Acquisition of Phage Sensitivity by Bacteria through Exchange of Phage Receptors.

Bacteriophages (phages) typically exhibit a narrow host range, yet they tremendously impact horizontal gene transfer (HGT). Here, we investigate phage dynamics in communities harboring phage-resistant (R) and sensitive (S) bacteria, a common scenario in nature. Using Bacillus subtilis and its lytic phage SPP1, we demonstrate that R cells, lacking SPP1 receptor, can be lysed by SPP1 when co-cultured with S cells. This unanticipated lysis was triggered in part by phage lytic enzymes released from nearby infected cells. Strikingly, we discovered that occasionally phages can invade R cells, a phenomenon we termed acquisition of sensitivity (ASEN). We found that ASEN is mediated by R cells transiently gaining phage attachment molecules from neighboring S cells and provide evidence that this molecular exchange is driven by membrane vesicles. Exchange of phage attachment molecules could even occur in an interspecies fashion, enabling phage adsorption to non-host species, providing an unexplored route for HGT. VIDEO ABSTRACT.

Bacteria elicit a phage tolerance response subsequent to infection of their neighbors.

Appearance of plaques on a bacterial lawn is a sign of successive rounds of bacteriophage infection. Yet, mechanisms evolved by bacteria to limit plaque spread have been hardly explored. Here, we investigated the dynamics of plaque development by lytic phages infecting the bacterium Bacillus subtilis. We report that plaque expansion is followed by a constriction phase owing to bacterial growth into the plaque zone. This phenomenon exposed an adaptive process, herein termed "phage tolerance response", elicited by non-infected bacteria upon sensing infection of their neighbors. The temporary phage tolerance is executed by the stress-response RNA polymerase sigma factor σX (SigX). Artificial expression of SigX prior to phage attack largely eliminates infection. SigX tolerance is primarily conferred by activation of the dlt operon, encoding enzymes that catalyze D-alanylation of cell wall teichoic acid polymers, the major attachment sites for phages infecting Gram-positive bacteria. D-alanylation impedes phage binding and hence infection, thus enabling the uninfected bacteria to form a protective shield opposing phage spread.

A mutant bacteriophage evolved to infect resistant bacteria gained a broader host range.

Bacteriophages (phages) are the most abundant entities in nature, yet little is known about their capacity to acquire new hosts and invade new niches. By exploiting the Gram-positive soil bacterium Bacillus subtilis (B. subtilis) and its lytic phage SPO1 as a model, we followed the coevolution of bacteria and phages. After infection, phage-resistant bacteria were readily isolated. These bacteria were defective in production of glycosylated wall teichoic acid (WTA) polymers that served as SPO1 receptor. Subsequently, a SPO1 mutant phage that could infect the resistant bacteria evolved. The emerging phage contained mutations in two genes, encoding the baseplate and fibers required for host attachment. Remarkably, the mutant phage gained the capacity to infect non-host Bacillus species that are not infected by the wild-type phage. We provide evidence that the evolved phage lost its dependency on the species-specific glycosylation pattern of WTA polymers. Instead, the mutant phage gained the capacity to directly adhere to the WTA backbone, conserved among different species, thereby crossing the species barrier.

Phage-Resistant Bacteria Reveal a Role for Potassium in Root Colonization.

Bacteriophage predation is an important factor in bacterial community dynamics and evolution. Phage-bacterium interaction has mainly been studied in lab cultures, while dynamics in natural habitats, and especially in the plant root niche, are underexplored. To better understand this process, we characterized infection of the soil bacterium Bacillus subtilis NCBI 3610 by the lytic phage SPO1 during growth in LB medium and compared it to root colonization. Resistance in vitro was primarily through modification of the phage receptor. However, this type of resistance reduced the ability to colonize the root. From a line that survived phage infection while retaining the ability to colonize the root, we identified a new phage resistance mechanism involving potassium (K+) ion influx modulation and enhanced biofilm formation. Furthermore, we show that potassium serves as a stimulator of root colonization among diverse growth-promoting bacilli species, with implications for plant health. IMPORTANCE Bacteriophage predation is an important factor in bacterial community dynamics and evolution. Phage-bacterium interaction has mainly been studied in lab cultures, while dynamics in natural habitats, and especially in the plant root niche, are underexplored. To better understand this process, we characterized infection of the soil bacterium Bacillus subtilis NCBI 3610 by the lytic phage SPO1 during growth in LB medium and compared it to root colonization. Resistance in vitro was primarily through modification of the phage receptor. However, this type of resistance reduced the ability to colonize the root. From a line that survived phage infection while retaining the ability to colonize the root, we identified a new phage resistance mechanism involving potassium (K+) ion influx modulation and enhanced biofilm formation. Furthermore, we show that potassium serves as a stimulator of root colonization among diverse growth-promoting bacilli species, with implications for plant health.

Plant immune system activation is necessary for efficient root colonization by auxin-secreting beneficial bacteria.

Although plant roots encounter a plethora of microorganisms in the surrounding soil, at the rhizosphere, plants exert selective forces on their bacterial colonizers. Unlike immune recognition of pathogenic bacteria, the mechanisms by which beneficial bacteria are selected and how they interact with the plant immune system are not well understood. To better understand this process, we studied the interaction of auxin-producing Bacillus velezensis FZB42 with Arabidopsis roots and found that activation of the plant immune system is necessary for efficient bacterial colonization and auxin secretion. A feedback loop is established in which bacterial colonization triggers an immune reaction and production of reactive oxygen species, which, in turn, stimulate auxin production by the bacteria. Auxin promotes bacterial survival and efficient root colonization, allowing the bacteria to inhibit fungal infection and promote plant health. Thus, a feedback loop between bacteria and the plant immune system promotes the fitness of both partners.

Interspecies nutrient extraction and toxin delivery between bacteria.

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.Bacteria can exchange nutrients and macromolecules through tubular membranous structures called nanotubes. Here, the authors show that Bacillus subtilis can kill and prey on Bacillus megaterium by delivering a toxin and extracting nutrients in a nanotube-dependent manner.

Type 2 diabetes and congenital hyperinsulinism cause DNA double-strand breaks and p53 activity in ß cells.

ß cell failure in type 2 diabetes (T2D) is associated with hyperglycemia, but the mechanisms are not fully understood. Congenital hyperinsulinism caused by glucokinase mutations (GCK-CHI) is associated with ß cell replication and apoptosis. Here, we show that genetic activation of ß cell glucokinase, initially triggering replication, causes apoptosis associated with DNA double-strand breaks and activation of the tumor suppressor p53. ATP-sensitive potassium channels (KATP channels) and calcineurin mediate this toxic effect. Toxicity of long-term glucokinase overactivity was confirmed by finding late-onset diabetes in older members of a GCK-CHI family. Glucagon-like peptide-1 (GLP-1) mimetic treatment or p53 deletion rescues ß cells from glucokinase-induced death, but only GLP-1 analog rescues ß cell function. DNA damage and p53 activity in T2D suggest shared mechanisms of ß cell failure in hyperglycemia and CHI. Our results reveal membrane depolarization via KATP channels, calcineurin signaling, DNA breaks, and p53 as determinants of ß cell glucotoxicity and suggest pharmacological approaches to enhance ß cell survival in diabetes.