Phage-encoded Shiga toxin (Stx) acts as a bacterial defense against the eukaryotic predator Tetrahymena thermophila. It is unknown how Stx enters Tetrahymena protozoa or how it kills them. Tetrahymena protozoa are phagocytotic; hence, Stx could gain entry to the cytoplasm through the oral apparatus or via endocytosis. We find that Stx2 can kill T. thermophila protozoa that lack an oral apparatus, indicating that Stx2 can enter these cells via endocytosis. As opposed to the lack of effect on mammalian phagocytes, Stx2 produced by bacteria encapsulated within phagocytotic vesicles is also capable of killing Tetrahymena. Addition of an excess of the carbohydrate binding subunits of Stx2 (StxB) and/or ricin (ricin B) blocks Stx2 cytotoxicity. Thus, regardless of whether Stx2 enters the cytoplasm by endocytosis or from the phagocytotic vesicle, this transport is mediated by a putative glycoconjugate receptor. Bacteriophage-mediated lysis of Stx-encoding bacteria is necessary for Stx toxicity in Tetrahymena; i.e., toxin released as a consequence of digestion of bacteria by Tetrahymena is harmless to the cell. This finding provides a rationale as to why the genes encoding Stx are found almost exclusively on bacteriophages; Stx must be released from the bacteria prior to the digestion of the cell, or it will not be able to exert its cytotoxic effect. It also suggests a reason why other bacterial exotoxins are also found only on temperate bacteriophages. Incubation of Tetrahymena with purified Stx2 decreases total protein synthesis. This finding indicates that, similar to mammalian cells, Stx2 kills Tetrahymena by inactivating its ribosomes.

The ferric uptake regulator (Fur) is the global iron biosensor in many bacteria. Fur functions as an iron-dependent transcriptional repressor for most of its regulated genes. There are a few examples where holo-Fur activates transcription, either directly or indirectly. Recent studies suggest that apo-Fur might also act as a positive regulator and that, besides iron metabolism, the Fur regulon might encompass other biological processes such as DNA synthesis, energy metabolism, and biofilm formation. Here, we obtained a genomic view of the Fur regulatory network in Bacillus subtilis using chromatin immunoprecipitation sequencing (ChIP-seq). Besides the known Fur target sites, 70 putative DNA binding sites were identified, and the vast majority had higher occupancy under iron-sufficient conditions. Among the new sites detected, a Fur binding site in the promoter region of the catDE operon is of particular interest. This operon, encoding catechol 2,3-dioxygenase, is critical for catechol degradation and is under negative regulation of CatR and YodB. These three repressors (Fur, CatR, and YodB) function cooperatively to regulate the transcription of catDE, with Fur functioning as a sensor of iron limitation and CatR as the major sensor of catechol stress. Genetic analysis suggests that CatDE is involved in metabolism of the catecholate siderophore bacillibactin, particularly when bacillibactin is constitutively produced and accumulates intracellularly, potentially generating endogenous toxic catechol derivatives. This study documents a role for catechol degradation in bacillibactin metabolism and provides evidence that catechol 2,3-dioxygenase can detoxify endogenously produced catechol substrates in addition to its more widely studied role in biodegradation of environmental aromatic compounds and pollutants.IMPORTANCE Many bacteria synthesize high-affinity iron chelators (siderophores). Siderophore-mediated iron acquisition is an efficient and widely utilized strategy for bacteria to meet their cellular iron requirements. One prominent class of siderophores uses catecholate groups to chelate iron. B. subtilis bacillibactin, structurally similar to enterobactin (made by enteric bacteria), is a triscatecholate siderophore that is hydrolyzed to monomeric units after import to release iron. However, the ultimate fates of these catechol compounds and their potential toxicities have not been defined previously. We performed genome-wide identification of Fur binding sites in vivo and uncovered a connection between catechol degradation and bacillibactin metabolism in B. subtilis Besides its role in the detoxification of environmental catechols, the catechol 2,3-dioxygenase encoded by catDE also protects cells from intoxication by endogenous bacillibactin-derived catechol metabolites under iron-limited conditions. These findings shed light on the degradation pathway and precursor recycling of the catecholate siderophores.

Osmotic stress is a significant physical challenge for free-living cells. Cells from all three domains of life maintain viability during osmotic stress by tightly regulating the major cellular osmolyte potassium (K+) and by import or synthesis of compatible solutes. It has been widely established that in response to high salt stress, many bacteria transiently accumulate high levels of K+, leading to bacteriostasis, with growth resuming only when compatible solutes accumulate and K+ levels are restored to biocompatible levels. Using Bacillus subtilis as a model system, we provide evidence that K+ fluxes perturb Mg2+ homeostasis: import of K+ upon osmotic upshift is correlated with Mg2+ efflux, and Mg2+ reimport is critical for adaptation. The transient growth inhibition resulting from hyperosmotic stress is coincident with loss of Mg2+ and a decrease in protein translation. Conversely, the reimport of Mg2+ is a limiting factor during resumption of growth. Furthermore, we show the essential signaling dinucleotide cyclic di-AMP fluctuates dynamically in coordination with Mg2+ and K+ levels, consistent with the proposal that cyclic di-AMP orchestrates the cellular response to osmotic stress. IMPORTANCE Environments with high concentrations of salt or other solutes impose an osmotic stress on cells, ultimately limiting viability by dehydration of the cytosol. A very common cellular response to high osmolarity is to immediately import high levels of potassium ion (K+), which helps prevent dehydration and allows time for the import or synthesis of biocompatible solutes that allow a resumption of growth. Here, using Bacillus subtilis as a model, we demonstrate that concomitant with K+ import there is a large reduction in intracellular magnesium (Mg2+) mediated by specific efflux pumps. Further, it is the reimport of Mg2+ that is rate-limiting for the resumption of growth. These coordinated fluxes of K+ and Mg2+ are orchestrated by cyclic-di-AMP, an essential second messenger in Firmicutes. These findings amend the conventional model for osmoadaptation and reveal that Mg2+ limitation is the proximal cause of the bacteriostasis that precedes resumption of growth.

Separate germline and somatic genomes are found in numerous lineages across the eukaryotic tree of life, often separated into distinct tissues (e.g., in plants, animals, and fungi) or distinct nuclei sharing a common cytoplasm (e.g., in ciliates and some foraminifera). In ciliates, germline-limited (i.e., micronuclear-specific) DNA is eliminated during the development of a new somatic (i.e., macronuclear) genome in a process that is tightly linked to large-scale genome rearrangements, such as deletions and reordering of protein-coding sequences. Most studies of germline genome architecture in ciliates have focused on the model ciliates Oxytricha trifallax, Paramecium tetraurelia, and Tetrahymena thermophila, for which the complete germline genome sequences are known. Outside of these model taxa, only a few dozen germline loci have been characterized from a limited number of cultivable species, which is likely due to difficulties in obtaining sufficient quantities of "purified" germline DNA in these taxa. Combining single-cell transcriptomics and genomics, we have overcome these limitations and provide the first insights into the structure of the germline genome of the ciliate Chilodonella uncinata, a member of the understudied class Phyllopharyngea Our analyses reveal the following: (i) large gene families contain a disproportionate number of genes from scrambled germline loci; (ii) germline-soma boundaries in the germline genome are demarcated by substantial shifts in GC content; (iii) single-cell omics techniques provide large-scale quality germline genome data with limited effort, at least for ciliates with extensively fragmented somatic genomes. Our approach provides an efficient means to understand better the evolution of genome rearrangements between germline and soma in ciliates.IMPORTANCE Our understanding of the distinctions between germline and somatic genomes in ciliates has largely relied on studies of a few model genera (e.g., Oxytricha, Paramecium, Tetrahymena). We have used single-cell omics to explore germline-soma distinctions in the ciliate Chilodonella uncinata, which likely diverged from the better-studied ciliates ~700 million years ago. The analyses presented here indicate that developmentally regulated genome rearrangements between germline and soma are demarcated by rapid transitions in local GC composition and lead to diversification of protein families. The approaches used here provide the basis for future work aimed at discerning the evolutionary impacts of germline-soma distinctions among diverse ciliates.

How to achieve protein diversity by genome and transcriptome processing is essential for organismal complexity and adaptation. The present work identifies that the macronuclear genome of Halteria grandinella, a cosmopolitan unicellular eukaryote, is composed almost entirely of gene-sized nanochromosomes with extremely short nongenic regions. This challenges our usual understanding of chromosomal structure and suggests the possibility of novel mechvanisms in transcriptional regulation. Comprehensive analysis of multiple data sets reveals that Halteria transcription dynamics are influenced by: (i) nonuniform nanochromosome copy numbers correlated with gene-expression level; (ii) dynamic alterations at both the DNA and RNA levels, including alternative internal eliminated sequence (IES) deletions during macronucleus formation and large-scale alternative splicing in transcript maturation; and (iii) extremely short 5' and 3' untranslated regions (UTRs) and universal TATA box-like motifs in the compact 5' subtelomeric regions of most chromosomes. This study broadens the view of ciliate biology and the evolution of unicellular eukaryotes, and identifies Halteria as one of the most compact known eukaryotic genomes, indicating that complex cell structure does not require complex gene architecture.