Nucleotide sequence analysis of the gene encoding the Caulobacter crescentus paracrystalline surface layer protein ANGUSGILCHRIST Department of Microbiology, University of British Columbia, Vancouver, B.C., Canada V6T 123 JAMES A . FISHER' Applied Biosystems, Foster City, CA 94404, U.S.A .
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AND
JOHN S M I T ~ Department of Microbiology, University of British Columbia, Vancouver, B.C., Canada V6T 1 2 3 Received July 22, 1991 Revision received November 7, 1991 Accepted November 12, 1991 GILCHRIST,A., FISHER,J. A., and SMIT,J. 1992. Nucleotide sequence analysis of the gene encoding the Caulobacter crescentus paracrystalline surface layer protein. Can. J . Microbiol. 38: 193-202. The entire nucleotide sequence of the rsaA gene, encoding the paracrystalline surface (S) layer protein (RsaA) of Caulobacter crescentus CBISA, was determined. The rsaA gene encoded a protein of 1026 amino acids, with a predicted molecular weight of 98 132. Protease cleavage of mature RsaA protein and amino acid sequencing of retrievable peptides yielded two peptides: one aligned with a region approximately two-thirds the way into the predicted amino acid sequence and the second peptide corresponded to the predicted carboxy terminus. Thus, no cleavage processing of the carboxy portion of the RsaA protein occurred during export, and with the exception of the removal of the initial methionine residue, the protein was not processed by cleavage to produce the mature protein. The predicted RsaA amino acid profile was unusual, with small neutral residues predominating. Excepting aspartate, charged amino acids were in relatively low proportion, resulting in an especially acidic protein, with a predicted pI of 3.46. As with most other sequenced S-layer proteins, RsaA contained no cysteine residues. A homology scan of the Swiss Protein Bank 17 produced no close matches to the predicted RsaA sequence. However, RsaA protein shared measurable homology with some exported proteins of other bacteria, including the hemolysins. Of particular interest was a specific region of the RsaA protein that was homologous to the repeat regions of glycine and aspartate residues found in several proteases and hemolysins. These repeats are implicated in the binding of calcium for proper structure and biological activity of these proteins. Those present in the RsaA protein may perform a similar function, since S-layer assembly and surface attachment requires calcium. RsaA protein also shared some homology with 10 other S-layer proteins, with the Campylobacter fetus S-layer protein scoring highest. Key words: Caulobacter crescentus, surface layer, nucleotide sequence, rsaA , calcium. GILCHRIST,A., FISHER,J. A., et SMIT,J . 1992. Nucleotide sequence analysis of the gene encoding the Caulobacter crescentus paracrystalline surface layer protein. Can. J . Microbiol. 38 : 193-202. Nous avons determine la sequence nucleotidique complete du gene rsaA qui code la proteine (RsaA) presente dans la couche de surface (S) paracrystalline chez Caulobacter crescentus CBISA. Ce gene rsaA code pour une proteine de 1026 acides amines et de poids moleculaire de 98 132. Le clivage par des proteases de la proteine RsaA mature et le sequencage des acides amines des peptides recouvrables a produit deux peptides : un s'allignant avec une region localisee approximativement jusqu'au deux tiers de la sequence peptidique predite et un deuxieme correspondant a l'extremite carboxyterminale predite: Ainsi aucune maturation par clivage de la portion carboxyle de la proteine RsaA n'a eu lieu durant l'exportation. A l'exception de l'elimination du residu methionine initial, la proteine n'a pas subi de maturation par clivage pour produire la proteine mature. La composition en acides amines predite pour RsaA etait inhabituelle a cause d'une predominance en petits residus neutres. Mise a part l'aspartate, la proportion des acides amines charges etait relativement faible expliquant ainsi que la proteine est particulierement acide avec un pI prevu a 3'46. Comme pour la plupart des autres proteines sequencees de la couche S, la RsaA ne contenait pas de residu de cysteine. Une recherche d'homologie dans la Swiss Protein Bank 17 n'a pas permis de detecter de sequence apparentee a la sequence predite pour RsaA. Cette proteine RsaA possedait cependant un degre d'homologie mesurable avec les proteines secretees par d'autres bacteries incluant des hemolysines. Une region precise de la proteine RsaA presente un intbet particulier par son homologie a des regions repetitives dans les residus glycine et aspartate rencontres dans certaines proteases et hemolysines. Ces repetitions jouent un r81e dans la fixation du calcium en contribuant a la structure et a l'activite biologique de ces proteines. Celles presentes dans la proteine RsaA pourraient jouer un r61e semblable puisque l'assemblage de la couche S et l'attachement en surface requierent du calcium. La proteine RsaA partage une certaine homologie avec dix autres proteines de la couche S mais c'est celle de Campylobacter fetus qui vient au premier rang. Mots clks : Caulobacter crescentus, couche de surface, sequence nucleotidjque, rsaA, calcium. [Traduit par la redaction]
'present address: Ribogene, Menlo Park, CA 94025, U.S.A. 2 ~ u t h o to r whom all correspondence should be addressed. Printed in Canada / lmprime au Canada
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CAN. J. MICROBIOL. VOL. 38, 1992
Introduction Paracrystalline surface arrays or S-layers have been described from species across the eubacterial and archaebacterial spectrum (Sleytr and Messner 1988b; Smit 1986). They consist of regularly arranged protein subunits that form two-dimensional arrays on the surface of the cell wall in Gram-negative and -positive bacteria; in archaebacteria they are external to the plasma membrane and often are the cell wall. Although the functions of most known S-layers have not been fully ascertained, they certainly relate to their position on the cell. To produce the large amount of protein required to sheath the cell, one can assume that much of a bacterium's energies are devoted to this endeavor. This, coupled with the fact that S-layers are often lost in laboratory culture, has led to proposals that they play specific and essential roles in their natural environments. Thus, functions may include steric protection of cells from external influences, working as molecular sieves, and preventing lytic enzymes, bacteriophages, parasitic bacteria, or foreign DNAs from contacting the hidden membranes. Caulobacter crescentus is a Gram-negative dimorphic bacterium that inhabits many aquatic environments (Anast and Smit 1988; MacRae and Smit 1991; Poindexter 1981). The major component of the C. crescentus S-layer is the apparent M, 105 000 protein (RsaA protein, formerly referred to as 130K); it is probably the most abundant protein of the cell, accounting for approximately 5% of total cell protein synthesis (Agabian et al. 1979; Smit and Agabian 1984). The protein forms a hexagonal array and, with the possible exception of an exopolysaccharide associated with the membranes (Ravenscroft et al. 1991) and lipopolysaccharide, is the outermost layer of the cell (Smit et al. 1981a). Subunit assembly of the S-layer occurs in two distinct ways: either as random addition of subunits within the preexisting array, or de novo assembly at the specific sites of stalk elongation or along the cell division plane (Smit and Agabian 1982). These latter cases of spatially restricted array formation are temporally regulated, occurring at specific stages of the life cycle. Since the RsaA protein is synthesized at a constant rate during the cell cycle (Agabian et al. 1979; Fisher et al. 1988) from a single-copy gene, it is difficult to propose how these two processes are coordinated. Indeed the entire transport journey across the inner and outer membranes, culminating with final addition to the S-layer supramolecule, is likely to prove a complex process. It is of interest to us to learn in molecular detail how the S-layer is exported, assembled, and attached to the cell surface; toward that end, primary sequence information was essential. The rsaA gene was cloned from C. crescentus CB15A (Smit and Agabian 1984) and the site of transcription initiation determined (Fisher et al. 1988). The site of translation initiation was also determined by alignment with protein sequencing of the first 21 amino acids of the mature protein. Unlike other sequenced S-layers, there was no cleaved signal leader peptide. This report also details the completion and clarification of sequence determination for the entire large protein and attempts to define functional regions. A likely region related to the role of calcium in surface attachment and self assembly was noted, but with respect to a mechanism of export, we learned that this protein has no clear analogy to other characterized exported
bacterial proteins. Eleven bacterial S-layer protein genes, including RsaA, have been sequenced and are here compared with each other.
Materials and methods Bacterial strains and growth media Caulobacter crescentus CBl5A (ATCC 19089) was grown at 30°C, with shaking, in a peptone yeast extract (PYE) medium (Poindexter 1964), supplemented with 20 mM MgCl, and 10 mM CaCl,. Escherichia coli DH5aF1 (Hanahan 1983) was used for all techniques requiring E. coli, except when nonmethylated DNA was required for subcloning, as in cases involving Bcn and ClaI restriction sites. In these cases, E. coli RB404 (Brent and Ptashne 1980), containing the dam-3 and dam-6 mutations, was used. L broth, TYP, 2 x YT, and M9 minimal media were used to grow E. coli at 37"C, where required (Sambrook et al. 1989). Gene cloning and related methods The preparation of plasmid DNA, fragment purifications, ligations, and other necessary genetic manipulations were carried out using standard methods (Sarnbrook et al. 1989). Plasrnids were prepared by either the boiling method of Holmes and Quigley (1981) or the alkaline technique of Birnboim and Doly (1979). Plasmids were introduced into E. coli in all cases by electrotransformation (Dower et al. 1988; Gilchrist and Smit 1991). Nucleotide sequence analysis of the S-layer gene DNA sequence analysis was performed by the dideoxy chain termination method (Sanger et al. 1977). Two methods were used to generate single-stranded template DNA: the M 13mp18 and M13mp19 single-stranded DNA phage system (Vieira and Messing 1987) and the pTZ plasmid vector system (United States Biochemical Corp., Cleveland, Ohio). Helper phage M 13K07 was used to produce single-stranded DNA from cells carrying pTZ-rsaA constructs. In both cases, single-stranded DNA phage were precipitated from supernatants by additions of polyethylene glycol to 3.3% and ammonium acetate to 430 mM. Precipitated phage were suspended in 4.5 M sodium perchlorate, and the liberated DNA was bound to glass filters, followed by extensive washing with 70% ethanol. Single-stranded template DNA was recovered from the filters in a solution of 1 mM Tris and 100 pM EDTA, and the DNA concentration was estimated by visual examination of bands obtained from agarose gel electrophoresis. Sequencing reactions were performed using the Sequenase kit (United States Biochemical Corp., Cleveland, Ohio) with [cY-~'s]~ATP, following the protocol supplied with the kit. Alternatively, T7 DNA polymerase, deoxy-NTPs (including 7-deaza GTP), and dideoxy-NTPs were purchased from Pharmacia (Baie d'Urfe, Que.) and sequencing reactions run according to supplied protocols. Reaction products were separated on 5-6% polyacrylamide-urea gels. The region extending from approximately 400 bp upstream of the beginning of rsaA to approximately 800 bp into the gene was reevaluated (see Fisher et al. 1988) by generating a series of overlapping, exonuclease I11 derived deletion clones of the original E,Ava fragment (Fisher et al. 1988) in pGEM-7zf (Promega, Madison, Wis.), using the procedure of Henikoff (1987). Sequence was generated from single-stranded template produced from this plasmid by standard methods (Vieira and Messing 1987). Free energies for mRNA secondary structure were calculated according to the method of Tinoco et al. (1973). Oligodeoxyribonucleotide primers (18-22 mers) were prepared by a service within the Biochemistry department of the University of British Columbia. They were assembled on an Applied Biosystems model 380B DNA synthesizer, using P-cyanoethylN,N-diisopropylamino phosphoramidites (Sinha et al. 1984). Oligonucleotides were purified by C 18 SEP-PAK minicolumns, as described in Atkinson and Smith (1984).
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GILCHRIST ET AL.
F I G . 1. Restriction maps of the C. crescentus chromosomal DNA region containing the rsaA gene and the DNA segments used for DNA sequencing. The closed bar indicates the rsaA coding region, with the direction of transcription being left to right. The entire coding region was sequenced contiguously in both directions. Restriction enzyme sites are indicated by upper case letters: H , HindIII; C, ClaI; B, BclI; A, AvaI; X, XhoI; P , PstI; Bm, BamHI. The positions at which oligonucleotide probes hybridized are marked with arrows. Those pointing down were used for sequencing in the 5' to 3' direction of the gene, while those pointing up were used for the 3 ' to 5 ' direction. The restriction enzyme sites were used to generate smaller fragments for sequencing. Those smaller fragments, as well as the complete HindIII to BamHI fragment, were cloned into one or more of pTZ18U, pTZ18R, pTZ19U, or pTZ19R for single-strand template production. As well, the entire HindIII to BamHI fragment was cloned into M13mp18 and M13mp19 for singlestrand template production. The AvaI site indicated was one of seven on the HindIII to BamHI fragment.
The Delaney sequence handling program (1983), which uses codon preferences to predict the proper reading frame, was used to confirm proper reading frame from sequence data. Many of the PCIGENE (Intelligenetics, Mountain View, Calif.) programs were used for sequence analysis (see below), including the FSTPSCAN program to search for homology with the Swiss Prot release 17, using the method of Myers and Miller (1988). As well, the FASTA program (Pearson and Lipman 1988) was used to scan the Swiss Prot release 15. Amino acid and peptide analysis of the RsaA protein RsaA protein was purified from aggregates composed of shed surface proteins and an insoluble red pigment produced by C. crescentus CB15 (Smit et al. 1981b), using gel filtration in the presence of sodium dodecyl sulfate, as previously described (Smit and Agabian 1984). Purified protein was hydrolyzed in 6 M HC1 at 110°C for 24 h and the amino acid composition evaluated with a Durrum D5OO amino acid analyzer. Peptides were generated from the same RsaA protein preparation by digestion at room temperature with V8 protease in 0.1 M ammonium carbonate buffer, pH 8.0. The digested protein was applied to a Aquapore RP300A reverse phase column, and peaks were eluted with a 0-100010 gradient of buffer A (0.1 % trifluoroacetic acid (TFA)) in buffer B (0.085% TFA in 70% acetonitrile). For those peaks retrievable in sufficient quantity, amino acid sequencing was done by sequential Edman degradations.
Results and Discussion Nucleotide sequence analysis Various fragments of the rsaA gene were subcloned into pTZ and M13 vectors to facilitate single-stranded sequencing (Fig. 1). The HindIII to BamHI 4.2-kb fragment was cloned into both M13mp18 and M13mp19 as well as pTZ18U, pTZ 19U, pTZ 18R, and pTZ 19R. ~ u iher t smaller subclones were put in the pTZ vectors. In these cases the universal and reverse primer annealing regions in the pTZ vectors were used to sequence the ends of the inserts. In addition, numerous sequence-derived oligonucleotide primers were generated, so that sequencing of much of the gene proceeded in a "walking" manner, such that sequence derived from one primer overlapped the hybridization region for the next (Sambrook et al. 1989). Both strands of the rsaA gene were fully sequenced 3 ' to the AvaI site indicated in Fig. 1, with all sequences overlapping. A previous study (Fisher et al. 1988) focussed on the 5 ' untranslated regions of the rsaA gene, but also included sequence and the predicted translation up to the AvaI site. During the course of the present study, using a predictive analysis of the .third position codon bias expressed in
TABLE1. Amino acid composition of the RsaA protein
Amino acid composition (mol%) from: Amino acid Ala Arg Asn ASP CYs Gln Glu G~Y His Ile Leu LYs Met Phe Pro Ser Thr T ~ P Tvr
DNA sequence
amino acid analysis
19.7
19.4
0.1 1.4
ndD 1.6
'Total Asn plus Asp; total from DNA sequence is 10.9. bnd, not determined. For the calculations this value was set to 0. 'Total Gln plus Glu; total from DNA sequence is 2.7.
Caulobacter (see below), we noted that several errors were likely present in the reported sequence, resulting in several regions where the predicted amino acid sequence was inaccurate. Accordingly, a major portion of the region 5 ' to the AvaI site was resequenced. The corrections resulted in a reading frame that was contiguous with the remainder of the gene and, using the Delaney reading frame predictive program, exhibited the strong codon bias present throughout the gene (see below). The entire gene sequence with flanking DNA is shown in Fig. 2. The open reading frame extended for 3081 nucleotides, coding for a protein of 1026 amino acids. The second and third forward frames contained 59 or 13 stop codons, respectively. The mature polypeptide, with its N-terminal methio-
CAN. J . MICROBIOL. VOL. 38, 1992
GCT~TCGACGTATGACGTTTGCTC~GCCATCGCTGCTCCCATGCGCGCCACTCGGTCGCAGGGGGTGTGGGATTTTTTTTG~ACAATCCTC -35 -10 re130K S.D.
M A Y T T A O L V T A Y T N A N L G K A P D A A T T L T L D A Y A T ATGGCCTATACGACGGCCCAGTTGGTGACTGCGTACACCAACGCCAACCTCGGCAAGGCGCCTGACGCCGCCACCACGCTGACGCTCGACGCGTACGCGA Q T Q T G G L S D A A A L T N T L K L V N S T T A V A I Q T Y Q F CTCAAACCCAGACGGGCGGCCTCTCGGACGCCGCTGCGCTGACCAACACCCTGAAGCTGGTCAACAGCACGACGGCTGTTGCCATCCAGACCTACCAGTT F T G V A P S A A G L D F L V D S T T N T N D L N D A Y Y S K F A CTTCACCGGCGTTGCCCCGTCGGCCGCTGGTCTGGACTTCCTGGTCGACTCGACCACCAACACCAACGACCTGAACGACGCGTACTACTCGAAGTTCGCT Q E N R F I N F S I N L A T G A G A G A T A F A A A Y T G V S Y A Q CAGGAAAACCGCTTCATCAACTTCTCGATCAACCTGGCCACGGGCGCCGGCGCCGGCGCGACGGCTTTCGCCGCCGCCTACACGGGCGTTTCGTACGCCC
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T V A T A Y D K I I G N A V A T A A G V D V A A A V A F L S R Q A AGACGGTCGCCACCGCCTATGACAAGATCATCGGCAACGCCGTCGCGACCGCCGCTGGCGTCGACGTCGCGGCCGCCGTGGCTTTCCTGAGCCGCCAGGC N I D Y L T A F V R A N T P F T A A A D I D L A V K A A L I G T I CAACATCGACTACCTGACCGCCTTCGTGCGCGCCAACACGCCGTTCACGGCCGCTGCCGACATCGATCTGGCCGTCAAGGCCGCCCTGATCGGCACCATC L N A A T V S G I G G Y A T A T A A M I N D L S D G A L S T D N A A CTGAACGCCGCCACGGTGTCGGGCATCGGTGGTTACGCGACCGCCACGGCCGCGATGATCAACGACCTGTCGGACGGCGCCCTGTCGACCGACAACGCGG G V N L F T A Y P S S G V S G S T L S L T T G T D T L T G T A N N CTGGCGTGAACCTGTTCACCGCCTATCCGTCGTCGGGCGTGTCGGGTTCGACCCTCTCGCTGACCACCGGCACCGACACCCTGACGGGCACCGCCAACAA D T F V A G E V A G A A T L T V G D T L S G G A G T D V L N W V Q CGACACGTTCGTTGCGGGTGAAGTCGCCGGCGCTGCGACCCTGACCGTTGGCGACACCCTGAGCGGCGGTGCTGGCACCGACGTCCTGAACTGGGTGCAA
A A A V T A L P T G V T I S G I E T M N V T S G A A I T L N T S S G GCTGCTGCGGTTACGGCTCTGCCGACCGGCGTGACGATCTCGGGCATCGAAACGATGAACGTGACGTCGGGCGCTGCGATCACCCTGAACACGTCTTCGG V T G L T A L N T N T S G A A Q T V T A G A G Q N L T A T T A A Q GCGTGACGGGTCTGACCGCCCTGAACACCAACACCAGCGGCGCGGCTCAAACCGTCACCGCCGGCGCTGGCCAGAACCTGACCGCCACGACCGCCGCTCA A A N N V A V D G G A N V T V A S T G V T S G T T T V G A N S A A AGCCGCGAACAACGTCGCCGTCGACGGGCGCGCCAACGTCACCGTCGCCTCGACGGGCGTGACCTCGGGCACGACCACGGTCGGCGCCAACTCGGCCGCT S G T V S V S V A N S S T T T T G A I A V T G G T A V T V A Q T A G TCGGGCACCGTGTCGGTGAGCGTCGCGAACTCGAGCACGACCACCACGGGCGCTATCGCCGTGACCGGTGGTACGGCCGTGACCGTGGCTCAAACGGCCG N
A
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GCAACGCCGTGAACACCACGTTGACGCAAGCCGACGTGACCGTGACCGGTAACTCCAGCACCACGGCCGTGACGGTCACCCAAACCGCCGCCGCCACCGC
G A T V A G R V N G A V T I T D S A A A S A T T A G K I A T V T L CGGCGCTACGGTCGCCGGTCGCGTCAACGGCGCTGTGACGATCACCGACTCTGCCGCCGCCTCGGCCACGACCGCCGGCAAGATCGCCACGGTCACCCTG G S F G A A T I D S S A L T T V N L S G T G T S L G I G R G A L T A GGCAGCTTCGGCGCCGCCACGATCGACTCGAGCGCTCTGACGACCGTCAACCTGTCGGGCACGGGCACCTCGCTCGGCATCGGCCGCGGCGCTCTGACCG T
P
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CCACGCCGACCGCCAACACCCTGACCCTGAACGTCAATGGTCTGACGACGACCGGCGCGATCACGGACTCGGAAGCGGCTGCTGACGATGGTTTCACCAC
I N I A G S T A S S T I A S L V A A D A T T L N I S G D A R V T I CATCAACATCGCTGGTTCGACCGCCTCTTCGACGATCGCCAGCCTGGTGGCCGCCGACGCGACGACCCTGAACATCTCGGGCGACGCTCGCGTCACGATC T S H T A A A L T G I T V T N S V G A T L G A E L A T G L V F T G G ACCTCGCACACCGCTGCCGCCCTGACGGGCATCACGGTGACCAACAGCGTTGGTGCGACCCTCGGCGCCGAACTGGCGACCGGTCTGGTCTTCACGGGCG
A G A D S I L L G A T T ~ A I V M G A G D D T V T V S S A T L G GCGCTGGCCGTGACTCGATCCTGCTGGGCGCCACGACCAAGGCGATCGTCATGGGCGCCGGCGACGACACCGTCACCGTCAGCTCGGCGACCCTGGGCGC G G S V N G G D G T D V L V A N V N G S S F S A D P A F G G F E T TGGTGGTTCGGTCAACGGCGGCGACGGCACCGACGTTCTGGTGGCCAACGTCAACGGTTCGTCGTTCAGCGCTGACCCGGCCTTCGGCGGCTTCGAAACC L R V A G A A A Q G S H N A N G F T A L Q L G A T A G A T T F T N V CTCCGCGTCGCTGGCGCGGCGGCTCAAGGCTCGCACAACGCCAACGGCTTCACGGCTCTGCAACTGGGCGCGACGGCGGGTGCGACGACCTTCACCAACG
A V N V G L T V L A A P T G T T T V T L A N A T G T S D V F N L T TTGCGGTGAATGTCGGCCTGACCGTTCTGGCGGCTCCGACCGGTACGACGACCGTGACCCTGGCCAACGCCACGGGCACCTCGGACGTGTTCAACCTGAC L S S S A A L A A G T V A L A G V E T V N I A A T D T N T T A H V CCTGTCGTCCTCGGCCGCTCTGGCCGCTGGTACGGTTGCGCTGGCTGGCGTCGAGACGGTGAACATCGCCGCCACCGACACCAACACGACCGCTCACGTC D T L T L Q A T S A K S I V V T G N A G L N L T N T G N T A V T S F GACACGCTGACGCTGCAAGCCACCTCGGCCAAGTCGATCGTGGTGACGGGCAACGCCGGTCTGAACCTGACCAACACCGGCAACACGGCTGTCACCAGCT
D A S A V T G T G S A V T F V S A N T T V G E V V 2 I B G G B G A TCGACGCCAGCGCCGTCACCGGCACGGCTCCGGCTGTGACCTTCGTGTCGGCCAACACCACGGTGGGTGAAGTCGTCACGATCCGCGGCGGCGCTGGCGC
* * * * R S L I G S B I A U R I L L G G A G A R I L Y Y I G G I R I E I G CGACTCGCTGACCGGTTCGGCCACCGCCAATGACACCATCATCGGTGGCGCTGGCGCTGACACCCTGGTCTACACCGGCGGTACGGACACCTTCACGGGT
* G I G B Q I F D I N A I G T S T A F V T I T D A A V G D K L D L V G GGCACGGGCGCGGATATCTTCGATATCAACGCTATCGGCACCTCGACCGCTTTCGTGACGATCACCGACGCCGCTGTCGGCGACAAGCTCGACCTCGTCG I S T N G A I A D G A F G A A V T L G A A A T L A Q Y L D A A A A GCATCTCGACGAACGGCGCTATCGCTGACGGCGCCTTCGGCGCTGCGGTCACCCTGGGCGCTGCTGCGACCCTGGCTCAGTACCTGGACGCTGCTGCTGC G D G S G T S V A K W F Q F G G D T Y V V V D S S A G A T F V S G CGGCGACGGCAGCGGCACCTCGGTTGCCAAGTGGTTCCAGTTCGGCGGCGACACCTATGTCGTCGTTGACAGCTCGGCTGGCGCGACCTTCGTCAGCGGC A
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GCTGACGCGGTGATCAAGCTGACCGGTCTGGTCACGCTGACCACCTCGGCCTTCGCCACCGAAGTCCTGACGCTCGCCTAAGCGAACGTCTGATCCTCGC CTAGGCGAGGATCGCTAGACTAAGAGACCCCGTCTTCCGAAAGGGAGGCGGGGTCTTTCTTATGGGCGCTACGCGCTGGCCGGCCTTGCCTAGTTCCGGT
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AND
JOHN S M I T ~ Department of Microbiology, University of British Columbia, Vancouver, B.C., Canada V6T 1 2 3 Received July 22, 1991 Revision received November 7, 1991 Accepted November 12, 1991 GILCHRIST,A., FISHER,J. A., and SMIT,J. 1992. Nucleotide sequence analysis of the gene encoding the Caulobacter crescentus paracrystalline surface layer protein. Can. J . Microbiol. 38: 193-202. The entire nucleotide sequence of the rsaA gene, encoding the paracrystalline surface (S) layer protein (RsaA) of Caulobacter crescentus CBISA, was determined. The rsaA gene encoded a protein of 1026 amino acids, with a predicted molecular weight of 98 132. Protease cleavage of mature RsaA protein and amino acid sequencing of retrievable peptides yielded two peptides: one aligned with a region approximately two-thirds the way into the predicted amino acid sequence and the second peptide corresponded to the predicted carboxy terminus. Thus, no cleavage processing of the carboxy portion of the RsaA protein occurred during export, and with the exception of the removal of the initial methionine residue, the protein was not processed by cleavage to produce the mature protein. The predicted RsaA amino acid profile was unusual, with small neutral residues predominating. Excepting aspartate, charged amino acids were in relatively low proportion, resulting in an especially acidic protein, with a predicted pI of 3.46. As with most other sequenced S-layer proteins, RsaA contained no cysteine residues. A homology scan of the Swiss Protein Bank 17 produced no close matches to the predicted RsaA sequence. However, RsaA protein shared measurable homology with some exported proteins of other bacteria, including the hemolysins. Of particular interest was a specific region of the RsaA protein that was homologous to the repeat regions of glycine and aspartate residues found in several proteases and hemolysins. These repeats are implicated in the binding of calcium for proper structure and biological activity of these proteins. Those present in the RsaA protein may perform a similar function, since S-layer assembly and surface attachment requires calcium. RsaA protein also shared some homology with 10 other S-layer proteins, with the Campylobacter fetus S-layer protein scoring highest. Key words: Caulobacter crescentus, surface layer, nucleotide sequence, rsaA , calcium. GILCHRIST,A., FISHER,J. A., et SMIT,J . 1992. Nucleotide sequence analysis of the gene encoding the Caulobacter crescentus paracrystalline surface layer protein. Can. J . Microbiol. 38 : 193-202. Nous avons determine la sequence nucleotidique complete du gene rsaA qui code la proteine (RsaA) presente dans la couche de surface (S) paracrystalline chez Caulobacter crescentus CBISA. Ce gene rsaA code pour une proteine de 1026 acides amines et de poids moleculaire de 98 132. Le clivage par des proteases de la proteine RsaA mature et le sequencage des acides amines des peptides recouvrables a produit deux peptides : un s'allignant avec une region localisee approximativement jusqu'au deux tiers de la sequence peptidique predite et un deuxieme correspondant a l'extremite carboxyterminale predite: Ainsi aucune maturation par clivage de la portion carboxyle de la proteine RsaA n'a eu lieu durant l'exportation. A l'exception de l'elimination du residu methionine initial, la proteine n'a pas subi de maturation par clivage pour produire la proteine mature. La composition en acides amines predite pour RsaA etait inhabituelle a cause d'une predominance en petits residus neutres. Mise a part l'aspartate, la proportion des acides amines charges etait relativement faible expliquant ainsi que la proteine est particulierement acide avec un pI prevu a 3'46. Comme pour la plupart des autres proteines sequencees de la couche S, la RsaA ne contenait pas de residu de cysteine. Une recherche d'homologie dans la Swiss Protein Bank 17 n'a pas permis de detecter de sequence apparentee a la sequence predite pour RsaA. Cette proteine RsaA possedait cependant un degre d'homologie mesurable avec les proteines secretees par d'autres bacteries incluant des hemolysines. Une region precise de la proteine RsaA presente un intbet particulier par son homologie a des regions repetitives dans les residus glycine et aspartate rencontres dans certaines proteases et hemolysines. Ces repetitions jouent un r81e dans la fixation du calcium en contribuant a la structure et a l'activite biologique de ces proteines. Celles presentes dans la proteine RsaA pourraient jouer un r61e semblable puisque l'assemblage de la couche S et l'attachement en surface requierent du calcium. La proteine RsaA partage une certaine homologie avec dix autres proteines de la couche S mais c'est celle de Campylobacter fetus qui vient au premier rang. Mots clks : Caulobacter crescentus, couche de surface, sequence nucleotidjque, rsaA, calcium. [Traduit par la redaction]
'present address: Ribogene, Menlo Park, CA 94025, U.S.A. 2 ~ u t h o to r whom all correspondence should be addressed. Printed in Canada / lmprime au Canada
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CAN. J. MICROBIOL. VOL. 38, 1992
Introduction Paracrystalline surface arrays or S-layers have been described from species across the eubacterial and archaebacterial spectrum (Sleytr and Messner 1988b; Smit 1986). They consist of regularly arranged protein subunits that form two-dimensional arrays on the surface of the cell wall in Gram-negative and -positive bacteria; in archaebacteria they are external to the plasma membrane and often are the cell wall. Although the functions of most known S-layers have not been fully ascertained, they certainly relate to their position on the cell. To produce the large amount of protein required to sheath the cell, one can assume that much of a bacterium's energies are devoted to this endeavor. This, coupled with the fact that S-layers are often lost in laboratory culture, has led to proposals that they play specific and essential roles in their natural environments. Thus, functions may include steric protection of cells from external influences, working as molecular sieves, and preventing lytic enzymes, bacteriophages, parasitic bacteria, or foreign DNAs from contacting the hidden membranes. Caulobacter crescentus is a Gram-negative dimorphic bacterium that inhabits many aquatic environments (Anast and Smit 1988; MacRae and Smit 1991; Poindexter 1981). The major component of the C. crescentus S-layer is the apparent M, 105 000 protein (RsaA protein, formerly referred to as 130K); it is probably the most abundant protein of the cell, accounting for approximately 5% of total cell protein synthesis (Agabian et al. 1979; Smit and Agabian 1984). The protein forms a hexagonal array and, with the possible exception of an exopolysaccharide associated with the membranes (Ravenscroft et al. 1991) and lipopolysaccharide, is the outermost layer of the cell (Smit et al. 1981a). Subunit assembly of the S-layer occurs in two distinct ways: either as random addition of subunits within the preexisting array, or de novo assembly at the specific sites of stalk elongation or along the cell division plane (Smit and Agabian 1982). These latter cases of spatially restricted array formation are temporally regulated, occurring at specific stages of the life cycle. Since the RsaA protein is synthesized at a constant rate during the cell cycle (Agabian et al. 1979; Fisher et al. 1988) from a single-copy gene, it is difficult to propose how these two processes are coordinated. Indeed the entire transport journey across the inner and outer membranes, culminating with final addition to the S-layer supramolecule, is likely to prove a complex process. It is of interest to us to learn in molecular detail how the S-layer is exported, assembled, and attached to the cell surface; toward that end, primary sequence information was essential. The rsaA gene was cloned from C. crescentus CB15A (Smit and Agabian 1984) and the site of transcription initiation determined (Fisher et al. 1988). The site of translation initiation was also determined by alignment with protein sequencing of the first 21 amino acids of the mature protein. Unlike other sequenced S-layers, there was no cleaved signal leader peptide. This report also details the completion and clarification of sequence determination for the entire large protein and attempts to define functional regions. A likely region related to the role of calcium in surface attachment and self assembly was noted, but with respect to a mechanism of export, we learned that this protein has no clear analogy to other characterized exported
bacterial proteins. Eleven bacterial S-layer protein genes, including RsaA, have been sequenced and are here compared with each other.
Materials and methods Bacterial strains and growth media Caulobacter crescentus CBl5A (ATCC 19089) was grown at 30°C, with shaking, in a peptone yeast extract (PYE) medium (Poindexter 1964), supplemented with 20 mM MgCl, and 10 mM CaCl,. Escherichia coli DH5aF1 (Hanahan 1983) was used for all techniques requiring E. coli, except when nonmethylated DNA was required for subcloning, as in cases involving Bcn and ClaI restriction sites. In these cases, E. coli RB404 (Brent and Ptashne 1980), containing the dam-3 and dam-6 mutations, was used. L broth, TYP, 2 x YT, and M9 minimal media were used to grow E. coli at 37"C, where required (Sambrook et al. 1989). Gene cloning and related methods The preparation of plasmid DNA, fragment purifications, ligations, and other necessary genetic manipulations were carried out using standard methods (Sarnbrook et al. 1989). Plasrnids were prepared by either the boiling method of Holmes and Quigley (1981) or the alkaline technique of Birnboim and Doly (1979). Plasmids were introduced into E. coli in all cases by electrotransformation (Dower et al. 1988; Gilchrist and Smit 1991). Nucleotide sequence analysis of the S-layer gene DNA sequence analysis was performed by the dideoxy chain termination method (Sanger et al. 1977). Two methods were used to generate single-stranded template DNA: the M 13mp18 and M13mp19 single-stranded DNA phage system (Vieira and Messing 1987) and the pTZ plasmid vector system (United States Biochemical Corp., Cleveland, Ohio). Helper phage M 13K07 was used to produce single-stranded DNA from cells carrying pTZ-rsaA constructs. In both cases, single-stranded DNA phage were precipitated from supernatants by additions of polyethylene glycol to 3.3% and ammonium acetate to 430 mM. Precipitated phage were suspended in 4.5 M sodium perchlorate, and the liberated DNA was bound to glass filters, followed by extensive washing with 70% ethanol. Single-stranded template DNA was recovered from the filters in a solution of 1 mM Tris and 100 pM EDTA, and the DNA concentration was estimated by visual examination of bands obtained from agarose gel electrophoresis. Sequencing reactions were performed using the Sequenase kit (United States Biochemical Corp., Cleveland, Ohio) with [cY-~'s]~ATP, following the protocol supplied with the kit. Alternatively, T7 DNA polymerase, deoxy-NTPs (including 7-deaza GTP), and dideoxy-NTPs were purchased from Pharmacia (Baie d'Urfe, Que.) and sequencing reactions run according to supplied protocols. Reaction products were separated on 5-6% polyacrylamide-urea gels. The region extending from approximately 400 bp upstream of the beginning of rsaA to approximately 800 bp into the gene was reevaluated (see Fisher et al. 1988) by generating a series of overlapping, exonuclease I11 derived deletion clones of the original E,Ava fragment (Fisher et al. 1988) in pGEM-7zf (Promega, Madison, Wis.), using the procedure of Henikoff (1987). Sequence was generated from single-stranded template produced from this plasmid by standard methods (Vieira and Messing 1987). Free energies for mRNA secondary structure were calculated according to the method of Tinoco et al. (1973). Oligodeoxyribonucleotide primers (18-22 mers) were prepared by a service within the Biochemistry department of the University of British Columbia. They were assembled on an Applied Biosystems model 380B DNA synthesizer, using P-cyanoethylN,N-diisopropylamino phosphoramidites (Sinha et al. 1984). Oligonucleotides were purified by C 18 SEP-PAK minicolumns, as described in Atkinson and Smith (1984).
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GILCHRIST ET AL.
F I G . 1. Restriction maps of the C. crescentus chromosomal DNA region containing the rsaA gene and the DNA segments used for DNA sequencing. The closed bar indicates the rsaA coding region, with the direction of transcription being left to right. The entire coding region was sequenced contiguously in both directions. Restriction enzyme sites are indicated by upper case letters: H , HindIII; C, ClaI; B, BclI; A, AvaI; X, XhoI; P , PstI; Bm, BamHI. The positions at which oligonucleotide probes hybridized are marked with arrows. Those pointing down were used for sequencing in the 5' to 3' direction of the gene, while those pointing up were used for the 3 ' to 5 ' direction. The restriction enzyme sites were used to generate smaller fragments for sequencing. Those smaller fragments, as well as the complete HindIII to BamHI fragment, were cloned into one or more of pTZ18U, pTZ18R, pTZ19U, or pTZ19R for single-strand template production. As well, the entire HindIII to BamHI fragment was cloned into M13mp18 and M13mp19 for singlestrand template production. The AvaI site indicated was one of seven on the HindIII to BamHI fragment.
The Delaney sequence handling program (1983), which uses codon preferences to predict the proper reading frame, was used to confirm proper reading frame from sequence data. Many of the PCIGENE (Intelligenetics, Mountain View, Calif.) programs were used for sequence analysis (see below), including the FSTPSCAN program to search for homology with the Swiss Prot release 17, using the method of Myers and Miller (1988). As well, the FASTA program (Pearson and Lipman 1988) was used to scan the Swiss Prot release 15. Amino acid and peptide analysis of the RsaA protein RsaA protein was purified from aggregates composed of shed surface proteins and an insoluble red pigment produced by C. crescentus CB15 (Smit et al. 1981b), using gel filtration in the presence of sodium dodecyl sulfate, as previously described (Smit and Agabian 1984). Purified protein was hydrolyzed in 6 M HC1 at 110°C for 24 h and the amino acid composition evaluated with a Durrum D5OO amino acid analyzer. Peptides were generated from the same RsaA protein preparation by digestion at room temperature with V8 protease in 0.1 M ammonium carbonate buffer, pH 8.0. The digested protein was applied to a Aquapore RP300A reverse phase column, and peaks were eluted with a 0-100010 gradient of buffer A (0.1 % trifluoroacetic acid (TFA)) in buffer B (0.085% TFA in 70% acetonitrile). For those peaks retrievable in sufficient quantity, amino acid sequencing was done by sequential Edman degradations.
Results and Discussion Nucleotide sequence analysis Various fragments of the rsaA gene were subcloned into pTZ and M13 vectors to facilitate single-stranded sequencing (Fig. 1). The HindIII to BamHI 4.2-kb fragment was cloned into both M13mp18 and M13mp19 as well as pTZ18U, pTZ 19U, pTZ 18R, and pTZ 19R. ~ u iher t smaller subclones were put in the pTZ vectors. In these cases the universal and reverse primer annealing regions in the pTZ vectors were used to sequence the ends of the inserts. In addition, numerous sequence-derived oligonucleotide primers were generated, so that sequencing of much of the gene proceeded in a "walking" manner, such that sequence derived from one primer overlapped the hybridization region for the next (Sambrook et al. 1989). Both strands of the rsaA gene were fully sequenced 3 ' to the AvaI site indicated in Fig. 1, with all sequences overlapping. A previous study (Fisher et al. 1988) focussed on the 5 ' untranslated regions of the rsaA gene, but also included sequence and the predicted translation up to the AvaI site. During the course of the present study, using a predictive analysis of the .third position codon bias expressed in
TABLE1. Amino acid composition of the RsaA protein
Amino acid composition (mol%) from: Amino acid Ala Arg Asn ASP CYs Gln Glu G~Y His Ile Leu LYs Met Phe Pro Ser Thr T ~ P Tvr
DNA sequence
amino acid analysis
19.7
19.4
0.1 1.4
ndD 1.6
'Total Asn plus Asp; total from DNA sequence is 10.9. bnd, not determined. For the calculations this value was set to 0. 'Total Gln plus Glu; total from DNA sequence is 2.7.
Caulobacter (see below), we noted that several errors were likely present in the reported sequence, resulting in several regions where the predicted amino acid sequence was inaccurate. Accordingly, a major portion of the region 5 ' to the AvaI site was resequenced. The corrections resulted in a reading frame that was contiguous with the remainder of the gene and, using the Delaney reading frame predictive program, exhibited the strong codon bias present throughout the gene (see below). The entire gene sequence with flanking DNA is shown in Fig. 2. The open reading frame extended for 3081 nucleotides, coding for a protein of 1026 amino acids. The second and third forward frames contained 59 or 13 stop codons, respectively. The mature polypeptide, with its N-terminal methio-
CAN. J . MICROBIOL. VOL. 38, 1992
GCT~TCGACGTATGACGTTTGCTC~GCCATCGCTGCTCCCATGCGCGCCACTCGGTCGCAGGGGGTGTGGGATTTTTTTTG~ACAATCCTC -35 -10 re130K S.D.
M A Y T T A O L V T A Y T N A N L G K A P D A A T T L T L D A Y A T ATGGCCTATACGACGGCCCAGTTGGTGACTGCGTACACCAACGCCAACCTCGGCAAGGCGCCTGACGCCGCCACCACGCTGACGCTCGACGCGTACGCGA Q T Q T G G L S D A A A L T N T L K L V N S T T A V A I Q T Y Q F CTCAAACCCAGACGGGCGGCCTCTCGGACGCCGCTGCGCTGACCAACACCCTGAAGCTGGTCAACAGCACGACGGCTGTTGCCATCCAGACCTACCAGTT F T G V A P S A A G L D F L V D S T T N T N D L N D A Y Y S K F A CTTCACCGGCGTTGCCCCGTCGGCCGCTGGTCTGGACTTCCTGGTCGACTCGACCACCAACACCAACGACCTGAACGACGCGTACTACTCGAAGTTCGCT Q E N R F I N F S I N L A T G A G A G A T A F A A A Y T G V S Y A Q CAGGAAAACCGCTTCATCAACTTCTCGATCAACCTGGCCACGGGCGCCGGCGCCGGCGCGACGGCTTTCGCCGCCGCCTACACGGGCGTTTCGTACGCCC
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T V A T A Y D K I I G N A V A T A A G V D V A A A V A F L S R Q A AGACGGTCGCCACCGCCTATGACAAGATCATCGGCAACGCCGTCGCGACCGCCGCTGGCGTCGACGTCGCGGCCGCCGTGGCTTTCCTGAGCCGCCAGGC N I D Y L T A F V R A N T P F T A A A D I D L A V K A A L I G T I CAACATCGACTACCTGACCGCCTTCGTGCGCGCCAACACGCCGTTCACGGCCGCTGCCGACATCGATCTGGCCGTCAAGGCCGCCCTGATCGGCACCATC L N A A T V S G I G G Y A T A T A A M I N D L S D G A L S T D N A A CTGAACGCCGCCACGGTGTCGGGCATCGGTGGTTACGCGACCGCCACGGCCGCGATGATCAACGACCTGTCGGACGGCGCCCTGTCGACCGACAACGCGG G V N L F T A Y P S S G V S G S T L S L T T G T D T L T G T A N N CTGGCGTGAACCTGTTCACCGCCTATCCGTCGTCGGGCGTGTCGGGTTCGACCCTCTCGCTGACCACCGGCACCGACACCCTGACGGGCACCGCCAACAA D T F V A G E V A G A A T L T V G D T L S G G A G T D V L N W V Q CGACACGTTCGTTGCGGGTGAAGTCGCCGGCGCTGCGACCCTGACCGTTGGCGACACCCTGAGCGGCGGTGCTGGCACCGACGTCCTGAACTGGGTGCAA
A A A V T A L P T G V T I S G I E T M N V T S G A A I T L N T S S G GCTGCTGCGGTTACGGCTCTGCCGACCGGCGTGACGATCTCGGGCATCGAAACGATGAACGTGACGTCGGGCGCTGCGATCACCCTGAACACGTCTTCGG V T G L T A L N T N T S G A A Q T V T A G A G Q N L T A T T A A Q GCGTGACGGGTCTGACCGCCCTGAACACCAACACCAGCGGCGCGGCTCAAACCGTCACCGCCGGCGCTGGCCAGAACCTGACCGCCACGACCGCCGCTCA A A N N V A V D G G A N V T V A S T G V T S G T T T V G A N S A A AGCCGCGAACAACGTCGCCGTCGACGGGCGCGCCAACGTCACCGTCGCCTCGACGGGCGTGACCTCGGGCACGACCACGGTCGGCGCCAACTCGGCCGCT S G T V S V S V A N S S T T T T G A I A V T G G T A V T V A Q T A G TCGGGCACCGTGTCGGTGAGCGTCGCGAACTCGAGCACGACCACCACGGGCGCTATCGCCGTGACCGGTGGTACGGCCGTGACCGTGGCTCAAACGGCCG N
A
V
N
T
T
L
T
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A
D
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A
A
A
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A
GCAACGCCGTGAACACCACGTTGACGCAAGCCGACGTGACCGTGACCGGTAACTCCAGCACCACGGCCGTGACGGTCACCCAAACCGCCGCCGCCACCGC
G A T V A G R V N G A V T I T D S A A A S A T T A G K I A T V T L CGGCGCTACGGTCGCCGGTCGCGTCAACGGCGCTGTGACGATCACCGACTCTGCCGCCGCCTCGGCCACGACCGCCGGCAAGATCGCCACGGTCACCCTG G S F G A A T I D S S A L T T V N L S G T G T S L G I G R G A L T A GGCAGCTTCGGCGCCGCCACGATCGACTCGAGCGCTCTGACGACCGTCAACCTGTCGGGCACGGGCACCTCGCTCGGCATCGGCCGCGGCGCTCTGACCG T
P
T
A
N
T
L
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A
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CCACGCCGACCGCCAACACCCTGACCCTGAACGTCAATGGTCTGACGACGACCGGCGCGATCACGGACTCGGAAGCGGCTGCTGACGATGGTTTCACCAC
I N I A G S T A S S T I A S L V A A D A T T L N I S G D A R V T I CATCAACATCGCTGGTTCGACCGCCTCTTCGACGATCGCCAGCCTGGTGGCCGCCGACGCGACGACCCTGAACATCTCGGGCGACGCTCGCGTCACGATC T S H T A A A L T G I T V T N S V G A T L G A E L A T G L V F T G G ACCTCGCACACCGCTGCCGCCCTGACGGGCATCACGGTGACCAACAGCGTTGGTGCGACCCTCGGCGCCGAACTGGCGACCGGTCTGGTCTTCACGGGCG
A G A D S I L L G A T T ~ A I V M G A G D D T V T V S S A T L G GCGCTGGCCGTGACTCGATCCTGCTGGGCGCCACGACCAAGGCGATCGTCATGGGCGCCGGCGACGACACCGTCACCGTCAGCTCGGCGACCCTGGGCGC G G S V N G G D G T D V L V A N V N G S S F S A D P A F G G F E T TGGTGGTTCGGTCAACGGCGGCGACGGCACCGACGTTCTGGTGGCCAACGTCAACGGTTCGTCGTTCAGCGCTGACCCGGCCTTCGGCGGCTTCGAAACC L R V A G A A A Q G S H N A N G F T A L Q L G A T A G A T T F T N V CTCCGCGTCGCTGGCGCGGCGGCTCAAGGCTCGCACAACGCCAACGGCTTCACGGCTCTGCAACTGGGCGCGACGGCGGGTGCGACGACCTTCACCAACG
A V N V G L T V L A A P T G T T T V T L A N A T G T S D V F N L T TTGCGGTGAATGTCGGCCTGACCGTTCTGGCGGCTCCGACCGGTACGACGACCGTGACCCTGGCCAACGCCACGGGCACCTCGGACGTGTTCAACCTGAC L S S S A A L A A G T V A L A G V E T V N I A A T D T N T T A H V CCTGTCGTCCTCGGCCGCTCTGGCCGCTGGTACGGTTGCGCTGGCTGGCGTCGAGACGGTGAACATCGCCGCCACCGACACCAACACGACCGCTCACGTC D T L T L Q A T S A K S I V V T G N A G L N L T N T G N T A V T S F GACACGCTGACGCTGCAAGCCACCTCGGCCAAGTCGATCGTGGTGACGGGCAACGCCGGTCTGAACCTGACCAACACCGGCAACACGGCTGTCACCAGCT
D A S A V T G T G S A V T F V S A N T T V G E V V 2 I B G G B G A TCGACGCCAGCGCCGTCACCGGCACGGCTCCGGCTGTGACCTTCGTGTCGGCCAACACCACGGTGGGTGAAGTCGTCACGATCCGCGGCGGCGCTGGCGC
* * * * R S L I G S B I A U R I L L G G A G A R I L Y Y I G G I R I E I G CGACTCGCTGACCGGTTCGGCCACCGCCAATGACACCATCATCGGTGGCGCTGGCGCTGACACCCTGGTCTACACCGGCGGTACGGACACCTTCACGGGT
* G I G B Q I F D I N A I G T S T A F V T I T D A A V G D K L D L V G GGCACGGGCGCGGATATCTTCGATATCAACGCTATCGGCACCTCGACCGCTTTCGTGACGATCACCGACGCCGCTGTCGGCGACAAGCTCGACCTCGTCG I S T N G A I A D G A F G A A V T L G A A A T L A Q Y L D A A A A GCATCTCGACGAACGGCGCTATCGCTGACGGCGCCTTCGGCGCTGCGGTCACCCTGGGCGCTGCTGCGACCCTGGCTCAGTACCTGGACGCTGCTGCTGC G D G S G T S V A K W F Q F G G D T Y V V V D S S A G A T F V S G CGGCGACGGCAGCGGCACCTCGGTTGCCAAGTGGTTCCAGTTCGGCGGCGACACCTATGTCGTCGTTGACAGCTCGGCTGGCGCGACCTTCGTCAGCGGC A
D
A
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GCTGACGCGGTGATCAAGCTGACCGGTCTGGTCACGCTGACCACCTCGGCCTTCGCCACCGAAGTCCTGACGCTCGCCTAAGCGAACGTCTGATCCTCGC CTAGGCGAGGATCGCTAGACTAAGAGACCCCGTCTTCCGAAAGGGAGGCGGGGTCTTTCTTATGGGCGCTACGCGCTGGCCGGCCTTGCCTAGTTCCGGT
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