1A or B, respectively) as an inoculum. Cells showed a diauxic(biphasic) pattern. The first growth phase of the diauxic profileoverlapped with the naphthalene growth profile (Fig. 1), andthe medium showed a characteristic olive-green color, indicatingthat naphthalene was utilized. As cells entered the second logphase, the glucose concentration declined (Fig. 1). Irrespectiveof the carbon source of the inoculum, cells grew slowly on glucose(Fig. 1). Varying the concentration of naphthalene (0.025 and0.05%) or glucose (0.25, 0.5, or 1%) in a double-carbon-sourcemedium yielded growth profiles with a shorter duration of thefirst log phase and a longer duration of the second log phase,respectively (data not shown). Cells showed a diauxic growthprofile on salicylate plus glucose (Fig. 2A), benzyl alcoholplus glucose (Fig. 2B), and benzoic acid plus glucose (Fig.2C) with utilization of salicylate (Fig. 2A) in the first andglucose (Fig. 2A, B, and C) in the second log phase. These resultssuggest that this strain utilizes aromatics in the first logphase and glucose in the second log phase.

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FIG. 1. Growth profile of P. putida CSV86 on 0.1% naphthalene plus 0.25% glucose ( ${triangledown}$ ), 0.1% naphthalene ( ${circ}$ ), or 0.25% glucose ( ${square}$ ). Cells grown on naphthalene (A) or glucose (B) were used as an inoculum. Solid symbols represent reducing sugar concentrations in the spent medium: ${blacktriangledown}$ for cells grown in naphthalene plus glucose and • for naphthalene-grown cells.

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FIG. 2. Growth profile of P. putida CSV86 on 0.1% aromatics plus 0.25% glucose ( ${triangledown}$ ), 0.1% aromatic compounds ( ${circ}$ ), or 0.25% glucose ( ${square}$ ). Aromatics used were salicylate (A), benzyl alcohol (B), and benzoate (C). Cells grown on the respective aromatic compound were used as an inoculum. Measured salicylate ( ${blacksquare}$ ) and glucose ( ${blacktriangledown}$ ) concentrations in the culture media are shown.

Organic acids are known to suppress utilization of aromatics(3-5, 13, 15, 21, 22, 26, 34). On naphthalene plus succinate(Fig. 3A) and salicylate plus succinate (Fig. 3B), cells didnot show a biphasic growth profile and utilized salicylate inthe log phase. Other combinations, such as naphthalene pluspyruvate, salicylate plus organic acid, and benzyl alcohol plusorganic acid, with cells grown either on aromatics or on organicacid as an inoculum gave similar results (data not shown). Whengrown on glucose plus succinate, cells showed a diauxic profile,with the first growth phase overlapping with the growth profileon organic acid alone, while the glucose concentration in themedium declined in the second log phase (Fig. 4). Inocula preparedeither on succinate (Fig. 4A) or on glucose (Fig. 4B) did notalter the organism's preference for organic acid utilization.Glucose plus pyruvate and glucose plus citrate gave similarprofiles (data not shown). Suppression of glucose utilizationby organic acids has been reported for several pseudomonads(3, 6, 17, 30, 32).

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FIG. 3. (A) Growth profile of P. putida CSV86 on 0.1% naphthalene plus 0.25% succinate ( ${triangledown}$ ), 0.1% naphthalene ( ${circ}$ ), or 0.25% succinate ( ${square}$ ). (B) Growth profile of P. putida CSV86 on 0.1% salicylate plus 0.25% succinate ( ${triangledown}$ ), 0.1% salicylate ( ${circ}$ ), or 0.25% succinate ( ${square}$ ). ${blacksquare}$ , salicylate concentration in the medium. Naphthalene- or salicylate-grown cells were used as an inoculum.

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FIG. 4. Growth profile of P. putida CSV86 on 0.25% succinate plus 0.25% glucose ( ${triangledown}$ ), 0.25% succinate ( ${circ}$ ), or 0.25% glucose ( ${square}$ ). Cells grown on succinate (A) or glucose (B) were used as an inoculum. ${blacktriangledown}$ , glucose concentration in the medium.

Enzyme activity profiles.

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Enzyme activity profiles.

On naphthalene plus glucose, cells showed maximum activity ofC23O in the first log phase and ZWF activity in the second logphase (Fig. 5A). Similarly, cells grown on benzyl alcohol plusglucose showed maximum activity of BADH and C12O in the firstlog phase and ZWF activity in the second log phase (Fig. 5B).The activities of 12DHNO and C23O (Fig. 5C) and of BADH andC12O (Fig. 5D) were maximal in log-phase cells grown on naphthaleneplus pyruvate and benzyl alcohol plus succinate, respectively.Similar results were observed with other combinations (datanot shown). The activity of ZWF from succinate-grown cells waslow (specific activity, 7). Cells grown on glucose plus succinateshowed significantly higher ZWF activity (specific activity,21) in the second log phase of the diauxic growth profile thanin the first log phase (specific activity, 11). These resultsindicated that succinate suppressed ZWF activity.

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FIG. 5. Activity profiles of the enzymes involved in utilization of aromatics and glucose from cells grown on 0.1% naphthalene plus 0.25% glucose (A), 0.1% benzyl alcohol plus 0.25% glucose (B), 0.1% naphthalene plus 0.1% pyruvate (C), and 0.1% benzyl alcohol plus 0.25% succinate (D). Cells grown on the respective aromatic (for A and B) or organic acid (for C and D) were used as inocula. Growth profiles on double carbon sources are indicated by ${triangledown}$ and specific activities of the enzymes by bars as indicated.

When naphthalene or benzyl alcohol was provided as the secondcarbon source to mid-log-phase glucose-grown cells, the activitiesof C23O (Fig. 6A), BADH, and C12O (Fig. 6E) increased significantlyand the activity of ZWF declined marginally. When glucose wasadded as the second carbon source to aromatic-grown cells, theactivities of C23O (Fig. 6B) and of BADH and C12O (Fig. 6F)reached a maximum by 3 and 1 h, respectively. The increase inZWF activity was followed by a concomitant decrease in the aromaticcompound-degrading enzymes. Addition of a protein synthesisinhibitor, chloramphenicol (Cm), along with the second carbonsource showed no further increase (induction) of aromatic- orglucose-utilizing enzymes when aromatics or glucose was addedas the second carbon source (Fig. 6C, D, G, and H). These resultssuggest that addition of glucose failed to induce ZWF or suppressthe aromatic degradation enzymes and that the increase in enzymeactivity (induction) was due to de novo synthesis of proteins.

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FIG. 6. Induction of key enzymes involved in the metabolism of aromatics and glucose in response to a second carbon source. Cells were grown to mid-log phase on glucose (0.25% for 18 h), naphthalene (0.1% for 6 h), or benzyl alcohol (0.1% for 6 h), and then the desired second carbon source (given below after the word "plus") was added either in the absence or in the presence of chloramphenicol (Cm; 100 µg/ml) at 0 h. Enzyme activities (bars) were monitored in the cell extracts. (A) Glucose plus naphthalene; (B) naphthalene plus glucose; (C) glucose plus naphthalene and Cm; (D) naphthalene plus glucose and Cm; (E) glucose plus benzyl alcohol; (F) benzyl alcohol plus glucose; (G) glucose plus benzyl alcohol and Cm; (H) benzyl alcohol plus glucose and Cm. Note that different scales are used on the y axis for each panel.

The data presented clearly show that P. putida CSV86 preferentiallyutilizes aromatic compounds over glucose. This is a novel propertyand has not been reported so far. This strain cometabolizesaromatics plus organic acids, and organic acids suppress glucoseutilization. Preferential utilization of aromatics by this straincould be due to either (i) inability of glucose to suppressthe aromatic-degrading enzymes, (ii) modulation of glucose uptake,(iii) suppression of glucose utilization enzymes by aromatics("reverse CCR") or organic acids produced during the degradationof hydrocarbon, or (iv) the combination of all or some of theseevents. The unusual carbon source preference by P. putida CSV86provides opportunities for bioremediation of aromatic compoundseven in the presence of simple carbon compounds such as glucoseand organic acids in the environment.

ACKNOWLEDGMENTS

A.B. thanks University Grants Commission, India, for the awardof a senior research fellowship.

FOOTNOTES

* Corresponding author. Mailing address: Biotechnology Group, School of Biosciences and Bioengineering, Indian Institute of Technology—Bombay, Powai, Mumbai 400 076, India. Phone: 91 22 2576 7836. Fax: 91 22 2572 3480. E-mail: pphale{at}iitb.ac.in.

REFERENCES

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