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Assembly of the Bacillus subtilis spore coat involves over 80 proteins which self‐organize into a basal layer, a lamellar inner coat, a striated electrodense outer coat and a more external crust. CotB is an abundant component of the outer coat. The C‐terminal moiety of CotB, SKRB, formed by serine‐rich repeats, is polyphosphorylated by the Ser/Thr kinase CotH. We show that another coat protein, CotG, with a central serine‐repeat region, SKRG, interacts with the C‐terminal moiety of CotB and promotes its phosphorylation by CotH in vivo and in a heterologous system. CotG itself is phosphorylated by CotH but phosphorylation is enhanced in the absence of CotB. Spores of a strain producing an inactive form of CotH, like those formed by a cotG deletion mutant, lack the pattern of electrondense outer coat striations, but retain the crust. In contrast, deletion of the SKRB region, has no major impact on outer coat structure. Thus, phosphorylation of CotG by CotH is a key factor establishing the structure of the outer coat. The presence of the cotB/cotH/cotG cluster in several species closely related to B. subtilis hints at the importance of this protein phosphorylation module in the morphogenesis of the spore surface layers., CotB and CotG are abundant components of the Bacillus subtilis spore coat, both possessing a series of serine‐/lysine‐rich repeats (SKR). CotG interacts with CotB promoting its polyphosphorylation in the SKR region by the CotH kinase. CotG, itself phosphorylated by CotH, is a key determinant of the striated pattern of the outer coat. Conservancy of the cotB/cotH/cotG cluster suggests that protein phosphorylation is an important mechanism in the morphogenesis of the spore surface across species.

Coelomocytes is the generic name for a collection of cellular morphotypes, present in many coelomate animals, and highly variable among echinoderm classes. The roles attributed to the major types of these free circulating cells present in the coelomic fluid of echinoderms include immune response, phagocytic digestion and clotting. Our main aim in this study was to characterize coelomocytes found in the coelomic fluid of Marthasterias glacialis (class Asteroidea) by using a combination of flow cytometry (FC), imaging flow cytometry (IFC) and fluorescence plus transmission electron microscopy (TEM). Two coelomocyte populations (P1 and P2) identified through flow cytometry were subsequently studied in terms of abundance, morphology, ultrastructure, cell viability and cell cycle profiles. Ultrastructurally, P2 diploid cells were present as two main morphotypes, similar to phagocytes and vertebrate thrombocytes, whereas the smaller P1 cellular population was characterized by low mitotic activity, a relatively undifferentiated cytotype and a high nucleus/cytoplasm ratio. In the present study we could not rule out possible similarities between haploid P1 cells and stem-cell types in other animals. Additionally, we report the presence of two other morphotypes in P2 that could only be detected by fluorescence microscopy, as well as a morphotype revealed via combined microscopy/FC. This integrative experimental workflow combined cells physical separation with different microscopic image capture technologies, enabling us to better tackle the characterization of the heterogeneous composition of coelomocytes populations.

The thymus generates central immune tolerance by producing self-restricted and self-tolerant T-cells as a result of interactions between the developing thymocytes and the stromal microenvironment, mainly formed by the thymic epithelial cells. The thymic epithelium derives from the endoderm of the pharyngeal pouches, embryonic structures that rely on environmental cues from the surrounding mesenchyme for its development. Here, we review the most recent advances in our understanding of the molecular mechanisms involved in early thymic organogenesis at stages preceding the expression of the transcription factor Foxn1, the early marker of thymic epithelial cells identity. Foxn1-independent developmental stages, such as the specification of the pharyngeal endoderm, patterning of the pouches, and thymus fate commitment are discussed, with a special focus on epithelial–mesenchymal interactions.

Coelomocytes is a generic name for a collection of cellular morphotypes, present in many coelomate animals, that has been reported as highly variable across echinoderm classes. The roles attributed to the major types of the free circulating cells present in the coelomic fluid of echinoderms include immune response, phagocytic digestion and clotting. The main aim of the present study is the thorough characterization of coelomocytes present in the coelomic fluid ofMarthasterias glacialis(class Asteroidea) through the combined use of flow cytometry (FC) and fluorescence plus transmission electron microscopy. Two coelomocyte populations (here named P1 and P2) were identified by flow cytometry and subsequently studied in terms of abundance, morphology, ultrastructure, cell viability and cell cycle profiles. Ultrastructurally, P2 diploid cells showed two main morphotypes, similar to phagocytes and vertebrate thrombocytes, whereas the small P1 haploid cellular population was characterized by a low mitotic activity, relatively undifferentiated cytotype and a high nucleus/cytoplasm ratio. These last cells resemble stem-cell types present in other animals. P1 and P2 cells differ also in cell viability and cell cycle profiles. Additionally, two other morphotypes were only detected by fluorescence microscopy and a third one when using a combined microscopy/FC approach.

Conjugative plasmids encode genes that enable them to transfer, by conjugation, from a given host cell to another cell. Conjugative transfer, despite being an important feature of conjugative plasmids, is not constitutive for most plasmids, the reason being that genes involved in horizontal transfer are mostly repressed. Only upon their transient de-repression are plasmids able to transfer horizontally. If host cells harbour multiple plasmids, their simultaneous transfer depends on simultaneous transient de-repression of all plasmids. If de-repression of different plasmids was random and independent events, simultaneous de-repression should be a rare event because the probability of simultaneous de-repression would be the product of the probabilities of de-repression of each plasmid. Some previous observations support this hypothesis, while others show that co-transfer of plasmids is more frequent than this reasoning indicates. Here, we show that co-transfer of multiple plasmids mainly results from non-independent events: the probability that all plasmids within a cell become de-repressed is much higher than if de-repression of plasmids genes were independent. We found a simple model for the probability of co-transfer: the plasmid having the lowest conjugation rates is the one who limits co-transfer. In this sense, cells receiving the plasmid with the lower transfer rate also receive the other plasmid. If de-repression happens simultaneously on co-resident plasmids, common cues may stimulate de-repression of distinct plasmids.

The avian thymus and parathyroids (T/PT) common primordium derives from the endoderm of the third and fourth pharyngeal pouches (3/4PP). The molecular mechanisms that govern T/PT development are not fully understood. Here we study the effects of Notch and Hedgehog (Hh) signalling modulation during common primordium development using in vitro, in vivo and in ovo approaches. The impairment of Notch activity reduced Foxn1/thymus-fated and Gcm2/Pth/parathyroid-fated domains in the 3/4PP and further compromised the development of the parathyroid glands. When Hh signalling was abolished, we observed a reduction in the Gata3/Gcm2- and Lfng-expression domains at the median/anterior and median/posterior territories of the pouches, respectively. In contrast, the Foxn1 expression-domain at the dorsal tip of the pouches expanded ventrally into the Lfng-expression domain. This study offers novel evidence on the role of Notch signalling in T/PT common primordium development, in an Hh-dependent manner.

In this chapter you will learn that, besides the initial oncogenic mutations that trigger tumorigenesis, cancer cells must have additional molecular changes and morphological modifications in order to metastasize and become malignant. One of the mechanisms that tumor cells may undergo to achieve this state is the Epithelial-Mesenchymal Transition (EMT). After giving a global perspective of what EMT is and of the main EMT activating factors, the particularities of EMT in tumors and the molecular mechanisms that underlie the tumor EMT phenotypic plasticity are adressed. Finally, the contribution of EMT studies for the development of a new generation of cancer therapies is discussed.

Plasmids are genetic elements that play a role in bacterial evolution by providing new genes that promote adaptation to diverse conditions. Plasmids are also known to reduce bacterial competitiveness in the absence of selection for plasmid-encoded traits. It is easier to understand plasmid persistence when considering the evidence that plasmid maintenance can improve during co-evolution with the bacterial host, i.e. the chromosome. However, bacteria isolated from nature often harbor diverse mobile elements: phages, transposons, genomic islands and even other plasmids. Recent interest has emerged on the role such elements play on the persistence and evolution of plasmids. Here, we mainly review interactions between different plasmids, but also discuss their interactions with other genetic elements. We focus on interactions that impact fundamental plasmid traits, such as the fitness effect imposed on their hosts and the transfer efficiency into new host cells. We illustrate these phenomena with examples concerning clinically relevant organisms and the spread of plasmids carrying antibiotic resistance genes and virulence factors.

Naturally occurring plasmids have medical importance given that they frequently code for virulence or antibiotic resistance. In many cases, plasmids impose a fitness cost to their hosts, meaning that the growth rate of plasmid-bearing cells is lower than that of plasmid-free cells. However, this does not fit with the fact that plasmids are ubiquitous in nature nor that plasmids and their hosts adapt to each other very fast - as has been shown in laboratory evolutionary assays. Even when plasmids are costly, they seem to largely interact in such a way that the cost of two plasmids is lower than the cost of one of them alone. Moreover, it has been argued that transfer rates are too low to compensate for plasmid costs and segregation. Several mechanisms involving interactions between plasmids and other replicons could overcome this limitation, hence contributing to the maintenance of plasmids in bacterial populations. We examine the importance of these mechanisms from a clinical point of view, particularly the spread of antibiotic resistance genes.

As shown in the previous article, two distinct conjugative plasmids sometimes interact within bacterial cells, implicating changes of transfer rates. In most cases of interactions within bacteria, the transfer of one of the plasmids decreases. Less frequently, the transfer rate of one of the plasmids increases. Here we analyse what happens if three distinct conjugative plasmids colonize the same bacterial cell. Our aim is to understand how interactions between two plasmids affect the transfer rate of the third plasmid. After showing that plasmids interact in 59 out of 84 possible interactions we show that, with some exceptions, if the transfer rate of a plasmid decreases in the presence of a second plasmid, a decrease is also observed in the presence of a third plasmid. Moreover, if the conjugation rate of a plasmid increases in the presence of another, an increase is also observed if there is a third plasmid in the cell. Both types of interactions are mostly independent of the third plasmid's identity, even if sometimes the third plasmid quantitatively distorts the interaction of the other two plasmids. There is a bias towards negative intensifying interactions, which provide good news concerning the spread conjugative plasmids encoding antibiotic-resistance genes and virulence factors.

Conjugative plasmids encode the genes responsible for the synthesis of conjugative pili and plasmid transfer. Expression of the conjugative machinery (including conjugative pili) may be costly to bacteria, not only due to the energetic/metabolic cost associated with their expression but also because they serve as receptors for certain viruses. Consequently, the presence of two plasmids in the same cell may be disadvantageous to each plasmid, because they may impose a higher fitness cost on the host. Therefore, plasmids may encode mechanisms to cope with co-resident plasmids. Moreover, it is possible that the transfer rate of a plasmid is affected by the presence of a distinct plasmid in the recipient cell. In this work, we measured transfer rates of twelve natural plasmids belonging to seven incompatibility groups in three situations, namely when: (i) donor cells contain a plasmid and recipient cells are plasmid-free; (ii) donor cells contain two unrelated plasmids and recipient cells are plasmid-free; and (iii) half of the cells contain a given plasmid and the other half contain another, unrelated, plasmid. In the third situation, recipient cells of a plasmid are the donor cells of the other plasmid. We show that there are more negative interactions (reduction of a plasmid's conjugative efficiency) between plasmids if they reside in the same cell than if they reside in different cells. However, if plasmids interacted intercellularly, the transfer rate of one of the plasmids was often higher (when the unrelated conjugative plasmid was present in the recipient cell) than if the recipient cell was plasmid-free - a positive effect. Experimental data retrieved from the study of mutant plasmids not expressing conjugative pili on the cell surface suggest that positive effects result from a higher efficiency of mating pair formation. Overall, our results suggest that negative interactions are significantly more frequent when plasmids occupy the same cell. Such interactions may determine how antibiotic resistance disseminates in bacterial populations.

We report evidence that the CotC polypeptide, a previously identified component of the Bacillus subtilis spore coat, is assembled into at least four distinct forms. Two of these, having molecular masses of 12 and 21 kDa, appeared 8 h after the onset of sporulation and were probably assembled on the forming spore immediately after their synthesis, since no accumulation of either of them was detected in the mother cell compartment, where their synthesis occurs. The other two components, 12.5 and 30 kDa, were generated 2 h later and were probably the products of posttranslational modifications of the two early forms occurring directly on the coat surface during spore maturation. None of the CotC forms was found either on the spore coat or in the mother cell compartment of a cotH mutant. This indicates that CotH serves a dual role of stabilizing the early forms of CotC and promoting the assembly of both early and late forms on the spore surface.

During the process of sporulation in the gram-positive soil bacterium Bacillus subtilis the developing spore is encased in a complex protein structure called the coat, which confers resistance to several physicochemical agents and contributes to the response of spores to the presence of germinants (7, 8, 15). The coat is formed by over 30 polypeptides, ranging in size from about 6 to about 70 kDa, which are assembled into a lamellar inner coat and a thick electron-dense outer coat (7, 8, 15). With only one possible exception (38), synthesis of the coat structural components is restricted to the mother cell chamber of the sporulating cell and is temporally governed by a cascade of transcription factors in the order σE, SpoIIID, σK, and GerE (7, 8, 15, 24, 35, 40). σE and SpoIIID drive synthesis of a class of morphogenetic proteins that (irrespective of their association with the final coat structure) appear to guide the assembly of several structural components into the spore coat (reviewed in references 7, 8, and 15). For instance, spores produced by a cotE mutant fail to assemble the electron-dense outer coat and the remaining coat structure appears to lack, in addition to CotE, several other abundant components (47). The results of a recent study indicate that specific regions in CotE are required for the assembly of different proteins and suggest that CotE might control the assembly of several outer-coat components by direct protein-protein interactions (2, 28). Most of the coat structural components are synthesized (under the control of σK and GerE) at a later stage in coat assembly, and it is only after σK is activated that assembly of the coat is unequivocally recognized by electron microscopy of sporulating cells (7, 8, 15). Activation of σK results in the expression of several genes coding for spore coat proteins and also results in transcription of the gerE gene (4), which encodes an ambivalent transcriptional regulator of coat gene expression. GerE acts together with σK to activate a late class of cot genes, but it also represses transcription of other cot genes (18, 19, 45, 46). These regulatory circuits suggest that the time and level of expression of the genes coding for coat structural components are important for the correct assembly of the coat structure (7, 8, 15). Proper assembly of the coat further relies on mechanisms such as translational control (34) and posttranslational modifications, including proteolytical processing of larger precursors, protein secretion, and protein cross-linking (reviewed in references 7, 8, and 15). These modifications may provide an additional level of control over the timing of assembly of specific components. For example, SafA is a morphogenetic protein of about 45 kDa produced under σE control from hour 2 of sporulation onwards but the main form of SafA detected in the coats is a smaller (approximately 30 kDa) species corresponding to the C-terminal region of the protein (32, 33). This smaller species is produced by internal translation initiation (34). In addition, the full-length and 30-kDa forms of SafA are processed by the YabG protease, which is produced under the control of σK (42, 43). The exact contribution of these mechanisms to the ordered assembly of the various coat components is poorly understood, and determination of their nature and contribution will ultimately rely on the functional and structural characterization of selected components (11, 29). To learn more about the mechanisms involved in the morphogenesis of the coat structure, we analyzed the assembly of the outer-coat protein CotB. The cotB gene was initially found (by reverse genetics) to encode an abundant spore coat component (6), later shown to be in the outer coat (47), which appears to be surface exposed (20). CotB has been utilized as a vehicle for the presentation of heterologous antigens at the spore surface, suggesting its potential use in vaccine development (9, 20). Thus, the study of the assembly of CotB may allow a more precise manipulation of CotB as a fusion partner for heterologous antigen presentation. Also, it will expand our knowledge of the protein-protein interactions underlying assembly of a complex multiprotein structure and may provide us with tools for nanoengineering applications involving the B. subtilis spore. The cotB gene forms a cluster with two cot genes, cotH and cotG (6, 30, 36). Expression of cotH is under the control of σK, whereas both cotG and cotB are expressed later under the dual control of σK and GerE (18, 30, 36, 46, 45). Assembly of CotB-66 was shown to require expression of both cotG and cotH (30, 36). We now show that cotB encodes a 46-kDa polypeptide (CotB-46) which is posttranslationally converted into a form of about 66 kDa (herein called CotB-66). This form of CotB (CotB-66) is equivalent to the 59-kDa protein previously reported by Donovan et al. (6). We show that formation of CotB-66 requires both cotG and cotH and that CotG does not accumulate in a cotH mutant. This suggests that the requirement for CotH or a CotH-dependent protein for CotB-66 formation results in part from its stabilization of CotG. We also found that CotB is present in complexes with CotG at the time when formation of CotB-66 is detected. Moreover, CotB was found to interact with itself and with CotG in a Saccharomyces cerevisiae two-hybrid assay. We suggest that formation of CotB-66 requires a direct interaction with CotG.

Summary PNPase and RNase II are the key regulatory exo-nucleases controlling mRNA decay in Escherichia coli The rnb transcripts were found to proceed through the terminator and PNPase was found to be involved in the 3’to 5’degradation of rnb mRNA. Analysis of these longer 3’termini revealed that they are located in UA-rich regions. Comparison of single and double mutants suggested that PNPase and RNase II could have different roles in the degradation of these unstructured regions. We have shown that RNase II levels can vary over a fivefold range in haploid cells and that its expression depends on PNPase levels. PNPase-deficient strains were found to have a 2–2.5-fold increase in RNase M activity, while PNPase-overproducing strains reduced the rnb message and RNase II levels. Conversely, the amount of PNPase in the rnb deletion strain was approximately twofold higher than that in the wild-type strain. These observations suggest that the two main exonu-cleases are inter-regulated through a fine tuning mechanism. We discuss the implications of these results with regard to mRNA degradation and cell metabolism.

The degradation of mRNA plays a central role in the control of protein synthesis. In Escherichia coli, the rnb gene encodes ribonuclease II (RNase II), one of the two main exonucleases involved in mRNA decay. We have constructed strain CMA201, in which the rnb promoter region and the gene were deleted from the chromosome and replaced by a tettr cassette. This is the first rnb absolute deletion mutant that shows the complete absence of rnb-specific mRNA. This strain has growth characteristics similar to the wild-type, even though it has no RNase II activity, and it should be useful in studies of mRNA metabolism.

We report evidence that CotC and CotU, two previously identified components of the Bacillus subtilis spore coat, are produced concurrently in the mother cell chamber of the sporulating cell under the control of σ K and GerE and immediately assembled around the forming spore. In the coat, the two proteins interact to form a coat component of 23 kDa. The CotU-CotC interaction was not detected in two heterologous hosts, suggesting that it occurs only in B. subtilis . Monomeric forms of both CotU and CotC failed to be assembled at the surface of the developing spore and accumulated in the mother cell compartment of cells mutant for cotE . In contrast, neither CotU nor CotC accumulated in the mother cell compartment of cells mutant for cotH . These results suggest that CotH is required to protect both CotU and CotC in the mother cell compartment of the sporangium and that CotE is needed to allow their assembly and subsequent interaction at the spore surface.

The Bacillus subtilis spore is encased in a multilayered protein coat that confers resistance to peptidoglycan-breaking enzymes and noxious chemicals and also influences the germination response. Biogenesis of the spore coat relies on the ordered synthesis and assembly of over 40 polypeptides (5, 10, 11). The assembly process is controlled at various levels. First, the time and degree of expression of the genes encoding the various coat components are under complex regulation. Most, if not all, components of the coat are produced in the mother cell chamber of the sporulating cell (5, 10, 11, 34). Some coat proteins are synthesized prior to engulfment of the prespore by the mother cell, and those are under the control of σE, whose activity is influenced by the GerR and SpoIIID ancillary transcription factors (5, 6, 9, 10, 11). However, the synthesis of many other coat proteins and the deposition of most of the coat structural components are governed by σK, which replaces σE following engulfment completion (5, 9, 10, 11). The activity of σK is modulated by the GerE transcription factor, itself the product of a σK-controlled gene, which can act both as an activator or a repressor (5, 6, 10, 11, 41). Second, the formation of the spore coat is controlled by a class of unique morphogenetic proteins which are required for the assembly of several structural components at the surface of the developing spore (5, 10, 11). Third, the process is also controlled by posttranscriptional and posttranslational mechanisms which include proteolysis, glycosylation, and cross-linking. Together, these mechanisms are thought to contribute to the proper structural organization and properties of the coat structure (5, 10, 11). The roles of several coat morphogenetic proteins have been examined in some detail (5, 10, 11, 22). However, our knowledge of the contribution of mechanisms such as protein cross-linking to the assembly process is still very limited. Nevertheless, about 30% of the total spore coat protein is refractory to extraction, forming an insoluble highly cross-linked fraction (5, 10, 30). Several coat proteins appear to be cross-linked, and the available evidence suggests that both reversible and irreversible cross-links occur within the coat (5, 10, 11, 12, 14, 20, 21, 42). In a classical study, Pandey and Aronson (30) have suggested the existence of o,o-dityrosine cross-links in purified coat material, an observation confirmed by more recent work (5). CotG and three other tyrosine-rich coat components, CotB, CotC, and CotU, have been recently shown to be present as multimers within the spore coat (12, 21, 14, 42). However, the nature of the cross-links in CotB, CotC, CotU, or CotG has not been reported, and even though other enzymes could, in principle, promote oxidative cross-linking of the coat structural components (see references 7 and 25 and references therein), a peroxidase activity associated with the B. subtilis spore has not yet been found. In contrast, a transglutaminase activity is detected in sporulating cells and spores (16, 18, 37), and ɛ-(γ-glutamyl)lysyl cross-links have been detected in B. subtilis spores and in purified coat material (17). Moreover, the gene (tgl) for a transglutaminase was cloned following the purification of a spore-associated activity (16, 37). The enzyme could cross-link α-casein and bovine serum albumin in vitro, and a cysteine residue at position 116 was found to be essential for the reaction (16). Tgl-like polypeptides are present in other Bacillus and related spore-forming species but absent from the genomes of other bacteria, suggesting a specific role in sporulation (Fig. ​(Fig.1).1). Recently, it was shown that the deletion of tgl prevents cross-linking of GerQ, a coat protein that is also involved in spore germination (29, 31, 32). However, it is not known whether GerQ is directly cross-linked by Tgl (31). Other polypeptides have been suggested to be cross-linked by a transglutaminase in the coats, including the lysine- and glutamine-rich CotX protein, a component of the coat insoluble fraction and CotM, a member of the α-crystallin family of small heat shock proteins (12, 39). FIG. 1. Tgl is present in other spore formers. The figure represents a Clustal W alignment (www.ebi.ac.uk) of the Tgl proteins found in the following sporeformers: Bs, B. subtilis 168 (NP-391005); Bl, Bacillus licheniformis DSM 13 (YP-080406); Gk, Geobacillus ... Transglutaminases intervene in key biological processes, such as cytoskeletal scaffolding, the control of protein-protein interactions, blood clotting, wound healing, apoptosis, development and morphogenesis, or the keratinization of skin (8, 23). Because the ɛ-(γ-glutamyl)lysyl isopeptide is stable and proteolysis resistant, it contributes to the rigidity and resistance to chemical, enzymatic, physical, and mechanical decay of various tissues and cellular structures (8, 23). Here, we have investigated the assembly and function of Tgl. Our results suggest that Tgl may be present at various levels within the coats and that its assembly is independent of its activity. We also found that in addition to GerQ, three other polypeptides are more extractable from spores of a tgl deletion mutant or from those with an inactive Tgl. The complete insolubilization of at least two of the Tgl-controlled polypeptides occurs several hours later than when the assembly of Tgl is first detected. Tgl is required for the structural integrity of the outer coat layers, and increased production of Tgl interferes with the assembly and structure of the coat and alters its germination properties.

In Escherichia coli, ribonucleases are effectors that rapidly modulate the levels of mRNAs for adaptation to a changing environment. Factors involved in the regulation of these ribonucleases can be relevant for mRNA stability. RNase II is one of the main ribonucleases responsible for exonucleolytic activity in E. coli extracts. We have identified and characterized a new E. coli gene, which was named gmr (gene modulating RNase II). The results demonstrate that a deletion of gmr can be associated with changes in RNase II levels and activity. Western analysis and exoribonuclease activity assays showed a threefold increase in RNase II in the gmr deletion strain. Gmr does not affect RNase II mRNA, but modulates RNase II at the level of protein stability. RNase II protein turnover is slower in the gmr deletion strain. We also show that RNase II levels change in different media, and that this regulation is abolished in a strain lacking gmr. The data presented here show that the regulation of ribonucleolytic activity can depend on growth conditions, and this regulation can be mediated by factors that are not RNases.

Summary: The rnb gene encodes ribonuclease II (RNase II), one of the two major Escherichia coli exonucleases involved in mRNA degradation. In this paper, the rnb transcript is characterized regarding its promoter and terminator regions. The combined results from S1 nuclease protection analysis, DNase I footprinting and gene fusions with IacZ have shown that rnb is expressed from two promoters. S1 nuclease protection analysis and DNA footprinting have shown that rnb has two promoters, P1 and P2. Transcriptional and translational IacZ reporter fusions, constructed to the rnb gene, revealed that P2, the rnb proximal promoter, is stronger than P1. However, P2 is not transcribed in vitro, suggesting that an additional factor is required in vivo. The 3' end of the rnb transcript mapped to a stem-loop structure immediately after the translated region.

Ribonuclease II (RNase II), encoded by the rnb gene, is one of the two major Escherichia coli exonucleases involved in mRNA degradation. Some of the ribonucleases implicated in this process have recently been shown to be inter-regulated. In this paper we studied the effects of the endonucleases RNase E and RNase III in rnb expression. We have shown that RNase E cleaves the rnb message internally: when this ribonuclease is inactivated rnb mRNA accumulates with a concomitant increase in RNase II activity. RNase III also affects RNase II expression but in an indirect way. We discuss these implications for the regulation of mRNA degradation.

Ribonuclease II (encoded by rnb) is one of the two main exonucleases involved in mRNA degradation in Escherichia coli. We report the precise physical mapping of rnb to 29 min on the chromosomal map in the vicinity of pyrF, and clarify the genetic and physical maps of this E. coli chromosomal region. The results were confirmed by the construction of a strain partially deleted for rnb.

Summary The Escherichia coli ribonuclease II (RNase II) is an exonuclease involved in mRNA degradation that hydrolyses single-stranded polyribonucleotides processively in the 3′ to 5′ direction. Sequencing of a 2.2 kb Msel–Rsal fragment containing the rnb gene revealed an open reading frame of 1794 nucleotides that encodes a protein of 598 amino acid residues, whose calculated molecular mass is 67 583 Da. This value is in good agreement with that obtained by sodium dodecyl sulphate/ polyacrylamide gel electrophoresis of polypeptides synthesized by expression with the T7 RNA polymerase/promoter system. This system was also used to confirm the correct orientation of rnb. Translation initiation was confirmed by rnb–lacZ fusions. The mRNA start site was determined by S1 nuclease mapping. Two E. coli mutants harbouring different rnb alleles deficient in RNase II activity were complemented with the expressed fragment carrying the rnb gene.

Bacterial endospores are encased in a complex protein coat, which confers protection against noxious chemicals and influences the germination response. In Bacillus subtilis , over 20 polypeptides are organized into an amorphous undercoat, a lamellar lightly staining inner structure, and an electron-dense outer coat. Here we report on the identification of a polypeptide of about 30 kDa required for proper coat assembly, which was extracted from spores of a gerE mutant. The N-terminal sequence of this polypeptide matched the deduced product of the tasA gene, after removal of a putative 27-residue signal peptide, and TasA was immunologically detected in material extracted from purified spores. Remarkably, deletion of tasA results in the production of asymmetric spores that accumulate misassembled material in one pole and have a greatly expanded undercoat and an altered outer coat structure. Moreover, we found that tasA and gerE mutations act synergistically to decrease the efficiency of spore germination. We show that tasA is the most distal member of a three-gene operon, which also encodes the type I signal peptidase SipW. Expression of the tasA operon is enhanced 2 h after the onset of sporulation, under the control of ς H . When tasA transcription is uncoupled from sipW expression, a presumptive TasA precursor accumulates, suggesting that its maturation depends on SipW. Mature TasA is found in supernatants of sporulating cultures and intracellularly from 2 h of sporulation onward. We suggest that, at an early stage of sporulation, TasA is secreted to the septal compartment. Later, after engulfment of the prespore by the mother cell, TasA acts from the septal-proximal pole of the spore membranes to nucleate the organization of the undercoat region. TasA is the first example of a polypeptide involved in coat assembly whose production is not mother cell specific but rather precedes its formation. Our results implicate secretion as a mechanism to target individual proteins to specific cellular locations during the assembly of the bacterial endospore coat.

Endospores of Bacillus subtilis are encased in a protein shell, known as the spore coat, composed of a lamella-like inner layer and an electron-dense outer layer. We report the identification and characterization of a gene, herein called cotH, located at 300 degrees on the B. subtilis genetic map between two divergent cot genes, cotB and cotG. The cotH open reading frame extended for 1,086 bp and corresponded to a polypeptide of 42.8 kDa. Spores of a cotH null mutant were normally heat, lysozyme, and chloroform resistant but were impaired in germination. The mutant spores were also pleiotropically deficient in several coat proteins, including the products of the previously cloned cotB, -C, and -G genes. On the basis of the analysis of a cotE cotH double mutant, we infer that CotH is probably localized in the inner coat and is involved in the assembly of several proteins in the outer layer of the coat.