Comparison of the Lipoprotein Gene among the Enterobacteriaceae

Transcription

1 rhe JVURNAI. VI HIULOCICAL CHEMISTRY Vol. 2%. No 5. I.%ur of March IO. pp s Prmled m I S A Comparison of the Lipoprotein Gene among the Enterobacteriaceae DNA SEQUENCE OF ERWINIA AMYLOVORA LIPOPROTEIN GENE* Hideo Yamagata, Kenzo Nakamura, and Masayori Inouye (Received for publication. August 19, 1980) From the Department of Biochemlstry, Stale University of New York at Stony Brook, Stony Brook, New York A DNA sequence of 816 base pairs encompassing the entire Erwinia amylovora lipoprotein gene was determined. Sequence comparison between E. amylouom, Escherichia coli, and Serratia marcescens suggests that the structure of the lipoprotein has been highly conserved under the constraint of efficient gene expression selecting promoter structure, mrna secondary structure, and codon usage in addition to the polypeptide function. The sequence also suggests that the lpp gene of the three bacteria diverged sequentially in the course of evolution. Boehringer Mannheim. Bacterial alkaline phosphatase was from Worthington Biochemical. T4 polynucleotide kinase was from P-L Biochemical. Nitrocellulose filter (BA85) was from Schleicher and Schuell. Restriction enzymes were from Bethesda Research Labora- Lipoprotein is a major outer membrane protein in Escherichia coli. There are approximately 7.2 X 10 molecules of tories and New England Biolabs. Preparation of DNA-Preparation of the E. amylolw-a DNA and lipoprotein per cell, which makes the lipoprotein the most phage DNA were described previously (3). abundant protein, numerically, in E. coli. One-third of the Cloning of the E. arnyloclora lpp Gene-Cloning of the E. amylooora lpp gene was carried out basically by following the methods lipoprotein molecules are covalently linked to the peptidogly- described previously (3). A 1-kb Hue I11 fragment of plasmid can (bound form as opposed to free form) and are believed to pken221 DNA carrying the S. marcescens lpp gene (4) labeled with contribute to the structural integrity of the cell envelope (1). I in citro bynick translation (5) wasused as the hybridization The lipoprotein is also a major envelope protein in various probe. Total E. amylovora DNA was cleaved with restriction enzyme other Gram-negative bacteria. DNA-RNA hybridization, as HindIII and fractionated on a preparative agarose gel. The fractions well as immunological tests, have revealed that the nucleotide containing the HindIII fragments which hybridized to the probe were combined, mixed with Hind111 cleaved A540 DNA (6). and treated sequence of the gene coding for the lipoprotein (lpp gene) is with T4 ligase. The ligated UNA was subsequently used to transfect highly conserved in enteric bacteria (2). Comparison of the E. coli K802 (3). Recombinant phages carrying the lpp genewere DNA sequences of the lpp genes in these bacteria should screened by the technique of plaque hybridization (7). Four out of allow us to determine which part of the gene structure has 1100 plaques examined gave positive hybridization. been highly conserved and which part of the gene structure Restriction Mapping-Restriction maps around the lpp gene of has been variable during evolution. The conserved regions A5401ppEa-1 and E. amylovora chromosome were constructed by Southern blot hybridization (8). Fine restriction mapping of the 1.3- would be expected to play important roles in the highly kb Barn HI-Kpn I fragment was carried out by labeling with P efficient expression of the lpp genes, in the process of secretion either uniformly by nick translation or at its 5 ends using polynucleand assembly of this protein into the outer membrane, or in the function of the protein. From the evolutionary point of otide kinase (9). DNA Sequencing-Sequencing procedures were that of Maxam view, comparison of the DNA sequence provides the ultimate and Gilbert (9). A thin sequencing gel system (0.04 X 20 X 40 cm) detail for comparative analysis of homologous genes. At this with 20 or 10%. polyacrylamide in 7 M urea (10) was used to separate cleavage products. point such comparative DNA sequence analysis has been carried out only for parts of trp operon and the lpp region. RESULTS AND DISCUSSION For the lpp region, the complete nucleotide sequences have been determined in E. coli and Serratia marcescens (3, 4). Cloning and Sequencing of the E. amylovora Ipp Gene- The E. amylovora lpp gene was cloned onto A540 vector as In this article, we present the complete nucleotide sequence of the lpp gene of Erwinia amylovora which is one of the described under Experimental Procedures. One of X phages which gave positive hybridization, designated as A540lppEamost distantly related to E. coli among the Enterobacteriaceae (2). This made possible for the first time the comparison 1, was further analyzed. As shown in Fig. 1A, A540IppEa-1 has insertions of two HindIII fragments, the size of which are 7.3 of the entire nucleotide sequences of homologous genes in kb and three different bacteria. This in turn enabled us 6.7 kb, respectively. The restriction map around the to postulate lpp gene on A540lppEa-1 DNA matched well with that on the an order for the evolutionary events which have led to the variation in these lpp genes. This comparison also revealed * This work was supported by Grant GM from the United States h c Health Service and Grant BC-67 from the American Cancer Society. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked aduertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. several interesting and unique features of the Ipp genes which are highly conserved in these bacteria. These features probably are relevant to the mechanism of expression of the gene or the secretion and assembly of the lipoprotein into the outer membrane EXPERIMENTAL PROCEDURES Materials-[ P]Orthophosphate, [u-. P]dCTP (>SO0 Ci/mmol), and [u- P]dATP (>300 Ci/rnmol) were obtained from Amersham and New England Nuclear. E. coli DNA polymerase I was from E. amylovora chromosome DNA indicating that the entire lpp gene was cloned into the phage vehicle (data not shown). A 1.3-kb Bum HI-Kpn I fragment ofa5401ppea-i DNA carry- ing the lpp gene was separated by 3.5% polyacrylamide gel electrophoresis. According to the strategy shown in Fig. lb, The abbreviation used is: kb, kilobase pair.

2 B A A '$40 ' J FIG. 1. Restriction maps and sequencing strategy. A, restriction cleavage mapping around the lpp gene on X540ZppEa-1. h540lppe:a-i DNA was digested with several restriction endonucleases and applied onto 0.7V or 1.5% agarose gels. DNA fragments separated on the agarose gels were denatured with alkali and transferred to a sheet of nitrocellulose filter as described by Southern (8). The fragments carrying lpp gene were detected by autoradiography after hybridization with "P-labeled I-kb HaeIII fragment of pken221 plasmid DNA carrying S. marcescen.s Ipp gene (4, 6). The structure of X540 was taken from Murray and Murray (6). B, DNA sequencing strategy. The open circle indicates the position of the "1' label at the 5' end. The singly labeled fragments which are shown by arrouls were obtained either by secondary restriction enzyme cleavage at the sites indicated by the cvrtical line 4 or by strand separation. The broken regions of arrou3.s indicate that the sequences of those regions which were not determined in these experiments. the nucleotide sequence of 816 base pairs encompassing the lpp gene was determined. Features of the Sequence of the E. amylouora lpp Gene as Compared to Those of E. coli and S. marcescens-fig. 2 shows the DNA sequence of the E. amylocora lpp gene as well as those of E. coli (3) and S. marcexens (4). Since the sequences of lpp genes of these three bacteria are highly conserved, the transcription initiation and termination sites in the E. amyloc'ora or the S. marcescen.s sequence were deduced from the DNA sequence of E. coli, for which the complete nucleotide sequence of the lipoprotein mrna had been previously determined (11). The features of the E. amylotlora lpp gene as compared to those of E. coli and S. marcescens are as follows: 1. From the transcription initiation site (base 1) to the transcription termination site (base 319), the nucleotide sequence of the lpp gene is very conserved (89% homology to the E. coli lpp gene and 88% homology to S. marcexens lpp gene). Specifically, the sequence of the 5' nontranslated region of the mrna is highly conserved (97%. homology to E. coli and 92"; homology to S. marcescen.s, Fig. 3Bj. 2. The promoter region (base -45 to -1) is also highly conserved (87% homology to E. coli and 93%) homology to S. marcescens, Fig. 3B) and has an extremely high A + T content (80%') as in E. coli (80%) and S. marcescens (78%), as shown in Fig. 3A. 3. In the DNA sequence corresponding to the NH?-terminal portion of the signal peptide, there are three contiguous base Erurinia nmylouora Lipoprotein Gene substitutions (base 44 to 46) as compared to E. coli resulting in two amino acid substitutions. The same change was also found in S. marcescens. 4. In the DNA sequence corresponding to the COOH-ter- minal portion of the signal peptide, there are two base substitutions (base 83 and 95) as compared to E. coli which do not cause changes in the amino acid sequence. There are no base additions or deletions relative to E. coli, as is the case for S. marcescens. 5. In the DNA sequence corresponding to the region between the NH2 terminus and the 51st amino acid residue of the mature lipoprotein (bases 99 to 251), there are 13 base substitutions resulting in three amino acid substitutions as compared to E. coli. No amino acid substitutions were found in this region of the S. marcescens sequence as compared to the E. coli sequence. 6. In the DNA sequence corresponding to the seven amino acid residues at the COOH terminus (bases 252 to 272j, there are six base changes as compared to E. coli resulting in three amino acid substitutions. 7. Nine stable stem and loop structures (I to IX) can be formed in the mrna region (Fig. 4). The structures I, 111, and VI11 are similar to those found in E. coli mrna (11) while the structures 111 and IV are similar to those found in the S. marcescens mrna (4). 8. The codon usage in E. amylouora lipoprotein mrna is similar to both those of E. coli and S. marcexens lipoprotein mrna. In spite of several possible codons for each amino acid, only a few codons are used (Table I). Constraints in the Evolution of Ipp Gene-In the DNA sequence, 220 bases upstream from the lpp promoter region, a possible COOH-terminal portion of a neighboring gene was found. This unknown gene has also been found in E. coli and S. marcescens (4). The amino acid sequence of this unknown gene product deduced from the DNA sequence is absolutely conserved among these three bacteria. This suggests that the DNA sequence from the unknown gene to the lpp gene in these bacteria evolved from the same ancestral sequence. The '3 homology as shown in Fig. 3B is remarkably similar for each genetic segment in any given pair. This might be evidence for different rates of evolution for different genes. The lower extent of homology found in the sequences between the two genes (Fig. 3B) suggests that constraints in this region have been lax, permitting substitution of about 509 of the bases. In contrast, the evolution of the COOHterminal portion of the neighboring gene seems to be under a constraint of the polypeptide function. With regard to the third bases in each triplet codons in this COOH-terminal gene fragment, 9 out of 13 third position bases were substituted between E. coli and E. amylovora while 6 out of 13 bases were substituted between E. amylovora and S. marcescens (Fig. 2). No substitutions were found in the bases at the first and second positions of these triplet codons. As a result, the codon usage in this unknown gene is more flexible than that found in the lpp gene. For example, three threonine residues in the unknown peptide deduced from the DNA sequence are coded by three different codons in E. amylorlora and S. marcexens and two different codons in E. coli. The evolution of the lpp gene also seems to be under the constraint of polypeptide function. In the sequence of 78 amino acid residues of the prolipoproteins, only 8 amino acid residues are altered either between E. amylorlora and E. coli or between E. amylouora and S. marcescens. Most of these alterations are localized in the NHr- or COOH-terminal portion of the signal peptide or in the COOH-terminal portion of the mature lipoprotein. The alterations in the signal peptide region do not change the fundamental properties of the signal

3 2196 Lipoprotein Gene amylovora Erwinia 3 Y B.

4 ~~ Erwinia amylovora Lipoprotein Gene 2197 peptide proposed in the loop model for the functions of the signal peptide, such as basic character at the NH?-terminal section and existence of a long hydrophobic region (12). In addition, the evolution of the lpp gene seems to be under a constraint with respect to its highly efficient expression. For most of the amino acid residues, one or two codons out of several possible codons are used in all three bacteria (Table I). As discussed previously (11, 13), almost all of those codons used correspond to major species among the isoaccepting trnas. The usage of the major isoaccepting species of trna for individual amino acids probably facilitates very efficient translation. The A + T richness of the promoter sequence is considered to destabilize the helix structure and facilitate the RNA polymerase-mediated strand unwinding necessary for the initiation of transcription (14, 15). The extremely high A 81 HOMOLOGY (%) E. c. - E % 49 E S. m. S. m.- E. c. + T content in the promoter region of the lpp genes is, therefore, likely to be essential for the efficient transcription of the Zpp gene. The Ipp mrnas of all three bacteria can form many stable hairpin stem and loop structures which may have relevance to their longer functional half-lives in comparison to other mrnas (16). Most of the base substitutions found among the sequences of lpp mrna regions of the three bacteria do not interfere with the secondary structures of the mrnas. For example, the secondary structures I, 111, and VI11 of E. amylovora (Fig. 4) are almost the same as those of E. coli I, 111, and IX (ll), respectively, and only one base substitution is located in the loop of the structure I (u to A as underlined in Fig. 4). Rather frequent base substitutions in the E. amylovora sequence as compared to that of E. coli around the translation termination site interfere with the formation of secondary structure similar to the structure I1 of E. coli mrna (11). However, the base substitutions are such that they enable the formation of another secondary structure (the structure I1 of E. amylouora). Thus, the lpp genes of the three bacteria seem to be highly conserved under the constraint of efficiency of gene expression which selects the promoter structure, mrna structure, and codon usage in addition to the constraint of the polypeptide function. In contrast to the lpp gene, polypeptide function probably played a major role in the evolution of the unknown gene neighboring the lpp gene and also trpa gene as reported by Nichols and Yanofsky (17). Sequential Divergence in the lpp Gene Evolution-In spite of the highly conserved nucleotide sequences from the pro- II BASE L POSITION I I NUMBER FIG. 3. Distribution of AT base pairs and the homology between the sequences around the lpp gene of E. coli, E. amylouora, and S. marcescens. The sequences shown in Fig. 2 are divided into 8 sections as indicated. A, AT content of each section is indicated on the top of the bar. Closed bars indicate the regions where AT contents are more than 75%. Shadowed burs indicate the reeions 0 ~~ ~ where AT content is 60 to 74%. B, homology of the sequence of each FIG. 4. Possible secondary structure of E. amylovora liposection is indicated on the top of the bar. Homology higher than 801 protein mrna. The AG values for the nine stem and loop structures is indicated by closed bars, and 60 to 79% homology by shadou>ed shown (I to ZX) were calculated according to Tinoco et al. (22, 23) to burs. Abbreviations: Ex., E. coli; E.u., E. amyloc,ora; and Sm., S. be -21.1, -8.8, -1.0, -6.8, -1.4, -1.4, -2.5, -1.6, and -1.8 kcal, marcescens. respectively. TABLE I Codon usage in E. amylor~ora lipoprotein mrna compared to those in E. coli und S. murcescens Values in parentheses are from S. murcescens and E. coli lipoprotein mkna (4, ll), respectively. - I'he UUU UCU Ser 3 (3.3) UAU Tyr UGIJ Cys UUC UCC 0 (3.2) UAC UGC 1 (1.1) 1 (1.1) UUA Leu 2 UCA (0.0) Term UAA 1 (1.1) Term IJGA UUG UCG UAG Trp ljgg Leu CUU 0 (1.0) CCU Pro CAU His 4 CGU Arg (3.3) CUC ccc CAC 1 (2.0) CGC 1 (1.1) CUA CCA CAA Gln 0 (3.0) CGA CUG 9 (6.9) CCG 6 CAG (3.5) CGG AUU Ile Thr ACU 1 AAU 4 Asn (2.4) (0.0) Ser AGU 1 AUC ACC 3 (2.2) (0.0) (7.6) 5 AAC (2.2) 2 AGC AUA ACA AAA Lys AGA 4 Arg (5.6) Met AUG 1 (2.3) ACG AAG AGG 1 (1.1) Val GUU 2 (3.3) GCU Ala GAU 5 Asp (7.8) GGU 1 Gly (2.2) 2 (1.2) GUC GCC 1 (0.0) GAC 7 (6.6) (2.1) 1 GGC GUA 3 (2.2) GCA 4 (4.3) GAA Glu GGA GUG 1 (1.1) GCG 1 (1.1) GAG GGG ~. _ Vi i VI

5 2198 Erwinia Gene Lipoprotein arnylovora moter region to the translation termination site of the lpp gene, there are several regions where rather drastic changes in the nucleotide sequences are found. These changes are assigned letters a to h in Fig. 2. They are caused by either base additions or deletions or substitutions of three contiguous bases, which do not easily revert. Between E. coli and E. amylovora, there are four such alterations (a, c, f, and g) and between S. marcescens and E. amylocwra there are also four (b, d, e and h), whereas between E. coli and S. marcescen-s there are as many as eight alterations (a to h). It should be noted that at all of these regions E. amylocrora always share a common sequence with one of the other two bacteria. This could be interpreted as follows. The E. amylo~xra gene is especially protected against mutations. As a result, these drastic mutations are found only in the E. coli or S. marce.vcens lpp genes. Alternatively, a more likely explanation is that first the E. coli (or S. marcescens) and E. amylo~1ora lineage diverged from the common ancestor of the three bacteria. The drastic mutations described above (b, d, e, and h) then occurred in the common ancestor of E. coli (or S. marcescens) and E. arnylouora. Secondly, the E. coli (or S. marcescens) lineage diverged from the common ancestor of E. coli (or S. marcescens) and E. amylovora and the drastic mutations (a, c, f and g) then occurred in the E. coli (or S. marcescens) lineage. It should be emphasized that the present data provide the first evidence for the sequential divergence of bacterial genomes based on the DNA sequences. With regard to single point mutations, it is difficult to find such evidence for sequential divergence of the lpp genes. This is probably because single point mutations are very easily reverted. Many phylogenetic trees of Enterobacteriaceae have been proposed such as the one based on immunological comparison of alkaline phosphatase (18) or comparison of ribosomal components (19). In most phylogenetic studies, Erwinia is regarded as more distantly related to E. coli than S. marcescens. Our results described here, however, indicate that E. amylor?ora lpp gene is more related to the E. coli lpp gene than the S. marcescens lpp gene is. DNA-DNA hybridization studies of tnaa, trp, thya, and lacz genes of Enterobacteriaceae also showed that the E. amylovora genes are more closely related to the E. coli genes than the 3. marce.scen.s genes are (20). Furthermore, recent DNA-DNA hybridization studies showed that E. amylovora is least related to other Erwiniae and more closely related to Escherichiae (21). Further analyses of the lpp genes of Enterobacteriaceae are in progress to understand the evolutional history of the bacteria. Acknoulledgments-We thank Dr. M. Kiley and Mr. L. Zwiebel for critical reading of the manuscript. KEFEHENCES 1. IliHienzo, J. M., Nakamura, K., and Inouye, M. (1978) Annu. Rets. Biochem. 47, Nakarnura, K., Pirtle, K. M., and Inouye, M. (1979) J. Bncteriol. 137, Nakamura, K., and Inouye, M. (1979) Cell 18, Nakarnura, K., and Inouye, M. (1980) Proc. Natl. Acacl..%I. 11. S. A. 77, Kigby, P. W. J.. Dieckmann. M., Hhodes, C., and Berg, 1. (1977) J. Mol. Biol. 113, Murray, K., and Murray, N. E. (1975) J. Mol. Biol. 98, Benton, W. I)., and Davis, K. W. (1977) Scwnce 196, Southern, E. M. (1975) J. Mol. Biol. 98, 50: Maxarn, A. M., and Gilhert, W. (1977) Proc. Naf1. Acrcrl. Scr. U. S. A. 74, Sanger, F., and Coulson, A. K. (1978) FEES Left. 87, I. Nakarnura, K., Pirtle, H. M., Pirtle, I. L., Takeishi, K.. and Inouye, M. (1980) J. Biol. Chenz. 255, Halegoua, S., and Inouye, M. (1979) in Bactcrircl Outer Menhranes (Inouye, M., ed) pp John Wilev & Sons, New York 13. Post, L. E., Strycharz, C. L)., Nornura, M., Lewis, H., and Dennis, P. 1. (1979) Proc. Natl. Acad. Sci. U. S. A. 76, Charnberlin, M. J. (1976) in RNA Polymerrcse (Losick, H., and Charnberlin. M., eds) pp Cold Spring Harhor Laboratory, Cold Spring Harbor, N. Y. 15. Vollenweider, H. J., Fiandt, M., and Szybalski, W. (1979) Science 205, Hirashirna, A,, Childs, G., and Inouve. M. (1973) J. Mol. Hid. 79, Nichols, B. l,, and Yanofsky, C. (1979) Proc. Nafl. Acad. Sei. I:. S. A. 76, Cocks, G. T.. and Wilson, A. C. (1972) J. Bacterid. 110, Hori, H. (1976) Mol. Gen. Genef. 145, Anilionis, A,, and Riley. M. (1980) J. Bacteriol. 143, Azad. H. H., and Kado, C. I. (1980) J. Gen. Microhiol. 120, 1 li- I Tinoco, I., Borer, P., Dengler, B., Levine, M., Uhlenbeck, O., Crothers, D., and Gralla, J., (1973) Nat. New Biol. 246, Borer, P. N., Dengler, B., Tinoco, I., Jr., and Uhlenbeck, 0. C. (1974) J. Mol. Bi01. 86,