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Nucleotide Sequence and Genetic Complementation Analysis oflepfromAzotobacter vinelandii
BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS ARTICLE NO.
239, 393–400 (1997)
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Nucleotide Sequence and Genetic Complementation Analysis of lep from Azotobacter vinelandii Carissa A. Jock, Lakshmidevi Pulakat, SaeHong Lee, and Narasaiah Gavini1 Department of Biological Sciences, Bowling Green State University, Bowling Green, Ohio 43403
Received August 21, 1997
The lep of Azotobacter vinelandii is an 852-base-pair open reading frame (ORF) which encodes a protein of 284 amino acid residues. The translated protein shares 75% homology with leader peptidase I isolated from Pseudomonas flourescens and 37% homology with leader peptidase I isolated from Escherichia coli. Five highly conserved regions found in the family of leader peptidase I proteins are conserved in A. vinelandii Lep. The putative membrane topology of the protein seems similar to that of E. coli leader peptidase I based on the hydrophobicity analysis of the predicted amino acid sequence. Southern blotting analysis of the A. vinelandii chromosome by probing with lep specific DNA revealed that lep is present as a single copy per the chromosome. A multicopy plasmid carrying A. vinelandii lep could complement a temperature sensitive lep mutant of E. coli strain IT41, suggesting that we have identified the functional copy of lep present on A. vinelandii genome. q 1997 Academic Press
Azotobacter vinelandii is a free-living soil bacterium widely known for its ability to fix nitrogen aerobically (1-4). It was speculated that this bacterium contains multiple chromosomes per cell with unique biology of cell maintenance and growth (5-7). Until recent, it was generally accepted that A. vinelandii could not uptake amino acids and it was proposed that the genes coding for amino acid transport proteins were not present within its genome (8). However, in a recent study, some amount of amino acid transport in A. vinelandii was reported suggesting that at least some amino acid transport proteins are present in this organism (9). Amino acid transport proteins are known to be located at the outer surface of the cell; therefore if amino acid transport proteins are synthesized in A. vinelandii, they need to undergo post translational protein processing in order to reach their appropriate destination. 1 Corresponding author and mailing address: Department of Biological Sciences, Bowling Green State University, Bowling Green, Ohio 43403. E-mail: [email protected].
In general, one to ten thousand different proteins are synthesized within the cytosol of a given cell in order for proper cell metabolism and viability. In both prokaryotic and eukaryotic cells, these newly synthesized proteins must reach specific intracellular locations for metabolic processes to be carried out effectively. One protein that plays a major role in protein processing is leader peptidase I. In the absence of leader peptidase I, the majority of pre-proteins accumulate on the periplasmic side of the inner membrane eventually leading to cell death (10-13). Therefore, the leader peptidases are essential for effective protein processing and cell viability in both prokaryotic and eukaryotic cells. Genes coding for leader peptidase I have been isolated and sequenced from a variety of organisms (14-21). The nucleotide and deduced amino acid sequences of the genes isolated from a variety of organisms share significant homology suggesting a common organization in their functional mechanistics. Here we report the nucleotide sequence analysis of lep isolated from A. vinelandii, genetic complementation analysis and comparison of predicted membrane topology to other leader peptidases. MATERIALS AND METHODS Bacterial strains, plasmids, and growth conditions. E.coli strains were normally grown at 377C in Luria broth or 2YT (22). The antibiotic ampicillin was used at final concentration of 50mg/ml wherever the selection was made. A. vinelandii strains were grown at 307C in modified Burk nitrogen-free (BN0) medium (23). When it was necessary to include fixed nitrogen in the medium, ammonium acetate (NH4OAc. H2O) was added to a final concentration of 400mg per mL. General molecular techniques. Restriction enzymes were purchased either from Boehringer Mannheim (Indianapolis, IN) or from Promega (Madison, WI). DNA sub-cloning, plasmid DNA isolations, restriction enzyme digestions, agarose gel electrophoresis, ligations and E. coli transformations were carried out as described in the laboratory manual (22, 24) or as suggested in the manufacturers instructions. Oligonucleotides used for sequencing were purchased from GIBCO BRL Life Technologies, Inc. (Gaithersbureg, MD). Radio-labeled material for sequencing ([35S]-dATP) was obtained from Dupont NEN (Boston, MA). Nucleotide sequencing was performed using a T7 Sequenase version 2.0 DNA sequencing kit purchased from USBAmersham Life Sciences, Inc. (Cleveland, OH). Nucleotide and amino acid sequence analysis was performed using MacVector 5.0 software
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FIG. 1. (a) A Partial Restriction Map of the 6.2kb EcoRI Fragment that Harbors the lep of A. vinelandii. The lep is located in the 1.3kb EcoRI-HindIII fragment and is marked by a box. Arrow represents the direction of translation. (b) Nucleotide sequence and the derived amino acid sequence of A. vinelandii lep gene. Numbering represents amino acids. Transmembrane domains are underlined. The highly conserved domains found in members of the leader peptidase I family are overlined (Boxes A-E). The potential catalytically active Serine and Lysine residues are marked by bold letters.
and sequence homology searches were conducted using NCBI BLAST search analysis (25). Amino acid alignments were done using the Clustal V multiple protein sequence alignment program via Netscape (26, 27). Hydropathy plot analysis was determined by utilizing the TMpred program via Netscape (28). Amino acid sequences were obtained through Swiss Protein Database and MacVector 5.0.
RESULTS AND DISCUSSION Sub-cloning of lep from pBG400 and nucleotide sequence analysis. Previously, a 6.2kb EcoRI restriction fragment of the A. vinelandii genome carrying lep op-
eron was identified (29). This fragment was cloned into the unique EcoRI site of pUC19 and the plasmid was designated pBG400. A partial restriction map of this 6.2kb EcoRI fragment is shown in the Fig. 1a. By cleavage with HindIII, the EcoRI-HindIII fragments of 1.3kb and 4.9kb were generated and then were subcloned by ligating into the EcoRI and HindIII sites of M13 mp18 and M13 mp19. The recombinant plaques were identified and were used to obtain pure, single stranded DNA to determine the nucleotide sequence. Initial nucleotide sequences were used for a homology
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FIG. 1—Continued
search using NCBI BLAST and it was found that the 1.3 kb EcoRI-HindIII fragment was a putative candidate for harboring the lep gene. The nucleotide sequences of both strands of this fragment were further verified with the help of standard or sequence specific primers and radio-labeled chain termination sequencing. It was found that the nucleotide sequence of the 1.3 kb EcoRI-HindIII fragment contained an open reading frame that could encode a protein of 284 amino acids that shared high homology with other known leader peptidases. This potential leader peptidase I from A. vinelandii has a predicted molecular weight of 32,037 daltons. The nucleotide and predicted amino acid sequence of A. vinelandii lep is given in figure (Fig. 1b) Comparison of A. vinelandii Lep to other known leader peptidases. Since the nucleotide and amino acid sequence comparisons have identified a similarity between our sequence and sequences of the members of leader peptidase I family, our next step was to further analyze the translated amino acid sequence to find trends in areas of conservation, membrane topology and catalysis. As shown in Fig. 2, the leader peptidase I of A. vinelandii shares 75% identity with the leader peptidase I from P. flourescens, 37% identity with the leader peptidase I of E. coli, and 28% identity with B. subtilis. Shared homology ranges from 24-27% when the leader peptidase I of A. vinelandii is compared to the Saccharomyces cerevisiae proteins Imp1 and Imp2
(data not shown). The identified members of the leader peptidase I family are known to have particular regions of highly conserved sequences(10). To identify if our nucleotide derived amino acid sequence contained the signature motif, a computer based motif search was performed. This analysis indicated that the A. vinelandii Lep sequence does contain the signature sequence of the leader peptidase I family. The first segment of the signature has a consensus sequence of [GS]-X-SM-X-[PS]-[AT]-[LF] which is identified as S-G-S-M-KP-T-L in A. vinelandii (Fig. 2). The second portion of the signature sequence (K-R-[LIVMSTA]-X-G-X-[PG]G-[DE]-X-[LIVM]-X-[LIVMFY]) is found in the A. vinelandii Lep sequence as K-R-V-V-G-L-P-G-D-H-I-R-Y. The third portion of the signature sequence, G-D-[NH]X-X-X-[SND]-X-X-[SG], is found as G-D-N-R-D-N-S-ND-S in the A. vinelandii Lep sequence. Furthermore, the two additional conserved regions that were recently identified among the leader peptidase family were also conserved in A. vinelandii Lep sequence (Fig. 2 Box A and Box C). The presence of this signature sequence in A. vinelandii Lep indicates that the protein encoded by the open reading frame located in the 1.3kb fragment is, most likely, a member of the leader peptidase I family. In both E. coli and B. subtilis (LepS), the catalytically active amino acid residues have been identified through experimentation. In both cases a serine and a lysine residue are implicated in the activity of the en-
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FIG. 2. Multiple Sequence Alignment results (adapted from ClustalV 1.7 homology alignment). The amino acid sequences used for the alignment are identified by accession number when available. The putative and identified SPases (Signal Peptidase I or Leader Petidase I) are as follows: A.v.Lep (this study): putative Leader Petidase I from A. vinelandii strain OP; S51921 Signal Peptidase I from Phormidium laminosum (18); D90904_35 (LepB) Leader Peptidase I from Synechocystis sp. strain:PCC6803 (38); D90899_84 (LepB) Leader Peptidase I from Synechocystis sp. strain:PCC6803 (38); U45883_2 (SipT) Type I Signal Peptidase from Bacillus subtilis (39); P41025 (Sip) Signal Peptidase I from Bacillus amyloliquefaciens Strain ATCC 23844 (40); P42668 (Sip) from Bacillus licheniformis Strain ATCC 9789 (41); P28628 (SipS) Signal Peptidase I from Bacillus subtilis Strain 168 / 6GM(AMY) (42); I40552 (SipP40) Signal Peptidase I from Bacillus subtilis (43); P37943 (SipP) Signal Peptidase I from Bacillus subtilis Plasmid PTA1015 (44); S22414 Signal Peptidase I from Pseudomonas fluorescens (16); P00803 (LepB) Signal Peptidase I from Escherichia coli Strain K12 (45); S12020 (Lep) Signal Peptidase I from Salmonella typhimurium (14); P44454 (LepB or HI0015) Signal Peptidase I from Haemophilus influenzae Strain RD / KW20 (17); P41027 (SipC) Signal Peptidase I from Bacillus caldolyticus (44); Q10789 (LepB or MTCY274.34C) probable Signal Peptidase I from Mycobacterium tuberculosis Strain H37RV (46); S66597 Leader Peptidase from Rhodobacter capsulatus (47); U33883_2 (SipS) Signal Peptidase from Bradyrhizobium japonicum Strain mutant 132 (48). The darkness of blocks reflects the significance of similarity - with black boxes representing ú90% conserved identity, dark gray boxes representing ú75% conserved identity and light gray boxes representing ú80% conserved functional similarity. Numbers on the right correspond to numbers of amino acid residues. Proteins in the family S26 (SPase I) share the following signature patterns: SPase I signature 1 (Accession PS00501) contains the putative active site serine, [GS]-x-S-M-x-[PS]-[AT]-[LF]; SPase I signature 2 (Accession PS00760) contains the putative active site lysine, K-R-[LIVMSTA](2)-G-x-[PG]-G-[DE]-x-[LIVM]-x-[LIVMFY]; and SPase I signature 3 (Accession PS00761) has an unknown function, [LIVMFYW](2)-x(2)-G-D-[NH]-x(3)-[SND]-x(2)-[SG]. A.v.Lep matches are shown in bold.
zyme (30-32). As with E. coli and B. subtilis, a serine (Serine 91) and a lysine (Lysine 146) located within Box B and Box D respectively, are conserved in A. vinelandii (Fig. 2). A conserved GDN sequence is also found within the Box E of the A. vinelandii leader peptidase I (Fig. 2). The hydropathy analysis of the translated amino acid sequence of A. vinelandii leader peptidase I indi-
cated that this protein has two putative membrane spanning regions (Fig. 3). The first hydrophobic domain, which corresponds to H1 in E. coli (33), spans residues 7 - 33 (Fig. 1b, Fig. 3 and Fig. 4). The second hydrophobic domain, which corresponds to H2 in E. coli (30), spans residues 65 - 85 in the sequence (Fig. 1b, Fig. 3 and Fig. 4). These regions are comparable to potential membrane-spanning regions of other leader
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FIG. 2—Continued
peptidases. A third region of hydrophobic residues can be identified in the region following Serine 91 in A. vinelandii; however, this region was not identified as a possible transmembrane-spanning region by our hydropathy plot analysis of the amino acid sequence of the protein (Fig. 4). This finding is consistent with the periplasmic location of H3 in E. coli (33). Therefore, we
propose that the membrane topology of A. vinelandii Lep is similar to the membrane topologies of E. coli and P. flourescens as opposed to B. subtilis and H. influenzae (Fig. 4). Previous studies on other leader peptidases have shown that the presence of positively charged amino acid residues also play a role in the control of the mem-
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FIG. 3. Hydropathy Plot Analysis of the Nucleotide Derived Amino Acid Sequence of A. vinelandii Leader Peptidase I. Peak one and peak two represent putative transmembrane domains and corresponds to residues 7-28 and 66-85 respectively. The analysis of the possible transmembrane domains was done using TMpred program (25).
brane topology of these integral inner membrane proteins (34, 35). The primary sequence of the A. vinelandii leader peptidase I shows that positively charged amino acid residues are present in the region immediately following H1 and preceding H2. The presence of the positively charged residues and the placement of the putative transmembrane domains further support the idea that the membrane topology of the leader peptidase I of A. vinelandii is similar to that of E. coli rather than to that of B. subtilis and H. influenzae (Fig. 4). Genetic complementation analysis. To further investigate the functional role of leader peptidase I in A. vinelandii, complementation studies using a conditional lethal E. coli strain, were performed. E. coli strain IT41 does not exhibit leader peptidase I activity at a non-permissive temperatures of 427C (36). It was shown that this mutation could be complemented by a plasmid carrying a functional lep gene. To determine if the A. vinelandii lep that we have obtained encode a functional leader peptidase I, we analyzed whether a plasmid carrying A. vinelandii lep can complement the mutation in E. coli strain IT41. The A vinelandii Lep ORF present in 1.3kb EcoRI-HindIII fragment is not preceded by any putative promoter. Therefore, we constructed a plasmid carrying A. vinelandii lep gene down stream to the lac promoter present in pUC19 (37). It was achieved by ligating the 1.3kb EcoRI-HindIII fragment into EcoRI-HindIII site of pUC19. This plasmid
was designated pBG401. A control plasmid in which the lep gene was placed in the opposite orientation downstream to the lac promoter was also constructed by ligating 1.3kb EcoRI-HindIII fragment into EcoRIHindIII sites of pUC18 and was designated pBG402. The E. coli strain IT41 was transformed with plasmids pGB401 and pBG402, and the ability of the trans-
FIG. 4. Putative Membrane Topology Comparisons of Leader Peptidase I Proteins. Transmembrane domains are indicated by boxes. Amino and carboxy termini are labeled. The position of the catalytically active serine residue is indicated by an asterisk. The putative membrane topology type for each organism of study is indicated below each topology diagram. The topology models are based on the published sequences and previously predicted models. A. vinelandii Lep is more similar to the Lep of E. coli type than Bacillus or Heamophilus types.
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Genetic Complementation Analysis of A. vinelandii lep Growth conditions used
E. coli strains (Plasmid)
307C (2YT Amp agar plates
427C (2YT Amp agar plates)
427C (2YT Amp /IPTG agar plates)
TG1(pUC19) TG1(pBG402) TG1(pBG401) IT41(pUC19) IT41(pBG402) IT41(pBG401)
/// /// /// /// /// ///
/// /// /// 000 000 000
/// /// /// 000 000 ///
Note. Cells of the E. coli strain IT41 that produces an inactive leader peptidase I at 427C and of a control strain TG1 were made competent and were transformed with plasmids pUC19, pBG401 (a pUC19 derivative that can encode A. vinelandii Lep due to the placement of the Lep encoding sequence under the control of lac promoter) and pBG402 (a pUC18 derivative in which the A. vinelandii Lep encoding sequence is placed in the opposite orientation of lac promoter). The growth of these cells were assessed at the permissive temperature of 307C and non-permissive temperature of 427C. Since the expression of A. vinelandii lep cloned in pUC plasmids is dependent on IPTG induction, only the IT41 cells that harbor pBG401 and incubated on 2YT agar plates supplemented with ampicillin (Amp) and IPTG were able to grow at the non-permissive temperature of 427C. ///, represents positive growth and 000 represents lack of growth.
formants to grow at 427C on 2YT ampicillin plates supplemented with IPTG was analyzed. While the E. coli strain IT41 carrying the pBG401 could grow at 427C, the E. coli strain IT41 carrying the pBG402 could not grow at that temperature (Table 1). This result suggested that the Lep protein, encoded by the nucleotide sequence present on 1.3kb EcoRI-HindIII fragment indeed is a functional leader peptidase I. Moreover, Southern blot analysis of the A. vinelandii chromosome indicated that the lep gene is present as a single copy on the chromosome (data not shown). Taken together these results suggest that we have cloned the functional lep gene of A. vinelandii. ACKNOWLEDGMENTS We greatly appreciate the help provided by Bryan Hausman and Ryan Schreiner during the preparation of this manuscript. We thank Vondolee M. Delgado-Partin and Ross E. Dalbey for providing us with the E. coli strain IT41. We also thank the members of Gavini and Pulakat laboratories at BGSU for their helpful discussions. This work is supported by Research Challenge Grant, Faculty Research Committee Grant and USDA grant to LP and NG.
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39. Tjalsma, H., Bolhuis, A., Bron, S., Venema, G., and van Dijl, J. M. (1996) EMBL/GENBANK/DDBJ DATA BANKS. 40. Hoang, V., and Hofemeister, J. (1994) EMBL/GENBANK/ DDBJ DATA BANKS. 41. Hoang, V., Birger, A., and Hofemeister, J. (1993) EMBL/GENBANK/DDBJ DATA BANKS. 42. Sorokin, A., Zumstein, E., Azevedo, V., Ehrlich, S. D., and Serror, P. (1993) Mol. Microbiol. 10, 385–95. 43. Meijer, W. J., Venema, G., and Bron, S. (1995) Nucleic Acids Res. 23, 612–619. 44. Meijer, W. J. J., De Jong, A., Wisman, G. B. A., Tjalsma, H., Venema, G., Bron, S., and van Dijl, J. M. (1995) Mol. Microbiol. 17, 621–631. 45. Blattner, F. R., Plunkett, G. I., Mayhew, G. F., Perna, N. T., and Glasner, F. D. (1997) EMBL/GENBANK/DDBJ DATA BANKS. 46. Connor, R., Churcher, C. M., Barrell, B. G., Rajandream, M. A., and Walsh, S. V. (1996) EMBL/GENBANK/DDBJ DATA BANKS. 47. Rauhut, R., Jaeger, A., Conrad, C., and Klug, G. (1996) Nucleic Acids Res. 24, 1246–1251. 48. Mueller, P., Ahrens, K., Keller, T., and Klaucke, A. (1995) Mol.Microbiol. 18(5), 831–840.
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Nucleotide Sequence and Genetic Complementation Analysis of lep from Azotobacter vinelandii Carissa A. Jock, Lakshmidevi Pulakat, SaeHong Lee, and Narasaiah Gavini1 Department of Biological Sciences, Bowling Green State University, Bowling Green, Ohio 43403
Received August 21, 1997
The lep of Azotobacter vinelandii is an 852-base-pair open reading frame (ORF) which encodes a protein of 284 amino acid residues. The translated protein shares 75% homology with leader peptidase I isolated from Pseudomonas flourescens and 37% homology with leader peptidase I isolated from Escherichia coli. Five highly conserved regions found in the family of leader peptidase I proteins are conserved in A. vinelandii Lep. The putative membrane topology of the protein seems similar to that of E. coli leader peptidase I based on the hydrophobicity analysis of the predicted amino acid sequence. Southern blotting analysis of the A. vinelandii chromosome by probing with lep specific DNA revealed that lep is present as a single copy per the chromosome. A multicopy plasmid carrying A. vinelandii lep could complement a temperature sensitive lep mutant of E. coli strain IT41, suggesting that we have identified the functional copy of lep present on A. vinelandii genome. q 1997 Academic Press
Azotobacter vinelandii is a free-living soil bacterium widely known for its ability to fix nitrogen aerobically (1-4). It was speculated that this bacterium contains multiple chromosomes per cell with unique biology of cell maintenance and growth (5-7). Until recent, it was generally accepted that A. vinelandii could not uptake amino acids and it was proposed that the genes coding for amino acid transport proteins were not present within its genome (8). However, in a recent study, some amount of amino acid transport in A. vinelandii was reported suggesting that at least some amino acid transport proteins are present in this organism (9). Amino acid transport proteins are known to be located at the outer surface of the cell; therefore if amino acid transport proteins are synthesized in A. vinelandii, they need to undergo post translational protein processing in order to reach their appropriate destination. 1 Corresponding author and mailing address: Department of Biological Sciences, Bowling Green State University, Bowling Green, Ohio 43403. E-mail: [email protected].
In general, one to ten thousand different proteins are synthesized within the cytosol of a given cell in order for proper cell metabolism and viability. In both prokaryotic and eukaryotic cells, these newly synthesized proteins must reach specific intracellular locations for metabolic processes to be carried out effectively. One protein that plays a major role in protein processing is leader peptidase I. In the absence of leader peptidase I, the majority of pre-proteins accumulate on the periplasmic side of the inner membrane eventually leading to cell death (10-13). Therefore, the leader peptidases are essential for effective protein processing and cell viability in both prokaryotic and eukaryotic cells. Genes coding for leader peptidase I have been isolated and sequenced from a variety of organisms (14-21). The nucleotide and deduced amino acid sequences of the genes isolated from a variety of organisms share significant homology suggesting a common organization in their functional mechanistics. Here we report the nucleotide sequence analysis of lep isolated from A. vinelandii, genetic complementation analysis and comparison of predicted membrane topology to other leader peptidases. MATERIALS AND METHODS Bacterial strains, plasmids, and growth conditions. E.coli strains were normally grown at 377C in Luria broth or 2YT (22). The antibiotic ampicillin was used at final concentration of 50mg/ml wherever the selection was made. A. vinelandii strains were grown at 307C in modified Burk nitrogen-free (BN0) medium (23). When it was necessary to include fixed nitrogen in the medium, ammonium acetate (NH4OAc. H2O) was added to a final concentration of 400mg per mL. General molecular techniques. Restriction enzymes were purchased either from Boehringer Mannheim (Indianapolis, IN) or from Promega (Madison, WI). DNA sub-cloning, plasmid DNA isolations, restriction enzyme digestions, agarose gel electrophoresis, ligations and E. coli transformations were carried out as described in the laboratory manual (22, 24) or as suggested in the manufacturers instructions. Oligonucleotides used for sequencing were purchased from GIBCO BRL Life Technologies, Inc. (Gaithersbureg, MD). Radio-labeled material for sequencing ([35S]-dATP) was obtained from Dupont NEN (Boston, MA). Nucleotide sequencing was performed using a T7 Sequenase version 2.0 DNA sequencing kit purchased from USBAmersham Life Sciences, Inc. (Cleveland, OH). Nucleotide and amino acid sequence analysis was performed using MacVector 5.0 software
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FIG. 1. (a) A Partial Restriction Map of the 6.2kb EcoRI Fragment that Harbors the lep of A. vinelandii. The lep is located in the 1.3kb EcoRI-HindIII fragment and is marked by a box. Arrow represents the direction of translation. (b) Nucleotide sequence and the derived amino acid sequence of A. vinelandii lep gene. Numbering represents amino acids. Transmembrane domains are underlined. The highly conserved domains found in members of the leader peptidase I family are overlined (Boxes A-E). The potential catalytically active Serine and Lysine residues are marked by bold letters.
and sequence homology searches were conducted using NCBI BLAST search analysis (25). Amino acid alignments were done using the Clustal V multiple protein sequence alignment program via Netscape (26, 27). Hydropathy plot analysis was determined by utilizing the TMpred program via Netscape (28). Amino acid sequences were obtained through Swiss Protein Database and MacVector 5.0.
RESULTS AND DISCUSSION Sub-cloning of lep from pBG400 and nucleotide sequence analysis. Previously, a 6.2kb EcoRI restriction fragment of the A. vinelandii genome carrying lep op-
eron was identified (29). This fragment was cloned into the unique EcoRI site of pUC19 and the plasmid was designated pBG400. A partial restriction map of this 6.2kb EcoRI fragment is shown in the Fig. 1a. By cleavage with HindIII, the EcoRI-HindIII fragments of 1.3kb and 4.9kb were generated and then were subcloned by ligating into the EcoRI and HindIII sites of M13 mp18 and M13 mp19. The recombinant plaques were identified and were used to obtain pure, single stranded DNA to determine the nucleotide sequence. Initial nucleotide sequences were used for a homology
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FIG. 1—Continued
search using NCBI BLAST and it was found that the 1.3 kb EcoRI-HindIII fragment was a putative candidate for harboring the lep gene. The nucleotide sequences of both strands of this fragment were further verified with the help of standard or sequence specific primers and radio-labeled chain termination sequencing. It was found that the nucleotide sequence of the 1.3 kb EcoRI-HindIII fragment contained an open reading frame that could encode a protein of 284 amino acids that shared high homology with other known leader peptidases. This potential leader peptidase I from A. vinelandii has a predicted molecular weight of 32,037 daltons. The nucleotide and predicted amino acid sequence of A. vinelandii lep is given in figure (Fig. 1b) Comparison of A. vinelandii Lep to other known leader peptidases. Since the nucleotide and amino acid sequence comparisons have identified a similarity between our sequence and sequences of the members of leader peptidase I family, our next step was to further analyze the translated amino acid sequence to find trends in areas of conservation, membrane topology and catalysis. As shown in Fig. 2, the leader peptidase I of A. vinelandii shares 75% identity with the leader peptidase I from P. flourescens, 37% identity with the leader peptidase I of E. coli, and 28% identity with B. subtilis. Shared homology ranges from 24-27% when the leader peptidase I of A. vinelandii is compared to the Saccharomyces cerevisiae proteins Imp1 and Imp2
(data not shown). The identified members of the leader peptidase I family are known to have particular regions of highly conserved sequences(10). To identify if our nucleotide derived amino acid sequence contained the signature motif, a computer based motif search was performed. This analysis indicated that the A. vinelandii Lep sequence does contain the signature sequence of the leader peptidase I family. The first segment of the signature has a consensus sequence of [GS]-X-SM-X-[PS]-[AT]-[LF] which is identified as S-G-S-M-KP-T-L in A. vinelandii (Fig. 2). The second portion of the signature sequence (K-R-[LIVMSTA]-X-G-X-[PG]G-[DE]-X-[LIVM]-X-[LIVMFY]) is found in the A. vinelandii Lep sequence as K-R-V-V-G-L-P-G-D-H-I-R-Y. The third portion of the signature sequence, G-D-[NH]X-X-X-[SND]-X-X-[SG], is found as G-D-N-R-D-N-S-ND-S in the A. vinelandii Lep sequence. Furthermore, the two additional conserved regions that were recently identified among the leader peptidase family were also conserved in A. vinelandii Lep sequence (Fig. 2 Box A and Box C). The presence of this signature sequence in A. vinelandii Lep indicates that the protein encoded by the open reading frame located in the 1.3kb fragment is, most likely, a member of the leader peptidase I family. In both E. coli and B. subtilis (LepS), the catalytically active amino acid residues have been identified through experimentation. In both cases a serine and a lysine residue are implicated in the activity of the en-
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FIG. 2. Multiple Sequence Alignment results (adapted from ClustalV 1.7 homology alignment). The amino acid sequences used for the alignment are identified by accession number when available. The putative and identified SPases (Signal Peptidase I or Leader Petidase I) are as follows: A.v.Lep (this study): putative Leader Petidase I from A. vinelandii strain OP; S51921 Signal Peptidase I from Phormidium laminosum (18); D90904_35 (LepB) Leader Peptidase I from Synechocystis sp. strain:PCC6803 (38); D90899_84 (LepB) Leader Peptidase I from Synechocystis sp. strain:PCC6803 (38); U45883_2 (SipT) Type I Signal Peptidase from Bacillus subtilis (39); P41025 (Sip) Signal Peptidase I from Bacillus amyloliquefaciens Strain ATCC 23844 (40); P42668 (Sip) from Bacillus licheniformis Strain ATCC 9789 (41); P28628 (SipS) Signal Peptidase I from Bacillus subtilis Strain 168 / 6GM(AMY) (42); I40552 (SipP40) Signal Peptidase I from Bacillus subtilis (43); P37943 (SipP) Signal Peptidase I from Bacillus subtilis Plasmid PTA1015 (44); S22414 Signal Peptidase I from Pseudomonas fluorescens (16); P00803 (LepB) Signal Peptidase I from Escherichia coli Strain K12 (45); S12020 (Lep) Signal Peptidase I from Salmonella typhimurium (14); P44454 (LepB or HI0015) Signal Peptidase I from Haemophilus influenzae Strain RD / KW20 (17); P41027 (SipC) Signal Peptidase I from Bacillus caldolyticus (44); Q10789 (LepB or MTCY274.34C) probable Signal Peptidase I from Mycobacterium tuberculosis Strain H37RV (46); S66597 Leader Peptidase from Rhodobacter capsulatus (47); U33883_2 (SipS) Signal Peptidase from Bradyrhizobium japonicum Strain mutant 132 (48). The darkness of blocks reflects the significance of similarity - with black boxes representing ú90% conserved identity, dark gray boxes representing ú75% conserved identity and light gray boxes representing ú80% conserved functional similarity. Numbers on the right correspond to numbers of amino acid residues. Proteins in the family S26 (SPase I) share the following signature patterns: SPase I signature 1 (Accession PS00501) contains the putative active site serine, [GS]-x-S-M-x-[PS]-[AT]-[LF]; SPase I signature 2 (Accession PS00760) contains the putative active site lysine, K-R-[LIVMSTA](2)-G-x-[PG]-G-[DE]-x-[LIVM]-x-[LIVMFY]; and SPase I signature 3 (Accession PS00761) has an unknown function, [LIVMFYW](2)-x(2)-G-D-[NH]-x(3)-[SND]-x(2)-[SG]. A.v.Lep matches are shown in bold.
zyme (30-32). As with E. coli and B. subtilis, a serine (Serine 91) and a lysine (Lysine 146) located within Box B and Box D respectively, are conserved in A. vinelandii (Fig. 2). A conserved GDN sequence is also found within the Box E of the A. vinelandii leader peptidase I (Fig. 2). The hydropathy analysis of the translated amino acid sequence of A. vinelandii leader peptidase I indi-
cated that this protein has two putative membrane spanning regions (Fig. 3). The first hydrophobic domain, which corresponds to H1 in E. coli (33), spans residues 7 - 33 (Fig. 1b, Fig. 3 and Fig. 4). The second hydrophobic domain, which corresponds to H2 in E. coli (30), spans residues 65 - 85 in the sequence (Fig. 1b, Fig. 3 and Fig. 4). These regions are comparable to potential membrane-spanning regions of other leader
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FIG. 2—Continued
peptidases. A third region of hydrophobic residues can be identified in the region following Serine 91 in A. vinelandii; however, this region was not identified as a possible transmembrane-spanning region by our hydropathy plot analysis of the amino acid sequence of the protein (Fig. 4). This finding is consistent with the periplasmic location of H3 in E. coli (33). Therefore, we
propose that the membrane topology of A. vinelandii Lep is similar to the membrane topologies of E. coli and P. flourescens as opposed to B. subtilis and H. influenzae (Fig. 4). Previous studies on other leader peptidases have shown that the presence of positively charged amino acid residues also play a role in the control of the mem-
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FIG. 3. Hydropathy Plot Analysis of the Nucleotide Derived Amino Acid Sequence of A. vinelandii Leader Peptidase I. Peak one and peak two represent putative transmembrane domains and corresponds to residues 7-28 and 66-85 respectively. The analysis of the possible transmembrane domains was done using TMpred program (25).
brane topology of these integral inner membrane proteins (34, 35). The primary sequence of the A. vinelandii leader peptidase I shows that positively charged amino acid residues are present in the region immediately following H1 and preceding H2. The presence of the positively charged residues and the placement of the putative transmembrane domains further support the idea that the membrane topology of the leader peptidase I of A. vinelandii is similar to that of E. coli rather than to that of B. subtilis and H. influenzae (Fig. 4). Genetic complementation analysis. To further investigate the functional role of leader peptidase I in A. vinelandii, complementation studies using a conditional lethal E. coli strain, were performed. E. coli strain IT41 does not exhibit leader peptidase I activity at a non-permissive temperatures of 427C (36). It was shown that this mutation could be complemented by a plasmid carrying a functional lep gene. To determine if the A. vinelandii lep that we have obtained encode a functional leader peptidase I, we analyzed whether a plasmid carrying A. vinelandii lep can complement the mutation in E. coli strain IT41. The A vinelandii Lep ORF present in 1.3kb EcoRI-HindIII fragment is not preceded by any putative promoter. Therefore, we constructed a plasmid carrying A. vinelandii lep gene down stream to the lac promoter present in pUC19 (37). It was achieved by ligating the 1.3kb EcoRI-HindIII fragment into EcoRI-HindIII site of pUC19. This plasmid
was designated pBG401. A control plasmid in which the lep gene was placed in the opposite orientation downstream to the lac promoter was also constructed by ligating 1.3kb EcoRI-HindIII fragment into EcoRIHindIII sites of pUC18 and was designated pBG402. The E. coli strain IT41 was transformed with plasmids pGB401 and pBG402, and the ability of the trans-
FIG. 4. Putative Membrane Topology Comparisons of Leader Peptidase I Proteins. Transmembrane domains are indicated by boxes. Amino and carboxy termini are labeled. The position of the catalytically active serine residue is indicated by an asterisk. The putative membrane topology type for each organism of study is indicated below each topology diagram. The topology models are based on the published sequences and previously predicted models. A. vinelandii Lep is more similar to the Lep of E. coli type than Bacillus or Heamophilus types.
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Genetic Complementation Analysis of A. vinelandii lep Growth conditions used
E. coli strains (Plasmid)
307C (2YT Amp agar plates
427C (2YT Amp agar plates)
427C (2YT Amp /IPTG agar plates)
TG1(pUC19) TG1(pBG402) TG1(pBG401) IT41(pUC19) IT41(pBG402) IT41(pBG401)
/// /// /// /// /// ///
/// /// /// 000 000 000
/// /// /// 000 000 ///
Note. Cells of the E. coli strain IT41 that produces an inactive leader peptidase I at 427C and of a control strain TG1 were made competent and were transformed with plasmids pUC19, pBG401 (a pUC19 derivative that can encode A. vinelandii Lep due to the placement of the Lep encoding sequence under the control of lac promoter) and pBG402 (a pUC18 derivative in which the A. vinelandii Lep encoding sequence is placed in the opposite orientation of lac promoter). The growth of these cells were assessed at the permissive temperature of 307C and non-permissive temperature of 427C. Since the expression of A. vinelandii lep cloned in pUC plasmids is dependent on IPTG induction, only the IT41 cells that harbor pBG401 and incubated on 2YT agar plates supplemented with ampicillin (Amp) and IPTG were able to grow at the non-permissive temperature of 427C. ///, represents positive growth and 000 represents lack of growth.
formants to grow at 427C on 2YT ampicillin plates supplemented with IPTG was analyzed. While the E. coli strain IT41 carrying the pBG401 could grow at 427C, the E. coli strain IT41 carrying the pBG402 could not grow at that temperature (Table 1). This result suggested that the Lep protein, encoded by the nucleotide sequence present on 1.3kb EcoRI-HindIII fragment indeed is a functional leader peptidase I. Moreover, Southern blot analysis of the A. vinelandii chromosome indicated that the lep gene is present as a single copy on the chromosome (data not shown). Taken together these results suggest that we have cloned the functional lep gene of A. vinelandii. ACKNOWLEDGMENTS We greatly appreciate the help provided by Bryan Hausman and Ryan Schreiner during the preparation of this manuscript. We thank Vondolee M. Delgado-Partin and Ross E. Dalbey for providing us with the E. coli strain IT41. We also thank the members of Gavini and Pulakat laboratories at BGSU for their helpful discussions. This work is supported by Research Challenge Grant, Faculty Research Committee Grant and USDA grant to LP and NG.
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