Division of labour and terminal differentiation in a novel Bacillus thuringiensis strain

Abstract

A major challenge in bacterial developmental biology has been to understand the mechanisms underlying cell fate decisions. Some differentiated cell types display cooperative behaviour. Cooperation is one of the greatest mysteries of evolutionary biology and microbes have been considered as an excellent system for experimentally testing evolution theories. Bacillus thuringiensis (Bt) is a spore-forming bacterium, which is genetically closely related to B. anthracis, the agent of anthrax, and to B. cereus, an opportunistic human pathogen. The defining feature that distinguishes Bt from its relatives is its ability to produce crystal inclusions in the sporulating cells. These toxins are solubilized after ingestion and are cooperative public goods in insect hosts. In this study, we describe a Bt strain LM1212 that presents the unique ability to terminally differentiate into crystal producers and spore formers. Transcriptional analysis based on lacZ and gfp reporter genes suggested that this phenotype is the consequence of a new type of cell differentiation associated with a novel regulation mode of cry gene expression. The differentiating crystal-producer phenotype has higher spore productivity than a typical Bt strain and is better able to compete with Cry toxin null 'cheaters'. Potentially, this division of labour provides additional fitness benefits in terms of spore viability or durability of Cry toxin.

Figure 1

Differentiation of crystal producers and spore formers in LM1212. The spores and crystals appear in distinct cells in LM1212. Pictures of (a) optical micrograph and (b) transmission electron microscopy (TEM). (c) TEM reveals the crystal lattice in LM1212 inclusions. S, spore; C, crystal toxin.

Figure 2

SDS–polyacrylamide gel electrophoresis (PAGE) and mass spectrometry (MS) analyses of LM1212 crystals. Purified LM1212 crystals were analysed by 10% SDS–PAGE. Eight bands (1–8, indicated by arrows) were selected for identification by matrix-assisted laser desorption/ionization–time of flight MS. All seven expected Cry-like proteins but no Cyt-like protein were detected. In addition, a NT32-like protein (4) and a putative protein (8) were found as components of LM1212 crystals. M, protein standard; Mix, mixture of truncated Cry-like proteins. Numbers in parentheses show peptide coverage (%) in MS analysis.

Figure 3

Transcriptional analysis of LM1212 cry genes. (a and c) Sequence and primer extension analyses of cry32Wa1 and cry35-like promoters. Primers used for cloning the promoter regions are underlined and primers for extension experiments are indicated by black italics. Putative translation start codons are boxed and transcription start sites (TSSs) are noted with bold lowercases. The putative −10 and −35 regions of the promoters are indicated by dashed lines. The sequence extension results are noted above the gels and TSSs are shown with high black lowercases. (b and d) The activity of cry32Wa1 and cry35-like promoters as assessed by lacZ fusions. The columns on the right of the diagrams indicate the β-galactosidase activity of T20 (LM1212: black columns and HD73: white columns). Data are an average of three independent assays. Error bar is the s.d. Cells were cultured in SSM medium. T0 is the end of logarithmic growth and Tn means n hours after T0.

Figure 4

The activity of LM1212 cry promoters is restricted to non-sporulating cells in both LM1212 and HD73 strains. We used a gfp reporter gene to assess the activity of the P32 and P35 promoters in LM1212 (a and b) and in HD73 (c and d). The activity of the sporulation-dependent cry gene promoter P_cry1Ac is restricted to the sporulating cells in LM1212 and HD73 (e and f). Non-sporulating cells in HD73 are indicated by yellow arrows. Left: GFP/FM-4-64 overlay. Right: bright-field image. Sporulating cells are indicated by arrows and green fluorescent protein-expressing cells are indicated by arrow heads. Cells were grown in SSM medium until T20 with erythromycin. The cell walls were stained with FM-4-64.

Figure 5

Productivity of LM1212 and relative fitness in competition with Bt strains. (A) The spore yield of LM1212 is intermediate between that of a canonical Bt strain and a cry null mutant, data are means±s.e. Different superscripts indicate significantly different means, according to post hoc treatment contrasts, after fitting generalized linear models. (B) LM(p35′Z) has a positive frequency-dependent fitness, but outcompetes HD73(p35′Z) over a wide range of initial conditions. (C) A Cry null strain, HD73Cry⁻(p35′Z), can outcompete LM(p35′Z), except when rare. Spores were heat treated at 65 °C for 25 min before plating. Lines are fitted statistical models.