Vol. 129, No. 3 Printed in U.S.A.

JOURNAL OF BACTERIOLOGY, Mar. 1977, p. 1448-1456 Copyright ©0 1977 American Society for Microbiology

Immunochemical Comparison of Phosphoribosylanthranilate Isomerase-Indoleglycerol Phosphate Synthetase Among the Enterobacteriaceae GREGORY R. REYES AND VICTOR ROCHA* Oakes College and the Board of Studies in Biology, University of California-Santa Cruz, Santa Cruz, California 95064

Received for publication 7 October 1976

The bifunctional enzyme of the tryptophan operon, phosphoribosylanthranilate isomerase-indoleglycerol phosphate synthetase (PRAI-InGPS; EC 4.1.1.48), was characterized by an immunochemical study of six representative members of the Enterobacteriaceae: Escherichia coli, Salmonella typhimurium, Enterobacter aerogenes, Serratia marcescens, Erwinia carotovora, and Proteus vulgaris. PRAI-InGPS was purified from E. coli, and antisera were prepared in rabbits. These antisera were utilized in quantitative microcomplement fixation allowing for a comparison of the overall antigenic surface structure of the various homologous enzymes. These data showed E. coli PRAI-InGPS and S. marcescens and E. carotovora PRAI-InGPS (taken as a group) to have an index of dissimilarity of approximately 10, whereas the other organisms had values intermediate. In addition, antiserum to E. coli tryptophan synthetase /2 subunit was used in microcomplement fixation to extend the previous comparison of this subunit (Rocha, Crawford, and Mills, 1972) to E. carotovora and P. vulgaris. Indexes of dissimilarity for E. coli compared to P. vulgaris orE. carotovora were 1.0 and 1.7, respectively. Agar immunodiffusion using PRAI-InGPS antisera showed significant cross-reaction amongE. coli, E. aerogenes, S. typhimurium, and P. vulgaris whereas the enzymes from S. marcescens and E. carotovora cross-reacted to a lesser extent, with the latter reaction being quite weak. Comparative enzyme neutralization using E. coli PRAI-InGPS antisera showed significant cross-reactions among the enzymes in that all were neutralized at least 25%. The data taken together indicate that the trpC gene products in the Enterobacteriaceae are a homologous group of proteins, that the genetic divergence of the trpC gene is basically the same as the trpA gene, and that both are less conserved than the trpB gene. Furthermore, the PRAI-InGPS enzyme active site appears to represent a more evolutionarily conserved region of the protein. These findings indicate that, with respect to PRAI-InGPS, similarity to E. coli among the organisms examined is in the following order: (E. aerogenes, S. typhimurium, P. vulgaris) > (S. marcescens, E. carotovora). In the Enterobacteriaceae the biosynthesis of amined by primary sequence (8, 10) and/or imtryptophan involves six chemical steps (Fig. 1). munochemical examination (14, 17), whereas The genes for the tryptophan pathway in those in three enteric bacteria (Escherichia coli, Salenteric bacteria that have been examined ap- monella typhimurium, and Enterobacter aeropear to be organized as a single operon on the genes) PRAI-InGPS has been examined by chromosome (cf. reference 2). In this pathway trypsin-chymotrypsin peptide mapping (12). In the biosynthesis of indoleglycerol phosphate addition, deoxyribonucleic acid-ribonucleic acid (InGP) is catalyzed by the bifunctional enzyme hybrids, using tryptophan messenger ribonuphosphoribosylanthranilate - isomerase- indole - cleic acid from several bacterial species, have glycerol phosphate synthetase (PRAI-InGPS; been examined (7). These examinations have EC 4.1.1.48) wherein phosphoribosylanthrani- provided a considerable amount of information late is isomerized to 1-(o-carboxyphenylamino)- concerning the evolution of the tryptophan syn1-deoxyribulose phosphate (CDRP) and subse- thetic pathway (2). To extend these studies, we undertook an immunochemical examination quently decarboxylated to form InGP (5, 19). The a and /2 subunits of tryptophan synthe- and comparison of PRAI-InGPS from a broad tase in various enteric bacteria have been ex- representative group of bacteria from the En1448

VOL. 129, 1977

HN- COOH

IMMUNOCHEMISTRY OF PRAI-InGPS 1449

Pyrus fate + Glutamine Gluta imate

HO

COOH PRPP

v

H

2

A

PRT

~H

H

e,i'S

PRAI

H2C=C-C00H Chorismic Acid

PRA

Anthranilic Acid H H

H H COOH 0 0

V(!) of-,-HC H C2 O®-ol HOCCC-CH H

H

00

GPS

c-c-CH

/C02

InGPS

OOL~ -Serine

Gly-3-P

HCOO

J2

H

HI

OOXHH TSH

H

TSA

"'

TS-

L- Tryptophan

InGP

CORP

NH2

GI

I

3-P

L

Serine

N

H

Indole FIG. 1. Pathway of tryptophan biosynthesis. Chorismic acid is the branch point compound in aromatic amino acid biosynthesis. Abbreviations: AS, anthranilate synthetase; PRT, phosphoribosyltransferase; PRPP, phosphoribosyl-5-pyrophosphate; PP, inorganic pyrophosphate; PRA, phosphoribosylanthranilate; PRAI, phosphoriboribosylanthranilate isomerase; CDRP, 1-(o-carboxyphenylamino)-I-deoxyribulose 5-phosphate; InGP, indoleglycerolphosphate; InGPS, indoleglycerolphosphate synthetase; Gly-3-P, glyceraldehyde3-phosphate; TS, tryptophan synthetase; TS-A and TS-B, A and B reactions oftryptophan synthetase (24). TABLE 1. Bacterial strains used Organism

Strain

E. coli E. coli S. typhimurium E. aerogenes S. marcescens E. carotovora P. vulgaris P. vulgaris a

Genotype

A2/F'A2 trpAED102 AtrpED102(Leaderi) C-44 trpA62-1 trp- tyr- pheE-7 trpEtrpER4 trpEtrpPV15 nic- trpEnicnicUCSC, University of California-Santa Cruz.

terobacteriaceae. Our data complement and extend those of McQuade and Creighton (12) and are consistent with their suggestion that the trpC gene products are a homologous group of proteins and that the trpC and trpA genes have not diverged at greatly different rates. Furthermore, we have extended the previous immunochemical comparison of the tryptophan synthetase /2 subunit (17) to Proteus vulgaris and Erwinia carotovora to further support the conclusion that the trpB cistron represents a strongly conserved cistron, in contrast to that of the trpA and trpC cistrons. MATERIALS AND METHODS Bacterial strains. Table 1 lists the bacterial strains utilized in this study along with their particular auxotrophic dependency. P. vulgaris PV 15 behaves like a trpE missense mutation and was constructed in our laboratory from the nic- strain provided by W. L. Belser using the method of Adelberg

Source

I. P. Crawford I. P. Crawford I. P. Crawford I. P. Crawford UCSCa UCSC UCSC W. L. Belser

(1). All auxotrophs were grown at 37°C in carboys and harvested using a Beckman zonal rotor adapted for continuous flow. The minimal salts medium of Vogel and Bonner (22) supplemented with either 0.05% (E. coli, E. aerogenes, S. typhimurium, Serratia marcescens) or 0.1% (E. carotovora, P. vulgaris) acid-hydrolyzed casein was used for both starter and large-scale cultures. Glucose was the carbon source and was used at 0.3%. Membrane filter (Millipore Corp.)-sterilized indole or tryptophan was supplied at a concentration of 5 ,g/ml. E. aerogenes 62-1 also received phenylalanine and tyrosine at 20 ,ug/ml. Cultures were allowed to derepress in stationary phase for a period of 2 to 6 h, after which time they were harvested and stored at -20°C. Bacterial extracts. Harvested bacteria were suspended in 0.1 M potassium phosphate buffer (KPB) (pH 7.0) containing 1 mM 2-mercaptoethanol. In the case of 2 subunit extracts, pyridoxal phosphate at a concentration of 10 ug/ml was added to the KPB buffer. Cells were then disrupted by sonic oscillation using a Branson Sonifier followed by centrifugation to remove cellular debris. Supernatants were then

1450

REYES AND ROCHA

treated with a 20% streptomycin sulfate solution (5 ml/100 ml of crude extract), stirred for 15 min, and centrifuged at 16,000 x g for 30 min to sediment the precipitated nucleic acids. The supernatants were then dialyzed overnight against two changes of original suspending buffer. After dialysis, the supernatants were centrifuged at 100,000 x g for 60 min. The supernatants were then dispensed into small portions and stored at -20°C. Extracts prepared in this manner were free of nonspecific anticomplementary material. Enzyme assays. Indole glycerol phosphate synthetase activity was measured by the increase in InGP from CDRP after periodate oxidation of the InGP to indole-3-aldehyde (5). Phosphoribosylanthranilate isomerase activity was measured spectrofluorometrically by the method of Crawford and Gunsalus (3). Tryptophan synthetase was measured by the time-dependent disappearance of indole in its conversion to tryptophan (20). One unit of enzyme is defined as that catalyzing the formation of 1.0 ,imol of product or the disappearance of 1.0 ,umol of substrate per min at 37°C. Specific activity is reported as enzyme units per milligram of protein where protein is measured by the method of Lowry et al. (11) with bovine serum albumin as standard. Enzyme purification. E. coli AtrpED102 was utilized for the purification of PRAI-InGPS as it produces very high levels of the enzyme upon derepression of the operon. The procedure employed is a modification of that previously described (5) with changes in the gel filtration step from G-100 to G-150 and the inclusion of a second G-150 gel filtration step prior to the final hydroxylapatite chromatography. Homogeneous enzyme was obtained as judged by sodium dodecyl sulfate (SDS)-polyacrylamide electrophoresis; however, slight aggregation was observed using polyacrylamide electrophoresis as evidenced by a second very minor immunochemically active band (not shown). Gel electrophoresis. SDS-polyacrylamide electrophoresis was performed as described by Weber and Osborn (23), whereas the procedure of Davis (6) was used for polyacrylamide electrophoresis. Antiserum preparation and immunochemistry. Antisera were prepared against the E. coli PRAIInGPS enzyme using protein isolated by the above described procedure. After an initial bleeding to obtain preimmune sera, each of two rabbits received 4 mg of antigen in complete Freund adjuvant injected intradermally in areas adjacent to lymph nodes. After 5 weeks each rabbit received 2 mg of PRAI-InGPS intravenously through the marginal ear vein. At 7 and 9 days after the boost, the rabbits were bled by cardiac puncture and the individual sera were labeled R-5-31876 and R-6-31876. Four weeks later the boost was repeated and sera R-5 and R-6 were obtained. In addition, a second-course E. coli PRAI-InGPS rabbit antiserum (R-912-3473) and a second-course E. coli tryptophan synthetase (2 subunit rabbit antiserum (R-2871) were generously supplied by S. E. Mills. All PRAI-InGPS antisera used in the study gave sharp single bands of identity against purified or crude preparations of PRAIInGPS (e.g., see Fig. 4, lower right), whereas crude

J. BACTERIOL. and pure preparations of the 12 subunit gave single sharp bands of identity against R-2871 (data not shown). Quantitative microcomplement fixation and enzyme neutralization experiments were performed as previously described (9, 14, 17). Immunodiffusion in agar gels was performed by the method of Ouchterlony (15).

RESULTS The bifunctional enzyme PRAI-InGPS from six enteric bacteria was compared by three separate criteria. First, to examine the overall antigenic surface structure of the proteins, we obtained quantitative microcomplement fixation curves with antisera prepared against purified E. coli PRAI-InGPS. Secondly, as a support to the microcomplement fixation data, especially with the more divergent proteins (e.g., S. marcescens and E. carotovora), we performed double-diffusion analysis in agar gels. Third, to examine a more restricted set of antigenic determinants, we performed comparative antiserum neutralization of PRAI-InGPS enzyme activity. A similar analysis of the tryptophan synthetase (2 subunits of P. vulgaris and E. carotovora was made using E. coli 2 antiserum.

Microcomplement fixation. The ratio of antiserum concentration required with homologous and cross-reacting antigens to give identical levels of complement fixation is termed the index of dissimilarity. Thus, the lower the ratio, the more antigenically similar are two proteins, with a value of 1.0 indicating identity within the resolution of the technique. The concentration of antiserum necessary for P. vulgaris PRAI-InGPS to show equivalent fixation with theE. coli enzyme is illustrated in Fig. 2. Antiserum R-5-31876 at dilutions of 1/800, 1/670 and 1/560 gave a straight line when the maximum complement fixation values at these antiserum dilutions (Fig. 2A) were plotted against the logarithms of these dilutions (Fig. 2B). In the same experiment, the E. coli enzyme was examined at an antiserum dilution of 1/2,100. Regression analysis was performed for the three heterologous maximum complement fixation points obtained with the three antiserum dilutions, and the equation of the line thus obtained was used to determnine the serum dilution necessary to fix complement at the level of the homologous antigen. As an illustration, it can be seen in Fig. 2B that identical fixation occurred with the P. vulgaris and the E. coli enzyme at antiserum dilutions of 1/ 593 and 1/2,100, respectively, giving an index of dissimilarity of 3.54 (2,100/593). Using the same approach, the PRAI-InGPS enzymes from the

IMMUNOCHEMISTRY OF PRAI-InGPS

VOL. 129, 1977

1451

100 z

100

0

z O 80

80

X

4 Iz w 60 w E

4

x

LL 60 z

w 2 40

C-)o

-° 0~

8 20

20 2 D

0l

4 0

1

2

3 45

vJ

10

80S O /400 SERUM DILUTION '1600

ENZYME UNITS (X 10-4)

FIG. 2. (A) Complement fixation by serum R-5-31876. Reaction with the homologous PRAI-InGPS enzyme of E. coli at an antiserum dilution of 1:2,100 (0) and cross-reaction with the heterologous PRAI-InGPS enzyme of P. vulgaris at antiserum dilutions of 1:560 (E), 1:670 (A), and 1:800 (A). (B) The values of maximum complement fixation determined for the P. vulgaris enzyme in A are plotted as a function of the logarithm of serum dilution, and linear regression performed on these points yields a straight line. The maximum complement fixation point for the E. coli enzyme from A is indicated by the arrow. The antiserum dilution necessary for the P. vulgaris enzyme to fix this amount of complement is obtained from the x-axis, enabling a determination of its index of dissimilarity.

other enteric bacteria were examined with three separate antisera and the data are presented in Table 2. From these data, the indexes of dissimilarity for the various enzymes were calculated and are presented in Table 3. In a similar manner, E. coli tryptophan synthetase 12

A2

antiserum (R-2871) was used to examine the subunits of P. vulgaris and E. carotovora,

and these data are presented in Tables 2 and 4. The (2 subunit data represent an extension of a previous study and provide the necessary information for a complete immunochemical comparison of the trpC and trpB gene products in these bacteria. From Table 3 it can be seen that, in complement fixation, PRAI-InGPS in the enteric bacteria form two general groups. E. coli, E. aerogenes, S. typhimurium, and closely associated P. vulgaris form one group with indexes of dissimilarity between 1 and 5. S. marcescens and E. carotovora with indexes of dissimilarity around 10 form a second group. The indexes of dissimilarity for the E. carotovora enzyme are to a slight extent more similar to those of the E. coli enzyme than are those of the S. marcescens enzyme. The indexes of dissimilarity for the PRAI-InGPS enzymes are quite similar to those of the subunit of tryptophan synthetase and are in sharp contrast to the values for the (32 subunit which do not differ a

by more than a factor of 2 (Table 4). It is important to note that the extent of cross-reaction among the enteric bacteria PRAI-InGPS enzymes is essentially the same regardless of the antiserum used, and therefore the validity of the homology is strongly supported. Thus, the amount of immunological cross-reaction among the PRAI-InGPS enzymes is significant and comparable to that of the corresponding a subunits, and both are much lower than that of the corresponding (2 subunits. Agar immunodiffusion analysis. To further examine the nature of the homology among the PRAI-InGPS enzymes, we examined the proteins by agar immunodiffusion analysis. Figure 3 shows the results of reacting an equal number of enzyme units against one of the antisera used in this study (R-6-31876). It can be clearly seen that E. coli, E. aerogenes, S. typhimurium, and P. vulgaris formed well defined precipitation bands with some slight spurring. However, the enzyme from S. marcescens did not form a clear sharp band and also a significant spur was observed. Finally, the E. carotovora enzyme produced a very faint band indicating little cross-reaction. Thus, by immunodiffusion analysis, S. marcescens and E. carotovora cross-react differently, with the latter being the poorer cross-reacting protein. The

1452

J. BACTERIOL.

REYES AND ROCHA

TABLE 2. Microcomplement fixation titrations of sera R-5-31876, R-6-31876, and R-2871, with different antigen preparations PRAI-InGPS source

E. coli

Serum

R-5 R-6

S. typhimurium

R-5

R-6

E. aerogenes

R-5

R-6 R-6

P. vulgaris

R-5 R-5 R-6

E. carotovora

R-5 R-6

R-6

S. marcescens

R-5

R-6

P. vulgaris

R-2871

E. carotovora

R-2871

Serum dilution

C'fmaxa

1:2,000 1:2,300 1:2,800 1:1,000 1:1,300 1:1,800 1:400 1:580 1:840 1:210 1:270 1:380 1:800

83 56 23 72 41 19 97 73 27 84 55 23 69 42 25 95 80 65 75 52 42 80 30 12 82 60 41 47 30 15 91 71 40 75 54 35 71 57 41 98 88 37 67 53 38 87 63 40 81 43 19

1:1,100 1:1,400 1:300 1:380 1:540 1:400 1:600 1:880 1:600 1:770 1:1,080 1:560 1:670 1:800 1:200 1:250 1:360 1:200 1:250 1:360 1:150 1:220 1:350 1:180 1:230 1:320 1:150 1:200 1:275 1:100 1:120 1:150 1:5,500 1:7,000 1:9,000 1:3,000 1:3,900 1:5,200

E. coli controlb

Serum dilution

C'fmaXa

1:2,100

73

1:900

83

1:1,800

94

1:900

92

1:950

85

1:2,100

77

1:2,100

75

1:1,000

45

1:1,900

96

1:1,000

86

1:1,000

84

1:1,900

97

1:1,000

87

1:5,500

83

1:7,000

42

a C'fmax, Maximum percentage of complement fixation obtained with the serum dilution and antigen indicated. b In the comparison of heterologous antigens, a homologous E. coli control curve was run at a dilution known to give appropriate fixation.

VOL. 129, 1977

IMMUNOCHEMISTRY OF PRAI-InGPS

1453

TABLE

3. Indexes of dissimilarity of the PRAIInGPS from 8iX species of Enterobacteriaceae Organism

Index of dissimilarity Serum Serum Serum R-912-3473 R-5-31876 R-6-31876

E. coli 1.0 1.0 1.0 E. aerogenes 2.9 3.1 3.0b S. typhimurium NDa 3.9 4.3 P. vulgaris ND 3.6b 5.0 E. carotovora ND 10.1 TV S. marcescens 8.6 11.8 13.3 a ND, Not determined. b Average values from two titrations outlined in Table 2.

TABLE 4. Indexes of dissimilarity for the trpA, trpB, and trpC gene products from a representative group of enteric bacteria Index of dissimilarity

Organism (3

subunita

a

subunit"

PRAI-InGPSc

E. coli 1.0 1.0 1.0 S. dysenteriae 1.0 1.1 NDe S. typhimurium 1.4 2.2 4.1 E. aerogenes 1.2 2.8 3.0 P. vulgaris 1.0d ND 4.3 E. carotovora ND 1.7d 9.0 S. marcescens 1.8 8.4 11.2 a Data for ,B subunit from reference 17. b Data for a subunit from reference 14. ' Indexes are the average values of those shown in Table 3. d Calculated from data in Table 2 using R-2871. e ND, Not determined.

PRAI-InGPS's of the remaining organisms cross-reacted to about the same extent and appeared to represent a distinct group. In contrast, the ,32 subunits from all the organisms examined gave strong precipitation bands when reacted against E. coli 132 antiserum R-2871 (data not shown). In particular, the cross-reactions of S. marcescens and E. carotovora with E. coli /32 antiserum were much more extensive than the cross-reaction of PRAI-InGPS from these organisms with E. coli PRAI-InGPS antiserum. These findings are in complete agreement with the corresponding complement fixation data. Enzyme antiserum neutralization. Neutralization of InGP synthetase activity of PRAIInGPS was found to be linearly proportional to antiserum concentration (Fig. 4). Neutralization assays were performed using two antisera (R-5-31876 and R-6-31876), and neutralization efficiencies were calculated from neutralization titers. Neutralization titers are defined as syn-

FIG. 3. Interaction of E. coli PRAI-InGPS antiserum (R-6-31876) (A) with PRAI-InGPS from six members of the Enterobacteriaceae. E. coli crude extract (E), the homologous antigen, showed various degrees of cross-reaction with E. aerogenes (1), S. typhimurium (2), P. vulgaris (3), S. marcescens (4), and E. carotovora (5) (very faint but clearly visible on original plate). The lower left portion shows the expected reaction of identity when the E. coli enzyme (E) is reacted against itself (E). The lower right portion shows the single line reaction of identity between E. coli crude extract (E) and purified E. coli PRAI-InGPS (PE).

thetase units neutralized per milliliter of antiserum, whereas neutralization efficiencies are defined as the ratio of homologous neutralization titer divided by heterologous neutralization titer (2). The results of these experiments are presented in Table 5 and clearly show extensive immunological cross-reaction among the PRAIInGPS enzymes of the organisms examined, suggesting antigenic similarities among the proteins at sites which cannot be blocked and still maintain enzymatic activity. For comparison, the neutralization efficiencies for the tryptophan synthetase a and 82 subunits reacting with appropriate antisera are presented in Table 6. In general, the immunological cross-reactions for the three enzymes in this test are similar and show similar relationships among the organisms examined.

1454

J. BACTERIOL.

REYES AND ROCHA

TABLE 6. Neutralization efficiencies for the trpA, trpB, and trpC gene products from a representative group of enteric bacteria Neutralization efficiency caerogenes

Organism

2.5

itb

a ~~~~~~~~~~~~~~~0

U

P.

vulgaris

2.0 E

E.coli

1.0

1

2

Antiserum

3

Added

5

4

(.41)

subunita /3

subunit

PRAI-InGPSc

E. coli 1.0 1.0 1.0 S. dysenteriae 1.0 1.02 NDd S. typhimurium 1.25 1.49 2.14 E. aerogenes 1.39 1.64 1.85 P. vulgaris ND ND 1.42 E. carotovora ND ND 2.08 S. marcescens 1.89 2.38 2.95 a Data for the a subunits are from reference 14. b Data for the / subunits are from reference 17. c Neutralization efficiencies are the average of the data obtained from both sera in Table 5. d ND, Not determined.

FIG. 4. Neutralization of PRAI-InGPS by antiserum R-6-31876. Crude extracts containing approximately 3 x 1O-3 synthetase units of PRAI-InGPS from E. coli (a), P. vulgaris (0), and E. aerogenes (O) were added to a constant saturating amount of CDRP substrate and various amounts of serum. rately probed with sufficient sensitivity to enaNeutralization titers (enzyme units neutralized/mil- ble the formulation of definite statements conliliter of antiserum) are the slopes of the lines which cerning evolutionary phenomena as they apply to homologous proteins among closely related were determined by linear regression.

organisms. Prager and Wilson, in their study of lysozymes, have firmly established this techTABLE 5. Neutralization titers and neutralization nique as a method by which to gauge evolutionefficiencies of sera R-5-31876 and R-6-31876 reacting ary distance between organisms (16). The a and with PRAI-InGPS from each of six species of 132 subunits of tryptophan synthetase in the Enterobacteriaceae Enterobacteriaceae have been examined by miSerum R-5-31876 Serum R-6-31876 fixation crocomplement (14, 17). In the case of a Organism subunits, there is a very strong correlation beNE" NTa NE" tween immunochemical cross-reaction data and E. coli 1.06 1.0 0.310 1.0 amino acid sequence data that appears to be of S. typhimurium NDc ND 0.145 2.14 value (10, 17). In the case of the /82 predictive E. aerogenes 0.592 1.79 0.163 1.91 subunit, work is currently in progress to estabP. vulgaris 0.812 1.31 0.203 1.53 lish the nature of the relationship between the E. carotovora 0.457 2.33 0.169 1.83 S. marcescens 0.298 3.56 0.133 2.34 very extensive immunochemical cross-reaction NT, Neutralization titer. Values given repre- data and the amino acid sequences of the ensent number of synthetase units neutralized per teric bacteria 832 subunits. In this regard, the primary sequence of the pyridoxyl peptide (23 milliliter of antiserum. b NE, Neutralization efficiency. Ratio of homoloresidues) of E. coli and S. marcescens has been shown to be completely conserved (V. Rocha, gous NT/heterologous NT for a given amount of antiserum. M. Deeley, and I. P. Crawford, 1976, Abstr. ND, Not determined. Annu. Meet. Am. Soc. Microbiol. 1976, K127, p. 157) and is consistent with the idea that the (32 subunits constitute a homologous group of DISCUSSION proteins that is much more conserved than the Comparison of the primary structure of pro- corresponding a subunits. The very strong teins from homologous genes or genetic regions cross-reaction in microcomplement fixation behas proven to be extremely useful in examining tween E. coli and P. vulgaris or E. carotovora the genetic divergence of species during evolu- observed in this study is supportive of this contion. There are many examples, including cyto- clusion and was predicted. chrome c, the lysozymes, the histones, and the Using E. coli PRAI-InGPS antisera, our comimmunoglobins among others (cf. references 13, plement fixation data show that E. coli, E. 18). Quantitative microcomplement fixation is aerogenes, and S. typhimurium form a group, a relatively easy procedure by which the overall with P. vulgaris closely associated. S. marcestertiary structure of a protein may be accu- cens and E. carotovora constitute a distinct NTa

a

c

VOL. 129, 1977

separate group with the latter being slightly more related to E. coli. However, indexes of dissimilarity of this magnitude should be interpreted with caution. The immunodiffusion data suggest that S. marcescens is more closely related to E. coli than is E. carotovora and that perhaps complement fixation cannot distinguish between the two enzymes. Thus, our complement fixation and immunodiffusion data show the following relationships among the enteric bacteria PRAI-InGPS enzymes relative to E. coli: E. coli > (E. aerogenes, S. typhimurium, P. vulgaris) > (S. marcescens, E. carotovora). The immunochemical relationship among the PRAI-InGPS's of these enteric bacteria is essentially the same as that observed for the a

subunit, with the exception that immunochemical and primary sequence data are not available for P. vulgaris and E. carotovora. Thus, it appears that the trpC gene products in the Enterobacteriaceae have on their surface about the same number of antigenic determinants in common (identical or cross-reacting) as do the a subunits from these organisms. The normal interpretation of this finding is that the trpC cistron is genetically diverging at approximately the same rate as the trpA cistron in these bacteria. It is interesting to note that both the bifunctional PRAI-InGPS and the a subunit (binding InGP and (2 subunits) have two known binding responsibilities and have similar evolutionary patterns whereas the (2 subunit with five binding responsibilities (pyridoxyl phosphate, indole, serine, a subunit, and other ,( subunits) appears to have experienced a much more conserved evolution. Binding responsibilities may play a principal role in determining the genetic divergence observed for these trp operon products. This view would be consistent with the notion that protein evolution is primarily a surface structure phenomenon, and that is precisely what microcomplement fixation measures (9). Neutralization efficiencies obtained by antiserum neutralization of enzyme activity using E. coli sera showed extensive cross-reaction among the organisms examined and were comparable to that observed for both the a and (32 subunits (Table 6). The normal interpretation of these data is that the active sites of these enzymes are experiencing similar evolutionary changes as would be expected for homologous enzymes. The similarity between the E. coli and P. vulgaris PRAI-InGPS was somewhat unexpected. TaxonomicallyP. vulgaris is considered to be a member of group III in the enteric

IMMUNOCHEMISTRY OF PRAI-InGPS

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bacteria and is generally thought to be rather different from E. coli (group I) (21). Its guanine plus cytosine content of 39 mol% is very low and quite different from that of E. coli at 50 mol%. The (2 subunit immunochemical data also show P. vulgaris to be very cosely related to E. coli. Thus with regard to (2 subunit and PRAI-InGPS immunochemical data, P. vulgaris is much more closely related to E. coli that either S. marcescens or E. carotovora, both of which are group II enteric bacteria. Perhaps the evolution of the tryptophan operon in P. vulgaris is not reflective of the overall genetic divergence of the entire chromosome of this organism. The amino acid sequence differences among the PRAI-InGPS enzymes would allow for unequivocal determination of the evolutionary relationships of the proteins. However, such data are currently unavailable. It is possible, though, to use the primary sequence and immunochemical data available for the a subunit, whereE. coli compared to Shigella dysenteriae, S. typhimurium, and S. marcescens shows indexes of dissimilarity of 1.1, 2.2 and 8.4 with corresponding amino acid sequence differences of 2, 13, and 37% (10, 17), to make some reasonable estimates of the percent amino acid sequence differences between E. coli and the other organisms used in this study. By this approach we would suggest that the primary sequence of E. aerogenes, S. typhimurium, and P. vulgaris PRAI-InGPS would differ from that of E. coli by approximately 15 to 20%, whereas S. marcescens and E. carotovora would have enzymes that differ from E. coli by approximately 40 to 45%. In support of these estimates, McQuade and Creighton have carried out comparative trypsin-chymotrypsin peptide studies on three enteric bacteria (E. coli, S. typhimurium, and E. aerogenes), and their maps indicate that the percent of peptide similarity among these PRAI-InGPS enzymes and the corresponding a subunits is approximately the same (4, 12). In this regard, we are presently performing comparative tryptic peptide mapping studies on the PRAI-InGPS enzymes of this representative group of bacteria from the Enterobacteriaceae to support our immunochemical data, and the results are the subject of a separate communication. ACKNOWLEDGMENTS This investigation was supported by the Research Committee of the University of California, Santa Cruz, and by a Public Health Service Minority Biomedical Support Grant (S06 RR08132-01) from the General Research Support Branch, Division of Research Resources, National Institutes of Health.

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