Cell division machinery drives cell-specific gene activation during differentiation in Bacillus subtilis.

When faced with starvation, the bacterium Bacillus subtilis transforms itself into a dormant cell type called a "spore". Sporulation initiates with an asymmetric division event, which requires the relocation of the core divisome components FtsA and FtsZ, after which the sigma factor σF is exclusively activated in the smaller daughter cell. Compartment-specific activation of σF requires the SpoIIE phosphatase, which displays a biased localization on one side of the asymmetric division septum and associates with the structural protein DivIVA, but the mechanism by which this preferential localization is achieved is unclear. Here, we isolated a variant of DivIVA that indiscriminately activates σF in both daughter cells due to promiscuous localization of SpoIIE, which was corrected by overproduction of FtsA and FtsZ. We propose that the core components of the redeployed cell division machinery drive the asymmetric localization of DivIVA and SpoIIE to trigger the initiation of the sporulation program.

Dual-function AzuCR RNA modulates carbon metabolism.

SignificanceWhile most small, regulatory RNAs are thought to be "noncoding," a few have been found to also encode a small protein. Here we describe a 164-nucleotide RNA that encodes a 28-amino acid, amphipathic protein, which interacts with aerobic glycerol-3-phosphate dehydrogenase and increases dehydrogenase activity but also base pairs with two mRNAs to reduce expression. The coding and base-pairing sequences overlap, and the two regulatory functions compete.

The ubiquitous yybP-ykoY riboswitch is a manganese-responsive regulatory element.

The highly structured, cis-encoded RNA elements known as riboswitches modify gene expression upon binding a wide range of molecules. The yybP-ykoY motif was one of the most broadly distributed and numerous bacterial riboswitches for which the cognate ligand was unknown. Using a combination of in vivo reporter and in vitro expression assays, equilibrium dialysis, and northern analysis, we show that the yybP-ykoY motif responds directly to manganese ions in both Escherichia coli and Bacillus subtilis. The identification of the yybP-ykoY motif as a manganese ion sensor suggests that the genes that are preceded by this motif and encode a diverse set of poorly characterized membrane proteins have roles in metal homeostasis.

A 2-dimensional ratchet model describes assembly initiation of a specialized bacterial cell surface.

Bacterial spores are dormant cells that are encased in a thick protein shell, the "coat," which participates in protecting the organism's DNA from environmental insults. The coat is composed of dozens of proteins that assemble in an orchestrated fashion during sporulation. In Bacillus subtilis, 2 proteins initiate coat assembly: SpoVM, which preferentially binds to micron-scale convex membranes and marks the surface of the developing spore as the site for coat assembly; and SpoIVA, a structural protein recruited by SpoVM that uses ATP hydrolysis to drive its irreversible polymerization around the developing spore. Here, we describe the initiation of coat assembly by SpoVM and SpoIVA. Using single-molecule fluorescence microscopy in vivo in sporulating cells and in vitro on synthetic spores, we report that SpoVM's localization is primarily driven by a lower off-rate on membranes of preferred curvature in the absence of other coat proteins. Recruitment and polymerization of SpoIVA results in the entrapment of SpoVM on the forespore surface. Using experimentally derived reaction parameters, we show that a 2-dimensional ratchet model can describe the interdependent localization dynamics of SpoVM and SpoIVA, wherein SpoVM displays a longer residence time on the forespore surface, which favors recruitment of SpoIVA to that location. Localized SpoIVA polymerization in turn prevents further sampling of other membranes by prelocalized SpoVM molecules. Our model therefore describes the dynamics of structural proteins as they localize and assemble at the correct place and time within a cell to form a supramolecular complex.

MicL, a new σE-dependent sRNA, combats envelope stress by repressing synthesis of Lpp, the major outer membrane lipoprotein.

In enteric bacteria, the transcription factor σ(E) maintains membrane homeostasis by inducing synthesis of proteins involved in membrane repair and two small regulatory RNAs (sRNAs) that down-regulate synthesis of abundant membrane porins. Here, we describe the discovery of a third σ(E)-dependent sRNA, MicL (mRNA-interfering complementary RNA regulator of Lpp), transcribed from a promoter located within the coding sequence of the cutC gene. MicL is synthesized as a 308-nucleotide (nt) primary transcript that is processed to an 80-nt form. Both forms possess features typical of Hfq-binding sRNAs but surprisingly target only a single mRNA, which encodes the outer membrane lipoprotein Lpp, the most abundant protein of the cell. We show that the copper sensitivity phenotype previously ascribed to inactivation of the cutC gene is actually derived from the loss of MicL and elevated Lpp levels. This observation raises the possibility that other phenotypes currently attributed to protein defects are due to deficiencies in unappreciated regulatory RNAs. We also report that σ(E) activity is sensitive to Lpp abundance and that MicL and Lpp comprise a new σ(E) regulatory loop that opposes membrane stress. Together MicA, RybB, and MicL allow σ(E) to repress the synthesis of all abundant outer membrane proteins in response to stress.

Stem-loops direct precise processing of 3' UTR-derived small RNA MicL.

Increasing numbers of 3'UTR-derived small, regulatory RNAs (sRNAs) are being discovered in bacteria, most generated by cleavage from longer transcripts. The enzyme required for these cleavages has been reported to be RNase E, the major endoribonuclease in enterica bacteria. Previous studies investigating RNase E have come to a range of different conclusions regarding the determinants for RNase E processing. To better understand the sequence and structure determinants for the precise processing of a 3' UTR-derived sRNA, we examined the cleavage of multiple mutant and chimeric derivatives of the 3' UTR-derived MicL sRNA in vivo and in vitro. Our results revealed that tandem stem-loops 3' to the cleavage site define optimal, correctly-positioned cleavage of MicL and probably other sRNAs. Moreover, our assays of MicL, ArcZ and CpxQ showed that sRNAs exhibit differential sensitivity to RNase E, likely a consequence of a hierarchy of sRNA features recognized by the endonuclease.

Protection against deleterious nitrogen compounds: role of σS-dependent small RNAs encoded adjacent to sdiA.

Here, we report the characterization of a set of small, regulatory RNAs (sRNAs) expressed from an Escherichia coli locus we have denoted sdsN located adjacent to the LuxR-homolog gene sdiA Two longer sRNAs, SdsN137 and SdsN178 are transcribed from two σ(S)-dependent promoters but share the same terminator. Low temperature, rich nitrogen sources and the Crl and NarP transcription factors differentially affect the levels of the SdsN transcripts. Whole genome expression analysis after pulse overexpression of SdsN137 and assays of lacZ fusions revealed that the SdsN137 directly represses the synthesis of the nitroreductase NfsA, which catalyzes the reduction of the nitrogroup (NO2) in nitroaromatic compounds and the flavohemoglobin HmpA, which has aerobic nitric oxide (NO) dioxygenase activity. Consistent with this regulation, SdsN137 confers resistance to nitrofurans. In addition, SdsN137 negatively regulates synthesis of NarP. Interestingly, SdsN178 is defective at regulating the above targets due to unusual binding to the Hfq protein, but cleavage leads to a shorter form, SdsN124, able to repress nfsA and hmpA.

Multiple factors dictate target selection by Hfq-binding small RNAs.

Hfq-binding small RNAs (sRNAs) in bacteria modulate the stability and translational efficiency of target mRNAs through limited base-pairing interactions. While these sRNAs are known to regulate numerous mRNAs as part of stress responses, what distinguishes targets and non-targets among the mRNAs predicted to base pair with Hfq-binding sRNAs is poorly understood. Using the Hfq-binding sRNA Spot 42 of Escherichia coli as a model, we found that predictions using only the three unstructured regions of Spot 42 substantially improved the identification of previously known and novel Spot 42 targets. Furthermore, increasing the extent of base-pairing in single or multiple base-pairing regions improved the strength of regulation, but only for the unstructured regions of Spot 42. We also found that non-targets predicted to base pair with Spot 42 lacked an Hfq-binding site, folded into a secondary structure that occluded the Spot 42 targeting site, or had overlapping Hfq-binding and targeting sites. By modifying these features, we could impart Spot 42 regulation on non-target mRNAs. Our results thus provide valuable insights into the requirements for target selection by sRNAs.

Altruistic feeding and cell-cell signaling during bacterial differentiation actively enhance phenotypic heterogeneity.

Starvation triggers bacterial spore formation, a committed differentiation program that transforms a vegetative cell into a dormant spore. Cells in a population enter sporulation non-uniformly to secure against the possibility that favorable growth conditions, which puts sporulation-committed cells at a disadvantage, may resume. This heterogeneous behavior is initiated by a passive mechanism: stochastic activation of a master transcriptional regulator. Here, we identify a cell-cell communication pathway that actively promotes phenotypic heterogeneity, wherein Bacillus subtilis cells that start sporulating early utilize a calcineurin-like phosphoesterase to release glycerol, which simultaneously acts as a signaling molecule and a nutrient to delay non-sporulating cells from entering sporulation. This produced a more diverse population that was better poised to exploit a sudden influx of nutrients compared to those generating heterogeneity via stochastic gene expression alone. Although conflict systems are prevalent among microbes, genetically encoded cooperative behavior in unicellular organisms can evidently also boost inclusive fitness.

The stoichiometry of the Escherichia coli Hfq protein bound to RNA.

The Escherichia coli RNA binding protein Hfq is involved in many aspects of post-transcriptional gene expression. Tight binding of Hfq to polyadenylate sequences at the 3' end of mRNAs influences exonucleolytic degradation, while Hfq binding to small noncoding RNAs (sRNA) and their targeted mRNAs facilitate their hybridization which in turn effects translation. Hfq binding to an A-rich tract in the 5' leader region of the rpoS mRNA and to the sRNA DsrA have been shown to be important for DsrA enhanced translation initiation of this mRNA. The complexes of Hfq-A(18) and Hfq-DsrA provide models for understanding how Hfq interacts with these two RNA sequence/structure motifs. Different methods have reported different values for the stoichiometry of Hfq-A(18) and Hfq-DsrA. In this work, mass spectrometry and analytical ultracentrifugation provide direct evidence that the strong binding mode of the Hfq hexamer (Hfq(6)) for A(18) and domain II of DsrA (DsrA(DII)) involve 1:1 complexes. This stoichiometry was also supported by fluorescence anisotropy and a competition gel mobility shift experiment using wild-type and truncated Hfq. More limited studies of Hfq binding to DsrA as well as to the sRNAs RprA, OxyS, and an 18-nt segment of OxyS were also consistent with 1:1 stoichiometry. Mass spectrometry of cross-linked samples of Hfq(6), A(18), and DsrA(DII) exhibit intensity corresponding to a ternary 1:1:1 complex; however, the small intensity of this peak and fluorescence anisotropy experiments did not provide evidence that this ternary complex is stable in solution.