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1 TI Food and Nutrition, PO Box 557, 6700 AN Wageningen, The Netherlands
2 NIZO Food Research, PO Box 20, 6710 BA Ede, The Netherlands
3 Radboud University Nijmegen Medical Centre/NCMLS, Centre for Molecular and Biomolecular Informatics, PO Box 9101, 6500 HB Nijmegen, The Netherlands
4 Wageningen University, Laboratory of Microbiology, PO Box 8033, 6700 EJ Wageningen, The Netherlands
Correspondence
Michiel Kleerebezem
Michiel.Kleerebezem{at}nizo.nl
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMETHODSRESULTSDISCUSSIONREFERENCES |
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Supplementary data showing the CLUSTAL_X alignment of sequences in the Bsh and Pva clusters, annotation of Gram-positive CBAH superfamily members in the phylogenetic tree, and HMM data are available with the online version of this paper.
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONREFERENCES |
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Bacterial Bsh activity has received much attention based on its postulated role in both positive and negative biological processes in the host. For example, bile salt hydrolysis may be involved in serum cholesterol lowering (Pereira & Gibson, 2002
). Bile salt deconjugation is the obligatory first reaction in further oxidation and dehydroxylation steps of bile salts by intestinal bacteria, and it includes the production of secondary bile salts that have been linked to various intestinal diseases, such as the formation of gallstones and colon cancer (Ridlon et al., 2006). Moreover, results of in vitro studies have suggested that bile salt deconjugation plays a role in mucin production and excretion in the intestinal lumen (Klinkspoor et al., 1999), and this could affect the nutritional environment encountered by the intestinal microbiota, and intestinal transit time (Shimotoyodome et al., 2000).
Based on the biological implications of Bsh activity, it is important to provide a correct annotation of bsh genes in bacterial DNA sequences. Notably, Bsh amino acid sequences resemble those of penicillin V acylase (Pva; EC 3.5.1.11), and they belong to the same enzyme superfamily of linear amide CN hydrolases [Pfam CBAH, PF02275 (http://pfam.sanger.ac.uk)]. Although both Bsh and Pva are capable of hydrolysing the same type of chemical bond, the overall chemical nature of their substrates is quite different (bile salts and penicillins, respectively; Fig. 1). The steroid moiety of bile salts is significantly more voluminous when compared with the corresponding moiety of penicillin V. Pva-encoding genes may at times be incorrectly annotated as Bsh, as has been found, for example, in Listeria monocytogenes (Begley et al., 2005), and this can lead to the unreliable prediction of the presence of Bsh activity in a particular bacterial strain.
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). For example, experimentally verified Bsh enzymes can be found for Enterococcus faecium (Wijaya et al., 2004), Bifidobacterium species (Kim et al., 2004; Tanaka et al., 2000), Clostridium species (Kirby et al., 1995), Lactobacillus species (Christiaens et al., 1992; Elkins & Savage, 1998; Lambert et al., 2007; Leer et al., 1993) and Lis. monocytogenes (Dussurget et al., 2002). Experimentally verified Pva-family proteins have been found for Bacillus species (Olsson & Uhlen, 1986; Rathinaswamy et al., 2005) and Lis. monocytogenes (Begley et al., 2005).
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; De Smet et al., 1995; Pereira et al., 2003; Tannock et al., 1994). In Lactobacillus-free mice, the Bsh activity in the ileal content has been found to be reduced by almost 90 % when compared with Lactobacillus-reconstituted mice (Tannock et al., 1989). Moreover, one of the commonly used criteria in selection of candidate probiotic strains is their ability to hydrolyse bile salts (Begley et al., 2006). There is a growing interest in the use of bile-salt-hydrolysing Lactobacillus strains in fermented milk products because they could potentially lead to reduction of serum cholesterol concentrations in humans (Pereira & Gibson, 2002). Consequently, the accuracy of annotation of bsh genes in lactobacilli deserves special attention.
In this study, a combination of in silico methods was employed to distinguish accurately between Bsh- and Pva-family members. All protein sequences of CBAH superfamily members in public databases were compared, and their functionality was predicted using phylogenetic profiling, sequence alignment and 3D-modelling techniques, incorporating experimentally verified Bsh and Pva proteins. In addition, hidden Markov models (HMMs) were constructed that enabled the distinction between Bsh and Pva proteins. Annotation of Bsh-family members in several Lactobacillus species is based mainly on sequence homology, and it has been found to be mostly incorrect, as exemplified for the four CBAH superfamily paralogues found in the model organism Lactobacillus plantarum WCFS1 (Kleerebezem et al., 2003; Lambert et al., 2007). In this work, we propose a refined annotation of Bsh- and Pva-family members in lactobacilli.
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METHODS |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONREFERENCES |
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) using BLASTP (Altschul et al., 1990), and, as seed, the sequence of the experimentally verified Bsh1 (lp_3536) of Lb. plantarum WCFS1 (Kleerebezem et al., 2003; Lambert et al., 2007). Multiple sequence alignments were created with MUSCLE (Edgar, 2004), using default settings (www.drive5.com/muscle). CLUSTAL_X (Thompson et al., 1997) was used to display multiple sequence alignments, and to create phylogenetic trees, and these trees were then displayed with LOFT (van der Heijden et al., 2007). HMMs were constructed from the multiple sequence alignments by using HMMER 2.3.2 (Durbin et al., 1998).
3D structure homology modelling.
The 3D structures used as templates for homology modelling were Bsh from Bifidobacterium longum [2hez.pdb (Protein Data Bank identification no.; www.rcsb.org/pdb/home/home.do)], Bsh from Clostridium perfringens in complex with reaction products taurine and deoxycholate (2bjf.pdb), and Pva in complex with diethane diol from Bacillus sphaericus (2pva.pdb). Homology modelling was performed with WHATIF/YASARA Twinset (www.yasara.com).
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RESULTS |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONREFERENCES |
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) (Table 1
). The superfamily tree shows that sequences derived from Gram-negative and Gram-positive organisms were clearly divided in two separate groups.
Focusing on the group of sequences derived from Gram-positive organisms, two clusters were identified (Fig. 2). The first cluster, hereafter called the Bsh cluster, consisted of sequences that appeared phylogenetically closely related, and contained all Bsh proteins that had been biochemically verified, including Bsh1 of Lb. plantarum WCFS1. The Bsh cluster contained 25 sequences, of which 23 are annotated in the public NCBI database (http://ncbi.nlm.nih.gov) as Bsh, and one is annotated as penicillin acylase (see supplementary Table S1, available with the online version of this paper). For one sequence, no function is annotated; however, this protein has been shown experimentally to be a Bsh (Dussurget et al., 2002). Furthermore, this cluster contained 10 sequences derived from lactobacilli, of which nine sequences are annotated as Bsh, and one sequence is annotated as Pva. The second cluster, hereafter called the Pva cluster, consisted of sequences that generally appeared to be less tightly related phylogenetically, as compared with those in the Bsh cluster. All experimentally verified Pva proteins were encompassed within the Pva cluster. This cluster contained 49 sequences, of which 11 are annotated as Pva, 30 are annotated as Bsh, one is annotated as an ABC transporter, and seven sequences lack a functional annotation (Table S1). Furthermore, eight sequences of lactobacilli were grouped in the Pva cluster, of which seven are annotated as Bsh, and one is annotated as Pva. The three paralogous proteins previously annotated as Bsh (Bsh2, Bsh3 and Bsh4; Kleerebezem et al., 2003), which were predicted to be present in addition to the experimentally verified Bsh1 (Lambert et al., 2007) in Lb. plantarum WCFS1, were grouped phylogenetically in the Pva cluster, with Bsh2 and Bsh3 being more closely related to each other than to Bsh4.
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3D protein homology modelling, and sequence alignment
The 3D structures of the crystallized Bsh of Bif. longum (Kumar et al., 2006) (2hez.pdb), Bsh of C. perfringens (Rossocha et al., 2005) (2bjf.pdb), and Pva of Bac. sphaericus (Suresh et al., 1999) (2pva.pdb) were superimposed, and the catalytic residues and binding pockets were compared (Fig. 3a, b). Based on multiple sequence alignment (see supplementary Fig. S1, available with the online version of this paper), the catalytic and binding-pocket residues of all Bsh and Pva family members were defined. The residues present in the substrate-binding pocket appeared to be more conserved in the Bsh cluster compared with the Pva cluster. Catalytic and putative substrate-binding residues of the Bsh and Pva clusters, and the four Lb. plantarum enzymes, are compared in Tables 2 and 3.
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, the substrate-binding pocket of Bif. longum Bsh is shown, with a deoxycholate molecule (as an example of the steroid moiety of bile salts) modelled in the same orientation as that in the C. perfringens 3D structure (2bjf.pdb). The side chains lining this pocket are shown to come mainly from three surface loops, and they are predominantly hydrophobic. The substrate-binding pocket is also hydrophobic in other sequences in the Bsh cluster (Table 2); this is in agreement with the hydrophobic nature of the steroid moiety of bile salts. As expected, the predicted catalytic and substrate-binding residues of the experimentally verified Bsh1 enzyme of Lb. plantarum WCFS1 are most similar to those of the Bif. longum and C. perfringens Bsh enzymes, as shown in Table 2 and Fig. 3(c).
Clear differences were found in the 3D structure of the substrate-binding pockets of proteins of the Bsh and Pva clusters. First, loop 3 differs in length and orientation, as illustrated in Fig. 3(b). In the Pva of Bac. sphaericus, loop 3 is folded inward, severely reducing the size of the binding pocket. Second, the side chains of loops 2 and 3 lining the binding pocket are far more variable in size and hydrophilicity/hydrophobicity in the Pva group (Table 3). The catalytic residues, however, appear to be highly conserved in the two respective groups. Five out of the six catalytic residues appear to be identical in both the Bsh and the Pva cluster, whereas the catalytic residue at position 82 in the 3D model was usually asparagine in the Bsh cluster, and tyrosine, or occasionally threonine (four out of 27), in the Pva cluster (Fig. S1). Predicted binding-pocket residues of Bsh2 and Bsh3 of Lb. plantarum WCFS1 appeared to resemble those of the 3D structure of Pva, as illustrated in Table 3 and Fig. 3(d), whereas the residues of Bsh4 appeared to fit less well to that of either Bsh of Bif. longum or Pva of C. perfringens.
Thus, using 3D modelling, the separation of Bsh family members in the Bsh and the Pva clusters, as found by phylogenetic profiling, was confirmed based on 3D models of the substrate-binding pocket and, consequently, the ability to accommodate bile salt molecules.
HMMs for distinction between Bsh and Pva family members
From the CLUSTAL_X alignments, separate and distinctive HMMs were constructed for the proteins of the Bsh and Pva clusters (see supplementary material SL1, available with the online version of this paper). Using the Bsh HMM to annotate the CBAH superfamily members, only proteins of the Bsh cluster were found as best hits. Likewise, only proteins of the Pva cluster were found as best hits using the Pva HMM. Subsequently, the two HMMs were used for a search of all publicly available sequenced genomes. Using the Bsh HMM, all best hits found in the sequenced genomes were already present in the Bsh cluster of the phylogenetic tree (Fig. 2), and no additional hits were found (E value <1.80x10–179). Furthermore, with the Bsh HMM, a clear separation was found in the E value of the members of the Bsh cluster (E value <1.80x10–179) and subsequent hits (E value >2.00x10–108), which were members of the Pva cluster (see supplementary material SL2, available with the online version of this paper). Likewise, using the Pva HMM, all best hits (E value <6.60x10–132) were found in the sequenced genomes consisting of members of the Pva cluster. Again, a clear separation was found in the E values of the Pva sequences (E value <6.60x10–132) and subsequent hits (E value >8.20x10–97), which were either members of the Bsh cluster or Gram-negative sequences not present in our phylogenetic tree. These findings strongly suggest that the HMMs presented here enable an accurate prediction of the functionality of any CBAH superfamily member, classifying it as either a Bsh enzyme (EC 3.5.1.24) or a Pva-related enzyme (EC 3.5.1.11).
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONREFERENCES |
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).
The construction of a phylogenetic tree is an excellent way of visualizing the relatedness of sequences, and it is more informative than the study of sequence homology alone. In our study, phylogenetic clustering clearly separated Gram-positive CBAH superfamily members into two groups: a Bsh cluster that is predicted to contain Bsh enzymes, and a Pva cluster, the members of which are predicted to be Pva-related proteins. Interestingly, it appeared that all strains that were represented in the Bsh cluster, and not in the Pva cluster, were typical gut-related bacteria (i.e. bifidobacteria, Enterococcus faecalis, E. faecium and Lactobacillus acidophilus), while strains represented in the Pva cluster, and not the Bsh cluster, were not typical gut-related bacteria. This finding is in good agreement with the reported correlation between the presence of Bsh activity in lactic acid bacteria and isolation from the intestine or faeces (Tanaka et al., 1999). The prediction of the functionality of the CBAH superfamily members by phylogenetic profiling was reinforced by the fact that the functionality of all experimentally verified Bsh and Pva proteins matched the in silico clustering.
Our results indicate that the sequences annotated as Bsh in the Pva cluster are likely to be incorrectly annotated in the public NCBI (http://ncbi.nlm.nih.gov) and KEGG (www.genome.jp/kegg) databases, and that they are probably Pva or Pva-related enzymes. Particularly for Lactobacillus spp., the refinement of the annotation of functionality of members of this family of enzymes could have an important impact on their anticipated influence on gastrointestinal bile salt metabolism and its cognate physiological consequences in the host. According to our current analysis, the present Pva annotation of one sequence, the Lactobacillus gasseri ATCC 33323 CBAH superfamily sequence [Swiss-Prot accession no. Q047A3 (www.expasy.ch/sprot/)], should be corrected to Bsh, while the sequences in the Pva cluster currently annotated as Bsh in Lactobacillus salivarius UCC118 (Swiss-Prot accession no. Q1WUK8), Lactobacillus brevis ATCC 367 (Swiss-Prot accession nos Q03NN7 and Q03P51), Lactobacillus sakei 23K (Swiss-Prot accession no. Q38Z70), and Lb. plantarum WCFS1 [Swiss-Prot accession nosQ890F5 (Bsh2); Q88SP0 (Bsh3), and Q88UC9 (Bsh4)], should be corrected to Pva related (Table S1). The reannotation of these sequences was further validated by experimental evidence of the presence of Bsh activity in bacterial strains. For example, for Lb. brevis ATCC 367, the original annotation predicted the presence of two Bsh sequences and one Pva sequence and, thus, this strain was expected to show Bsh activity. However, our reannotation predicted the presence of Pva-related sequences only; thus, Lb. brevis ATCC 367 should, according to our annotation, show no Bsh activity. Indeed, using a Bsh plate assay that has been described earlier (Dashkevicz & Feighner, 1989), Lb. brevis ATCC 367 showed no Bsh activity, suggesting that our reannotation of the Bsh sequences to Pva-related sequences was correct (data not shown).
Furthermore, the members of the Pva and Bsh clusters were not only phylogenetically different but also structurally different. 3D modelling and sequence alignment showed clear differences in the 3D structure of the substrate-binding pockets of Bsh and Pva enzymes, as illustrated in Fig. 3. In particular, one loop of the substrate-binding pocket (loop 3) in Pva is folded inwards as compared with the Bsh enzymes. As a consequence, it is unlikely that bile salts can be accommodated in the substrate-binding pocket of Pva (Fig. 1). Compared with members of the Bsh cluster, the members of the Pva cluster were found to be less related to each other during phylogenetic analysis. This finding was reflected in a higher variability in the residues of the binding pocket of members of the Pva cluster, as compared with members of the Bsh cluster, and this suggests that there is considerable variation in putative penicillin derivatives or similar molecules that can be bound. As expected, the predicted substrate-binding pocket of the experimentally verified Bsh1 of Lb. plantarum (Lambert et al., 2007) structurally resembled the Bsh enzymes of Bif. longum (Kumar et al., 2006) and C. perfringens (Rossocha et al., 2005). The limited degree of variation in the residues of the binding pocket, and thus a more strict substrate preference, for members of the Bsh cluster is reflected by the fact that Bsh1 of Lb. plantarum WCFS1 is able to hydrolyse bile salts, but not penicillin V or penicillin G (Lambert et al., 2008).
In addition, the paralogues Bsh2, Bsh3 and Bsh4 were clustered in the Pva group during phylogenetic analysis, with Bsh2 and Bsh3 being more related to each other than to Bsh4. This is in agreement with the finding that the predicted substrate-binding pocket of Bsh2 and Bsh3 appeared to show a greater structural resemblance to that of the Pva enzyme of Bac. sphaericus (Suresh et al., 1999) than that of Bsh4. It is possible that Bsh4 encodes an enzyme related to Pva that is capable of acylating a substrate other than penicillin V.
Notably, one of the catalytic residues appeared to differ between the Bsh and Pva cluster. This finding facilitates accurate prediction of the functionality of the Bsh family members as Bsh or Pva (-related) from their amino acid sequences. Novel and discriminative HMMs were constructed to distinguish between Bsh- and Pva-family members. Thereby, our analyses defined the discriminative characteristics of the Pva and Bsh enzyme families, despite the fact that this did not appear to be feasible using the available conserved motif databases. For example, the conserved domain search tool (Marchler-Bauer et al., 2005) provided by NCBI (www.ncbi.nlm.nih.gov/Structure/cdd/cdd.shtml) aims at detection and prediction of conserved domains in proteins, and includes domains imported from SMART, Pfam and COG databases. However, this tool attributes ‘penicillin V acylase, also known as conjugated bile salt acid hydrolase’ to the best-conserved domain found in all experimentally verified Bsh and Pva proteins used here (Table 1), and thereby fails to distinguish between Bsh-like and penicillin-acylase-like domains. Analogously, SMART, Pfam and COG databases do not provide a correct discriminative domain description for all experimentally verified Bsh and Pva proteins (data not shown).
In conclusion, we have provided evidence that various Pva-related sequences are wrongly annotated as Bsh in various Gram-positive bacteria, including several lactobacilli. For intestinal lactobacilli in particular, the capacity to deconjugate bile acids has been suggested to be of importance for their survival capacity in the gastrointestinal tract of mammals, and it is proposed to strongly influence gut physiology. Therefore, appropriate annotation of the Bsh enzymes encoded in the genomes of Lactobacillus and other lactic acid bacteria is important to predict their in situ functionality in the gastrointestinal tract. The methodology presented here provides the in silico tools to effectively distinguish the genes encoding the important Bsh enzymes from those encoding closely related enzymes with Pva-like activities.
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ACKNOWLEDGEMENTS |
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Edited by: J. M. van Dijl
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REFERENCES |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONREFERENCES |
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Received 15 January 2008;
revised 14 April 2008;
accepted 24 April 2008.
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