Folia Microbiol. 34,185-194 (1989)

Production of Acid and Alkaline Phosphatasesby Myxococcus coralloidesF. GONzALEZ*, J. MUNOZ, J.M. ARIAS and E. MONTOYA

Departmento de Microbiologia, Facultad de Ciencias, Universidad de Granada, 18071 Granada, Spain

Received October 17, 1988

ABSTRACT. Acid and alkaline phosphatase of Myxococcus coralloides were examined during vegeta­tive growth in a liquid medium. Two extracellular phosphatases and two cell-bound phosphatases, acidand alkaline in both cases, were produced. The phosphatase production was unaltered by the presence ofhigh concentrations of inorganic phosphate. Both enzymes were produced constitutively. These twohydrolases were released into the growth medium during the exponential growth phase (approximately10 % of total activity). The production of these enzymes was modified by the presence of organic acidsand metal ions in the medium.

Myxobacteria produce a number of extracellular compounds such as antibiotics,lytic enzymes and polysaccharides (Rosenberg and Varon 1984). They secretea variety of enzymes that hydrolyze proteins, nucleic acids, fatty acid esters,polysaccharides and peptidoglycan (Shimkets 1984).

Phosphatases are enzymes that release phosphate from phosphate esters and theirrole is to supply phosphate under conditions of inorganic phosphate deprivation.These enzymes are widely found in bacteria. Although the phosphatases arerepressed by orthophosphate in many bacteria (Shah and Blobel1967; Cheng et a1.1970; Nesmeyanova et a1. 1981; Von Tigerstrom 1984), they are constitutive inother cases (Mau-Haui and Blumenthal 1961 ; Cheng and Costerton 1973; Poirierand Holt 1983).

We have reported on acid and alkaline phosphatase (EC 3.1.3.2 and 3.1.3.1)activities during the life cycle of Myxococcus coralloides (Gonzalez et a1. 1987). Thispaper describes the phosphatase production by M. coralloides during its vegetativegrowth in liquid medium. The effect of inorganic phosphate and other substances onphosphatase production are also reported.

MATERIAL AND METHODS

Organism and culture conditions. Myxococcus coralloides strain D obtained inour laboratory (Arias and Montoya 1978), was grown in cr liquid medium(containing potassium phosphate, ~O mmol/L) as described by Arias et a1. (1983).

*Corresponding author.

'186 F. GONZALEZ et al. Vol. 34

The CT liquid medium was modified by the elimination of phosphates (low-Pi-CT medium) and by the addition of potassium phosphate (20 mmol/L; high-Pi-CT medium). A defined amino acid medium (the M1 medium of Witkin and Rosenberg 1970) was supplemented with (g/L) aspartate (500), L-glutamate (2000), (NI-I4)2504 (60), starch (100), dj enkolic acid (17), pantothenic acid (33), spermidinq (33), nicotinamide (3) and biotin (3). The concentration of organic acids (as sodium salts) and metal ions (as sulfates, except arsenate added as a primary sodium salt) used in the CT medium was 2 g/L and 1 mmol/L, respectively. All media were adjusted to pH 6.5. Growth was measured by following absorbance at 650 nm. The amino acids were obtained from Sigma; all the other chemicals were from commercial sources.

Analytical and enzyme assays. Protein was determined by the C0omassie blue method (Sedmak and Grossberg i977). Inorganic phosphate concentration was determined by the method of Ames (1966). The supernatant or cell-bound phosphatase activity was assayed as follows: a portion of the supernatant or cell (1 mL) was treated with toluene, and 0.6 mL was added to 2.4 mL 4-nitrophenyl- phosphate (3 mmol/L, in Tris-HCl, 50 mmol/L, at pH 8.5 for alkaline phosphatase or in sodium acetate, 50 retool/L, at pH 4.5 for acid phosphatase). The mixture was incubated for 30 min at 28 ~ The reaction was stopped by adding NaOH (1 tool/L, 1 mL) and the cells were removed by centrifugation and absorbance at 410 nm was determined. A unit of activity was defined as an increase of 1.0 A41o unit produced per h. The specific activities (U per mg protein) shown are the mean of at least three different assays from separate cultures. Puromycin was obtained from Sigma Chemical Co. (USA).

Replacement culture and puromycin treatment. The effect of inorganic phosphate on phosphatase activities was studied in a system based on a replacement culture. Exponentially growing cells (absorbance 0.3-0.5 at 650 nm) were harvested by centrifugation, washed once in sterile distilled water and transferred, at the same absorbance, to fresh medium. The cell-growth and enzyme activities were assayed 0, 30, 60 and 90 min after change of medium. The media used were the low-Pi-CT medium and the high-Pi-CT medium.

Recovery of phosphatase activity after puromycin treatment was studied as follows. Puromycin (20 mg/L) was added to exponentially growing cells (absorbance 0.3-0.5 at 650 nm). The cell growth and enzyme activities were assayed at different times after the puromycin addition. When the enzyme activities decreased about 5 -10 fold the cells were harvested by centrifugation, washed twice in a fresh medium without antibiotic and transferred to a fresh medium. The cell growth and enzyme activities were assayed 0, 15, 30, 45, 60 and 120 min after the change of medium. The media used were again the low-Pi-CT medium and the high-Pi-CT medium.

RESULTS

The growth of M. coralloides and the production of acid and alkaline phosphatase

1989 PRODUCTION OF PHOSPHATASES BY M. coralloides 157

in a low-Pi-CT medium, a high-Pi-CT medium and a defined medium are shown in Table I. Cell growth was not affected by the addition of potassium phosphate (20 mmol/L), although in the high-Pi-CT medium the lysis phase was delayed. Nevertheless, the growth rate in a defined medium was slower and the maximum absorbance reached was only 0.45. Two extracellular phosphatases and two cell-as- sociated phosphatases were produced. Prior to studying the production of phosphat- ases, the activity of extraceUular and cell-bound phosphatase was tested at dif- ferent pH values. They were assayed using the culture supernatants and toluene- treated cells as enzyme sources. In both cases the acid activity was optimal at pH 4.5, the alkaline at pH 8.5. These pH values were conserved for posterior assays. All assays were carried out in buffer (Tris or acetate) with 4-nitrophenylphosphate (3 mmol/L) as substrate.

The specific activities of cell-bound acid and alkaline phosphatases after growth in the low-Pi-CT medium was similar to that observed in the high-Pi-CT medium. In the low-Pi-CT medium, the inorganic phosphate content was reduced by the omission of phosphate salts; Casitone served as the sole source of inorganic phosphate. This culture medium was analyzed for inorganic phosphate and found to contain a concentration of 1.5 mmol/L. When potassium phosphate (20 mmol/L) was added to the CT-medium (high-Pi-CT medium), there was no inhibition of enzyme production. In these media the cell-associated phosphatases were produced throughout the growth phase and the specific activity increased during growth, and decreased in the lysis phase (after 50 h of cultivation). With respect to total activity, the cell-bound phosphatases increased in parallel with the increase in cell culture density.

The possible presence of organic phosphate in Casitone was circumvented by using the defined medium. In this medium, with various levels of inorganic phosphate, no inhibition of phosphatase activity was apparent.

The specific activities of extracellular enzymes were greater during the first hours of growth and decreased afterwards in the phase growth. Besides the specific activities increased during the lysis phase, probably due to cell lysis; thus in the high-Pi-CT medium this increase was smaller since in this medium lysis was delayed. In the low-Pi-CT medium supernatant acid and alkaline phosphatase was 10 and 13 %, respectively, of the total activity (supernatant and cell-bound activity). The addition of phosphate (20 mmol/L) had little effect on extracellular enzymes, increased the acid one 1.3-fold and decreased the alkaline one 1.3-fold. Thus the percentage of total activity was 13 % for acid and 9 % for alkaline phosphatase. These changes in the specific activities were not caused by differences in the amount of extracellular protein since the total extracellular protein per cell was constant throughout growth (Gonzfilez et al. 1987).

When inorganic phosphate was increased 20-fold there was no decrease in phosphatase production; thus both the acid and the alkaline phosphatase were constitutive (nonrepressible).

Shifts between high-Pi-CT and low-Pi-CT media had no significant effect on

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specific activities (Table II). The changes shown correspond to 60 min after thechange of medium. No changes were observed after this time. Variations in cell massduring culture in fresh medium for 60-90 min were not significant. In addition, westudied the recovery of phosphatase activity after incubation with an inhibitor ofprotein synthesis (20 mglL of puromycin; Table III). The orthophosphate concen­tration does not appear to be an important factor in the recovery of phosphataseactivities. The increase in cell mass during the time of incubation without antibioticwas not significant. These results agree with our initial studies of production. .

Table IV summarizes the effects of some organic acids and ions on phosphataseproduction. Supplementation of cr medium with succinate, Fe3+ and Zn2+ had littleor no effect on phosphatase production. Likewise, cell-bound acid phosphatase wasnot affected by the addition of citrate, malate and Cu2+, or the cell-bound alkalinephosphatase by the copper ion. However, the cell-bound acid phosphatase activityincreased 1.8-fold under the effect of AsOl-. Citrate and arsenate increased thecell-bound alkaline phosphatase production 1.8- and 2.6-fold, respectively. Malate,citrate and copper ion increased the supernatant acid phosphatase activity 4-fold,1.9-fold and 2-fold, respectively. Likewise, malate and copper ion increased thesupernatant alkaline phosphatase activity about 3-fold; citrate and arsenate in­creased this activity about 2-fold. The addition of malate to crmedium decreasedthe cell-bound alkaline phosphatase activity 2-fold. In all cases the assays werecarried out with an exponentially growing culture (absorbance 0.3-0.4 at 650 nm).Thus the differences in cell mass were not significant.

TABLE IV. Effects of organic acids and metal ions on phosphatase production of M. coralloidu·

Addition

None

CitrateMalateSuccinate

As0 43­

Cu2 +Fe3 +

Zn2+

Acid phosphatase Alkaline phosphatasespecific activity specific acti\'ity

Cell Supernatant Cell Supernatant

9.22 0.83 4.72 0.62

9.48 1.61 8.77 1.218.96 3.32 2.25 2.249.49 0.90 4.86 0.58

17.5 1.06 12.2 1.249.68 1.66 4.67 1.899.12 0.89 4.48 0.608.63 0.78 4.6U 0.65

.Carrieu out with culture in exponential growth, at the same absorbance (U.3 - 0.4 at 650 nm).The basal medium was CT. The concentrations of organic acids and metal ions were 2 giL andI mmollL, respectively.

DISCUSSION

M. coraHoides acid and alkaline phosphatase appeared to be nonrepressible by

192 F. GONZALEZ et al. Vol. 34

orthophosphate which contrasts with other bacteria. Escherichia coli alkaline phosphatase was decreased 300-fold in the presence of inorganic phosphate (2.3 mmol/L) (Done et al. 1965) and in Pseudomonas aeruginosa there was a 500-fold decrease in alkaline phosphatase activity in the presence of inorganic phosphate (7 retool/L) (Cheng et al. 1970). In Bactedonema and Actinomyces, alkaline phosphatase was inhibited by Na2HPO4 (10 retool/L) (Franker et al. 1978). However, constitutive phosphatase has been reported in other bacteria, e.g. gram-negative rumen bacteria (Cheng and Costerton 1973) and various strains of E. coli, Staphylococcus aureus and Neurospora crassa (Mau-Haui and Blumenthal 1961).

Among gliding bacteria, phosphatases from Capnocytophaga species are pro- duced constitutively (Poirier and Holt 1983). Lysobacter enzymogenes, a non-fruit- ing gliding bacterium, produces two alkaline phosphatases: extracellular and cell-associated. The production of both phosphatases, especially the extracellular enzyme, is reduced by inorganic phosphate (Von Tigerstrom 1984).

Because phosphate starvation causes a derepression of phosphatases, it was suggested that orthophosphate itself is the corepressor. However, phosphatases are not repressed in other bacteria, thus phosphate deprivation does not seem to be an important factor. Phosphatase synthesis is regulated by organic acids (Hochberg and Sargent 1973), nucleotide species (Wilkins 1972), metal depletion or metal addition (Arnold and Garrison 1973 ; Hochberg and Sargent 1973 ; Spencer et al. 1981 ; Arnold et at. 1983). Moreover, mutations that decrease phosphate transport lead to constitutive expression of these enzymes, even though intracellular phosphate concentrations may be high (Willsky et al. 1973). Thus, the transport of phosphate rather than the absolute concentration of phosphate may act as a signal for these enzymes (Gottesman 1984). Arsenate inhibits inorganic phosphate transport (Willsky and Malamy 1980). This ion-increased phosphatase production in M. coralloides could be due to a decrease in inorganic phosphate in the ceils caused by arsenate inhibition on phosphate transport.

The constancy of the amount of extracellular protein produced per cell, in contrast to the variations in enzyme activities in the broth of different media, has also been described for other secreting bacteria (Coleman 1981; Nicaud et al. 1984). Although the phosphatase production by M. coralloides was not affected by phosphate concentration, this ion delayed the lysis phase during its vegetative growth (Fern~ndez-Vivas et al. 1983) and inhibited the process of fructification at high concentration (Gonzfilez et al. 1987).

Myxobacteria produce and secrete a number of extracellular enzymes in large amounts and these enzymes have a nutritional role. Cells that live in a dense swarm accumulate a high local concentration of hydrolytic enzymes that support a faster growth rate by providing the cells with more nutrients (Rosenberg et al. 1977). It is reasonable to suggest that constitutive enzyme production and secretion would be advantageous for degradation of nutritional substrates in myxobacterial swarms.

This work was supported by a grant from Comision Asesora de Investigacion Cientifica y Tecnica.

1989

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PRODUCfION OF PHOSPHATASES BY M. coralloides 193

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