Previous work identified gp56, encoded by the lytic bacteriophage SP01, as responsible for inhibition of Bacillus subtilis cell division during its infection. Assembly of the essential tubulin-like protein FtsZ into a ring-shaped structure at the nascent site of cytokinesis determines the timing and position of division in most bacteria. This FtsZ ring serves as a scaffold for recruitment of other proteins into a mature division-competent structure permitting membrane constriction and septal cell wall synthesis. Here we show that expression of the predicted 9.3-kDa gene product 56 (gp56) of SP01 inhibits later stages of B. subtilis cell division without altering FtsZ ring assembly. GFP-tagged gp56 localizes to the membrane at the site of division. While its localization doesn't interfere with recruitment of early division proteins, gp56 interferes with the recruitment of late division proteins, including Pbp2b and FtsW. Imaging of cells with specific division components deleted or depleted and two-hybrid analysis suggest that gp56 localization and activity depends on its interaction with FtsL. Together these data support a model where gp56 interacts with a central part of the division machinery to disrupt late recruitment of the division proteins involved in septal cell wall synthesis. IMPORTANCE Studies over the past decades have uncovered bacteriophage-encoded factors that interfere with host cell shape or cytokinesis during viral infection. Phage factors that cause cell filamentation that have been investigated to date all act by targeting FtsZ, the conserved prokaryotic tubulin homolog that composes the cytokinetic ring in most bacteria and some groups of archaea. However, the mechanisms of several phage factors that inhibit cytokinesis, including gp56 of bacteriophage SP01 of Bacillus subtilis, remain unexplored. Here, we show that unlike other published examples of phage inhibition of cyotkinesis, gp56 blocks B. subtilis cell division without targeting FtsZ. Rather, it utilizes the assembled FtsZ cytokinetic ring to localize to the division machinery and block recruitment of proteins needed for the septal cell wall synthesis.

15Previous work identified gp56, encoded by the lytic bacteriophage SP01, as responsible for 16 inhibition of Bacillus subtilis cell division during its infection. Assembly of the essential tubulin-17 like protein FtsZ into a ring-shaped structure at the nascent site of cytokinesis determines the 18 timing and position of division in most bacteria. This FtsZ ring serves as a scaffold for 19 recruitment of other proteins into a mature division-competent structure permitting membrane 20 constriction and septal cell wall synthesis. Here we show that expression of the predicted 9.3-21 kDa gene product 56 (gp56) of SP01 inhibits latter stages of B. subtilis cell division without 22altering FtsZ ring assembly. GFP-tagged gp56 localizes to the membrane at the site of division. 48 complement. To achieve this, FtsZ assembly at mid-cell and subsequent division are highly 49 precise, with less than a 1% margin of error, suggesting a highly regulated process (2, 3). 50Blocking FtsZ assembly prevents membrane invagination and septal cell wall synthesis, leading 51 to filamentous, multinucleated cells and eventual cell death (4). 52 53As a conserved protein that is essential for division in most bacteria, FtsZ is an appealing target 54 of study for both physiologically relevant modes of its regulation and for potential development 55 of novel antibiotics (5-7). Included among cellular regulators of FtsZ assembly are proteins 56 encoded in regions of the E. coli genome that originally derived from phage, now turned 57 inactive. Cells have co-opted several of these so-called cryptic phage genes for increased host 58 fitness under particular conditions. These include dicB and dicF of cryptic phage Qin (aka phage 59 Kim) and the kilR (orfE) gene of cyptic phage Rac (8). The RNA product of dicF binds to ftsZ 60 mRNA to inhibit its translation (9), while the DicB peptide interacts with FtsZ inhibitor MinC 61 (10) to target ring assembly independently of its normal regulator MinD, but dependent on ZipA 62 (11). Transient division inhibition by cryptic DicB benefits the host by inhibiting phage receptor 63 proteins ManYZ, enhancing immunity to bacteriophage lambda infection by up to 100-fold (12). 64The KilR peptide of Rac inhibits E. coli division through an unknown Min-independent 65 mechanism that also causes increased loss of rod shape (13). 66 67 Functional bacteriophages also appear to encode factors that transiently block host cell division 68 during infection. Expression of the 0.4 gene of T7 phage or kil of lambda phage both lead to E. 69 coli cell filamentation through direct interference with FtsZ assembly by their protein products 70 (14-16). In both cases, temporary inhibition of host cytokinesis by the phage prior to host lysis 71 results in a subtle competitive advantage for the virus, although the specific nature of these 72 advantages remains unclear. 73 74 Although all of the above factors come from phage that infect E. coli, it is likely that cytokinesis 75 serves as a target for phage in the majority of other bacte...