Regulation of Extracellular Protease Secretion in Pseudomonas ...

JouRNAL or BACruOLOGY, Sept. 1975, p. 954-961 Copyright X) 1975 American Society for Microbiology

Vol. 123, No. 3 Printed in U.S.A.

Regulation of Extracellular Protease Secretion in Pseudomonas maltophilia ROBERT S. BOETHLING Department of Bacteriology, University of California, Los Angeles, California 90024

Received for publication 6 May 1975

Cells grown in minimal medium and harvested in late exponential phase secreted extracellular protease linearly when suspended at high density in fresh medium. If the cells were suspended with 0.2% (wt/vol) yeast extract and no additional carbon source, the rate of exoenzyme production was increased several-fold. When pyruvate, L-malate, succinate, or a-ketoglutarate was added, repression of exoenzyme secretion was observed. The most effective repressor was a-ketoglutarate. These compounds were also preferred substrates for growth of Pseudomonas maltophilia. The data suggest that exoenzyme secretion is controlled by a mechanism similar to catabolite repression. In support of this was the observation that a-ketoglutarate repressed exoenzyme secretion preferentially with respect to total protein synthesis. The addition of inhibitors that affect protein synthesis indicated that exoenzyme secretion is several times more sensitive than is total protein synthesis. The addition of chloramphenicol and rifamycin-SV to actively secreting cell suspensions suggested that de novo protein synthesis is required, but that exoenzyme secretion may be supported for at least 30 min in the absence of messenger synthesis. Rifamycin-insensitive protease secretion could be reversed by either a-ketoglutarate or chloramphenicol, suggesting that a-ketoglutarate is coupled to a post-transcriptional control mechanism. Extracellular enzymes of microbial origin have been studied extensively with respect to their isolation and characterization. The regulation of these enzymes has received less consideration. The biosynthesis of extracellular enzymes has been investigated most intensively in Bacillus amyloliquefaciens, and a detailed mechanism for the synthesis and release of exoenzymes by this organism has been proposed (2). The model suggests that translation occurs at specialized sites associated with the cytoplasmic membrane and is maintained under specific conditions by a large pool of exoenzyme messenger ribonucleic acid (mRNA). The pool of mRNA is the result of a positive imbalance of transcription over degradation of the messenger. This model now appears to be correct in its basic features. There is ample indirect evidence to suggest that the synthesis of exoenzymes may occur on polysomes associated with the cytoplasmic membrane (2, 8, 19, 22). It is relevant in this context that Cancedda and Schlesinger (3) have provided direct evidence that the periplasmic alkaline phosphatase of Escherichia coli is synthesized on polysomes associated with the membrane. The secretion of extracellular en-

zymes in the absence of messenger synthesis has been observed in Pseudomonas lemoignei, a gram-negative organism, in addition to the genus Bacillus, and so appears to be a physiologically meaningful and perhaps key aspect of exoenzyme secretion in prokaryotes (2, 7, 9, 20, 22). A critical problem in the regulation of exoenzyme secretion was defined by Pollock (17), who observed that a common feature of many systems was the repression of exoenzyme secretion early in the growth cycle, followed by derepression in late exponential or early stationary phase. The details of the mechanism by which the control of exoenzyme production is achieved are not clear. Repression of extracellular protease secretion by free amino acids has been observed in many systems (10, 14) and may be accompanied by induction by peptides or other proteinaceous substrates (12, 13). In other systems, protease secretion seems to be induced by amino acids (6, 15). Apparent regulation of exoenzyme secretion by catabolite repression is also commonly observed (6, 22, 24). Unfortunately, in those systems tested exogenous cyclic adenosine 5'-monophosphate (cAMP) has been with954

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PROTEASE SECRETION BY P. MALTOPHILIA

out effect (22, 24). Coleman has proposed a model which attempts to provide a detailed explanation for the maximal rate of exoenzyme secretion which is observed after the end of exponential growth in B. amyloliquefaciens (4). Preliminary results have been offered (5, 23), but in general there are insufficient data to support the model. In this communication, some aspects of the regulation of the secretion of an extracellular protease produced by Pseudomonas maltophilia (1) are reported. Secretion appears to be regulated by a mechanism similar to catabolite repression, and under the conditions used may be supported for approximately 30 min in the absence of messenger synthesis. In addition, a probable effect of the repressor at the translational or post-translational level is reported.

MATERIALS AND METHODS Bacteria and culture conditions. The organism was isolated and maintained as previously described (1). For most experiments cells were grown in the following minimal medium: K,HPO4,*3H20 and KH,PO0 each 500 mg/liter; (NH4)HP04, 800 mg/liter; MgSO447H20, 200 mg/liter; CaCl22H,O, 53 mg/liter; MnSO4.H20, 0.85 mg/liter; ferric citrate reagent, 10 MM; disodium succinate, 50 mM; L-methio-

nine, 0.5 mM. The ferric citrate reagent was made as a 4 mM stock solution from 1.57 mg of FeNH4(SO j, * 12H20 per ml and 2.36 mg of sodium citrate 5H,O per ml. L-Methionine was added from a concentrated sterile solution after autoclaving. The pH of the medium was 7.2. In several experiments the cells were grown in a medium which had been modified by the substitution of the indicated carbon source for succinate. Cultures of 400 ml in 1-liter flasks were incubated until late exponential phase (usually about 20 h) with shaking at 30 C in a New Brunswick psychrotherm incubator. Growth was followed turbidimetrically at 540 nm, and the cells were collected by centrifugation when the culture had reached an optical density of 200 Klett units. The culture fluid was discarded and the pellet was washed twice with 40 mM K2HPO4KH2PO4, pH 6.9. Washed cells were suspended in a secretion medium which was identical in composition to the growth medium described above, except that disodium succinate was replaced as indicated in individual experiments. The cells were suspended to a density of about 500 Klett units (0.75 mg of cell protein/ml), and 5- to 20-ml volumes of suspension in 125-ml flasks were incubated at 30 C in a reciprocal shaker bath. . Enzyme assay. Proteolytic activity was determined by a modification of the method of Kunitz (11). The assay mixtures contained 0.2 to 1.0 ml of culture fluid and water to a volume of 2.0 ml, and 1.0 ml of a solution of casein (Hammarsten) at 10 mg/ml in 50 mM glycine buffer (pH 10). The enzyme and casein solutions were preincubated at 37 C separately for 5

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min, and the assay was initiated by the addition of casein solution to enzyme solution. After 15 min at 37 C the assay was terminated by plunging the tubes into ice and immediately adding 2.0 ml of 10% (wt/vol) trichloroacetic acid. After at least 1 h, the precipitated casein was removed by filtration with Whatman GF/C disks, and the absorbance was measured at 280 nm. Samples of culture fluid were obtained from cell suspensions at intervals by centrifugation at 12,000 x g for 10 min. Blank values were obtained for each assay by the addition of trichloroacetic acid before casein solution, and 1 unit of activity is defined as that amount sufficient to produce an increase in absorbance at 280 nm of 1.0. Protein synthesis. Total protein synthesis was measured by incorporation of either L- ["IC Ileucine or L- ['IC lphenylalanine into trichloroacetic acid-insoluble material. Cell suspensions contained labeled amino acid at 0.1 gCi/ml and 0.2% (wt/vol) yeast extract, without additional carbon source. Samples of 1.0 ml were removed at various times, added to equal volumes of 20% (wt/vol) trichloroacetic acid, and placed in a boiling water bath for 10 min. The tubes were then cooled to room temperature and the insoluble residues were collected on 0.65-Am membrane filters (Millipore Corp.). The filters were washed with a few milliliters of 5% (wt/vol) trichloroacetic acid and dried in an oven at 90 C. Radioactivity was determined by liquid scintillation counting in a Beckman liquid scintillation spectrometer. The filters were counted in vials with 10 ml of a toluene-based scintillation fluid. None of the carbon sources or antibiotics used in the study inhibited protease activity at the concentrations used under normal conditions. RNA synthesis. RNA synthesis was measured by incorporation of [3H]uracil into trichloroacetic acidinsoluble material. Cell suspensions contained 0.2% (wt/vol) yeast extract, without additional carbon source. At 75 min, [8H ]uracil was added to a final concentration of 0.8 ;Ci/ml. Samples of 1.0 ml were then removed at various times and added to equal volumes of a solution containing 20% (wt/vol) trichloroacetic acid and 2 mM unlabeled uracil. The tubes were prechilled at 0 C and maintained in the cold for about 30 min after the addition of cell suspension. The insoluble material was collected by filtration with Whatman GF/C disks and washed with a few milliliters of a solution containing 5% (wt/vol) trichloroacetic acid and 1 mM unlabeled uracil. The filters were dried and counted as described. Reagents. Sodium pyruvate, a-ketoglutarate, L-alanine, chloramphenicol, and actinomycin D were obtained from Sigma Chemical Co. [8H ]uracil (5.6 Ci/mmol, generally labeled) was purchased from New England Nuclear Corp., and L- [4C Ileucine (316 mCi/ mmol, uniformly labeled) and L-["4Clphenylalanine (460 mCi/mmol, uniformly labeled) from Schwarz/ Mann. Yeast extract was obtained from Difco, puromycin and casein (Hammarsten) from Nutritional Biochemicals, rifamycin-SV from Calbiochem, and Handifluor liquid scintillation counting solution from Mallinckrodt. All other chemicals were of reagent grade.

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RESULTS Control of protease secretion by carbon When cells in minimal medium were harvested in late exponential phase and resuspended at high density in medium of the same composition, protease secretion was approximately linear for at least 4 h (Fig. 1). During this period the optical density of the suspension increased by more than 50%, and the pH increased from 7.2 to 8.7. The change in pH was representative of that observed in the original culture. In addition, at no time was a large pool of protease activity associated with the cells (data not shown). It was reported previously that protease secretion in a complex medium containing yeast extract and succinate was not detected until early stationary phase (1). These results suggested that secretion might be subject to control by amino acid repression. When the cells were resuspended in medium containing yeast extract and no additional carbon source or yeast extract and succinate, however, the rate of protease secretion in the presence of yeast extract alone was significantly higher than that observed when both were present (Fig. 2B). Moreover, the lag before protease secretion began was shorter with yeast extract alone. The omission of both yeast extract and succinate effectively prevented secretion. The apparent repression of protease secretion by sources.

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FIG. 2. Effects of various carbon sources on protease secretion by succinate-grown cells. The cells were resuspended with 0.2% (wt/vol) yeast extract and various compounds, all at 25 mM. (A) Optical density in Klett units of the cell suspensions. Symbols: (0) yeast extract; (x) yeast extract plus succinate. (B) Protease activity. Symbols: (0) yeast extract; (0) yeast extract plus pyruvate; (0) yeast extract plus L-malate; (x) yeast extract plus succinate; (A) yeast extract plus a-ketoglutarate.

succinate could not be accounted for as growth inhibition, since the increase in optical density of the suspensions was considerably greater with succinate and yeast extract than with yeast extract alone (Fig. 2A). The data suggested that protease secretion might be subject to catabolite repression. The effects of other carbon sources on protease secretion by succinate-grown cells were therefore tested (Fig. 2B). In the presence of yeast extract, both L-malate and succinate had similar effects, but pyruvate was less effective. Of those tested, the most effective repressor was a-ketoglutarate. The increase in optical density of the cell suspensions was enhanced in all cases by the addition of the carbon source to resuspension medium containing yeast extract. The repression of protease secretion by tricarboxylic acid cycle intermediates and pyruvate may be HOURS related to the fact that these compounds are FIG. Secretion of extracellular protease by 1. washed cells. The cells were resuspended with 25 mM preferred substrates for growth of this organism succinate as the sole carbon source. Symbols: (0) (21). The incomplete repression by pyruvate and protease activity; (0) optical density in Klett units; malate might have been the result of defects in (0) pH of the medium.

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transport, since the cells were grown in minimal repressive effect, but was in fact stimulatory to medium containing only succinate as carbon protease secretion (Fig. 3C). source. To rule out this possibility, cells were Repression of protease secretion by a-kegrown in minimal media in the presence of toglutarate. The effect of a-ketoglutarate when various carbon sources and resuspended with added at time zero suggested the possibility yeast extract with and without the correspond- that protease secretion by active cell suspening carbon source. Representative experiments sions might be inhibited at any time by the are shown in Fig. 3. It can be seen that the addition of this compound to the medium. This relationship of enzyme secretion with the addi- proved to be the case (Fig. 4). The ability of tional carbon source to that without was quali- a-ketoglutarate to repress protease secretion tatively similar for both pyruvate and malate to when added to actively secreting suspensions that observed with succinate-grown cells. In was exploited to demonstrate that repression by addition, it is evident that L-alanine had no a-ketoglutarate is specific for protease secretion (Fig. 5). When a-ketoglutarate was added at 60 min to a cell suspension containing yeast extract and no additional carbon source, protease secretion was effectively suppressed while total 4 A protein synthesis was stimulated. Effects of inhibitors of transcription and 3 translation on protease secretion and protein synthesis. The results of studies with other have shown that extracellular enzyme systems 2 secretion is more susceptible to inhibitors that affect protein synthesis than is total protein synthesis (2, 8, 19, 22). It has been suggested (2) that this observation may be explained by the existence of two classes of ribosomes, one located at the periphery of the cell, perhaps associated with the cytoplasmic membrane, and the other more or less uniformly distributed in the cytoplasm. According to this hypothesis, protease synthesis occurs preferentially on membrane-associated polysomes. To test this idea, the effects of various inhibitors on the secretion of extracellular protease and protein synthesis were determined by resuspending cells in the presence of varying concentrations of each antibiotic. Samples of cell suspension were removed at intervals and prepared for measurement of protein synthesis, or centrifuged and assayed for protease activity. From this information, values for 50% inhibition of both parameters were calculated, and these data appear in Table 1. With the exception of actinomycin D, a differential effect was observed in each case. The effect was least pronounced with rifamycin-SV, but nevertheless constituted a more than threefold difference in sensitivity. 1 2 3 4 Effects of chloramphenicol and rifon protease secretion. Chloramamycin-SV HOURS was added at various times to actively phenicol FIG. 3. Effect of various carbon sources on secretion by cells grown on the corresponding compounds. The secreting cell suspensions (Fig. 6). Inhibition of cells were resuspended with 0.2% (wt/vol) yeast protease synthesis was prompt and essentially complete regardless of the time of addition of extract with (0) or without (0) the indicated carbon the antibiotic. This suggests that secretion of source. All carbon sources were present in the resuspension medium at 25 mM. (A) L-Malate; (B) pyru- the enzyme involves de novo protein synthesis vate; (C) L-alanine. and is not a result of the release of preformed

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completely inhibited the secretion of extracellular protease. When added to actively secreting cells, however, a lag of approximately 30 min

was observed before the accumulation of protease was effectively inhibited. It was possible that protease secretion was not

inhibited immediately because the cells initially were not permeable to the drug. This possibility was tested as follows: rifamycin and [3HJuracil were added to an active cell suspension, and protease secretion and the incorporaTABLE 1. Effect of inhibitors on protease secretion and total protein synthesisa

2 HOURS FIG. 4. Effect of a-ketoglutarate on protease secretion. The cells were resuspended with 0.2% (wt/vol) yeast extract, and a-ketoglutarate was added at various times to a final concentration of 25 mM. Symbols: (0) no addition; (0) plus a-ketoglutarate.

Concn of inhibitor required for 50% inhibition (pg/ml)

Inhibitor

Chloramphenicol Puromycin Rifamycin-SV Actinomycin D

Protease secretion

Total protein synthesis

0.27 1.8 3.5 40

1.8 30 12 40

a Washed cells were resuspended in basal medium with 0.2% (wt/vol) yeast extract, 0.1 ACi of L- ["C Jleucine or L- [14C ]phenylalanine per ml, and varying concentrations of antibiotic. Samples were removed at intervals and prepared for measurement of total protein synthesis, or assayed for protease activity. Values of percentage of inhibition for each concentration of inhibitor were calculated from the rates of protease secretion and "IC-labeled amino acid incorporation during the linear phase, from 60 to 120 min. The uncertainty associated with these values is approximately 10%.

30

60 90 120 150 MINUTES FIG. 5. Preferential inhibition of protease secretion by a-ketoglutarate. The cells were resuspended with

0.2% (wt/vol) yeast extract and 0.1 pCi of L-['4C]leu-

cine per ml. At 60 min the cell suspension was divided and a-ketoglutarate was added to one portion to a final concentration of 25 mM. Symbols: (0) no addition; (0) plus a-ketoglutarate; (solid line) protease activity; (dashed line) [14C lleucine incorporated.

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HOURS The effect of rifamycin-SV, an inhibiFIG. 6. Effect of chloramphenicol on protease secretor of RNA synthesis, was similarly determined tion. cells were resuspended with 0.2% (wt/vol) by the addition of the antibiotic at various yeast The extract, and chloramphenicol was added at times to cell suspensions (Fig. 7). It can be seen various times to a final concentration of 100 ug/ml. that the addition of rifamycin at zero time Symbols: (-) no addition; (0) plus chloramphenicol. enzyme.

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70 2 3 HOURS FIG. 7. Effect of rifamycin-SVon protease secretion. The cells were resuspended with 0.2% (wt/vol) yeast extract, and rifamycin-SV was added at various times to a final concentration of 100 ;g/ml. Symbols: (@) no addition; (0) plus rifamycin.

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chloramphenicol and a-ketoglutarate behaved identically. DISCUSSION The tricarboxylic acid cycle intermediates succinate, malate, and a-ketoglutarate were effective repressors of extracellular protease secretion in cell suspensions containing basal medium and yeast extract. Pyruvate was relatively less effective. These compounds are also preferred substrates for growth of P. maltophilia in minimal medium (21). Of those tested, a-ketoglutarate was by far the most effective repressor. The correlation between growth rate and repression of protease secretion did not hold within this group of compounds, however, since the generation times in minimal media were

1

tion of radioactive uracil into total cellular RNA followed subsequently in the rifamycincontaining suspension and in the control (Fig. 8). It is evident that total RNA synthesis was inhibited immediately by at least 90%. Although it is possible that the residual RNA synthesis is in fact that which provides mRNA for protease synthesis, this must be considered unlikely in view of the fact that protease synthesis was inhibited by 50% at a concentration of rifamycin less than one-third of that required for similar inhibition of total protein synthesis (Table 1). The data suggest that, during the phase of active secretion, protease synthesis may be supported for approximately 30 min in the absence of additional mRNA synthesis. Inhibition of rifamycin-insensitive protease secretion by a-ketoglutarate. The prompt inhibition of protease secretion by a-ketoglutarate was strikingly similar to that observed with chloramphenicol, and unlike the effect of rifamycin. These results suggested that a-ketoglutarate inhibits protease secretion at the level of translation, or possibly at a posttranslational step in the secretion process. If this is the case, a-ketoglutarate and chloramphenicol, a known inhibitor of translation, should inhibit rifamycin-insensitive protease secretion when added with rifamycin to actively secreting cells. The results of such an experiment are shown in Fig. 9. Although inhibition by a-ketoglutarate was not complete, extensive inhibition was observed, and most importantly, were

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60 120 180 MINUTES FIG. 8. Effect of rifamycin-SV addition on RNA synthesis and protease secretion. The cells were resuspended with 0.2% (wt/vol) yeast extract. At 75 min, ['Hjuracil was added to give 0.8 ACi/ml. The cell suspension was then divided, and rifamycin-SV was added to one portion to a final concentration of 100 ,gg/ml. Symbols: (-) no addition; (0) plus rifamycin. (A) ['H]uracil incorporated; (B) protease activity.

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2 3 HOURS FIG. 9. Inhibition of rifamycin-insensitive protease secretion by a-ketoglutarate and chloramphenicol. The cells were resuspended with 0.2% (wt/vol) yeast extract. At 90 min the cell suspension was divided into four portions which were amended as follows. Symbols: (0) no addition; (0) rifamycin-SV, 100 ,gg/ml final concentration; (0) rifamycin plus a-ketoglutarate, 25 mM final concentration; (x) rifamycin plus chloramphenicol, 100 Aglml final concentration. 1

similar for all four (5 to 6 h). Although repression by a-ketoglutarate was nearly complete when the compound was present from zero time, the repression was temporary, since protease secretion was eventually observed at the rate characteristic of succinate. This might be expected if it is assumed that a-ketoglutarate is assimilated at least in part by conversion to succinate. The effect of L-alanine differed from the effects of the tricarboxylic acid cycle intermediates and pyruvate in that L-alanine was stimulatory rather than repressive with respect to protease secretion. The data suggest that protease secretion is controlled by a mechanism similar to catabolite repression. Apparent regulation of extracellular protease secretion by catabolite repression has also been observed for Vibrio parahaemolyticus by Tanaka and Iuchi (24). In the latter case, control did not appear to be mediated by a system similar to that operative in the lactose operon of E. coli and involving cAMP (16, 18), since cAMP did not overcome repression of protease secretion but did reverse repression of an intracellular enzyme. The reversal of repression by dicarboxylic acids was attempted in the present system with exogenously provided cAMP, but was unsuccessful. This failure may

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have been a result of the inability of the cells to transport cAMP. In any event, the ability of a-ketoglutarate to repress protease secretion when added to suspensions of actively secreting cells provided an opportunity to demonstrate that repression was specific for protease secretion vis-a-vis total protein synthesis. When added to active cell suspensions, a-ketoglutarate caused a prompt inhibition of protease secretion, but significantly enhanced uptake of L- ["C ]leucine into total protein. A complication in the study of exoenzyme secretion in prokaryotes has been the difficulty in clearly differentiating synthesis, permeation, and release (17). Data presented here concerning the effects of various inhibitors that affect protein synthesis support the contention that these processes are closely linked to one another. When chloramphenicol was added at various times to actively secreting cell suspensions, inhibition of protease secretion was prompt and nearly complete, suggesting that protease synthesis is tightly coupled to permeation and release. In addition, the hypothesis that exoenzyme synthesis is confined to membrane-associated ribosomes (2) was supported by the differential effects of several antibiotics on protease secretion and protein synthesis. The evidence is not conclusive, since Paigen and Williams (16) have pointed out that the synthesis of a number of intracellular enzymes that are subject to catabolite repression is inhibited preferentially by certain antibiotics. This point is especially cogent in view of the probable regulation of protease secretion by a catabolite repression-like mechanism. The effect of the addition of rifamycin-SV to cell suspensions suggests that protease secretion is supported by a pool of messenger during the phase of active secretion. In B. amyloliquefaciens a similar phenomenon appears to be the result of an imbalance of transcription and degradation of the messenger, and not mRNA that is intrinsically stable (2, 7). In the present example the data do not permit a conclusion as to which of the two is correct. The similar effects of chloramphenicol and a-ketoglutarate when added to actively secreting cell suspensions suggest that a-ketoglutarate may be coupled to control of exoenzyme secretion at the translational or post-translational level. This concept is supported by the ability of a-ketoglutarate to inhibit rifamycininsensitive protease secretion, an ability shared by chloramphenicol. If the effect of a-ketoglutarate on protease secretion is a reflection of a mechanism similar to cAMP-mediated catabolite repression, this finding is anomalous, since

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catabolite repression is primarily thought to be a transcriptional control (16, 18). For this reason it would seem likely either than repression is not mediated by cAMP alone, or that it is mediated by an independent mechanism. The data presented here do not rule out the possibility that a-ketoglutarate and chloramphenicol inhibit transport or release, and concomitant activation, of an inactive enzyme precursor. ACKNOWLEDGMENTS This investigation was supported in part by Public Health Service grant AM 11148 from the National Institute of Arthritis, Metabolism and Digestive Diseases to J. Lascelles. During this investigation the author was a Public Health Service predoctoral trainee, supported by Public Health Service grant GM 01297 from the National Institute of General Medical Sciences. The helpful criticism and suggestions of J. Lascelles are gratefully acknowledged. LITERATURE CITED 1. Boethling, R. S. 1975. Purification and properties of a serine protease from Pseudomonas maltophilia. J. Bacteriol. 121:933-941. 2. Both, G. W., J. L. McInnes, J. E. Hanlon, B. K. May, and W. H. Elliott. 1972. Evidence for an accumulation of messenger RNA specific for extracellular protease and its relevance to the mechanism of enzyme secretion in bacteria. J. Mol. Biol. 67:199-217. 3. Cancedda, R., and M. J. Schlesinger. 1974. Localization of polyribosomes containing alkaline phosphatase nascent polypeptides on membranes of Escherichia coli. J. Bacteriol. 117:290-301. 4. Coleman, G. 1967. Studies on the regulation of extracellular enzyme formation by Bacillus subtilis. J. Gen. Microbiol. 49:421-431. 5. Coleman, G., and D. A. Stormonth. 1975. Stimulation of the differential rate of exoenzyme formation in Bacillus amyloliquefaciens by streptolydigin, an inhibitor of RNA chain elongation. J. Gen. Microbiol. 86:194-196. 6. Daatselaar, M. C. C., and W. Harder. 1974. Some aspects of the regulation of the production of extracellular proteolytic enzymes by a marine bacterium. Arch. Microbiol. 101:21-34. 7. Glenn, A. R., G. W. Both, J. L. McInnes, B. K. May, and W. H. Elliott. 1973. Dynamic state of the messenger RNA pool specific for extracellular protease in Bacillus amyloliquefaciens: its relevance to the mechanism of enzyme secretion. J. Mol. Biol. 73:221-230. 8. Glew, R. H., and E. C. Heath. 1971. Studies on the extracellular alkaline phosphatase of Micrococcus sodonensis. II. Factors affecting secretion. J. Biol. Chem. 246:1566-1574.

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9. Gould, A. R., B. K. May, and W. H. Elliott. 1973. Accumulation of messenger RNA for extracellular enzymes as a general phenomenon in Bacillus amyloliquefaciens. J. Mol. Biol. 73:213-219. 10. Hofsten, B., and C. Tjeder. 1965. An extracellular proteolytic enzyme from a strain of Arthrobacter. I. Formation of the enzyme and isolation of mutant strains without proteolytic activity. Biochim. Biophys. Acta 110:576-584. 11. Kunitz, M. 1946. Crystalline soybean trypsin inhibitor. II. General properties. J. Gen. Physiol. 30:291-310. 12. Litchfield, C. D., and J. M. Prescott. 1970. Regulation of proteolytic enzyme production by Aeromonas proteolytica. I. Extracellular endopeptidase. Can. J. Microbiol. 16:17-22. 13. Litchfield, C. D., and J. M. Prescott. 1970. Regulation of proteolytic enzyme production by Aeromonas proteolytica. II. Extracellular aminopeptidase. Can. J. Microbiol. 16:23-27. 14. May, B. K., and W. H. Elliott. 1968. Characteristics of extracellular protease formation by Bacillus subtilis and its control by amino acid repression. Biochim. Biophys. Acta 157:607-615. 15. McDonald, I. J., and A. K. Chambers. 1966. Regulation of proteinase formation in a species of Micrococcus. Can. J. Microbiol. 12:1175-1185. 16. Paigen, K., and B. Williams. 1970. Catabolite repression and other control mechanisms in carbohydrate utilization, p. 251-324. In A. H. Rose and J. F. Wilkinson (ed.), Advances in microbial physiology, vol. 4. Academic Press Inc., London. 17. Pollock, M. R. 1963. Exoenzymes, p. 121-178. In I. C. Gunsalus and R. Y. Stanier (ed.), The bacteria, vol. 4. Academic Press Inc., New York. 18. Rickenberg, H. V. 1974. Cyclic AMP in prokaryotes. Annu. Rev. Microbiol. 28:353-369. 19. Sargent, M. G., and J. 0. Lampen. 1970. A mechanism for penicillinase secretion in Bacillus licheniformis. Proc. Natl. Acad. Sci. U.S.A. 65:962-969. 20. Semets, E. V., A. R. Glenn, B. K. May, and W. H. Elliott. 1973. Accumulation of messenger ribonucleic acid specific for extracellular protease in Bacillus subtilis 168. J. Bacteriol. 116:531-534. 21. Stanier. R. Y., N. J. Palleroni, and M. Doudoroff. 1966. The aerobic pseudomonads: a taxonomic study. J. Gen. Microbiol. 43:159-271. 22. Stinson, M. W., and J. M. Merrick. 1974. Extracellular enzyme secretion by Pseudomonas lemoignei. J. Bacteriol. 119:152-161. 23. Stormonth, D. A., and G. Coleman. 1974. Cellular changes accompanying the transition form minimal to maximal rate of extracellular enzyme secretion by Bacillus amyloliquefaciens. J. Appl. Bacteriol. 37:225-237. 24. Tanaka, S., and S. Iuchi. 1971. Induction and repression of an extracellular proteinase in Vibrio parahaemolyticus. Biken J. 14:81-96.