Polysaccharide utilization locus and CAZYme genome repertoires reveal diverse ecological adaptation of Prevotella species

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Polysaccharide utilization locus and CAZYme genome repertoires reveal diverse ecological adaptation of Prevotella species Tomaˇz Accetto ∗ , Gorazd Avguˇstin University of Ljubljana, Biotechnical Faculty, Animal Science Department, Groblje 3, 1230 Domˇzale, Slovenia

a r t i c l e

i n f o

Article history: Received 27 May 2015 Received in revised form 14 July 2015 Accepted 17 July 2015 Keywords: Prevotella Polysaccharide utilization locus Carbohydrate acting enzyme Ecology Bioinformatics

a b s t r a c t The results of metagenomic studies have clearly established that bacteria of the genus Prevotella represent one of the important groups found in the oral cavity and large intestine of man, and they also dominate the rumen. They belong to the Bacteroidetes, a phylum well-known for its polysaccharide degrading potential that stems from the outer membrane-localized enzyme/binding protein complexes encoded in polysaccharide utilization loci (PULs). Dozens of Prevotella species have been described, primarily from the oral cavity, and many of them occur simultaneously at the same sites, but research on their ecological adaptation has been neglected. Therefore, in this study, the repertoires of PULs and carbohydrate acting enzymes (CAZYmes) found in Prevotella genomes were analyzed and it was concluded that the Prevotella species were widely heterogeneous in this respect and displayed several distinct adaptations with regard to the number, source and nature of the substrates apparently preferred for growth. © 2015 Published by Elsevier GmbH.

Introduction The anaerobic bacterial species of the genus Prevotella are members of the large bacterial phylum Bacteroidetes that encompasses polysaccharide and protein degraders found in seawater, fresh water and soils, as well as the animal gut [56]. The Bacteroidetes are special in several ways: the base composition and spacing of their promoters are distinct [6], the Shine-Dalgarno sequence is not used in translation initiation [1], and their capability to degrade a wide array of carbohydrates depends on the starch utilization system-like (Sus-like) multiprotein complexes that bind and partially degrade substrates prior to their transport into the periplasm where final breakdown occurs [37]. The central elements of the Sus-like systems are encoded by the paralogues of Bacteroides thetaiotaomicron VPI-5482 susC and susD whose products were shown to account for >60% of starch binding, while SusC also transfers oligosaccharides across the outer membrane [37]. The three SusD-like proteins with hitherto known structures, the B. thetaiotaomicron VPI-5482 proteins SusD, BT1043 and BT3984, are structural homologues but their binding strategies differ: for SusD it is thought that the overall helical shape of amylose is recognized, while for BT1043 the specific hydrogen bond interactions are

Abbreviations: PUL, polysaccharide utilization locus; CAZYme, carbohydrate acting enzyme. ∗ Corresponding author. Tel.: +386 13203869. E-mail address: [email protected] (T. Accetto).

crucial [5,30,31]. susC and susD are usually the central elements of larger gene clusters, the polysaccharide utilization loci (PULs), that are also formed of genes coding for carbohydrate active enzymes (CAZYmes) [34], response regulators and transporters, which all contribute to efficient degradation of a single substrate [37]. The genomes of gut-dwelling B. thetaiotaomicron VPI-5482 and B. ovatus ATCC 8483 contain 88 and 110 PULs, respectively [38]. The PULs are inducible and transcriptional profiling has revealed the substrates of numerous B. thetaiotaomicron VPI-5482 and B. ovatus ATCC 8483 PULs [36,38]. These bacteria occupy distinct niches in the gut and they both degrade starch, storage polysaccharides and pectin, although B. thetaiotaomicron VPI-5482 cannot degrade hemicelluloses, while B. ovatus ATCC 8483 cannot use host mucin Oglycans for growth, which is mirrored in their PUL repertoires [38]. These repertoires are plastic due to lateral gene transfer, which is attested to by a recent transfer of PULs specialized for the degradation of porphyran from a marine bacteroidete into the gut-dwelling B. plebeius 17135 isolated in Japan, probably due to consumption of non-sterile red algae [27]. According to Bergey’s Manual [32], the genus Prevotella contains 34 species and at least a further 13 species have been described in recent years [2,15–19,26,33,44–46,53,57]. The majority of prevotellas have been isolated from the oral cavity of mammals, whereas other species inhabit the rumen, large bowel and urogenital tract, and some species have been described from clinical specimens only [2,15–19,26,32,33,44–46,53,57]. While some oral species are considered true pathogens (e.g. Prevotella intermedia contributes to periodontitis [10]), other oral species may be

http://dx.doi.org/10.1016/j.syapm.2015.07.007 0723-2020/© 2015 Published by Elsevier GmbH.

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opportunistic pathogens as they are frequently isolated from a variety of polymicrobial abscesses [7]. However, the ruminal and large bowel prevotellas are symbionts that contribute to polysaccharide breakdown [25]. It is noteworthy that Prevotella are widespread in oral cavity surfaces, account for more than 10% of all bacteria in saliva [48] and, on average, 11 Prevotella species may be found at the same time in healthy gingiva alone [40], yet the niches they occupy in the oral ecosystem remain obscure. In order to gain an insight into their roles in the oral microbiota using genomic data, we cataloged, annotated and compared the PUL and CAZYme genomic repertoires of 39 Prevotella species that were already sequenced. A clear grouping of Prevotella species emerged that reflected the number, origin and variability of the polysaccharides most likely used for growth by different species. Furthermore, the rumen/bowel and urogenital tract species provided examples of Prevotella adaptation to non-oral habitats. Materials and methods The genomes The Prevotella genomic data were obtained in February 2014 from the EBI and GenBank. The strains and degrees of their genome sequence completion are listed in Table S1. Genomes that contained only sequence data were annotated using Prokka 1.7 [47]. The Hallella seregens ATCC 51272 and Alloprevotella rava F0323 genomes were included since H. seregens was recently reclassified as Prevotella dentalis [59] and A. rava belongs to a recently proposed related genus that now also encompasses the former Prevotella tannerae [20]. Identification of susCD-like loci in Prevotella genomes and clustering of Prevotella SusD-like proteins on the basis of their amino acid similarity Since all hitherto described PULs contain susC and susD in tandem [36], we searched for putative PULs in Prevotella genomes using SusD-like proteins as proxies with HMMERv3 hmmsearch [22] and the SusD.hmm, SusD-like 2.hmm and SusD-like 3.hmm models downloaded from PFAM [24]. Structures of B. thetaiotaomicron VPI-5482 proteins SusD, SusD-like BT1043 and BT3984 have already been resolved and they display similar folds, although the amino acid identities are low and blastp [8] searches fail to detect O-mucin specialized BT1043 as a match to starch-binding SusD [5,30,31]. Therefore, we reasoned that SusD-like proteins from PULs with the same target substrate in various Prevotella genomes should exhibit significant amino acid identity, whereas the hits to SusD-like proteins binding other substrates would be insignificant. SusD-like proteins were thus compared among themselves by a blastp search using blast 2.2.27+ [8], and hits exhibiting >50% coverage and >40% amino acid identity were retained. The proteins exhibiting high amino acid identity were clustered with mcl [58] using an inflation parameter of 1.5. The SusD-like protein profiles were then generated by noting the presence/absence of a specific SusD-like protein group in the individual Prevotella genome.

B. ovatus ATCC 8483 and P. bryantii B1 4 proteins. If a significant match was found, the matched protein was searched for among the proteins known to be coded in PULs whose substrates had been identified in transcriptomic profiling [14,36,38], thereby revealing the putative substrate of the Prevotella PUL. Additionally, the protein family data [24,35] for the susCD-like neighborhoods were used for evaluation of the conclusions obtained using the blastp search. SusD-like protein phylogeny reconstruction The maximum likelihood tree was reconstructed by MEGA6 [54] using a JTT amino acid substitution matrix and 1000 bootstrap replicates. CAZYme and MEROPS annotation of Prevotella genomes The CAZYme repertoires of Prevotella species were obtained using the CAZYme family hmm models available in dbCAN release 3.0 [62]. The Prevotella proteins were searched for against the dbCAN hmm models using HMMERv3 hmmscan [22] and the suggested E-value cutoff was applied [62]. The peptidase content of Prevotella genomes was estimated using the Merops peptidase database [43] compilation of peptidases available in merops scan.lib (downloaded in May 2014) used in Merops batch-BLAST (http://merops.sanger.ac.uk/cgi-bin/batch blast). The searches were carried out locally using blastp 2.2.27+ [8] and an E-value cutoff of 1e−10. Hierarchical clustering of Prevotella species according to their SusD-like protein and CAZYme repertoires Clustering was carried out with R package pvclust [52] using average linkage and 10,000 bootstrap replicates. For CAZY repertoires, the distance measure chosen was Manhattan, since it is known that enzymes from the same CAZY family may act on several substrates or in different ways on the same substrate [50]. Thus, the Manhattan coefficient would capture both the variability of CAZYme families in a genome as well as the number of enzymes in a family. The CAZY glycoside hydrolases, polysaccharide lyases and carbohydrate esterases were included in the analysis. For SusDlike protein profiles, the binary distance metric was chosen, since we wanted to emphasize the degree of shared SusD-like proteins that may reflect the common substrates bound by Prevotella species and the index should not be affected by the total number of SusDlike proteins per genome, which in fact varied greatly. The SusD-like protein repertoire comparison was based on groups with more than one member. Ordination analysis of annotated SusD-like protein and CAZYme Prevotella repertoires Non-metric multidimensional analysis was performed on data contained in Table S3 using R package Vegan [42]. The distance measure chosen was Bray–Curtis. Results

Assigning putative substrates to identified Prevotella PULs Diversity of Prevotella SusD-like protein repertoires The results obtained through transcriptomic profiling of B. thetaiotaomicron VPI-5482 [36,38], B. ovatus ATCC 8483 [38] and Prevotella bryantii B1 4 [14] were used to infer most probable substrates for the identified Prevotella PULs. SusD-like protein groups that contained five or more members were manually investigated. For each member in a group, the immediate genome neighborhood of the susCD-like genes was inspected using Artemis [9] and searched using blastp against the B. thetaiotaomicron VPI-5482,

Almost 1200 full-length SusD-like proteins were detected in 50 Prevotella genomes, and there was a wide variation in their number per genome. A distinct reduction in the number of SusDlike proteins to four or less was seen in several Prevotella species (i.e. P. corporis, P. disiens, P. intermedia, P. micans, P. nigrescens, P. pallens and P. tannerae) (Table 1). The SusD-like proteins were divided by mcl into 274 groups of which 99 contained at least

Please cite this article in press as: T. Accetto, G. Avguˇstin, Polysaccharide utilization locus and CAZYme genome repertoires reveal diverse ecological adaptation of Prevotella species, Syst. Appl. Microbiol. (2015), http://dx.doi.org/10.1016/j.syapm.2015.07.007

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Table 1 The characteristics of Prevotella genomes relating to degradation of polysaccharides and peptides. The color scheme is the same as in Fig. 1, except for the group with a reduced number of SusD-like proteins (red). GAG: glycosaminoglycan, R: rumen, F: non-host-associated, G: gut, O: oral, U: urogenital tract, C: clinical strain.

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five members. The SusD-like protein repertoires of the individual Prevotella species, excluding those with the obviously reduced SusD-like protein number, were compared and their similarity is shown in Fig. 1A. Several statistically significant groups of species with similar repertoires were apparent and are color highlighted in Fig. 1A. While the group colored in violet was composed exclusively of urogenital tract strains, the oral strains were found in all other

groups, including the gray one that also contained strains from the rumen, gut and non-host associated environments (see Table 1). Annotation of Prevotella susCD clusters The putative substrates were predicted for the 480 out of 1196 susCD clusters identified in Prevotella using SusD-like proteins

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Fig. 1. Pvclust hierarchical clustering of the Prevotella SusD-like protein (A) and CAZYme (B) repertoires. The numbers in red represent approximately unbiased bootstrap probabilities. The color scheme of high probability clusters in (A) is also applied in (B). The red rectangle outlines the species with a distinctively diminished number of CAZYmes. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)

as proxies, and they are summarized in Table 1 and detailed in Table S2. For most susCD clusters, the predictions were based on the Bacteroides and Prevotella transcriptomic profiling [14,36,38] data blast transfer to Prevotella genomes studied. The reliability of this approach was corroborated by considering the protein family data of the clusters that were universally in agreement with predicted substrates. While the majority of the annotated susCD clusters were associated with genes whose products are known to take part in plant or host glycan breakdown, some of the clusters were surprisingly associated with peptidases (Table S2). All the Prevotella genomes, with the exception of P. pleuritidis, P. timonensis and P. nanceiensis, possessed the archetypal starch degrading PUL found in B. thetaiotaomicron VPI-5482. Additionally, three defined mcl groups of susCD clusters were found that may also be involved in starch degradation (Table S2), since: (i) SusD-like proteins from all three groups clustered together with the SusD of the archetypal starch PUL in the SusD-like phylogenetic tree (Fig. S1), and

(ii) they contained enzymes from CAZY families GH13 and GH97 that are known to encode starch degrading enzymes. Some quite widespread Prevotella susCD clusters could not be annotated using the results of the Bacteroides transcription profiling. Interestingly, the SusD-like proteins from the four largest such mcl defined groups with a total of 64 members formed a coherent group in the SusD-like phylogenetic tree reconstruction (Fig. S1; clusters 3, 5, 11 and 14) implying similar target substrate. The SusD-like phylogenetic tree also suggested that: (i) O-glycan and starch binding SusD-like proteins formed distinct separate lineages from other SusD-like proteins, and (ii) the SusD-like proteins coded in susCD tandem repeats, which are found frequently in xylan (mcl groups 25 and 27, see also [14]) and rhamnogalacturonan (mcl groups 37 and 47) PULs, were quite diverged and possibly bind to distinct parts of these complex glycans. The coloring in Table 1 highlights the Prevotella species with similar SusD-like protein repertoires, as seen in Fig. 1A. While

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the groups in gray and violet seemed specialist, possessing PULs targeting, in addition to starch, almost exclusively plant or host derived glycans, respectively, the blue group was predicted to be more generalist, proficient in both plant and host glycan degradation. For some species, the share of the annotated

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susCD clusters was quite low, which could lead to bias or even false conclusions of the analyzed Prevotella species reliance on host or plant glycans. Therefore, this led us to assay also the CAZYme content and its variability in Prevotella genomes.

Table 2 Number of CAZYmes in Prevotella genomes that target the main glycans encountered in the mammalian intestine. The color scheme is the same as in Fig. 1, except for the group with a reduced number of SusD-like proteins (red). GAG: glycosaminoglycan.

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CAZYme and peptidase repertoires As in the case of SusD-like proteins, the number of CAZYmes in Prevotella genomes varied widely (Table 1) and there was a significant correlation between the two (Pearson’s r = 0.78). The variability of the Prevotella genome CAZYme content can be seen in Fig. 1B. Similar groups of species as in the SusD-like protein repertoire comparison (Fig. 1A) were observed and oral Prevotella strains were again found in all but the urogenital group. Two differences could be observed when comparing the dendrograms: (i) the group encompassing P. denticola and P. multiformis in the SusD-like protein comparison was larger and also included P. melaninogenica and P. veroralis strains, and (ii) the group composed of rumen, gut, freeliving and oral species (highlighted in gray) was also larger but included H. seregens, P. dentalis and P. maculosa. All these strains were placed in the close vicinity of respective groups already in the SusD-like protein dendrogram but the connection was below the statistical significance cutoff. Prevotella genomes with reduced SusD-like protein numbers also showed a reduction in CAZYme numbers to less than 15 per megabase (Mb) and formed a coherent group in the CAZYme comparison, with the exception of P. micans that possessed a slightly larger and diverse CAZYme repertoire with approximately 20 enzymes per Mb (Table S4). The evident CAZYme number reduction was also seen in the otherwise SusD-like protein richer urogenital group of strains and, in particular, in P. marshii that possessed only 14 CAZYmes per megabase but nine SusD-like proteins. The number of CAZYmes in Prevotella genomes targeting the main glycan classes encountered in mammalian intestine can be seen in Table 2. The CAZY families consisting of members possessing diverse substrate specificities (e.g. GH2 and GH3) were not included in the comparison (see discussion). All the Prevotella genomes, even those lacking the archetypal SusD (see Table 1), seemed to possess the enzymes needed for starch breakdown. As mentioned above, there was a strong correlation between SusD-like proteins and CAZYmes: for SusD-like proteins and CAZYmes targeting plant glycans Pearsons’s r = 0.89 and in the case of N-, O-glycans and GAGs Pearson’s r = 0.74. For the latter, there were outliers containing numerous CAZYmes but no or only a few SusD-like proteins (A. rava F0323, P. ruminicola 23, P. buccalis ATCC 35310). This suggested that the SusD-like protein annotation was less efficient for host glycans, and novel SusD-like binding proteins not found in Bacteroides may reside in Prevotella. The number of peptidases encoded in Prevotella genomes was found to be fairly constant per Mb and

comparable to the number found in marine Bacteroidetes [23]. The number was also not reduced in species with diminished SusD-like proteins and CAZYme repertoires.

Discussion Overall, the usage of SusD-like hmms for SusD-like proteins and PUL discovery in Prevotella proved effective, since: (i) the identified SusD-like protein number was consistent with previous studies because 105 SusD-like proteins were detected in B. thetaiotaomicron VPI-5482 (not shown) compared to 103 reported previously [36], and (ii) the SusD-like proteins were always found in tandem with SusC-like proteins and presumably had no function on their own, unlike the SusC-like proteins which are part of the TonBdependent receptor family [41] that also transfer siderophores and other small molecules besides carbohydrates across the Gramnegative outer membrane. Thus, SusD-like proteins were suitable PUL indicators. Although we were able to assign the target substrate to less than half of all Prevotella SusD-like proteins found (and consequently PULs), the major SusD-like protein groups were well covered, since only six out of the 25 largest groups remained without annotation and four of them clustered together with a high bootstrap confidence in the SusD-like protein phylogenetic tree (Fig. S1), which suggested a common substrate. The most prevalent SusD-like protein group targeted starch; however, it is noteworthy that only three out of the 25 largest groups targeted hemicellulose (xylan and mannan) or pectin (arabinan). Thus, regarding the genus as a whole, the Prevotella seem to be quite host glycan and plant storage polysaccharide oriented. However, this is based on the fact that Prevotella isolation was biased in favor of oral sites. Recent molecular ecological studies show that Prevotella may be quite common in the hindgut of man, chimpanzee, cow and swine, as well as in the rumen [3,13,21,28,29,39]. Interestingly, six out of the 25 largest SusD-like protein groups were consistently associated with peptidases, thus providing evidence for the hypothesis that SusCDlike proteins may also be instrumental in protein degradation and peptide transfer across the Gram-negative outer membrane. Alternatively, since only a small share of total peptidases was present in such PUL resembling susCD centered operons it is possible that these peptidases act as helper enzymes in mucin O-glycan degradation. However, no CAZYmes were encoded in these genomic regions.

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0.6

Alloprevotella rava F0323 P.timonensis CRIS 5C−B1

0.0

P.multisaccharivorax DSM17128

−0.2

NMDS2

0.2

0.4

P.marshii DSM16973

−0.4

Hallella seregens ATCC51272 P.dentalis DSM3688

−0.6

P.bergensis DSM17361

−1.0

−0.5

0.0

0.5

NMDS1 Fig. 2. A non-metric multidimensional ordination analysis of the annotated SusD-like protein and CAZYme Prevotella genome profiles, as presented in Tables 1 and 2. Colors of the proposed adaptation groups: 1, plant glycan group – gray; 2, plant glycan and host glycan group – blue; 3, group centered on host glycans but capable of degrading plant glycan to a limited extent – green, orange and the neighboring elements; 4, urogenital group targeting host glycan – violet; 5, group with extensively reduced capability for degrading carbohydrates other than starch – red. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)

Since the share of annotated SusD-like proteins in some Prevotella genomes was low, we also evaluated their CAZYme repertoires. These clustered quite similarly to the SusD-like protein repertoires and were in agreement with the predicted substrates of Prevotella PULs (i.e. the genomes enriched in plant glycan-specific SusD-like proteins were also enriched in CAZYmes degrading plant glycans). In this study, we mainly sought to document the overall dependence of specific Prevotella species on hostor plant-derived glycans. Therefore, not all dominant Prevotella CAZY families were included in the analysis, since some quite frequent families (e.g. GH2, GH3 and GH5; Table S5) contained members acting on diverse plant and host glycan substrates. Given that the majority of characterized enzymes in these families degrade plant glycans [34], the number of plant degrading CAZYmes on which the Prevotella groups were defined may be slightly underestimated in Table 2 and Table S3. In agreement with this, the plant polysaccharide-degrading Prevotella (gray and blue groups, including also P. dentalis, see below) actually showed an enrichment of enzymes from these families compared to other Prevotella species (Mann–Whitney U-test, for GH2, GH3 and GH5; p < 0.0002). Based on SusD-like protein and CAZYme repertoires, as well as their clustering and substrates, it seems quite clear that Prevotella species were heterogeneous and could be divided into at least five groups of distinct predicted ecological adaptation (Fig. 2). The first group consisted of oral, gut, rumen and non-host associated strains (the gray colored group), which apparently degrade mainly the plant storage and structural polysaccharides. Surprisingly, P. ruminicola 23, which also belonged to this group, contains a large gene cluster targeting host glycans (see Table S2, mcl group 82, centered on SusD-like protein PRU 1161), which was unique among the rumen strains. Loosely connected to this group were also P. dentalis and H. seregens. The second group, colored blue, consisted of oral and gut strains that seemed capable of efficiently exploiting both the plant polysaccharides as well as host-secreted glycans. The third group was comprised of other Prevotella strains possessing the capability to degrade both plant and host glycans but with the distinction that the plant degradation capability seemed

less pronounced given the total number of SusD-like proteins and CAZYmes involved in plant polysaccharide breakdown. P. multisaccharivorax seemed to be a border line case between the second and third groups. The fourth group consisted of urogenital strains of P. amnii and P. bivia (violet) that appeared to rely almost exclusively on host glycans besides starch. The strains of the fifth group, comprising P. corporis, P. disiens, P. intermedia, P. micans, P. nigrescens, P. pallens and P. tannerae, exhibited an even stronger reduction in numbers of both the SusD-like proteins and CAZYmes, and were predicted to rely mainly on peptides/proteins besides starch since the peptidase numbers were unchanged. This is also in agreement with their reported life-styles, since P. intermedia is known for its role in periodontitis, may invade eukaryotic cells and may also be dispersed to other body sites [49]. A similar situation seems to be the case for P. nigrescens and P. tannerae [51,60,61], while for other species we can only predict that they would occupy the same niche, since data on their ecology is unavailable. The predicted ability of prevotellas to occupy distinct niches, similar to that postulated for B. thetaiotaomicron and B. vulgatus [38], is in agreement with the observation of multiple Prevotella species at the same oral sites [40]. These sites may thus be colonized by strains possessing different glycan preferences. Also, the ongoing genome sequencing of numerous rumen Prevotella strains performed by the Hungate 1000 project [11] will no doubt offer the possibility to expand the spectrum of recognized Prevotella ecological adaptations in the future. The Prevotella grouping proposed above is an initial assessment that calls for future experimental verification using (co)culture growth experiments in minimal media supplemented with defined glycan substrates and analysis of target susCD loci both at the transcriptional as well as the protein level. To our knowledge, there are few systematic studies linking the CAZY and SusCD repertoires to actual growth on specific substrates [12,36,38], although with ongoing studies yielding new monospecific (possessing only one substrate) CAZY subfamilies [4] and the high interest in Bacteroidetes PULs [55], the glycan predilections of newly isolated Bacteroidetes will presumably be easier to discern in the future.

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Acknowledgements This work was supported by the Slovenian Research Agency (research project P4-0097). Appendix A. Supplementary data Supplementary data associated with this article can be found, in the online version, at http://dx.doi.org/10.1016/j.syapm.2015.07. 007. References [1] Accetto, T., Avguˇstin, G. (2011) Inability of Prevotella bryantii to form a functional Shine-Dalgarno interaction reflects unique evolution of ribosome binding sites in Bacteroidetes. PLoS ONE 6 (8), e22914, http://dx.doi.org/10. 1371/journal.pone.0022914. [2] Alauzet, C., Mory, F., Carlier, J.-P., Marchandin, H., Jumas-Bilak, E., Lozniewski, A. (2007) Prevotella nanceiensis sp. nov., isolated from human clinical samples. Int. J. Syst. Evol. Microbiol. 57 (10), 2216–2220, http://dx.doi.org/10.1099/ijs.0. 65173-0. 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