A genomic signature and the identification of new sporulation genes

Abstract

Bacterial endospores are the most resistant cell type known to humans, as they are able to withstand extremes of temperature, pressure, chemical injury, and time. They are also of interest because the endospore is the infective particle in a variety of human and livestock diseases. Endosporulation is characterized by the morphogenesis of an endospore within a mother cell. Based on the genes known to be involved in endosporulation in the model organism Bacillus subtilis, a conserved core of about 100 genes was derived, representing the minimal machinery for endosporulation. The core was used to define a genomic signature of about 50 genes that are able to distinguish endospore-forming organisms, based on complete genome sequences, and we show this 50-gene signature is robust against phylogenetic proximity and other artifacts. This signature includes previously uncharacterized genes that we can now show are important for sporulation in B. subtilis and/or are under developmental control, thus further validating this genomic signature. We also predict that a series of polyextremophylic organisms, as well as several gut bacteria, are able to form endospores, and we identified 3 new loci essential for sporulation in B. subtilis: ytaF, ylmC, and ylzA. In all, the results support the view that endosporulation likely evolved once, at the base of the Firmicutes phylum, and is unrelated to other bacterial cell differentiation programs and that this involved the evolution of new genes and functions, as well as the cooption of ancestral, housekeeping functions.

Fig 1

Phylogenetic analysis of the response regulator Spo0A (A) and the cell-type-specific σ⁷⁰ factors that control endosporulation in B. subtilis (B) revealed that these regulatory factors are specific to endosporulating species. The trees were constructed from amino acid sequences in the species data set that contained the same structural domain architecture as each of the types of regulators and were inferred by maximum likelihood (ML) and neighbor joining (NJ) (see Materials and Methods for details). Tree scales are in evolutionary distances according to the Jones-Taylor-Thornton (JTT) amino acid substitution model. Asterisks indicate nodes supported by a bootstrap value higher than 70%.

Fig 2

The phylogenetic extent of the core endosporulation machinery reveals that a substantial part of the machinery is restricted to endosporulating species. Proteins are grouped according to the regulatory protein that controls their production (top), species are shown on the NCBI taxonomic tree (left), and lifestyle is denoted by color, with endosporulators, exosporulators, mycobacteria, and nonendosporulators shown in blue, pink, green, and red, respectively. The presence of an orthologue for a given protein in a given species is indicated by a black dot.

Fig 3

Genomic signature for endosporulation. (A) The signature is defined as those genes present in 90% of endosporulating bacteria and in no more than 5% (inner circle) or 10% (outer circle) of the remaining bacterial species. Note that the first gene of the spoIIIA operon is only found at the 10% cutoff. Genes with an established function in sporulation, genes coding for the RNA polymerase σ factors that control gene expression during sporulation and genes encoding global transcriptional regulators are shown, as is one gene, tepA, with a predicted function in protein secretion but which has not yet been implicated in sporulation. Also shown are genes with no assigned function. The positions of the genes are shown in degrees in the B. subtilis 168 chromosome. (B) Percentage of the minimal core in genomes, showing that endosporulating organisms all have a high proportion of this signature, and that all mycobacteria and exosporulating organisms have less than 20% of this signature. Those organisms not known to sporulate but that show a proportion of the signature comparable to the known endosporulators are predicted to be able to form endospores.

Fig 4

Identification of new endosporulation genes and their functional analysis in B. subtilis. (A) The region of the ytaF, ylzA, ylmC, and ymxH genes in the B. subtilis chromosome, with possible promoters and transcriptional terminators represented by broken arrows and stem-loop structures, respectively. The red lines below the physical map represent the inserts in the plasmids used to disrupt the indicated genes by means of a single-reciprocal (ytaF) or a double-crossover (ylmC, ymxH, and ylzA) event. The green lines represent the sequences transcriptionally fused to the gfp gene. (B) Impacts of insertional mutations in the indicated genes on the ability of the resulting strains to form spores, as assessed by comparing the titers of heat-resistant spores to the total (viable) cell count. The results presented are the averages of three independent experiments, and are shown, for each mutant, as the percentage of the sporulation level obtained for the congenic reference (Spo⁺) strain MB24 in DSM. (C) Quantitative analysis of the stage at which the indicated mutations affect sporulation, as determined by fluorescence (following staining with FM 4-64) and phase-contrast microscopy of cells collected at hour 8 of sporulation in DSM. The wild type is shown as a reference. Stage II, septation completed; stage III, engulfment completed.

Fig 5

ylmC, ytaF, and ylzA are expressed during spore development. (A) Expression of transcriptional fusions of ylmC, ytaF, and ylzA to gfp, inserted at the nonessential amyE locus of a wild-type strain or the indicated mutant, during sporulation in DSM. P_ylzA* refers to a fusion bearing two substitutions in the −10 promoter element of a putative σ^F-dependent promoter (see the text for details). Samples were withdrawn from cultures at the represented times (in hours) after the onset of sporulation (or T0). The cells were stained with the membrane dye FM4-64 (middle column for each strain), prior to observation by fluorescence microscopy. The arrowheads point to the position of the mother cell (white) and forespore (yellow) compartments in the selected cells. Bar, 2 μm. (B) Schematic representation of the expression patterns found for the ylmC, ytaF, and ylzA genes; pale green denotes weak expression, and the darker green indicates stronger accumulation of GFP.