Bacterial and fungal chitinase chiJ orthologs evolve under different selective constraints following horizontal gene transfer

Abstract

Background: Certain bacteria from the genus Streptomyces are currently used as biological control agents against plant pathogenic fungi. Hydrolytic enzymes that degrade fungal cell wall components, such as chitinases, are suggested as one possible mechanism in biocontrol interactions. Adaptive evolution of chitinases are previously reported for plant chitinases involved in defence against fungal pathogens, and in fungal chitinases involved in fungal-fungal interactions. In this study we investigated the molecular evolution of chitinase chiJ in the bacterial genus Streptomyces. In addition, as chiJ orthologs are previously reported in certain fungal species as a result from horizontal gene transfer, we conducted a comparative study of differences in evolutionary patterns between bacterial and fungal taxa.

Findings: ChiJ contained three sites evolving under strong positive selection and four groups of co-evolving sites. Regions of high amino acid diversity were predicted to be surface-exposed and associated with coil regions that connect certain α-helices and β-strands in the family 18 chitinase TIM barrel structure, but not associated with the catalytic cleft. The comparative study with fungal ChiJ orthologs identified three regions that display signs of type 1 functional divergence, where unique adaptations in the bacterial and fungal taxa are driven by positive selection.

Conclusions: The identified surface-exposed regions of chitinase ChiJ where sequence diversification is driven by positive selection may putatively be related to functional divergence between bacterial and fungal orthologs. These results show that ChiJ orthologs have evolved under different selective constraints following the horizontal gene transfer event.

Figure 1

Phylogenetic relationships of Streptomyces ChiJ and related proteins. Phylogenetic analyses were performed using maximum likelihood methods as implemented in PhyML-aLRT, based on an alignment of chitinase amino acid sequences. Branch support values (approximate likelihood-ratio test probabilities (> 0.95) / bootstrap proportions (> 70%)) are associated with nodes. The bar marker indicate the number of amino acid substitutions per 100 amino acids. Streptomyces ChiA, ChiB, ChiJ and fungal ChiJ orthologs are indicated. Sequence identifiers include protein name or locus ID.

Figure 2

Homology model of Streptomyces sp. Mg1 ChiJ chitinase. A. Catalytically important residues are marked in red. Amino acids under strong and weak positive selection are shown in royal blue and dark green respectively. Variable regions from reverse conservation analysis are coloured in salmon and marked I-VII (I = 44–47, II = 75–80, III = 110–111, IV = 123–127, V = 178–185, VI = 200–203, VII = 312–327). B. Secondary structure elements of the enzyme are coloured differently. Side chains of co-evolving amino acids are shown in balls and sticks. Co-evolving groups of residues are illustrated in the same colour. Chito-tetraose is modelled to show the substrate-binding cleft.

Figure 3

Reverse conservation analysis of ChiJ orthologs. Amino acid conservation was estimated using Rate4Site, based on a Clustal X alignment of ChiJ Streptomyces orthologs, and plotted as W mean scores in arbitrary units (solid line). Amino acid conservation was estimated in a similar way for fungal orthologs in a previous work [10], and included in the figure (dashed line). Horizontal line indicates a 0.5 standard deviation cut-off. The x-axis represent residue position, asterisks (*) indicate positions of predicted catalytic residues, diamonds (◊) indicate predicted substrate-interacting residues, boxed P indicate residues under strong (Bayes factor ≥ 50) positive selection, P indicate residues under weak (Bayes factor 10–49) positive selection, boxed C interconnected by horizontal lines indicate co-evolving residue groups and vertical dashed lines indicate identical residues. The position of highly variable regions successfully visualised by homology modelling are indicated (I through VII). Magnifications illustrate residue S score distribution of the selected region. The position of a highly variable region in the fungal orthologs is indicated by A.