The metalloprotease gene of Serratia marcescens strain SM6. Sharon C. Braunagel and Michael J. Benedik*. Department of Biology, Texas A & M University, ...
Mol Gen Genet (1990) 222:446-451 © Springer-Verlag 1990
The metalloprotease gene of S e r r a t i a m a r c e s c e n s strain SM6 Sharon C. Braunagel and Michael J. Benedik* Department of Biology,Texas A & M University,CollegeStation, TX 77843-3258, USA Received October 16, 1989 / March 28, 1990
Summary. Utilizing the DNA sequence of the metalloprotease from Serratia strain E-15, we isolated and sequenced the homologous gene from Serratia strain SM6. These two genes are similar at both the DNA and protein sequence level. Expression of the protease gene in Escherichia coli was achieved by use of the lac promoter. This resulted in the production and excretion of an immunologically detectable but inactive protein of slightly higher molecular weight than that from Serratia. We introduced the cloned gene into previously described protease mutants. The observed pattern of protease expression suggested that these mutations fall into three classes. Key words: Protease - Extracellular proteins - Secretion - Enterobacteriaceae - Gene cloning
The bacterium Serratia marcescens is known to excrete a variety of degradative enzymes into the surrounding media. These activities include a nuclease and several proteases, chitinases, and lipases (Braun and Schmitz 1980; Bromke and Hammel 1979; Eaves and Jeffries 1963; Heller 1979; Monreal and Reese 1969; Gikskov et al. 1988). Analysis of a variety of different Serratia strains reveals variation in the number of different proteases ranging from one to four or more (Grimont et al. 1977). Of these, the major extracellular protease has been characterized by a number of groups (Aiyappa and Harris 1976; Braun and Schmitz 1980). It is a zinc metalloprotease with a wide pH optimum of 6.0-9.0. Schmitz and Braun (1985) showed that two forms of the protein could be found extracellularly. The major form was an enzyme of 51 kda and the minor form was of about 53 kda. Both forms appear rapidly during pulse chase experi*Present address and offprint requests to: M.J. Benedik, Department of Biochemicaland BiophysicalSciences,Universityof Houston, Houston, TX 77204-5500, USA
ments. During normal growth no intracellular form was detectable by immunological methods, although a 52 ka form could be found intracellularly after the addition of protein secretion inhibitors. Further studies on the extracellular forms demonstrated that the major form I could be converted to two other forms, II and III, each of which has a slightly higher apparent molecular weight. N- and C-terminal sequencing revealed identical sequences at both ends of forms I and II. In addition to the various metalloprotease forms, there exists in Serratia a serine protease of molecular weight 41 kda (Yanagida et al. 1986). The gene for the metalloprotease has been cloned from S. marcescens strain E-15 and sequenced by Nakahama et al. (1986). They found that the metalloprotease gene encoded a protein of 470 amino acids with a molecular weight of 50632 daltons. The sequence that they reported was almost identical with partial amino acid sequences reported by Lee et al. (1985) and Schmitz and Braun (1985). Analysis of the DNA sequence suggests that the mature protein-coding region is preceded by a coding region for a pre-sequence. There are three possible Met initiation codons of which two are preceded by reasonable Shine-Dalgarno sequences. Based on protein size, Nakahama et al. (1986) suggested that the third Met codon is used, resulting in a pre-sequence consisting of 16 amino acids. Although this N-terminal sequence is presumably processed from the mature form, it does not resemble a classical procaryotic signal sequence. Whether it plays a role in protease export or zymogen activation is not clear. When a plasmid carrying this gene is introduced into Serratia, increased protease levels relative to control strains could be found in the supernatant. When this same plasmid was transformed into Escherichia coli no enzymatic activity was detected in the supernatant. However an intracellular band of 51 kda was detected immunologically by Western blot analysis. In the present study we have cloned and sequenced the metalloprotease gene from S. marcescens strain SM6 because the homologous gene described above (Nakaha-
447
ma et al. 1986) was not made available to us. We have used the cloned gene to analyze its expression in E. coli and to characterize our previously described protease mutants (Hines et al. 1988). Isolation and sequencing of the protease gene To isolate the gene of the metalloprotease from S. marcescens strain SM6, we screened a previously described genomic library in 2EMBL3 (Ball et al. 1987). Phages were plated on the E. coli strain LE392 and probed with the synthetic oligonucleotide QCTGTAGACGACCTGTTGCATTATCATGAGCGGGGC chosen from the sequence of the metalloprotease of Nakahama et al. (1986). DNA was prepared from two independent plaques that hybridized to the probe. Both presented different restriction patterns, however each had in common a 3.8 kb HindIII-EcoRI fragment. From one of these a 5 kb EcoRI fragment was purified and subcloned into the vector pUC18 (Vieira and Messing 1982) to make pUC18-prtE7. Figure 1 shows an approximate restriction map of this region. Two further sets of subclones from which the upstream region was removed were constructed using convenient restriction sites. The 3.8 kb HindIII-EcoRI fragment was subcloned into pUC18 and pUC19 to make pUC18-prtHE and pUC19-prtHE respectively. The prtHE fragment carries the entire predicted protease gene and downstream flanking region, but only about 90 bases of the upstream region. A slightly longer 3.9 kb ClaI-HindIII fragment which carries about 190 bases of the upstream region was also used to create pUC18-prtCE and pUC19-prtCE. Figure 2 shows the sequence of the protease gene from the ClaI to EcoRI restriction sites. The sequence is similar to the sequence from Nakahama et al. (1986) and the two protease genes are almost identical. There is about 95% identity at the DNA level and 97% at the peptide level. The only significant difference is a 2base deletion at position 1234 followed by a single base deletion at 1249 in the Nakahama et al. (1986) sequence relative to our sequence. This results in a frameshifted region of 5 amino acids with a single amino acid deletion. The region in our sequence downstream of the protease gene from bases 3180 to 3840 has strong homology to part of the E. coli fts YEX operon sequence (EMBL accession number X04398). This region o f f t s Y E X con-
EcoRI
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2kb Sall B a m H l
l
I
3kb
,
Sail
Z,kb
5kb EcoRl
I
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) prtCE ~prtHE
Fig. 1. Restriction map of the EcoRI fragment carrying the metalloprotease gene from Serratia rnarcescens strain SM6. The lines indicate the lengths of the D N A segments referred to in the text. The open box indicates the protease reading frame
tains an unidentified open reading frame and does not include the fts YEX genes. Protease expression in S. marcescens The level of protease expression in S. marcescens can be easily monitored by halo size on skim milk plates (Hines et al. 1988). Introducing either pUC18-prtE7 or pUC18-prtCE into the wild-type strain SM6 generates transformants with increased halo size. However pUC18-prtHE has no noticeable effect on halo size in SM6. All these plasmids were also introduced into the protease mutants of SM6 which we have described previously (Hines et al. 1988). Some of these mutants (prt: :Tn5-1, prt: :Tn5-2, prt: :Tn5-3 and prt: :Tn5-9) express no detectable protease, while others (prt: : Tn5-4, prt: : Tn5-6, prt: : Tn5-7 and prt: : Tn5-8) express reduced levels of protease. Transforming all eight mutants with the plasmid pUC18-prtE7 restores protease expression to wild-type or nearly wild-type levels. The plasmid pUC18-prtHE has no effect on protease activity on indicator plates when transformed into these mutants except in one case, prt: : Tn5-9, where protease activity was partially restored. The plasmid pUC18-prtCE restores protease activity to all the mutants, however the halo size was always slightly smaller than when these mutants carried the pUC18-prtE7 plasmid. The above analysis allowed us to classify the mutants into three groups. The three mutants prt::Tn5-1, prt: :Tn5-2 and prt::Tn5-3 fall into one class and express no protease unless they carry the pUC18-prtE7 or prtCE plasmids. The mutant prt:: Tn5-9 also express no protease but activity is partially restored when it carries any protease plasmid including pUC18-prtHE; it therefore constitutes a second class. The four reduced expression mutants fall into the third class. One mutant from each class was chosen for further analysis. Plasmids carrying the prtCE and prtHE clones in both pUC18 and pUC19 were transformed into each mutant and protease levels determined (Table 1). Protease expression from the pUC18 plasmids must come from a promoter carried on the fragment, however in pUC19 the protease gene can be expressed from the plasmid lac promoter. No inducer is required since lac repressor is not present in the S. marcescens strains. The smaller fragment prtHE can complement one class of mutants efficiently only if expressed from a plasmid promoter. This supports the assignment of a promoter upstream of the HindIII site but downstream of the ClaI site since prtCE complements these mutants regardless of vector. However the suggested promoter sequence of Nakahama et al. (1986) lies just upstream of the Clal site. We would argue that a functional promoter must lie between these two positions. Based on this analysis it is suggested that the class of negative mutants (prt: : Tn5-1, prt : : Tn5-2, prt: : Tn53) with similar complementation patterns probably represent mutations in the protease structural gene itself. However the single unusual negative mutant, prt: : Tn59, clearly represents a different locus. It can be partially complemented by the prtHE fragment even in the
448 A
C
ATCGATTACCATTTTT~TGGTGATCTTATTTGcTGATATATATGCATT~TTcTAC~T~cAcACTGCCGGT~cGGCGCAT~GCCCCTTcCAGC~GcTT~GGTTTATT~CCGTG 1
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A T C
120
C
GcTTA~GGGGAGGTTATGTcTATcTGTcTGATTGATATc~TCAGGT~TGAGTGG~TCG~Cc~TGc~TCTACTAA~AAGGC~TTGAAATTACTG~TcCAGCCTTGCGG~CGCG 121
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+ .........
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S
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+ ......... L
A
A
+ .........
+
A
240 3~
N F T
C
T
AC~CCGGCTACGATGCTGTAGACGAcCTGCTGcATTATCATGAGCGGGGT~CGGGATTCAGATT~TGGC~GGATTCATTTTCT~cGAGC~GCTGGGCTGTTTATTACCCG~GAG 241
.........
3
6
T
T
+ ......... G
Y
D
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+ ......... V
D
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+ .........
L
L
H
Y
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H
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N
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I
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-+ ......... N
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Q
+ .........
A
G
L
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+ .........
I
T
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+ .........
+
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360 75
~cCAAACcT~CGGTTAC~GGTATTTGGCcAGcCGGTCAAATT~CTTCTCcTTCCCGGACTAT~GTTcTCTTCCACC~CGTCGCCGGCGACACCGGGCTGAGC~GTTCAGc 361
.........
7
6
N
Q
+ ......... T
W
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G
+ ......... Y
K
V
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F
G
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V
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L
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F
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G
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D
+ ......... T
G
L
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+
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480 115
C
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GCGG~CAGCAGcAGCAGGCT~GCTGTCGCTGCAGTC~TGGGCCGACGTTGCC~TATCAcCTTCACCGAAGTGGCGGCCGGTCAAAAGGCC~TATCACCTTCGGC~TTACAGcCAG 4S1 I
......... I
6
A
E
+ ......... Q
Q
Q
+ .........
Q
A
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L
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S
L
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A
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A
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A
C
N
+ .........
I
T
F
G
G
+ .........
N
Y
S
+ .........
+
Q
600 155
C
G
GATCGTCCCGGCCACTATGATTATGGTACCCAGG~CTACGCcTTCcTGCCG~CACCATTTGGCAGGGC~AGGATTTGGGCGGCCAGACCTGGTAT~CGTC~C~TCC~CGTG~G 601 1
......... 5
6
D
R
+ ......... P
G
H
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Y
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G
+ .........
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F
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N
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G
G
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+ .........
N
V
N
Q
T
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720 195
A
CG
CATCcGGCGAC~G~GACTACGGCCGC~AGACGTTCACCCATGAGATTGGCCATGCGCTGGGcCTGAGCCAcCCGGGCGACTAC~CGCCGGTGAGGGC~CCCGACcTA~CGACGTC 721 1
......... 9
6
H
P
+ ......... A
T
E
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D
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Q
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F
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840 235
R T
C
T
C
T T
T C
A
C
ACCTATGCGG~GATACCCGCCAGTTCAG~CTGATGAGCTACTGGAGTGAAACC~CACCGGTGGCGAC~CGGCGGTCACTATGCCGCGGCTCCGTTGCTGGATGACATTG~CGCCATT 841 2
......... 3
6
T
Y
+ ......... A
E
D
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T
R
Q
A
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S
L
M
S
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Y
W
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N
T
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T
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D
N
G
+ ......... G
H
Y
A
+ ......... A
A
P
L
+ ......... L
D
D
I
+ ......... A
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+ .........
+
I
960 275
T
CAGCATCTGTATGGcGCC~CCCGTCGACCCGCACCGGCGACACCGTGTACGGCTTT~CTCC~CACCGGTCGTGACTTCCTCAGCACCACCAGC~TTCGcAGAAAGTGATCTTTGCG 961 2
......... 7
6
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H
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Y
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A
N
P
S
+ .........
T
R
T
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D
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+ ......... G
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T
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+ ......... D
F
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S
+ ......... T
T
S
N
+ ......... S
Q
K
V
+ ......... I
F
+ .........
+
A
1080 315
L C
G
A
C
GCCTGGGATGCGGGTGGC~CGATACCTTCGAcTTCTcCGGTTATACcGCT~CCAGCG~ATC~C~TG~TGAGAAATCGTTCTCCGACGTGGGCGGCcTG~GGGc~CGTCT~GATA 1081 3
......... 1
6
A
W
+ ......... D
A
G
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G
N
D
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F
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+ ......... D
V
G
G
+ ......... L
K
G
N
+ ......... V
S
+ .........
+
!
1200 355
T
GcCGcCGGTGTGA~CATTGAG~CGCCATTGGCGGTTCCGGc~TGACGTGATCGTCGGC~CGCGGCC~C~CGTGCTGAAAGGCGGCGCGGGT~CGA~GTGCTGTT~GGCGGcGGc 1201 3
......... 5
6
A
A
+ ......... G
V
T
+ .........
I
E
N
A
+ .........
I
G
G
S F
G
+ .........
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N
D
A
Q
T
V R
+ ......... I
V
G
N
+ ......... A
A
N
N
+ ......... V
L
K
+ .........
G
G
A
G
N
+ ......... D
V
L
F
+ ......... G
G
+ .........
+
G
1320 395
L
C
G
C
A
T
GGGGCGGATG~CTGTGGGG~GGTGCCGGCAAAGACATCTTTGTGTTCTCTGCCGCCAGCGATTCCGCACCGGGTGCTTCCGA~TGGATCCGCGAcTT~CAGAAAGGGATCGAC~GATC 1321 3
......... 9
6
G
T
A
+ ......... D
E
L
+ .........
W
G
T
G
A
+ .........
G
C
K
C
D
i
G
+ .........
F
V
F
S
C
+ ......... A
A
S
D
+ ......... S
A
P
G
+ ......... A
S
T
D
W
+ ......... I
R
G
D
F
+ ......... Q
K
G
I
+ ......... D
K
+ .........
+
I
1440 435
A
A
GACcTGTCGTTCTTC~TAAAG~%GCG~TAGCAGTGATTTCATCcACTTCGTCGATCACTTCAGCGGCACGGCCGGTGAGGCGCTGCTGAGCTAC~CGCGTCCAGC~CGTGACCGAT 1441 4
......... 3
6
D
L
+ ......... S
F
F
+ .........
N
K
E
A
+ .........
N
S
S
D
+ .........
F
I
H
F
+ ......... V
D
H
F
+ ......... S
G
T
A
+ ......... G
Q
E
A
L
+ ......... L
S
Y
N
+ ......... A
S
S
N
+ ......... V
T
T T
T
+ .........
+
D
A
1560 475
N
T
T
G
G
TTGT~GGTG~CAT~GGCGGG~ATCAGGcGccGGA~TT~cTGGTGAAAATCGTCGGc~AGGTAGAcGTCG~CACGGACTTTATCGTGT~AcAG~CGG~cGc~CGGCGCAGT~TCG 1561 4
......... 7
6
L
S
+ ......... V
N
I
G
+ ......... G
H
Q
A
+ ......... P
D
F
L
+ ......... V
K
I
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+ ......... V
A
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D
+ ......... F
I
V
+ .........
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*
1680 515
CC ~ A C GC G~cGGG~GTGATG~AG~GC~cGGTATGAA~GGTACTTTAGCA~GCAccG~TTTGGCGGcGGGTGG~ATGATGGTGAcGAGTG~GGTGATGGCcGG~AGTTTGGcATTG~CGA~CG~GCA TG
1681
.........
+ .........
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A
A
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G
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A
CA
+ .........
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G
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C
+ .........
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GT
+
1800
C
GTCGCTGGCGGGAC~TGGCAGGTGG~CAGCG~cGGC~TGCCAAATCGAGTTTCTGG~GCATG~AAAGCGAGACC~CGG~TATCAGCTGGTGGAT~GGC~CAGTGTTTGCA 1801
.........
+ .........
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1920
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2040
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2160
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2280
+ . . . . . . . . .
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2400
A A C G G A G A GAGCGTGTTTGCGGCGG~GTCGTGGGCTGGCGCCCGG~TCCGGACGGCATCGC~CTG~TGCGGGCGGATGGCAGCACG~TGGCGTTCTT~TCG~G~GAGGCAT~TGTACCGC~T~AGC 1921
.........
+ .........
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+ .........
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TGGGTGCGGATGATGCTTTGACGTTGAAAGCGCTGGCTTGATG~GAAACGGGTTCGGCGCGCCG~CCCG~GGTAGGTCTCTACAGGTAGAGCG~CGCACGATCAGG~GTGGCCGGA 2041
.........
+ .........
+ .........
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+- ........
G~GT~cAGAAAGCCACGATGGCGTCCGC~GCG~GG~GGTG~GCGGTAGCGGTTGAGCAG~cAAA~CA~GTTGGCCAGCAGCAG~AGCGAGGTGCCGGT~AGcAGCGAG~GCCG~ 2161
.........
+ .........
+ .........
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+ .........
+ .........
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ATCGGTGCTGCGCAGGAAATATTGTTGGCCGGCCAGCCAGA~CATCAGCAGCGTCATGGCCACGTAGGT~CGATCGGCCAGCGCATCT~TTCCAGCCGGGTCCAGA~GGTGGCCAGCAG 2281
. . . . . . . . .
2401
. . . . . . . . .
+ . . . . . . . . .
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cAGTGCGcCGATCGCCAGCAGCGCCAGcGGCAGCGG~CAG~CAGGCTG~cGTCATcTGG~TGGCGAAACTCAGGGTGTACAGCAGGTGGGAcAGGAAAAAGGCG~GATGG~ATACAG + . . . . . . . . .
+ . . . . . . . . .
+ . . . . . . . . .
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+
2520
CACcCGTTCGCGCGGC~CAGGAGC~TGCGTCGCCGATCAGCGTCGCCAGCAGGCCGAGTACGATCAGAT~cC~G~AGCGCCGAGCACCGG~GCCTGCCAGGcc~CAGC~AGcAG 2521
. . . . . . . . .
+ . . . . . . . . .
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+ . . . . . . . . .
+ . . . . . . . . .
+ . . . . . . . . .
+
2640
CAGCAGCGTGACGGGTTTG~CAC~CAGCGCTGCCAGCGCGGCCCAcGGT~GAGGCGTCGACG~CAGCC~C~GGAAAAG~TACGGC~GG~GGCC~CTCATT~TGTCTT~CT 2641
. . . . . . . . .
+ . . . . . . . . .
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+ . . . . . . . . .
+ . . . . . . . . .
+
2760
TATGTATGAGGGAGCCGC~C~CCGGCTCCAAACTGTCACC~TGTCACTTCTTCATTTCAGTGTAGGTAGCCTGC~GGCGATcGAC~TGCTTTCTTGAG~TGCTATGTTTA~GCTG 2761
. . . . . . . . .
+ . . . . . . . . .
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+ . . . . . . . . .
+ . . . . . . . . .
+ . . . . . . . . .
+
2880
ATTTAGCCGCG~GGAAATGGGAAACCGATGAGT~GCCACCGTTATTTTTTGTCGCCGTGATTGCGTTGATCGCCGTGCTGGCCACGc~CGcTAcTTC~GCAGCGG~AGcAGGAGG~ 2881
.........
+ .........
+ .........
+ .........
+ .........
+ .........
+ .........
+ .........
+ .........
+ .........
+ .........
+ .........
+
3000
+
3120
GGAAAA~GATCGCGCGCCGATG~G~AGT~TG~AGGTGA~GGTGAGCGACAAA~GcGcGGTGCCGGTCACCAA~GA~c~G~G~G~CGCAG~GCGAGC~GTTGGTc~TG~GATG~TT 3001
Fig. 2
.........
+ .........
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449 3121
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+ . . . . . . . . .
+ . . . . . . . . .
+ . . . . . . . . .
+ . . . . . . . . .
+ . . . . . . . . .
+ . . . . . . . . .
+ . . . . . . . . .
+ . . . . . . . . .
+
3240
3241
. . . . . . . . .
+ . . . . . . . . .
+ . . . . . . . . .
+ . . . . . . . . .
+ . . . . . . . . .
+ . . . . . . . . .
+ . . . . . . . . .
+ . . . . . . . . .
+ . . . . . . . . .
+ . . . . . . . . .
+ . . . . . . . . .
+ . . . . . . . . .
+
3360
3361
.........
+ .........
+ .........
+ .........
+ .........
+ .........
+ .........
+ .........
+ .........
+ .........
+ .........
+ .........
+
3480
+ .........
+ .........
+ .........
+ .........
+
3600
+ .........
+ .........
+ .........
+ .........
+
3720
+ .........
+ .........
+ .........
+ .........
+
3848
AGAGCAGGAAGCCCCACAGCcACAGCATCAGCAGGCGGCCCAGATTAATCAACATGGTcGGTTTTCTCTTGCGAAcGAAGATAAAGGCGATAGGCCACCTGGCCGGCGACCTTCTCCCG~ 3481
.........
+ .........
+ .........
+ .........
+ .........
+ .........
+ .........
+ .........
TGCAGCTGCCAGCTGGCAGGCACTCCGCCGCGGCGCTTTCCGCCTCGGCTTCTACATAGATCCAGGCCTCGTCGGCCAGCCAGCCGCGTTGCTCCAGCAACAGGGCGTCTCCGCCAGCAG 3601
.........
+ .........
+ .........
+ .........
+ .........
+ .........
+ .........
+ .........
GCCTTTGCGAAACGGCGGGTCGAGGAACACCACGTCGAACGGTTGGCCATCGCCCGCCAGCCAGCTCAGCGCGTTGGTGTTGATAACCACCCCTTTGCCCTGCAGCAGCGCCAGATTTTT 3721
.........
+ .........
+ .........
+ .........
+ .........
+-
+ .........
+ .........
+ .........
+ .........
TTCCAGCTGTGCGCCACCGGCCGTTCGAATT 3841
.........
+ .........
3871
Fig. 2. The DNA sequence from the ClaI to EcoRI restriction sites is shown. The sequence was determined using the dideoxy method (Sanger et al. 1977) from templates made by shbcloning convenient restriction fragments or by using synthetic oligonucleotide primers. Computer analysis was aided by programs from the University of Wisconsin Genetics Computer Group (Devereux et al. 1984). The region from positions I to 1984 is compared with the protease
from S. marcescens strain E-15. The differences in the DNA sequence are shown above the sequence and differences in the predicted amino acid sequence are shown below. Translation is shown from the first Met codon (lower case), although Nakahama et al. (1986) propose the third Met (start of upper case) as the actual start. Open triangles indicate bases missing from the Nakahama et al. (1986) sequence
Table 1. Levels of metalloprotease expression from protease mutants
activity in any of the cell fractions, supernatant or intracellular fraction (Neu and Heppel 1965). However immunologically detectable protein could be found with anti-protease serum after SDS-polyacrylamide gel electrophoresis (PAGE) and Western transfer f r o m the supernatants of cells carrying certain protease clones. The full-length clone, prtET, does not appear to be expressed in E. coll. The protease gene p r o m o t e r m a y not function, possibly due to lack of a positive activator and the upstream region of this D N A fragment might well terminate transcription initiated f r o m the plasmid lac p r o m o t er. This is similar to our observations o f nuctease expression in E. coli (Ball et al. 1987). Surprisingly the shorter clone, prtHE, also expressed no detectable protein in E. coli even from the lac promoter. The supernatants of cultures of E. coil containing either of the p r t H E clones in vectors pBGS18 or 19 (Spratt et al. 1986) did not have any detectable protein by Western blot analysis (Fig. 3A) nor did the supernatant from pBGS18-prtCE containing cells (not shown). However if p r t C E is expressed f r o m the lac p r o m o t e r in pBGS19 and induced with isopropyl-/?-D-thiogalactopyranoside (IPTG), a considerable a m o u n t of protease peptide was found in the supernatant Western blotting (Fig. 3). This result is the same if the pUC19 vector is used instead o f p B G S I 9 (not shown) and is in contrast to the observations of N a k a h a m a et al. (1986) who detected protease protein only in the periplasm of E. coli. Although assigning a p r o m o t e r to the region between the ClaI and H i n d I I I sites could explain the pattern o f protease expression seen in S. marcescens, we are unable to explain the requirement of that region for production of the extracellular peptide in E, coli. There is a stop codon just beyond the H i n d I I I site so we cannot assign a translational start to this region. The role of this region m a y be identical for expression of the protease in both E. coIi and S. rnarcescens. Possibly this region has a regulatory role, perhaps to stabilize the message or to increase the efficiency of translation, in addition to carrying a promoter. In the report of N a k a h a m a et al. (1986) the inactive
SM6 Plate
prt: :Tn5-1
prt: :Tn5-6
prt: :Tn5-9
+ + + +
-
+
-
0.062
0.002
0.046
0.002
0.063
0.002
0.048
0.027
0.065
0.058
0.057
0.054
0.061
0.056
0.071
0.056
0.068
0.054
0.054
0.063
phenotype
Protease levels pUC18prtHE pUC19prtHE pUC18prtCE pUC10prtCE
The protease activities of wild type SM6 and of a representative of each class of Tn5-induced protease mutants are shown with or without the diffeent protease plasmids. The plate phenotype designates relative halo size on skim milk plates. The numerical values represent protease activity measured using the azocasein assay described in Hines et al. (1988). No lac repressor is present in these Serratia marcescens strains pUC18 vector which has no effect on the other negative mutants, and is fully complemented by all the other constructs tested. We certainly have no direct evidence to explain the nature of this mutation but an attractive hypothesis is that another gene exists on our plasmids in the 2 kb region downstream of the protease gene; a n u m b e r of open reading frames (ORFs) do exist in this region. In a similar case a downstream O R F was recently identified as a m a t u r a t i o n protein for the extracellular lactococcal serine protease ( H a a n d r i k m a n et al. 1989). Experiments to test this prediction are underway. Expression in E. coli
All of the gene constructs were separately transformed into JM101. There was no detectable enzymatic protease
450 A
1
2
3
Figure 3B also confirms that the m u t a n t strains
4
prt: :Tn5-1 and prt: :Tn5-9 do not m a k e or secrete any
protease --130kDa -- 75kDa
- - 50 k Da
B
1
2
3
4
5
protein,
either
active
or
inactive,
while
prt: :Tn5-6 does secrete some. These data, based on
6
75 kDa - -
50kDa--
39kDa--
Fig. 3A and B. Western blots of SDS-PAGE demonstrating pres-
ence of protease protein were performed using anti-protease serum and developed with alkaline phosphatase-conjugated second antibodies. A Lane 1, negative control consisting of JM101 carrying the pBGSI9 vector; lanes 2 and 3, JM101 containing pBGSI9 with the EcoRI-HindlII or the Eco-RI-ClaI insert respectively; lane 4, SM6 positive control. B The unmarked lane is prestained molecular weight markers; lane 1, purified metalloprotease; lane 2, JMI01PhoA c containing the protease clone pBGS19-prtCE; lane 3, SM6 positive control; lane 4, mutant strain prt::Tn5-1; lane 5, mutant prt: :Tn5-6; lane 6, mutant prt: :Tn5-9. All samples are supernatant fractions from fresh stationary cultures, and equal volumes were loaded in each lane from equivalent cell densities
protease zymogen was detected intracellularly in E. coli. We were unable to analyze the periplasmic or intracellular fractions because J M I 0 1 has a m a j o r band detected by our antibody in the same region masking the position where the protease would be expected. This caused concern as the b a n d that we saw after Western blotting o f the supernatant fraction of E. coli (Fig. 3) could be due to cell lysis and indiscriminate release of intracellular proteins. To eliminate this possibility the protease clone p B G S I 9 - p r t C E was transformed into a derivative of JM101 which expresses alkaline phosphatase constitutively. The supernatant and cellular fractions of these transformants were assayed for phosphatase activity. The supernatant contained only 6% of the total phosphatase activity. Fig. 3 B shows a Western blot of the purified protease, and supernatants from JM101 p h o A ~ (pBGS19-prtCE) and SM6. The gel was loaded with equal volumes of supernatant f r o m cultures at equivalent cell densities and shows similar intensities of the reactive band in JM101 and the S. marcescens SM6 control. It was concluded that cell lysis is not sufficient to account for the equivalent levels of extracellular protease found.
Western blot and antibody binding, are consistent with the original plate assays and the protease activity assays (Table 1). There is a slight difference in the molecular weight between the control SM6 protease band and that of protease proteins present in E. coli (Fig. 3). The protein released into the supernatant of the E. coli transformant is always slightly larger (2-3 kda) than the wild type. This suggests incomplete or incorrect processing of the protease peptide in E. coll. Whether or not this is the reason why no protease activity is observed remains to be determined. In both this report (Fig. 3 A) and that of N a k a h a m a et al. (1986) no functional protease could be detected in E. coli; the peptide found was of slightly larger molecular weight than the mature protease detected in Serratia. This suggests incomplete or incorrect processing of the protease zymogen in E. coli. Although m a n y proteases are autocatalytically processed, such as the serine protease of S. marcescens (Yanagida et al. 1986), others are known to require accessory genes. A m a t u r a t i o n protein is needed for the extracellular lactococcal serine protease ( H a a n d r i k m a n et al. 1989) and accessory genes which have been identified for the extracellular proteases of Erwinia chrysanthemi function either in expression or excretion of the protease (Wandersman et al. 1987). Although a Serratia accessory gene is required for maturation of the metalloprotease to yield an active protease, complete m a t u r a t i o n is not essential for extracellular secretion. Acknowledgements. This work was supported by grant GM36891 from the National Institutes of Health, a Biomedical Research Support Grant from the NIH through Texas A & M University, and the Texas Advanced Research Program. Some of the equipment used in this work was purchased with funds from an NSF equipment grant BBS-8703784 awarded to M. Benedik and others. We thank Dr. Greg Shipley and Tim Ball for their helpful advice and Shari Fernandez for technical assistance.
References
Aiyappa PS, Harris JO (1976) The extracellular metalloprotease of Serratia marcescens: I. Purification and characterization. Mol Cell Biochem 13:95-100 Ball TK, Saurugger PN, Benedik MJ (1987) The extracellular nuclease gene of Serratia marcescens and its secretion from Escherichia coli. Gene 57:183-192 Braun V, Schmitz G (1980) Excretion of a protease by Serratia marcescens. Arch Microbiol 124:55 61 Bromke BJ, Hammel JM (1979) Regulation of extracellular protease formation by Serratia marcescens. Can J Microbiol 25 :4252 Devereux J, Haeberli P, Smithies O (1984) A comprehensive set of sequence analysis programs for the VAX. Nucleic Acids Res 12:387 395 Eaves GN, Jeffries CD (1963) Isolation and properties of an exocellular nuclease ofSerratia marcescens. J Bacteriol 85:273 278 Gikskov M, Olsen L, Molin S (1988) Cloning and expression in Escherichia coli of the gene for extracellular phospholipase A1 from Serratia liquefaciens. J Bacteriol 170:5855-5862
451 Grimont PAD, Grimont F, Dulong De Rosnay HLC (1977) Characterization of Serratia marcescens, S. liquefaciens, S. plymuthica and S. marinorubra by electrophoresis of their proteinases. J Gen Microbiol 99:301-310 Haandrikman AJ, Kok J, Laan H, Soemitro S, Ledeboer AM, Konings WN, Venema G (1989) Identification of a gene required for maturation of an extracellular lactococcal serine proteinase. J Bacteriol 171:2789-2794 Heller K (1979) Lipolytic activity copurified with the outer membrane of Serratia marcescens. J Bacteriol 140:1120-1122 Hines DA, Saurugger PN, Ihler GM, Benedik MJ (1988) Genetic analysis of extracellular proteins of Serratia marceseens. J Bacteriol 170:4141-4146 Lee IS, Wakabayashi S, Matsubara H, Miyata K, Tomoda K (1985) Serratia protease. Amino acid sequence of the N-terminal half including a zinc binding site and of the C-terminal peptide. Fed Proc 44:1057 Monreal J, Reese ET (1969) The chitinase of Serratia mareescens. Can J Microbiol 15 : 689-696 Nakahama K, Yoshimura K, Marumoto R, Kikuchi M, Lee IS, Hase T, Matsubara H (1986) Cloning and sequencing of Serratia protease gene. Nucleic Acids Res 14:5843-5855 Neu HC, Heppel LA (1965) The release of enzymes from
Escherichia coli by osmotic shock and during the formation
of spheroplasts. J Biol Chem 240:3685-3692 Sanger F, Nicklen S, Coulson AR (1977) DNA sequencing with chain-terminating inhibitors. Proc Natl Acad Sci USA 74:5463-5467 Schmitz G, Braun V (1985) Cell-bound and secreted proteases of Serratia marcescens. J Bacteriol 161 : 1002-1009 Spratt BG, Hedge PJ, te Hessen S, Edelman A, Broome-Smith JK (1986) Kanamycin resistant vectors that are analogues of pUC8, p u c g , pEMBL8 and pEMBL9. Gene 41 : 337-342 Vieira J, Messing J (1982) The pUC plasmids, an M13mp7 derived system for insertion mutagenesis and sequencing with synthetic universal primers. Gene 19:259-268 Wandersman C, Delepelaire P, Letoffe S, Schwartz M (1987) Characterization of Erwinia chrysanthemi extracellular proteases: Cloning and expression of the protease genes in Escherichia coli. J Bacteriol 169:5046-5053 Yanagida N, Uozumi T, Beppu T (1986) Specific excretion of Serratia marcescens proteases through the outer membrane of Escherichia coli. J Bacteriol 166:937-944 C o m m u n i c a t e d by J. Lengeler
