Distribution of HIP1 motifs per ORF in PCC 7942. The X-axis shows the categories of genes, according to their number of HIP1 motifs. The Y-axis shows how many PCC 7942 genes belong to each category. The distribution is reminiscent of a random process, with most ORFs having none or a few copies of HIP1, and a few ORFs having several ones.

Identification of putative alien (PA), highly expressed (PHX) and moderately highly expressed (PX) genes. For each gene belonging to the HIP1^less set (having to few HIP1 motifs for its size) we calculated the codon usage bias against two set of genes: a) the mean codon usage of all genes in the genome B(F|C); and b) the codon usage of ribosomal proteins B(F|RP) which are expected to be highly expressed. Genes with a codon usage suggesting a high (PX) and very high (PHX) level of expression are shown in blue and green respectively. Genes with a codon usage suggesting a xenolog origin (PA) are shown in red. The rest of the genes belonging to the HIP1^less set that didn't meet the criteria to be included in PA, PX or PHX sets are shown in gray.

Taxonomic classification of proteins having an unusual phylogenetic relationship. Proteins having an unusual phylogenetic relationship are classified at phylum level. As shown, most of them are related to proteobacteria.

Identification of xenologs in PCC 7942 by combining different sources of information. Cases of horizontally transferred genes into the genome of PCC 7942 were identified by having at least two of the following properties: G+C content deviating more than 1 SD from the mean value of all genes; low number of HIP1 motifs relative to gene length well as an unusual codon usage (PA genes); and an unusual phylogenetic relationship. ORFan genes were also considered.

Conserved genes in PCC 7942. a) Number of best reciprocal BLAST hits between PCC 7942 and each of the following genomes: P. marinus SS 120, the chromatophore from P. chromatophora and the set of cyanobacterial conserved genes from CyOGs database; b) the set of conserved genes in PCC 7942 is defined as the union of the tree above datasets, plus those conserved in 34 out of 35 genomes (this last comparison added 66 genes to the dataset). Overall, the set of conserved genes comprises 54% of the genome of PCC 7942; c) the set of conserved genes (or paleome) is enriched in genes with functional annotation.

The role of horizontal gene transfer in the evolution of sensory systems in PCC 7942. Several proteins containing diguanylate cyclase and other domains involved in signal transduction mechanisms are predicted to be xenologs. Here we show a) a neighbor-joining tree reconstructed using the diguanylate-cyclase domain (86 sites, Poisson correction, 100 bootstrap replicates, branches with bootstraps below 50 are collapsed) from these proteins. Those proteins predicted to be xenologs are colored with red branches in the tree; b) domain structure of proteins following Pfam database [52].

Phylogenomic tree of cyanobacteria. Neighbor-Joining tree reconstructed from a matrix of 231 concatenated protein families conserved among sequenced cyanobacteria (63,065 amino acid positions, Poisson distance correction). Bootstrap values (100 replications) for Neighbor-Joining and Parsimony, and quartet puzzling support values for Maximum-Likelihood are shown in nodes in the following order: Neighbor-Joining/Maximum-Likelihood/Parsimony.

Distribution of HIP1 motifs per ORF in PCC 7942 versus ORF length. Each dot in the figure represents one gene from the PCC 7942 genome. They are sorted by their number of coded amino acids (X-axis) and by the number of HIP1 motifs in their coding sequence (Y-axis). Black triangles show the mean of the size distribution for each category (0 HIP1 motifs, 1 HIP1 motifs... etc); black circles indicate the positions separated by one standard deviation of the mean; green dots represent ORFs with a codon usage reminiscent of highly expressed genes (PHX); blue dots, genes with codon usage similar to (but not as pronounced as) highly expressed genes (PX); and red dots putative alien genes (PA); gray dots, other genes.

Number of proteins in PCC 7942 with an unusual phylogenetic relationship. For each protein coded in the genome of PCC 7942 we did blast searches against NCBI nr database to identify top scoring best hits. Then, for each protein having at least 4 hits we reconstructed a Neighbor-Joining tree (100 bootstrap replications). We then identified whether the nearest neighbor in the tree belonged to a cyanobacteria or not. When it was not possible to use the tree to identify the nearest neighbor (because the immediate ancestral node of PCC 7942 protein did not share a unique co-descendent OTU; or the bootstrap value was lower than 50; or because there were not at least four homologs in the database) we used the BLAST best hit criteria to decide whether or not the query protein has an unusual phylogenetic relationship. As shown, most proteins do not have its nearest homolog outside cyanobacteria (1177 + 1035 = 2212); however 253 proteins have an unusual phylogenetic relationship (10 of them to protists endowed with plastids). *Number of proteins in which the phylum identified was the same with BLAST and with Neighbor-Joining.

Distribution of G+C content among ORFs in PCC 7942. Each dot corresponds to an ORF in PCC 7942. The X axis indicated gene size translated into amino acids. Y axis indicated G+C content deviation from the mean. Mean and ± 1SD are shown by gray and doted lines respectively.

Coverage of sequences from the metagenomes of two microbialites (Río Mesquitez and Pozas Azules) found in the region of Cuatro Cienegas along the genome of PCC 7942. X axis indicates the nucleotide position along the PCC 7942 genome. Left axis indicates the percent of similarity of aligned metagenomic sequences to the genome of PCC 7942 (blue and red dots correspond to alignments to forward and reverse strands to S. elongatus DNA). The position of these alignments along the genome are also shown at the bottom of the figure to differentiate regions of coverage from non-coverage; right axis indicates a relative measure of coverage of sequences from the metagenomes on the genome of PCC 7942 (the green line at the bottom corresponds to the metagenome from Río Mesquites; while the red line to the metagenome from Pozas Azules). Coverage was calculated for windows of 10,000 nucleotides (sliding 500 nt.) as: (number of aligned nucleotides along the genome)/10,000. The blue rectangle indicates a large region of no coverage along the genome. Larger coverage peaks correspond mostly to genes classified as PX or PHX according to codon usage. The following genes contribute to the largest coverage peaks (1) FtsH peptidase (Synpcc7942_0297, PX); (2) F0F1 ATP synthase subunit alpha (Synpcc7942_0336, PX); (3) photosystem II 44 kDa subunit reaction center protein (Synpcc7942_0656, PHX); (4) ATPase, E1-E2 type (Synpcc7942_1082, PX), glycosyltransferase (Synpcc7942_1083), glycogen branching enzyme (Synpcc7942_1085, PX), ATPase (Synpcc7942_1089, PX); (5) protochlorophyllide reductase iron-sulfur ATP-binding protein (Synpcc7942_1419), light-independent protochlorophyllide reductase subunit N (Synpcc7942_1420, PX), putative carboxysome assembly protein (Synpcc7942_1421); (6) two component transcriptional regulator (Synpcc7942_1453); (7) F0F1 ATP synthase subunit beta (Synpcc7942_2315, PX).

Functional classification of xenologs and conserved genes in PCC 7942. Following COG database 1,921 genes from PCC 7942 are classified into functional categories. Of them, 1,665 genes are classified in a single category. Here we show whether these genes are identified as core genes, xenologs or none of them. Xenologous genes are particularly abundant in the categories signal transduction mechanisms and cell wall/membrane/envelope biogenesis.

Genomic islands and conserved genes in PCC 7942. Starting from the outermost circle: First two circles: xenologs and ORFans are shown in red and blue respectively (both DNA strands are shown); third circle: regions of atypical three nucleotide composition (χ ² analyses, see methods) are shown as purple peaks; fourth circle: genes not classified as xenologs nor core genes are shown in gray (for simplicity both chains of DNA are shown as one); fifth circle, core genes in green (both DNA strands are shown). The genome was drawn using Genomeviz [53].