Fleming Building, Institute of Pharmacy, Chemistry and Biomedical Sciences, University of Sunderland, Sunderland SR2 3SD, UK1
Department of Biomedical Sciences, University of Bradford, UK2
Author for correspondence: Iain C. Sutcliffe. Tel: +44 191 515 2995. Fax: +44 191 515 3747. e-mail: iain.sutcliffe{at}sunderland.ac.uk
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ABSTRACT |
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Keywords: Streptococcus pyogenes, Bacillus subtilis, genomics, YidC, exported proteins
Abbreviations: Lpp, lipoprotein; MSD, membrane-spanning domain; SBP, substrate-binding protein
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INTRODUCTION |
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The archetypal bacterial Lpp is the murein lipoprotein of Escherichia coli characterized by Braun and co-workers (reviewed in Braun & Wu, 1994) . Structural studies revealed that a diacylglycerol moiety is thioether linked to an N-terminal cysteine of this Lpp and that this lipid group serves to orientate the protein by anchoring it to the inner leaflet of the outer membrane. Chemically identical lipid modifications have since been proposed, primarily on the basis of protein sequence analysis (see below), for a great many proteins in both Gram-positive and Gram-negative bacteria, although relatively few have received extensive biochemical characterization.
Biosynthesis of bacterial lipoprotein of the Braun type proceeds via a well conserved pathway that is apparently unique to prokaryotes (Fig. 1). Following signal-peptide-directed export of the prolipoprotein (proLpp), the enzyme prolipoprotein diacylglycerol transferase (Lgt) uses phospholipid substrates and catalyses the addition of a diacylglycerol lipid unit onto the thiol of a crucial conserved cysteine which is located within a lipobox motif at the cleavage region of the proLpp signal peptide (Sankaran et al., 1995
; Qi et al., 1995
). Subsequently, the signal peptide is removed by a specific lipoprotein signal peptidase II (Lsp) enzyme which cleaves within the lipobox to release the lipidated cysteine as the N-terminus of the mature Lpp (Braun & Wu, 1994
; Sankaran & Wu, 1995
). These two steps have been confirmed to be necessary and sufficient for protein lipidation in Gram-positive bacteria (Zhao & Wu, 1992
; Witke & Götz, 1995
; Leskelä et al., 1999
; Tjalsma et al., 1999a
, b
; Bengtsson et al., 1999
; Petit et al., 2001
). A third step, wherein the Lpp N terminus is further modified by addition of an amide-linked fatty acid, is apparently not conserved as homologues of the enzyme lipoprotein aminoacyl transferase are not found in the genomes of low G+C Gram-positive bacteria (Tjalsma et al., 1999a
and our unpublished observations).
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Molecular genetic studies, and genome sequencing projects in particular, have identified a large number of putative Lpp genes on the basis of the presence of possible lipobox sequences at the N termini of translated protein sequences. Indeed, putative Lpps may represent at least 0·58·0% of the bacterial proteome (Chambaud et al., 1999 ; Tjalsma et al., 1999a
; Haake, 2000
). However, it remains possible that a significant proportion of these putative Lpps are false-positives, misidentified due to the coincident presence of a cysteine within the signal sequences of exported proteins or proteins targeted for insertion into plasma membranes. Thus it is desirable to develop a method for more accurately assigning sequences as putative Lpps. In this context it is significant that the lipobox amino acids are apparently subject to subtle taxon-specific restrictions. For example, the lipobox consensus defined from a dataset of 26 proven spirochaetal Lpps is distinctive in comparison to that of E. coli Lpps (Haake, 2000
). Likewise, the lipobox sequences of a subset of Gram-positive bacterial Lpp (proven and putative) were more restrictive than that described in the PS00013 pattern at the -3 and -2 positions (Sutcliffe & Russell, 1995
). To investigate this further we have defined a dataset of Gram-positive bacterial Lpps for which there is experimental evidence to support lipidation, and performed sequence analyses to create a revised pattern for the identification of Gram-positive bacterial putative Lpps. This pattern has been applied to the genome of the human pathogen Streptococcus pyogenes, for which a novel inventory of putative Lpps is presented herein.
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METHODS |
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Protein primary sequence analyses.
Membrane spanning domains (MSDs) in protein sequences were predicted using TMpred (Hofmann & Stoffel, 1993 ; http://www.ch.embnet.org/software/TMPRED_form.html), set to a minimum length of 14 aa for the hydrophobic domains. Signal peptide analyses were performed using the refined hidden Markov model version 2.0 of SignalP (Nielsen et al., 1997
; Nielsen & Krogh, 1998
; http://www.cbs.dtu.dk/services/SignalP-2.0/). Although Lpp signal peptide cleavage sites are not predicted, the graphical display from this service was particularly useful as it provided an indication of the lengths of predicted signal peptide n- and h-regions relative to the position of the possible lipobox cysteine. Where sequence features required further clarification, other methods for predicting transmembrane domains were also applied including TopPred2 (Claros & von Heijne, 1994
; http://bioweb.pasteur.fr/seqanal/interfaces/toppred.html) and DAS (Cserzo et al., 1997
; http://www.sbc.su.se/
miklos/DAS/).
Sequences homologous to the putative Lpps were identified by BLAST searches (Altschul et al., 1997 ) using the National Center for Biotechnology Information tBLASTn server (http://www.ncbi.nlm.nih.gov/BLAST/), typically with the sequence filtering option switched off and the Expect value set at 0·001.
Access to sequence annotation of the S. pyogenes and Streptococcus pneumoniae genomes was obtained via the Entrez Genomes facility at the National Center for Biotechnology Information (http://www.ncbi.nlm.nih.gov/PMGifs/Genomes/micr.html). Finally, other general searches were performed using the PEDANT genome sequence analysis tool (Frishman et al., 2001 ; http://pedant.gsf.de/).
Criteria for exclusion of sequences as false-positives unlikely to encode Lpps.
The N-terminal features of putative Lpps were individually analysed to determine whether they might be considered false-positives using the following criteria. Firstly, TMpred was used to determine the position of the most N-terminal MSD and also the number of additional MSDs. Those sequences where either an MSD was clearly absent or the most N-terminal MSD clearly extended beyond the lipobox cysteine (e.g. sequences likely to direct insertion of integral membrane proteins) were considered possible false-positives. The detection using TMpred of additional MSDs beyond the lipobox cysteine was not considered sufficient justification for excluding sequences, since both the CtaC and the QoxA proven Lpps have two additional MSDs predicted beyond their N-terminal lipid anchors (Bengtsson et al., 1999 ; Sakamoto et al., 2001
; Antelmann et al., 2001
), as does the Mycoplasma pneumoniae F0F1 ATPase (Pyrowolakis et al., 1998
).
Sequences were also analysed using SignalP. Those sequences where an h-region was typically predicted to end within 2 aa of the lipobox cysteine were retained as possible Lpps. Cumulatively, those sequences where signal peptide features were absent altogether and/or the lipobox cysteine was clearly internal rather than terminal to an h-region/MSD were considered to be likely false-positives. Those sequences where further clarification was needed were investigated using various other servers for predicting transmembrane regions (notably TopPred2 and DAS) and a consensus taken as to the position of the putative lipobox cysteine relative to the length of the first predicted MSD.
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RESULTS AND DISCUSSION |
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Given that certain proven Lpps had signal peptide sequences features clearly contradictory to those described by the PS00013 pattern, and that additional discrimination against false-positives is likely to result from the derivation of a taxon-specific pattern, a modified pattern was defined for the proven Lpp signal peptide sequences of Gram-positive bacteria (Table 2). This pattern, <[MV]-X(0,13)-[RK]-{DERKQ}(6,20)-[LIVMFESTAG]-[LVIAM]-[IVMSTAFG]-[AG]-C (using Prosite syntax), was termed G+LPP (Table 3
). To test the utility of this pattern, it was applied to a search of the genome of B. subtilis, as signal peptide dependent export has been extensively studied in this organism (Tjalsma et al., 2000
). A set of 103 putative Lpps of B. subtilis were identified by a pattern search with PS00013, whereas only 61 probable Lpps and the six proven Lpps of this organism were identified using the G+LPP pattern. The G+LPP pattern gave a greater discrimination (four rather than sixteen hits) against sequences considered false-positives using the criteria described herein (data not shown). Cumulatively, it was considered that the advantage of using the G+LPP pattern was the greater confidence that could be placed in the predictions that conforming sequences are indeed likely to be Lpps.
Application of the G+LPP pattern to analysis of the S. pyogenes genome
S. pyogenes sequences in the SWISS-PROT/TrEMBL database containing the PS00013 pattern were identified and compared to those identified in a similar pattern search with G+LPP (Table 4). The PS00013 search identified 36 sequences of which nine (25%) were excluded as unlikely Lpps (false-positives) using the criteria described herein. The G+LPP pattern search again proved more discriminatory than PS00013 in that 26 sequences were identified, of which only one (4%) was considered as an unlikely Lpp. Thus eight out of nine (89%) of the unlikely Lpps initially identified using PS00013 were excluded (Table 4
). Both searches identified the only previously identified proven Lpp, the putative acid phosphatase LppC (Gase et al., 1997
; Malke, 1998
). Similarly, both searches also identified the Lbp laminin-binding protein (Terao et al., 2002
) and three previously identified SBPs (Podbielski et al., 1996
; Podbielski & Leonard, 1998
; Janulczyk et al., 1999
), all of which have previously been considered putative Lpps. Notably, however, the search with G+LPP identified an additional putative SBP sequence (Spy0903) that had been excluded using PS00013 on account of the extended n-region features of this signal sequence.
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A second major functional category of S. pyogenes Lpps were those that may be involved in protein export and extracytoplasmic folding. Thus two homologues (Spy0247 and Spy0351) were identified of B. subtilis SpoIIIJ (Errington et al., 1992 ), a probable Lpp that has recently been suggested to be associated with the export and processing of integral membrane proteins (Tjalsma et al., 2000
; Luirink et al., 2001
; Yen et al., 2001
). Both Spy0247 and Spy0351 are predicted to encode proteins with multiple MSDs in addition to the predicted N-terminal lipid anchor, as in SpoIIIJ (Luirink et al., 2001
; Yen et al., 2001
). Likewise, two homologous putative Lpps of the SpoIIIJ family have been identified in the pneumococcal genome (Tettelin et al., 2001
). The functional significance of the probable N-terminal lipidation of these SpoIIIJ family proteins is difficult to predict given that they are also integral membrane proteins. However, the lipid anchor may serve to correctly orientate an N-terminal domain and it is noted that several proven Lpps also have additional MSDs (see Methods).
In addition to the above, at least two probable Lpps (Spy1390 and Spy2037) may function as extracytoplasmic folding catalysts in S. pyogenes. Both proteins are homologous to the recently described pneumococcal putative maturase PpmA (Overweg et al., 2000a , b
) and to the proven Lpps PrsA of B. subtilis and PrtM of Lactococcus lactis (Haandrikman et al., 1991
; Kontinen & Sarvas, 1993
). Collectively these proteins belong to the type C family of peptidyl-prolyl isomerases (Rudd et al., 1995
) which appear to direct correct protein folding. Thus these Lpps may act as folding catalysts or chaperones during protein export, although it is not clear whether this function is likely to be general or linked to the export of specific proteins. Since chaperones are likely to be required to interact with their cognate proteins as they emerge from the cytoplasmic membrane, the predicted localization of Lpps immediately adjacent to the external face of the membrane is consistent with this role. A further putative Lpp, peptidyl-prolyl isomerase, is discussed below.
Spy1558 is a member of the family of thioredoxin proteins and two homologous thioredoxin family members were identified in the pneumococcal genome (Tettelin et al., 2001 ). Both Spy1558 and its pneumococcal putative Lpp homologue SP0659 are situated adjacent to genes encoding putative exported peptide methionine sulfoxide reductases. These enzymes repair the damage to proteins that results from the oxidation of methionine residues to methionine sulfoxide and this catalytic activity requires an associated thioredoxin regenerating system (Lowther et al., 2000
). This may therefore be the role of the adjacent Lpp.
In addition to Lpps belonging to the functional categories described above, three surface enzymes (LppC acid phosphatase; Spy0210, which contains a domain suggesting it may be a protease of the transglutaminase family; and Spy0857, a putative peptidoglycan hydrolase), two conserved hypothetical proteins and an ORF with no significant homologues were identified as probable Lpps.
Three putative Lpp sequences were identified that conformed with the PS00013 pattern but not the G+LPP pattern and, as such, warrant further consideration. Of these, the putative pullulanase Spy1972 is unusual in the length of its signal sequence n-region and also contains a C-terminal LPXTG motif that may provide an alternative wall-anchoring mechanism (Janulczyk & Rasmussen, 2001 ). The signal peptide features of Spy1361 were consistent with an h-region preceding the possible lipobox but this sequence was excluded by G+LPP due to the presence of a glutamine residue within the h-region. Spy1361 was noted to contain a 300 aa domain homologous to the leucine-rich repeat region of L. monocytogenes internalin A that is preceded by an N-terminal domain containing four histidine-triad motifs (HXXHXH) which is homologous to PhtE of S. pneumoniae (Adamou et al., 2001
). The signal peptide features of the third protein Spy2066, a putative dipeptidase, are ambiguous. Thus without further evidence for the Lpp nature of these proteins, it is prudent to consider these three proteins as possible rather than probable Lpps.
Possible false-negatives not identified by pattern searching the S. pyogenes genome
A combination of strategies was used to locate possible Lpp sequences missed using either of the above pattern searches (i.e. possible false-negatives). These included analyses of the S. pyogenes genome annotation, searches for homologues of pneumococcal Lpps, searches through PEDANT and very low stringency BLAST searches (E=100) using a representative selection of the signal sequences for the probable Lpps identified herein (Table 4). These analyses identified six possible false-negatives that warrant further consideration.
Four additional SBPs that may be possible Lpps were identified. One such sequence was Spy0163 which, as noted above, is a paralogue of Spy1228. This sequence was revealed by BLAST searches with Spy1228 and the pneumococcal putative Lpp SP0845 but found to be annotated without five potential N-terminal amino acids, including an alternative start methionine and two lysines that would complete the signal peptide n-region. This revised signal peptide for Spy0163 matches the G+LPP pattern and both the sequence motifs identified by Rosati et al. (1999) are present. Secondly, Spy1592 was identified as a putative SBP. The signal sequence of this protein contains a lipobox sequence which was excluded by the pattern searches because of an asparagine at the -4 position. However, the corresponding ORF (99% amino acid identity) in the S. pyogenes Manfredo genome (http://www.sanger.ac.uk/Projects/S_pyogenes/) contains a serine at this position, suggesting this putative SBP may indeed be a Lpp in some strains. Likewise, the putative amino acid SBP Spy0778 and Spy1306, a homologue of pneumococcal MalX, were excluded as their possible lipobox sequences contained proline in the -4 position. As this is incompatible with both PS00013 and the G+LPP patterns, these SBPs should not be considered to be Lpps until proven otherwise, particularly as some SBPs (including the Spy0713 AdcA metal binding protein homologue) are evidently not Lpps (Turner et al., 1999
; Claverys, 2001
).
The S. pyogenes genome also contains an additional possible Lpp, peptidyl-prolyl isomerase of the cyclophilin family, Spy0457, which is highly homologous to the pneumococcal SP0771 putative Lpp. Again, this sequence was excluded by the pattern searches as the putative lipobox sequence contains an asparagine at the -4 position. As with Spy1592, the corresponding ORF (99% amino acid identity) in the S. pyogenes Manfredo genome contains the acceptable amino acid serine at this position and so this protein is likely to be a Lpp, at least in some strains of S. pyogenes. As discussed above, peptidyl-prolyl isomerase Lpp may assist in the folding of exported protein(s).
Finally, Spy2033 was identified as a possible false-negative. This sequence has been shown to contain a functional signal sequence directing either protein export or cell surface localization (Gibson & Caparon, 2002 ). The deposited Spy2033 sequence has an abnormally long 64 aa signal sequence. However, it is notable that the h-region ends in a putative lipobox cysteine which is preceded by sequence consistent with a canonical Lpp signal peptide (M41KFKKVLVIPALALAATCFLTAC63). The alternative start at M41 is also consistent with sequence alignments of Spy2033 with its homologue, the Streptococcus cristatus putative Lpp TptA. Thus it may be that the annotation of this sequence needs verification.
Conclusions
It is evident from the data herein that the identification of putative Lpp genes has been greatly facilitated by computer-assisted sequence analysis methods. Pattern searches such as those described here are useful in application to the published bacterial genomes (and other deposited sequences) and, with appropriate software [e.g. MacVector (Janulczyk & Rasmussen, 2001 )], can also be applied to the publicly available unfinished genome sequences. Searches with the PS00013 pattern are useful in identifying possible Lpps, although more extensive analysis (Table 4
and unpublished observations) suggests that a significant proportion of these are unlikely to be Lpps (i.e. they are false-positives). This lack of stringency associated with the PS00013 pattern was recognized during the annotation of the S. pneumoniae genome as putative Lpps were initially identified using PS00013, then the dataset refined using a novel hidden Markov model (Tettelin et al., 2001
). Moreover, the ISREC ProfileScan Server (http://hits.isb-sib.ch/cgi-bin/hits_motifscan) for pattern matching is noted to consider PS00013 as a low specificity pattern that matches very frequently. The present analysis demonstrates the benefits of employing a taxon-specific approach to increase the stringency of the pattern used for Lpp identification. The G+LPP pattern described herein allows probable Lpps of Gram-positive bacteria (excluding Mollicutes) to be identified with greater confidence. Moreover, the G+LPP pattern identifies proven and putative Lpp signal sequences that are not detected by searching with the PS00013 pattern. Application of the G+LPP pattern to the genomes of S. pyogenes and B. subtilis confirms that putative Lpps represent an abundant (at least
1·5% of the proteome) class of cell envelope proteins. Functional predictions suggest that the Lpps of S. pyogenes may contribute to the virulence of this important pathogen and so these proteins may represent targets for novel therapeutic interventions. However, it must be emphasized that in silico analyses such as these are of importance primarily as a foundation for experimental analyses.
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ACKNOWLEDGEMENTS |
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Received 11 January 2002;
revised 5 March 2002;
accepted 14 March 2002.