Vol. 172, No. 5
JOURNAL OF BACTERIOLOGY, May 1990, p. 2343-2350
0021-9193/90/052343-08$02.00/0 Copyright © 1990, American Society for Microbiology
Secretion and Expression of the Pasteurella haemolytica Leukotoxin SARAH K. HIGHLANDER,* MICHAEL J. ENGLER, AND GEORGE M. WEINSTOCK Department of Biochemistry and Molecular Biology, The University of Texas Medical School, 6431 Fannin, Houston, Texas 77030 Received 15 September 1989/Accepted 22 January 1990
The PasteureUa haemolytica leukotoxin gene cluster (iktCABD) is homologous to the Escherichia coli hemolysin locus (hlyCABD). Since the cloned leukotoxin (LktA) is not secreted from E. coli cells, a heteroplasmid complementation system was developed that permits secretion of the leukotoxin from cells expressing the hemolysin transport proteins ElyB and HlyD. We observed that the secreted leukotoxin protein had weak hemolytic activity when activated by either the HlyC or LktC proteins and that LktC expressed in E. coli could confer weak hemolytic activity upon hemolysin. Thus, it appears that the accessory proteins of the leukotoxin and hemolysin gene clusters are functionally similar, although their expression in E. coli is not equivalent. Northern (RNA) blot analysis of the P. haemolytica leukotoxin gene cluster revealed a major 3.5-kilobase transcript that includes the lktC and lktA genes. The start site for this transcript mapped to a cytosine residue 30 nucleotides upstream from the putative start of IktC; a similar initiation site was observed in E. coli, although adjacent cytosine and adenine residues were also utilized. The 3.5-kilobase transcript terminated near the rho-independent terminator structure between iktA and lktB, but transcription may continue, via antitermination or de novo transcription initiation, into the downstream lktB and lktD genes. We propose that the lack of LktB and LktD function in E. coli is a result, at least in part, of poor lktBD transcription and suggest that a P. haemolytica-specific regulator is required for optimal expression of the leukotoxin genes.
In a previous study, we found that the LktA protein was produced in an E. coli host but was not secreted by LktB and LktD, yet the leukotoxin could be secreted when a complete hemolysin locus was present in the same cell (10). Here, we extend these observations by examining the function of cognate secretion and activation proteins on the leukotoxin in E. coli. The HlyC and HlyBD proteins were able to complement hemolytic activity and secretion of the leukotoxin, respectively, and the LktC protein weakly activated HlyA hemolysis. In contrast, the LktB and LktD proteins were not active and their genes were poorly expressed in E. coli. Transcriptional mapping of the leukotoxin gene cluster indicated that transcription of lktB and lktD is weak in P. haemolytica; an lktBD message was undetectable in E. coli. In contrast, the lktC and lktA genes were found to be contained within a single transcriptional unit whose promoter is weakly recognized in E. coli. These observations suggest that while the hemolysin and leukotoxin gene clusters encode proteins with similar functions, expression of the leukotoxin gene cluster may require a positive regulator not present in E. coli.
Pasteurella haemolytica secretes a 102-kilodalton leukotoxin that is believed to be involved in the pathogenesis of a severe bovine pneumonia, commonly called shipping fever (2, 23). This disease, which develops following stress to the animal, is a subject for vaccine development studies (6, 7). The leukotoxin is ruminant specific and acts to compromise the primary immune response of the bovine lung by lysing macrophages and other leukocytes (2, 18, 23). Since P. haemolytica is normally a nonpathogenic commensal bacterium of cattle, it is possible that leukotoxin expression is responsible for development of the disease state. A four-gene cluster which encodes the leukotoxin (LktA) and proteins required for its activation (LktC) and secretion (LktB, LktD) has been cloned in Escherichia coli (4, 10, 16, 25), and their nucleotide sequences were determined (10, 17, 25). DNA sequence comparisons revealed that the leukotoxin gene cluster is homologous to the hemolysin determinant of E. coli (10, 17, 24, 25) and has genes analogous to the hlyC, hlyA, hlyB, and hlyD genes for hemolysin. The HlyC protein is required to activate the hemolytic phenotype of HlyA (21), while HlyB and HlyD are required to secrete the HlyA protein from the cell (27). Similarly, LktC may be required for the leukotoxic activity of LktA (17), and it is presumed, by analogy, that the LktB and LktD proteins are responsible for secreting LktA from P. haemolytica cells. The leukotoxin determinant is similar in sequence to toxin genes from Morganella morganii, Proteus vulgaris, Proteus mirabilis (15, 28), and Actinobacillus actinomycetemcomitans (13) and to a portion of the Bordetella pertussis adenylate cyclase toxin (8). Each of these toxins requires accessory proteins for secretion and possesses a series of glycine-rich amino acid repeats within the toxin structural protein. This observation has led to the designation of RTX (repeat toxin) for this family of toxins (25). *
MATERIALS AND METHODS Bacterial strains and plasmids. The strains and plasmids used were described in a previous study (10). P. haemolytica and E. coli strains were grown in brain-heart infusion (Difco Laboratories, Detroit, Mich.) broth containing ampicillin (100 ,ug/ml) at 30 or 37°C. Overnight, stationary-phase cultures (-400 Klett units) were harvested by low-speed centrifugation for preparation of supernatants and whole-cell lysates. When needed, the lac inducer isopropyl-3-D-thiogalactopyranoside was added to mid-log-phase cultures (-150 Klett units) to 0.5 mM, and growth was continued to stationary phase before cell harvest. Hemolytic activity was detected by plating bacteria on tryptose-soy agar plates containing 5% sheep blood (Remel, Lenexa, Kans.), colonies were lifted, and hemolysis was scored visually. Plasmid
Corresponding author. 2343
2344
HIGHLANDER ET AL.
preparation and manipulation and E. coli transformation were done as described previously (10). The GenBank/ EMBL accession number for the leukotoxin sequence (10) is M24197. Protein secretion analysis. Secreted proteins were detected in filtered, cell-free supernatants by Western immunoblotting, as described previously (10). Blots were probed with bovine serum from a heifer that had recovered from pasteurellosis. RNA preparation. RNA was prepared from late-log-phase cells (-300 Klett units) by the hot phenol-sodium dodecyl sulfate (SDS) method described by Peck and Wang (22). Half of each sample was treated with DNase 1 (150 ,ug/ml) for 60 min at 37°C and then phenol extracted and ethanol precipitated. Northern (RNA) blots. RNA samples were electrophoresed at 8.5 V/cm in 1.2% agarose gels containing 1.1% formaldehyde with a MOPS (morpholinepropanesulfonic acid) buffer system (26). The gel was washed with 0.1 N NaOH for 15 min, neutralized with 1 M Tris hydrochloride (pH 7.2) for 30 min, and then equilibrated with 1Ox SSC (1.5 M NaCl, 0.15 M sodium citrate, pH 7.2) for 30 min. RNA was transferred, by capillary action, to GeneScreen Plus (New England Nuclear Products, Boston, Mass.) membranes in 20x SSC overnight (26). The RNA was fixed to the membrane by exposure to two 15-W germicidal UV light bulbs for 2 min at a distance of 12 cm (12). Blots were hybridized to radiolabeled DNA fragments (10) in 1 M NaCl-50% formamide-1% SDS-100 ,g of salmon sperm DNA per ml at 42°C. Unbound probe was removed by two brief washes in 2x SSC-0.5% SDS at room temperature. Primer extensions. Oligonucleotide primers were synthesized by using an Applied Biosystems (Foster City, Calif.) 380A DNA synthesizer and were labeled at their 5' ends with polynucleotide kinase and [-y-32P]ATP (1). Primer (20 ng) was annealed to 10 or 20 ,ug of whole-cell RNA in 1 M NaCl-0.16 M HEPES (N-2-hydroxyethylpiperazine-N'-2ethanesulfonic acid) (pH 7.5)-0.3 mM EDTA by heating for 2 min at 85°C, followed by slow cooling to less than 45°C. Extensions were performed at either 37 or 45°C with 1,000 U of Moloney murine leukemia virus reverse transcriptase (Bethesda Research Laboratories, Gaithersburg, Md.) per reaction mix, as recommended by the supplier. Fragment size was determined by comparison with the mobility of bands generated from dideoxy sequencing of M13mpl8, as previously described (10). Si nuclease protection. DNA fragments were purified from acrylamide gels (19) and end labeled with [y-32P]ATP by polynucleotide kinase phosphate exchange in a buffer composed of 50 mM imidazole hydrochloride (pH 6.5), 10 mM MgCl2, 0.1 mM EDTA, 0.1 mM spermidine, 50 mM dithiothreitol, and 0.3 mM ATP. Fragments were annealed to 250 jig of P. haemolytica RNA in 0.4 M NaCl-40 mM 1.4piperazinediethanesulfonic acid (pH 6.5)-i mM EDTA-50% formamide overnight at 45°C and then digested with 90 U of S1 nuclease (Sigma Chemical Co., St. Louis, Mo.) as described by Kassavetis and Geiduschek (11). The mobilities of protected fragments were compared with the mobilities of end-labeled 1-kilobase (kb) molecular weight markers (Bethesda Research Laboratories, Gaithersburg, Md.) on 7 M urea-4% polyacrylamide gels.
RESULTS Secretion and activation of the leukotoxin by cognate hemolysin proteins. In E. coli, the cloned P. haemolytica leuko-
J. BACTERIOL.
toxin protein is not secreted into the medium, even when the secretion genes lktB and lktD are present on a subclone (10). When a plasmid carrying the hemolysin locus, pSF4000 (29), and plasmid subclones carrying the leukotoxin gene, lktA, were comaintained, secretion of the leukotoxin from E. coli was observed (10). This suggested that some function of the hemolysin gene cluster could complement a leukotoxin function that was defective or unexpressed on the leukotoxin subclones in E. coli. To localize this function, a series of deletions of pSF4000 were constructed and tested for their ability to complement secretion of the LktA protein in E. coli. Deletions were designed with consideration of the location of the four hemolysin open reading frames and the hlyCABD promoter on the plasmid (7, 28, 30). The leukotoxin subclones and pSF4000 derivatives used for these experiments are shown in Fig. 1. The vector replicons (pBS+ for leukotoxin and pACYC184 for hemolysin) are compatible and can be stably maintained in the same cell. Heteroplasmid strains were constructed by transforming a leukotoxin subclone into a strain carrying a hemolysin locus deletion. A Western blot of cell supernatants from the various heteroplasmid strains constructed is also shown. The results of the complementation experiments and the hemolytic phenotypes of the heteroplasmid strains are listed in Tables 1 and 2, respectively. LktA secretion required the presence of the hly promoter sequence, located within the 937-base-pair SmaI fragment (29, 30), as well as the hlyB and hlyD open reading frames (e.g., pSH305, pSH309, and pSH312 complemented LktA secretion). The data show that neither the HlyC nor LktC protein is required for secretion of the leukotoxin in E. coli (e.g., pSH305 plus pSH218) and are consistent with the assignment of transport functions to the HlyB and HlyD proteins within the hemolysin system (27). The HlyC protein is required to activate the hemolysin protein (21), and LktC has been reported to be required for leukotoxin activation (16). When heteroplasmid strains were plated on sheep blood agar plates, it was observed that HlyC+ plasmids could confer weak hemolytic activity to secreted leukotoxin (e.g., pSH309 plus pSH218). Expression of lktC also activated weak hemolysis by the leukotoxin, and LktC was found to activate the HlyA protein from pSH312, but in this case LktC could not restore the characteristic large-zone hemolytic phenotype to HlyA+ cells. Several of the possible heteroplasmid combinations could not be stably maintained. In most cases the incompatible pairs were those in which cognate C genes were present on each plasmid. The HlyC and HlyBD proteins can provide functions of hemolytic activation and secretion of LktA in E. coli, yet the cognate proteins from the leukotoxin gene cluster do not function well, if at all, in this host. Either the Lkt proteins are inactive or the cloned gene cluster is not efficiently expressed. It was expected that the leukotoxin would be secreted from a strain carrying an LktB+ LktD+ plasmid, such as pSH209, but secretion was not observed even when transcription was driven by the lac promoter on this plasmid, and large quantities of LktA were produced (10). For this reason, we evaluated the relative levels of Lkt transcript within P. haemolytica and E. coli. The IkiCA transcript. RNAs prepared from P. haemolytica or from E. coli strains carrying various plasmid subclones of the leukotoxin genes were probed for the presence of lkt transcripts on Northern blots. When the 1.4-kb BglII-PstI fragment of pSH228, spanning the lktC-lktA junction (Fig. la) was used as a probe, a ca. 3.5-kb transcript was detected in RNA prepared from P. haemolytica PHL101 (Fig. 2a).
VOL. 172, 1990
P. HAEMOLYTICA LEUKOTOXIN
BglII I
TX.---i JLI/d -
a
BglII
PstI I
pSH224 ULktC)
2345
-,--..x -, -: ---i- Cy--
i
pSH228 (LktCA) pSH218
tLktA)
pSH209
ILktABD)
pMC232 tLktA)
b
Ptac F
SmiaI NsiI I hTPL. I
5SmaI
pSF4000
NsiI
NsiI
I
I
I
I
BglII
BamHI pSH305
(H1yBD)
-H
i
pSH307
pSH309 {HyCBD)
I
I
.2.~~~~~~~~~~
pSH312
tHlyABD)
C
HlyA LktA
J,
mommow omowml*
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-
u'..,.'!-'i .::. .".:::,!
..,
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0
FIG. 1. Functional complementation studies. (a) Abbreviated restriction map of leukotoxin fragments, carried by the ColEl-derived ampicillin-resistant vector plasmids pBS+ (pSH209, pSH218, pSH224, and pSH228) or pTac2 (pMC232). Leukotoxin transcription is controlled by the lkt promoter on pSH224 and pSH228 (see Fig. 2), by the lac promoter on pSH209, by a cryptic plasmid promoter on pSH218, and by the tac promoter on pMC232. (b) The hemolysin plasmids are derivatives of the chloramphenicol-resistant plasmid clone pSF4000 (pl5A replicon) (30). Plasmid pSH305 was constructed by partial BamHI and then BglII digestion and ligation of the compatible ends. pSH307 and pSH309 were derived from partial Nsil digestion and ligation. Plasmid pSH312 was created by cleaving pSF4000 at the BamHI site, filling the resulting ends with DNA polymerase, and ligating to form a frame shift (asterisk). Cells carrying insertion or deletion plasmids were nonhemolytic on sheep blood agar. (c) Western blot of secreted proteins. Culture supernatants of E. coli strains containing a P. haemolytica leukotoxin subclone (plasmids pSH209, pSH218, or pSH228) and an E. coli hemolysin plasmid (pSF4000, pSH305, pSH307, pSH309, or pSH312) were electrophoresed on SDS-polyacrylamide, transferred to nitrocellulose, and probed with bovine serum from an animal that had recovered from pasteurellosis. The locations of the 110-kDa hemolysin (HlyA) and 102-kDa leukotoxin (LktA) proteins are shown.
2346
J. BACTERIOL.
HIGHLANDER ET AL. TABLE 1. Secretion of leukotoxin from heteroplasmid E. coli strains Leukotoxin secretion' with hemolysin plasmid:
Leukotoxin
plasmid
pACYC184 (vector)
pSH209 (lktABD) pSH218 (lktA) pSH228 (IktCA)
NT NT
pSF4000
(hIyCABD)
pSH305 (hlyBD)
pSH307 (hlyC
pSH3O9 (hlyCBD)
pSH312 (hlyABD)
+ + +
NM
+
-
+
NT
NM
NM NT +
+ +
-
NM
a Symbols: +, secretion of leukotoxin; -, no secretion of leukotoxin; NT, not tested; NM, heteroplasmid strain could not be maintained.
Transcripts of similar length were detected in E. coli strains carrying plasmid pMC232 (P,aclkt'CA subclone) (M. Chidambaram and M. J. Engler, Abstr. Annu. Meet. Am. Soc. Microbiol. 1989, D215, p. 118) or pSH228 (lktCA subclone), although the intensity of the band observed for the latter was very weak. No larger RNAs were detected with this probe, suggesting that the 3.5-kb species represents a predominant lktCA transcript that begins upstream of IktC and terminates near the 3' end of 1ktA. An inverted repeat structure, tl, followed by a poly(T) tract was present in this region and was probably responsible for terminating this message (Fig. 2b) (10, 17). Based on the transcript length and the position of the putative terminator, it was predicted that the lktCA promoter would map within 200 nucleotides of the lktC ATG start codon. Primer SH25, homologous to nucleotides +41 to +61 of the sequence, was used for extensions of PHL101, E. coli(pSH228), and E. coli(pBS+) RNAs, using reverse transcriptase (Fig. 3). The extended primer produced a single 91-nucleotide (nt) band with PHL101 RNA as the template, while pSH228 RNA yielded the 91-nt plus minor 92- and 93-nt products. No band was observed with pBS+ RNA as the template. The 91-nt extension product places the start of the lktCA transcript at cytosine residue -30 relative to the putative LktC start codon. Transcription on pSH228 also initiated at this residue, but could also begin at the cytosine and adenine residues immediately preceding it. The relative amounts of extension product seen are believed to be representative of the total amount of lktCA message present within each RNA sample and indicate that the concentration of this transcript is much lower in E. coli than in P. haemolytica, even though the genes are carried on a multicopy vector in E. coli. A second primer (SH31), homologous to nucleotides +74 to +96 within the DNA sequence, was used for primer extensions that confirmed the location of the 5' end (not shown). Transcription initiation at cytosine residues is uncommon (9), yet the start site was positioned immediately downstream of an E. coli consensus -10 promoter sequence (10, 20). No consensuslike -35 sequence was obvious, but several adenine-rich sequences lay upstream of this region. These sequences of CA6(C/T)A were TABLE 2. Hemolytic phenotypes of heteroplasmid strains Phenotypea with hemolysin plasmid:
Leukotoxin plasmid
pSH218 (lktA) pSH228 (lktCA) pSH224 (lktC) pBS+ (vector)
pSF4000
(hlyCABD) +L NM NT +L
pSH305
(hlyBD) _ +S -
pSH309
(hIyCBD) +S NM NM
pSH312
(hlyABD) NT +S +S
a Symbols: +, hemolysis; +L, large-zone hemolysis (characteristic of E. coli hemolysin); +S, small-zone hemolysis (characteristic of P. haemolytica hemolysin); -, no hemolysis; NT, not tested; NM, heteroplasmid strain could not be maintained.
phased at 10-base intervals and formed a site of static DNA bending within the promoter region (S. K. Highlander, unpublished observation). lktBD transcription. The failure to observe complementing LktBD secretion activity in E. coli could be due to a gene expression or protein activity defect in this host. We attempted to examine and compare the levels of lktBD transcript in P. haemolytica and in E. coli, but the results of these analyses were not definitive. When the ClaI-BgII fragment (Fig. 2b and 4b) was used as a probe specific for the lktA-lktB junction, the 3.5-kb transcript plus 7.5-kb and 2.0-kb RNAs were detected in P. haemolytica as well as in E. coli when it carried the Ptaclkt'CA plasmid pMC232 (Fig. 4a). An XbaI probe, specific for the lktB and IktD genes (Fig. 4b), hybridized to ca. 6- and 4.5-kb RNAs from P. haemolytica, but did not hybridize to RNA from E. coli strains carrying either pMC232 or the PlaclktABD subclone pSH209. Note that the right half of the blot was treated with an equivalent amount of probe, yet the hybridization signals specific for the lktBD region were much weaker, indicating a lower concentration of lktBD transcripts with respect to IktCA message. None of the probes detected transcripts encoded by the hemolysin plasmid pSF4000 (data not shown). While a 7.5-kb transcript was consistent with a full-length message that includes the four leukotoxin genes, this transcript was not detected with upstream (1.4-kb BglIIPstI fragment, Fig. 2) or downstream (2.1-kb XbaI) probes. In addition, this transcript was also observed with plasmid pMC232, which does not contain the complete IktB and lktD genes and is only 7.1 kb in size (M. Chidambaram, personal communication). The 6.0- and 4.5-kb transcripts detected with the downstream probe could be indicative of other messages that initiate within this region. To determine whether transcription of IktB and/or lktD was initiating within regions immediately upstream of these genes, we attempted to map start sites by primer extension, as described above. Extension with primer SH27, homologous to nucleotides +3567 to +3596, showed a predominant 117-nt band plus a stepwise ladder of smaller species below it when the polymerization was performed at 37°C (Fig. 5a); when the reverse transcriptase reaction was repeated at 45°C, the 117-nt band and laddering disappeared (not shown). We hypothesized that the 117-nt fragment resulted from reverse transcriptase pausing near nucleotide +3480 within the lktB open reading frame. Consistent results were obtained with a primer (SH29) homologous to nucleotides +3510 to +3539: this primer produced a 60-nt band plus a stepwise ladder at 37°C (Fig. 5a). While reverse transcriptase pausing might be expected near the putative terminator, tl, visual inspection failed to reveal a potential secondary structure immediately adjacent to nt +3480, although stemloop structures involving the predicted ribosome-binding site (10) were detected adjacent to the tl dyad. Si mapping of this region also gave equivocal results. The 5'-end-labeled ClaI-XbaI fragment protected RNA species of 490, 380, and
P. HAEMOLYTICA LEUKOTOXIN
VOL. 172, 1990
Bg/ I1-PstI 2
1
3
b
4
a
Ps
Bg
Ps
ti
3.5 kb
P1
2347
C
Xb Bg
I
I
I
pSH228I-_ LktC 3.5kb
.
LktA
...........-s.....LktB'
23S
I
A1TGGCAACTCTATATTGTTTCACACATTATAGAGTrGCCGTAITrAIT
16S
tl 1kb
FIG. 2. (a) Northern blot of P. haemolytica and E. coli RNAs. RNAs were probed with the 32P-labeled 1.4-kb BglII-PstI fragment of pSH228. Lane 1, P. haemolytica RNA; lane 2, pSH228 RNA (IktCA); lane 3, pMC232 RNA (lkt'CA, isopropyl-p-D-thiogalactopyranoside induced); lane 4, pBS+ RNA (vector). The locations of the cross-hybridizing 23S and 16S rRNAs are noted. (b) Abbreviated restriction map of the lktCA region on pSH228 showing the LktC, LktA, and truncated LktB open reading frames. The positions of the lktCA promoter and putative terminator site, tl, are indicated. C, CMaI; Bg, BglII; Ps, PstI; and Xb, XbaI.
290 bp from Si digestion (Fig. 5b). The 490-bp fragment is consistent either with an RNA that initiates near the terminator dyad or with a longer species that is cleaved within the single-stranded region of the stem-loop structure by Si nuclease. The 380- and 290-bp fragments could arise from new transcripts that initiate downstream, within the lktB open reading frame, from a longer message that is processed at sites within this region, or from additional transcripts that initiate on the noncoding DNA strand. Finally, primer extension with an oligomer specific to the 5' end of lktD failed to reveal an lktD transcription initiation site (not shown), and Si mapping with the 1,139-bp EcoRV fragment (see map, Fig. 4b) that overlaps the lktB-lktD junction failed to protect GATC
1
2
3
a specific RNA species that was smaller than the probe fragment (Fig. Sb), suggesting that there is not an individual transcription unit for IktD that begins immediately upstream of the gene.
DISCUSSION The transport proteins, B and D, of the hemolysin and leukotoxin gene clusters have very similar amino acid sequences (10, 25): the LktB and HlyB proteins are 80% alike, and the LktD and HlyD proteins have 60% identity. We have demonstrated that the amino acid similarities are manifested as functional similarities by developing a heterologous se-
b
-120 -130 -140 -160 -150 ATAACTTTAA AACACTCCTT TTTCTCTTCT GATTATATAA AAGACA&A AT_A CAATTTA TATTGAAATT TTGTGAGGAA AAAGAGAAGA CTAATATATT TTCTGTTTTT TATGTTAAAT
a
-170
0.
120 -110
-100
-60
-70
-80
-90
AGCTACSARA AACAk52/ja2a2AcA~MX AACGACAA TAAGATCGAG TAATGATTAT
110
TCGATGTTTT TTGTTGTTTT TTGTTGTTTT TTGTGCTGTT ATTCTAGCTC ATTACTAATA
100
90
80
70
+1 -10 -20 -30 or------------ lktCA transcript-----------ATTATGTThT A&TTTTTGAC CTAATTTAGA ATAATTATCG AGTGCAAATT ATG AAT CAA TAATACAATA TTAAAAACTG GATTAAATCT TATTAATAGC TCACGTTTAA TAC TTA GTT N M LktC: Q
-50
91
10
-40
20
30
40
50
60
TCT TAT TTT AAC TTA CTA GGA AAC ATT ACT TGG CTA TGG ATG AAC TCC TCCC AGA ATA AAA TTG AAT GAT CCT TTG TAA TGA ACC GAT ACC TAC TTG AGG AGGG S L W M N N I T S N L L F G W Y S
vD290 X-z E-4 U
~ ~ ~ ~ ~~~~~~~~~~~~~~~~~0'E-4 70 100 _ E
80,
2
. &
90
E-
S
m
U
:E
I
~~~~ fi> ~~~~~~~~~~~~~-4 4 ~
200
Z
Ln)
s
*
ts> ~~~~~~~~~~~~~~~-4
200
E4
a|E-4 04' ,4r ¢E ), 8 E-4 @0 = ( 04E-4 , U
JOURNAL OF BACTERIOLOGY, May 1990, p. 2343-2350
0021-9193/90/052343-08$02.00/0 Copyright © 1990, American Society for Microbiology
Secretion and Expression of the Pasteurella haemolytica Leukotoxin SARAH K. HIGHLANDER,* MICHAEL J. ENGLER, AND GEORGE M. WEINSTOCK Department of Biochemistry and Molecular Biology, The University of Texas Medical School, 6431 Fannin, Houston, Texas 77030 Received 15 September 1989/Accepted 22 January 1990
The PasteureUa haemolytica leukotoxin gene cluster (iktCABD) is homologous to the Escherichia coli hemolysin locus (hlyCABD). Since the cloned leukotoxin (LktA) is not secreted from E. coli cells, a heteroplasmid complementation system was developed that permits secretion of the leukotoxin from cells expressing the hemolysin transport proteins ElyB and HlyD. We observed that the secreted leukotoxin protein had weak hemolytic activity when activated by either the HlyC or LktC proteins and that LktC expressed in E. coli could confer weak hemolytic activity upon hemolysin. Thus, it appears that the accessory proteins of the leukotoxin and hemolysin gene clusters are functionally similar, although their expression in E. coli is not equivalent. Northern (RNA) blot analysis of the P. haemolytica leukotoxin gene cluster revealed a major 3.5-kilobase transcript that includes the lktC and lktA genes. The start site for this transcript mapped to a cytosine residue 30 nucleotides upstream from the putative start of IktC; a similar initiation site was observed in E. coli, although adjacent cytosine and adenine residues were also utilized. The 3.5-kilobase transcript terminated near the rho-independent terminator structure between iktA and lktB, but transcription may continue, via antitermination or de novo transcription initiation, into the downstream lktB and lktD genes. We propose that the lack of LktB and LktD function in E. coli is a result, at least in part, of poor lktBD transcription and suggest that a P. haemolytica-specific regulator is required for optimal expression of the leukotoxin genes.
In a previous study, we found that the LktA protein was produced in an E. coli host but was not secreted by LktB and LktD, yet the leukotoxin could be secreted when a complete hemolysin locus was present in the same cell (10). Here, we extend these observations by examining the function of cognate secretion and activation proteins on the leukotoxin in E. coli. The HlyC and HlyBD proteins were able to complement hemolytic activity and secretion of the leukotoxin, respectively, and the LktC protein weakly activated HlyA hemolysis. In contrast, the LktB and LktD proteins were not active and their genes were poorly expressed in E. coli. Transcriptional mapping of the leukotoxin gene cluster indicated that transcription of lktB and lktD is weak in P. haemolytica; an lktBD message was undetectable in E. coli. In contrast, the lktC and lktA genes were found to be contained within a single transcriptional unit whose promoter is weakly recognized in E. coli. These observations suggest that while the hemolysin and leukotoxin gene clusters encode proteins with similar functions, expression of the leukotoxin gene cluster may require a positive regulator not present in E. coli.
Pasteurella haemolytica secretes a 102-kilodalton leukotoxin that is believed to be involved in the pathogenesis of a severe bovine pneumonia, commonly called shipping fever (2, 23). This disease, which develops following stress to the animal, is a subject for vaccine development studies (6, 7). The leukotoxin is ruminant specific and acts to compromise the primary immune response of the bovine lung by lysing macrophages and other leukocytes (2, 18, 23). Since P. haemolytica is normally a nonpathogenic commensal bacterium of cattle, it is possible that leukotoxin expression is responsible for development of the disease state. A four-gene cluster which encodes the leukotoxin (LktA) and proteins required for its activation (LktC) and secretion (LktB, LktD) has been cloned in Escherichia coli (4, 10, 16, 25), and their nucleotide sequences were determined (10, 17, 25). DNA sequence comparisons revealed that the leukotoxin gene cluster is homologous to the hemolysin determinant of E. coli (10, 17, 24, 25) and has genes analogous to the hlyC, hlyA, hlyB, and hlyD genes for hemolysin. The HlyC protein is required to activate the hemolytic phenotype of HlyA (21), while HlyB and HlyD are required to secrete the HlyA protein from the cell (27). Similarly, LktC may be required for the leukotoxic activity of LktA (17), and it is presumed, by analogy, that the LktB and LktD proteins are responsible for secreting LktA from P. haemolytica cells. The leukotoxin determinant is similar in sequence to toxin genes from Morganella morganii, Proteus vulgaris, Proteus mirabilis (15, 28), and Actinobacillus actinomycetemcomitans (13) and to a portion of the Bordetella pertussis adenylate cyclase toxin (8). Each of these toxins requires accessory proteins for secretion and possesses a series of glycine-rich amino acid repeats within the toxin structural protein. This observation has led to the designation of RTX (repeat toxin) for this family of toxins (25). *
MATERIALS AND METHODS Bacterial strains and plasmids. The strains and plasmids used were described in a previous study (10). P. haemolytica and E. coli strains were grown in brain-heart infusion (Difco Laboratories, Detroit, Mich.) broth containing ampicillin (100 ,ug/ml) at 30 or 37°C. Overnight, stationary-phase cultures (-400 Klett units) were harvested by low-speed centrifugation for preparation of supernatants and whole-cell lysates. When needed, the lac inducer isopropyl-3-D-thiogalactopyranoside was added to mid-log-phase cultures (-150 Klett units) to 0.5 mM, and growth was continued to stationary phase before cell harvest. Hemolytic activity was detected by plating bacteria on tryptose-soy agar plates containing 5% sheep blood (Remel, Lenexa, Kans.), colonies were lifted, and hemolysis was scored visually. Plasmid
Corresponding author. 2343
2344
HIGHLANDER ET AL.
preparation and manipulation and E. coli transformation were done as described previously (10). The GenBank/ EMBL accession number for the leukotoxin sequence (10) is M24197. Protein secretion analysis. Secreted proteins were detected in filtered, cell-free supernatants by Western immunoblotting, as described previously (10). Blots were probed with bovine serum from a heifer that had recovered from pasteurellosis. RNA preparation. RNA was prepared from late-log-phase cells (-300 Klett units) by the hot phenol-sodium dodecyl sulfate (SDS) method described by Peck and Wang (22). Half of each sample was treated with DNase 1 (150 ,ug/ml) for 60 min at 37°C and then phenol extracted and ethanol precipitated. Northern (RNA) blots. RNA samples were electrophoresed at 8.5 V/cm in 1.2% agarose gels containing 1.1% formaldehyde with a MOPS (morpholinepropanesulfonic acid) buffer system (26). The gel was washed with 0.1 N NaOH for 15 min, neutralized with 1 M Tris hydrochloride (pH 7.2) for 30 min, and then equilibrated with 1Ox SSC (1.5 M NaCl, 0.15 M sodium citrate, pH 7.2) for 30 min. RNA was transferred, by capillary action, to GeneScreen Plus (New England Nuclear Products, Boston, Mass.) membranes in 20x SSC overnight (26). The RNA was fixed to the membrane by exposure to two 15-W germicidal UV light bulbs for 2 min at a distance of 12 cm (12). Blots were hybridized to radiolabeled DNA fragments (10) in 1 M NaCl-50% formamide-1% SDS-100 ,g of salmon sperm DNA per ml at 42°C. Unbound probe was removed by two brief washes in 2x SSC-0.5% SDS at room temperature. Primer extensions. Oligonucleotide primers were synthesized by using an Applied Biosystems (Foster City, Calif.) 380A DNA synthesizer and were labeled at their 5' ends with polynucleotide kinase and [-y-32P]ATP (1). Primer (20 ng) was annealed to 10 or 20 ,ug of whole-cell RNA in 1 M NaCl-0.16 M HEPES (N-2-hydroxyethylpiperazine-N'-2ethanesulfonic acid) (pH 7.5)-0.3 mM EDTA by heating for 2 min at 85°C, followed by slow cooling to less than 45°C. Extensions were performed at either 37 or 45°C with 1,000 U of Moloney murine leukemia virus reverse transcriptase (Bethesda Research Laboratories, Gaithersburg, Md.) per reaction mix, as recommended by the supplier. Fragment size was determined by comparison with the mobility of bands generated from dideoxy sequencing of M13mpl8, as previously described (10). Si nuclease protection. DNA fragments were purified from acrylamide gels (19) and end labeled with [y-32P]ATP by polynucleotide kinase phosphate exchange in a buffer composed of 50 mM imidazole hydrochloride (pH 6.5), 10 mM MgCl2, 0.1 mM EDTA, 0.1 mM spermidine, 50 mM dithiothreitol, and 0.3 mM ATP. Fragments were annealed to 250 jig of P. haemolytica RNA in 0.4 M NaCl-40 mM 1.4piperazinediethanesulfonic acid (pH 6.5)-i mM EDTA-50% formamide overnight at 45°C and then digested with 90 U of S1 nuclease (Sigma Chemical Co., St. Louis, Mo.) as described by Kassavetis and Geiduschek (11). The mobilities of protected fragments were compared with the mobilities of end-labeled 1-kilobase (kb) molecular weight markers (Bethesda Research Laboratories, Gaithersburg, Md.) on 7 M urea-4% polyacrylamide gels.
RESULTS Secretion and activation of the leukotoxin by cognate hemolysin proteins. In E. coli, the cloned P. haemolytica leuko-
J. BACTERIOL.
toxin protein is not secreted into the medium, even when the secretion genes lktB and lktD are present on a subclone (10). When a plasmid carrying the hemolysin locus, pSF4000 (29), and plasmid subclones carrying the leukotoxin gene, lktA, were comaintained, secretion of the leukotoxin from E. coli was observed (10). This suggested that some function of the hemolysin gene cluster could complement a leukotoxin function that was defective or unexpressed on the leukotoxin subclones in E. coli. To localize this function, a series of deletions of pSF4000 were constructed and tested for their ability to complement secretion of the LktA protein in E. coli. Deletions were designed with consideration of the location of the four hemolysin open reading frames and the hlyCABD promoter on the plasmid (7, 28, 30). The leukotoxin subclones and pSF4000 derivatives used for these experiments are shown in Fig. 1. The vector replicons (pBS+ for leukotoxin and pACYC184 for hemolysin) are compatible and can be stably maintained in the same cell. Heteroplasmid strains were constructed by transforming a leukotoxin subclone into a strain carrying a hemolysin locus deletion. A Western blot of cell supernatants from the various heteroplasmid strains constructed is also shown. The results of the complementation experiments and the hemolytic phenotypes of the heteroplasmid strains are listed in Tables 1 and 2, respectively. LktA secretion required the presence of the hly promoter sequence, located within the 937-base-pair SmaI fragment (29, 30), as well as the hlyB and hlyD open reading frames (e.g., pSH305, pSH309, and pSH312 complemented LktA secretion). The data show that neither the HlyC nor LktC protein is required for secretion of the leukotoxin in E. coli (e.g., pSH305 plus pSH218) and are consistent with the assignment of transport functions to the HlyB and HlyD proteins within the hemolysin system (27). The HlyC protein is required to activate the hemolysin protein (21), and LktC has been reported to be required for leukotoxin activation (16). When heteroplasmid strains were plated on sheep blood agar plates, it was observed that HlyC+ plasmids could confer weak hemolytic activity to secreted leukotoxin (e.g., pSH309 plus pSH218). Expression of lktC also activated weak hemolysis by the leukotoxin, and LktC was found to activate the HlyA protein from pSH312, but in this case LktC could not restore the characteristic large-zone hemolytic phenotype to HlyA+ cells. Several of the possible heteroplasmid combinations could not be stably maintained. In most cases the incompatible pairs were those in which cognate C genes were present on each plasmid. The HlyC and HlyBD proteins can provide functions of hemolytic activation and secretion of LktA in E. coli, yet the cognate proteins from the leukotoxin gene cluster do not function well, if at all, in this host. Either the Lkt proteins are inactive or the cloned gene cluster is not efficiently expressed. It was expected that the leukotoxin would be secreted from a strain carrying an LktB+ LktD+ plasmid, such as pSH209, but secretion was not observed even when transcription was driven by the lac promoter on this plasmid, and large quantities of LktA were produced (10). For this reason, we evaluated the relative levels of Lkt transcript within P. haemolytica and E. coli. The IkiCA transcript. RNAs prepared from P. haemolytica or from E. coli strains carrying various plasmid subclones of the leukotoxin genes were probed for the presence of lkt transcripts on Northern blots. When the 1.4-kb BglII-PstI fragment of pSH228, spanning the lktC-lktA junction (Fig. la) was used as a probe, a ca. 3.5-kb transcript was detected in RNA prepared from P. haemolytica PHL101 (Fig. 2a).
VOL. 172, 1990
P. HAEMOLYTICA LEUKOTOXIN
BglII I
TX.---i JLI/d -
a
BglII
PstI I
pSH224 ULktC)
2345
-,--..x -, -: ---i- Cy--
i
pSH228 (LktCA) pSH218
tLktA)
pSH209
ILktABD)
pMC232 tLktA)
b
Ptac F
SmiaI NsiI I hTPL. I
5SmaI
pSF4000
NsiI
NsiI
I
I
I
I
BglII
BamHI pSH305
(H1yBD)
-H
i
pSH307
pSH309 {HyCBD)
I
I
.2.~~~~~~~~~~
pSH312
tHlyABD)
C
HlyA LktA
J,
mommow omowml*
...%--eil-
-
u'..,.'!-'i .::. .".:::,!
..,
a I
0
FIG. 1. Functional complementation studies. (a) Abbreviated restriction map of leukotoxin fragments, carried by the ColEl-derived ampicillin-resistant vector plasmids pBS+ (pSH209, pSH218, pSH224, and pSH228) or pTac2 (pMC232). Leukotoxin transcription is controlled by the lkt promoter on pSH224 and pSH228 (see Fig. 2), by the lac promoter on pSH209, by a cryptic plasmid promoter on pSH218, and by the tac promoter on pMC232. (b) The hemolysin plasmids are derivatives of the chloramphenicol-resistant plasmid clone pSF4000 (pl5A replicon) (30). Plasmid pSH305 was constructed by partial BamHI and then BglII digestion and ligation of the compatible ends. pSH307 and pSH309 were derived from partial Nsil digestion and ligation. Plasmid pSH312 was created by cleaving pSF4000 at the BamHI site, filling the resulting ends with DNA polymerase, and ligating to form a frame shift (asterisk). Cells carrying insertion or deletion plasmids were nonhemolytic on sheep blood agar. (c) Western blot of secreted proteins. Culture supernatants of E. coli strains containing a P. haemolytica leukotoxin subclone (plasmids pSH209, pSH218, or pSH228) and an E. coli hemolysin plasmid (pSF4000, pSH305, pSH307, pSH309, or pSH312) were electrophoresed on SDS-polyacrylamide, transferred to nitrocellulose, and probed with bovine serum from an animal that had recovered from pasteurellosis. The locations of the 110-kDa hemolysin (HlyA) and 102-kDa leukotoxin (LktA) proteins are shown.
2346
J. BACTERIOL.
HIGHLANDER ET AL. TABLE 1. Secretion of leukotoxin from heteroplasmid E. coli strains Leukotoxin secretion' with hemolysin plasmid:
Leukotoxin
plasmid
pACYC184 (vector)
pSH209 (lktABD) pSH218 (lktA) pSH228 (IktCA)
NT NT
pSF4000
(hIyCABD)
pSH305 (hlyBD)
pSH307 (hlyC
pSH3O9 (hlyCBD)
pSH312 (hlyABD)
+ + +
NM
+
-
+
NT
NM
NM NT +
+ +
-
NM
a Symbols: +, secretion of leukotoxin; -, no secretion of leukotoxin; NT, not tested; NM, heteroplasmid strain could not be maintained.
Transcripts of similar length were detected in E. coli strains carrying plasmid pMC232 (P,aclkt'CA subclone) (M. Chidambaram and M. J. Engler, Abstr. Annu. Meet. Am. Soc. Microbiol. 1989, D215, p. 118) or pSH228 (lktCA subclone), although the intensity of the band observed for the latter was very weak. No larger RNAs were detected with this probe, suggesting that the 3.5-kb species represents a predominant lktCA transcript that begins upstream of IktC and terminates near the 3' end of 1ktA. An inverted repeat structure, tl, followed by a poly(T) tract was present in this region and was probably responsible for terminating this message (Fig. 2b) (10, 17). Based on the transcript length and the position of the putative terminator, it was predicted that the lktCA promoter would map within 200 nucleotides of the lktC ATG start codon. Primer SH25, homologous to nucleotides +41 to +61 of the sequence, was used for extensions of PHL101, E. coli(pSH228), and E. coli(pBS+) RNAs, using reverse transcriptase (Fig. 3). The extended primer produced a single 91-nucleotide (nt) band with PHL101 RNA as the template, while pSH228 RNA yielded the 91-nt plus minor 92- and 93-nt products. No band was observed with pBS+ RNA as the template. The 91-nt extension product places the start of the lktCA transcript at cytosine residue -30 relative to the putative LktC start codon. Transcription on pSH228 also initiated at this residue, but could also begin at the cytosine and adenine residues immediately preceding it. The relative amounts of extension product seen are believed to be representative of the total amount of lktCA message present within each RNA sample and indicate that the concentration of this transcript is much lower in E. coli than in P. haemolytica, even though the genes are carried on a multicopy vector in E. coli. A second primer (SH31), homologous to nucleotides +74 to +96 within the DNA sequence, was used for primer extensions that confirmed the location of the 5' end (not shown). Transcription initiation at cytosine residues is uncommon (9), yet the start site was positioned immediately downstream of an E. coli consensus -10 promoter sequence (10, 20). No consensuslike -35 sequence was obvious, but several adenine-rich sequences lay upstream of this region. These sequences of CA6(C/T)A were TABLE 2. Hemolytic phenotypes of heteroplasmid strains Phenotypea with hemolysin plasmid:
Leukotoxin plasmid
pSH218 (lktA) pSH228 (lktCA) pSH224 (lktC) pBS+ (vector)
pSF4000
(hlyCABD) +L NM NT +L
pSH305
(hlyBD) _ +S -
pSH309
(hIyCBD) +S NM NM
pSH312
(hlyABD) NT +S +S
a Symbols: +, hemolysis; +L, large-zone hemolysis (characteristic of E. coli hemolysin); +S, small-zone hemolysis (characteristic of P. haemolytica hemolysin); -, no hemolysis; NT, not tested; NM, heteroplasmid strain could not be maintained.
phased at 10-base intervals and formed a site of static DNA bending within the promoter region (S. K. Highlander, unpublished observation). lktBD transcription. The failure to observe complementing LktBD secretion activity in E. coli could be due to a gene expression or protein activity defect in this host. We attempted to examine and compare the levels of lktBD transcript in P. haemolytica and in E. coli, but the results of these analyses were not definitive. When the ClaI-BgII fragment (Fig. 2b and 4b) was used as a probe specific for the lktA-lktB junction, the 3.5-kb transcript plus 7.5-kb and 2.0-kb RNAs were detected in P. haemolytica as well as in E. coli when it carried the Ptaclkt'CA plasmid pMC232 (Fig. 4a). An XbaI probe, specific for the lktB and IktD genes (Fig. 4b), hybridized to ca. 6- and 4.5-kb RNAs from P. haemolytica, but did not hybridize to RNA from E. coli strains carrying either pMC232 or the PlaclktABD subclone pSH209. Note that the right half of the blot was treated with an equivalent amount of probe, yet the hybridization signals specific for the lktBD region were much weaker, indicating a lower concentration of lktBD transcripts with respect to IktCA message. None of the probes detected transcripts encoded by the hemolysin plasmid pSF4000 (data not shown). While a 7.5-kb transcript was consistent with a full-length message that includes the four leukotoxin genes, this transcript was not detected with upstream (1.4-kb BglIIPstI fragment, Fig. 2) or downstream (2.1-kb XbaI) probes. In addition, this transcript was also observed with plasmid pMC232, which does not contain the complete IktB and lktD genes and is only 7.1 kb in size (M. Chidambaram, personal communication). The 6.0- and 4.5-kb transcripts detected with the downstream probe could be indicative of other messages that initiate within this region. To determine whether transcription of IktB and/or lktD was initiating within regions immediately upstream of these genes, we attempted to map start sites by primer extension, as described above. Extension with primer SH27, homologous to nucleotides +3567 to +3596, showed a predominant 117-nt band plus a stepwise ladder of smaller species below it when the polymerization was performed at 37°C (Fig. 5a); when the reverse transcriptase reaction was repeated at 45°C, the 117-nt band and laddering disappeared (not shown). We hypothesized that the 117-nt fragment resulted from reverse transcriptase pausing near nucleotide +3480 within the lktB open reading frame. Consistent results were obtained with a primer (SH29) homologous to nucleotides +3510 to +3539: this primer produced a 60-nt band plus a stepwise ladder at 37°C (Fig. 5a). While reverse transcriptase pausing might be expected near the putative terminator, tl, visual inspection failed to reveal a potential secondary structure immediately adjacent to nt +3480, although stemloop structures involving the predicted ribosome-binding site (10) were detected adjacent to the tl dyad. Si mapping of this region also gave equivocal results. The 5'-end-labeled ClaI-XbaI fragment protected RNA species of 490, 380, and
P. HAEMOLYTICA LEUKOTOXIN
VOL. 172, 1990
Bg/ I1-PstI 2
1
3
b
4
a
Ps
Bg
Ps
ti
3.5 kb
P1
2347
C
Xb Bg
I
I
I
pSH228I-_ LktC 3.5kb
.
LktA
...........-s.....LktB'
23S
I
A1TGGCAACTCTATATTGTTTCACACATTATAGAGTrGCCGTAITrAIT
16S
tl 1kb
FIG. 2. (a) Northern blot of P. haemolytica and E. coli RNAs. RNAs were probed with the 32P-labeled 1.4-kb BglII-PstI fragment of pSH228. Lane 1, P. haemolytica RNA; lane 2, pSH228 RNA (IktCA); lane 3, pMC232 RNA (lkt'CA, isopropyl-p-D-thiogalactopyranoside induced); lane 4, pBS+ RNA (vector). The locations of the cross-hybridizing 23S and 16S rRNAs are noted. (b) Abbreviated restriction map of the lktCA region on pSH228 showing the LktC, LktA, and truncated LktB open reading frames. The positions of the lktCA promoter and putative terminator site, tl, are indicated. C, CMaI; Bg, BglII; Ps, PstI; and Xb, XbaI.
290 bp from Si digestion (Fig. 5b). The 490-bp fragment is consistent either with an RNA that initiates near the terminator dyad or with a longer species that is cleaved within the single-stranded region of the stem-loop structure by Si nuclease. The 380- and 290-bp fragments could arise from new transcripts that initiate downstream, within the lktB open reading frame, from a longer message that is processed at sites within this region, or from additional transcripts that initiate on the noncoding DNA strand. Finally, primer extension with an oligomer specific to the 5' end of lktD failed to reveal an lktD transcription initiation site (not shown), and Si mapping with the 1,139-bp EcoRV fragment (see map, Fig. 4b) that overlaps the lktB-lktD junction failed to protect GATC
1
2
3
a specific RNA species that was smaller than the probe fragment (Fig. Sb), suggesting that there is not an individual transcription unit for IktD that begins immediately upstream of the gene.
DISCUSSION The transport proteins, B and D, of the hemolysin and leukotoxin gene clusters have very similar amino acid sequences (10, 25): the LktB and HlyB proteins are 80% alike, and the LktD and HlyD proteins have 60% identity. We have demonstrated that the amino acid similarities are manifested as functional similarities by developing a heterologous se-
b
-120 -130 -140 -160 -150 ATAACTTTAA AACACTCCTT TTTCTCTTCT GATTATATAA AAGACA&A AT_A CAATTTA TATTGAAATT TTGTGAGGAA AAAGAGAAGA CTAATATATT TTCTGTTTTT TATGTTAAAT
a
-170
0.
120 -110
-100
-60
-70
-80
-90
AGCTACSARA AACAk52/ja2a2AcA~MX AACGACAA TAAGATCGAG TAATGATTAT
110
TCGATGTTTT TTGTTGTTTT TTGTTGTTTT TTGTGCTGTT ATTCTAGCTC ATTACTAATA
100
90
80
70
+1 -10 -20 -30 or------------ lktCA transcript-----------ATTATGTThT A&TTTTTGAC CTAATTTAGA ATAATTATCG AGTGCAAATT ATG AAT CAA TAATACAATA TTAAAAACTG GATTAAATCT TATTAATAGC TCACGTTTAA TAC TTA GTT N M LktC: Q
-50
91
10
-40
20
30
40
50
60
TCT TAT TTT AAC TTA CTA GGA AAC ATT ACT TGG CTA TGG ATG AAC TCC TCCC AGA ATA AAA TTG AAT GAT CCT TTG TAA TGA ACC GAT ACC TAC TTG AGG AGGG S L W M N N I T S N L L F G W Y S
vD290 X-z E-4 U
~ ~ ~ ~ ~~~~~~~~~~~~~~~~~0'E-4 70 100 _ E
80,
2
. &
90
E-
S
m
U
:E
I
~~~~ fi> ~~~~~~~~~~~~~-4 4 ~
200
Z
Ln)
s
*
ts> ~~~~~~~~~~~~~~~-4
200
E4
a|E-4 04' ,4r ¢E ), 8 E-4 @0 = ( 04E-4 , U
