Sample selection for diversity analyses
During the duration of the whole experiment (112 days), a significant relationship between leaf bacterial biomass and phenol oxidase activity was observed, suggesting a link between bacteria and degradation of phenols in leaves (Fig. 1). To investigate the potential role of phenol-degrading bacteria, three dates were selected for molecular analysis of the largest subunit of multicomponent phenol hydroxylases (LmPHs). The three dates corresponded to three distinct stages throughout the leaf decomposition sequence: a initial stage (day 7), coinciding with the initiation of bacterial colonization; a midterm stage (day 58), coinciding with increasing bacterial biomass and phenol oxidase activity and the maximum fungal biomass (Artigas et al., 2011); and a late stage (day 112), when the maximum bacterial biomass was measured although phenol oxidase slightly decreased (Fig. 1).
Figure 1. Linear regression analysis between phenol oxidase activity and biomass of bacteria accumulated in Platanus acerifolia leaves throughout the decomposition process at the Fuirosos stream (data obtained from Artigas et al., 2011). The R square (r2) and probability (P) values after the linear regression analysis are shown. The initial (day 7), midterm (day 58), and late (day 112) stage samples selected for the analysis of LmPH genes are indicated in arrows.
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Phylogenetic analysis of bacterial phenol hydroxylases
The LmPH gene was amplified by PCR from the three leaf litter decomposition stages, and a total number of 148 good quality sequences were obtained from cloning experiments. The estimated rarefaction curves in each sample approached saturation, indicating a good coverage of LmPH gene richness (Fig. 2). All subsequent analyses were performed using an OTU-based approach of the deduced amino acid sequences at a 0.1 cutoff level. The analysis of sequences from the three stages resulted in 16 different OTUs, nine of them being specific for either the initial or the midterm stage. OTU 14 was the most abundant and contained LmPH sequences from the initial (11 sequences), the midterm (22), and late (33) stages. The second most abundant OTU 3 (12 sequences) was exclusively composed of sequences from the initial stage. Other highly represented OTUs, such as OTUs 15 and 16, grouped exclusively sequences from the midterm and late decomposition stages.
Figure 2. Rarefaction curves for amino acid-derived LmPH sequences at a cutoff value of 10% dissimilarity obtained for the three leaf litter decomposition stages (●) initial, (○) midterm, and (▼) late. Pairwise distances among sequences were conducted using the Poisson correction model. The coding data were translated assuming a standard genetic code table. All positions containing gaps and missing data were eliminated. There were a total of 138 amino acid positions in the final sequences.
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The potential functional differences between communities over the course of leaf decomposition were investigated by deducing kinetic properties of bacterial phenol hydroxylases. LmPH genes can be assigned to different functional groups according to changes at selected positions of the amino acid sequence (Futamata et al., 2001). Key amino acid residues at positions 217, 252, and 253 (position numbering based on the Pseudomonas sp. CF600 dmpN gene sequence) may facilitate the prediction of theoretical Michaelis–Menten semi-saturation constants for most uncultured microorganisms (Viggor et al., 2008). Most of the retrieved sequences (86) belonged to the betaproteobacteria low-Ks LmPH group, previously defined by Futamata et al. (2001) and grouped separately into clusters A and E (Fig. 3). LmPH sequences in cluster A showed significant similarities (> 80%) to phcN, tbc1D, and afpN genes from Comamonas testosteroni, Burkholderia cepacia, and Alcaligenes faecalis, respectively. On the other hand, cluster E contained LmPH sequences with high similarity with phenol-degrading genes from Comamonas sp. and Alicycliphilus sp. Sequences from the three stages appeared in both clusters, although those from the late stage were less abundant in cluster E.
Figure 3. Neighbor-joining phylogenetic tree based on the derived amino acid sequences of LmPH genes from samples of Platanus acerifolia leaves at different stages of leaf decomposition at the Fuirosos stream. Bootstrap values (> 50%) based on 1000 trials are indicated at nodes. Phylogenetic trees were reconstructed using neighbor-joining and maximum-likelihood methods yielding similar tree topologies. Phylogenies were reconstructed using the amino Poisson correction and complete deletion methods. Reference LmPH sequences (bold) were obtained from blast searches within the GenBank reference genomic sequences database. Clusters A, B, C, D, and E were defined according to potential activity of deduced amino acid residues at positions 217, 252, and 253 based on the Pseudomonas sp. CF600 dmpN gene sequence (accession number P19732). Terminal tree nodes containing LmPH sequences obtained in this study have been collapsed and indicated by the OTU number. The number of sequences of the initial (INI), midterm (MID), and late (LATE) samples in every OTU is indicated.
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All sequences in cluster B except one (LATE13_E10) were retrieved from the initial and midterm stage samples. Sequences in this cluster exhibited high sequence diversity and grouped into eight different OTUs. Higher similarities (84–94%) were found to LmPH sequences retrieved from noncultured microorganisms from benzene-contaminated soils or trichloroethylene-contaminated aquifers. Maximum similarities to isolated representatives (< 84%) were obtained with Methylibium petroleiphilum strain PM1, a Betaproteobacterium that degrades methyl tert-butyl ether (Hristova et al., 2007). Although degradation of phenolic compounds has not been studied in detail in PM1, exposure of this strain to MTBE induces additional pathways for the degradation of aromatics such as benzene, toluene, and xylene. In a recent study employing PCR-denaturing gradient gel electrophoresis (DGGE) analysis of reverse-transcribed rRNA, active M. petroleiphilum was shown to accumulate in soils contaminated with penta-chlorophenol (Cáliz et al., 2011).
The specific Variovorax group (cluster C) was also represented by two sequences obtained from the midterm stage (sequences MID06_F3 and MID06_G7, OTU 7). Nevertheless, these two sequences were < 85% similar to Variovorax sp. HAB30. The ecological relevance of Variovorax sp. relies in the presence of a characteristic LmPH type, corresponding to highly active phenol-degrading enzymes with high semi-saturation constants according to determinations of kinetic parameters using isolated cultures (Futamata et al., 2005). Cluster D grouped sequences belonging to Gammaproteobacteria with a high-Ks LmPH, including Pseudomonas putida relatives. A single sequence from the initial stage (sequence INI06_A3, OTU 1) was found in cluster D. Interestingly, this sequence contained the typical signature of low-Ks phenol hydroxylases at amino acid positions 252 and 253, and position in the high-Ks group should be confirmed by incubation experiments with isolated cultures.
Changes in the phenol-degrading bacterial community
The number of bacterial OTUs remained at relatively low values (from 5 to 10) in the three samples analyzed. The bacterial community at the initial and midterm stages of decomposition showed a greater richness, greater diversity (Shannon's H′), and greater evenness (E) of LmPH gene compared to the late stage (Table 1). The significant decrease in richness and diversity values suggests a major specificity of phenol-degrading bacteria in the late-stage community. The results from the phenol-degrading bacterial community analysis showed a highest degree of specialization at the late decomposition stage. All LmPH genes obtained at the late stage, except for one, grouped in clusters A and E together with sequences belonging to known high-affinity phenol degraders (Watanabe et al., 1996). On the contrary, at the initial stage, the lower bacterial biomass and weaker phenol oxidase activity may indicate that decomposition of the large recalcitrant plant molecules had not yet begun (Fig. 1, Artigas et al., 2011). At this first stage, bacterial communities are supposed to be defined by environmental conditions of the stream and random colonization of the leaf surface (Harrop et al., 2009; Marks et al., 2009).
Table 1. Main community indicators: observed richness (S), estimated richness (SChao, and SAce), Shannon's diversity index (H′), and evenness (E′) of the phenol-degrading bacterial communities in Platanus acerifolia leaves at the initial (day 7), midterm (day 58), and late (day 112) stages of leaf decomposition at the Fuirosos stream
|Decomposition stage|| S || S Chao || S Ace ||H′||E′|
|Initial||9||9.5||10.2||1.93 ± 0.19||0.74|
|Midterm||10||10||10.3||1.85 ± 0.28||0.46|
|Late||5||5||5.7||0.98 ± 0.28||0.40|
|Total||16||16.5||16.8||2.06 ± 0.15||0.73|
Differences in the community composition of potential phenol-degrading bacteria were tested from the tree topology using UniFrac and parsimony tests. All pairwise comparisons between samples were highly significant indicating a changing bacterial community at the three degradation stages (Table 2). UniFrac distances ranged from 0.298 to 0.607 and were higher between the initial and late stage samples. UniFrac tests have been previously used as a semi-quantitative determination of the similarities between the bacterial communities on the phyllosphere of Populus deltoides sampled at different times (Redford et al., 2010). According to our estimations, major changes in the phenol-degrading bacterial community may occur between the initial and midterm stages of leaf decomposition. At the midterm, the greatest community richness and diversity was found and coincided with increasing phenol oxidase activity and maximum fungal biomass (Artigas et al., 2011). The LmPH sequences from this stage were scattered throughout the phylogenetic tree (in clusters A, B, C, and E), and their corresponding enzymes exhibit different kinetic properties. It is known that bacteria and fungi have complementary roles in leaf litter degradation. Bacteria are thought to increase their contribution only after leaf material has been partially broken down (Baldy et al., 1995), whereas fungi, especially aquatic hyphomycetes, have been recognized as dominant, in terms of both activity and biomass, during early decomposition (Gulis & Suberkropp, 2003; Romaní et al., 2006). However, bacteria may make a greater contribution to leaf litter decomposition particularly when fungal activity is compromised by unfavorable conditions (Pascoal & Cassio, 2004; Kubartova et al., 2009).
Table 2. Values of diversity (weighted and unweighted UniFrac and parsimony tests) of pairwise comparisons of the phenol-degrading bacterial communities in Platanus acerifolia leaves at the initial (day 7), midterm (day 58), and late (day 112) stages of leaves decomposition at the Fuirosos stream
|Unweighted UniFrac test score||n.a.||n.a.||n.a.||0.580**|
|Weighted UniFrac test score||0.473**||0.298**||0.607**||n.a.|
|Parsimony test score||13*||25*||8**||n.a.|
In conclusion, by analyzing the LmPH gene from different leaf decomposition stages, we have shown that the bacterial community changes significantly over the course of leaf litter degradation in streams. During early decomposition, the bacterial community is rather complex and potentially exhibits a low degree of metabolic specialization in view of the deduced enzyme kinetics. As decomposition progresses, the phenol-degrading bacterial community is dominated by suspected low-Ks type bacteria, with a high similarity to Alcaligenes spp., Comamonas sp., and Ralstonia sp, suggesting a gradual selection of specialized phenol degraders as decomposition progressed. To the best of our knowledge, this work represents the first specific analysis of any functional gene marker and of bacterial and fungal origin, used for investigating microbial communities during the leaf litter decomposition process in streams. Time series analyses of bacterial and fungal communities in leaf litter decomposition have previously been performed using either DGGE or terminal-restriction fragment length polymorphism (T-RFLP) of amplified SSU rRNA fragments (Das et al., 2007; Marks et al., 2009; Kelly et al., 2010), although no general conclusions can be derived from these studies. The relative presence of general and specialized microorganisms on leaf surfaces during litter decomposition has been proposed as a major determinant of diversity (Das et al., 2007). Moreover, leafs of different plant species have been shown to bear specific fungal and bacterial communities during the decomposition process in riverine environments (Marks et al., 2009). In this work, we show that the use of functional genes, as the bacterial LmPH gene, as a proxy to study microbial diversity of relevant microorganisms in leaf litter decomposition is possible. We are confident that the use of other functional genetic markers of bacteria, and its extension to the study of fungi, will provide additional and interesting results to support the idea of changing microbial communities in the process of litter decomposition and increase our understanding of how microorganism interacts in ecosystem processes.