Comparing the distribution of expression and network coherence for single- and multi-species methods for the B. subtilis-B. anthracis pairing. A comparison of the expression and network coherence for the different MS and SS methods. For brevity, we present here only the results from full data methods (FD) from the B. subtilis-B. anthracis pairing (the results for the other pairings and expression only (EO) methods can be found in Additional file 1). Abbreviations are given for each method; a key to these abbreviations can be found in Table 1. Across the three comparisons, no method outperformed all other methods as judged by all three metrics, with the MSCM results performing competitively with the others. (a) The distributions of the residual values from each method for the pairing of B. subtilis and B. anthracis. We also show, next to each distribution (in gray), the residuals from randomly shuffled (bi)clusters that match the size distribution for each method with n = 1000 for the number of copies of the original set of (bi)clusters (same number of genes, conditions and (bi)clusters). Most methods tested were significantly better than random for both organisms; the exceptions being MSISA, QUBIC, and Coalesce (COAL). In addition, this plot illustrates the tendency of MSKM to allow an organism with a considerably larger expression dataset to dominate the analysis. (b) The distributions of the average absolute correlation from each method for the pairing of B. subtilis and B. anthracis are displayed to allow comparison between methods that identify inversely correlated biclusters (MSISA, QUBIC) and those that do not. As in (a), we also display the results from a randomly shuffled distribution next to each method in gray (n = 1000). In all cases, with the exception of QUBIC for B. subtilis, the method was significantly higher than random. (c) The distributions of the association P-values (-log10) from each method compared.

Comparison of the fraction of biclusters with significant GO and KEGG annotation enrichments for the single and multi-species methods for the B. subtilis-B. anthracis pairing. (a) GO terms. For all multi-species methods there is a consistent increase from the shared to elaboration step, with the percentage of elaborated biclusters with significant GO term enrichments consistently greater than those from the single species optimization. (b) KEGG pathways. For both of the multi-species biclustering methods (MSCM and MSISA), there is a consistent increase in percentage from the shared to elaborated optimizations, similar to the GO term enrichments, with a similarly large increase for the refined MSISA biclusters for B. anthracis. The two k-means clustering variants showed either negligible increase or even a decrease between the shared and elaboration steps.

Conserved motility modules active in all three organisms and motility assays. (a) We show a schematic of the flagellar apparatus for B. subtilis showing the location of 26 flagellar proteins, two motor proteins (MotA and MotB) and two transcriptional regulators (FlgM and SigD) (using gene names from B. subtilis). (b) The left panel shows the presence (blue)/absence (white) of the corresponding genes in the genomes of B. anthracis Sterne (BAS), B. cereus ATCC 14579 (BC), L. monocytogenes EGD-e (LMO) and B. subtilis 168 (BSU). In B. anthracis Sterne, motB, fliM, fliF, and flgL are represented by two colors indicating a gene coding for a truncated protein due to a frameshift mutation that introduces a premature stop codon. The right panel shows the gene presence in the main flagellar bicluster resulting from each of the three pairwise multi-species biclusterings. Indicated are genes of the flagellar apparatus - included in the bicluster core (red), in the elaboration of the bicluster (orange), and not included in the bicluster (gray). B. subtilis and L. monocytogenes are both known to be flagellated and motile. B. anthracis Sterne is non-motile, but our results indicate a bicluster enriched for genes involved in flagellar biosynthesis. (c) Swimming motility was assayed on 0.3% agar plates for B. cereus ATCC 14579, B. anthracis Sterne, B. subtilis PY79, and B. subtilis PY79 ΔmotAB::tet (strain DS219). B. cereus and B. subtilis are motile [51,68]. A deletion of motAB in B. subtilis impairs motility [69,91]. The assay shows that B. anthracis Sterne is not motile under the conditions tested.

Schematic overview of the multiple-species method. (a) Shared-space bicluster seeds are generated by calculating the pairwise correlation of the gene pairs to a randomly selected gene pair. (b) The shared-space multi-species optimization, where orthologous gene pairs are iteratively added or dropped from the bicluster according to the multi-species multi-data score. (c) When completed, shared-space biclusters are separated into their respective species, and further optimized during the elaboration step. During this step the genes from the original shared-space bicluster are prevented from being dropped, as indicated by the boxes surrounding these genes (represented as black circles). OC, orthologous core (the set of actively expressed orthologous genes shared between a group of organisms on which we run our multi-species biclustering).

Comparison of the size, coverage and overlap for single and multi-species methods for the B. subtilis-B. anthracis pairing (full data results only, where applicable). For brevity, we present here only the results from full data methods (FD) from the B. subtilis-B. anthracis pairing (results for the other pairings and EO methods can be found in Additional file 1). (a) The distribution of the number of genes in the (bi)clusters from the different methods. There is a consistent increase in the median size between the shared and elaboration steps (this is most extreme in the case of the MSISA method). For both organisms, Coalesce (COAL) and QUBIC produced the next largest biclusters, in terms of the number of genes. (b) The distribution of the number of conditions in the biclusters from the different biclustering methods only. We do not show this for the MSKM and BMSKM results as these methods use all conditions. For both organisms, the MS/SS cMonkey methods produced the biclusters with the most conditions. The MSISA method produced the biclusters with the least number of conditions. (c) The coverage of the total expression data matrix by the (bi)clusters from the different methods is displayed. The elaborated results of the MSKM and BMSKM methods achieve perfect coverage, by definition. The MSISA and QUBIC biclusters had the smallest coverage of any of the methods, while the Coalesce biclusters achieved coverages comparable with the SSCM biclusters. (d) The distribution of all pairwise, non-zero overlaps between the (bi)clusters from the different methods; overlap in terms of the overlap of expression matrix elements, rather than genes. By definition, the MSKM and BMSKM clusters have no overlap, while the MSISA and QUBIC biclusters had the greatest. Of the biclustering methods, Coalesce had the least overlap. Coalesce identifies more distinct biclusters with greater numbers of genes, but fewer conditions; and the SS/MS cMonkey methods identify biclusters that are slightly more overlapped than does Coalesce, with fewer genes, but covering more conditions.

Expression profiles of three partially conserved sporulation biclusters, identified by the multi-species analysis of B. subtilis and B. anthracis. Bicluster 84 (blue line) is composed primarily of genes involved in metabolic functions during sporulation, bicluster 82 (green line) includes primarily genes encoding spore coat proteins, and bicluster 32 (red line) contains genes involved in spore cortex formation and activation of the σ factors required for the latest stages of sporulation. (a) The B. anthracis biclusters display distinct profiles, revealing a temporal aspect not present in the B. subtilis dataset. The B. subtilis biclusters all follow the same expression profile (that is, similar expression over nearly every experimental condition included in the dataset), as shown for (b) only sporulation experimental conditions (with abscissa corresponding to: 1) hour 2 sigF; 2) hour 2.5 sigE; 3) hour 3.5 gerR; 4) hour 3.5 spoIIID; 5) hour 4 sigG; 6) hour 4.5 sigK; 7) hour 5 spoVT; 8) hour 5.5 gerE; 9) hour 6.5 gerE) and (c) all experimental conditions within the three biclusters.