JOURNAL OF BACTERIOLOGY, Apr. 1989, p. 2202-2208 Vol. 171, No.

4
0021-9193/89/042202-07$02.00/0
Copyright © 1989, American Society for Microbiology

Streptokinase Mutations Relieving Escherichia coli K-12 (prlA4) of

Detriments Caused by the Wild-Type skc Gene
JORG MULLER, HILMER REINERT, AND HORST MALKE*
Central Institute of Microbiology and Experimental Therapy, Academy of Sciences of the German Democratic Republic,
Jena 6900, German Democratic Republic
Received 8 September 1988/Accepted 13 January 1989

A novel phenotype is described for Escherichia coli K-12 carrying the prlA4 allele determining a membrane
component of the protein export mechanism. It is manifest as transformation deficiency for plasmids containing
the cloned group C streptococcal streptokinase gene, skc. Streptokinase plasmid mutations relieving the prl4
strain of this deficiency fell into three classes. Class 1 included skc::ISS insertions, with IS5 integrated in a
region encoding the Skc signal sequence and inactivating skc. Class 2 included IS] insertions leaving skc intact
but reducing skc expression, presumably by altering the function of the skc promoter as judged by an insertion
site close to the -35 region. The most interesting class, 3, included skc deletions removing the entire signal
sequence or a tetrapeptide from its hydrophobic core. The tetrapeptide deletion reduced the size, hydropho-
bicity, and predicted alpha-helicity of the central region of the Skc signal sequence but facilitated the export of
mature Skc in both the wild type and the prL44 mutant. These findings indicate that the incompatibility between
prlA4 and skc is related to deleterious effects of the Skc signal sequence. The tetrapeptide deletion may function
by altering the conformation of the signal sequence so as to render interaction with both the PrIA wild-type
protein and the PrlA4 mutant protein less detrimental to the export mechanism. These findings also provide an
explanation for the difficulties encountered in cloning streptokinase genes in E. coli plasmids and maintaining
their structural stability.

Streptokinase (Skc), the most important procaryotic plas-
minogen activator, is produced as a secreted 47-kilodalton
(kDa) protein by many pathogenic streptococci of different
serogroups. The Skc gene, skc, from Streptococcus equisi-
milis H46A (group C) has been cloned, sequenced, and
expressed in several heterologous gram-positive and gram-
negative bacterial species under both homologous and het-
erologous expression signals (30-33). When studying skc
expression in Escherichia coli, we made several findings
indicating that the gene product interferes with the normal
physiology of this host. Among these findings are pro-
nounced mucoidicity of cells carrying wild-type skc, incom-
plete export of Skc into the periplasmic space, structural
instability of Skc plasmids designed for high-level skc
expression, difficulties encountered in cloning Skc genes
from additional streptococcal serogroups in E. coli plasmids,
and unsuccessful attempts to express heterologous genes
under control of the skc expression-secretion signals. Al-
though the available evidence indicates that gram-positive
signal peptides are recognized and processed in E. coli (4),
these findings suggest that the deleterious skc effects are
caused by a defective secretion mechanism of the heterolo-
gous gene product. On the basis of studies with a prlA
mutant of E. coli, in this report we present direct support for
this suggestion and describe a novel phenotype for the
mutated prlA gene.
The prlA (or sec 1) gene is a promoter-distal component of
the 50S ribosomal protein operon at 73 min on the E. coli
chromosome (13, 22). Its gene product (49 kDa), although
being translationally coupled to the upstream genes of the
operon (21), is not a ribosomal protein but an integral
cytoplasmic membrane protein containing several hydropho-
bic segments presumed to span the membrane (1, 8). Exten-
sive mutational analyses have shown that prlA mutations are
*
Corresponding author.
2202
pleiotropic and affect protein localization by being export
defective or suppressing the secretion defects in signal
sequence mutants (13, 35, 37) and secA mutants (3, 6) with
defects in another peripheral cytoplasmic membrane protein
(92 kDa) specifically involved in protein export (38). The
evidence accumulated in these studies indicates that the
PrIA protein is part of the E. coli secretion mechanism, one
property of which is specific interaction with signal peptides
of the exported proteins (2, 5, 35, 43). We expanded these
findings by showing that the prlA4 allele prevents E. coli
from forming viable skc transformants at high levels of skc
expression and identified skc mutations capable of relieving
the lethal effects of skc.
MATERIALS AND METHODS
Bacterial strains, bacteriophage, and plasmids. The bacte-
rial strains, phage, and plasmids used are described in Table
1.
Media, enzymes, and chemicals. E. coli strains were cul-
tured aerobically at 37°C in LB medium (28) when used for
Skc production and grown in 2 x YT medium (Amersham
M13 Cloning and Sequencing Handbook) to obtain compe-
tent cells. For recognition of recombinant M13 phage, we
used soft-agar overlays consisting of 2x YT broth supple-
mented with 0.7% agar, 0.33 mM isopropyl-3-D-thiogalacto-
pyranoside, and 0.02% 5-bromo-4-chloro-3-indolyl-,-D-ga-
lactoside. S. equisimilis was grown at 37°C without agitation
in brain heart infusion broth (Difco Laboratories). If re-
quired, selective antibiotics were added to the media at the
following final concentrations (micrograms per milliliter):
ampicillin and erythromycin, 100; chloramphenicol, 50; tet-
racycline, 12.5. Restriction enzymes, T4 DNA ligase, DNA
polymerase I and its Klenow fragment, and the deoxy- and
dideoxynucleoside triphosphates were obtained from Boehr-
inger or Bethesda Research Laboratories. The M13 17-base
primer was synthesized at our institute, and [oa-32P]dATP
VOL. 171, 1989 STREPTOKINASE MUTATIONS 2203

TABLE 1. Bacterial strains, phage, and plasmids

Designation Relevant properties Source or
reference
Bacteria
S. equisimilis H46A Group C strain carrying skc 10
E. coli JM109 recAl endAl gyrA96 thi hsdR17 supE44 relAl A(lac-proAB) (F' traD36 proAB 47
lacPZ AM15) X-
E. coli SE2060 araD139 A(lac)U169 rpsL relA thi lamBS60 F- 14
E. coli SE6004 araD139 A(lac)U169 rpsL relA thi lamBS60 prlA4 F- 14
Phage M13mp18 and mp19 DNA sequencing vectors 47
Plasmids
pMF5 Tcr Skc+; pBR322 carrying skc as PstI fragment in sense orientation relative 30
to bla promoter
pSM752 Tcr Emr Skc+; E. coli-Streptococcus shuttle plasmid pMF5::pSM7 cointegrate 31
pMM191 Apr Skc+; pUC19 carrying skc in PstI site in sense orientation relative to This study
lacPO
pMM192 Apr Skc+; pUC19 carrying skc in Pstl site in antisense orientation relative to This study
lacPO
pN10-9 Apr Skc+ lacPOZ' 'skc; pUC8 carrying truncated 'skc missing codons 1-42 32
and fused in frame with lacZ'
pKM1 Apr Skc+ lacPOZ' 'skc; pUC9 carrying truncated 'skc missing codons 1-39 25
and fused in frame with lacZ'
pMM97 Skc+ pMM191::ISJ This study
pMM304 Skc- skc::IS5; pMM191 derivative This study
pMM247 Skc+ A(skc)-247; pMM191 derivative This study
pMM305 Skc+ A(skc)-305; pMM191 derivative This study
pKM1832 Apr SpeA+; pUC18 carrying speA encoding streptococcal pyrogenic exotoxin 46; C. Klessen
type A
pKM934 Apr Skc+ speA' 'skc; pUC9 carrying in-frame fusion between 5' end of speA' C. Klessen
and 'skc missing codons 1-39
pTG29 Cmr Amy'; carrying IS] as sequenced by Johnsrud (24) 45

(ca. 3,000 Ci/mmol) was purchased from Amersham. Human
plasminogen was prepared from blood plasma by lysine-
Sepharose affinity chromatography (11). Standard Skc
(Awelysin; specific activity, 55,000 U/mg of protein) came
from Arzneimittelwerk Dresden, Dresden, German Demo-
cratic Republic.
Recombinant DNA techniques. For large-scale plasmid
isolation, CsCl-ethidium bromide density gradient equilib-
rium centrifugation was used as described by Maniatis et al.
(34). The method of Holmes and Quigley (20) was used for
minipreparation of E. coli plasmids. Digestion of plasmid
DNA with restriction enzymes, isolation of specific DNA
fragments and their ligation, and agarose gel electrophoresis
and polyacrylamide gel electrophoresis- of DNA were per-
formed by standard procedures (34). It should be noted that
streptococcal DNA fragments, in particular the 450-base-
pair (bp) Hinfl fragment, containing the transcriptional con-
trol signals of skc migrated irregularly in polyacrylamide gels
because of their high poly(A) and poly(T) cluster contents,
which caused DNA bending. For precise sizing of the DNA
fragments covering this region, the gels had to be run at 60°C
to prevent DNA bending (12). The calcium chloride proce-
dure (34) was used to produce and transform 0.2-ml samples
of competent E. coli cells with plasmid or recombinant
DNA. In Southern hybridization experiments (34), plasmid
pTG29 (45) served as the source of IS] probe DNA. The 1.3-
and 0.8-kilobase EcoRI-PstI fragments containing, respec-
tively, the 588-bp right portion and the 180-bp left portion of
IS] were nick translated with the DNA polymerase I holo-
enzyme with [a-32P]dATP and hybridized at 68°C to inser-
tion mutant pMM191 and appropriate control DNA cut with
different restriction enzymes. Nucleotide sequence analysis
was performed by the dideoxy-chain termination method
(Amersham M13 Cloning and Sequencing Handbook) in
M13 vectors introduced into JM109. The IS5 insertion in
pMM191 was identified by sequencing restriction fragments,
one terminus of which corresponded to the EcoRI site
internal to IS5 (17, 41). skc deletion mutations were identi-
fied by sequencing of the HincII-Sau3AI fragment (size in
wild-type skc, 653 bp) containing the skc region encoding the
signal'peptide.
Distribution of Skc activity. Extracellular Skc was assayed
in chloroform-treated, cell-free culture supernatant fluids
obtained by centrifugation of liquid LB cultures. Periplasmic
protein was prepared by the minishock procedure as de-
scribed by Hazelbauer and Harayama (19). The cytoplasmic
protein fraction was obtained by subjecting the osmotically
shocked cells to sonication in 0.5 mM magnesium chloride at
0°C with four intermittent 20-s pulses at maximal output.
Assay of Skc activity. Skc-producing colonies were de-
tected by the casein-plasminogen overlay techniq4e as pre-
viously described (30). Quantitation of Skc in samples ob-
tained after cell fractionation was conducted in well plates
prepared from a medium containing 1% agarose, 10% skim
milk, 50 mM Tris hydrochloride (pH 8.1), 150 mM NaCI, and
10 ,ug of human plasminogen per ml. A series of Skc
solutions was used as standards, and after incubation for 2 to
16 h at 37°C, the sizes of the caseinolysis zones produced by
the samples were compared with those of the standards. The
area of the lysis zone could be correlated with the amount of
Skc; e.g., the caseinolysis zone produced by 1 U of Skc
corresponds to 10 ng of protein. To ensure that extracellular
Skc was not formed by cell lysis, the activity of a cytoplas-
mic marker protein (3-galactosidase) was assayed in culture
broths by hydrolysis of o-nitrophenyl-,-D-galactopyranoside
(36). Skc activity in samples 'subjected to sodium dodecyl
2204 MULLER ET AL. J. BACTERIOL.

TABLE 2. Transformation efficiencies of E. coli SE6004 (prIA4)
for Skc and SpeA plasmids containing wild-type or
truncated streptococcal genes
Transformation
Plasmid
Plasmid Gene ~~~~~~~~~efficiency
SE6004/SE2060 (ratio of
transformants)"
pMF5 skc
pSM752 skc
pMM191 skc
pMM192 skc
pKM1 'skc
pN10-9 'skc
pKM1832 speA
pKM934 speA' 'skc
a
Specific transformation yields determiped with SE2060 as the recipient
ranged from 5 x 104 to 5 x 105 transformants per ,g of DNA.
sulfate-10% polyacrylamide gel electrophoresis (27) was
detected zymographically by treating the gels with 2% Triton
X-100 and washing them in water before overlaying them
with the assay medium. In all assays, the specificity of the
reaction was controlled by omitting plasminogen.
RESULTS
Transformability of E. coli SE6004 (prlA4) by plasmids
containing wild-type and mutant skc genes. With the finding in
mind that priA mutations reduce the spegificity of the E. coli
protein export mechanism and restore secretion of proteins
with defective signal sequences (14), transformation experi-
ments were designed to answer the question of whether or
not a strain carrying prlA4 is capable of transporting wild-
type Skc more efficiently to the periplasmic space than does
an isogenic counterpart carrying the priA wild-type allele.
Four recombinant plasmids containing the complete skc
gene, including its control regions for transcription and
translation, were used to transform strains SE2060 and
SE6004 to antibiotic resistance and Skc production in com-
parable experiments. Surprisingly, SE6004 carrying the
prIA4 allele yielded no transformants with three of the
plasmid DNAs that contained skc in the sense orientation
relative to lacPO (pMM191) or the bla promoter (pMF5 and
pSM752) of the vectors which contribute to skc expression
(30). The fourth plasmid, pMM192, carrying skc in the
antisense orientation relative to lacPO, yielded SE6004
transformants reduced in number by 3 orders of magnitude
compared with the transformation efficiency of SE2060
(Table 2). To verify that the differential transformation yields
were caused by skc, the insert-free vectors were similarly
transformed into the two strains and found to transform
them at about the same frequencies (5 x 106 to 6 x 106
transformants per ,ug of DNA). (It should be noted that
vectors containing skc transformed SE2060 1 to 2 orders of
magnitude less efficiently than did the corresponding insert-
free vectors.)
To identify possible skc regions deleterious to the prlA4
<10-5 mutant, skc deletion mutants retaining Skc activity were
<10-5 used to transform SE2060 and SE6004 in comparable exper-
3 x 10-O
1 iments. Plasmids carrying skc deletions removing 39 (pKM1)
1 or 41 (pN10-9) codons from the 5' end of the skc coding
1 region and fused in frame to lacZ' transformed the pair of
1 strains at about equal efficiencies to both ampicillin resis-
tance and Skc production (Table 2). These deletions re-
moved the entire 26-codon signal sequence from skc, sug-
gesting that this region prevented the prlA4 mutant from
yielding viable transformants when exposed to plasmids
carrying wild-type skc.
Of interest was the question of whether the prlA4 mutant
is normally transformable by recombinant plasmids carrying
a different streptococcal gene specifying an exported pro-
tein. For this experiment, the speA gene (46) cloned into
pUC18 was chosen and found to transform strains SE2060
and SE6004 normally and at about equal efficiencies. Also, a
plasmid constructed by fusing the truncated 'skc gene in
frame to the transcriptional control region and the entire
signal sequence of speA was capable of transforming the two
strains with indistinguishable frequencies (Table 2). Thus,
the significantly reduced or missing transformability of the
prlA4 mutant was specific for plasmids encoding the signal
sequence of skc.
Selection and physical characterization of pMM191 mutants
capaible of transforming SE6004. When competent cells of
SE6004 (prlA4) were exposed to high concentrations of
pMM191 DNA (1 to 4 p.g/109 cells) in 14 independent
experiments, 17 ampicillin-resistant transformants were ob-
tained, 13 of which were Skc negative whereas the rest were
Skc positive. Compared with the transformation yield of
SE2060, these numbers amounted to a transformation fre-
quency of only 0.004%. Three of the Skc-positive clones and
one of the Skc-negative clones were chosen for further
study. When used to retransform SE6004, their plasmid
DNAs transformed this strain as efficiently as that of the
isogenic priA wild type, suggesting the presence of muta-
tions that created transformability. Restriction analysis,
Southern hybridization, and DNA sequencing confirmed this
suggestion, with the following results (Fig. 1).

PiI
-10 RBS I s
pMM 97 | IS1
proceg site
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 2B 29
pMM 191 (WT) ATGAAA AAT TACTTATCTTGGGATGrTTGCACTGCTGTTTGCA CTA ACATWACAGICAATCTGTCCMGC1JAA T CTGGA
M K N Y L S F G M F A L L F A L T F G T V N S V QA I A G
V,/-/12bp7/
pMM 247, pMM 305

1 13 14 15 16 oRIW93)
pMM 304 ATG CTG m GCACTAAJGAAGGT I.S 5 Acc1ATTCTCATMWArGAACA.
FIG. 1. Physical structure of pMM191 mutations relieving SE6004 of transformation deficiency for the wild-type (WT) plasmid. The
orientations of IS1 and IS5 are showvn by their indicative asymmetric PstI and EcoRI sites, respectively. Note the two possible signal sequence
deletions (hatched bars) leading to the same genotype. In pMM304, the duplicated 4-bp sequence at the IS5 integration site is in boldface.
RBS, Ribosome-binding site.
VOL. 171, 1989 STREPTOKINASE MUTATIONS 2205

Two of the plasmids turned out to represent insertion TABLE 3. Expression and location of Skc specified by
mutants carrying known insertion elements. Mutant plasmid wild-type and mutant pMM191
pMM191::ISl (Skc positive) carried IS] at a site close to the Skc activity
-35 region of skc (around nucleotide position 755 by the
numbering convention for the PstI fragment containing skc E. coli strain Total Cyto- Peri- Extra- Growth
[33]), as judged by PstI-and-Hinfl analysis in conjunction (U/ml) plasmic plasmic cellular (GD54)
with the identification of IS] by Southern hybridization. The
probe containing the 180-bp left portion of IS] hybridized to Early-stationary-phase
the 0.92-kilobase PstI fragment of pMM191::ISI, while the cultures
588-bp right portion of IS hybridized to the 2.42-kilobase SE2060(pMM191) 550 92 4 4 0.69
PstI fragment (Southern blot not shown). The sizes of the SE2060(pMM97) 85 94 4 2 0.66
SE6004 prlA4(pMM97) 65 95 2 3 0.64
two HinfI junction fragments between the PstI insert and IS] SE2060(pMM247) 2,020 47 6 47 0.61
(830 and 240 bp) confirmed this finding, which served to SE6004 prlA4(pMM247) 950 74 5 21 0.72
locate and determine the orientation of IS]. Furthermore,
flanking the IS] insertion site in the target DNA were two 24-h cultures
closely spaced regions (TAACATAATGGTT and AAACCT SE2060(pMMi91) 170 46 1 53 1.4
AATGATT; spacing, 16 bp) homologous to the consensus SE6004 prIA4(pMM97) 160 50 1 50 1.3
sequence (YAANNNNTTGATW) required for binding of SE2060(pMM247) 500 10 1 90 1.5
the integration host factor involved in IS] insertion (18). SE6004 prlA4(pMM247) 650 30 1 70 1.5
Note that the bases at positions 2 (A), 9 (T), and 10 (G) of the " OD_40 (optical density at 540 nm) = 1.0 corresponds to 3 x 108 to 5 x 108
target DNA were identical to the three bases which Gamas et cells per ml.
al. (18) found always to be present when comparing 15
different integration host factor-binding sites in E. coli bac-
teriophage and plasmids. In further agreement with the IS] lost the mucoidicity characteristic of SE2060 colonies
analysis of these researchers (18), the two streptococcal carrying wild-type pMM191.
DNA sites were embedded in A+T-rich regions (33). Skc specified by pMM247 in either host was expressed at
Mutant plasmid pMM191::IS5 (Skb negative) carried IS5 substantially higher levels than that encoded by wild-type
in the middle of the region coding for the hydrophobic pMM191 present in SE2060. In addition, export of the
stretch of the signal peptide, as revealed by nucleotide pMM247-encoded protein was facilitated in both SE2060 and
sequencing of the DNA around the ihsertion site and Hinfl SE6004, and the latter host was slightly less efficient than the
analysis of the mutant DNA (Fig. 1). In agreement with the priA wild-type strain (Table 3). This was found for both
known consensus target site sequence (CWAR) for IS5 early-stationary-phase cultures and cultures incubated for a
insertion (17), the host sequence where IS5 was inserted was prolonged time in the stationary phase (24-h cultutes). No
CTAA. There was no in-frame ATG provided by IS5 in an extracellular P-galactosidase activity was detectable in con-
open frame which might have served as a gratuitous trans- trol cultures with stPains carrying either pMM191 or
lation initiation codon for the interrupted skc gene, explain- pMM247, indicating that extracellular Skc did not result
ing the Skc-negative phenotype. from cell lysis. Also, viable-count and optical density mea-
As also determined by DNA sequencing, the remaining surements did not reveal cell lysis. The activity of authentic
two pMM191 mutants, pMM247 and pMM305, carried in- Skc was assayed in reconstruction experiments after mixture
frame deletions within skc, both removing a tetrapeptide with cell-free culture supernatant fluid and periplhsmic and
from the hydrophobic core of the signal sequence (Fig. 1). cytoplasmic cell fractions and found unaffected, proving that
Although they represent independent occurrences, it is the specific activity of Skc was not influenced by cellular
worth noting that they had the same genotype. Interestingly, location.
two different but overlapping deletion events (covering skc Processing of wild-type and mutant Skc. We used sodium
codons 10 to 13 and 12 to 15) can be envisaged to cause the dodecyl sulfate-polyacrylamide gel electrophoresis, fol-
mutant genotype (Fig. 1). However, because of the presence lowed by zymography of the gels, to detect and size Skc-
of identical codons for the repeated amino acids Phe and Ala active bands and found that the culture supernatant fluids of
in the nonoverlapping regions, we were unable to determine SE2060 and SE6004 bearing pMM191 or pMM247 contained
which events actually occurred. Skc activity at 47 kDa, corresponding to mature Skc as
Expression and cellular location of Skc specified by secreted by S. equisimilis H46A (Fig. 2). In agreement with
pMM191 and derived mutants. Skc expressed by E. coli is the data on Skc distribution, this finding showed that dele-
normally found in the medium, the periplasm, and the tion of the hydrophobic tetrapeptide from the signal se-
cytoplasm, and the distribution of the total activity depends quence did not prevent maturation of the protein in either the
on the age of the culture (30). It was of interest, therefore, to prlA wild-type or mutant strain and that extracellular Skc
determine the level of expression and the distribution of the underwent signal peptide processing before passing through
protein specified by wild-type and mutant plasmids when the outer membrane into the medium by an undefined
present in SE2060 or SE6004 (prlA4) (Table 3). mechanism. It should also be noted that the cytoplasmic
Skc specified by pMM191::ISl was expressed at substan- protein fractions from either strain, when subjected to so-
tially lower levels than that of wild-type pMM191, with no dium dodecyl sulfate-polyacrylamide gel electrophoresis,
qualitative differences regarding its distribution in the two did not reveal the presence of any pre-Skc. (In fact, the
host strains. This result suggests that ISf insertion near the skc-coded preprotein was seen only in the cytoplasm of S.
-35 region of skc impaired the function of the skc promoter. sanguiis, S. lactis, and Bacillus subtilis [Fig. 2, lane 6].)
If it was completely nonfunctional, Skc might have been However, we cannot exclude the possibility that in E. coli
expressed under the promoter known to exist at either end of cell disintegration by sonication caused pre-Skc to be proc-
IS] (29). The low level of expression may also explain the essed. Figure 2 also shows that all supernatant fluid samples
finding that SE2060 and SE6004 colonies carrying pMM191:: produced a 44-kDa band with Skc activity. As shown previ-
2206 MULLER ET AL.
1 2 3 4 5 6
kDa
' Wi..
^ beta-chain secondary structure in the central segment. A
00...
50-
47 -
44 -
.wt h,>~~~~~~~~~~~~~"Im..
FIG. 2. Sodium dodecyl sulfate-polyacrylamide gel electropho-
resis, followed by Skc assay of culture samples containing unpro-
cessed (50 kDa), processed (47 kDa), and degraded (44 kDa) Skc.
Lanes: 1, ctiltdre supernatant fluid from SE6004(pMM247); 2, cul-
ture supernatant fluid from SE2060(pMM247); 3, culture superna-
tant fluid from SE2060(ptJC19) (control); 4, culture supernatant fluid
from SE2060(pMM191); 5, culture supernatant fluid from S. equisi-
milis H46A; 6, cytoplasmic fraction from B. subtilis IH6140
(pSM752) showing unprocessed Skc. Note that the intensity of
caseinolysis does not reflect expression levels, because to ensure
clarity the samples were adjusted to contain 25 to 50 U of Skc per
lane.
ously by proteih.sequencing (23), this band corresponds to
an active degradation product of the mnature protein having
lost 31 or 32 C-terminal amino acids.
DISCUSSION
We have shown that the prlA4 allele renders E. coli
nontransfortnable for recombinant plasmids designed for
high-level Skc production or causes severely reduced trans-
formation yields of plasinids in which skc is less highly
expressed. This phenotype is novel in that priA mutations
have not previously been knowh to be incompatible with
either hbmologous or heterologous genes encoding exported
proteins. Of the three classes of mutations found to relieve
incompatibility, skc deletions removing either the entite
signal sequence (pKM1 and pN10-9) or a specific part of its
hydrophobic core (pMM247 and pMM305), are the most
interesting ones. A similar 12-bp in-frame deletion is known
for the lamB gene of E. coli, but unlike the situation in skc,
this mutation (lamBS78) blocks export of the LamB outer-
membrane protein in a prlA wild-type strain by preventing
the hydrophobic region of the signal sequence from forming
an alpha-helical conformation (7, 16). The tetrapeptide dele-
tion in pre-Skc drastically reduces the hydrophobicity of the
central segment of the signal sequence and also decreases its
size, as shown by a comparison of the calculated amino acid
side-chain volumes of the 10-residue central segments from
wild-type and deleted signal sequences (Table 4). As judged
by the -rules of Chou and Fasman (9), the most probable
conformation of the wild-type Skc signal peptide is an
TABLE 4. Hydrophobicity and size of segments from the signal peptides of Skc and A(Skc)-247
Signal Segment sequence'
peptide
N M
Skc KNYLSFGMFA MFALLFALIE
A(Skc)-247 KNYLSFGMFA FGMFALTFGT
a As described by Sjostrom et al. (44), the wild-type and mutant signal peptide amino acid sequences were divided, from the N terminus to the C terminus,
excluding the N-terminal M, into three overlapping (underlined) 10-residue segments (N, M, and C).
b Calculated by using the hydrophobicity scale of Kyte and Doolittle (26).
Calculated on the basis of the data of Seydel and Schaper (42).
J. BACTIERIOL.
alpha-helix. The tetrapeptide deletion reduces the potential
for alpha-helix formation and increases the propensity for
similar analysis of the SpeA signal peptide revealed that its
most probable conformation is a beta-sheet. Since genes
under the speA promoter are expressed in E. coli at levels
similar to those under the skc promoter (unpublished data),
compatibility between prlA4 and speA does not seem to be
the consequence of low-level speA expression but. is attrib-
utable to distinct conformational properties of the speA
signal peptide. After completion of these experimnents, we
learned that the staphylokinase signal peptide (Sak) is not
processed in a prIA4 strain and that signal peptide mutations
that strengthen beta-turd probability suppress this deficiency
(40). Thus, certain conformational features of both signal
peptides, Skc and Sak, may be required for the peptides to
be processed in a prlA4 strain. The wild-type Skc signal
peptide may interact with the PrlA4 protein, but its failure to
be processed may lethally jam the cellular export mecha-
nism, preventing essential membrane proteins from reaching
their final destination. Overproduction lethality has been
proposed previously to explain the maltose sensitivity phe-
notype of certain lamB-lacZ and malE-lacZ fusions that
determine hybrid proteins that become stuck in the cytoplas-
mic membrane (4, 15, 39). The tetrapeptide deletion in the
signal sequence of Skc not only creates secretability in a
prlA4 strain but also facilitates Skc export in the wild type.
Thus, the increased level of Skc expression in SE2060
(pMM247) compared with that of SE2060(pMM191) (Table
3) may be a result of restoration of the nornial physiology of
the E. coli host by virtue of a functional secretion mecha-
nism.
Besides extending the knowledge of the pleiotropic nature
of priA mutations, the present results have some bearing on
the problem of cloning skc and possibly other streptococcal
genes in E. coli. As regards the former, three more Skc genes
originating from group A and G streptococci have been
sequenced and proved to be difficult to clone in E. coli
plasmids (T. T. Huang, H. Malke, and J. J. Ferretti, Mol.
Microbiol., in press; F. Walter, M. Siegel, and H. Malke,
Nucleic Acids Res., in press). Although their gene products
show various amino acid sequence dissimilarities when
compared with one another or Skc, their signal sequences
are highly conserved and have properties similar to those
discussed here for the signal sequence of Skc. These findings
may help researchers to overcome problems in expressing
these important streptococcal ptoteins in E. coli. They also
explain the physical basis of the structural alterations of skc
that invariably accumulate in E. coli cultures containing
plasmids designed for high skc expression. Finally, the Skc
signal sequence carrying A(skc)-247 has been successfully
Total hydrophobicityb (cm3/mol)c
C N M C N M C
TFGTVNSVQA +3.2 +24.6 +3.4 385 402 292
TFGTVNSVQA +3.2 +13.7 +3.4 385 335 292
VOL. 171, 1989
used under control of the skc promoter to express heterolo-
gous genes in E. coli (J. Muller, unpublished data).
ACKNOWLEDGMENTS
We thank Jon Beckwith for providing the prIA strains, Joe Ferretti
for the speA gene, Christian Klessen for SpeA plasmids, and Marita
Siegel and Friedrich Walter for help with DNA sequencing and
hybridization, respectively.
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