JOURNAL OF BACTERIOLOGY, Aug. 1991, p. 4683-4691 0021-9193/91/154683-09$02.00/0 Copyright © 1991, American Society for Microbiology
Vol. 173, No. 15
Sequence and Transcriptional Regulation of comlOlA, a Locus Required for Genetic Transformation in Haemophilus influenzae THOMAS G. LARSON't AND SOL H. GOODGAL2* Graduate Group in Biochemistry' and Department of Microbiology,2 University of Pennsylvania School of Medicine, Philadelphia, Pennsylvania 19104 Received 11 December 1990/Accepted 21 May 1991
A 2.8-kb EcoRI-BgM fragment cloned from the wild-type Haemophilus influenzae Rd chromosome is shown to increase the transformability of the Com-101 mutant through trans complementation. Deletion and sequence
analyses indicate that the active region of the clone carries a 687-bp open reading frame. A 0.3-kb insertion in the corresponding EcoRI-Bgll fragment of the ComA101 chromosome is shown to be a partial (331-bp) duplication of this open reading frame. The wild-type sequence produces a peptide of a size that is consistent with the sequence data when this sequence is expressed in Escherichia coli with a T7 promoter-based transcription vector. RNA hybridization analysis using a DNA probe derived from the open reading frame suggests that the sequence is transiently expressed during competence development. On the basis of these observations, it is proposed that the open reading frame corresponds to the comlOlA gene.
Competence development in most known examples of naturally transforming bacteria involves the regulated expression of specific genes. The process generally involves global changes in the organism's metabolism that are coordinated with the specific expression of the functions required for genetic competence. The actual regulatory events are poorly understood in most organisms; however, some significant details of competence regulation in gram-positive Bacillus subtilis (see reference 8 for a review) have recently emerged. The com (competence) genes of B. subtilis have been grouped into two classes: the "early" genes and the "late" genes (2). The early genes are transcribed constitutively (2), although some of them may be upregulated preceding the onset of competence (8), while the late genes are expressed only in competence-inducing medium (1, 8, 19). Additionally, expression of the early genes is required for expression of the late genes (1, 8, 19) as well as for expression of other genes that do not appear to be related to competence (21, 40). Competence in gram-negative Haemophilus influenzae also appears to be regulated (9) by genetically encoded functions (7) that might belong to classes similar to those found in B. subtilis. The accompanying report describes the cloning of linked loci capable of increasing the transformation frequency of Com- phenotypes in H. influenzae (13). This report focuses on one of these loci, comlOlA, in greater detail. The wild-type clone of the comlOlA locus is shown to increase the transformability of the Com-101 mutant through trans complementation, and the comlOlA open reading frame is defined through sequence and deletion analysis. Finally, transcription of the comlOlA locus is shown to be transient: like the late class of competence genes in B. subtilis, the H. influenzae comlOlA gene is expressed after competence induction, supporting the assertion that the comlOlA locus encodes a competence-specific product.
MATERIALS AND METHODS Bacterial strains and plasmids. The origins and phenotypes of H. influenzae Rd1967, Com-78, Com-101, and Rec-1 are described in the accompanying paper (13). The E. coli lysogen HMS174(DE3) was a gift of W. Studier (33). E. coli DH5aMCR was obtained from Bethesda Research Laboratories. The plasmids used in this study are presented in Table 1. The construction and properties of the plasmids pER194 and pTGL6 are described in the accompanying paper (13). The vector pHK was a gift of G. Barcak (3). Plasmids pTZ18U and pTZ19U (17) were obtained from United States Biochemicals. Plasmids pUC18 (41) was obtained from Bethesda Research Laboratories. Plasmids pLysS and pLysE were gifts of W. Studier (34). All other plasmids were constructed in this study and will be described below. Bacterial growth and competence development. H. influenzae cultures were grown and preserved as described elsewhere (13). Chromosomal transformations were performed by using cultures made competent by the MIV (9) or aerobic-anaerobic procedure as described in the accompanying paper (13). Plasmid transformations were performed by using the saline-glycerol procedure described by Stuy and Walter (35) with the modifications described in the accompanying paper (13). Plasmid and chromosomal markers were selected as before. E. coli strains were generally grown in Luria-Bertani (LB) broth, plated on LB agar (15), and preserved by freezing in LB broth plus 10% glycerol at -70°C. Cultures used to prepare plasmid or competent cells were grown in TB medium (37). E. coli cultures were made competent by using the CaCl2 technique (15). 4-Methylumbelliferyl-f3-D-galactoside (MUG) was used for the detection of Lac- transformants (42). Transformed cultures were spread on LB agar containing 100 ,ug of MUG and 100 pLg of ampicillin per ml. After 14 to 16 h at 37°C, Lac' colonies were easily distinguishable from Lac- colonies when illuminated with longwave UV light because of the fluorescence of the hydrolyzed MUG. The use of isopropyl-,-D-thiogalactopyranoside was not necessary when MUG was used to test for insertions in plasmids pUC18, pTZ18U, and pTZ19U. Enzymes. Restriction enzymes, calf intestine alkaline
* Corresponding author. t Present address: Roche Institute of Molecular Biology, Nutley, NJ 07110-1199.
4683
4684
J. BACTERIOL.
LARSON AND GOODGAL TABLE 1. Plasmids used in this study
or DsrpinSource reference escnption
Plasmid
designation
pER194
pJl-8fQ(EcoRI::H. influenzae Rd 13.3kb)
pHK pLysE pLysS pTGL6 pTGL6-101 pTGL43
Kmr Tcr Cmr Cmr pHKfQ(EcoRI-MluI::pER194 7.8kb) pHKfQ(EcoRI-MluI::H. influenzae Rd Com-101 3.lkb) pUC18fQ(EcoRI-BglII/BamHI::pTGL6 2.8kb) pTGL43AO.4kb from BglII-BamHI site pTGL43AO.6kb from BglII-BamHI site pTGL43AO.8kb from BglII-BamHI site pTGL43A1.O8kb from BglII-BamHI site pTGL43D4N3AO.2kb from EcoRI site pTGL43D4N3AO.4kb from EcoRI site pTGL43D4N3AO.6kb from EcoRI site pTGL43D2N13fQ(HindIII::pHK 6.7kb)
pTGL43D2N13 pTGL43D2N15 pTGL43D3N2 pTGL43D4N3 pTGL43D4N3:D3Nl pTGL43D4N3:D12Nl pTGL43D4N3:D12N3 pTGL43D2N13HK pTGL43D2Nl5HK pTGL43D3N2HK pTGL43D4N3HK pTGL43D4N3:D3NlHK pTGL43D4N3:D12NlHK pTGL43D4N3:D12N3HK pTGL54 pTGL81 pTGL81HK pTGL115 pTGL136 pTGL136HK pTGL153 pTGL153HK pTGL180 pTGL180HK pTGL200 pTGL201 pTZ18U pTZ19U pUC18
pTGL43D2Nl5fQ(HindIII::pHK 6.7kb) pTGL43D3N2fl(HindIII::pHK 6.7kb) pTGL43D4N3Qk(HindIII::pHK 6.7kb) pTGL43D4N3:D3NlQf(HindIII::pHK 6.7kb) pTGL43D4N3:D12NlQf(HindIII::pHK 6.7kb) pTGL43D4N3:D12N3fQ(HindIII::pHK 6.7kb) pUC18fQ(EcoRI-BglII/BamHI::pTGL6-101 3.lkb) pTGL43AHindIII::0.2kb pTGL81Q(HindIII: :pHK 6.7kb) pUC18fQ(HincII/DraI::pTGL43 0.28kb) pUC18fQ(EcoRI-HincII/EcoRI-DraI::pTGL43 1.lkb) pTGL136fQ(HindIII::pHK 6.7kb) pUC18fQ(EcoRI-HincIIIEcoRI-SspI::pTGL43 1.5kb) pTGL153fl(HindIII: :pHK 6.7kb) pTGL43D4N3AO.8kb from EcoRI site pTGL180Qf(HindIII::pHK 6.7kb) pTZ18Ql(EcoRI-HindIII::pTGL43D4N3:D12N1O lkb) pTZ19fQ(EcoRI-HindIII: :pTGL43D4N3:D12N1O lkb) Apr, bacteriophage T7 promoter Apr, bacteriophage T7 promoter Apr
phosphatase, T4 DNA ligase, Klenow fragment of E. coli DNA polymerase I, and E. coli DNA polymerase I were obtained from the sources previously identified (13) and used as described elsewhere (13). Bal 31 exonuclease was obtained from Bethesda Research Laboratories, and RNAsefree DNAse was obtained from Worthington; both enzymes were used as described below. DNA preparation. H. influenzae chromosomal DNA was prepared for genetic transformations by using Marmur's method (16). Preparation of H. influenzae chromosomal DNA in agarose beads and its subsequent digestion with restriction enzymes has been described elsewhere (11). Plasmid DNA preparations were purified by using the alkaline lysis procedure (5, 15) followed by precipitation with polyethylene glycol (14). Small-scale plasmid preparations used for restriction analysis were made by using the abbreviated alkaline-lysis minipreparation procedure described by Morelle (20); for H. influenzae plasmid preparations, the previously described modifications were incorporated (13). Purification of the 13.3-kb insert from EcoRI-digested pER194 by using agarose gel electrophoresis and electroelution has been described elsewhere (13), and the EcoRI-BglII subfragments of pTGL6 and pTGL6-101 were prepared by using the same procedure. Restriction fragments from other plasmids were prepared by separating digests on 2% lowmelting-point agarose minigels (FMC GTG Low-Melting Agarose) and excising the desired bands. The DNA was
13 3 33 33 This This This This This This This This This This This This This This This This This This This This This This This This This This This This This 17 17 41
study study study study study study study study study study
study study study study study study study study study study study study study study study study study study study
recovered from the agarose by hot-phenol extraction at 65°C (modified from the procedure described in reference 31) followed by precipitation with 2-propanol and ammonium acetate (15). Cloning of a mutant copy of the pTGL6 insert. An EcoRI digest of Com-101 chromosomal DNA prepared from cells embedded in agarose beads was separated on a 1% agarose gel. DNA comigrating with the 13.3-kb EcoRI fragment of pER194 was purified from the gel by electroelution. A 1-,ug sample of the recovered DNA was subsequently digested with MluI and ligated into 1 pug of MluI-EcoRI-digested pHK vector in 100 ,ul of reaction mixture. The ligation reaction mixture was concentrated by ethanol precipitation, and the DNA was transformed into the Rec-1 strain of H. influenzae. Colonies that were kanamycin resistant (to 10 ,ug/ml) but tetracycline sensitive (to 5 ,ug/ml) were grown to stationary phase in 1 ml of supplemented brain heart infusion containing kanamycin at 37°C without shaking. A 10-,ul volume from each culture was lysed by adding it to 100 pl of 0.4 N NaOH-10 mM EDTA and heating the mixture to 40°C for 20 min. The samples were then applied to a nylon membrane (Hybond-N; Amersham) by using a slot-blotting apparatus. The membrane was hybridized overnight at 65°C and washed according to the instructions of the manufacturer. The probe was prepared from the pER194 insert and labeled as described elsewhere (13). Subcloning of EcoRI-BglIl fragments from pTGL6 and
VOL. 173, 1991
pTGL6-101 into pUC18. Plasmids pTGL6 and pTGL6-101 were digested with EcoRI and BgIII. The desired fragments were gel purified, ligated to EcoRI-BamHI-digested pUC18 vector, and transformed into DH5aMCR. Plasmid DNA was prepared from several Lac- colonies and tested for the presence of the desired insert by restriction mapping. Construction of subclones and deletions. Subclones of pTGL43 and pTGL54 were constructed by isolating DraI and SspI restriction fragments and ligating them to calf intestine alkaline phosphatase-treated HinclI digests of pUC18. Nested, unidirectional deletions of the pTGL43, pTGL43D4N3, and pTGL136 plasmid inserts were generated by using Bal31 exonuclease (22, 27, 28). The ligated DNAs from the subclones and deletion reactions were transformed into E. coli DH5ScMCR as described above. Plasmids from Lac- colonies were evaluated for the desired inserts by restriction analysis. Assay for increase in transformability of Com-78 and Com-101 mutants. The plasmids to be tested were first isolated and grown in the H. influenzae Rec-1 strain so that they could be amplified under conditions that do not permit recombination. The amplified plasmids were then transferred to Com-78 and Com-101 hosts, and the chromosomal transformation frequencies of these strains were evaluated as described in the accompanying paper (13). E. coli plasmids to be tested for the ability to complement the com mutations were digested with HindIII and ligated into a calf intestine alkaline phosphatase-treated HindIll digest of the pHK vector that had been grown in E. coli DH5otMCR. Figure 1 details the construction of one such plasmid, pTGL81HK. The ligated DNA was transformed into DH5aMCR, and the desired constructions were verified by restriction analysis. The appropriate plasmids were transformed into the H. influenzae Rec-1 strain and tested for their abilities to increase the transformability of the Com-78 and Com-101 strains as described above. DNA sequencing. The entire pTGL43 insert and the relevant portion of the pTGL54 insert were sequenced by using the dideoxy chain termination reaction method (29) with bacteriophage T7 DNA polymerase (36). Sequencing reactions with double-stranded DNA were performed by using the Sequenase kit (U.S. Biochemicals) as directed by the manufacturer, with minor modifications (39). The supplied universal primer was used for the forward reactions, and the New England BioLabs -48 reverse M13 sequencing primer (catalog no. 1233) was used for the reverse reactions. The reactions were separated by using the buffer-gradient gel procedure described by Biggins et al. (4). When this system was used, 300 to 400 nucleotides could be read from each template. When required, more nucleotides were read by using a standard 4% gel run under the conditions described by Parkison and Cheng (25). Reaction mixtures for these gels were prepared by using the extended sequencing conditions recommended in the Sequenase manual. Computer analysis of the sequence data was performed by using the PC Gene and IG Suite software packages (both from Intelligenetics). Transcriptional analysis of ORF2. A 200-ml culture of Rdl967 was grown in freshly prepared supplemented brain heart infusion with shaking at 36°C. At an optical density at 650 nm of 0.16, the culture was chilled in an ice bath, harvested, and suspended in an equal volume of freshly prepared MIV medium (9). After the culture was harvested and suspended a second time, 50 ml was set aside in an ice bath and the remainder was shaken at 36°C. Fifty-milliliter volumes were removed from the culture after 50, 100, and 150 min and transferred to an ice bath. After each sample
SEQUENCE AND TRANSCRIPTION OF comlOlA
4685
BamH I/BgI 11 Hind ill B.I
Hind
pTGL43 5.5 kb EcoRI
Hindill
pTGL81 5.2kb
+
Hi
H
EcoRI
Il
FIG. 1. Construction of pTGL81HK from pTGL43 and pHK. Abbreviations: Ap, ampicillin resistance marker; Kn, kanamycin resistance marker; Ori, origin of replication from pUC18; Rep, origin of replication from pHK; Tc, tetracycline resistance marker. The double line indicates H. influenzae chromosomal DNA, and the single line represents plasmid DNA. All other plasmids designated with the suffix HK (Table 1) were constructed by the same strategy, using deletions of the pTGL43 plasmid that retained the same unique HindIll site found in pTGL81. was removed and thoroughly chilled, the cells were recovered by centrifugation at 5,000 rpm in an SS-34 rotor (Sorvall) at 0°C for 10 min. The cell pellet was suspended immediately in 1 ml of ice-cold physiological saline and added to 9 ml of a phenol-buffer solution that had been preheated to 80°C and shaken vigorously. The phenol-buffer solution was prepared by mixing 5 ml of phenol saturated with LET buffer (100 mM LiCl, 10 mM EDTA, 10 mM Tris [pH 7.8]; adapted from reference 26) with 4 ml of LET buffer containing 10 mM sodium azide and 10 mM 2-mercaptoethanol. The mixture of cells and buffer was shaken vigorously, and the tubes were incubated at 80°C for 30 min with occasional shaking. When the 80°C incubation was complete, each tube was cooled to room temperature and 5 ml of chloroform-isoamyl alcohol (24:1) was added. The tubes were shaken vigorously for 5 min and then incubated for 10 min in an ice bath. The resulting emulsion was separated by centrifugation, and the aqueous phase was phenol-chloroform extracted at room temperature two additional times. The nucleic acids were precipitated by adding LiCl to a final concentration of 250 mM and then 1 volume of 2-propanol. The resulting pellets
4686
J. BACTERIOL.
LARSON AND GOODGAL
suspended in diethylpyrocarbonate-treated H20, and the RNA was selectively precipitated by adding sodium acetate to a final concentration of 3 M and incubating the mixture at -20°C overnight (10). The resulting precipitate was collected by centrifugation. The crude RNA was precipitated twice with ethanol and resuspended in diethylpyrocarbonate-treated H20 (15). The concentration of RNA was estimated photometrically, and the samples were stored at -200C. The crude RNA samples were prepared for hybridization analysis by treating them with DNAse (10). The samples were phenol-chloroform extracted two times and chloroform extracted once, and the RNA was precipitated twice with ethanol. Equivalent amounts of RNA from each time point were denatured by incubation in formamide-formaldehyde buffer at 50°C for 30 min and separated on an agaroseformaldehyde gel using MOPS (morpholine propanesulfonic acid) buffer (30). The RNA was transferred to a positively charged nylon membrane (GeneScreen Plus; Du Pont) by capillary blotting, and Northern (RNA) hybridization was carried out under the stringent conditions recommended by the manufacturer of the membrane. The probe was prepared by nick translation (15) of gel-purified pTGL115 insert and had a specific activity of approximately 5 x 107 cpm/,ug. Translation of the ORF2 gene product. The 1-kb fragment containing ORF2 was ligated into the pTZ18U and pTZ19U vectors so that it could be transcribed by the T7 promoter carried on these plasmids. The insert was removed from plasmid pTGL180 by digestion with EcoRI and HindlIl, purified from a low-melting-point agarose gel, and ligated into EcoRI-HindlIl digests of pTZ18U and pTZ19U. The ligated DNAs were transformed into DH5aMCR. The desired plasmids were recovered and designated pTGL200 (pTZ18U vector) and pTGL201 (pTZ19U vector); in pTGL201, the fragment is in the proper orientation for the ORF2 sequence to be transcribed by the T7 promoter, and in pTGL200, the insert is in the opposite orientation relative to the T7 promoter. The plasmids were transformed into HMS194(DE3), HMS194(DE3) containing pLysS, and HMS194(DE3) containing pLysE. Cultures were grown and samples were prepared as recommended by Studier et al. (34). Samples were separated by using discontinuous sodium dodecyl sulfate-polyacrylamide gel electrophoresis (12), and the protein was visualized by silver staining (6). Nucleotide sequence accession number. The complete sequence of the 2.8-kb EcoRI-BglII insert of pTGL43 has been submitted to GenBank and has received the accession number M59751. were
RESULTS Cloning and activity of a mutant allele of a gene that rescues Com-78 and Com-101 phenotypes. To determine the mechanism by which the gene present on the 7.8-kb EcoRI-MluI fragment cloned in pTGL6 increases the transformation activities of the Com-78 and Com-101 strains, the corresponding mutant locus was cloned from the Com-101 chromosome. An EcoRI digest of the Com- 101 chromosome was size fractionated to obtain fragments of approximately the same size as the wild-type 13.3-kb EcoRI chromosomal fragment cloned in the pER194 plasmid. The purified DNA was further digested with MluI, ligated to the pHK vector, and transformed into the H. influenzae Rec-1 strain. Of 200 kanamycin-resistant, tetracycline-sensitive isolates, 1 contained plasmid DNA that hybridized to the purified pER194 insert. Plasmid prepared from this isolate contained an insert
TABLE 2. Effects of pTGL6 and pTGL6-101 on transformability of Com-78 and Com-101 hostsa % of wild-type (Rd1967)
Fold increase over
transformation frequency
mutant frequency rqec
Plasmid
pTGL6 pTGL6-101
mtn
Com-78
Com-101
Com-78
Com-101
75 0.55
38 0.13
180 1.3
475 1.6
a Transformation of erythromycin resistance was performed by using the aerobic-anaerobic method.
of approximately the same size as the one in pTGL6 and was designated pTGL6-101. Restriction analysis demonstrated that the pTGL6 and pTGL6-101 plasmids are virtually identical except that the 2.8-kb EcoRI-BglII fragment of pTGL6 measures 3.1 kb in pTGL6-101 because of the presence of the 0.3-kb insert previously observed in the Com-101 chromosome (13). The pTGL6-101 plasmid was found not to increase the chromosomal transformation frequency of the Com-78 or Com-101 strain (Table 2). Complementation activity of the 2.8-kb EcoRI-Bglll fragment and its deletions. To facilitate further analysis of the 2.8-kb EcoRI-BglII fragment, it was subcloned into E. coli by inserting it into pUC18 to give pTGL43. The biological activity of the subcloned fragment was verified by reintroducing it into the Com- mutants. For this purpose, a shuttle plasmid, pTGL81HK (Fig. 1), was constructed by using E. coli DH5oMCR as a host; attempts to use other hosts for this construction failed, possibly because of MCR incompatibility. The pTGL81HK plasmid was then introduced into the H. influenzae Rec-1 strain and tested for the ability to increase the transformability of Com-78 and Com-101 hosts. The plasmid was found to increase the transformation frequency of both strains to a value exceeding that of the wild-type parental strain (Table 3). A set of plasmids containing nested deletions of the pTGL43 insert were prepared next and reintroduced into H. influenzae by using the strategy described above to determine how small a fragment could be and still bring about complementation. Deleting up to 1.08 kb from the BglIl site did not interfere with complementation, while deleting more than 1.28 kb from the BglII site abolished complementation (Fig. 2, Table 3). The smallest complementing derivative from the set of BglII end deletions (pTGL43D4N3) was then used to generate a set of deletions from the EcoRI end of the fragment. All of the derivatives prepared by deletions from the EcoRI site retained the ability to increase the transformTABLE 3. Effects of pTGL43 derivatives on transformability of Com-78 and Com-101 hostsa Plasmid
None
pTGL81 HK pTGL43 D4N3 HK pTGL153HK pTGL180HK
% of wild-type (Rdl967) transformation frequency Com-101 Com-78
0.6 300 100 0.06 40
0.01 300 100 0.006 60
a Transformation to erythromycin resistance was performed by using the aerobic-anaerobic method. See Fig. 2 for a map of the deletions and their relationship to ORF2.
VOL. 173, 1991
SEQUENCE AND TRANSCRIPTION OF comlOlA
ORFI 1113bp
B
H
ORF2 687bp
D D
S
Il
ability of the two mutants (Fig. 2, Table 3), indicating that the central 1 kb of the 2.8-kb EcoRI-BglII fragment is essential for complementation of the mutants. Sequence analysis of the wild-type EcoRI-BglII fragment. Starting from the BglII site, 100% of the 5'-strand and 80% of the 3'-strand sequence was determined for the 2.8-kb EcoRIBglII fragment by using a combination of subclones and nested deletions. The entire sequence of the smallest region found to complement the com mutations was determined in both directions (Fig. 3). Translation of the sequence for the 2.8-kb fragment starting from the BglII site reveals three contiguous open reading frames of 1,113, 687, and 594 bp (Fig. 2). ORF2 and ORF3 are in the same reading frame, while ORFi is in a different frame. The three complete open reading frames are followed by the beginning of a possible fourth open reading frame that has been truncated at the EcoRI site. The second, third, and possible fourth open reading frames all are preceded by apparent ribosomebinding sites, but the first open reading frame is not. An additiornal open reading frame of 201 bp is embedded in the ORFi sequence but in a different reading frame from ORF1, ORF2, and ORF3. This open reading frame is nrot preceded by an apparent ribosome-binding site. No other open reading frames of more than 200 bp were found in the sequence when translated from the BglII site. Only one possible open reading frame of more than 200 bp was found when the complementary strand was translated. This 387-bp open reading frame also is embedded in the ORFi sequence. It
ORF3 594bp
S,I D D
Dupicatd i Conm-1O1
ORF I R 387bp
81_ 43D2N1 3
43D2N1 5 43D3N2 43D4N3
153 136
4304N3 D3Ni 43D4N3 D1 2N1 43D4H3 D1 2N3 180
FIG. 2. Deletion analysis of complementation by the pTGL43 insert. Restriction site abbreviations: B, BglII; D, DraI; E, EcoRI; H, Hindlll; S, SspI. The bar below the map indicates the region duplicated in the Com- 101 chromosome. The probable open reading frames as determined by sequence analysis are indicated by arrows. The numbers to the left of the diagram refer to the TGL-series plasmid containing the particular deletion (Table 1). The chromosomal transformation frequencies of the Com-78 and Com-101 mutants containing selected plasmids are given in Table 3. Symbols: II1111, complements both Com-78 and Com-101; LI, no complementation.
40
50
I It t I t A-COT CTATAMAA0cA
I
20
10 1
COCC
30
I
201
1
I
s0
70
60
t
t1 I
I
)))>>> 151
4687
tI t
90 It
. .0
TTTTTTTAAT O-CC (
Vol. 173, No. 15
Sequence and Transcriptional Regulation of comlOlA, a Locus Required for Genetic Transformation in Haemophilus influenzae THOMAS G. LARSON't AND SOL H. GOODGAL2* Graduate Group in Biochemistry' and Department of Microbiology,2 University of Pennsylvania School of Medicine, Philadelphia, Pennsylvania 19104 Received 11 December 1990/Accepted 21 May 1991
A 2.8-kb EcoRI-BgM fragment cloned from the wild-type Haemophilus influenzae Rd chromosome is shown to increase the transformability of the Com-101 mutant through trans complementation. Deletion and sequence
analyses indicate that the active region of the clone carries a 687-bp open reading frame. A 0.3-kb insertion in the corresponding EcoRI-Bgll fragment of the ComA101 chromosome is shown to be a partial (331-bp) duplication of this open reading frame. The wild-type sequence produces a peptide of a size that is consistent with the sequence data when this sequence is expressed in Escherichia coli with a T7 promoter-based transcription vector. RNA hybridization analysis using a DNA probe derived from the open reading frame suggests that the sequence is transiently expressed during competence development. On the basis of these observations, it is proposed that the open reading frame corresponds to the comlOlA gene.
Competence development in most known examples of naturally transforming bacteria involves the regulated expression of specific genes. The process generally involves global changes in the organism's metabolism that are coordinated with the specific expression of the functions required for genetic competence. The actual regulatory events are poorly understood in most organisms; however, some significant details of competence regulation in gram-positive Bacillus subtilis (see reference 8 for a review) have recently emerged. The com (competence) genes of B. subtilis have been grouped into two classes: the "early" genes and the "late" genes (2). The early genes are transcribed constitutively (2), although some of them may be upregulated preceding the onset of competence (8), while the late genes are expressed only in competence-inducing medium (1, 8, 19). Additionally, expression of the early genes is required for expression of the late genes (1, 8, 19) as well as for expression of other genes that do not appear to be related to competence (21, 40). Competence in gram-negative Haemophilus influenzae also appears to be regulated (9) by genetically encoded functions (7) that might belong to classes similar to those found in B. subtilis. The accompanying report describes the cloning of linked loci capable of increasing the transformation frequency of Com- phenotypes in H. influenzae (13). This report focuses on one of these loci, comlOlA, in greater detail. The wild-type clone of the comlOlA locus is shown to increase the transformability of the Com-101 mutant through trans complementation, and the comlOlA open reading frame is defined through sequence and deletion analysis. Finally, transcription of the comlOlA locus is shown to be transient: like the late class of competence genes in B. subtilis, the H. influenzae comlOlA gene is expressed after competence induction, supporting the assertion that the comlOlA locus encodes a competence-specific product.
MATERIALS AND METHODS Bacterial strains and plasmids. The origins and phenotypes of H. influenzae Rd1967, Com-78, Com-101, and Rec-1 are described in the accompanying paper (13). The E. coli lysogen HMS174(DE3) was a gift of W. Studier (33). E. coli DH5aMCR was obtained from Bethesda Research Laboratories. The plasmids used in this study are presented in Table 1. The construction and properties of the plasmids pER194 and pTGL6 are described in the accompanying paper (13). The vector pHK was a gift of G. Barcak (3). Plasmids pTZ18U and pTZ19U (17) were obtained from United States Biochemicals. Plasmids pUC18 (41) was obtained from Bethesda Research Laboratories. Plasmids pLysS and pLysE were gifts of W. Studier (34). All other plasmids were constructed in this study and will be described below. Bacterial growth and competence development. H. influenzae cultures were grown and preserved as described elsewhere (13). Chromosomal transformations were performed by using cultures made competent by the MIV (9) or aerobic-anaerobic procedure as described in the accompanying paper (13). Plasmid transformations were performed by using the saline-glycerol procedure described by Stuy and Walter (35) with the modifications described in the accompanying paper (13). Plasmid and chromosomal markers were selected as before. E. coli strains were generally grown in Luria-Bertani (LB) broth, plated on LB agar (15), and preserved by freezing in LB broth plus 10% glycerol at -70°C. Cultures used to prepare plasmid or competent cells were grown in TB medium (37). E. coli cultures were made competent by using the CaCl2 technique (15). 4-Methylumbelliferyl-f3-D-galactoside (MUG) was used for the detection of Lac- transformants (42). Transformed cultures were spread on LB agar containing 100 ,ug of MUG and 100 pLg of ampicillin per ml. After 14 to 16 h at 37°C, Lac' colonies were easily distinguishable from Lac- colonies when illuminated with longwave UV light because of the fluorescence of the hydrolyzed MUG. The use of isopropyl-,-D-thiogalactopyranoside was not necessary when MUG was used to test for insertions in plasmids pUC18, pTZ18U, and pTZ19U. Enzymes. Restriction enzymes, calf intestine alkaline
* Corresponding author. t Present address: Roche Institute of Molecular Biology, Nutley, NJ 07110-1199.
4683
4684
J. BACTERIOL.
LARSON AND GOODGAL TABLE 1. Plasmids used in this study
or DsrpinSource reference escnption
Plasmid
designation
pER194
pJl-8fQ(EcoRI::H. influenzae Rd 13.3kb)
pHK pLysE pLysS pTGL6 pTGL6-101 pTGL43
Kmr Tcr Cmr Cmr pHKfQ(EcoRI-MluI::pER194 7.8kb) pHKfQ(EcoRI-MluI::H. influenzae Rd Com-101 3.lkb) pUC18fQ(EcoRI-BglII/BamHI::pTGL6 2.8kb) pTGL43AO.4kb from BglII-BamHI site pTGL43AO.6kb from BglII-BamHI site pTGL43AO.8kb from BglII-BamHI site pTGL43A1.O8kb from BglII-BamHI site pTGL43D4N3AO.2kb from EcoRI site pTGL43D4N3AO.4kb from EcoRI site pTGL43D4N3AO.6kb from EcoRI site pTGL43D2N13fQ(HindIII::pHK 6.7kb)
pTGL43D2N13 pTGL43D2N15 pTGL43D3N2 pTGL43D4N3 pTGL43D4N3:D3Nl pTGL43D4N3:D12Nl pTGL43D4N3:D12N3 pTGL43D2N13HK pTGL43D2Nl5HK pTGL43D3N2HK pTGL43D4N3HK pTGL43D4N3:D3NlHK pTGL43D4N3:D12NlHK pTGL43D4N3:D12N3HK pTGL54 pTGL81 pTGL81HK pTGL115 pTGL136 pTGL136HK pTGL153 pTGL153HK pTGL180 pTGL180HK pTGL200 pTGL201 pTZ18U pTZ19U pUC18
pTGL43D2Nl5fQ(HindIII::pHK 6.7kb) pTGL43D3N2fl(HindIII::pHK 6.7kb) pTGL43D4N3Qk(HindIII::pHK 6.7kb) pTGL43D4N3:D3NlQf(HindIII::pHK 6.7kb) pTGL43D4N3:D12NlQf(HindIII::pHK 6.7kb) pTGL43D4N3:D12N3fQ(HindIII::pHK 6.7kb) pUC18fQ(EcoRI-BglII/BamHI::pTGL6-101 3.lkb) pTGL43AHindIII::0.2kb pTGL81Q(HindIII: :pHK 6.7kb) pUC18fQ(HincII/DraI::pTGL43 0.28kb) pUC18fQ(EcoRI-HincII/EcoRI-DraI::pTGL43 1.lkb) pTGL136fQ(HindIII::pHK 6.7kb) pUC18fQ(EcoRI-HincIIIEcoRI-SspI::pTGL43 1.5kb) pTGL153fl(HindIII: :pHK 6.7kb) pTGL43D4N3AO.8kb from EcoRI site pTGL180Qf(HindIII::pHK 6.7kb) pTZ18Ql(EcoRI-HindIII::pTGL43D4N3:D12N1O lkb) pTZ19fQ(EcoRI-HindIII: :pTGL43D4N3:D12N1O lkb) Apr, bacteriophage T7 promoter Apr, bacteriophage T7 promoter Apr
phosphatase, T4 DNA ligase, Klenow fragment of E. coli DNA polymerase I, and E. coli DNA polymerase I were obtained from the sources previously identified (13) and used as described elsewhere (13). Bal 31 exonuclease was obtained from Bethesda Research Laboratories, and RNAsefree DNAse was obtained from Worthington; both enzymes were used as described below. DNA preparation. H. influenzae chromosomal DNA was prepared for genetic transformations by using Marmur's method (16). Preparation of H. influenzae chromosomal DNA in agarose beads and its subsequent digestion with restriction enzymes has been described elsewhere (11). Plasmid DNA preparations were purified by using the alkaline lysis procedure (5, 15) followed by precipitation with polyethylene glycol (14). Small-scale plasmid preparations used for restriction analysis were made by using the abbreviated alkaline-lysis minipreparation procedure described by Morelle (20); for H. influenzae plasmid preparations, the previously described modifications were incorporated (13). Purification of the 13.3-kb insert from EcoRI-digested pER194 by using agarose gel electrophoresis and electroelution has been described elsewhere (13), and the EcoRI-BglII subfragments of pTGL6 and pTGL6-101 were prepared by using the same procedure. Restriction fragments from other plasmids were prepared by separating digests on 2% lowmelting-point agarose minigels (FMC GTG Low-Melting Agarose) and excising the desired bands. The DNA was
13 3 33 33 This This This This This This This This This This This This This This This This This This This This This This This This This This This This This 17 17 41
study study study study study study study study study study
study study study study study study study study study study study study study study study study study study study
recovered from the agarose by hot-phenol extraction at 65°C (modified from the procedure described in reference 31) followed by precipitation with 2-propanol and ammonium acetate (15). Cloning of a mutant copy of the pTGL6 insert. An EcoRI digest of Com-101 chromosomal DNA prepared from cells embedded in agarose beads was separated on a 1% agarose gel. DNA comigrating with the 13.3-kb EcoRI fragment of pER194 was purified from the gel by electroelution. A 1-,ug sample of the recovered DNA was subsequently digested with MluI and ligated into 1 pug of MluI-EcoRI-digested pHK vector in 100 ,ul of reaction mixture. The ligation reaction mixture was concentrated by ethanol precipitation, and the DNA was transformed into the Rec-1 strain of H. influenzae. Colonies that were kanamycin resistant (to 10 ,ug/ml) but tetracycline sensitive (to 5 ,ug/ml) were grown to stationary phase in 1 ml of supplemented brain heart infusion containing kanamycin at 37°C without shaking. A 10-,ul volume from each culture was lysed by adding it to 100 pl of 0.4 N NaOH-10 mM EDTA and heating the mixture to 40°C for 20 min. The samples were then applied to a nylon membrane (Hybond-N; Amersham) by using a slot-blotting apparatus. The membrane was hybridized overnight at 65°C and washed according to the instructions of the manufacturer. The probe was prepared from the pER194 insert and labeled as described elsewhere (13). Subcloning of EcoRI-BglIl fragments from pTGL6 and
VOL. 173, 1991
pTGL6-101 into pUC18. Plasmids pTGL6 and pTGL6-101 were digested with EcoRI and BgIII. The desired fragments were gel purified, ligated to EcoRI-BamHI-digested pUC18 vector, and transformed into DH5aMCR. Plasmid DNA was prepared from several Lac- colonies and tested for the presence of the desired insert by restriction mapping. Construction of subclones and deletions. Subclones of pTGL43 and pTGL54 were constructed by isolating DraI and SspI restriction fragments and ligating them to calf intestine alkaline phosphatase-treated HinclI digests of pUC18. Nested, unidirectional deletions of the pTGL43, pTGL43D4N3, and pTGL136 plasmid inserts were generated by using Bal31 exonuclease (22, 27, 28). The ligated DNAs from the subclones and deletion reactions were transformed into E. coli DH5ScMCR as described above. Plasmids from Lac- colonies were evaluated for the desired inserts by restriction analysis. Assay for increase in transformability of Com-78 and Com-101 mutants. The plasmids to be tested were first isolated and grown in the H. influenzae Rec-1 strain so that they could be amplified under conditions that do not permit recombination. The amplified plasmids were then transferred to Com-78 and Com-101 hosts, and the chromosomal transformation frequencies of these strains were evaluated as described in the accompanying paper (13). E. coli plasmids to be tested for the ability to complement the com mutations were digested with HindIII and ligated into a calf intestine alkaline phosphatase-treated HindIll digest of the pHK vector that had been grown in E. coli DH5otMCR. Figure 1 details the construction of one such plasmid, pTGL81HK. The ligated DNA was transformed into DH5aMCR, and the desired constructions were verified by restriction analysis. The appropriate plasmids were transformed into the H. influenzae Rec-1 strain and tested for their abilities to increase the transformability of the Com-78 and Com-101 strains as described above. DNA sequencing. The entire pTGL43 insert and the relevant portion of the pTGL54 insert were sequenced by using the dideoxy chain termination reaction method (29) with bacteriophage T7 DNA polymerase (36). Sequencing reactions with double-stranded DNA were performed by using the Sequenase kit (U.S. Biochemicals) as directed by the manufacturer, with minor modifications (39). The supplied universal primer was used for the forward reactions, and the New England BioLabs -48 reverse M13 sequencing primer (catalog no. 1233) was used for the reverse reactions. The reactions were separated by using the buffer-gradient gel procedure described by Biggins et al. (4). When this system was used, 300 to 400 nucleotides could be read from each template. When required, more nucleotides were read by using a standard 4% gel run under the conditions described by Parkison and Cheng (25). Reaction mixtures for these gels were prepared by using the extended sequencing conditions recommended in the Sequenase manual. Computer analysis of the sequence data was performed by using the PC Gene and IG Suite software packages (both from Intelligenetics). Transcriptional analysis of ORF2. A 200-ml culture of Rdl967 was grown in freshly prepared supplemented brain heart infusion with shaking at 36°C. At an optical density at 650 nm of 0.16, the culture was chilled in an ice bath, harvested, and suspended in an equal volume of freshly prepared MIV medium (9). After the culture was harvested and suspended a second time, 50 ml was set aside in an ice bath and the remainder was shaken at 36°C. Fifty-milliliter volumes were removed from the culture after 50, 100, and 150 min and transferred to an ice bath. After each sample
SEQUENCE AND TRANSCRIPTION OF comlOlA
4685
BamH I/BgI 11 Hind ill B.I
Hind
pTGL43 5.5 kb EcoRI
Hindill
pTGL81 5.2kb
+
Hi
H
EcoRI
Il
FIG. 1. Construction of pTGL81HK from pTGL43 and pHK. Abbreviations: Ap, ampicillin resistance marker; Kn, kanamycin resistance marker; Ori, origin of replication from pUC18; Rep, origin of replication from pHK; Tc, tetracycline resistance marker. The double line indicates H. influenzae chromosomal DNA, and the single line represents plasmid DNA. All other plasmids designated with the suffix HK (Table 1) were constructed by the same strategy, using deletions of the pTGL43 plasmid that retained the same unique HindIll site found in pTGL81. was removed and thoroughly chilled, the cells were recovered by centrifugation at 5,000 rpm in an SS-34 rotor (Sorvall) at 0°C for 10 min. The cell pellet was suspended immediately in 1 ml of ice-cold physiological saline and added to 9 ml of a phenol-buffer solution that had been preheated to 80°C and shaken vigorously. The phenol-buffer solution was prepared by mixing 5 ml of phenol saturated with LET buffer (100 mM LiCl, 10 mM EDTA, 10 mM Tris [pH 7.8]; adapted from reference 26) with 4 ml of LET buffer containing 10 mM sodium azide and 10 mM 2-mercaptoethanol. The mixture of cells and buffer was shaken vigorously, and the tubes were incubated at 80°C for 30 min with occasional shaking. When the 80°C incubation was complete, each tube was cooled to room temperature and 5 ml of chloroform-isoamyl alcohol (24:1) was added. The tubes were shaken vigorously for 5 min and then incubated for 10 min in an ice bath. The resulting emulsion was separated by centrifugation, and the aqueous phase was phenol-chloroform extracted at room temperature two additional times. The nucleic acids were precipitated by adding LiCl to a final concentration of 250 mM and then 1 volume of 2-propanol. The resulting pellets
4686
J. BACTERIOL.
LARSON AND GOODGAL
suspended in diethylpyrocarbonate-treated H20, and the RNA was selectively precipitated by adding sodium acetate to a final concentration of 3 M and incubating the mixture at -20°C overnight (10). The resulting precipitate was collected by centrifugation. The crude RNA was precipitated twice with ethanol and resuspended in diethylpyrocarbonate-treated H20 (15). The concentration of RNA was estimated photometrically, and the samples were stored at -200C. The crude RNA samples were prepared for hybridization analysis by treating them with DNAse (10). The samples were phenol-chloroform extracted two times and chloroform extracted once, and the RNA was precipitated twice with ethanol. Equivalent amounts of RNA from each time point were denatured by incubation in formamide-formaldehyde buffer at 50°C for 30 min and separated on an agaroseformaldehyde gel using MOPS (morpholine propanesulfonic acid) buffer (30). The RNA was transferred to a positively charged nylon membrane (GeneScreen Plus; Du Pont) by capillary blotting, and Northern (RNA) hybridization was carried out under the stringent conditions recommended by the manufacturer of the membrane. The probe was prepared by nick translation (15) of gel-purified pTGL115 insert and had a specific activity of approximately 5 x 107 cpm/,ug. Translation of the ORF2 gene product. The 1-kb fragment containing ORF2 was ligated into the pTZ18U and pTZ19U vectors so that it could be transcribed by the T7 promoter carried on these plasmids. The insert was removed from plasmid pTGL180 by digestion with EcoRI and HindlIl, purified from a low-melting-point agarose gel, and ligated into EcoRI-HindlIl digests of pTZ18U and pTZ19U. The ligated DNAs were transformed into DH5aMCR. The desired plasmids were recovered and designated pTGL200 (pTZ18U vector) and pTGL201 (pTZ19U vector); in pTGL201, the fragment is in the proper orientation for the ORF2 sequence to be transcribed by the T7 promoter, and in pTGL200, the insert is in the opposite orientation relative to the T7 promoter. The plasmids were transformed into HMS194(DE3), HMS194(DE3) containing pLysS, and HMS194(DE3) containing pLysE. Cultures were grown and samples were prepared as recommended by Studier et al. (34). Samples were separated by using discontinuous sodium dodecyl sulfate-polyacrylamide gel electrophoresis (12), and the protein was visualized by silver staining (6). Nucleotide sequence accession number. The complete sequence of the 2.8-kb EcoRI-BglII insert of pTGL43 has been submitted to GenBank and has received the accession number M59751. were
RESULTS Cloning and activity of a mutant allele of a gene that rescues Com-78 and Com-101 phenotypes. To determine the mechanism by which the gene present on the 7.8-kb EcoRI-MluI fragment cloned in pTGL6 increases the transformation activities of the Com-78 and Com-101 strains, the corresponding mutant locus was cloned from the Com-101 chromosome. An EcoRI digest of the Com- 101 chromosome was size fractionated to obtain fragments of approximately the same size as the wild-type 13.3-kb EcoRI chromosomal fragment cloned in the pER194 plasmid. The purified DNA was further digested with MluI, ligated to the pHK vector, and transformed into the H. influenzae Rec-1 strain. Of 200 kanamycin-resistant, tetracycline-sensitive isolates, 1 contained plasmid DNA that hybridized to the purified pER194 insert. Plasmid prepared from this isolate contained an insert
TABLE 2. Effects of pTGL6 and pTGL6-101 on transformability of Com-78 and Com-101 hostsa % of wild-type (Rd1967)
Fold increase over
transformation frequency
mutant frequency rqec
Plasmid
pTGL6 pTGL6-101
mtn
Com-78
Com-101
Com-78
Com-101
75 0.55
38 0.13
180 1.3
475 1.6
a Transformation of erythromycin resistance was performed by using the aerobic-anaerobic method.
of approximately the same size as the one in pTGL6 and was designated pTGL6-101. Restriction analysis demonstrated that the pTGL6 and pTGL6-101 plasmids are virtually identical except that the 2.8-kb EcoRI-BglII fragment of pTGL6 measures 3.1 kb in pTGL6-101 because of the presence of the 0.3-kb insert previously observed in the Com-101 chromosome (13). The pTGL6-101 plasmid was found not to increase the chromosomal transformation frequency of the Com-78 or Com-101 strain (Table 2). Complementation activity of the 2.8-kb EcoRI-Bglll fragment and its deletions. To facilitate further analysis of the 2.8-kb EcoRI-BglII fragment, it was subcloned into E. coli by inserting it into pUC18 to give pTGL43. The biological activity of the subcloned fragment was verified by reintroducing it into the Com- mutants. For this purpose, a shuttle plasmid, pTGL81HK (Fig. 1), was constructed by using E. coli DH5oMCR as a host; attempts to use other hosts for this construction failed, possibly because of MCR incompatibility. The pTGL81HK plasmid was then introduced into the H. influenzae Rec-1 strain and tested for the ability to increase the transformability of Com-78 and Com-101 hosts. The plasmid was found to increase the transformation frequency of both strains to a value exceeding that of the wild-type parental strain (Table 3). A set of plasmids containing nested deletions of the pTGL43 insert were prepared next and reintroduced into H. influenzae by using the strategy described above to determine how small a fragment could be and still bring about complementation. Deleting up to 1.08 kb from the BglIl site did not interfere with complementation, while deleting more than 1.28 kb from the BglII site abolished complementation (Fig. 2, Table 3). The smallest complementing derivative from the set of BglII end deletions (pTGL43D4N3) was then used to generate a set of deletions from the EcoRI end of the fragment. All of the derivatives prepared by deletions from the EcoRI site retained the ability to increase the transformTABLE 3. Effects of pTGL43 derivatives on transformability of Com-78 and Com-101 hostsa Plasmid
None
pTGL81 HK pTGL43 D4N3 HK pTGL153HK pTGL180HK
% of wild-type (Rdl967) transformation frequency Com-101 Com-78
0.6 300 100 0.06 40
0.01 300 100 0.006 60
a Transformation to erythromycin resistance was performed by using the aerobic-anaerobic method. See Fig. 2 for a map of the deletions and their relationship to ORF2.
VOL. 173, 1991
SEQUENCE AND TRANSCRIPTION OF comlOlA
ORFI 1113bp
B
H
ORF2 687bp
D D
S
Il
ability of the two mutants (Fig. 2, Table 3), indicating that the central 1 kb of the 2.8-kb EcoRI-BglII fragment is essential for complementation of the mutants. Sequence analysis of the wild-type EcoRI-BglII fragment. Starting from the BglII site, 100% of the 5'-strand and 80% of the 3'-strand sequence was determined for the 2.8-kb EcoRIBglII fragment by using a combination of subclones and nested deletions. The entire sequence of the smallest region found to complement the com mutations was determined in both directions (Fig. 3). Translation of the sequence for the 2.8-kb fragment starting from the BglII site reveals three contiguous open reading frames of 1,113, 687, and 594 bp (Fig. 2). ORF2 and ORF3 are in the same reading frame, while ORFi is in a different frame. The three complete open reading frames are followed by the beginning of a possible fourth open reading frame that has been truncated at the EcoRI site. The second, third, and possible fourth open reading frames all are preceded by apparent ribosomebinding sites, but the first open reading frame is not. An additiornal open reading frame of 201 bp is embedded in the ORFi sequence but in a different reading frame from ORF1, ORF2, and ORF3. This open reading frame is nrot preceded by an apparent ribosome-binding site. No other open reading frames of more than 200 bp were found in the sequence when translated from the BglII site. Only one possible open reading frame of more than 200 bp was found when the complementary strand was translated. This 387-bp open reading frame also is embedded in the ORFi sequence. It
ORF3 594bp
S,I D D
Dupicatd i Conm-1O1
ORF I R 387bp
81_ 43D2N1 3
43D2N1 5 43D3N2 43D4N3
153 136
4304N3 D3Ni 43D4N3 D1 2N1 43D4H3 D1 2N3 180
FIG. 2. Deletion analysis of complementation by the pTGL43 insert. Restriction site abbreviations: B, BglII; D, DraI; E, EcoRI; H, Hindlll; S, SspI. The bar below the map indicates the region duplicated in the Com- 101 chromosome. The probable open reading frames as determined by sequence analysis are indicated by arrows. The numbers to the left of the diagram refer to the TGL-series plasmid containing the particular deletion (Table 1). The chromosomal transformation frequencies of the Com-78 and Com-101 mutants containing selected plasmids are given in Table 3. Symbols: II1111, complements both Com-78 and Com-101; LI, no complementation.
40
50
I It t I t A-COT CTATAMAA0cA
I
20
10 1
COCC
30
I
201
1
I
s0
70
60
t
t1 I
I
)))>>> 151
4687
tI t
90 It
. .0
TTTTTTTAAT O-CC (
