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...