1 Center for Molecular and Biomolecular Informatics, 6525ED, Nijmegen, The Netherlands
2 Wageningen Centre for Food Sciences, 6700 AN Wageningen, The Netherlands
3 Nestlé Research Center, Nestec SA, 1000 Lausanne 26, Switzerland
4 NIZO food research, 6710 BA Ede, The Netherlands
Correspondence
Jos Boekhorst
J.Boekhorst{at}cmbi.kun.nl
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ABSTRACT |
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Details of the size and location of conserved gene clusters in L. plantarum and L. johnsonii may be found in Supplementary Table S1; the number of proteins of L. plantarum and L. johnsonii for all COG classes in Supplementary Table S2; a KEGG comparison of major differences between L. plantarum and L. johnsonii in Supplementary Table S3; L. johnsonii and L. plantarum API 50 test results in Supplementary Table S4; the redundancy of enzymes involved in pyruvate metabolism in Supplementary Table S5; gene clusters encoding functionally related proteins present in L. plantarum but not in L. johnsonii and vice versa in Supplementary Table S6; lists of proteins unique to either L. plantarum or L. johnsonii in Supplementary Tables S7S12; lists of proteins involved in the biosynthesis of polysaccharides, bacteriocins and prophages in Supplementary Table S13 with the online version of this paper at http://mic.sgmjournals.org.
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INTRODUCTION |
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Recently, the genomes of two members of the genus Lactobacillus have been completely sequenced: Lactobacillus plantarum WCFS1 (Kleerebezem et al., 2003) and Lactobacillus johnsonii NCC533 (Pridmore et al., 2004
). L. johnsonii NCC533, isolated from human faeces, has been extensively studied for its probiotic activities, including immunomodulation (Haller et al., 2000a
, 2000b
) and interaction with the human host (Ibnou-Zekri et al., 2003
). L. plantarum WCFS1 was isolated from human saliva. L. plantarum is a versatile bacterium that is found in a variety of ecological niches, ranging from vegetable and plant fermentations to the human gastrointestinal tract. This flexibility of L. plantarum is reflected by its relatively large genome size, a large number of proteins involved in regulation and transport functions, and a high metabolic potential (Kleerebezem et al., 2003
).
In order to expand our understanding of the molecular evolution, diversity, function and adaptation of lactobacilli to specific environments, we have performed a whole-genome comparison of L. plantarum and L. johnsonii. In addition, we compared the proteins of these two organisms to the draft sequences of other LAB genomes (Klaenhammer et al., 2002). We provide a first comprehensive view of differences on the genome level in lactobacilli, and evidence for large genetic diversity in this genus. We identify features underlying the large difference in genome size and gene content in lactobacilli, and provide a first insight into the set of genes and functions which could be specific for lactic acid bacteria. This knowledge provides numerous leads for targeted experimental verification of unique or common physiological properties.
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METHODS |
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Genome comparisons.
Orthologous relationships were detected by a previously described method (Snel et al., 2002) using the Smith & Waterman sequence comparison algorithm (Smith & Waterman, 1981
) against the NCBI Clusters of Orthologous Group (COG) database (Tatusov et al., 2001
). The functional classification provided by the COG database was used for the functional comparison of L. plantarum and L. johnsonii on a genome-wide scale.
Homology relationships were established using BLASTP (Altschul et al., 1990) and Smith & Waterman sequence comparison. Homologues were detected with a threshold of 1E10; a gene was considered organism specific when it had no Smith & Waterman hits at all, or only hits with an e-score higher than 1E10 to proteins of other organisms in the non-redundant proteins databases (SWISS-PROT, TrREMBL and TrEMBL updates) (Boeckmann et al., 2003
) or the LAB genomes taken from the ERGO database. Proteins were considered LAB-specific when they did not have a Smith & Waterman hit with an e-score lower than 1E10 in a search against SWISS-PROT, TrREMBL, TrEMBL updates and the LAB sequences taken from the ERGO database.
Whole genomes were compared at the nucleotide level using the Dotter software (Sonnhammer & Durbin, 1995) with default values. A bidirectional best-hit approach was used to identify genome synteny at the protein level. The results of this analysis were visualized using the Artemis Comparison Tool (http://www.sanger.ac.uk/Software/ACT/).
Transporter classification was preformed according to the TC-DB scheme (Busch & Saier, 2002). All proteins were searched against the TC-DB Database Release 1.5.1 using BLASTP with a threshold of 10E4, followed by manual curation: false positive hits were removed manually when clear evidence suggested that they were not related to transport function.
Signal peptides were predicted using SignalP (Nielsen et al., 1997).
Base deviation analysis of genes was performed by calculating a chi-squared index based on the expected and observed frequency for each nucleotide (Tettelin et al., 2001).
Synchronizing annotation.
The two genomes compared in this study were initially analysed using different ontologies and annotations (Kleerebezem et al., 2003; Pridmore et al., 2004
). To facilitate functional comparison of L. plantarum and L. johnsonii, the annotation of proteins found to be homologous, but having different annotations in the two genomes, was manually verified and corrected where necessary. This resulted in an improved annotation of both genomes, in particular for the functional class regulation and for the assignment of EC numbers, and made automated detection of functional differences possible.
Reconstruction of metabolic pathways.
EC numbers were extracted from the genome annotations and manually curated. They were then automatically mapped onto the Kyoto Encyclopedia of Genes and Genomes (KEGG) metabolic pathways (Kanehisa et al., 2002) for visualization and identification of differences in metabolism between L. plantarum and L. johnsonii. In cases of predicted missing key enzymes in one of the two organisms, a further effort was made to identify homologous candidate enzymes by extensive manual searches with BLASTP and HMMER (Eddy, 1996
; Sonnhammer et al., 1998
).
Sugar utilization.
API 50 analysis of sugar utilization was performed using the supplier's protocol (BioMérieux Benelux). Additional sugar fermentation profiles were obtained from the literature (Fujisawa et al., 1992; Kleerebezem et al., 2003
).
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RESULTS AND DISCUSSION |
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A dot plot comparison at the protein level of the genomes of L. plantarum and L. johnsonii (Fig. 2) shows no large-scale conservation of gene order, but only conservation of genes in clusters, confirming the relatively large phylogenetic distance between L. plantarum and L. johnsonii. The lack of large-scale gene order conservation between L. plantarum and L. johnsonii is in strong contrast to the whole chromosome alignment of L. johnsonii and L. gasseri, which shows a high degree of conservation and synteny over the whole genome (Pridmore et al., 2004
). L. johnsonii and L. plantarum share only 28 large regions of conserved gene order, ranging in size from 7 (arbitrarily defined as minimum) to 75 genes, and encoding nearly 550 conserved proteins. Details of the size and location of these clusters may be found in Supplementary Table S1 with the online version of this paper at http://mic.sgmjournals.org.
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A similar synteny analysis for L. johnsonii NCC533 or L. plantarum WCFS1 with E. faecalis showed lower conservation, but many of the same clusters could be identified (data not shown). The number of conserved clusters, as well as the number of syntenic genes in the clusters, is smaller than in the johnsonii/plantarum comparison, but the degree of overall conservation corroborated well the fact that the genus Enterococcus is closely related to but distinct from Lactobacillus (Klein, 2003). Very limited synteny could be detected with L. lactis or streptococci (data not shown).
Phylogenetic trees based on 16S RNA (Fig. 1) or highly conserved genes support the relatively large phylogenetic distance between L. plantarum and L. johnsonii suggested by the protein dot plot. An unrooted tree based on the atpD gene (part of the highly conserved ATP synthase cluster) shows L. johnsonii to be closely related to L. gasseri, but also shows a relatively large distance between L. johnsonii and L. plantarum (Siezen et al., 2004
), in agreement with Fig. 1
. The phylogenetic distance between L. plantarum and L. johnsonii is in fact similar to the distance between L. plantarum and E. faecalis. These findings re-emphasize the difficulties in establishing the taxonomy of lactobacilli, and show that the current classification of the Lactobacillus genus, based on morphology and lactic acid production, is not always supported by phylogenetic relationships based on sequence homology and genome synteny.
Functional comparison of proteomes
The percentage of the total number of proteins of L. plantarum and L. johnsonii belonging to selected COG functional classes is shown in Fig. 4. Only classes displaying large differences between the two organisms are shown; Supplementary Table S2 shows the number of proteins of L. plantarum and L. johnsonii for all COG classes. This overview gives an indication of the differences in focus on metabolism and other cellular functions of these bacteria. Compared to L. johnsonii, L. plantarum has a relatively high number of proteins for carbohydrate, amino acid and lipid metabolism. Due to its smaller genome size, L. johnsonii has a higher percentage of genes involved in core functions' such as replication and translation.
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The mapping of enzymic functions on the metabolic pathways provided by the KEGG database resulted in the identification of a set of enzymes required for known biochemical pathways. The main differences between L. johnsonii and L. plantarum are listed in Supplementary Table S3. The classes and metabolic pathways that display striking differences between the two organisms will be described in some detail below.
The L. plantarum genome encodes 268 proteins predicted to be involved in the metabolism and transport of amino acids, while the L. johnsonii genomes encodes only 125. L. plantarum encodes the enzymes required for the biosynthesis of all amino acids, with the exception of leucine, isoleucine and valine. In contrast, L. johnsonii is predicted to be incapable of synthesizing most, if not all, of the 20 standard amino acids. This reflects the environmental niches in which the bacteria live. L. johnsonii typically is found only in the gut, although recent reports (Guan le et al., 2003; Meroth et al., 2003
) suggest that L. johnsonii might also occur in other nutrient-rich environments, where it can take up amino acids and peptides from its environment. To this end, L. johnsonii has an extracellular, cell-bound proteinase to liberate these peptides from proteinaceous substrates, and more intracellular peptidases for degradation of imported peptides than L. plantarum (Kleerebezem et al., 2003
; Pridmore et al., 2004
). In contrast, L. plantarum is also found in other environments, such as on plants and plant-derived materials, where amino acids and peptides are not as readily available, and hence has retained more amino acid biosynthetic capability.
While the L. plantarum genome encodes 90 proteins predicted to be involved in the transport and metabolism of vitamins and cofactors, the L. johnsonii genome encodes only 30. For instance, all the enzymes necessary for the biosynthesis of folate are present in L. plantarum. In contrast, L. johnsonii has only a few enzymes that could have a function in this pathway, but all of these enzymes could also have functions in other processes. This suggests that L. plantarum is capable of synthesizing its own folate, while L. johnsonii is not, which has recently been confirmed experimentally (Sybesma et al., 2003).
Both L. johnsonii and L. plantarum have the capacity to synthesize pyrimidines de novo. However, only the L. plantarum genome encodes the proteins essential for de novo synthesis of purines from phosphoribosyl pyrophosphate; L. johnsonii needs inosine, which can be converted to IMP in a single enzymic step. This is consistent with the observation that L. johnsonii needs to obtain purines or their precursors from its environment (Elli et al., 2000).
The L. plantarum genome encodes 13 proteins predicted to be involved in the biosynthesis of fatty acids, while the L. johnsonii genome encodes only one. However, the route by which L. johnsonii acquires fatty acids is still unknown.
L. plantarum can utilize a much wider variety of sugars than L. johnsonii (Supplementary Table S4). This corroborates the observation that many more proteins involved in the uptake, interconversion and degradation of sugars are encoded by the L. plantarum genome than by the L. johnsonii genome: the L. plantarum genome encodes 342 proteins of the COG class carbohydrate transport and metabolism, while the L. johnsonii genome encodes only 196.
L. plantarum has a more versatile pyruvate metabolism than L. johnsonii (Fig. 5). Both L. plantarum and L. johnsonii can convert pyruvate to L- and D-lactate, but L. johnsonii lacks the pyruvate dehydrogenase complex and other enzymes required for the conversion of pyruvate to acetate, acetaldehyde and acetyl-coenzyme A. Moreover, L. plantarum has a much higher redundancy of enzymes involved in pyruvate metabolism (Supplementary Table S5). L. plantarum is a facultative heterofermentative organism, capable of mixed-acid fermentation forming lactate, formate and/or acetate depending on environmental conditions, while L. johnsonii is an obligate homofermentative organism, capable of homolactic fermentation only. The lack of the pyruvate dehydrogenase complex in L. johnsonii is consistent with the anaerobic environment in the gastrointestinal tract
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The most notable electrochemical potential-driven transporters in L. johnsonii are two conjugated bile saltproton symporters (LJ0057 and LJ0058), which have been found to be unique proteins of the Lactobacillus acidophilus group of organisms (Pridmore et al., 2004). Striking differences are found in the number of multidrug/oligosaccharidyl-lipid/polysaccharide flippase superfamily, the auxin-efflux carrier family and the drug/metabolite transporter superfamily of transporters in L. plantarum. The L. plantarum genome encodes 5, 4 and 11 proteins belonging to these families, respectively, whereas the L. johnsonii genome encodes only one protein of each family. Primary active transporters (mainly ABC transporters: 147 and 105 proteins in L. plantarum and L. johnsonii, respectively) represent the largest group of transporters in both lactobacilli. In L. johnsonii and L. plantarum, 16 and 25 complete PEP-dependent, phosphoryl transfer-driven group translocators (phosphotransferase system, PTS) systems were identified, respectively, including multiple systems for the uptake of glucose, mannose, fructose, and
-glucosides, and single systems for cellobiose, sucrose, and galactitol.
Extracellular proteins
Extracellular proteins are considered to be important for interaction of bacteria with their environment, for example in adhesion and communication. This makes them of special interest in the case of lactobacilli, because they may be involved in hostmicrobe and microbemicrobe interactions, such as in the gastrointestinal tract or on plant materials. Putative extracellular proteins of L. plantarum and L. johnsonii were identified by the presence of a Sec-pathway-dependent signal peptide. Both proteins that are secreted into the environment and proteins that become attached to the cell surface fall into this category. The latter were identified by searching for cell-anchoring domains, such as the N-terminal lipoprotein motif for anchoring to the cell membrane (Sutcliffe & Russell, 1995) and the C-terminal LPxTG motif for anchoring to peptidoglycan (Navarre & Schneewind, 1999
). The L. plantarum and L. johnsonii genomes are predicted to encode 211 (Kleerebezem et al., 2003
) and 117 putative extracellular proteins, respectively. Nearly 90 % of these proteins in both species are predicted to contain at least one type of cell-wall anchoring domain.
A comparison of the putative extracellular proteins encoded in both genomes is summarized in Table 4. The set of extracellular proteins of known function is very similar in both lactobacilli, although L. plantarum has more paralogues for several of these known functions. However, the majority (5565 %) of putative extracellular proteins are of unknown function (Table 4
). Some of these are present in both lactobacilli, either as single copies of orthologues, or as multiple copies (paralogues) belonging to different families. Two families of putative cell-surface hydrolases (CSH-1 and CSH-2) are detected which have sequence characteristics of lipases or esterases (Anthonsen et al., 1995
; Wong & Schotz, 2002
). It is striking to note that the majority of extracellular proteins with unknown function are not shared by L. plantarum and L. johnsonii, but only occur in one of the two bacteria.
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Regulators
Regulatory proteins play an important role in the adaptation of an organism to different environments. The L. plantarum genome is predicted to encode 264 regulators (9·4 % of all proteins), while the L. johnsonii genome has only 114 putative regulators (6 %), as summarized in Supplementary Table S6. This agrees with the general observation that large genomes have a relatively high number of proteins involved in transcription and regulation (Konstantinidis & Tiedje, 2004; van Nimwegen, 2003
).
Besides the difference in genome size, the different lifestyles of L. plantarum and L. johnsonii also contribute to this difference. The number of proteins predicted to be involved in the regulation of sugar and energy metabolism is especially high in L. plantarum. This is in agreement with the differences found in sugar metabolism in the two organisms: L. plantarum can utilize a much wider variety of sugars than L. johnsonii (Supplementary Table S4). L. plantarum with its free-living lifestyle needs to be capable of dealing with many different environmental circumstances (Boneca et al., 2003), and apparently has both the metabolic capacity and the regulatory machinery to deal with adaptation to different niches, while L. johnsonii does not need a complex regulatory apparatus because of the relatively stable environment in the gastrointestinal tract.
LAB-specific and unique genes
A Smith & Waterman homology search was used to identify proteins unique to either L. plantarum or L. johnsonii, and proteins unique to LAB (Table 5). The table lists the number of proteins that are present in either L. plantarum or L. johnsonii and in at least one other LAB, but without homologues in organisms not considered as LAB. It also lists the number of proteins found to be unique to either L. plantarum or L. johnsonii. The individual proteins for these categories can be found in Supplementary Tables S7S12. The result of this analysis depends of course on the number of genomes available at the time of comparison, and is only preliminary, since many of the LAB genomes in the ERGO database were less than 100 % complete at the time of this analysis.
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Many of the proteins present in L. plantarum but absent in L. johnsonii, or vice versa, are grouped in clusters on the genome. A large number of these clustered unique genes encode functionally related proteins, such as those involved in the biosynthesis of polysaccharides, bacteriocins and prophages (Supplementary Table S13). In L. plantarum, such clusters frequently have a high base-deviation index (BDI), suggesting horizontal transfer (Kleerebezem et al., 2003). In L. johnsonii however, only the polysaccharide biosynthesis cluster (LJ10271047) has a high BDI.
Most of the proteins predicted to be LAB specific are of unknown function (Supplementary Tables S7S12). The identification of structural features, such as signal peptides, transmembrane helices and cell-wall anchors, and conserved domains/motifs in these proteins, such as those involved in the binding of ATP, DNA and carbohydrates, could be used to predict their function and to identify potentially interesting targets for future research. In this way, the preliminary analysis of LAB-specific genes described here can serve as a starting point for a more comprehensive study of LAB-specific proteins and gene clusters, once the complete genome sequences of many LAB species become available (Klaenhammer et al., 2002).
Concluding remarks
The ability of L. plantarum to survive in many different environments is reflected by the much more elaborate metabolic, regulatory and transport machinery compared to that of L. johnsonii. The differences between L. plantarum and L. johnsonii, both in genome organization and in gene content, are exceptionally large for two bacteria of the same genus (Suyama & Bork, 2001). Similar differences have been reported only in streptococci (Tettelin et al., 2002
). This low degree of synteny between L. plantarum and L. johnsonii suggests that they are only marginally more related to each other than to other Gram-positive bacteria. These findings emphasize the difficulty in taxonomic classification of lactobacilli.
Overall, the genome-wide comparison of two complete Lactobacillus genomes has provided unique information on the relatedness and differences between the two species. This has led to insight into the genomic adaptation to ecological niches of L. plantarum and L. johnsonii, and provides leads for targeted experimental studies.
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ACKNOWLEDGEMENTS |
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Received 9 June 2004;
revised 23 July 2004;
accepted 29 July 2004.
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