Vol. 133, No. 3

JOURNAL OF BACTERIOLOGY, Mar. 1978, P. 1089-1095

0021-9193/78/0133-1089$02.00/0 Copyright © 1978 American Society for Microbiology

Printed in U.S.A.

Evolution of Ribosomal Proteins in Enterobacteriaceae HIROSHI HORI* AND SYOZO OSAWA

Department of Biophysics and Biochemistry, Research Institute for Nuclear Medicine and Biology, Hiroshima University, Hiroshima, Japan 734

Received for publication 27 October 1977

The evolution of ribosomal proteins of about 70 bacterial strains belonging to the family Enterobacteriaceae has been studied by use of previously reported data (S. Osawa, T. Itoh, and E. Otaka, J. Bacteriol. 107:168-178, 1971) and those obtained in this paper. The proximity of the bacteria was quantified by cochromatographing the differentially labeled ribosomal proteins from two strains on a column of carboxymethyl cellulose in various combinations. They were then classified into 12 groups (=species?) according to their ribosomal protein compositions and were placed in a phylogenic tree.

It was previously suggested that ribosomal protein composition is a useful characteristic for studies of phylogenic taxonomy of bacteria (12). By analyzing the ribosomal proteins of 60 strains in the genus Escherichia and its related enterobacteria with carboxymethyl cellulose chromatography, these strains were classified into seven groups according to their ribosomal protein compositions, Escherichia coli, Shigella, Salnonella, Arizona, E. freundii, E. paraintermedia, and E. adecarboxylata. The similarity matrix of ribosomal proteins of these groups suggests that (i) E. coli and Shigella or Salmonella and Arizona are very close; (ii) E. coliShigella, Salmonella-Arizona, E. freundii, E. adecarboxylata, and E. paraintermedia are rather remote from one another; and (iii) E. freundii is more closely related to SalmonellaArizona than to E. coli. Since the publication of the above work, considerable progress has been made in the construction of a phylogenic tree covering both eucaryotes and procaryotes using 5S RNA sequences of various organisms (4, 5). Using a procedure similar to that for 5S RNAs, we have constructed a phylogenic tree of the enterobacteria from the data of the ribosomal protein compositions previously published, together with some new data on Klebsiella, Enterobacter, and Aerobacter. MATERIALS AND METHODS Bacterial strains. Bacterial strains whose ribosomal protein compositions were newly analyzed are listed with asterisks in Table 1. For the strains already analyzed and reported previously, only a summary is given in the table. For details, see Osawa et al. (12). Classification of ribosome groups and types. In the previous paper (12), the ribosomes whose protein compositions revealed more than 90% similarity

were classifed into groups and further differentiated into types to distinguish ribosomes with small differences in their protein composition. The groups recognized were E. coli (types C, B, K, KW, and D), E. freundii (types f-f, f-m, and f-i), E. paraintermedia (types p and j), E. adecarboxylata, Shigella, Salmonella, and Arizona. Data in Table 2 of Osawa et al. (12) on the differentiation of the protein components in various ribosome groups have been used in this paper with the following modifications or additions. (i) The protein names used previously were converted to the standard names according to Wittmann (17); (ii) some chromatographic protein peaks in the previous paper were dropped because it has been shown that they contain more than one protein; and (iii) new data on some Klebsiella and Enterobacter strains that are described in Table 1 were added (Table 2).

Carboxymethyl cellulose column chromatography of ribosomal proteins. The labeling of ribosomal proteins with [3H]- or ["4C]lysine, the preparation of labeled 30S and 50S ribosomal subunits by sucrose gradient centrifugation, and the simultaneous analyses of two ribosomal protein samples by carboxymethyl cellulose column chromatography were the same as described previously (13). Fraction of differentiated proteins in ribosomal components. The fraction of differentiated proteins between two species (Pd) was estimated by dividing the number of different protein peaks (dpp) by the number of proteins compared (npp), i.e., Pd = dpp/npp. Here "differentiated proteins" means the pro-

teins which differ chromatographically from each other between two species. Mean fraction of differentiated proteins. Supposing chromatographically distinguishable allelic proteins A, A', and A" from three bacteria, it is possible that protein A' differs from A by one chance, while A" differs from A by more than one, etc. Therefore the fraction of the differentiated proteins (pd) should be converted to the mean fraction of differentiated proteins (Kpp). This was done using the following two equations according to Zuckerkandl and Pauling (18) and Kimura (7), respectively:

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TABLE 1. Strains used and classification of ribosomes according to their protein compositionsa Ribo-

Ribosome group (species?)

E. coli

some

No. of

Bacterial strain (representative)

type (subgroup)

_

C

E. coli C

B K KW D

E. coli B(H) E. coli Q-13 E. coli K-12 W3637 Paracolobactrum coliforme 48859

Shigella

Shigella dysenteriae ATCC 13313

Salmonella

Salmonella typhimurium LT7 Arizona arizonae ATCC 13314 Citrobacter freundii ATCC 8090 Citrobacter freundii ATCC 6750 Paracolobactrum intermedium CSDH 49761

Arizona E. freundii

f-f f-rn

f-i

E. adecarbox-

a

E. adecarboxylata ATCC 23216

p

E. intermedia AHU 1412 Aerob. aerogenes IFO 3317* E. intermedia ATCC 21073*

ylata

E. paraintermedia

j

Klebsiella

oz

K

ozanae

strains examined e 22 2 6 1 1

RESULTS

Co-chromatographic patterns of ribosomal proteins. Ribosomal proteins labeled with [3H]lysine from one strain were simultaneously chromatographed with those labeled with

[I4C]lysine from another strain in various com-

1

ATCC 11296* pn

K. pneumoniae ATCC

1

13883*

Ent. aerogenes

ka

K. aerogenes ATCC 13882*

1

ea

Ent. aerogenes ATCC

1

13048*

Ent. cloacae

cl

Ent. cloacae ATCC

1

aa

13047* Aerob. aerogenes Illinois*

1

a Abbreviations used: ATCC, American Type Culture Collection; CSDH, Connecticut State Department of Health; AHU, Faculty of Agriculture, Hokkaido University, Sapporo; IFO, Institute for Fermentation, Osaka. The 5S RNA sequences of E. coli K-12, S. typhimurium, and Aerob. aerogenes Illinois were known (see ref. 4). * Indicates strains whose ribosomal protein compositions were newly analyzed.

Kpp =-ln( 1.0-pd) ak = pd/[( 1.0- Pd)npp] as is the standard error of Kpp.

smallest values. The value of 1/2 Kpp of the pair was taken to settle the branching point between them. The branching between two or more pairs was determined from the average number of 1/2 Kp, between the pairs. Motility test. The motility of the organisms was checked by stab culture. The bacteria were inoculated into nutrient soft agar (0.5%) with an inoculating needle and incubated for 24 h at 370C. Motile organisms migrated from the line of inoculation with growth, forming a diffuse turbid zone.

(1)

(2)

Construction of the phylogenic tree. The Kpp values were used for the construction of the phylogenic tree. All pairs of organisms were rearranged in order of increasing Kpp values, and the pair to be fonned first was decided simply by choosing the pair with the

binations to detect the difference. According to the grouping criterion previously settled, bacterial strains showing more than 90% similarity were considered to be of the same group, and the newly examined bacteria were classified into the following seven groups. (i) E. paraintermedia group ribosomes. The ribosomes from Aerobacter aerogenes IFO 3317 were indistinguishable from those of E. paraintermedia p previously reported (12), and therefore were included in E. parainternedia group ribosomes. (ii, iii, and iv) Klebsiella aerogenes, K pneumonwne and K ozanae group ribosomes. Klebsiella strains examined here were K. pneumoniae ATCC 13883, K. aerogenes ATCC 13882, and K. ozanae ATCC 11296. Figure 1 shows the comparison between K. pneumoniae and K. aerogenes 30S subunit proteins. It is seen that the protein composition is very similar between the two species compared, the difference being observed only in the S2 and S9 protein peaks. One 50S and two 30S components were different between K. pneumoniae and K. aerogenes. Two 30S and two 50S components were distinguishable between K. aerogenes and K. ozanae. At least three 50S and 30S components differed between K. pneumoniae and K. ozanae. The difference indexes of K. aerogenesK. pneumoniae, K. aerogenes-K. ozanae, and K. pneumoniae-K. ozanae are 12, 15, and 23%, respectively (Table 3). Therefore, three ribosome groups, K. aerogenes, K. pneumoniae, and K. ozanae groups, were recognized here according to the criterion mentioned before. These are rather closely related to one another. (v) Enterobacter aerogenes group ribosomes. The ribosomal proteins from Ent. aerogenes ATCC 13048 (type strain) are considerably different from the other bacteria so far described. Thus the ribosomes of this species were considered to represent an independent Ent. aerogenes ribosome group. To characterize

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VoIL. 133, 1978

TABLE 2. Differentiation of ribosomal proteins and motilities in enterobacteriaa RIBOSOME GROUP S OR TYPE 2 E.COLI cC B E,COLI

S S S S 3 4 5 7

S 8

S

S

S

SS

S

S

9 10 11 15 16 17 20

L 1

L 3

L 4

L L L LL L L L L L 5 11 13 20 22 XA XB XC 25 27 *XK

I- _

EB

E.COLI K E,COLI KW

EK EK

KW

E.COLI D SHIGELLA

ED

SG

FF FF FF FF FF PFF PFF AD FF AD PEA APKO KO APKP KO APKO AP- EL KO AKO AP- EL AZ

ARIZONA

SALMONELLA E,FREUNDII FF E.FREUNDII FM E.FREUNDII Fl E,PARAINT, P E,PARAINT. J E,ADECARBOXY, ENT,AEROGENES KL,OZANAE KL,PNEUMONIAE KL,AEROGENES ENT,CLOACAE A.AEROGENES(l)

PP PPP JA O P O L L

PP PP PP PP EL EL

EA KO EA EA EL EL

FF FF FF PP PP pp EA EA EA EA EA EA

FF FF FF FF FF FF FF FF FF FF FF FF FF Fl FF FF FF PP FF FM PP PJ FF AD AD AD AD AD FF FF EA EA PP EA KO PP KO KO KO PP KO KD PP KO KO EL EL EL EL EL KO EL EL EL A! Al

FF FF FF FF FF PP PP PP EA EA EA EA EL

FF

SA SA FF FM

FF SA SA FF FF FF FF FF FF PP FF PP FF PP FF PP FF PP FF DPP EA FF PP FF FF PP FF K- FF PP FF FF PP FF EL EL PP FF EL EL EL PP FF EL

ED SG

ECB ECK + ECW ECD

SHl

AZ S- ARI FF S- SAL EFF FF FF EFM EFI FF PP P- EPP PJ P- EPJ FF EAD EA EA ENA KO EA KOZ PJ EA KPN PJ EA KAE EL EL EL EL ENC EL EL Al EL AAE

+ + +

+ + -

+ + -

+

+

Abbreviations: *, Motility test (+, motile; -, nonmotile); **, abbreviation for strains. Names of ribosomal proteins (S2-L27) are according to international code. LXA, LXB, and LXC probably correspond to L15/L21, L23, and L24, respectively. Components with no symbol indicate those indistinguishable from E. coli C. The specific symbol is given to the group-specific component in the order of appearance in the table. The symbol with - is given for the component which is probably distinguishable from E. coli C. a

FIG. 1. Chromatography on carboxymethyl cellulose columns of 30S ribosomalproteins from K. pneumoniae and K. aerogenes. ( - - ) [3H]lysine-labeled K. pneumoniae proteins. (-----0-----) ['4C]lysine-labeled K. aerogenes proteins. The explanations for protein names are the same as in Table 2, footnote a.

TABLE 3. Difference matrix of enterobacteriaa ECB ECK ECW ECD SHI 0.04 0.04 0,08 0.08 0,08 E,COLI C E,COLI B 0.08 0,12 0.12 0.12 c 0.04 0.04 0.12 0.12 0,08 0.04 E,COLI K c 0,12 0.04 c E,COL! KW 0.08 0,17 0.17 0.08 0.17 0.12 0.12 0.15 E.COLt 0 c SHI1ELLA 0.12 0.12 0,15 0.15 0.08 c AR I ZONA 0.42 0.42 0.46 0,38 0,46 0.38 c SALMONELLA 0.35 0,38 0,38 0,42 0.35 0.42 E.FREUNOII FF 0.42 0,42 0.46 0,50 0,46 0.46 E,FREUNDII FM 0.42 0,42 0,46 0.50 0,46 0.46 E,FREUNDII F! 0.42 0,42 0,46 0.50 0.46 0.46 E.PARAINT. P 0.58 0.58 0.58 0.58 0.62 0.62 E.PARAINT, J 0.58 0.58 0.58 0,58 0.62 0.62 E .ADECARBOXY, 0.65 0,65 0.69 0.73 0.65 0.73 ENT,AEROGENES 0.69 0.69 0.69 0.69 0.73 0.73 KL .OZANAE 0.69 0.69 0.69 0.69 0.73 0,73 KL, PNEUMON I AE 0.69 0,69 0.69 0,69 0.73 0.73 KL, AEROGENES 0.62 0.62 0,62 0,62 0.65 0,65 ENT, CLOACAE 0.88 0,88 0.88 0.88 0.88 0,88 A,AEROGENES (!) III0.92 0,92 0.92 0.92 0.92 0.92 ECC

AR! SAL 0,49 0.42 0,55 0.49 0,55 0,49 0,62 0.55 0.49 0.42 0.62 0.55 0.17 0.15 0.35 0,27 0,35 0.27 0.35 0.27 0.65 0,62 0.65 0,62 0.62 0.58 0.77 0.77 0,73 0,77

0,77 0,81 0,69 0,73

0.85 0.92 0.88 0.92

EFF

EFM

EFI

0.55 0.55 0.5

0.55 0.55 0.55 0.62 0.62 0.62 0.69 0,69 0.69 0.62 0,62 0.62 0.62 0.62 0.62 0.42 0.42 0.42 0.31 0.31 0,31 0.04 0.04 0.04 0.04

EPP EPJ EAD 0,86 0.86 1.06 0,86 0.86 1.06 0,86 0.86 1.18 0,86 0.86 1.31

0.96 0.96 0.96 0.96 1,06 1.06 0,96 0.96 0.77 0.77 0.69 0.77 0.77 0.77 0.04 0,04 0.12 0,54 0.50 0.54 0.54 0.54 0.54 0.12 0.50 0.50 0.50 0,58 0.58 0.69 0,69 0.69 0,46 0.46 0.69 0.69 0.69 0,54 0.54 0.73 0.73 0.73 0,50 0.46 0.65 0.65 0.65 0,46 0.42 0,92 0,92 0.92 0,81 0.81 0.92 0,92 0.92 0.81 0.81

ENA 1.18 1.18 1.18 1.18

KOZ

1.18 1.18 1.18 1.18 1.31 1.31 1.31 1.47 1.18 1.18

KPN

1.18 1.18 1.18 1.18 1.06 1.31 1.31 1.31 1.31 1.31 0.96 1.47 1.47 1.65 0,86 1.47 0.69 1.18 1.31 0.69 1.18 1.31 0,69 1.18 1.18 1.31 0.86 0.62 0.77 0.69 0,86 0.62 0,77 0.62 1.65 1.87 1.87 0.49 0.37 0.81 0.26 0.85 0.38 0,85 0.31 0.23 0.81 0.27 0.15 0.12 0,88 0.73 0.65 0.65 0.88 0.73 0.69 0.69

KAE 0,96 0.96 0,96 0,96 1,06

ENC 2.16 2.16 2.16 2.16 2.16

AAE

25 6

2.56 2.56 2.56 2.56 1,06 2.16 2.56 1.18 1.87 2.16 1,31 2.56 2,56 1,06 2.56 2,56 1,06 2.56 2,56 1,06 2.56 2,56 0.62 1.65 1.65 0.55 1.65 1.65 1,65 2.16 2,16 0,31 1.31 1.31 0.17 1.06 1.18 0.12 1.06 1.18 0,96 1.06 0,62 0,12 0.65 0.12

a Lower left half of the table: Fraction of differentiated proteins between two species (Pd). Upper right half: Estimated evolutionary distance (Kpp). Components designated by a minus sign in Table 2 were treated tentatively as being different from E. coli C.

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this group, ribosomal proteins of Ent. aerogenes other species belonging to Enterobacter is Ent. were compared with those of E. coli, E. freundii, cloacae (14). The chromatographic profiles of E. paraintermedia, K. pneumoniae ATCC Ent. cloacae ATCC 13047 (type strain) were, 13883, Aerob. aerogenes Illinois, and Ent. cloa- however, very different from those of Ent. aercae ATCC 13047. Some of the components were ogenes, K. aerogenes, E. paraintermedia, and indistinguishable from those of E. coli, E. freun- E. coli. The difference indexes between Ent. dii (shown by the symbol FF in Table 2), or E. cloacae and the others so far examined were paraintermedia (shown by PP). Some others estimated to be 92 to 62% (Table 3). Figure 3 is were specific to this group (shown by EA). Fig- the comparison between Ent. cloacae ATCC ure 2 is the comparison of Ent. aerogenes 50S 13047 and Ent. aereogenes ATCC 13048 50S proteins with those of K. aerogenes. The com- subunit proteins. It is seen that the composition positions were rather similar, four components is quite different between the two species. (vii) Aerob. aerogenes group ribosomes. (Li, L5, Lii, and L25) being specific to Ent. The ribosomal proteins from Aerob. aerogenes aerogenes. (vi) Ent cloacae group ribosomes. An- Illinois revealed only a 12% difference from those

200 FIG. 2. Chromatography on carboxymethyl cellulose columns of 5OS ribosomalproteins from Ent. aerogenes and K. aerogenes. ( -0 - ) 3H-labeled Ent. aerogenes proteins. (---- -----) '4C-labeled K. aerogenes proteins. The explanations for protein names are the same as in Table 2, footnote a. 50

100

150

FIG. 3. Chromatography on carboxymethyl cellulose columns of 508 ribosomal proteins from Ent. cloacae and Ent. aerogenes. ( - - ) 3H-Ilabeled Ent. cloacae proteins. (----_-----) 14C-labeled Ent. aerogenes proteins.

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from Ent. cloacae ATCC 13047 and a difference of 81% from those from Aerob. aerogenes IFO 3317 (Table 3). The results described above were summarized in Table 2 together with those of the previous paper (12), which were slightly modified according to the reasons described in Materials and Methods. The number of proteins listed was 26 (npp = 26). Difference matrix of enterobacteria. Using the data in Table 2, the fraction of differentiated proteins (pd) and the mean fraction of differentiated proteins (Kpp) of each pair (see Materials and Methods) are described in Table 3. Phylogenic tree. The phylogenic tree of enterobacteria was constructed from the Kpp values in Table 3 by the method described in Materials and Methods (Fig. 4). Points A and B in Fig. 4, respectively, show the approximate time of divergence between E. coli and Salmonella [>(3.7 ± 2.6) x 107 years ago; Fig. 4A] and between E. coli and Aerob. aerogenes Illinois [;(5.4 ± 3.2) X 107 years ago; Fig. 4B]. These data were according to Hori (5), who estimated the values from the 5S RNA sequences. The branching order of the bacteria from E. coli stem may be summarized as first Ent. cloacae-Aerob. aer-

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ogenes, then Klebsiella-Ent. aerogenes-E. paraintermedia, E. adecarboxylata, Salmonella-Arizona-E. freundii, and Shigella. It should be noted, incidentally, that two Enterobacter species, Ent. aerogenes and Ent. cloacae, are widely separated from each other, although they have been taxonomicaily classified into the same genus. Motility. The results of motility tests are summarized in Table 2. Aerob. aerogenes Ilinois, the ribosome of which is close to Ent. cloacae, was motile, like Ent. cloacae. On the other hand, Aerob. aerogenes IF03317, which belongs to the E. paraintermedia group in its ribosomal protein composition, was nonmotile, like Klebsiella and E. paraintermedia.

DISCUSSION In the previous papers (4, 5), it was shown that 5S RNA is a useful molecule for the construction of a phylogenic tree for widely separated organisms, such as between Kingdoms Fungi and Animalia or between E. coli and Bacilli, etc. However, this molecule is not suited for the phylogenic studies of closely related species such as those in the Enterobacteriaceae, because 5S RNA is a molecule of low Pauling unit and has no nucleotide substitution for mil-

FIG. 4. Phylogenic tree of enterobacteria. The Kpp values were taken as the relative time scale in the abscissa. Points A and B show divergence times between E. coli and Salmonella typhimurium, -(3.7 ± 2.6) x 1i6 years ago, and between E. coli and Aerob. aerogenes, -(5.4 ± 3.2) x 107 years ago, respectively (5). (I - - 1) Range of the standard error of Kpp (ok in equation 2). (j-----°-----1) Range of 1/2 Kpi, of all pairs at the branching point.

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lions of years (ca. 0.4 Pauling [6]). For instance, the 5S RNA sequence of Salmonella typhimurium differs from that of E. coli by two base substitutions in 120 nucleotide length. The sequence of Aerob. aerogenes also contains only three base changes as compared with the E. coli sequence. Therefore the differences among the related species are so small that it is difficult to establish reliable relationships. In the present paper, ribosomal proteins are employed for studies on the phylogeny in enterobacteria. It is well known that, in spite of the diversification of amino acid sequences in ribosomal proteins in the bacterial kingdom (6), the essential structure and function of the ribosomes remain the same. Moreover, the proteins are exchangeable among widely separated species of bacteria. Thus the bulk of amino acid exchanges in ribosomal proteins are assumed to be nearly neutral and might not be due to selections towards different environments. We have therefore chosen 26 chromatographically separable ribosomal protein peaks, each of which contains only a single protein, and then compared them among different enterobacteria. The number of different peaks between two given strains varies from 0 to 23. This number would be sufficient for further calculation to establish approximate phylogenic relationships, even though the average Pauling unit of ribosomal proteins is small (ca. 0.2 Pauling [6]) as in the case of 5S RNA. Since we are dealing with 26, or roughly one half, of the ribosomal proteins, one wonders about the possible biasing of the results. For example, certain proteins may be more strongly conserved than others. However, the Pauling units of the N-terminal regions of several 30S ribosomal proteins are not very much different except for S14 (6); we assume here that the Pauling units are approximately the same among ribosomal proteins. Generally the number of amino acid substitutions in the homologous proteins is compared to estimate phylogenic relationships, since the rate of amino acid substitution during evolution (Kaa) is assumed to be proportional to the number of years that have elapsed since the evolutionary divergence of the two polypeptides from their common ancestor (7). In this study, however, the Kpp values derived from the chromatographic differences of ribosomal proteins were used instead of Kaa, which directly stems from the amino acid substitution data. The probability of the occurrence of a net charge difference between two homologous proteins due to a single amino acid substitution has been theoretically and empirically estimated to be 0.3 or 0.4 (10). Thus it is reasonable to assume that the chro-

matographic difference of ribosomal proteins in our case is, on average, due to about three amino acid exchanges. Kpp is then roughly proportional to Kaa. The result of this study is in general agreement with those of intergeneric recombination (2), DNA hybridization (3, 9, 15), immunology of alkaline phosphatase (1, 16), and sequences of tryptophan synthetase subunits (8). In Bergey's Manual of Determinative Bacteriology, 8th ed. (11, 14), the generic name "Aerobacter" was rejected and all strains in it were renamed either as "motile" Enterobacter or "nonmotile" Klebsiella. Generally speaking, this classification by motility is consistent with that by ribosomal protein compositions. The nonmotile E. paraintermedia, K. ozanae and K. pneumoniae, and K. aerogenes are related, whereas motile Ent. cloacae and Aerob. aerogenes Illinois are very close; the motile bacteria are far from the nonmotile bacteria. However, our chromatographic studies indicate that the motile Ent. aerogenes is much more closely related to the nonmotile Klebsiella than to the motile Ent. cloacae. ACKNOWLEDGMENTS We thank S. Hino, Faculty of Science, Hiroshima University, and S. Burke, Department of Microbiology, University of Illinois, who kindly supplied the strains Aerob. aerogenes IFO 3317 and Aerob. aerogenes Illinois, respectively. We are also greatly indebted to T. Ohta, National Institute of Genetics, Mishima, Japan, for her helpful discussions. This work was supported by grants from the Ministry of Education of Japan (no. 022121 and 121419). H.H. was supported by fellowships from the Japan Society for the Promotion of Science (no. 105 and 15).

LITERATURE CITED 1. Cocks G. T., and A. C. Wilson. 1972. Enzyme evolution in the Enterobacteriaceae. J. Bacteriol. 110:793-802. 2. Demerec, M., and N. Ohta. 1964. Genetic analysis of Salnonella typhimurium x E. coli hybrids. Proc. Natl.

Acad. Sci. U.S.A. 52:317-323. 3. Falkow, S., and R. V. Citarela. 1965. Molecular homology of F-merogenote DNA. J. Mol. Biol. 12:138-151. 4. Hori, H. 1975. Evolution of 5sRNA. J. Mol. Evol. 7:7546. 7:7546. 5. Hori, H. 1976. Molecular evolution of 5sRNA. Mol. Gen. Genet. 145:119-123. 6. Hori, H., K. Higo, and S. Osawa. 1977. The rates of evolution in some ribosomal components. J. Mol. Evol. 9:191-201. 7. Kimura, M. 1969. The rate of molecular evolution considered from the standpoint of population genetics. Proc. Natl. Acad. Sci. U.S.A. 63:1181-1188. 8. Li, S.-L., G. R. Drapeau, and C. Yanofsky. 1973. Amino terminal sequence of the tryptophan synthetase alpha chain of Serratia marcescens. J. Bacteriol. 113:1507-1508. 9. McCarthy, B. J., and E. T. Bolton. 1963. An approach to the measurement of genetic relatedness among or-

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ganisms. Proc. Natl. Acad. Sci. U.S.A. 50:156-164. 10. Nei, M., and R. Chakraborty. 1973. Genetic distance and electrophoretic identity of proteins between taxa. J. Mol. Evol. 2:323-328. 11. 0rskov, I. 1974. Genus VI. Klebsiella, p. 321-324. In R. E. Buchanan and N. E. Gibbons (ed.), Bergey's manual of determinative bacteriology, 8th ed. The Wililams and Wilkins Co., Baltimore. 12. Osawa, S., T. Itoh, and E. Otaka. 1971. Differentiation of the ribosomal protein compositions in the genus Escherichia and its related bacteria. J. Bacteriol. 107:168-178. 13. Osawa, S., E. Otaka, T. Itoh, and T. Fukui. 1969. Biosynthesis of 50S ribosomal subunit in E. coli. J. Mol. Biol. 46:321-351. 14. Sakazaki, R. 1974. Genus VII. Enterobacter, p. 324-325. In R. E. Buchanan and N. E. Gibbons (ed.), Bergey's

manual of determinative bacteriology, 8th ed. The Wiland Wilkins Co., Baltimore. Schildkraut, C., J. Marmur, and P. Doty. 1961. The formation of hybrid DNA molecules and their use in studies of DNA homologies. J. Mol. Biol. 3:595-617. Steffen, D. L., G. T. Cocks, and A. C. Wilson. 1972. Micro-complement fixation in Klebsiella classification. J. Bacteriol. 110:803-808. Wittmann, H. G. 1974. Purification and identification of Escherichia coli ribosomal proteins, p. 93-114. In M. Nomura, A. Tissieres, and P. Lengyel (ed.), Ribosomes. Cold Spring Harbor Laboratory, Cold Spring Harbor, N.Y. Zuckerkandl, E., and L. Pauling. 1965. Evolutionary divergence and convergence in proteins, p. 97-166. In V. Bryson and H. J. Vogel (ed.), Evolving genes and proteins. Academic Press Inc., New York. liams

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18.

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