Construction and Analysis of Two Genome-Scale Deletion Libraries for Bacillus subtilis

Summary Surface properties, such as adhesion and hydrophobicity, constrain dispersal of bacterial spores in the environment. In Bacillus subtilis, these properties are influenced by the outermost layer of the spore, the crust. Previous work has shown that two clusters, cotVWXYZ and cgeAB, encode the protein components of the crust. Here, we characterize the respective roles of these genes in surface properties using Bacterial Adherence to Hydrocarbons assays, negative staining of polysaccharides by India ink and Transmission Electron Microscopy. We showed that inactivation of crust genes caused increases in spore relative hydrophobicity, disrupted the spore polysaccharide layer, and impaired crust structure and attachment to the rest of the coat. We also found that cotO, previously identified for its role in outer coat formation, is necessary for proper encasement of the spore by the crust. In parallel, we conducted fluorescence microscopy experiments to determine the full network of genetic dependencies for subcellular localization of crust proteins. We determined that CotZ is required for the localization of most crust proteins, while CgeA is at the bottom of the genetic interaction hierarchy.

Section: Methodsmentioning

confidence: 99%

Contributions of crust proteins to spore surface properties in Bacillus subtilis

Shuster

Khemmani

Abe

et al. 2019

“…Sporulation efficiency was determined in 24–30 h cultures as the total number of heat‐resistant (80°C for 20 min) colony‐forming units (CFUs) compared to wild‐type heat‐resistant CFUs. Insertion‐deletion mutants were from the Bacillus knock‐out (BKE) collection (Koo et al ., ) or were generated by isothermal assembly (Gibson, ) of PCR products followed by direct transformation into B . subtilis .…”

Section: Methodsmentioning

confidence: 99%

The Bacillus subtilis germinant receptor GerA triggers premature germination in response to morphological defects during sporulation

Ramírez-Guadiana

Meeske

Wang

et al. 2017

Self Cite

During sporulation in Bacillus subtilis, germinant receptors assemble in the inner membrane of the developing spore. In response to specific nutrients these receptors trigger germination and outgrowth. In a transposon-sequencing screen, we serendipitously discovered that loss of function mutations in the gerA receptor partially suppress the phenotypes of >25 sporulation mutants. Most of these mutants have modest defects in the assembly of the spore protective layers that are exacerbated in the presence of a functional GerA receptor. Several lines of evidence indicate that these mutants inappropriately trigger activation of GerA during sporulation resulting in premature germination. These findings led us to discover that up to 8% of wild-type sporulating cells trigger premature germination during differentiation in a GerA-dependent manner. This phenomenon was observed in domesticated and undomesticated wild-type strains sporulating in liquid and on solid media. Our data indicate that the GerA receptor is poised on a knife's edge during spore development. We propose that this sensitized state ensures a rapid response to nutrient availability, but also elicits premature germination of spores with improperly assembled protective layers resulting in the elimination of even mildly defective individuals from the population.

“…Additionally, metal starvation was induced by the addition of EDTA at the concentrations indicated. Markerless in-frame deletion mutants were constructed from BKE strains as described previously (Koo et al, 2017). Briefly, BKE strains were acquired from the Bacillus Genetic Stock Center, chromosomal DNA was extracted, and the mutation, containing an erm cassette, was transformed into our wild-type (WT) strain 168.…”

Section: Strains and Growth Conditionsmentioning

confidence: 99%

Bacillus subtilis FolE is sustained by the ZagA zinc metallochaperone and the alarmone ZTP under conditions of zinc deficiency

Chandrangsu

Huang

Gaballa

et al. 2019

Summary Bacteria tightly regulate intracellular zinc levels to ensure sufficient zinc to support essential functions, while preventing toxicity. The bacterial response to zinc limitation includes the expression of putative zinc metallochaperones belonging to subfamily 1 of the COG0523 family of G3E GTPases. However, the client proteins and the metabolic processes served by these chaperones are unclear. Here, we demonstrate that the Bacillus subtilis YciC zinc metallochaperone (here renamed ZagA for ZTP activated GTPase A) supports de novo folate biosynthesis under conditions of zinc limitation, and interacts directly with the zinc‐dependent GTP cyclohydrolase IA, FolE (GCYH‐IA). Furthermore, we identify a role for the alarmone ZTP, a modified purine biosynthesis intermediate, in the response to zinc limitation. ZTP, a signal of 10‐formyl‐tetrahydrofolate (10f‐THF) deficiency in bacteria, transiently accumulates as FolE begins to fail, stimulates the interaction between ZagA and FolE, and thereby helps to sustain folate synthesis despite declining zinc availability.