A predicted physicochemically distinct sub-proteome associated with the intracellular organelle of the anammox bacterium Kuenenia stuttgartiensis

Abstract

Background: Anaerobic ammonium-oxidizing (anammox) bacteria perform a key step in global nitrogen cycling. These bacteria make use of an organelle to oxidize ammonia anaerobically to nitrogen (N2) and so contribute approximately 50% of the nitrogen in the atmosphere. It is currently unknown which proteins constitute the organellar proteome and how anammox bacteria are able to specifically target organellar and cell-envelope proteins to their correct final destinations. Experimental approaches are complicated by the absence of pure cultures and genetic accessibility. However, the genome of the anammox bacterium Candidatus "Kuenenia stuttgartiensis" has recently been sequenced. Here, we make use of these genome data to predict the organellar sub-proteome and address the molecular basis of protein sorting in anammox bacteria.

Results: Two training sets representing organellar (30 proteins) and cell envelope (59 proteins) proteins were constructed based on previous experimental evidence and comparative genomics. Random forest (RF) classifiers trained on these two sets could differentiate between organellar and cell envelope proteins with ~89% accuracy using 400 features consisting of frequencies of two adjacent amino acid combinations. A physicochemically distinct organellar sub-proteome containing 562 proteins was predicted with the best RF classifier. This set included almost all catabolic and respiratory factors encoded in the genome. Apparently, the cytoplasmic membrane performs no catabolic functions. We predict that the Tat-translocation system is located exclusively in the organellar membrane, whereas the Sec-translocation system is located on both the organellar and cytoplasmic membranes. Canonical signal peptides were predicted and validated experimentally, but a specific (N- or C-terminal) signal that could be used for protein targeting to the organelle remained elusive.

Conclusions: A physicochemically distinct organellar sub-proteome was predicted from the genome of the anammox bacterium K. stuttgartiensis. This result provides strong in silico support for the existing experimental evidence for the existence of an organelle in this bacterium, and is an important step forward in unravelling a geochemically relevant case of cytoplasmic differentiation in bacteria. The predicted dual location of the Sec-translocation system and the apparent absence of a specific N- or C-terminal signal in the organellar proteins suggests that additional chaperones may be necessary that act on an as-yet unknown property of the targeted proteins.

Figure 1

Performance comparison of the RF model trained on different types of input data. 500 RF models with randomly generated P1 and P2 sets, to correct for class A and P inbalance, were trained on each of the following 6 types of data: the full-length amino acid sequences, the signal peptides (SP) and the mature protein amino acid sequences, each analyzed with either the residue frequency of single amino acids or the frequency of 2 adjacent amino acids. When the 6 top-performing models of each input type are compared, the model trained with full-length protein sequences with the 2 adjacent amino acids combination shows the highest overall accuracy (89%) and A protein recall (90%).

Figure 2

Signal peptide predictions on the whole proteome of K. stuttgartiensis. Signal peptide predictions on the whole proteome (4663 proteins) of K. stuttgartiensis by fifteen signal peptide prediction algorithms. The y-axis shows the number of proteins predicted to carry a signal peptide. Abbreviations: G+: predict option of Gram-positive; G-: predict option of Gram-negative; Euk: predict option of Eukaryote. The number of predicted Tat and Type IV prepilin substrates using TatFind and PilFind were nine and ten, respectively.

Figure 3

Experimental validation of signal peptides in the Candidatus Kuenenia stuttgartiensis proteins. Identification of cleavage sites from seven Candidatus Kuenenia stuttgartiensis mature proteins. The peptides that were identified in the tryptic digest are coloured red; their N-terminal sides were non-tryptic. Underlined sequences represent the putative signal peptides, and the putative SPase 1 recognition sites adjacent to the non-tryptic side of the peptides are printed in bold. The left column indicates whether the protein is present in either the A or the P training set.

Figure 4

Sequence composition of the signal peptides of the anammoxosome and cell envelope protein sets. Weblogos of the signal peptides of protein sets A (anammoxosomal) and P (cell envelope) are shown. Both the hydrophobic h-regions (residues -6 to -17) and the signal peptidase AxA consensus (residues -1 to -3) preceding the cleavage site are clearly visible. The weblogos were created from sequences aligned to the cleavage site, using Weblogo [47].

Figure 5

Physicochemical differences between anammoxosomal and cell envelope proteins. Two physicochemical parameters are plotted against each other: GRAVY index (grand average of hydropathy) and aliphatic index (relative volume occupied by aliphatic side chains of I, L, V, and A), which can both be calculated from amino acid compositions. These two parameters separate sets A and P into two largely distinct clusters. Purple dots: set P. Blue dots: set A.