Small proteins, here defined as proteins of 50 amino acids or less in the absence of processing, have traditionally been overlooked due to challenges in their annotation and biochemical detection. In the past several years however, increasing numbers of small proteins have been identified either through the realization that mutations in “intergenic” regions actually are within unannotated small protein genes, or through the discovery that some small, regulatory RNAs (sRNAs) encode small proteins. These insights together with comparative sequence analysis indicate that tens if not hundreds of small proteins are synthesized in a given organism. This review will summarize what has been learned about the functions of several of these bacterial small proteins, most of which act at the membrane, illustrating the astonishing range of processes in which the small proteins act and pointing to several general conclusions. Important questions for future studies of these overlooked proteins also will be discussed.

Summary Although prokaryotes ordinarily undergo binary fission to produce two identical daughter cells, some are able to undergo alternative developmental pathways that produce daughter cells of distinct cell morphology and fate. One such example is a developmental program called sporulation in the bacterium Bacillus subtilis, which occurs under conditions of environmental stress. Sporulation has long been used as a model system to help elucidate basic processes of developmental biology including transcription regulation, intercellular signaling, membrane remodeling, protein localization, and cell fate determination. This review highlights some of the recent work that has been done to further understand prokaryotic cell differentiation during sporulation and its potential applications.

Bacterial proteins often localize to distinct sites within the cell, but the primary cues that dictate localization are largely unknown. Recent evidence has shown that positive membrane curvature can serve as a cue for localization of a peripheral membrane protein.Here we report that localization of the peripheral membrane protein DivIVA is determined in whole or in part by recognition of negative membrane curvature and that regions of the protein near the N and C terminus are important for localization. DivIVA, which is a cell division protein in Bacillus subtilis, localizes principally as a ring at nascent septa and secondarily to the less negatively curved, inside surface of the hemispherical poles of the cell. When cytokinesis is prevented, DivIVA redistributes itself to, and becomes markedly enriched at, the poles. When the rod-shaped cells are converted into spheres (protoplasts) by treatment with lysozyme, DivIVA adopts a largely uniform distribution around the cell. Recognition of membrane curvature by peripheral membrane proteins could be a general strategy for protein localization in bacteria.geometric cue ͉ protein localization

Proteins in bacteria often deploy to particular places within the cell, but the cues for localization are frequently mysterious. We found that the peripheral membrane protein SpoVM recognizes a geometric cue in localizing to a particular membrane during sporulation in Bacillus subtilis. Sporulation involves an inner cell maturing into a spore and an outer cell nurturing the developing spore. SpoVM is produced in the outer cell where it embeds in the membrane that surrounds the inner cell but not in the cytoplasmic membrane of the outer cell. We found that SpoVM localized by discriminating between the positive curvature of the membrane surrounding the inner cell and the negative curvature of the cytoplasmic membrane. Membrane curvature could be a general cue for protein localization in bacteria.Proteins often localize to particular positions within bacteria, sometimes in a dynamic manner. A striking but mysterious example of subcellular localization occurs during spore formation in Bacillus subtilis when SpoVM (VM) localizes to a particular patch of membrane (1). How VM discriminates between different membrane surfaces in the same cell is unknown.During sporulation, the cell divides asymmetrically to create mother cell and forespore compartments. Next, the mother cell engulfs the forespore, enveloping it with inner and outer membranes (Fig. 1A). Following engulfment a protein coat is deposited around the outer forespore membrane (2). Coat assembly depends on VM, a 26-residue peptide that is produced in the mother cell (3). VM is an amphipathic α-helix (4) that inserts into the membrane with its long axis parallel to the membrane and its hydrophobic face buried in the lipid bilayer (5). During engulfment, VM localizes to the membrane that tracks around the forespore, eventually decorating the entire surface of the forespore, as visualized using a fusion to the Green Fluorescent Protein (VM-GFP; Fig 1C) (1). Proline 9 (P9; Fig. 1B) is critical for this localization (1), as substitution of P9 with alanine (VM P9A -GFP) resulted in localization to both the cytoplasmic and the outer forespore membranes (Fig. 1D).Following engulfment, the outer forespore membrane becomes topologically isolated from the cytoplasmic membrane. We wondered if VM would adhere to the outer forespore membrane after isolation. We engineered cells to produce VM-GFP in response to an inducer and triggered synthesis of the fusion protein after engulfment. To monitor topological isolation, we stained the membranes with a membrane permeable dye, which stains all membranes, and a membrane impermeable dye, which can only access the engulfment membrane before membrane fusion (6). VM-GFP localized almost exclusively to the outer forespore membrane even when the forespore was topologically isolated (Fig. 1G-I). As a control, VM P9A -GFP synthesized after * Publisher's Disclaimer: This manuscript has been accepted for publication in Science. This version has not undergone final editing.Please refer to the complete version of record at http://www....

The assembly of the cell division machinery at midcell is a critical step of cytokinesis. Many rod-shaped bacteria position septa using nucleoid occlusion, which prevents division over the chromosome, and the Min system, which prevents division near the poles. Here we examined the in vivo assembly of the Bacillus subtilis MinCD targeting proteins DivIVA, a peripheral membrane protein that preferentially localizes to negatively curved membranes and resembles eukaryotic tropomyosins, and MinJ, which recruits MinCD to DivIVA. We used structured illumination microscopy to demonstrate that both DivIVA and MinJ localize as double rings that flank the septum and first appear early in septal biosynthesis. The subsequent recruitment of MinCD to these double rings would separate the Min proteins from their target, FtsZ, spatially regulating Min activity and allowing continued cell division. Curvature-based localization would also provide temporal regulation, since DivIVA and the Min proteins would localize to midcell after the onset of division. We use time-lapse microscopy and fluorescence recovery after photobleaching to demonstrate that DivIVA rings are highly stable and are constructed from newly synthesized DivIVA molecules. After cell division, DivIVA rings appear to collapse into patches at the rounded cell poles of separated cells, with little or no incorporation of newly synthesized subunits. Thus, changes in cell architecture mediate both the initial recruitment of DivIVA to sites of cell division and the subsequent collapse of these rings into patches (or rings of smaller diameter), while curvature-based localization of DivIVA spatially and temporally regulates Min activity.

Pathogenic yersiniae secrete 14 Yop proteins via the type III pathway. Synthesis of YopQ occurs when the type III machinery is activated by a low-calcium signal, but not when the calcium concentration is above 100 M. To characterize the mechanism that regulates the expression of yopQ, mutants that permit synthesis of YopQ in the presence of calcium were isolated. Yersiniae bearing deletion mutations in yopN, tyeA, sycN, or yscB synthesized and secreted YopQ in both the presence and the absence of calcium. In contrast, yersiniae with a deletion in yopD or lcrH synthesized YopQ in the presence of calcium but did not secrete the polypeptide. These variants displayed no defect in YopQ secretion under low-calcium conditions, revealing that yopD and lcrH are required for the regulation of yopQ expression. Experiments with transcriptional and translational fusions to the npt reporter gene suggest that yopD and lcrH regulate yopQ expression at a posttranscriptional step. YopD and LcrH form a complex in the bacterial cytosol and bind yopQ mRNA. Models that can account for posttranscriptional regulatory mechanisms of yop expression are discussed.

SummarySpore formation in Bacillus subtilis involves the formation of a thick, proteinaceous shell or coat that is assembled around a specialized membrane known as the outer forespore membrane. Here we present evidence that the assembling coat is tethered to the outer forespore membrane by a 26-amino-acid peptide called SpoVM, which is believed to form an amphipathic helix. We show that proper localization of SpoVM is dependent on SpoIVA, a morphogenetic protein that forms the basement layer of the spore coat, and conversely, that proper localization of SpoIVA is dependent on SpoVM. Genetic, biochemical and cytological evidence indicates that this mutual dependence is mediated in part by contact between an amino acid side-chain located near the extreme C-terminus of SpoIVA and an amino acid side-chain on the hydrophilic face of the SpoVM helix. Evidence is also presented that SpoVM adheres to the outer forespore membrane via hydrophobic, amino acid side-chains on the hydrophobic face of the helix. The results suggest that the SpoVM helix is oriented parallel to the membrane with the hydrophobic face buried in the lipid bilayer.

Summary Biofilm formation in Bacillus subtilis requires the differentiation of a subpopulation of cells responsible for the production of the extracellular matrix that structures the biofilm. Differentiation of matrix-producing cells depends, among other factors, on the FloT and YqfA proteins. These proteins are present exclusively in functional membrane microdomains of B. subtilis and are homologous to the eukaryotic lipid raft-specific flotillin proteins. In the absence of FloT and YqfA, diverse proteins normally localized to the membrane microdomains of B. subtilis are not functional. Here we show that the absence of FloT and YqfA reduces the level of the septal-localized protease FtsH. The flotillin homologues FloT and YqfA are occasionally present at the midcell in exponentially growing cells and the absence of FloT and YqfA negatively affects FtsH concentration. Biochemical experiments indicate a direct interaction between FloT/YqfA and FtsH. Moreover, FtsH is essential for the differentiation of matrix producers and hence, biofilm formation. This molecular trigger of biofilm formation may therefore be used as a target for the design of new biofilm inhibitors. Accordingly, we show that the small protein SpoVM, known to bind to and inhibit FtsH activity, inhibits biofilm formation in B. subtilis and other distantly related bacteria.