Summary Organization and segregation of replicated chromosomes are essential components of cell division in all organisms. The Bacillus subtilis partitioning protein Spo0J (ParB) has been implicated in both processes but how it functions remains unclear. We have discovered that Spo0J bound to its origin-proximal binding sites (parS) recruits the Structural Maintenance of Chromosomes (SMC) complex to the origin. We demonstrate that the subcellular localization of the SMC complex is disrupted in the absence of Spo0J or the parS sites. Furthermore, the SMC complex co-localizes with Spo0J at the origin and insertion of parS sites near the terminus targets SMC to this position leading to defects in chromosome organization and segregation. Finally, we show that purified SMC binds Spo0J-coated DNA with higher affinity than naked DNA. These data are consistent with a model in which recruitment of SMC to the origin by Spo0J-parS organizes the origin region and promotes efficient chromosome segregation.

Swarming colonies of the bacterium Proteus mirabilis are capable of self-recognition and territorial behavior. Swarms of independent P. mirabilis isolates can recognize each other as foreign and establish a visible boundary where they meet; in contrast, genetically identical swarms merge. The ids genes, which encode self-identity proteins, are necessary but not sufficient for this territorial behavior. Here we have identified two new gene clusters: one (idr) encodes rhs-related products, and another (tss) encodes a putative type VI secretion (T6S) apparatus. The Ids and Idr proteins function independently of each other in extracellular transport and in territorial behaviors; however, these self-recognition systems are linked via this type VI secretion system. The T6S system is required for export of select Ids and Idr proteins. Our results provide a mechanistic and physiological basis for the fundamental behaviors of self-recognition and territoriality in a bacterial model system.

The FtsK/SpoIIIE family of ATP-dependent DNA transporters mediates proper chromosome segregation in dividing bacteria. In sporulating Bacillus subtilis cells, SpoIIIE translocates much of the circular chromosome from the mother cell into the forespore, but the molecular mechanism remains unclear. Using a new assay to monitor DNA transport, we demonstrate that the two arms of the chromosome are simultaneously pumped into the forespore. Up to 70 molecules of SpoIIIE are recruited to the site of DNA translocation and assemble into complexes that could contain 12 subunits. The fusion of the septal membranes during cytokinesis precedes DNA translocation and does not require SpoIIIE, as suggested by analysis of lipid dynamics, serial thin-section electron microscopy, and cell separation by protoplasting. These data support a model for DNA transport in which the transmembrane segments of FtsK/SpoIIIE form linked DNA-conducting channels across the two lipid bilayers of the septum.

We announce the complete genome sequence for Proteus mirabilis strain BB2000, a model system for self recognition. This opportunistic pathogen contains a single, circular chromosome (3,846,754 bp). Comparisons between this genome and that of strain HI4320 reveal genetic variations corresponding to previously unknown physiological and self-recognition differences.

The ligand binding pocket of Cys-loop receptors consists of a number of binding loops termed A-F. Here we examine the 5-HT 3 receptor loop A residues Asn-128, Glu-129 and Phe-130 using modelling, mutagenesis, radioligand binding and functional studies on HEK 293 cells. Replacement of Asn-128 results in receptors that have wild type [ 3 H]granisetron binding characteristics but large changes (ranging from a five-fold decrease to a 1500-fold increase) in the 5-HT EC 50 when compared to wild type receptors. Phe-130 mutant receptors show both increases and decreases in K d and EC 50 values, depending on the amino acid substituted. The most critical of these residues appears to be Glu-129; its replacement with a range of other amino acids results in non-binding and non-functional receptors. Lack of binding and function in some, but not all, of these receptors is due to poor membrane expression. These data suggest that Glu-129 is important primarily for receptor expression, although it may also play a role in ligand binding; Phe-130 is important for both ligand binding and receptor function, and Asn-128 plays a larger role in receptor function than ligand binding. In light of these results, we have created two new homology models of the 5-HT 3 receptor, with alternative positions of loop A. In our preferred model Glu-129 and Phe-130 contribute to the binding site, while the location of Asn-128 immediately behind the binding pocket could contribute to the conformation changes that result in receptor gating. This study provides a new model of the 5-HT 3 receptor binding pocket, and also highlights the importance of experimental data to support modelling studies.