¢Tene. 86 (1990) 71-79 Elsevier
Comparative expression of the pC194 cat g e m in Streptococcus p n e u m o n i a e , B a c i l l u s subtili$ and E s c h e r i c h i a coil
(Chloramphenicol acetyltransferase; promoter utilization; regulation of cat expression; plasmid rearrangements; recombinant DNA)
Sara Ballester', Juan C. Alonsob, Paloma L6pez" and Manuel Espinosa" ° Cenwo de InvestigacionesBiol6gicas,C.S. L C., E-28006 Madlqd (Spain)and bMax-Planck-lnstitutflirMolelculare6enetik,D-1000 Berlin33 (F.R.G.) Tel.(030)8307274 Received by M. galas: 7 April 1989 Revised: 31 July 1989 Accepted: 18 August 1989
The expression of the cat gene of the staphylococcal plasmid pC 194 present in the pLS 1-pC194 composite plasmid pJS37 was lower in Streptococcus pneumoniae and Escherichia coli than in Bacillus subtilis. Different transcription start points (and, by inference, different promoter utilization) of the cat mRNA synthesized in S. pneumoniae or B. subtOis were detected. Plasmid pJS37 is prone to deletion formation when host cells are grown in the presence of chloramphenicol (Cm). The analysis of the expression of the cat gene carried by the deleted derivatives of pJS37 has shown that a new promoter for the synthesis of cat mRNA is involved in the selective advantage conferred to the host by those deleted plasmids. Characterization of either in vivo or in vitro deleted plasmids has shown that the nucleotid~: sequence that could encode for a putative leader peptide is required for the Cm-induced pC194 cat gene expression.
The best known mechanism that confers Cm resistance to Gram + bacteria is the enzymatic acetylation of the antibiotic by CAT (Shaw~ 1983); cat genes specifyingthis enzyme are found in various bacterial chromosomes and plasmids. In general, the expression of Gram + cat genes is inducible by subinhibitory concentrations of Cm, while the expression of Gram- genes is constitutive (Shaw, 1983). Two inducible cat genes are well characterized: one of the Correspondence to: Dr. M. Espinosa, Vel~quez 144, E-28006 Madrid (Spain) Tel. (!)2611800; Fax (1)2627518 Abbreviations: B., Bacillus; bp, base pair(s); CAT, Cm acetyltransferase; cat, gene encoding CAT; ccc, covaiently closed circular; Cm, chloramphenicol; EtdBr, ethidium bromide; kb, kilobase(s) or 1000bp; nt, nucleofide(s); ORF, open reading frame; PI, P2, PT, promoters; RBS, ribosome-binding site(s); $., Streptococcus; SD, Shine and Dalgarno; tsp, transcription start point(s); wt, wild type; [ ], denotes plasmid-carrier state. 0378-1119/90/503.50 © 199•, ElsevierScience Publishers B.V.(BiomedicalDivision)
staphylococcal plasmid pUB 112 (Dick and Matzura, 1988, and references therein) and the other in B. pumOus, cat-86 (Duvall et al., 1987). Regulation of cat gene expression occurs at the post-transcriptional level in pC194 (Byeon and Weisblum, 1984),as wellas in other cat genes (Ambulos et al., 1985). The translational regulation of the cat genes resides in an inverted repeat that includes their postulated RBS (Shine and Dalgarno, 1974), SD2. In the absence of Cm, a secondary structure in the cat mRNA is formed blocking SD2 (Ambulos etal., 1984; Bruckner and Matzura, 1985; Nicholson et al., 1985). An additional leader region, consisting of SD1 and a short ORF is involved in the inducibility of the cat gene of pUBII2 (Bruckner and Matzura, 1985; Bruckner et al., 1987; Dick and Matzura, 1988) and the cat-86 gene (Harwood et al., 1987; Duvall et al., 1987; Alexievaet al., 1988). It has been proposed that Cm-modified ribosomes translating the leader peptide are stalled by the inductor just upstream from the regulatory stem-loop,destabilizing it. Similarregu-
72 latory elements have been proposed for the rest of the Gram + cat genes (Kin, et al., 1988). However, from previous data it seemed that this general picture could not be extended to pC194 because: (i) the plasmid cat leader peptide would end just before the stem-loop (Bruckner and Matzura, 1985) and (ii) the SD1 and the first two nt of the start codon for the putative leader peptide would be located upstream from the tsp of the cat mRNA (Byeon and Weisblum, i984). It would thus appear that the pC194 cat 8erie, although inducible, would be an exception among the Gram + cat genes. Furthermore, no information is available about the role of the putative leader peptide in the inducibility of the pC194 cat geae. We have previously shown that plasmid pJS37 (composed by pC194 and the streptococcal plasmid pLS 1) exhibited structural instability in S. pneumoniae and B. subtilis when challenged with Cm, due to selection of plasmid deietions that conferred selective advantages to their hosts (Ballester et al., 1986; 1989). In this work, we have extended the observations on plasmid instability to E. coli, in which pJS37 also replicates, because it contains the pLS 1 replicon (Lacks et al., 1986). We have studied the expression of the pC194 cat gene borne by pJS37 and by its deleted derivatives in the three bacterial hosts. The selective advantage conferred to the host by the deleted plasmids is due to juxtaposition of an upstream promoter to the cat gene. The utilization of cat gene promoters by B. subtllis and by 8. pneumonlae is different. In addition, we have obtained information about the involvement of the proposed leader peptide in pC194 cat gene expression.
RESULTS A N D DISCUSSION (a) Plasmid alterations that Increase cat gene expression The pC194 replication origin was shown to function in $. pneumoniae and a plasmid with a restriction pattern
identical to the parental one was isolated (Ballester et al., 1986), This fmding apparently contrasted with unsuccessful attempts at transferring pCl94 to pneumococcus (Barany and Tomasz, 1980). The pC194 isolated from S, pneumonlae had a tenfold increase in its copy number, as compared to a derivative from pJS37 lacking the pLS 1 replication functions. This derivative (pJS370) lacks the small Pstl fragment of pJS37 (thus, it lacks the or/(+) and repA and repB genes of the pLSI moiety; (Fig. 2A) and was isolated by selection for tetracycline resistance. Furthermore, pJS370 has an inactive pLS l-or/(-) since it is placed in the nonfunctional orientation (del Solar et al., 1987). The copy number ofpJS370 in $. pneumonlae was estimated to be 20 per 8enome equivalent, and its CAT activity was six- to
TABLE I Specific CAT activities conferred by various plasmids to the indicated bacterial hosts Strain a
CAT specific activities (units)c A
pJS37 pC194cop pJS370 pJS31 pJSI pJS 12 pJS81 pJS8 pJS 16 pJSSI pJS2 pJS ! 7
0.1 0.1 0.1 0.1 0.1 21 0.1 27 115 315 530 45
20 62 10 88 76 245 50 450 984 302 547 47
60 230 22 260 199 448 130 930 5744 322 552 46
61 230 24 440 368 920 125 3100 6050 310 540 46
pJS37 pJS31 pJSl
ND NF NF
ND NF NF
625 650 4800
ND ND ND
pJS37 pJS3l pJS2
NF NF NF
NF NF NF
31 188 455
ND ND ND
$. pneumoniae 708
B. subtilis MB 11
E. coil C600
a Bacterialstrains used were:$. pneumonlae 708 (end.l exo.2 tn.l hex.4 maiMS94), E, toll C600 (thr-Ithi.l leu.6 laeYl tonA21 sup£44) wad It, subtllls MB11 (/ys-3hisH2 metBl0). b Plasmidsused were: pCI94 (Horinouchiand Weisblum, 1982),pLSI (Lacks et al,, 1986), pJS37 and its derivatives (Ballester et al,, 1986; 1989 and this work). Cultures of$. pneumonlae and B. subtll~ were ~'own and transformed as described (Espinosa et aL, 1982). £. cell was grown in L broth and transformed according to Kushner (1978). ° A, uninduced cultures; B, cultures induced with 0.3 pg On/ml; C,
culturesinducedas in B and grownin the presenceof 2.5 Fg Cm/ml(for $. pneumonlae) and 10pg Cm/ml(for B. sublYb and £. co//); D, cultures induced as in B and grown at the highestpermissibleCm concentration for each strain. For CAT activitydeterminations,cultures were ip'own with 10Pg Cm/ml (for B. ~bd//,v and £. co/i) or at 2.5 Pg Cm/ml for $. pneumonlae. Crude extracts were prepared from 1.5-mlcultures in a 200-pl vol, of 50 mM Tris. HCI pH 7.8/50pM #-mercaptoethanol.To lyse B. subUlb and E, cell cells, iysozyme(125p&/ml)was added; for $. pneumonlaethe lysiswas carriedout by sodiumdeoxycholateat 0.! %. After 10rain at 30°C the lysateswerepassed through a 25-gauseneedle four times.The enzymaticactivitiesweredeterminedby the colorimetric method of Shaw (1975). ND, not determined; NF, experimentnot Feasibledue to plasmidloss. tenfold lower than that of the $. pneumoniae.established pC194 (Table I). It seemed that a pC194 copy-number mutant (pC194cop) was selected in the pneumococcal host. We have not identified the mutation, but it appeared to be conditioned to the host, since the copy numbeo:~ of the mutant and wt plasmids were identical in B. subtilis.
oo .t I 0.1
7"2 56 3.9 2.7 2I
T i m e (h)
T O AA-
Fig. 1. PlasmidpJS37 structural instabilityin E. coll. (A) Growth curves in mediumcontainingCm at the concentrationsof 10 (open circles),20 (solid circles), 50 (open triangles)and 100pg/ml (solid triangles).(B) Analysisof the plasmidcontent of the cultures. Lanes !-3 and 5 correspondto cultures grown at 10, 20, 50 and 100/AgCm/ml,respectively.Lane4, ¢cc monomersof plasmid standard (crude extracts of E. coil V517; Macrinaet al., 1978); their size (in kb) is indicatedon the right.The positionsof the ©c¢monomersof plg37 and pJS2 are indicatedon the let1.Electrophoresisin ! % egarose was©onduetedfor 2 h at 80 V.BandswererevealedbystainingwithEtdBr.Total DNAextracts containingchromosomaland plasmidDNAwereprepared as described(Lackset al., 1986).(C) The nt sequencearoundthe borders of the deletionofpJS2. Upper strand, sequenceat the border in pLSI moiety; lowerstrand, sequenceat the borderin pC194moiety.Numbersindicatent positionsin pJS37.The resultingsequenceis underlined.Plasmidswerepurified ae¢ordin8to Currier and Nester (1976). Restrictionenzymes(NewEnglandBiolabsor BoehringerMannheim)wereused as specifiedby their suppliers. The nt sequencedeterminations were performed by the chemicalmethod of Maxam and Gilbert (1980).
pC 19~op is also able to replicate and express Cm resistance in $. thermophilus host in which the wt plasmid failed to be established (J.F. Viret, personal communication). These results indicate that the expression of cat in streptococci is severely impaired and that mutant plasmids are readily isolated when selection for Cm resistance is applied. Isolation ofhigh-copy-number plasmids from pLS 1 derivatives that carry the pC194 cat gene (pJS3, pJS5; Fig. 2) support the low expression of cat in these bacteria. Indirect evidence of poor cat gene expression in E. coli C600 (in conjunction with our failure to introduce pC194 into this host) led us to isolate derivatives of pJS37 from E. coli using the positive selection with Cm, for the generation of deletions enhancing cat expression. E. coli C600[pJS37] cultures were grown in the presence of increasing amounts of Cm. As reported for S. pneumoniae
(Ballester et al., 1986), the E. coil cultures exhibited longer lag periods as the Cm concentration increased (Fig. IA). The generation time also augmented with increasing amounts of Cm, from the usual doubling time of 35 min (at 10 pg Cm/ml) to 50 and 90 min for 20 and 50 pg Cm/ml, respectively. The cultures grown in the presence of 100/~g Cm/ml showed the longest lag period (9 h), but had a 35-min doubling time. If deleted plasmids were to be selected at 100/~g Cm/ml, we should expect this behavior because only plasmids conferring selective advantage to their host would allow a normal growth. Analysis of the DNA content of the cultures showed that a deleted derivative (pJS2) was indeed selected in the culture grown at 100/~g Cm/ml (Fig. 1B). Determination of the nt sequence at the deletion endpoints of pJS2 showed that: (i) the deletion occurred (without significant homology) between nt
r e p A repB ~:>C::=:>H I L
8 Nc I I
Nc P I I
NA I I , ,
r 1 I
ApJS51 ApJS31 ApJS| , ApJS2 A pJSg| ~pJS!2 , ApJSI6 , ApJSI7
(2752-6123) ( 2 7 4 8 - 5875) (294? -5875) (3016-6128) (3430 - 587~) (3637 - 5966) (2920-6026) (2908 - 6 0 9 6 )
,,,o I pT , . . . . 1. . . . . . L "" 5'-~ATTT~AAA---'SUP--TA~ . . . . . . ÷CAAO'TTTA~ . . ...~CA~'TTTT~ . . . . . . .
I ". "" ~AOT~CAAA~. . . . . . ÷T TA
T T TAAAA ,,- 181~--~ T"AATATTG ACT T T TAAA~--'~'G~TTGA T TC AAT GAAGAA AG C AGACA AGTA~G~ATATCAAATGAACT T T AA~ - 3' SOl Me t L y | L y | A I oA|pL ytEnd
Fig. 2. Deletions generated in plasmid pJS37. (A) Map of pJS37 and location of the deletions in its derivatives. Double and single line indicate pLSl
and pCI94 moieties,respectively.Arrowsindicate relevantgene products and their respective 5' to 3' orientations; filledportions represent promoters. Only relevantrestriction sites are depicted (H, Hindlll; N¢, Noel; P, PsH; B, Bell; E, £coRl; N, Nail; A, A¢¢I). Lines belowthe map of pJS37 indicate the region of deletions with their respective names and coordinates indicated on the right. The !~S37 derivatives were described previously(Ballester et al., 1986; 1989)or in this work. One of these derivatives,pJS31, is identical to pJS3, but the latter ©ontalns a C-, A transversion at nt 743 of pLSI which results in a rivet'oldincrease in the plasmid copy number (del Solar et al,, 1989).Another derivative,pJSSI, has a restriction pattern identical to pJSS, but the latter carries an uncharacterized high-copy-numbermutation within the pLSl replicon. For the isolationofpJSlT, 7/411of plasmid pJS81 DNA was linearized with Bell and treated with 1 unit or BAL 31 for various periods of time (2 to 9 rain) at 37°C. The ends were filled-in,ligated and I #g oI'DNAwas used to transformcompetent$, ~neumonlae70Scells.A similarexperimentwas perrormedto construct pJS 16frompJS8.Transformants were selectedwith2.S ~g Cm/ml.(a) Partial nt sequencesofplasmld pJS37 showingthe deletionend.points for the constitutivedeletedderivativesp~S$1, pJSl7 and pJS2 (arrows). PT, P! and !'2, promoters; SDI and SD2, proposed RBS for the translation of the leader l~ptide and CAT, respectively. Numbers correspond to coordinates of i~S37, nt sequence was determined as in legend of Fig. I.
3016 and 6125 of pJS37 (Fig. IC), and (ii)the regulatory stem-loop structure of the cat mRNA is eliminated, renderins the cat expression constitutive (Table I; Fig. 2B). Selection for high Cm resistance in Gram + bacteria harboring pJS3? allowed us the isolation era number ofplasmids with intema! deletions (Ballester et al., 1986; 1989). The extent of the deletions is shown in Fig. 2A and the at sequence at the deletion endpoints has either been reported (Ballester et al., 1986; 1989) or is shown here (Fig. 2B). The rearrangement either placed the putative upstream PT prometer next to the cat gene (pJS31, pJSl, pJSSl and pJSl2) or replaced cat promoters (Pi and P?; Leonhardt and Alonso, 1988) with PT. The latter deletions (pJSSl, pJS2) eliminated the regulatory stem-loop for cat expression (Fig. 2B). The resulting rearrangements led to the isolation
of plasmids that synthesized high levels of CAT (Table I). In $. pneumoniae and E. coli all plasmids tested specified for CAT activities higher than pJS37. However, B. subt!lb[pJS31 ] did not exceed the elevated CAT activity encoded by pJS37 in this host. B. subtilis[pJS3?] exhibited a ten- to 20-fold higher CAT activity than that detected in S. pneumoniae [pJS3?] and E. coli[pJ$37] (Table I). The CAT activities conferred by pJS31 and pJSl to S. pneumonlae were very similar, unlike for B. subtilis (Table I). These differences are neither ascribable to a different PT promoter utilization nor to a new promoter being generated (not shown). Although we do not have a clear explanation for this f'mding, the influence of additional factors on cat expression (such as different m R N A stabilities) cannot be ruled out.
75 OJ) Characterization of constitative pC194 cat gene mntants
The possible role of the putative leader peptide of the pC194 cat gene was next investigated. By BAL 31 deletions from pJSgl and analysis of several derivatives, an in vitro deleted plasmid, pJS17, was selected; nt sequence determination showed that the deletion extended from nt 2908 to 6096 (coordinates of pJS37; Fig. 2B). Consequently, pJS 17 (as pJS2 and pJS51) lacks promoters PI and P2 and the putative leader peptide (Fig. 2B). However, the stemloop structure that could block the translation of CAT in the absence of Cm remained intact in pJS 17, contrary to pJS2 and pJS51 (Fig. 2B). In spite of this, cat expression was constitutive in pJS 17, but the levels of CAT present in cells harboring this plasmid were low compared with the other constitutive plasmids, pJS51 and pJS2 (Table I). Significant transcriptional differences among these plasmids were discarded (not shown). Constitutive cat expression in pJS51 and pJS2 is consistent with the hypothesis that the blocking ofthe SD2 sequence by formation ofthe regulatory stem-loop structure represses the pC194 cat expression (Ambulos et al., 1984). However, pJS51 and pJS2 have also lost the proposed leader peptide, unlike pJSl7. It has been reported for cat-86 that the highest gene induction occurs when the ribosomes are stalled at Lys just before the stem-loop structure (Alexieva et al., 1988). The last residue ofthe proposed leader peptide ofpCl94 is also Lys, and its codon is located just before the stem-loop structure (Fig. 2B). Our results indicate that the inducibility of the pC194 cat depends on both an intact stem-loop structure and DNA sequences in the leader peptide region. Whether the synthesis of putative peptide or sequences before the stem-loop are required for the inducibility of the pC194 cat is still an unsolved question. However, our data point to the formation of the stem-loop leading to a block in the cat expression that would be abolished by the synthesis of the leader peptide. We favor this possibility because it considers the pC 194 cat gene as belonging to the same category as the pUBll2-cat and the cat-86 genes (see below).
that for B. subtilb Oane 6) the intensity detected for P2 was about two times higher than for PI. In contrast, P2 utilization by S. pneumoniae Oane 5) was three times lower than that of PI. Furthermore, only PI is used in vitro for cat mRNA synthesis by the RNA polymerase of E. co6 (Byeon and Weisblum, 1984). These results show that in both S. pneumoniae and E. coli, the ORF for the putative leader peptide of cut (Bruckner and Matzura, 1985) is not contalned within most of the cat mRNA molecules (those synthesized from PI), which cannot be translated even upon induction. We have predicted (Ballester et al., 1986) that, in the deleted plasmids selected by challenging S. pneumoniae[pJS37] with Cm, a readthrough transcription from the proposed tet promoter occurred (PT, Fig. 2A). To test if the putative PT was used by S. pneumoniae, we
c S G ,° C
"t • " ~' I r Q
P~, -,- 25?
• " ' Q i~l i. ,,
l , i 0,4
P2 Pl I
r,.,',,',.,-,.,-,~230 nt ~
¢o..._tmRNA I I
(c) Identification of the 5' terminus for cat mRNA synthesis
,-.A~..,,.,257 nt ~
Promoter utilization was analyzed by S 1 mapping of the 5' end of the mRNA produced from the pC194 cat gene in S. pneumoniae or B. subtilis. Total RNAs prepared from cells harboring pC194cop were hybridized with the BglIDdel fragment of pC194cop (nt 974 to 1424, coordinates from Horinouchi and Weisblum, 1982). This fragment contains promoters PI and 1)2 (Byeon and Weisblum, 1984; Leonhardt and Alonso, 1988) and the 5' end of the cat. Two protected fragments (Fig. 3) of 230 and 257 nt (corresponding to promoters PI and P2) were observed for both bacteria. Densitometric scanning ofthe bands showed
Fig. 3. Utilization of cat promoters Pl and 1'2 by S. pneumonlae (lane 5) and B. subtilb (lane 6) detected by nuelease S 1 mapping. The sizes of the protected fragments and of the probe DNA are indicated on the right and in the scheme showed below. Total RNA was prepared according to Aiba et al. (1981), with the modifications described for S.pneumonlae and B. subtgb (L6pez et ai., 1989). The S ! mapping of the 5' end oftranscripts was performed essentially as described (Lbpez et al., 1989). Conditions (RNA concentrations, amounts of formamide, temperatures of hybridization and amounts of nuelease S 1) were optimized for each transcript. Samples containing 3000 epm were applied to an 6% polyaerylamide sequencing gel. Electrophoresis and further manipulations ofthe gel were performed according to Maxam and Gilbert (1980). The Maxam and Gilbert reactic,ns of the 5'-32P-labeled (*) Bgll-Ddel fragment used as probe (schem~!ically depicted below) are shown in lanes I-4.
c o t mRNA
A C +G +C G T
3 4 5 6 tB
9.- ..... HM|
at . . . . .
,.s. . . . . . HpaZ
" ' - 14ht ,41 tet mRNA
~ *"" ~
145-- .~, "
273 a t . . . . . .
4ant cot mRNA
so_ , P!
Me t L y s L y s A I o A s p L y s [ n d
Fig.4. Utilizationof the PT promoter.(A) Identificationof the PT promoterofpLSl usedby $. pneumonias(lane 1) by nuclease Sl mapping.Guanine sequencingladder(lane 2). (B) Utilizationofthe PT promoterof pJS3 by $. pneumonias(lane7), £. coil(lane 8) and B. ~btilis (lane9). The Maxamand Gilbert(1980)reactionsofthe fragmentused as probe wereusedas standards(lanes 3-6). The sizesofthe protectedfralpnents,3=plabel(*)and position of the PT promoterin the probes are indicated.(C) Partial nt sequenceof pJS37 showingthe up (bent arrows) of cat mRNAfrompromoters,P7', P2 and PI. The -3Y and -10 regionsofthe three promotersare boxed.The proposed SDI and leaderpepttde are indicated.The reiion deleted in all pJS37 derivativesis sbr~wn.Coordinatesof pJS37 are also indicated. Samplesfor nuclease SI mappingwere prepared and analyzedin an 8% (A) or 6% (B) polyacrylamidesequencinggel as in Fig.3, legend;nt sequencewas determinedas in Fig, I, legend. determined the tsp of the tct mRNA synthesized in $. pneumonias 708[pLS1]. A fragment corresponding in size to the predicted tsp was observed (Fig. 4A; coordinates 1390 to 1757 from pLS 1; Lacks et al., 1986). To test if a new promoter or P T reads through the cat gene, total RNA from 5. pneumonlae[pJS31 ] was used; cat mRNA of three different sizes corresponding to transcripts from Pl, i'2 and a third longer fragment were observed (not shown). The precise tsp of the latter transcript was identified (Fig. 4B), showing the use of P T in $. pneumonias (lane 7), in E. coli (lane 8) and in B. subtilis (lane 9). Initiation of cat mRNA from PT was also observed for pJSl, pJS2, pJSSl, pJSl7 and pJS81 (not shown). It thus seems that transcriptional readthrough renders $. pneumonias resistant to Cm. Since the majority of the cat mRNA was synthesized from P2 in B. subtilis, transcription from P T would not substantially lead to an increase in CAT levels in cells harboring pJS31, in contrast to those carrying pJS37 (Table I). Based on the facts that (a) in E. coil and S. pneumonias, PI is mainly utilized and cat expression is poor; (b)in
B, subtOis, P2 is used and CAT levels are high; (e) enhancement ofcat expression in $. pneumonlae and E. coli requires the utilization of an upstream promoter (PT) and (d) pJS 17 specified for low and constitutive levels of CAT, we propose that the synthesis ofthe putative leader peptide, rather than the sequence before the stem-loop structure (Figs. 2B and 4C), is required for an efficient expression of the pC194 cat in the three bacterial species. This is consistent with the results obtained for plasmid pUB 112 (Dick and Matzura, 1988) and for cat-86 (Duvall et al., 1987; Alexieva et al., 1988). Results showing poor cat expression in £. coli have been demonstrated for other inducible cat genes (Bruckner et al., 1984; Shaw et al., 1985; Harwood et al., 1987). The cat promoter utilization of S. pneumoniae and E. coli is another example of the 8ene similarities reported for both bacterial species (Lacks et al., 1987; l~pez et el., 1989). Furthermore, B. sub~//s shows a differential utilization of some staphylococcal promoters as compared to E. coli (Hudson and Stewart, 1986) and to $. pneumoniae (our
77 (d) Determination of the mutation present in pJS8
transcription from P T would increase upon reduction ofthe stability of this putative terminator. This hypothesis was tested by in vitro deletion ofthe proposed terminator structure. To this end, one BAL 31-treated pJS8 derivative (pJSl6) was chosen for further characterization. Analysis of the nt sequence at the deletion endpoints showed that these ends are located at coordinates 2920 and 6026 of pJS37 (Fig. 5). The genetic organization in pJSl6, in comparison with the parental pJSS, changed at the regulatory elements of cat expression. First, the putative terminator located between P T and cat was removed. Determination of the CAT-specific activity of S. pneumoniae 708[pJS16] showed a sixfold increase in the enzymatic activity at 2.5 Fg Cm/ml, as compared to S. pneumoniae 708[pjS8] (Table I). Second, the -35 region of P2 was eliminated, but the deletion junction generated a new -35 region, located 16 bp upstream from the -10 region of P2. The new promoter 1'3 (Fig. 5) was functional (and as poorly used as 1)2), as judged by S 1 mapping of the cat mRNA present in $. pneumoaiae 708[pJS 16] (not shown). These results support the finding that the alteration of the proposed secondary structure in pJS8 (or its removal in pJSl6) resulted in a more efficient transcription ofcat mRNA from PT, as compared to the wt
Among the deleted derivatives of pJS37, plasmids with eightfold differences in CAT activities, but with identical deletion endpoints were isolated (pJS81 and pJSS; Table I). This result suggested to us that factors other than the conjunction of an upstream promoter could be involved in the cat expression by the deleted derivatives. To understand these differences, the nt sequence of the entire transcriptional unit ofpJS8 was determined. Plasmid pJS8 differed from pJSS1 only by a transition (G--, A) at nt 5952 of pJS37. This nt is included in a putative 13-bp inverted repeat that would allow the formation of a stem-loop structure followed by various T residues (Fig. 5) resembling the signals for termination of transcription (Rosenberg and Court, 1979). It has been suggested that such structure would function as a terminator for the transcription of the incA mRNA, involved in the post-transcriptional regulation of repH gene expression (Alonso and Tailor, 1988). The predicted free energy for the wt structure would be -66.1 kJ/mol; while for the mutant structure present in pJS8, the free energy would be -35.2 kJ/mol (Tinoco et al., 1973). One way to explain the different CAT levels conferred by pJS8 and pJS81 is by assuming that readthrough
TT T T TA TA AT CG IIq/
~~-~L......... ..... pJS81
TGATCA ........ CACATATTCTT
5'- ATTTTAATTTGGCTTTGCATTTTATC 5'' ATATTTTTAAAAT~TATATTTATGTT Q020
, I Fill.5. UpstreamreBionofcat genesfrom IxlS81,pJS8 and pJSl6. (UpperIXm)Structureof a putative transcriptionterminatorpresent in pJS81.The G --,A mutationin pJS8is indicated,as wellas the positionsofthe I'T, 1'2 and PI promoters.The indicateduniqueBall siteofpJS81was usedto linearize the plasmidand to construct pJSl6 by BAL31treatment. Below,boxed,is shownthe nt sequencemound the borders of the deletion ofpJSl6. Upper strand, sequenceat the borderin pLSI moiety;lowerstrand, sequenceat the borderin pC194moiety.Numbersindicatent positionsin pJS37.Underlined is the resultingsequence.(Lowerlint) Promoter structure in pJSl6. The new P.~promotergeneratedby the deletionis indicated as well as its position in relation to the position of promoters ,el and PT.
78 (e) Conclusions By quantitative measurements of CAT-specific activity, we have shown that the pC 194 cat gene expression is dependent on the bacterial host. A different promoter usage for the pC194 cat gene transcription between S. pneurnoniae and B. subt///s was observed. The majority of the transcription occurs from PI in the former host and from P2 in the latter. The poor expression of the pC194 cat in $. pneumoniae allowed us to develop an accelerated process of natural selection for plasmid mutations which increase the Cm resistance of the host cells. This positive selection permitted the rescue of various kinds ofplasmid alterations: (1) copy number mutations that increase the gene dosage; (2) deletions which couple a new promoter to the cat gene for the synthesis of cat mRNA molecules containing all the
regulatory elements needed for the inducible gene expression; (3)deletions eliminating the inverted repeat involved in the masking of the SD2 sequence of the cat structural gene; and (4)mutations improving the cat transcription from the P T promoter by lowering the efficiency of a possible transcription terminator located between the P T promoter and the cat gene. Our results indicate that, similarly to other cat genes from Gram + bacteria, translation of the leader peptide in pC 194 is required for gene induction. If this is the case, we may rule out the observation that the pC194 cat gene is an exception among other Gram ÷ genes
(Bruckner and Matzura, 1985).
Thanks are due to Prof. T.A. Trautner for helpi~l discussions and to J.F. Viret for communicating to us results prior to publication. The exchange program between CSIC and Max-Planck-Gesellschat~ supported visits between both laboratories. We thank Maite Alda for technical assistance, A. Hurtado and J.C. FernAndez for the art work, and Wendy Newton for corrections in the manuscript. Research at CIB was supported by Grant BIO88.0449 of CICYT to M.E,
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