Disruption of Two-component System LytSR Affects Forespore Engulfment in Bacillus thuringiensis

Abstract

Two-component regulatory systems (TCSs) play pivotal roles in bacteria sensing many different stimuli from environment. Here, we investigated the role of the LytSR TCS in spore formation in Bacillus thuringiensis (Bt) subsp. kurstaki HD73. lacZ gene fusions revealed that the transcription of the downstream genes, lrgAB, encoding two putative membrane-associated proteins, is regulated by LytSR. The sporulation efficiency of a lytSR mutant was significantly lower than that of wild-type HD73. A confocal microscopic analysis demonstrated that LytSR modulates the process of forespore engulfment. Moreover, the transcription of the lytSR operon is regulated by the mother-cell transcription factor SigE, whereas the transcription of the sporulation gene spoIIP was reduced in the lytSR mutant, as demonstrated with a β-galactosidase activity assay. These results suggest that LytSR modulates forespore engulfment by affecting the transcription of the spoIIP gene in Bt.

SpoVG Is Necessary for Sporulation in Bacillus anthracis

“…Confocal laser-scanning microscopy was performed as previously described [23]. Briefly, cells were stained with the membrane dye FM4-64 (final concentration 100 µM; Molecular Probes, Inc., Eugene, OR, USA).…”

Cell death dependent on holins LrgAB repressed by a novel ArsR family regulator CdsR

“…A comparison of the amino acid sequences of available LrgA homologs revealed that LrgA (HD73_RS29150) of B. thuringiensis HD73 shares 62%, 99%, and 100% similarity with LrgA (YsbA, BSU28910) of B. subtilis 168, LrgA (BC5439) of B. cereus ATCC 14579, and LrgA (BA5690) of B. anthracis Ames, respectively. In our previous study, we found that deletion of lrgAB did not significantly affect sporulation efficiency by the method of heating cells for elimination of non-sporulating cells [44]. In this study, we directly counted the number of sporulating cells in the microscopy images.…”

“…A 1032-bp fragment preceding the start codon of cdsR (cdsR fragment A) was amplified by PCR, utilizing HD73 genomic DNA as the template and cdsR-AF and cdsR-AR as the forward and reverse primers, respectively. Furthermore, the primers cdsR-BF and cdsR-BR were used to amplify a 1015-bp fragment downstream of the 96th codon of cdsR (cdsR fragment B), whereas the primers cdsR-KmF and cdsR-KmR were utilized to amplify a 1473-bp kanamycin resistance gene cassette (kana) derived from pMAD-ΔlrgAB [44] plasmid. The cdsR A, kana, and cdsR B fragments were ligated through overlapping PCR using the primers cdsR-AF and cdsR-BR.…”

“…The PlrgAB and gfp fragments were used as templates to jointly amplify the PlrgAB-gfp gene fusion fragment (1226-bp) using the PlrgAB-gfp-F/lrgAB-gfp-R primers. The resulting PCR product was inserted into the plasmid pHT304 through the EcoRI and PstI sites, leading to the generation of the plasmid pHT304-PlrgAB-gfp, which, in turn, was introduced into both the HD73 and ΔlrgAB mutant strains [44].…”

Identification and Functional Characterization of Two Homologous SpoVS Proteins Involved in Sporulation of Bacillus thuringiensis

“…The vital membrane dye FM 4-64 (Molecular Probes, Inc., Eugene, OR, USA) or MitoTracker green FM (ThermoFisher, USA) was dissolved in dimethyl sulfoxide ( 35 ). To assess polar septa and engulfment, 1-ml amounts of cells cultured to a designated time point in SSM medium were pelleted and resuspended in 0.02 to 0.05 ml H 2 O. Aliquots (2 μl) of the cell suspensions were placed on slides, stained with FM 4-64 (100 μM) and MitoTracker green FM (MTG; 100 nM) for 1 min ( 47 ), and then scanned with a confocal laser scanning microscope (Leica TCS SL; Leica Microsystems, Wetzlar, Germany) ( 24 ). Each strain was scanned independently at least three times, and each scan was then viewed in at least five fields.…”