Gene, 64 (1988) 155-164 155

Elsevier

GEN 02341

Construction and properties of a new insertion vector, pJDC9, that is protected by transcriptional
terminators and useful for cloning of DNA from Streptococcus pneumoniae

(Molecular cloning; recombinant DNA; promoters; gene disruption)

JawDer Chen and Donald A. Morrison

Laborator) for Cell, Molecular. and Developmental Biology, University of Illinois at Chicago, Chicago, IL 60680 (U.S.A.)

Received 27 October 1987

Revised 21 December 1987
Accepted 22 December 1987
Received by publisher 19 January 1988

SUMMARY

A new Escherichia coZi plasmid cloning vector, pJDC9, was constructed by replacing the TcR determinant
of pMB9 with the erythromycin-resistance ermB determinant and the IacZcr gene of pUC19. Efficient
transcriptional terminator signals were positioned at both ends of lacZa. Evidence is presented that protection
of the vector by terminator signals enabled cloning of many fragments of DNA from Streptococcus pneumoniae
that were unstable in vectors lacking such protection, including pBR322. At the pneumococcal ma1 locus, three
promoter sites required such protection, while overexpression of the malX protein appeared to be lethal despite
such protection.

INTRODUCTION examined in detail, the cause of instability has been

traced to promoter activity, either by showing
A substantial fraction of genes cloned from increased stability after mutations that reduce pro-
S. pneumoniae (pneumococcus) have been found to moter activity (Stassi and Lacks, 1982), or by de-
cause plasmid instability when inserted in common monstrating increased stability in vectors protected
E. colt’ plasmid vectors (Stassi and Lacks, 1982; by transcriptional terminators adjacent to the in-
Stassi et al., 1982; Martin et al., 1985; Prats et al., serted pneumococcal DNA (Chen and Morrison,
1985 ; Chandler and Morrison, 1987b; Chen and 1987; Chandler and Morrison, 1987a). This phe-
Morrison, 1987). In several cases that have been nomenon appears to be quite common among

Correspondence to: Dr. D.A. Morrison, LCMDB, M/C 067, for colicin El immunity; kb, 1000 bp; LB, Luria-Bertani broth;
University of Illinois, P.O. Box 4348, Chicago, IL 60680 (U.S.A.) MCS, multiple cloning site; PolIk, Klenow (large) fragment of
Tel. (312)996-6839. E. coli DNA polymerase I; R, resistant; ‘, sensitive; t,, termina-
tor of oop RNA in phage I (Hayes and Szybalski, 1973); T, TZ,
Abbreviations: Ap, ampicillin; blu, gene encoding p-lactamase; tandem terminators of the E. coli r&l operon; Tc, tetracycline;
bp, base pair(s); CAT, Cm acetyl transferase; cat, gene encoding ret, gene for resistance to Tc; XGal, 5-bromo-4-chloro-indolyl-
CAT; cat ~, promoterless cat; Cm, chloramphenicol; dhfr, gene /I-D-galactoside.
encoding dihydrofolate reductase; Er, erythromycin; imm, gene

0378-I 119/88/$03.50 0 1988 Elsevier Science Publishers B.V. (Biomedical Division)

156

pneumococcal genes (Chen and Morrison, 1987). MATERIALS AND METHODS

Fragments recognized as strong promoters in E. coli
were unusually common in digests of pneumococcal (a) Bacterial strains, plasmids, culture media, and
DNA and E. cofi vectors protected by efficient tran- genetic procedures
scriptional terminators were shown to be superior
cloning vehicles for random pneumococcal DNA E. co/i host strains were DHl {Hanah~, 1983)
fragments. Nonetheless, the successful cloning of the and JM103 (Messing et al., 1981). Plasmids used in
pneumococcal gene (Zyt) for N-acetyl-muramyl-t- this work are listed in Table I. Culture conditions
alanyl amidase in pBR322 by Garcia et al. (1985; were as described previously (Chen and Morrison,
1986) shows that there are exceptional genes which 1987). Selection of E. coli transformants was done
are quite stable without such terminator protection. by plating on LB agar supplemented with Ap (50
Here we describe the construction of an E. coii yg/ml), Tc (12.5 pg/ml), or Er (I mgiml).
cloning vector that was used in the earlier experi-
ments and that is protected by bidirectional tran- (b) DNA manipulations
scriptional terminator signals, The value of this vec-
tor for cloning pneumococcal DNA is directly de- Methods for cloning, ligation, and transformation
monstrated in two ways. First, we show that it allows were as described previously (Chen and Morrison,
cloning of strong promoters from the maltosac- 1987). In addition, Sl nuclease digestion, PolIk
charide utilization locus, maI, that cause instability in treatment, and Southern hybridization, were per-
other vectors. Second, we show that random formed as described elsewhere (Chen, 1987) essen-
pneumococcal fragments up to 12 kb in size cloned tially according to Maniatis et al. (1982). Enzymes
as intact inserts in this vector are unstable in other were obtained from International Biotechnologies
vectors with fewer protective terminator signals. Inc., Bethesda Research Laboratories, New England

TABLE I

Plasmids used in this study

_
Plasmids A Genotype Source or reference
.--
M 13mp9 M/3, IacZa Messing and Vieira (1982)
pBR322 hliz, tet Sutcliffe (1979)
pDS1 blu,dhfr,cat- Stueber and Bujard (1982)
pDSl(r,l + ) Ha, dhfr,cat-, t,l + Stueber and Bujard (1982)
pDSl(t,2+) bin, dhji cat , to2 + Stueber and Bujard (1982)
pJDC2 imm, ermB pR29, pMB9
pJDC3 imm, ermB M 13mp9, pJDC2
pJDC4 imm, ermB, t,,l + pJDC3, pDSl(t,l + )
pJDC6 imm, ermB, t,l + 1,2 + pJDC4, pDSl(t,Z + )
pJDC7 imm, ermB, t-1 + , T, T2 pJDC4, pKK232-8
pJDC8 imm, ermB, to1 + , T, T, pJDC7, pJDC6
pJDC9 imm, ermB, t,l +, T,T,, IacZa pJDC8, pUCI9
pJDClO imm, ermB, IacZa pJDC3, pUC19
pJDC95 1 pJDC9, 12-kb insert pJDC9, CP1200
@DC952 pJDC9. 9-kb insert pJDC9, CP1200
pJDC953 pJDC9, 8-kb insert pJDC9, CP1200
pKK232-8 bla, T, T,, cat Brosius (1984)
pLS70 malXMP, tet Stassi et al. (1982)
pMB9 imm, tef Rodriguez et al. (I 976); Bolivar et al. (1977)
pR29 bla, ermB, cat Claverys et al. (1984)
puc19 biu, IucZa Yanisch-Perron et al. (1985)

I’ Source of piasmids used in construction are described in MATERIALS AND METHODS, sections e and d.
 157

Biolabs, Amersham, or Boehringer-Mannheim, and One of these t, copies was transferred to pJDC7, to
were employed under reaction conditions recom- recreate a unique Hind111 site, as follows. A Hind111
mended by the suppliers. 32P-labeled nucleotides digest of pJDC7 and a HphI digest of pJDC6 were
were obtained from Amersham. Vectors were treated with S 1 nuclease and PolIk to generate blunt
routinely treated with alkaline phosphatase ends. Both were then digested with BamHI. The
(Boehringer-Mannheim) to increase the insertion small t, fragment from pJDC7 was ligated with the
frequency. When blunt ends were required, DNA larger fragment from pJDC6. Among the ErR trans-
fragments were treated with nuclease S 1 and repaired formants of DHl was one carrying the expected
with PolIk. plasmid structure, identical to that of pJDC7 except
for elimination of the second Hind111 site, between
(c) Construction of plasmid pJDC9 t,, and ermB. This plasmid was named pJDC8.
The IacZa determinant of pUC19 was incor-
The ErR determinant of pAMB1, ermB (Brehm porated into pJDC8 as follows. A HaeII digest of
et al., 1987), released from pR29 (Claverys et al., pUC19 was treated with Sl nuclease and PolIk to
1984) by digestion with ClaI, was cloned in pMB9, generate blunt ends. Plasmid pJDC8 was digested
replacing the TcR CZaI fragment of that vector, to with XmaI + HindHI; the ends of the resulting frag-
form a 5.3-kb plasmid, named pJDC2. The EcoRI- ments were filled with PolIk. A mixture of these
Hind111 MCS fragment of M13mp9 was ligated with products was ligated, and used to transform JM103.
an EcoRI + Hind111 digest of pJDC2, replacing the Among the plasmids in 80 ErR Lac + transformants,
small EcoRI-Hind111 fragment of pJDC2 (and three exhibited the structure expected for replace-
eliminating one C/a1 site) to form a plasmid named ment of the MCS of pJDC8 by the 1acZcc fragment
pJDC3. The phage 1 oop RNA terminator, t, (Hayes of pUC19, with a single SmaI site remaining near
and Szybalski, 1973), obtained on a 104-bp Hind111 ermB. One was named pJDC9.
fragment from pDSl(t,l + ), was inserted at the
Hind111 site of pJDC3, in the orientation to stop (d) Construction of pJDCl0
transcription proceeding from the MCS toward
ermB, forming the plasmid named pJDC4 (5.4 kb). The HaeII fragment of pUC19 containing 1acZcc
An EcoRI digest of pJDC4 was made blunt-ended and the MCS was inserted in place of the MCS of
by a fill-in reaction with PolIk. A Hi&I digest of pJDC3 to form pJDC10. The only differences
pKK232-8 was similarly treated with PolIk, and the between pJDC9 and pJDCl0 thus lie in the termina-
1221-bp fragment containing the tandemly repeated tors flanking the MCS and at the SmaI site near the
rrnB terminators (as well as loo-130 bp of non- pMB9 origin.
repeated flanking sequences at each end) was
recovered from a preparative agarose gel on a DEAE
membrane. Ligation of these two molecules and
transformation of DHl yielded ErR transformants,
4 y0 of which contained plasmids with the rrnB termi- RESULTSANDDISCUSSION
nators inserted in the orientation to stop transcrip-
tion proceeding from the MCS away from ermB. (a) Structure and history of pJDC9
One of these plasmids was retained and named
pJDC7. The steps by which plasmid pJDC9 (Fig. 1) was
A second copy of to was added to pJDC4 at the constructed from pMB9, pKK232-8, pUC19, pR29,
EcoRI site, as an inverted repeat of the first; in this M13mp9, pDSl(t,l +), and pDSl(t,,2+) are de-
case, t, was obtained as the 104-bp XbaI fragment scribed in MATERIALS AND METHODS, section c.
from pDS l(t,2 + ), made blunt-ended by treatment The partial map information presented in Fig. 1
with Sl nuclease and PolIk, and extended with reflects the limited knowledge of the structure of the
EcoRI linkers. The resulting plasmid, pJDC6, components of pJDC9. For several regions [t,, and
carried two inverted copies of t, on EcoRI and rrnB terminator fragments, the ermB gene (Brehm
Hind111 fragments, flanking the MCS of M13mp9. et al., 1987) within the CZaI ErR fragment, and
 1 kb
t i

wi? -Wi , kCZ tQ Er

I I

Pvull
5080

Fig. 1. Physical map of pJDC9. The relative locations of the segments containing the E. coli rrnB tandemly repeated terminators (T, T,),
lacZr, the I oop RNA terminator (t,), and the ErR determinant (Er) are shown at the top of the figure. Below, the horizontal lines
represent map segments, from bp i-3575, showing the relative positions of restriction sites (shown by vertical bars), for the indicated
restriction enzymes. Ten unique restricti sites are located in the MCS: BarnHI. EcoRI. HkzdIII, &WI, PstI, Sali, SmaI, SphI, ,%tI.
and Xbai. A unique ClaI site is also located at the errriB-pMB9 junction. The five PvuII fragments (322, 370, 670, 2100, and 3480 bp)
were identified on I Onagarose gels.

lucZa], the nucleotide sequence is known. The (b) Analysis of the pneum~occal mai locus: pro-
precise nucleotide sequence produced at joints moter activity correlates with instability in
involving S 1 digestion (at both ends of the facZa and Escherichia coli plasmid vectors
to fragments) is not known, except that the predicted
loss of a restriction site was confirmed in all such The 35kb PstI fragment containing most of the
cases. Finally, map information for the pMB9 mal locus (Fig. 2) has been shown to be unstable
portion of the plasmid is limited. A detailed listing of when inserted into pBR322, while a deletion causing
known restriction site positions is available on down-promoter mutations for both malX and malM
request. increased its stability in the same vector (Stassi and
 159

1.0 2.0 3.0 kb

I I / I I

w 312 , 1616 3470

I
2382 PlJCl9
t 1460
t 1656

I 3470 ,_
' I
1 1087 j 312 ts_ 1616
I
2382+
w pKK232-8
t
t 1656

913 ws
3470 j pJDC4
3470
I I pJDC6
1 3470

c 1087 I 312 L
, - 1616

L 1753 , pJDC9

100 121
737 316 , 913 J l2471 1 667 1
I I I pBR322

Fig. 2. Subcloning of pneumococcal malH4P fragments in various vectors. A map of the portion of the divergently transcribed ndXMP
operon cloned in pLS70 is shown at the top of the figure. Open segments: putative promoter regions. Solid black segments:
maHMP-coding regions (see X, M and P). Below, each horizontal line bounded by vertical bars represents a sub-fragment of maLl’MP
for which subcloning in the indicated vector was attempted. The size (bp) of each sub-fragment tested is shown with an arabic number.
These sub-fragments were released by digestion with the indicated restriction enzymes from pLS70 or other clones indicated in
RESULTS AND DISCUSSION, section b, separated by preparative electrophoresis, recovered on DEAE paper (Pettersson et al.,
1973), and ligated with the cloning vectors indicated on the right. After transformation of E. coli competent cells, recombinant plasmids
were extracted from transformants and the insert of each clone was examined by restriction digestion and electrophoresis on agarose
or polyacrylamide gels. Thick lines, successful subcloning of an intact fragment. Thin lines, failure to subclone a fragment intact. Vertical
bars, restriction site as shown on map, above. Small arrows, site and orientation of putative divergent promoters (Stassi et al., 1982).

Lacks, 1982). Stassi and Lacks (1982) proposed that
strong promoter activity had been responsible for the
cloning problem, but pointed out that deleterious
effects of the m&X protein expressed at high levels
could also explain their results. In support of the
strong-promoter hypothesis, we have presented evi-
dence that a small (3 16-bp) fragment containing the
intact m&X promoter and upstream DNA is stable
when cloned in pDSl(t,l + ) and other terminator
vectors, but not in pDS 1, which lacks the terminator
protection (Chen and Morrison, 1987). The ability of
transcriptional terminators to stabilize plasmids car-
rying this fragment directly implicates its promoter
activity as a source of cloning difficulty. Here, we
describe an analysis of the remainder of the cloned
ma1 locus both for sources of instability in E. coli
plasmid vectors and for promoter activity expressed
in E. coli, although the sites of promoters in this locus
have not yet been precisely identified in either host.
A 3470-bp fragment of the pneumococcal ma1
locus was purified after digestion ofthe streptococcal
plasmid pLS70 with PstI. This fragment, or portions
of it obtained by further digestion by appropriate
restriction enzymes, was ligated with dephos-
phorylated preparations of several vectors (pUC19,
pKK232-8, pJDC9, or pBR322) in attempts to
obtain stable recombinant clones in E. coli. Fig. 2 divergent promoters, but their activity is not directly
shows the results, including the few cases which demonstrated here, because of its instability in
yielded stable recombinant clones of the expected pKK232-8. An additional phenomenon, the failure
structure, as well as the many combinations which to clone the malX promoter when it is combined with
yielded either no transformants, or transformants in a substantial part of the mulX gene, even in termina-
which the insert had been deleted or otherwise tor vectors, is consistent with the lethality of trun-
rearranged. The pattern observed is consistent with cated fragments of the ma& protein suggested previ-
the hypothesis that each of three sites (located in the ously by Stassi and Lacks (1982).
316-bp, 667-bp, and 913-bp Hind111 fragments)
expressing promoter activity in E. co/i (detected as (c) Cloning of other pneumococcal genes between
activation of the pKK232-8 cat gene) is stably cloned flanking terminator signals
only if the vector is protected by a transcriptional
termination signal, and that fragments which contain Chen and Morrison (1987) showed that in shotgun
the malX promoter combined with the first 405 bp or cloning of pneumococcal DNA use of pJDC9 as a
more of the malX gene cannot be maintained in any vector significantly improves the yields of apparently
of the E. co/i vectors tested, even with such pro- intact cloned fragments, as compared with vectors
tection. pBR322 or pUC19. However, the criterion used to
This interpretation was reinforced by a set of define an intact fragment in that study was only the
direct recloning experiments. The clones of mu1 presence in the recombinant plasmids of a full-length
HindHI fragments obtained in the vector pJDC9, vector with an insert flanked by the expected
above, were used as sources of individual purified junction sites; the inserts were not directly compared
segments of the ma1 locus. Each fragment was to the pneumococcal chromosome. Nor was it
released from its vector by Hind111 digestion, and shown that the particular fragments cloned were
ligated with dephosphorylated vectors pBR322 or unstable in other vectors. We now present more
pKK232-8. As indicated in the lower part of Fig. 2, detailed data for several large pneumococcal frag-
in the case of pKK232-8, Cm selection retrieved ments cloned in pJDC9, showing that they derive
transformants only for the promoter fragments from pneumococcus, that they are free from internal
described above (316, 667, and 913 bp), and each deletions, and that they become unstable on transfer
such insert was intact. Conversely, in the case of to vectors less protected by terminators.
pBR322, recombinants with intact inserts were To examine apparently intact inserts for possible
readily obtained, but only for the four fragments that internal rearrangements, selected pJDC9 shotgun
did not display promoter activity in pKK232-8 (737, clones obtained in the previous study were further
247, 121, and 100 bp); for the other fragments, analysed by Southern DNA hybridization. Nine
recombinants never contained intact inserts. Thus, plasmids containing pneumococcal DNA inserts in
fragments that are unstable in E. coli vectors unless the Hind111 site of pJDC9 were randomly chosen for
bounded by terminators express promoter activity in analysis. The plasmids, pJDC951-959, contained
E. coli. insertsofsizes 12,9,8, 5.1,3.8,3.5,2.7,2.4,and2.3
The results displayed in Fig. 2 also appear to kb, respectively. For each clone, chromosomal DNA
reveal promoter activity in two fragments of the ma1 from S. pneumoniae strain CP1200 was digested with
locus where none had been predicted previously. Wind111 and fractionated on a 0.7y0 agarose gel in
One, the 667-bp Hind111 fragment, includes the 5’ parallel with plasmid digests. After transfer to nitro-
end of ma/P, and sequences extending upstream into celluiose, lanes were probed with j2P-labeled plas-
malA4. It expressed promoter activity when cloned in mid DNA. As shown in Fig. 3, each cloned fragment
pKK232-8 and required a terminator-protected vec- hybridized with a single chromosomal fragment of
tor for stable cloning. The other is the 312-bp DraI the same size. The three clones with the largest
fragment lying between the putative malX and ma&4 inserts were also double-digested to allow more
promoters identified by Lacks et al. (1982). As this sensitive detection of small deletions. Southern
fragment was stably cloned only in pJDC9, the most hybridization of the double digests (Fig. 4) also
economical hypothesis is that it contains strong revealed no differences between the cloned frag-
 161

A B C
P c P c P c
.
.
+ _)
.4*/-v
_)
‘ ,.-

D E F
P c P c P c

G t-i I
P c P c P c

-* . .

Fig. 3. Composite of nine Southern hybridization analyses of the integrity of pneumococcal recombinant plasmids. For each 0.7”,
agarose gel, a plasmid DNA lane (P) and a CP1015 chromosomal digest lane (C) are shown. For each panel, the “P-labeled DNA used
as a probe was prepared from the plasmid indicated for that panel. Arrows indicate the 7-kb Hind111 vector fragments. Bands appearing
in digests of both plasmid and chromosome are indicated by dots. Panels show results for plasmids (and insert sizes) as follows: (A)
pJDC951 (12 kb); (B) pJDC952 (9 kb); (C) pJDC953 (8 kb); (D) pJDC954 (5.1 kb); (E) pJDC955 (3.8 kb); (F) pJDC956 (3.5 kb); (G)
pJDC957 (2.7 kb); (H) pJDC958 (2.4 kb); and (I) pJDC959 (2.3 kb). (Note that pJDC955 appears to hybridize to several different
chromosomal fragments.)

ments and the intact chromosome. The combined (d) Dependence of the stability of large pneumo-
hybridization data indicate that this sample of coccal fragments in pJDC9 on terminator signals
pneumococcal DNA cloned in pJDC9 was
maintained stably in E. coli without internal To determine whether the stability of the pJDC9
rearrangement. clones depended on protection by terminators, the
large intact pneumococcal DNA inserts cloned in
pJDC95 l-953 were directly transferred into pJDC 10
and into the promoter probe vector, pKK232-8. The
162

A H HE HIP H/S H/K

PCPCPCPCPC

Fig. 4. Internal structure of pneumococcal DNA in plasmids pJDCY51, pJDC952, and pJDC953. Southern hybridization analysis of
double digests; composite photograph of three 0.7 “/,, agarose gels (Tris borate-EDTA buffer, Maniatis et al., 1982). In each panel,
chromosomal DNA and plasmid DNA digests are compared after digestion with Hind111 (H), Hind111 + EcoRI (H/E), Hind111 + PstI
(H/P), Hind111 + SmaI (H/S), and Hind111 + KpnI (H/K). Probes were 32P-labeled DNA of the indicated plasmids. Plasmids analysed
were (A) pJDC951, (B) pJDC952, and (C) pJDC953 (see Table I). Arrows specify position of Hind111 vector fragment. Dots indicate
bands common to plasmid and chromosome digests.

vector pJDC10 was constructed in essentially the pneumococcal DNA in pJDC9 was previously
same way as pJDC9, but without the two terminators shown to be higher than in pJDCl0 (Chen and
flanking IucZcc. pJDCl0 was the control vector of Morrison, 1987).
choice here as it would minimize vector-specific Hind111 fragments from pJDC951-953 were puri-
variations in stability, but recloning in pKK232-8 fied and ligated with dephosphorylated pJDC10.
was also examined as that vector had proved useful Among approx. 1000 ErR transformants obtained
in cloning of the corn locus (Chandler and Morrison, from 0.4 pg of the ligated DNA mixture in each
1987). The efficiency of shotgun cloning with transformation reaction, only 5 “/, were Lac ~. The
 163

structures of the resulting pJDC10 clones were one terminator or none at all. At the corn (Chandler
examined by comparing Hind111 digests of the and Morrison, 1987) locus of pneumococcus, speci-
source pJDC9 clones and the pJDCl0 ErRLacP fic fragments have also been identified which express
subclones. Aberrant plasmids were found for all promoter activity in E. coli, and which are stable in
three cases. This result indicates directly that sta- E. co/i plasmid vectors with the protection of down-
bility of pneumococcal DNA with the protection of stream terminator signals, but are unstable in related
double terminators in pJDC9 is superior to that unprotected vectors.
without such protection in pJDCl0 clones.
Transfer of the same large cloned fragments from
pJDC95 l-953 to pKK232-8, with selection for CmR,
ACKNOWLEDGEMENTS
often led to rearrangements of the insert (not shown),
suggesting that bidirectional terminators may be
This work was supported in part by NIH research
permitting the cloning of fragments with strong
grant No. A119875.
bidirectional promoters.

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