Production of active Serratia marcescens metalloprotease from ...

Vol. 174, No. 7

JOURNAL OF BACTERIOLOGY, Apr. 1992, p. 2361-2366

0021-9193/92/072361-06$02.00/0 Copyright © 1992, American Society for Microbiology

Production of Active Serratia marcescens Metalloprotease from Escherichia coli by o-Hemolysin HlyB and HlyD YOUSIN SUH AND MICHAEL J. BENEDIK*

Department of Biochemical and Biophysical Sciences, University of Houston, Houston, Texas 77204-5934 Received 9 December 1991/Accepted 30 January 1992

Serratia

abundant extracellular metalloprotease. The gene for this protease had no functional protease could be found. However, the protease gene carries the LXGGXGND repeat motif found in at-hemolysin and other proteins secreted by homologous systems. Using a dual-plasmid complementation system, we show that the a-hemolysin hIyB and hlyD transport determinants are sufficient to allow secretion and activation of a functional metalloprotease species from E. coli, as are the comparable protease secretion functions of Erwinia chrysanthemi. However, strains expressing protease with the hlyBD transport system are unstable and rapidly lose the ability to produce functional protease. marcescens

produces

an

previously been cloned and expressed in Escherichia coli, in which

A number of extracellular proteins are secreted into the medium by the gram-negative bacterium Serratia marcescens, including a nuclease, a chitinase, lipases, and proteases. Of these, the most abundant protein is a zinc metalloprotease (1). This enzyme is produced during the growth of a culture and reaches its maximal level as the cell enters stationary phase (5, 7). It is inducible by the addition of proteinaceous substrates to the medium, by certain amino acids such as leucine, or by a dense bacterial culture in which the concentration of proteins released by the cells is presumably sufficient to induce its expression (5). Its regulation, however, is not coordinated with that of other extracellular proteins of S. marcescens (3). The genes encoding most of these proteins have been cloned and sequenced. The nuclease (2), chitinase (18), phospholipase (12), and serine protease (30) genes are all expressed in Escherichia coli. However, the gene for the metalloprotease, which has also been cloned, does not express functional protease in E. coli; only a higher-molecular-weight zymogen can be found (6, 23). The major form of the metalloprotease is found as a species with a molecular weight of 51,000 (1, 5). A minor form with a molecular weight of 53,000 was also observed previously (25). The gene for metalloprotease from S. marcescens E-15 was sequenced previously (23), and a protein of 470 amino acids (Mr = 50,632) was predicted. A similar gene with 95% identity was also cloned and sequenced from the S. marcescens strain SM6 (6). The DNA sequence predicts a preprotein which could initiate at any one of three ATG start codons in frame with the peptide sequence. However, none of the three potential start sites leads to an amino-terminal domain which resembles a classical signal peptide. It is not clear which start codon is used in vivo. The B and C proteases of Erwinia chrysanthemi have also been cloned and sequenced previously (9). These proteases are about 60% related to the S. marcescens metalloprotease. Both groups of protease have a striking repeat motif of LXGGXGND. This motif has been found in a number of toxins, such as a-hemolysin, leukotoxin, and cyclasin (4), all of which are secreted extracellularly by similar transport systems. Secretion of the a-hemolysin of E. coli requires the *

products of the hemolysin genes hlyB and hlyD (14) and of the E. coli tolC gene (28). Only the carboxyl-terminal region of a-hemolysin is required for its secretion (24); nevertheless, this LXGGXGND motif, which is outside the essential carboxyl-terminal domain of a-hemolysin, appears to be predictive for secretion with a hemolysinlike system. The transport genes for the E. chrysanthemi proteases have recently been cloned and sequenced (21). Three genes which are homologs of hlyB, hlyD, and tolC appear to be required. On the basis of the similarities between the E. chrysanthemi and the S. marcescens proteases, it appeared worthwhile to test the capability of the oa-hemolysin transport system to support the secretion of the S. marcescens metalloprotease from E. coli. During the preparation of this paper, similar findings were published elsewhere (22). There are some differences between our findings and those of Letoffe et al., which are addressed in this report. MATERLALS AND METHODS

Bacterial strains and plasmids. JM101 (27) was used as the E. coli host in this work. Strain SM6 is our wild-type S. marcescens strain and was also the source of the metalloprotease gene used in this study. Cloning of the protease gene has previously been described (6), as have most of the protease subclones used here. To review, pUC18prtE7 carries a 5-kb EcoRI fragment containing the entire protease gene. A subclone, pUC19prtCE, carries a 3.7-kb ClaIEcoRI fragment. The ClaI site is about 150 bases upstream of the protease gene. A slightly shorter derivative, pUC19 prtHE, is deleted for 100 bases of this upstream DNA. About 2 kb of DNA is present downstream of the protease gene in all of these constructions. Expression of pUC18prtE7 is not isopropyl-3-D-thiogalactopyranoside (IPTG)-inducible and presumably comes from the protease promoter. The pUC19prtCE and pUC19prtHE clones all transcribe the protease gene from the plasmid lac promoter. The pUC18 prtCE and pUC18prtHE clones are in the inverse orientation and are therefore not transcribed from the lac promoter. Two additional subclones were generated for this work. The DNA downstream of the protease gene was removed from pUC19prtCE by digestion at a BspMII site 310 bases downstream of the protease gene and the EcoRI

Corresponding author. 2361

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SUH AND BENEDIK

site defining the 3' end of the insert. The ends were made blunt with the Klenow fragment of DNA polymerase I in the presence of all four deoxynucleoside triphosphates (dNTPs), and the plasmid was recircularized by T4 DNA ligase. This deletion derivative is called pUC19prtCEA16. A lower-copynumber derivative (pBH207) was created in pBH20 (17), which is pBR322 carrying the lac promoter in front of the EcoRI site pointing toward the tetracycline resistance gene. The ClaI-BglII fragment carrying protease and the downstream region was inserted into pBH20 digested with ClaI and BamHI, such that it was expressed from the lac promoter. The plasmid pSH305 was kindly provided by Sarah Highlander. It was derived from pSF4000, which carries the entire hemolysin gene cluster cloned into the p15 replicon plasmid pACYC184. A BamHI deletion was created to remove the hemolysin genes hlyA and hlyC to generate pSH305 (15). This plasmid, which confers chloramphenicol resistance and supplies the essential transport genes hlyB and hlyD for a-hemolysin secretion, is compatible with ColEl replicons used for the protease clones. The E. chrysanthemi protease secretion functions were supplied on the plasmid pGSD6, which is the pLAFR3 cosmid carrying the entire protease gene cluster from which the prtABC protease genes were deleted (8). This plasmid supplies the protease secretion functions and includes the E. chrysanthemi protease inhibitor gene but itself does not express protease. Media. All cultures were routinely grown in Luria-Bertani medium supplemented with 100 ,ug of ampicillin or 30 ,ug of chloramphenicol per ml as required. Protease indicator medium (skim milk plates) was Luria-Bertani agar with 1% nonfat dry powdered milk as previously described (16). Relative levels of protease activity were determined by measuring halo sizes produced on the skim milk plates. Western blotting (immunoblotting). Protein samples were separated by electrophoresis on a discontinuous 12% polyacrylamide gel (19). For Western blots, the proteins were transferred to Immobilon P membranes (Millipore), probed with rabbit antiprotease serum, and developed with alkaline phosphatase conjugated goat anti-rabbit second antibody. RESULTS Complementation of secretion functions in E. coli. The metalloprotease gene from S. marcescens has been shown not to express functional protease in E. coli (6, 23), although inactive zymogen can be found by immunoblotting. To test whether the a-hemolysin secretion factors encoded by the hlyB and hlyD genes in plasmid pSH305 and their E. chrysanthemi equivalents in plasmid pGSD6 can serve to secrete the metalloprotease, these plasmids were transformed into E. coli JM101 carrying the compatible plasmid pUC18prtE7, and cells were selected for both ampicillin resistance and either chloramphenicol resistance (for pSH305) or tetracycline resistance (for pGSD6). Dual-plasmid-carrying colonies were then transferred to skim milk agar to test for protease production as shown in Fig. 1. A halo where the milk protein had been cleared could be observed only for cells carrying both the protease plasmid and a plasmid with the secretion functions. The controls showed no halo even after 1 week of incubation. Role of upstream DNA in protease expression. Using halo size as a relative indication of protease production, we were able to analyze the level of protease produced from the different constructs previously described (6). The plasmid

FIG. 1. Fresh transformants carrying the designated plasmids were transferred onto skim milk plates freshly after transformation onto drug-containing plates and incubated for 24 h at 37°C. Top row from left to right is JM101(pSH305, pUC18prtE7) and JM101 (pGSD6, pUC19prtE7). Bottom row from left to right is JM101 (pUC18prtE7, pACYC184), JM101(pSH305, pUC18), and JM101

(pGSD6, pUC18).

carrying the prtE7 insert has about 1.2 kb of DNA upstream of the protease gene, as mapped in Fig. 2. This appears to include the promoter region which allows protease transcription in E. coli from this clone. The prtCE and prtHE plasmids have most of the upstream DNA deleted as shown (Fig. 2); the prtHE clone has an additional 100 bp deleted after the ClaI site. The sequence of the ClaI-EcoRI fragment, along with the construction of these plasmids, has already been presented elsewhere (6). In pUC19, both can be transcribed under control of the lac promoter and therefore should not require a functional protease promoter. As shown in Table 1, the halo size of JM1O1(pSH305) carrying pUC19prtCE was larger than that of JM101 (pSH305) carrying pUC18prtE7, which expresses protease only from its own promoter (or some other promoter on the insert). However, no protease from cells carrying pUC19prtHE was observed. Similarly, when the prtCE insert was reversed with respect to the lac promoter by cloning into pUC18, no protease was observed. The same general ratios were observed when the E. chrysanthemi secretion functions were provided by pGSD6, with the

Eaa

I

I

ia

Hindill

-k~ .

Sall

BmN

I I

BepMI Sall

~I

E

r

-Bo,' \prtE7

prtCEAI 6 FIG. 2. Restriction map of the EcoRI fragment constituting prtE7. The open box designates the protease reading frame. Underneath are diagrammed the regions contained in the other plasmid derivatives.

S. MARCESCENS PROTEASE CAN USE HlyB AND HlyD IN E. COLI

VOL. 174, 1992

TABLE 1. Relative protease expression observed with different plasmidsa Halo sizeb With Without IPTG IPTG

Strain

JM101(pSH305) JM101(pUC18prtE7) JM101(pSH305, pUC18prtE7) JM101(pSH305, pUC18prtCE) JM101(pSH305, pUC19prtCE) JM101(pSH305, pUC18prtHE) JM101(pSH305, pUC19prtHE) JM101(pSH305, pUC19prtCEA16) JM101(pSH305, pBH207) JM101(pGSD6) JM101(pGSD6, pUC18prtE7) JM101(pGSD6, pUC18prtCE) JM101(pGSD6, pUC19prtCE) JM101(pGSD6, pUC18prtHE) JM101(pGSD6, pUC19prtHE) JM101(pGSD6, pUC19prtCEA16)

+ ++

+ C

+ + +C + +/++ ++

+/-

+/-

++++

++c

+/-

+l-

+ ++++

+C

+ +C

a Colonies were newly transferred from a transformation onto skim milk plates with antibiotics for the maintenance of one or both plasmids as appropriate and incubated for 16 h. IPTG was added at 10-3 M. These results are a summary from at least three transformants each from at least two independent transformation experiments. b Numbers of plus signs indicate relative halo sizes. +/-, a very small but detectable halo; +, a halo of about 0.6 cm; + +, a halo of about 1.0 cm; + + +, a halo of about 1.2 cm; ++++, a halo of 1.4 cm or larger. c Colonies were small, had a mottled morphology, and looked sick.

exception that the halos were generally larger. The prtHE plasmids did show small halos in this background, unlike with the pSH305 secretion functions. A more surprising observation was the inhibition of protease expression upon induction of the lac promoter by IPTG with pUC19prtCE. Increasing the amount of IPTG added to the plate reduced protease expression from pUC19prtCE plus pSH305 until no halo was observed at 10-3 M IPTG. A similar inhibition was observed with pUC19prtCE and pGSD6, although some protease activity was still observed with this pair. The largest halo size was observed with no inducer. Intermediate levels of IPTG resulted in intermediate halo sizes (data not shown). Because pUC plasmids are such high-copy-number plasmids, they would be expected to titrate the LacI repressor protein, even when overexpressed from the lacIq allele present in JM101. This would lead to partial induction of lac promoter expression which allows expression from pUC19prtCE without IPTG. The colonies which grew on the IPTG plates also had unusual morphology and appeared small and mottled. Role of downstream sequences. The DNA fragment in the prtCE clones carries about 2 kb of downstream DNA. A subclone which deleted this downstream region was conS.M. E.c.

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structed in order to probe whether it had any role in protease expression or secretion. The plasmid pUC19prtCE was digested with restriction endonucleases BspMII and EcoRI, and the overhangs were filled in with the small subunit of DNA polymerase I in the presence of all four dNTPs and subsequently ligated. Colonies were picked and analyzed for loss of the downstream DNA and restoration of the EcoRI site, which would be expected from this event. One such plasmid clone, pUC19prtCEA16, was chosen for further analysis. Not only did this clone express functional protease as shown in Table 1, but it actually produced a larger halo than its parent plasmid pUC19prtCE when complemented with pSH305. However, it too was increasingly inhibited by the addition of higher concentrations of IPTG. The protease locus from E. chrysanthemi (20) carries a gene expressing a protease inhibitor which is active on the S. marcescens protease as well. The sequence of the E. chrysanthemi inhibitor (as revised in reference 21) is compared with that of an open reading frame downstream of the S. marcescens protease gene (the region deleted in prtCEA16) in Fig. 3. The putative S. marcescens inhibitor has about 40% identity with that of E. chrysanthemi. The latter has been shown to be a periplasmic enzyme whose gene sequence predicts a typical signal sequence. A nonhomologous signal sequence is also predicted by the S. marcescens open reading frame. This reading frame is from nucleotides 1706 to 2050 of our previously published sequence (6; EMBL accession no. X55521). The BspMII site is at position 1960, and the A16 deletion removes the last 28 amino acids. The sequence of Nakahama et al. (23) does not include this reading frame. Plasmids deleted for the inhibitor did not have any noticeable growth inhibition unless expression was induced by IPTG. This is in contrast to the findings of Letoffe et al. (22), who report that deletion of the downstream region results in severe growth inhibition. The IPTG inhibition that we observe is with both prtCE and prtCEAl16, showing that it is most likely due to overexpression of protease. It should also be noted that the experiments of Letoffe et al. were done in E. coli strains in which the lac promoter is not tightly regulated. The pGSD6 plasmid also carries the E. chrysanthemi protease inhibitor, which has been shown to inhibit the S. marcescens protease (20). Therefore, the fact that no difference in the halo sizes of pUC19prtCEA16 and its parent when complemented by pGSD6 was observed (Table 1) is not surprising, since the inhibitor is supplied by pGSD6 in this case. Growth curves of protease-carrying strains. We consistently observed that pSH305 dual-plasmid-carrying strains which express protease were very unstable, even without IPTG. After a few passages, halo sizes became variable and colonies frequently lost the ability to express protease. To determine whether there was a growth inhibition caused by protease expression, a growth curve in the presence or

1 MRGTLARTALAAGGMMVTSAVMAGSLALPTAQSLAGQWQVADSERQCQIEFLAHEQSETNGYQLV 65 1 MRQLIIATLLSA.... LSGGdCMA4SLRLPSAAELSGQWVLSGAEQHCDIR.LNTDVLDGTTWKLA 60 TmAtUre

116 S.m. 66 DRQQCLQSVFAAEVVGWRPAPDGIALLRADGSTLAFFSR.....EASVPQSAGCG E.C. 61 GDTACLQKLLPEAPVGWRPTPDGLTLTQADGSAVAFFSRNRDRYEHKLVDGSVRTLKKKA 120

FIG. 3. The predicted S. marcescens (S.m.) protease inhibitor protein sequence compared with that of the E. chrysanthemi (E.c.) inhibitor. Symbols: I, exact matches; :, conservative substitutions; ., less conservative substitutions. The start of the mature E. chrysanthemi inhibitor protein is marked by an arrow.

SUH AND BENEDIK

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10:

1

1:

2

3

4

5

6

7

FIG. 5. Western blot developed with antiprotease

serum.

Cul-

tures were grown, with antibiotics as appropriate, until anA6w value of 1 to 1.5 was reached. Cells were removed by centrifugation, and the supernatants were collected by ethanol precipitation. Supernatant lanes (1 to 3) contain the equivalent of 0.2 ml of culture

supernatant, except for SM6 (lane 4), which was 0.025 ml. The cell fractions (lanes 5 to 7) were prepared by sonication followed by ethanol precipitation to concentrate; the equivalent of 0.01 ml per lane was loaded. Lanes: 1, JM101(pGSD6); 2, JM101(pUC19prtCE A16); 3, JM101(pGSD6, pUC19prtCEA16); 4, SM6; 5, JM101(pG SD6, pUC19prtCEA16); 6, JM101(pUC19prtCEA16); 7, JM101(pG SD6).

0.1 :

0

30

60

95 120 Time

165

190

220

FIG. 4. Growth curve of JM101(pSH305, pUC19prtCEA16). A fresh transformant was transferred into Luria-Bertani broth containing both ampicillin and chloramphenicol. After the culture attained visible turbidity, it was split and i0-3 M IPTG was added to one flask. Samples were removed, and the A was measured either directly or with dilutions if the A600 was >1. El, IPTG-uninduced cells; 0, IPTG-induced cells. y axis, A600-

absence of IPTG from a fresh transformant was determined. In Fig. 4, this curve is shown for JM101(pSH305, pUC19prtCEA16) with or without IPTG induction. There is an obvious inhibition of growth in the presence of IPTG. Once a colony has lost the ability to produce protease, it is not recovered, despite constant selection for both plasmids. This instability was not as dramatic with pGSD6 as it was with pSH305 but could still be detected. Subcloning to lower-copy-number plasmid. The protease gene was subcloned to a lower-copy-number plasmid in the hope that this would reduce the instability observed with the clones in the pUC plasmid series. The vector pBH20 is a derivative of pBR322 which carries the lac promoter positioned just in front of and directed toward the EcoRI site of pBR322 (17). The protease gene was subcloned as a ClaIBgII fragment into the ClaI and BamHI sites of pBH20, respectively. This derivative, pBH207, produced only a very tiny halo around the colony when introduced into JM101(pSH305) but could be induced to express a larger halo upon IPTG induction, as shown in Table 1. Therefore, with this construct, protease expression was regulated as expected. The instability observed with the pUC protease plasmids was not seen with colonies which had never been induced by IPTG; however, colonies grown on IPTG plates still segregated non-protease-expressing colonies. The protease expressed from E. coli is extracellular. Culture supernatants and cell extracts were prepared for sodium dodecyl sulfate-polyacrylamide gel electrophoresis separation by ethanol precipitation of the cell-free supernatant and sonication of the cell pellet followed by precipitation. After electrophoresis, the proteins were transferred to Immobilon P membranes for immunodetection. Fig. 5 shows a Western blot developed with antiprotease serum. The control lanes carrying only pGSD6 (lanes 1 and 7) show no band. Cells expressing only protease without the secretion functions from pUC19prtCEA16 (lanes 2 and 6) show only the larger immature band, which is inactive. The small amount of

extracellular immature protease seen is probably due to cell lysis. Cells carrying both plasmids and expressing functional protease have both the mature active protease band and that of the immature species (lane 3). A significant level of immature species is also found intracellularly in this strain (lane 5), suggesting that secretion from E. coli with the pGSD6 plasmid is not very efficient. The E. coli strains were grown without IPTG induction for this blot. It is clear from lane 3 that the larger protein band, representing the inactive zymogen, is also efficiently secreted. It appears that activation of the zymogen is the limiting factor in our system, even at this uninduced level of protease expression. Supernatants from S. marcescens do not show the larger immature species, although breakdown products are readily seen on this blot (lane 4). This autodigestion was also observed by Letoffe et al. (22). In Fig. 6 are shown only the supernatant fractions of JM101 carrying pUC19prtCEA16 with or without the a-hemolysin secretion functions of plasmid pSH305. A small amount of processed extracellular protease can be seen in lane 2, but the majority of the protease is the unprocessed zymogen. Lane 4 shows the protease expressed with no secretion functions; this lane was overloaded fourfold relative to lane 2 in order to visualize the small amount of zymogen found in the growth medium, presumably from cell lysis. Again, these results are from uninduced cells. When JM101(pSH305, pUC19prtCEA16) is IPTG induced, we observe a decrease in extracellular protease (of both sizes) and accumulation of protease intracellularly (data not shown), suggesting that IPTG inhibition is due to saturation of the a-hemolysin secretion system. DISCUSSION In this work, we have demonstrated that the secretion functions of the ao-hemolysin system, which are the products

1

2

3

4

FIG. 6. Western blot developed with antiprotease serum. Cultures were grown, with antibiotics as appropriate, until anA6 value of 1 to 1.5 was reached. Cells were removed by centrifugation, and the supernatants were collected by ethanol precipitation. Lanes: 1,

JM101(pSH305); 2, JM101(pSH305, pUC19prtCEA16); 3, SM6; 4, JM101(pUC19prtCEA16). About fourfold more supernatant was added to lane 4 in order to visualize the band.

VOL. 174, 1992

S. MARCESCENS PROTEASE CAN USE HlyB AND HlyD IN E. COLI

of the genes hlyB and hlyD (14), are sufficient for the production of active metalloprotease when the S. marcescens protease gene is expressed in E. coli. This is a reasonable finding in view of the recent report that the E. chrysanthemi proteases utilize accessory genes which are related to the a-hemolysin secretion proteins (21) and that the E. chrysanthemi proteases are about 60% homologous to the S. marcescens metalloprotease (29). The present work extends that observation, showing that the o-hemolysin transport proteins can functionally substitute for the S. marcescens counterparts. This result differs from that of Letoffe et al. (22), who report that the E. coli a-hemolysin secretion functions did not support the activation and secretion of the S. marcescens protease. The explanation for this discrepancy is probably quite simple. In our hands, the dual-plasmid system was quite unstable and variants which lost the ability to produce protease arose rapidly, despite constant selection for both plasmids. If we had not tested primary transformants, we would likely not have detected protease production with pSH305. Additionally, the primary clone in the work of Letoffe et al. had its upstream region fixed at the HindIII site, which is functionally equivalent to our prtHE clone. In our hands, this clone was poor at expressing protease and no activity was detected with the a-hemolysin secretion functions of pSH305. As Letoffe et al. observed, activity could be detected when the E. chrysanthemi secretion functions were used in place of those of a-hemolysin. The E. chrysanthemi secretion functions on plasmid pGSD6 were more stable in a dual-plasmid system with the S. marcescens protease than was the a-hemolysin system on pSH305. Related to this is the observation that when protease is expressed from the high-copy-number pUC plasmid, induction of the lac promoter by IPTG results in colonies unable to express protease. The inhibition observed with overexpression does not occur in S. marcescens. When these plasmids are introduced into wild-type S. marcescens, even without any lac repressor present, there is an increase in protease production over the normal levels (data not presented). This suggests a gradation of functionality of the secretion systems for their specific target protein. The a-hemolysin is worst suited for the S. marcescens protease, the E. chrysanthemi protease secretion factors function well with the S. marcescens protease but still show some inhibition at high levels of protease expression, and no inhibition is observed in S. marcescens. A real determination of whether there is a difference between the S. marcescens and the E. chrysanthemi secretion functions will, however, require the cloning of the S. marcescens secretion functions and their expression in a dual-plasmid system in E. coli to rule out the possibility that the defect is E. coli specific. A significant observation is that the S. marcescens protease is inactive in its intracellular form (or extracellularly leaked form; 6), even in the absence of the inhibitor protein, but that the process of transport by hlyB and hlyD or its homologs is requited to cause its activation, although not with 100% efficiency. Presumably, this is an autocatalytic event, but there is no direct evidence for this. The hemolysin requires another gene product, that of hlyC, which is missing in our system, for its activation (24). We previously reported finding the larger inactive zymogen in the growth medium of E. coli without any secretion functions (6). Although this observation holds, we now ascribe the release of this zymogen to cell lysis. Significantly more protein is found extracellularly in the presence of either the a-hemolysin or the E. chrysanthemi secretion functions than in their absence (Fig. 5). This supports the conclusion that protease

2365

secretion is mediated by the HlyB and HlyD proteins or their homologs but, additionally, that protease activation requires the protease to be properly secreted. Whether activation is affected by these secretion factors or is an indirect result of protease being secreted cannot yet be distinguished. There remains a mystery regarding protease expression from the prtHE clones. Clearly the prtE7 clone expresses protease from some endogenous promoter. It is not oriented correctly to be expressed from the lac promoter and shows no IPTO induction or inhibition. However, the prtCE clones only express prqtease from the lac promoter. When in the inverse orientation (pUC18), no protease expression in E. coli is observed. What is surprising is the observation that the prtHE clones expressed little or no protease (with pGSD6 or pSH305, respectively), regardless of orientation. It is unlikely that this clone lacks the translational start signal. There are three possible Met codons in frame from which protease translation could start. Nakahama et al. (23) assigned the third Met as the likely start codon. The ClaI used to define the 5' end of the prtCE clones lies 136 bases upstream of the first Met and 187 bases upstream of the third Met. Cleearly this clone includes all of the relevant upstream DNA for efficient translation. The HindIII site lies 38 bases upstream of the first Met and 89 bases upstream of the third Met. This too should carry sufficient DNA for translation, since transcription can be provided from the lac promoter in the pUC19 clone. There are no other possible Met starts further upstream because a TAA stop codon is found in the DNA just after the HindIII site in the protease reading frame. Therefore, translation must start after the HindIII site. This dilemma remains unresolved. There has been a report of a specific protease inhibitor made by E. chrysanthemi (20). In this paper, we present evidence of a similar inhibitor whose gene lies immediately downstream of the S. marcescens protease. The prtCEA16 clone deletes almost 2 kb of downstream DNA in the protease gene clone of prtE7 and prtCE. This clone clearly expressed more protease in our dual-plasmid system than its parent plasmid. The simplest explanation is that we have deleted part or all of a protease inhibitor gene which lies downstream of the protease structural gene. There is an open reading frame in this region'which is partially homologous to that of the inhibitor from E. chrysanthemi. Letoffe et al. (22) suggest that the protease inhibitor is essential for cell viability, presumably because it inhibits activation of any intracellular protease; however, it is not essential for expression of the E. chrysanthemi proteases in either E. chrysanthemi or E. coli. Our results suggest that the observations made with the E. chrysanthemi proteases also hold true for E. coli. The inhibitor is not essential for cell viability, as is shown by our prtCEA16 clone, and no active protease can be detected when the protease gene is expressed in E. coli in the absence of secretion functions (6). The S. marcescens protease represents another member of a family of proteins which are secreted in a signal peptide-independent manner utilizing secretion systems related to that of cx-hemolysin. Among these are the proteases from E. chrysanthemi and Pseudomonas aeruginosa (4, 22), the toxins of Bordetella pertussis and Pasteurella haemolytica (13, 15, 26), colicin V (11), and the NodO protein of Rhizobium leguminosarum (10). The secretion functions for these proteins are also clearly functionally related, as shown by cross complementation in the present work and in that of others (13, 15, 26).

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SUH AND BENEDIK

ACKNOWLEDGMENTS

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