Checkpoint kinase 1 (CHK1) is a key component of the ATR (ataxia telangiectasia-mutated and Rad3-related)-dependent DNA damage response pathway that protect cells from replication stress, a cell intrinsic phenomenon enhanced by oncogenic transformation. Here, we show that CHK1 is overexpressed and hyperactivated in T-cell acute lymphoblastic leukemia (T-ALL). CHEK1 mRNA is highly abundant in patients of the proliferative T-ALL subgroup and leukemia cells exhibit constitutively elevated levels of the replication stress marker phospho-RPA32 and the DNA damage marker γH2AX. Importantly, pharmacologic inhibition of CHK1 using PF-004777736 or CHK1 short hairpin RNA-mediated silencing impairs T-ALL cell proliferation and viability. CHK1 inactivation results in the accumulation of cells with incompletely replicated DNA, ensuing DNA damage, ATM/CHK2 activation and subsequent ATM- and caspase-3-dependent apoptosis. In contrast to normal thymocytes, primary T-ALL cells are sensitive to therapeutic doses of PF-004777736, even in the presence of stromal or interleukin-7 survival signals. Moreover, CHK1 inhibition significantly delays in vivo growth of xenotransplanted T-ALL tumors. We conclude that CHK1 is critical for T-ALL proliferation and viability by downmodulating replication stress and preventing ATM/caspase-3-dependent cell death. Pharmacologic inhibition of CHK1 may be a promising therapeutic alternative for T-ALL treatment.

Two highly similar RNA polymerase sigma subunits, σF and σG, govern the early and late phases of forespore-specific gene expression during spore differentiation in Bacillus subtilis. σF drives synthesis of σG but the latter only becomes active once engulfment of the forespore by the mother cell is completed, its levels rising quickly due to a positive feedback loop. The mechanisms that prevent premature or ectopic activation of σG while discriminating between σF and σG in the forespore are not fully comprehended. Here, we report that the substitution of an asparagine by a glutamic acid at position 45 of σG (N45E) strongly reduced binding by a previously characterized anti-sigma factor, CsfB (also known as Gin), in vitro, and increased the activity of σG in vivo. The N45E mutation caused the appearance of a sub-population of pre-divisional cells with strong activity of σG. CsfB is normally produced in the forespore, under σF control, but sigGN45E mutant cells also expressed csfB and did so in a σG-dependent manner, autonomously from σF. Thus, a negative feedback loop involving CsfB counteracts the positive feedback loop resulting from ectopic σG activity. N45 is invariant in the homologous position of σG orthologues, whereas its functional equivalent in σF proteins, E39, is highly conserved. While CsfB does not bind to wild-type σF, a E39N substitution in σF resulted in efficient binding of CsfB to σF. Moreover, under certain conditions, the E39N alteration strongly restrains the activity of σF in vivo, in a csfB-dependent manner, and the efficiency of sporulation. Therefore, a single amino residue, N45/E39, is sufficient for the ability of CsfB to discriminate between the two forespore-specific sigma factors in B. subtilis.

The Bacillus subtilis SpoVE integral membrane protein is essential for the heat resistance of spores, probably because of its involvement in spore peptidoglycan synthesis. We found that an SpoVE-yellow fluorescent protein (YFP) fusion protein becomes localized to the forespore during the earliest stages of engulfment, and this pattern is maintained throughout sporulation. SpoVE belongs to a well-conserved family of proteins that includes the FtsW and RodA proteins of B. subtilis. These proteins are involved in bacterial shape determination, although their function is not known. FtsW is necessary for the formation of the asymmetric septum in sporulation, and we found that an FtsW-YFP fusion localized to this structure prior to the initiation of engulfment in a nonoverlapping pattern with SpoVE-cyan fluorescent protein. Since FtsW and RodA are essential for normal growth, it has not been possible to identify loss-of-function mutations that would greatly facilitate analysis of their function. We took advantage of the fact that SpoVE is not required for growth to obtain point mutations in SpoVE that block the development of spore heat resistance but that allow normal protein expression and targeting to the forespore. These mutant proteins will be invaluable tools for future experiments aimed at elucidating the function of members of the SEDS ("shape, elongation, division, and sporulation") family of proteins.Bacterial shape is determined by an extracellular structure composed of peptidoglycan (PG), a rigid polymer of repeated subunits of a disaccharide peptide monomer. This giant macromolecule is found on the outside of the cytoplasmic membrane of nearly all eubacteria. A series of essential and highly conserved enzymes convert UDP-GlcNAc to lipid II, the UDPmuramyl pentapeptide (47). To form mature PG, lipid II is translocated across the cytoplasmic membrane via an uncharacterized mechanism and is added to the preexisting cell wall through transpeptidation and transglycosylation reactions mediated by members of the PBP (penicillin binding protein) family of proteins. Little is known about the mechanism of lipid II transport across the cytoplasmic membrane other than that it is probably dependent on a protein(s) since it does not occur spontaneously across lipid bilayers (46).The integral membrane proteins RodA and FtsW are members of what has been termed the SEDS ("shape, elongation, division, and sporulation") family of integral membrane proteins (16, 18) that are present in all cell wall-containing bacteria. Several lines of evidence are consistent with the participation of RodA and FtsW in the translocation of lipid II during cell elongation and cell division, respectively (17, 19), although this hypothesis has not been subjected to a direct test. For instance, depletion of RodA leads to a block in lateral cell growth (16), although RodA is not strictly essential for cell viability. Null mutants exhibit slow growth and small cell diameters and are viable in minimal medium (7). Also, temperature-sensitive Escheri...

The Bacillus subtilis murB gene, encoding UDP-N-acetylenolpyruvoylglucosamine reductase, a key enzyme in the peptidoglycan (PG) biosynthetic pathway, is embedded in the dcw (for "division and cell wall") cluster immediately upstream of divIB. Previous attempts to inactivate murB were unsuccessful, suggesting its essentiality. Here we show that the cell morphology, growth rate, and resistance to cell wall-active antibiotics of murB conditional mutants is a function of the expression level of murB. In one mutant, in which murB was insertionally inactivated in a merodiploid bearing a second xylose-inducible PxylA-murB allele, DivIB levels were reduced and a normal growth rate was achieved only if MurB levels were threefold that of the wild-type strain. However, expression of an extra copy of divIB restored normal growth at wild-type levels of MurB. In contrast, DivIB levels were normal in a second mutant containing an in-frame deletion of murB (⌬murB) in the presence of the PxylA-murB gene. Furthermore, this strain grew normally with wild-type levels of MurB. During sporulation, the levels of MurB were highest at the time of synthesis of the spore cortex PG. Interestingly, the ⌬murB PxylA-murB mutant did not sporulate efficiently even at high concentrations of inducer. Since high levels of inducer did not interfere with sporulation of a murB ؉ PxylA-murB strain, it appears that ectopic expression of murB fails to support efficient sporulation. These data suggest that coordinate expression of divIB and murB is important for growth and sporulation. The genetic context of the murB gene within the dcw cluster is unique to the Bacillus group and, taken together with our data, suggests that in these species it contributes to the optimal expression of cell division and PG biosynthetic functions during both vegetative growth and spore development.Peptidoglycan (PG) is a critical component of the eubacterial cell envelope, providing mechanical resistance to withstand osmotic pressure (55) and functioning as a major determinant of cell shape, which ultimately also relies on topological information imposed by the actin cytoskeleton (6,10,33,54). PG is also intimately involved in the cell division process and, in spore-forming bacteria, is additionally required for a cytoskeleton-like role during engulfment of the prespore by the mother cell, as well as for the formation of the spore cortex, a modified layer of PG, essential for spore heat resistance (reviewed in reference 17).In all eubacteria examined to date, many of the genes involved in PG biosynthesis and cell division are grouped in the highly conserved dcw (for "division and cell wall") cluster (Fig. 1A). A correlation between bacterial cell shape and the arrangement of genes within the dcw cluster has been suggested (51). Specifically, it appears that rod-shaped bacteria have retained a more conserved and compact dcw cluster, and that transitions to other bacterial shapes have involved rearrangements and loss of gene order conservation within the cluster (51). Other f...

SummaryThe initiation of the developmental process of sporulation in the rod-shaped bacterium Bacillus subtilis involves the activation of the Spo0A response regulator. Spo0A then drives the switch in the site of division septum formation from midcell to a polar position. Activated Spo0A is required for the transcription of key sporulation loci such as spoIIG , which are negatively regulated by the Soj protein. The transcriptional repressing activity of Soj is antagonized by Spo0J, and both proteins belong to the wellconserved Par family of partitioning proteins. Soj has been shown to jump from nucleoid to nucleoid via the cell pole. The dynamic behaviour of Soj is somehow controlled by Spo0J, which prevents the static association of Soj with the nucleoid, and presumably its transcriptional repression activity. Soj in turn is required for the proper condensation of Spo0J foci around the oriC region. The asymmetric partitioning of the sporangial cell requires DivIB and other proteins involved in vegetative (medial) division. We describe an allele of the cell division gene divIB ( divIB80 ) that reduces the cellular levels of DivIB, and affects nucleoid structure and segregation in growing cells, yet has no major impact on cell division. In divIB80 cells Spo0J foci are not correctly condensed and Soj associates statically with the nucleoid. The divIB80 allele prevents transcription of spoIIG , and arrests sporulation prior to the formation of the asymmetric division septum. The defect in Spo0A-dependent gene expression, and the Spo -phenotype can be suppressed by expression of divIB in trans or by deletion of the soj-spo0J locus. However, deletion of the spo0J-soj region does not restore the normal cellular levels of DivIB. Therefore, the reduced levels of DivIB in the divIB80 mutant are sufficient for efficient cell division, but not to sustain a second, earlier function of DivIB related to the activity of the Spo0J/ Soj system of chromosome segregation.