The transcription factor Spo0A is a master regulator for entry into sporulation in Bacillus subtilis and also regulates expression of the virulent B. subtilis phage /29. Here, we describe a novel function for Spo0A, being an inhibitor of DNA replication of both, the /29 genome and the B. subtilis chromosome. Binding of Spo0A near the /29 DNA ends, constituting the two origins of replication of the linear /29 genome, prevents formation of /29 protein p6-nucleoprotein initiation complex resulting in inhibition of /29 DNA replication. At the B. subtilis oriC, binding of Spo0A to specific sequences, which mostly coincide with DnaA-binding sites, prevents open complex formation. Thus, by binding to the origins of replication, Spo0A prevents the initiation step of DNA replication of either genome. The implications of this novel role of Spo0A for phage /29 development and the bacterial chromosome replication during the onset of sporulation are discussed.

Phage phi29 is a virulent phage of Bacillus subtilis with no known lysogenic cycle. Indeed, lysis occurs rapidly following infection of vegetative cells. Here, we show that phi29 possesses a powerful strategy that enables it to adapt its infection strategy to the physiological conditions of the infected host to optimize its survival and proliferation. Thus, the lytic cycle is suppressed when the infected cell has initiated the process of sporulation and the infecting phage genome is directed into the highly resistant spore to remain dormant until germination of the spore. We have also identified two host-encoded factors that are key players in this adaptive infection strategy. We present evidence that chromosome segregation protein Spo0J is involved in spore entrapment of the infected phi29 genome. In addition, we demonstrate that Spo0A, the master regulator for initiation of sporulation, suppresses phi29 development by repressing the main early phi29 promoters via different and novel mechanisms and also by preventing activation of the single late phi29 promoter.

Little is known about the organization or proteins involved in membrane-associated replication of prokaryotic genomes. Here we show that the actin-like MreB cytoskeleton of the distantly related bacteria Escherichia coli and Bacillus subtilis is required for efficient viral DNA replication. Detailed analyses of B. subtilis phage 29 showed that the MreB cytoskeleton plays a crucial role in organizing phage DNA replication at the membrane. Thus, phage double-stranded DNA and components of the 29 replication machinery localize in peripheral helix-like structures in a cytoskeleton-dependent way. Importantly, we show that MreB interacts directly with the 29 membrane-protein p16.7, responsible for attaching viral DNA at the cell membrane. Altogether, the results reveal another function for the MreB cytoskeleton and describe a mechanism by which viral DNA replication is organized at the bacterial membrane.Bacillus subtilis ͉ phage 29 G enes of the mreB family encode homologues of eukaryotic actin (1, 2) that form a cytoskeleton in most non-spherical bacteria (3-6). MreB proteins form filamentous structures following a helical path around the inner surface of the cytoplasmic membrane (1). These actin-like filaments are continuously remodelled during cell-cycle progression (7-11). Evidence is accumulating that the bacterial MreB cytoskeleton plays key roles in several important cellular processes such as cell shape determination, chromosome segregation, and cell polarity (1,3,8,(12)(13)(14)(15)(16). Whereas Gram-negative bacteria have a single mreB gene, Gram-positive bacteria often have multiple mreB homologues. Bacillus subtilis encodes 3 MreB isoforms: MreB, Mbl,.For decades, evidence has been provided that replication of phage DNA, like that of other prokaryotic genomes, occurs at the cytoplasmic membrane (for review see 20). However, little is known about the proteins or their organization in membraneassociated replication of viral genomes in bacteria. Phages 29 and SPP1 infect the Gram-positive bacterium B. subtilis, and phage PRD1 infects the Gram-negative bacterium Escherichia coli. Whereas PRD1 and 29 use the protein-primed mechanism of DNA replication, phage SPP1 replicates its DNA initially via the theta mode and later via a rolling circle mode [reviewed in (21)]. Here we show a key role for the MreB cytoskeleton in phages replicating by different modes in the distantly related bacteria E. coli and B. subtilis. Thus, the efficiency of replication of phage PRD1, and that of phages SPP1 and 29, is severely affected in the absence of an intact cytoskeleton.The underlying mechanism by which the cytoskeleton leads to efficient phage DNA replication was analyzed in detail for B. subtilis phage 29, whose DNA replication has been well characterized in vitro. The 29 genome consists of a linear doublestranded DNA (dsDNA) with a terminal protein (TP) covalently linked at each 5Ј end that is the primer for the initiation of phage DNA replication. Hence, initiation of 29 DNA replication occurs via a so-called protein-prim...

Regulating the expression of individual miRNAs (microRNAs) is important for cell development and function. The up- or down-regulation of the processing of specific miRNA precursors to the mature active form represents one tool to control miRNA concentration and is mediated by proteins that recognize the terminal loop of the RNA precursors. Terminal loop recognition is achieved by the combined action of several RNA-binding domains. The proteins can then regulate the processing by recruiting RNA enzymes, changing the RNA structure and preventing or enhancing the accessibility and processing activity of the core processing complexes. The present review focuses on how terminal loop-binding proteins recognize their RNA targets and mediate their regulatory function(s), and highlights how terminal loop-mediated regulation relates to the broader regulation of mRNA metabolism.

SummaryThe host of the lytic bacteriophage f29 is the sporeforming bacterium Bacillus subtilis. When infection occurs during early stages of sporulation, however, f29 development is suppressed and the infecting phage genome becomes trapped into the developing spore. Recently, we have shown that Spo0A, the key transcriptional regulator for entry into sporulation, is directly responsible for suppression of the lytic f29 cycle in cells having initiated sporulation. Surprisingly, we found that f29 development is suppressed in a subpopulation of logarithmically growing culture and that spo0A is heterogeneously expressed during this growth stage. Furthermore, we showed that kinC and, to a minor extent, kinD, are responsible for heterogeneous expression levels of spo0A during logarithmical growth that are below the threshold to activate sporulation, but sufficient for suppression of the lytic cycle of f29. Whereas spo0A was known to be heterogeneously expressed during the early stages of sporulation, our findings show that this also occurs during logarithmical growth. These insights are likely to have important consequences, not only for the life cycle of f29, but also for B. subtilis developmental processes.

Localization of the miRNA-induced silencing complex to GW/P bodies by GW220/TNGW1 may regulate the fate of target mRNAs.

RNA granules have been observed in different organisms, cell types and under different conditions, and their formation is crucial for the mRNA life cycle. However, very little is known about the molecular mechanisms governing their assembly and disassembly. The aggregation-prone LSCRs (low-sequence-complexity regions), and in particular, the polyQ/N-rich regions, have been extensively studied under pathological conditions due to their role in neurodegenerative diseases. In the present review, we discuss recent in vitro, in vivo and computational data that, globally, suggest a role for polyQ/N regions in RNA granule assembly.

The phi29 family of phages is divided in three groups. Members of groups 1 and 2 infect the spore-forming bacterium Bacillus subtilis. Previous studies showed that group 1 phage phi29 adapts its infection strategy to the physiological state of the host. Thus, the lytic cycle of phi29 is suppressed when cells are infected during the early stages of sporulation and the infecting genome becomes trapped into the spore. A major element of this adaptive strategy is a very sensitive response to the host-encoded Spo0A protein, the key regulator for sporulation activation, which is directly responsible for suppression of phi29 development. Here we analysed if this adaptation is conserved in phage Nf belonging to group 2. The results obtained show that although Nf also possesses the alternative infection strategy, it is clearly less sensitive to Spo0A-mediated suppression than phi29. Sequence determination of the Nf genome revealed striking differences in the number of Spo0A binding site sequences. The results provide evidence that the life style of two highly related phages is distinctly tuned by differences in binding sites for a host-encoded regulatory protein, being a good example of how viruses have evolved to optimally exploit features of their host.