Vol. 58, No. 2

INFECTION AND IMMUNITY, Feb. 1990, p. 480-488 0019-9567/90/020480-09$02.00/0 Copyright ©D 1990, American Society for Microbiology

Molecular Characterization of the Clostridium difficile Toxin A Gene C. H. DOVE, S.-Z. WANG, S. B. PRICE,t C. J. PHELPS, D. M. LYERLY, T. D. WILKINS, AND J. L. JOHNSON* Department of Anaerobic Microbiology, Virginia Polytechnic Institute and State University, Blacksburg, Virginia 24061 Received 10 July 1989/Accepted 21 October 1989

The gene encoding the toxin A protein of Clostridium difficile (strain VPI 10463) was cloned and sequenced. The coding region of 8,133 base pairs has a mol% G+C of 26.9 and encodes 2,710 amino acids. The deduced polypeptide has a molecular mass of ca. 308 kilodaltons. Nearly a third of the gene, at the 3' end, consists of 38 repeating sequences. The repeating units were grouped into two classes, I and II, on the basis of length and the low levels of DNA sequence similarities between them. There were seven class I repeating units, each containing 90 nucleotides, and 31 class II units, which, with two exceptions, were either 60 or 63 nucleotides in length. On the basis of DNA sequence similarities, the class II repeating units were further segregated into subclasses: 7 class IIA, 13 class IIB, 5 class IIC, and 6 class IID. The dipeptide tyrosine-phenylalanine was found in all 38 repeating units, and other amino acid sequences were unique to a specific class or subclass. This region of the protein has epitopes for the monoclonal antibody PCG-4 and includes the binding region for the Galoa1-3Gal01-4GlcNAc carbohydrate receptor. Located 1,350 base pairs upstream from the toxin A translation start site is the 3' end of the toxin B gene. Between the two toxin genes is a small open reading frame, which encodes a deduced polypeptide of ca. 16 or 19 kilodaltons. The role of this open reading frame is unknown.

Clostridium difficile is the major causative agent of pseudomembranous colitis in humans (2). The organism produces two toxins, designated toxin A and toxin B (1, 32, 33). They are both cytotoxic and lethal for animals, although toxin B is about 1,000-fold more cytotoxic than toxin A for most cell lines. Both toxins appear to be produced in all toxigenic strains; however, the toxicity of strains may vary by several orders of magnitude (20, 32). The actions of these toxins appear to be quite complex and at present are not understood. Although toxin A has a direct toxic effect on the intestinal mucosa, toxin B does not cause a significant response when given intragastrically to hamsters, unless it is initially mixed with a small amount of toxin A (19). Alternatively, toxin B is also toxic if it is given to hamsters with bruised (injured) ceca. The results are consistent with the initial binding and primary tissue damage being caused by toxin A or by mechanical injury, followed by the entry of toxin B. Investigators in a number of laboratories have worked on the isolation and physical properties of the toxins (1, 2, 27, 30, 32). Both toxins have been purified to homogeneity (18, 32). An interesting and controversial property of the toxins has been their molecular weights. Initial molecular weight estimations obtained by using native proteins have ranged from 440,000 to 600,000 for toxin A and 360,000 to 500,000 for toxin B (1, 30, 32). However, in later studies there has been controversy as to whether the toxins dissociate into smaller subunits under denaturing conditions. Under these conditions, size estimations for the toxins range from 300,000 (18) to 50,000 (27, 30) and down to 42,500 and 16,000 (29). These discrepancies have been summarized by Lyerly et al. (16) and are difficult to explain (35). Perhaps in some of the isolation procedures a smaller contaminating protein copurified with the toxins and tended to mask the toxins in the polyacrylamide gels. The best approach to resolve this controversy is to clone and sequence the toxin genes.

Several investigators have begun cloning these genes. Muldrow and his collaborators (26) have reported the cloning of a 0.3-kilobase-pair (kb) fragment of the toxin A gene in the lambda bacteriophage expression vector gtll. The expressed peptides reacted with toxin A polyvalent antisera. When the cloned fragment from toxin A was used as a labeled probe, it reacted with a PstI-generated fragment of C. diJficile DNA, which they estimated as 4.5 kb. We have cloned a 4.7-kb PstI fragment into a plasmid vector (28). This fragment has an internal PstI site which is protected from digestion in the C. difficile DNA. When this fragment is expressed, the peptide reacts with both toxin A affinitypurified polyclonal antisera and with the monoclonal antibody PCG-4 (17). Preliminary results on the sequencing of this fragment have shown that there are many repeating sequence units within the fragment (14). Eichel-Streiber et al. (8) have recently cloned portions of the 4.7-kb PstI fragment into a plasmid expression vector and obtained an expression product that also reacted with toxin A antisera. Wren et al. (36) have reported the cloning of toxin A in lambda phage. The clone expressed a protein that caused elongation of Chinese hamster ovary cells, and this protein had an estimated molecular weight of 235,000. In this study, we have completed the cloning and sequencing of toxin A and its flanking regions. Nearly one-third of the gene (from the 3' end) consists of a series of repeating units which appear to code for the receptor portion of the toxin. MATERIALS AND METHODS Bacteria, bacteriophages, and plasmids. DNA isolated from C. difficile VPI 10463 was used for cloning. Plasmids pBR322, pUC18, and pUC19 were used for the primary cloning of C. difficile DNA fragments. Subclones in the M13 phages mpl8 and mpl9 were used for DNA sequencing. The plasmids, phages, and Escherichia coli host strains JM109, DH5ot, and DHScF' were all obtained from Bethesda Research Laboratories, Inc. E. coli strain Chi 1776 was purchased from the American Type Culture Collection. Enzymes and radiolabeled compounds. Restriction endonu-

Corresponding author. t Present address: Department of Microbiology and Immunology, *

University of Kentucky Medical Center, Lexington, KY 40536.

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CHARACTERIZATION OF THE C. DIFFICILE TOXIN A GENE

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clease enzymes were purchased from Bethesda Research Laboratories, Inc., International Biotechnologies, Inc., or Promega Biotec. Endonucleases III and VII, E. coli DNA polymerase I, and the polymerase I Klenow fragment were obtained from Bethesda Research Laboratories, Inc. DNA ligase and calf alkaline phosphatase were purchased from Boehringer Mannheim Biochemicals. The T7 DNA polymerase, Sequenase, was purchased from U.S. Biochemical. The labeled nucleotide triphosphate [a-35S]dATP was obtained from New England Nuclear. Random primer labeling kits were obtained from International Biotechnologies, Inc. All of the enzymes were used according to the instructions provided by the manufacturers. DNA isolations. High-molecular-weight C. difficile DNA was isolated by using a variation of the Marmur procedure (13, 22). The harvested cells (12) were suspended in 50 mM Tris-1 mM EDTA buffer (pH 8.0). After 50 ,ug of lysozyme per ml was added, the cell suspension was incubated at 37°C until the cells were susceptible to sodium dodecyl sulfate disruption. At the time of disruption, EDTA was added to a final concentration of 40 mM, proteinase K was added to a final concentration of 30 to 50 ,ug/ml, and ,B-mercaptoethanol was added to a concentration of 1% to inhibit endogenous nuclease activity in the lysate. Plasmid DNA and the replicating-form DNA of M13 phage were isolated by the Birnboim and Doly alkaline lysis procedure (4). DNA preparations used for probe fragment generation and nested deletions were further purified by CsCl centrifugation. Specific DNA restriction fragments were separated from others on low-melting-point agarose, and the individual bands were cut from the gel for use in the random priming labeling procedure. When restriction fragments of C. difficile DNA were needed in a particular size range (i.e., for cloning by chromosome walking), the gel was cut at the lower size range and this part was removed. A well was then cut into the gel at the upper size range, the polarity of the electrophoresis unit was reversed, and the fragments were electroeluted into the well. This tended to concentrate the fragments entering the well and resulted in a lower eluate volume. The fragments were then ethanol precipitated. Primary cloning. Of the four primary clones used in this study (Fig. 1), three were cloned by the chromosome walking approach. The 2.6-kb PstI fragment of pCD11 was used as a probe for cloning pCD11L, the 0.5-kb HindIII-EcoRI fragment of pCD11L was used as a probe for cloning pCD17,

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which was then used as a probe to clone pCD19, and pCD11 was used to detect clone pCD11R-6. The cloning was carried out under EK-2, BL-2 containment with E. coli Chi 1776 as host. Cells were made competent by the Hanahan procedure (11, 21). Toxicity assays. Lysates from each primary clone were checked for animal and cell toxicity. Mouse lethality tests were performed by injecting five 8-week-old BALB/c mice (Dominion Laboratories, Dublin, Va.) intraperitoneally with 200 ,ul of lysate and observing them for illness or death. Cytotoxicity was checked in the Chinese hamster ovary (CHO) cell assay by following a procedure previously described (7). Lysates of E. coli Chi 1776 transformed with pUC18 were used as negative controls. DNA sequencing and sequence analysis. Both strands of the DNA were sequenced by using the dideoxy-chain termination procedure developed by Sanger et al. (3, 31). DNA fragments were cloned into M13, and nested deletions were generated in replicating-form DNA by using the exonuclease III and exonuclease VII procedure (37). Restriction sites used for subcloning were sequenced across by using oligonucleotide primers and double-stranded sequencing. Synthetic oligonucleotide primers were also used for filling occasional sequence gaps not covered by the nested deletions. Sequence analysis was done by using the Pustell programs from International Biotechnologies, Inc., and the Sequence Analysis Software Package from the Genetics Computer Group, University of Wisconsin. The data bases that were searched included the GenBank data base and the National Biomedical Research Foundation Protein Sequence Data Base. Unweighted Pair Group cluster analysis was done by using the NTSYS-pc programs (F. J. Rohlf, Exeter Publishing, Ltd.). N-terminal sequencing. Toxin A was purified from culture filtrates of C. difficile VPI 10463 by sequential ammonium sulfate precipitation, ion-exchange chromatography, and precipitation at pH 5.6 as previously described (18). The highly purified protein was denatured with a final concentration of 2.5% sodium dodecyl sulfate-5% 2-mercaptoethanol at 100°C for 2 min and subjected to sodium dodecyl sulfatepolyacrylamide gel electrophoresis. After electrophoresis, the protein was transferred to polyvinylidene difluoride membranes by electroblotting and the N-terminal amino acid

INFECT. IMMUN.

DOVE ET AL.

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MAT ATA BAT ATA TAT TNT MIT MAA TM MAA MCA TCI 11A TCT MAA MAC MCT ATA TNT MAC MAAMT BIT MGA MAT BIT ITA TA GAG CCT ATA TAT MAT CCT BAT MBC BIT MAA BAT ATA E El 1E I V B E I T I F ISD CVW K T IllS NV V V E P KIST I F S B PSD T ICSE

5001

TCT MCT TCA CTA BAT TNT TCC TAT MAA CAT ClT TAT MA ATA MAT MGA TAT ATA MAT MAA GTA TIG ATA BCA CAT BAT TrA TAT MCA MCT ITA ATA MAT AIT MAT AC MAT TAT TAT TCA E NE I ITS V P IL K L I A P L SLITT IL SF11 INE I NET S T TB6 I SERT

1121

MAT MAG TM TMC CCT MA AlT ATA BIT CIT AMC CCA MAT MCA TC CM MAA MAA ITA MAT ATA MAT ITA MAT MgT TCT tCT TIT GMA TAT MAA 211 TCT MCA GMA SMA MCT MAC TNT AIT N F IBI S IT NE L S S ISIF E 1KV W Elf I I PCt I I V L E PENT FEN K K V E

141

ITA BIT MGA TMC ITA MAA MAMT MAT MAA MAA ATA ITA CMA MAA ATA MGA AlTC MAA BIT AltC ITA TCT MAT MCT CMA tCA TTT MAT MAA AlT MCT ATA BAT TNT MAA BAT ATT MAA MAA K N LIE I F E KEN I K Q B TQ I E K NER NI DSF KSD K K I L L IEII ElT L V

1211

CTA TCA ITA SM TAT ATA AlTG MGT MAT TNT MAA lTCA TNT MAT TCT GMAMT MAA ITA BAT MGA MAT CAT ITA GOA TNT MAA ATA ATA BAT MAT MAA MCT TAT TMC TAT BAT MAA BAT MCT C I I IS SI E B SE IN K T B E F K K S F NL I ES E El L DES I N ES F S L L

1441

MCT AlT MAT BIT MAA MAA TAT TAT TNT BAT ATA MAT E I I I S T I K K F NE

1401

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1721

MT ATn MAT MT GMA TA ABA GMA ATA TCT AT I * I SE K YE K I I

MAA ITA BIT MAA MA ITA AlTC MAT ATA MAT MAT lTCA ITA TFC TAT lIT BAT CCT ATA MAA ITT AMC ITA ITA MCT K

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5911

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4ae1

TNT MAA TAT TNT MCA CCT OCT MAT MCT TAT MAT MAT AMC ATA MAA BIT CMG OCT ATA GIT TAT CMA MGT MAA TTC TTA MCT TTI MAT SIT MAA MAA E B K K Q A IQI I T L E EI C L T TI I V A P A E KF F F CE

4201

211 CMA MCT AIT BAT MOT MAA MAA TAT TMC TNT MAT MCT AMC MCT MCT MA MCA MCT MCT A TM CM MCT AlT BAT SIT MAA V Q E T E TA I A V Q I IF A I TI T K K T W SB6 K ID SI W

1321

TMG CMA MCT AIT BAT SIT MAA MAA TAT TMC ITTf MAT MCT AMC MCT MCT ATA MCT ICA MCT SIT TAT MCA AIT AIT MAT Ill I T I I E V Q T I DI6K K I T FEN T E TA I A W

4441

GTNT MfAA SM CCT MAT M ITT GMA TAT TNT MCA CCT MCT MAT AB BAT MCT AMC AMC ATA MA BIT CMA MCT BIT MAA CAT TNT TAT TNT MAT MCT BAT SIT AIT AlT C" ATA SM12 S E E I Q C I E I El A E SI I EI I V I H I I C A A F F P A T I F K P F T K F

1111

MAA TAT TMC TNT MAT CCT MAT

4411

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I

El

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PEE

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MAA TAT TMC TNT MAT MCT AMC MCT MCT MA MCA MCT MCT E TA C A A TB K I I F E T

N

A

A

V

I L

AlA CIT TMC CMA MAT MA TNC ITA MCT Ill MAT SIT MAA MAA TAT TMC TNT BIT MCT MAC TCA MAA MCA BIT MCT a"211AMA AIT AIT AMC MAT MAA V E I I E E K BIS K A V TB El K K I IF I L I Q E I F L T L

K

I

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FE0

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SM ATT CIT CMA MAT GSA TAT AIT MCT ATT MAA MGA MAT MAT TlT TAT TNT BAT MCT A S I I T I IE EEN F T F El I L Q

maD

MAT MAT MAA TCT MAA AlT GTA MCA SM ITA TNT MAA SGA CCT MAT a" ITT MAG TAT TNT MCA CCT MCT MAT MCT CMC MAT MAT AMC ATA MAA BIT CMG MCT ATA lIT TMC CMG AMC MAA I I Q I HE E E I A I V E E P F ElT El9 F C I F A P A KII 9 E K C S K E V TI4 V

421

TM CMA ACC AIT BAT SIT MAA MAA TAT TMC TNT MAT CIT AMC MCT MCT MAA MCA MT MCT I A T E L E TA V Q A T I W D5GK K I I F

7141

SMA TM CMA MCT AIT MAT SIT MAA MAA TAT TMC TNT MAT CIT AMC MCT MCT MA MCA MCT MCT SMA TM CMA MCT AIT BAT SIT MAA MAA TAT TMC ITT MAT MCT AMC MCT ITC ATA 5CC A I I FE T ElT F V Q I I E L E TA A TI T IVW Q CA W DSIK K I T DI4K K I I F

7111

TCA MCT SIT TAT MCA MGT AIT MAT SIT MAA CAT TIT TAT TNT MAT MCT BAT SIT AIT AlT CMG AlA SMA 121 TNT MAA GOA CCT MAT GOA TNT MAA TMC TNT MCA CCT MCT MAT AMB BAT S F I I A El I El F P A HF I F K P SIV I F ElT DIG I ITS El6 K SIB6 EQ9 I

7241

AMC MBA CT1 CM MT ATT MAT BIT I I SI6 T I6LE

7401

TM CMA MCT AIT MAT SIT MAA MAA TMC TM ITT MAT MCT AMC MCT TCT ATA MCT TCA MCT BIT TAT MCA AIT ATT I I I A I I I I K SIB V Q El I I F EYE TIS K W

7121

MCT MT MAA CAT TNT TAT TNT MT MCT BAT BAT AIT AlT CMG ATA SMA 121 TNT MAA SMA CCT MAT SMA TNT MAA TMC TNT MCA CAT MCT MAT MCA MAT MCT MAA MAT ATA MAA BIT CMA A EI T F F A P A E S E GI Q F C I I I B V II6 P SI I EQ HF Dl6 FEN T II6 K

711

MCT AlA CIT TAT CMA MAT MGA ITC CIA TAT ITrA CAT MAAMT ATA TAT TAT TNT SIT MAT MAT TCA MAA MB MCT MCT SIT TMG ITA MCT AIT BAT BIT MAT MGA TAT TMC ITC MAG CCT 1 1 Q E E F L IV L H S E I I I F B E E I K A A TB V V I I S B E E I I F C P I A

7711

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70I

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051

MCT AIT MAT BIT MAA GTA TAT TMC TlT AlT CCT MAT MCT MCT AlT MCT MA MCT BIT SMA CIT ITTC GM AIT MAT SIT lIT AlA TAT TTC TNT SIT ITT BAT SM ITA MAA 6CC CCT GMB

B121

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FIG. 2--Continued

V

K

A

at"

P

1

483

484

INFECT. IMMUN.

DOVE ET AL. Ii

'567145760

Al

I4

6817-6906

A2

I3

6475-6564

A4

I5

7219-7308

A3

I6

7558-7647

As

I7

7831-7920

A6

I2

6073-6162

A7 B,

90

80

100

B2

Percent DNA Sequence Similarity

B6

FIG. 3. Nucleotide sequence similarity cluster analysis of the class I repeating sequences.

83 B50

sequence was

determined by previously described methods

Bs

(23). RESULTS Primary clones of toxin A. Relationships between the five primary clones, each containing a portion of the C. difficile toxin A gene, are shown in Fig. 1. Also included in the figure is a partial restriction map of this 15-kb region of the C. difficile genome. Clone pCD11 has been partially characterized and shown to contain a carbohydrate binding region and antigenic epitopes which react with the monoclonal antibody PCG-4 (28). Clone pCD11R-6, in addition to containing the entire pCD11 insert and most of the pCD11L insert, contains the last 80 bases of the toxin A gene and approximately 4.1 kb of additional sequences downstream from the toxin A gene. The downstream region contains two open reading frames (ORFs) and part of the third, one of which is shown in Fig. 1. All of these ORFs read in the direction opposite that of the toxin A gene (data not shown). Clone pCD11L contains an additional 1.5 kb of sequence upstream of the pCD11 insert. Clone pCD17 was used as a probe for cloning pCD19. Clone pCD19 codes for the 5' end of toxin A, a small ORF that could code for a 16- or 19-kilodalton (kDa) protein and 1.2 kb of toxin B. These clones were not toxic for mice or CHO cells. The clone immediately upstream from the pCD19 insert was found to contain the remainder of the toxin B gene, and we have since been able to reconstruct the intact gene in a plasmid. The recombinant protein expressed by this plasmid is cytotoxic to tissue cells, is lethal to mice, and has immunological identity with toxin B (D. M. Lyerly and J. L. Johnson, unpublished data). Nucleotide and amino acid sequences for toxin A. The nucleotide sequence and the deduced amino acids for the toxin A gene are shown in Fig. 2 (GenBank accession number, M30307). The open reading frame is 8,133 nucleotides long and codes for 2,710 amino acids. The gene contains 26.9 mol% G+C, and the deduced protein has a molecular mass of 308,103 Da. The amino acid sequence of the N-terminal end of toxin A was determined by microanalysis after electrophoresis under denaturing conditions (23), and the first 10 amino acids agree with the first 10 deduced amino acids of the toxin A open reading frame, indicating that there are no posttranslational modifications involving a signal peptide. An interesting property of this gene is the repeating sequences at the 3' end. A total of 2,551 nucleotides, or 31.5% of the gene, are in 38 contiguous repeating units. This region extends from nucleotides 5,545 to 8,106. The repeating units were grouped into two classes, I and II, on the basis of the low levels of DNA sequence similarities between

B9 B7 B,,

B13

B12 D2 C1

C3 C5

C4 C2

Ds D4

50

60

70

80

90

100

Percent DNA Sequence Similarity

FIG. 4. Nucleotide sequence similarity cluster analysis of the class II repeating sequences.

them. There are 7 class I and 31 class II repeating units. Each of the class I repeats is 90 nucleotides long, and the class II repeats are either 60 or 63 nucleotides long, with the one exception being 66 nucleotides long. The class II repeats have been subdivided into 7 class IIA, 13 class IIB, 5 class IIC, and 6 class IID repeats. Nucleotide sequence similarities among the class I repeats are shown in Fig. 3. Similarities ranged from 73 to 98%, with the average values in the cluster analysis being 80% or greater. Nucleotide sequence similarities among the class II repeats are shown in Fig. 4. With the exception of class IID, clustering within each subclass is high, being 70% or higher for class IIA, 65% or higher for class IIB, and 76% or higher for class IIC repeats. The class IID repeats are a diverse collection, in that all are very distinct. Two of them fit closer to the class IIB cluster and one fits closer to the class IIC group than to the others in class IID. This is also the only group in w/hich there is any size variation; repeat unit class IID4 has an extra AAA codon, while IIDs has one fewer codon. The deduced amino acid residues for the repeated se-

CHARACTERIZATION OF THE C. DIFFICILE TOXIN A GENE

VOL. 58, 1990

485

Class I peptides

LIG

FIK G P D

I1 I2

1891-1920

M Q

2025-2054

V K

I3

2159-2188

IG V FIS T S NI G F E Y F A P A N T M Q IIG V FIK G P NI G F E Y F A P A N T

I4

2273-2302

M V TIG V FIK G P NI G F E Y F A P A N T H N N N I E G Q A I

V

G F E Y F A P A N T Q N N N I E G Q A I Y N N N I E G Q A I

D A N N I E G Q A I

I5

2407-2436

M Q IG

I6

2520-2549

N Q IG V

I7

2611-2640

F|K G P D G F E Y F A P A N T D A N N I E G Q A I P Q I[ VF|K G S NIG F E Y F A P A N T D A N N I E G Q A I

CONSENSUS

NQ I G V F K G P N G F E Y F A P A N T D A N N I E G Q A I

FIK

V

G P NI G F E Y F A P A N T D A N N I E G Q A I

FIG. 5. Deduced amino acid sequences for the class I repeating units. Unit designations (I1 to 17) are listed in order from the N-terminal to C-terminal direction. The inclusive amino acid residue numbers are given for each unit, and the conserved amino acids are boxed. CLASS IIA

PEPTIDES

Al

1921-1940

V Y Q S K F L T

N G K

KFY

Y F D N N S

A2

2055-2074

V Y Q S K F L T L N G K

KIY

Y

A3

2189-2208

L Y Q N E F L T L N G K KIY Y Fl G S D S

A4

2303-2322

V Y Q N K F L T L N G K KIY Y Fl D N D S

As

2437-2456

L Y Q N K F L T

N G K KIY Y F G S D S

A6

2550-2569

R Y Q N R FL

H D N I|

A7

2641-2660

R Y Q N R FL H

L G K I

CONSENSUS

V Y Q N K F L T L N G K K Y Y F G N N S

CLASS IIB

Fl D

N N S

F G N N S

Y

G N N S

PEPTIDES

B,

1849-1869

N L V T G W Q T

N G K KYYF1D IN T G

B2

1941-1961

K A V T G W R II

N N E KIY Y

B3

2075-2095

K A V T G

B4

2096-2116

E A A T G W Q T I D G K KI| Y

Bs

2117-2137

E A A T G W Q T

B6

2209-2229

K A V T G W R I

B7

2323-2343

K A VIT GtW Q

Bs

2344-2364

E A A T G W Q T

B9

2365-2385

E A A T G W Q T

w

FIN

P N N A

Q TIIID S K KIY Y FIN T N T A

FIN T N T A

N N K K|Y Y

TuIID

FIN

D G K KIY Y FIN I

P N N A

G K KIY Y FIN L N T A L

N

T

A

D G K KIY Y FIN T N T F

B,o

2457-2477

K A V T G L R T

I

D G K KI|

B,,

2478-2498

V A V T G W Q T

I

N G K KIY Y FIN T N T S

B,2 B,3

2570-2590

K A A T G W V T

2661-2681

K A V

CONSENSUS

K A V T G W Q T I D G K K Y Y F N T N T A

T G

W Q T

Y

FIN T N T A

D G N RIY Y FIE P N T A N G K V|

YF|M P D T A

CLASS IIC

C,

1870-1890

A A L T S YK

C2

2004-2024

I A F N G Y K T I D G

C3

2138-2158

I A S T G Y T I I N G K H F Y F N T D G I

C4

2386-2406

I A S T G Y T S I N G K H F Y F N T D G I

C5

2499-2519

I A S T G Y T I I S G K H F Y F N T D G I

CONSENSUS

I A S T G Y T I I N G K H F Y F N T D G I

I I N G K H F Y F N N D G V K

H F Y F D S D C V

CLASS IID

D,

1962-1982

I A A V G L Q V I D N N K Y Y F N P D T A

D2

1983-2003

I I S K G W Q T V N G S R Y Y F D T D T A

D3

2230-2250

I A A I H L C T I NN D K Y Y F S Y D G I

D,

2251-2271

D5

2591-2611

M G A N G Y K T I D N K N F Y F R N G L

D6

2682-2702

M A A A G G L F E I D G V I Y F F G V D G

CONSENSUS

I A A

quences are shown in Fig. 5 and 6. The inclusive amino acid residue numbers are given for each repeat unit, and the conserved amino acid residues within each class or class subgroup are boxed. Seventy percent of the amino acids in the class I peptides are conserved among the units, while less than 50% are conserved within each of the class II subgroups. The dipeptide tyrosine-phenylalanine (YF) is the most conserved and can be found in all 38 repeat units. It represents residues 14 and 15 in the class I units and residues 15 and 16 in the class II repeats, except for unit IID4. Base differences in the regions of conserved amino acids involved the codon's third base as expected, whereas switching from one amino acid to another in a given position usually involved a total codon change or at least two of the bases. A hydropathic index plot for the deduced toxin A protein and a map of the repeat units are shown in Fig. 7. There is no evidence for a signal peptide at the amino-terminal end; this finding is in agreement with the lack of posttranslational modification of the N-terminal end of the protein. The only strongly hydrophobic region in the deduced protein is from residues 1,050 to 1,100. There appears to be a periodicity in the hydropathic index within each repeat region. However, the repeat region is for the most part hydrophilic. The 160 bases immediately upstream from the toxin A translation initiation site are shown in Fig. 2. There appears to be a ribosomal binding site (GGAGGT) starting six bases upstream of the initiation codon. Since we do not know where transcription initiates, it is difficult to predict promoter regions, although there are several TA-rich areas in the region of 160 bases upstream (Fig. 2). Other than these, there do not appear to be any other unique structures, such as inverted or tandem repeats. Small protein. A small ORF (ca. 500 base pairs; Fig. 8; GenBank accession number, 30308) is located 122 bases downstream from the stop codon of the toxin B gene. Although the deduced amino acid sequence begins with the first start codon, there are two additional ATG codons at the amino acid residue positions 25 and 27. There appear to be ribosomal binding sites in the -10 regions of the first (GGTGGA) and third (GGAGGC) ATG codons. The deduced protein would have a molecular mass of 18,798 Da by using the longer sequence and a 15,878-Da molecular mass by using the shorter sequence. The pl values for the two peptides are 9.22 and 9.11, respectively. The hydropathic

L Q N G Y I T I E R N N F Y F D A N N E S K

-

G

-

-

T I

-

N

-

-

Y Y F

-

-

D

-

-

FIG. 6. Deduced amino acid sequences for the class II repeating units. Unit designations are made in the same manner as for the class I units.

486

INFECT. IMMUN.

DOVE ET AL.

RESIDUES x

w z

FI 0 0

0

b

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1000

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