To monitor inaccuracy in gene expression in living cells, we designed an experimental system in the bacterium Bacillus subtilis whereby spontaneous errors can be visualized and quantified at a single-cell level. Our strategy was to introduce mutations into a chromosomally encoded gfp allele, such that errors in protein production are reported in real time by the formation of fluorescent GFP molecules. The data reveal that the amount of errors can greatly exceed previous estimates, and that the error rate increases dramatically at lower temperatures and during stationary phase. Furthermore, we demonstrate that when facing an antibiotic threat, an increase in error level is sufficient to allow survival of bacteria carrying a mutated antibiotic-resistance gene. We propose that bacterial gene expression is error prone, frequently yielding protein molecules that differ slightly from the sequence specified by their DNA, thus generating a cellular reservoir of nonidentical protein molecules. This variation may be a key factor in increasing bacterial fitness, expanding the capability of an isogenic population to face environmental challenges.Bacillus subtilis | translational errors | variations in living cells | translation fidelity D NA is duplicated with remarkable fidelity to ensure that accurate genetic information is transmitted from one generation to the next. This information is passed from DNA to RNA and from RNA to protein during gene expression; however, the accuracy of these downstream events is relatively less understood. Although RNA and proteins are generally considered short-lived noninherited molecules, several diseases are now known to arise from errors occurring during transcription and translation (1-3), implying that faithful transfer of genetic information from DNA to proteins is crucial for maintaining proper cellular functions.

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Summary We have previously described the existence of membranous nanotubes, bridging adjacent bacteria, facilitating intercellular trafficking of nutrients, cytoplasmic proteins, and even plasmids, yet components enabling their biogenesis remain elusive. Here we reveal the identity of a molecular apparatus providing a platform for nanotube biogenesis. Using Bacillus subtilis ( Bs ), we demonstrate that conserved components of the flagellar export apparatus (FliO, FliP, FliQ, FliR, FlhB, and FlhA), designated CORE, dually serve for flagellum and nanotube assembly. Mutants lacking CORE genes, but not other flagellar components, are deficient in both nanotube production and the associated intercellular molecular trafficking. In accord, CORE components are located at sites of nanotube emergence. Deleting CORE s of distinct species established that CORE-mediated nanotube formation is widespread. Furthermore, exogenous CORE s from diverse species could restore nanotube generation and functionality in Bs lacking endogenous CORE . Our results demonstrate that the CORE-derived nanotube is a ubiquitous organelle that facilitates intercellular molecular trade across the bacterial kingdom.

Bacteria have developed various mechanisms by which they sense, interact, and kill other bacteria, in an attempt to outcompete one another and survive. Here we show that Bacillus subtilis can kill and prey on Bacillus megaterium. We find that Bacillus subtilis rapidly inhibits Bacillus megaterium growth by delivering the tRNase toxin WapA. Furthermore, utilizing the methionine analogue L-azidohomoalanine as a nutrient reporter, we provide evidence of nutrient extraction from Bacillus megaterium by Bacillus subtilis. Toxin delivery and nutrient extraction occur in a contact-dependent manner, and both activities are abolished in the absence of the phosphodiestrase YmdB, shown previously to mediate intercellular nanotube formation. Furthermore, we detect the localization of WapA molecules to nanotubes. Thus, we propose that Bacillus subtilis utilizes the same nanotube apparatus in a bidirectional manner, delivering toxin and acquiring beneficial cargo, thereby maximally exploiting potential niche resources.

Highlights d Host-attached EPEC extract nutrients directly from infected host cytoplasm d Host nutrient extraction (HNE) requires CORE proteins of the EPEC injectisome d CORE supports the formation of protruding membranous nanotubes to enable HNE d Distant CORE genes restored HNE capacity in EPEC lacking its native CORE

Bacterial spores can remain dormant for years, yet they possess a remarkable potential to rapidly resume a vegetative life form. Here, we identified a distinct phase at the onset of spore outgrowth, designated the ripening period. This transition phase is exploited by the germinating spore for molecular reorganization toward elongation and subsequent cell division. We have previously shown that spores of different ages, kept under various temperatures, harbor dissimilar molecular reservoirs (E. Segev, Y. Smith, and S. Ben-Yehuda, Cell 148:139 -149, 2012). Utilizing this phenomenon, we observed that the length of the ripening period can vary according to the spore molecular content. Importantly, the duration of the ripening period was found to correlate with the initial spore rRNA content and the kinetics of rRNA accumulation upon exiting dormancy. Further, the synthesis of the ribosomal protein RplA and the degradation of the spore-specific protein SspA also correlated with the duration of the ripening period. Our data suggest that the spore molecular cargo determines the extent of the ripening period, a potentially crucial phase for a germinating spore in obtaining limited resources during revival.

SummaryWhen grown on a solid surface, bacteria form highly organized colonies, yet little is known about the earliest stages of colony establishment. Following Bacillus subtilis colony development from a single progenitor cell, a sequence of highly ordered spatiotemporal events was revealed. Colony was initiated by the formation of leading-cell chains, deriving from the colony center and extending in multiple directions, typically in a “Y-shaped” structure. By eradicating particular cells during these early stages, we could influence the shape of the resulting colony and demonstrate that Y-arm extension defines colony size. A mutant in ymdB encoding a phosphodiesterase displayed unordered developmental patterns, indicating a role in guiding these initial events. Finally, we provide evidence that intercellular nanotubes contribute to proper colony formation. In summary, we reveal a “construction plan” for building a colony and provide the initial molecular basis for this process.

Linkages between the outer membrane of Gram-negative bacteria and the peptidoglycan layer are crucial for the maintenance of cellular integrity and enable survival in challenging environments1–5. The function of the outer membrane is dependent on outer membrane proteins (OMPs), which are inserted into the membrane by the β-barrel assembly machine6,7 (BAM). Growing Escherichia coli cells segregate old OMPs towards the poles by a process known as binary partitioning, the basis of which is unknown8. Here we demonstrate that peptidoglycan underpins the spatiotemporal organization of OMPs. Mature, tetrapeptide-rich peptidoglycan binds to BAM components and suppresses OMP foldase activity. Nascent peptidoglycan, which is enriched in pentapeptides and concentrated at septa9, associates with BAM poorly and has little effect on its activity, leading to preferential insertion of OMPs at division sites. The synchronization of OMP biogenesis with cell wall growth results in the binary partitioning of OMPs as cells divide. Our study reveals that Gram-negative bacteria coordinate the assembly of two major cell envelope layers by rendering OMP biogenesis responsive to peptidoglycan maturation, a potential vulnerability that could be exploited in future antibiotic design.