Usefulness of the European Epidemic Intelligence Information System in the management of an outbreak of listeriosis, Belgium, 2011.

A cluster of time-linked cases and the identification of a clonal strain suggest the occurrence of an outbreak of listeriosis in Belgium in 2011, presumably due to the consumption of hard cheese made with pasteurised milk and produced by a Belgium manufacturer. The outbreak clone was identified as Listeria monocytogenes serovar 1/2a, sensitive to arsenic and cadmium and of multilocus sequence typing MLST-type 37. Food investigation of this outbreak was facilitated by the European Epidemic Intelligence Information System and data exchanged between French and Belgium listeriosis surveillance systems.

[Follow-up of the first case of Mycobacterium ulcerans infection documented by PCR, genotyping and culture in the Republic of Congo-Brazzaville]. / Suivi du premier cas d'infection à Mycobacterium ulcerans confirmé par culture, PCR et génotypage République du Congo-Brazzaville.

This article presents follow-up data from the first patient in whom Mycobacterium ulcerans infection (MUI) was documented by PCR, genotyping and culture in the Republic of Congo-Brazzaville. Findings show the importance of regular clinical and microbiological evaluation for the disseminated form of the disease. The patient was probably infected in Pointe Noire where MUI has been described but never documented. Culture of specimens collected before antibiotic treatment showed that the bacterium was sensitive to the antibiotics being administered (streptomycin and rifampin) and was identical to isolates from Atlantic-coast regions of West Africa where MUI is endemic. The patient was treated with streptomycin and rifampin for 12 weeks in association with surgery. During treatment clinical examination was performed every day and microbiological analysis every two weeks. The duration of follow-up from the end of specific antibiotic treatment was 26 months. Medical treatment failed to prevent bone involvement and fistulae that were treated by surgery. However medical treatment may have limited dissemination of the disease. Serial microbiological evaluation was useful to detect bone involvement in this patient, but persistent positive gene amplification is not a proof of active disease. This study confirms that MUI is still endemic in the region of Pointe Noire. This finding underlines the need to optimize epidemiologic surveillance, laboratory diagnostic capabilities, and therapeutic management in the Republic of Congo-Brazzaville.

Identification of a new variable number tandem repeat locus in Mycobacterium ulcerans for potential strain discrimination among African isolates.

Intra-species discrimination in the highly clonal pathogen Mycobacterium ulcerans was studied in a diverse collection of isolates by PCR amplification of a short sequence repeat locus containing heterogeneous arrays of tri-nucleotide repeats with an ACC consensus pattern. Post-amplification analysis indicated excellent typeability and identified five M. ulcerans alleles, including a unique Angolan type differentiated for the first time among a genetically conserved cluster of African isolates. These results are consistent with previously investigated independent markers, and provide an additional locus for variable number tandem repeat-based typing of M. ulcerans.

Bacillus subtilis locus encoding a killer protein and its antidote.

We have isolated mutations that block sporulation after formation of the polar septum in Bacillus subtilis. These mutations were mapped to the two genes of a new locus, spoIIS. Inactivation of the second gene, spoIISB, decreases sporulation efficiency by 4 orders of magnitude. Inactivation of the first gene, spoIISA, has no effect on sporulation but it fully restores sporulation of a spoIISB null mutant, indicating that SpoIISB is required only to counteract the negative effect of SpoIISA on sporulation. An internal promoter ensures the synthesis of an excess of SpoIISB over SpoIISA during exponential growth and sporulation. In the absence of SpoIISB, the sporulating cells show lethal damage of their envelope shortly after asymmetric septation, a defect that can be corrected by synthesizing SpoIISB only in the mother cell. However, forced synthesis of SpoIISA in exponentially growing cells or in the forespore leads to the same type of morphological damage and to cell death. In both cases protection against the killing effect of SpoIISA can be provided by simultaneous synthesis of SpoIISB. The spoIIS locus is unique to B. subtilis, and since it is completely dispensable for sporulation its physiological role remains elusive.

Septation, dephosphorylation, and the activation of sigmaF during sporulation in Bacillus subtilis.

Cell-specific activation of transcription factor sigmaF during sporulation in Bacillus subtilis requires the formation of the polar septum and the activity of a serine phosphatase (SpoIIE) located in the septum. The SpoIIE phosphatase indirectly activates sigmaF by dephosphorylating a protein (SpoIIAA-P) in the pathway that controls the activity of the transcription factor. By use of a SpoIIE-GFP fusion protein in time-course and time-lapse experiments and by direct visualization of septa in living cells, we show that SpoIIE is present in the predivisional sporangium, where it often localizes near both cell poles in structures known as E-rings. We also present evidence consistent with the view that SpoIIE is present in both progeny cells after polar division. These findings are incompatible with a model for the control of sigmaF activity in which the phosphatase is simply sequestered to one cell. Instead, we conclude that the function of SpoIIE is subject to regulation, and we present evidence that this occurs in two stages. The first stage, which involves the phosphatase function of SpoIIE, depends on the cell division protein FtsZ and could correspond to the FtsZ-dependent assembly of SpoIIE into E-rings. The second stage occurs after the dephosphorylation of SpoIIAA-P and is dependent on the later-acting, cell-division protein DivIC. Evidence based on the use of modified and mutant forms of the phosphatase protein indicates that SpoIIE blocks the capacity of unphosphorylated SpoIIAA to activate sigmaF until formation of the polar septum is completed.

The SpoIIE phosphatase, the sporulation septum and the establishment of forespore-specific transcription in Bacillus subtilis: a reassessment.

Making a spore in Bacillus subtilis requires the formation of two cells, the forespore and the mother cell, which follow dissimilar patterns of gene expression. Cell specificity is first established in the forespore under the control of the sigma F factor, which is itself activated through the action of the SpoIIE serine phosphatase, an enzyme targeted to the septum between the two cells. Deletion of the 10 transmembrane segments of the SpoIIE protein leads to random distribution of SpoIIE in the cytoplasm. Activation of sigma F is slightly delayed and less efficient than in wild type, but it remains restricted to the forespore in a large proportion of cells and the bacteria sporulate with 30% efficiency. Overexpression of the complete SpoIIE protein in a divIC mutant leads to significant sigma F activity, indicating that the septum requirement for activating sigma F can be bypassed. In contradiction to current models, we propose that genetic asymmetry is not created by unequal distribution of SpoIIE within the sporangium, but by exclusion of an inhibitor of SpoIIE from the forespore. This putative inhibitor would be a cytoplasmic molecule that interacts with SpoIIE and shuts off its phosphatase activity until it disappears specifically from the forespore.

Transient gene asymmetry during sporulation and establishment of cell specificity in Bacillus subtilis.

Sporulation in Bacillus subtilis is initiated by an asymmetric division generating two cells of different size and fate. During a short interval, the smaller forespore harbors only 30% of the chromosome until the remaining part is translocated across the septum. We demonstrate that moving the gene for sigmaF, the forespore-specific transcription factor, in the trapped region of the chromosome is sufficient to produce spores in the absence of the essential activators SpoIIAA and SpoIIE. We propose that transient genetic asymmetry is the device that releases SpoIIE phosphatase activity in the forespore and establishes cell specificity.

Localization of the sporulation protein SpoIIE in Bacillus subtilis is dependent upon the cell division protein FtsZ.

SpollE is an integral membrane protein that governs the establishment of cell-specific gene transcription during the process of sporulation in Bacillus subtilis. Synthesis of SpollE commences shortly after the onset of sporulation, after which the protein localizes at sites of potential cell division near both ends of the sporangium. We now show that, within the limits of resolution of immunofluorescence microscopy, this bipolar pattern of localization observed in early-sporulating cells was superimposable with the bipolar pattern of localization of the cell division protein FtsZ. The localization of SpollE was dependent upon FtsZ because little or no localization was observed along the length of filaments that were generated by depleting sporulating cells for the cell division protein. In contrast, SpollE and FtsZ were found to co-localize at regularly spaced intervals in filaments generated by the use of a temperature-sensitive mutant of the cell division gene divlC. Finally, in cells engineered to synthesize SpollE during growth, SpollE localized at the mid-cell position, coincident with the position of FtsZ, which exhibits a medial pattern of localization in cells undergoing binary fission. These results suggest that the bipolar pattern of localization of SpollE is dictated by the sporulation-induced switch in the position of FtsZ or of other, FtsZ-associated, cell division proteins. Thus, it appears that B. subtilis has co-opted the cell division machinery as a means of localizing a cell fate determinant to the polar septum during sporulation.

SpoIIQ, a forespore-expressed gene required for engulfment in Bacillus subtilis.

A crucial step in converting an actively growing Bacillus subtilis cell into a dormant spore is the formation of a cell within a cell. This unusual structure is created by a phagocytosis-like process in which the larger mother cell progressively engulfs the adjacent smaller forespore. Only mutations blocking engulfment at an early stage and affecting genes expressed in the mother cell have been identified. Here we describe a new locus, spoIIQ, which is transcribed in the forespore and which encodes a membrane-bound protein required at a late stage of engulfment. Immunofluorescence microscopy analysis have shown that SpoIIQ is initially targeted to the septum at the boundary between the two cells and then spreads around the entire membrane of the forespore. Septum targeting requires only the first 52 residues of SpoIIQ as well as unidentified forespore-specific components. Electron-microscopy studies of cells engineered to activate the mother-cell program of gene expression independently of the forespore indicate that other as yet uncharacterized genes are involved in engulfment and that this morphological process is driven from both sides of the forespore envelope.

Plasmids for ectopic integration in Bacillus subtilis.

Plasmids have been constructed that allow integration by a double recombination event at the thrC locus of the Bacillus subtilis (Bs) chromosome. These plasmids can be used either for construction of merodiploid strains and complementation analysis, or for construction of transcriptional fusions to the Escherichia coli lacZ gene. The plasmids contain an antibiotic (An) marker selectable in Bs, as well as an additional An marker outside of the region that can recombine into the chromosome. When used in conjunction with recipient strains containing a third An marker at their thrC locus, these plasmids allow easy identification of transformants issued from a marker exchange event without additional Campbell-type integration. The existing plasmids used for ectopic integration at the amyE locus have been modified similarly.

SpoIIE governs the phosphorylation state of a protein regulating transcription factor sigma F during sporulation in Bacillus subtilis.

Cell-specific activation of the transcription factor sigma F during sporulation in Bacillus subtilis is controlled by a regulatory pathway involving the proteins SpoIIE, SpoIIAA, and SpoIIAB. SpoIIAB is an antagonist of sigma F, and SpoIIAA, which is capable of overcoming SpoIIAB-mediated inhibition of sigma F, is an antagonist of SpoIIAB. SpoIIAA is, in turn, negatively regulated by SpoIIAB, which phosphorylates SpoIIAA on serine 58. SpoIIAA is also positively regulated by SpoIIE, which dephosphorylates SpoIIAA-P, the phosphorylated form of SpoIIAA. Here, isoelectric focusing and Western blot analysis were used to examine the phosphorylation state of SpoIIAA in vivo. SpoIIAA was found to be largely in the phosphorylated state during sporulation in wild-type cells but a significant portion of the protein that was unphosphorylated could also be detected. Consistent with the idea that SpoIIE governs dephosphorylation of SpoIIAA-P, SpoIIAA was entirely in the phosphorylated state in spoIIE mutant cells. Conversely, overexpression of spoIIE led to an increase in the ratio of unphosphorylated SpoIIAA to SpoIIAA-P and caused inappropriate activation of sigma F in the predivisional sporangium. We also show that a mutant form of SpoIIAA (SpoIIAA-S58T) in which serine 58 was replaced with threonine was present exclusively as SpoIIAA-P, a finding that confirms previous biochemical evidence that the mutant protein is an effective substrate for the SpoIIAB kinase but that SpoIIAA-S58T-P cannot be dephosphorylated by SpoIIE. We conclude that SpoIIE plays a crucial role in controlling the phosphorylation state of SpoIIAA during sporulation and thus in governing the cell-specific activation of sigma F.

Molecular genetics of sporulation in Bacillus subtilis.

The process of sporulation in the bacterium Bacillus subtilis proceeds through a well-defined series of morphological stages that involve the conversion of a growing cell into a two-cell-chamber sporangium within which a spore is produced. Over 125 genes are involved in this process, the transcription of which is temporally and spatially controlled by four DNA-binding proteins and five RNA polymerase sigma factors. Through a combination of genetic, biochemical, and cell biological approaches, regulatory networks have been elucidated that explicitly link the activation of these sigma factors to landmark events in the course of morphogenesis and to each other through pathways of intercellular communication. Signals targeting proteins to specific subcellular localizations and governing the assembly of macromolecular structures have been uncovered but their nature remains to be determined.

Antibiotic-resistance cassettes for Bacillus subtilis.

The genes encoding resistance to four different antibiotics (erythromycin, kanamycin, tetracycline and spectinomycin) were cloned in the polylinker of various Escherichia coli plasmid vectors. These cassettes can be inserted into cloned Bacillus subtilis (Bs) genes and used to create tagged chromosomal disruptions after recombination into Bs and selection in the presence of the appropriate antibiotic.

Localization of protein implicated in establishment of cell type to sites of asymmetric division.

Asymmetric division in Bacillus subtilis generates progeny cells with dissimilar fates. SpoIIE, a membrane protein required for the establishment of cell type, was shown to localize near sites of potential polar division. SpoIIE initially localizes in a bipolar pattern, coalescing at marks in the cell envelope at which asymmetric division can take place. Then, during division, SpoIIE becomes restricted to the polar septum and is lost from the distal pole. Thus, when division is complete, SpoIIE sits at the boundary between the progeny from which it dictates cell fate by the activation of a cell-specific transcription factor.

Activation of cell-specific transcription by a serine phosphatase at the site of asymmetric division.

Cell fate is determined by cell-specific activation of transcription factor sigma F after asymmetric division during sporulation by Bacillus subtilis. The activity of sigma F is governed by SpoIIAA, SpoIIAB, and SpoIIE, a membrane protein localized at the polar septum. SpoIIAB binds to and inhibits sigma F, and SpoIIAA inhibits SpoIIAB, which prevents SpoIIAB from binding to sigma F. SpoIIAB is also a serine kinase that inactivates SpoIIAA. Here, it is demonstrated that SpoIIE dephosphorylates SpoIIAA-P and overcomes SpoIIAB-mediated inhibition of sigma F. The finding that SpoIIE is a serine phosphatase links asymmetric division to the pathway governing cell-specific gene transcription.

Extracellular signal protein triggering the proteolytic activation of a developmental transcription factor in B. subtilis.

We present biochemical evidence for an intercellular signal transduction pathway in B. subtilis. This pathway governs the conversion of the proprotein pro-sigma E to mature transcription factor sigma E. Proteolytic processing is mediated by the membrane protein SpollGA and is triggered by the inferred extracellular signal protein SpollR. A factor in conditioned medium from B. subtilis cells engineered to produce SpollR during growth triggered processing in protoplasts of B. subtilis cells that had been engineered to produce SpollGA and pro-sigma E. The factor was also detected in, and partially purified from, extracts of SpollR-producing cells of E. coli. We speculate that SpollGA is both a receptor and a protease and the SpollR interacts with SpollGA on the outside of the cytoplasmic membrane, activating the intracellular protease domain of SpollGA.

Transcription of spoIVB is the only role of sigma G that is essential for pro-sigma K processing during spore formation in Bacillus subtilis.

Activation of pro-sigma K processing in the mother cell at late stages of sporulation in Bacillus subtilis requires the presence of active sigma G in the forespore. Placing the spoIVB gene under the control of sigma F, the early forespore transcription factor, allows sigma K to become active in the absence of sigma G. Therefore, transcription of spoIVB is the only role of sigma G that is essential for the signaling pathway between sigma G and sigma K.

Cell-type specificity during development in Bacillus subtilis: the molecular and morphological requirements for sigma E activation.

Development in Bacillus subtilis involves the formation of two cell types with activation of the transcription factors sigma F in the forespore and sigma E in the mother cell. Activation of sigma E is due to the processing of the inactive precursor pro-sigma E, which requires the putative protease SpoIIGA and the presence of active sigma F. We have introduced missense mutations altering the promoter recognition properties of sigma F. These mutations abolish pro-sigma E processing, suggesting that sigma F is involved through its transcriptional activity and that the processing machinery responds to a signal generated by the product(s) of some unidentified gene(s) transcribed in the forespore. The role of the septum in transducing this signal was investigated. Induction of sigma F during exponential growth in cells producing SpoIIGA and pro-sigma E led to a high level of processing and sigma E activity. Moreover, pro-sigma E was efficiently processed in a mutant strain blocked prior to septation and synthesizing sigma F in active form at the onset of sporulation. Therefore, the sporulation septum is not required for induction of pro-sigma E processing and pro-sigma E can be processed in the same cell in which sigma F is active. These results suggest that some unknown mechanism must exist to prevent sigma E from becoming active in the forespore.

Cell-cell signaling pathway activating a developmental transcription factor in Bacillus subtilis.

Transcription in the mother cell at early stages of sporulation in Bacillus subtilis is controlled by sigma E, a sigma factor that is synthesized in the predivisional cell as an inactive larger precursor, pro-sigma E. Activation of sigma E depends on sigma F, the factor that governs transcription in the forespore. Genetic experiments have indicated that transduction of the activation signal from the forespore to the mother cell requires the products of some genes belonging to the sigma F-controlled regulon. We have identified and characterized a sigma F-dependent gene, csfX, encoding a protein necessary and sufficient for triggering processing of pro-sigma E. The CsfX protein contains a typical amino-terminal signal sequence suggesting that, although synthesized in the forespore, it may act across the septum to activate the membrane-bound enzyme that is responsible for pro-sigma E processing in the mother cell.

Identification and characterization of the Bacillus subtilis spoIIP locus.

We have identified an additional sporulation gene, named spoIIP, in the region of the Bacillus subtilis chromosome located immediately downstream of the gpr gene (227 degrees on the genetic map). A null mutation of spoIIP arrests sporulation at an early stage of engulfment (stage IIii), a phenotype similar to that already described for spoIID and spoIIM mutants. This gene encodes a 401-residue polypeptide, which is predicted to be anchored in the membrane, most of the protein being localized outside the cytoplasm. The spoIIP gene is transcribed from a promoter located in the interval between the gpr and the spoIIP reading frames. This promoter has the structural and genetic characteristics of a sigma E-dependent promoter. Transcription of spoIIP is abolished by a mutation in spoIIGB, the gene encoding sigma E, and can be induced during exponential growth in cells engineered to produce an active form of sigma E. Plasmid integration-excision experiments leading to the formation of genetic mosaics during sporulation indicate that as with SpoIID and SpoIIM, SpoIIP is required only in the mother cell. Disruption of spoIIP had little or no effect on the expression of sigma F- and sigma E-controlled regulons but inhibited transcription from sigma G-dependent promoters and abolished transcription from promoters under the control of sigma K. We propose that, together with SpoIID and SpoIIM, the SpoIIP protein is involved in the dissolution of the peptidoglycan located in the sporulation septum.