Identification of proteins likely to be involved in morphogenesis, cell division, and signal transduction in Planctomycetes by comparative genomics

Abstract

Members of the Planctomycetes clade share many unusual features for bacteria. Their cytoplasm contains membrane-bound compartments, they lack peptidoglycan and FtsZ, they divide by polar budding, and they are capable of endocytosis. Planctomycete genomes have remained enigmatic, generally being quite large (up to 9 Mb), and on average, 55% of their predicted proteins are of unknown function. Importantly, proteins related to the unusual traits of Planctomycetes remain largely unknown. Thus, we embarked on bioinformatic analyses of these genomes in an effort to predict proteins that are likely to be involved in compartmentalization, cell division, and signal transduction. We used three complementary strategies. First, we defined the Planctomycetes core genome and subtracted genes of well-studied model organisms. Second, we analyzed the gene content and synteny of morphogenesis and cell division genes and combined both methods using a "guilt-by-association" approach. Third, we identified signal transduction systems as well as sigma factors. These analyses provide a manageable list of candidate genes for future genetic studies and provide evidence for complex signaling in the Planctomycetes akin to that observed for bacteria with complex life-styles, such as Myxococcus xanthus.

Fig 1

The class Planctomycetia is split into two distinct orders. Shown is a maximum likelihood phylogenetic tree of planctomycetal 16S rRNA gene sequences. Bootstrap values are shown only for deep branches, and different Escherichia coli sequences were used as an outgroup, represented by an arrow (GenBank accession numbers of the E. coli sequences used are GU594315, GU594305, GU594306, GU594304, GU594302, and GU594316). “Ca. Kuenenia stuttgartiensis,” “Candidatus Kuenenia stuttgartiensis.”

Fig 2

The planctomycetal core genome. The planctomycetal core genome was determined by comparing all eight sequenced Planctomycetes species against each other by using the BLAST algorithm with a coverage of >60% and an E value cutoff of 1e−5 as parameters. The Venn diagram visualizes the 564 clusters fulfilling these criteria. After in silico subtraction, using proteins encoded by Escherichia coli (GenBank accession number U00096) and Bacillus subtilis (accession number AL009126), 450 planctomycetal core genome clusters were eliminated, and 114 planctomycete-specific clusters remained. Genes within these 114 clusters fulfill two criteria: they are conserved among planctomycetes and absent in the genomes of the two most intensively studied model organisms, E. coli and B. subtilis.

Fig 3

Selected putative P. limnophilus operons. Putative P. limnophilus operon 1 (PO1) to PO5 are shown, which fulfill two criteria: (i) they contain one gene that was evaluated as being putatively involved in cell division or compartmentalization based on a manual inspection of the P. limnophilus core genome, and (ii) they harbor at least one more core genome member. Core genome-related genes are shown as red outlined arrows. Identifiers for all genes are given, and selected putative domains are highlighted (vWF, von Willebrand factor; FHS, Forkhead associated).

Fig 4

Content and synteny of dcw operon genes among planctomycetes. The cell division-related (gray) and peptidoglycan synthesis-related (black) genes arranged in the dcw operons of E. coli and B. subtilis are shown in comparison to homologous genes from planctomycetal genomes. Genes of unknown function are shown in white. Genes with weak similarity to the E. coli query are labeled in gray (W, mraW; Z, mraZ; L, ftsL).

Fig 5

Putative cell division-related planctomycetal operons. The putative Plabr and Isop operons fulfilled two criteria: (i) they contain a cell division- or peptidoglycan synthesis-related dcw gene, and (ii) at least one additional core genome member is present within the same putative operon. In a second round, corresponding P. limnophilus genes were identified, which turned out to be localized on 5 different putative operons (PO6 to PO10). Selected protein domains are highlighted, while core genome members are boxed in red.

Fig 6

Domain architectures of special 2CSs and ECFs in planctomycetes. The typical domain architecture is shown based on results from the SMART database (http://smart.embl-heidelberg.de/). (A) Novel type of RRs in Planctomycetes species consisting of an N-terminal Ser/Thr/Tyr protein kinase domain (Pfam designation, Pkinase) and a C-terminal receiver domain. (B) Typical C-terminal extensions of the ECF01-Gob proteins. Standard ECFs (shown on the top) are comprised of only two regions, the σ2 (Pfam designation, Sigma70_r2) and σ4 (Pfam designation, Sigma70_r4) domains, with a length of about 200 amino acids. The C-terminal extensions of ECF01-Gob ECFs in planctomycetes contain up to 1,000 amino acids, which, among other domains (see the text for details), contain putative transmembrane regions (shown in blue rectangles) as well as secretin and WD40 domains with different arrangement styles. The scale bar represents the protein length in amino acids.

Fig 7

Phylogenetic tree and classification of ECF sigma factors from planctomycetes. Shown is a phylogenetic tree of all ECF sequences extracted from the MiST2 database, generated by the least-squares distances method. A subsequent group analysis of unclassified planctomycetal ECFs was done based on their phylogenetic distances, genomic context conservation, or the presence of promoter motifs, which resulted in the assignment of the novel groups ECF45, ECF46, ECFSTK1, ECFSTK2, ECFSTK3, and ECFSTK4 as well as the ECF01-like groups ECF01-Gob and ECF01-P. The corresponding sequence information is shown in Table S5 in the supplemental material. Different groups are shown in different colors and are highlighted correspondingly in the tree map. (A) Abundance and distribution of ECFs in the different planctomycete species. Abbreviations: Rba, Rhodopirellula baltica SH1; Pst, Pirellula staleyi DSM 6068; Pma, Planctomyces maris DSM 8797; Pli, Planctomyces limnophilus DSM 3776; Pbr, Planctomyces brasiliensis DSM 5305; Ipa, Isosphaera pallida ATCC 43644; Gob, Gemmata obscuriglobus UQM 2246; Bma, Blastopirellula marina DSM 3645. (B) Phylogenetic tree of ECFs in planctomycete species. Eight ECFs were excluded because of their phylogenetic distance from other ECFs. These proteins are highlighted in Table S5 in the supplemental material.