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Regulation of chromosome inheritance is essential to ensure proper transmission of genetic information. To accomplish accurate genome segregation, cells organize their chromosomes and actively separate them prior to cytokinesis. In Bacillus subtilis the Spo0J protein is required for accurate chromosome segregation and it regulates the developmental switch from vegetative growth to sporulation. Spo0J is a DNA-binding protein that recognizes at least eight identified parS sites located near the origin of replication. As judged by fluorescence microscopy, Spo0J forms discrete foci associated with the oriC region of the chromosome throughout the cell cycle. In an attempt to determine the mechanisms utilized by Spo0J to facilitate productive chromosome segregation, we have investigated the DNA binding activity of Spo0J. In vivo we find Spo0J associates with several kilobases of DNA flanking its specific binding sites (parS) through a parS-dependent nucleation event that promotes lateral spreading of Spo0J along the chromosome. Using purified components we find that Spo0J has the ability to coat non-specific DNA substrates. These 'Spo0J domains' provide large structures near oriC that could potentially demark, organize or localize the origin region of the chromosome.

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.

Bacterial cells are much smaller and have a much simpler overall structure and organization than eukaryotes. Several prominent differences in cell organization are relevant to the mechanisms of chromosome segregation, particularly the lack of an overt chromosome condensation/decondensation cycle and the lack of a microtubule-based spindle. Although bacterial chromosomes have a rather dispersed appearance, they nevertheless have an underlying high level of spatial organization. During the DNA replication cycle, early replicated (oriC) regions are localized towards the cell poles, whereas the late replicated terminus (terC) region is medially located. This spatial organization is thought to be driven by an active segregation mechanism that separates the sister chromosomes continuously as replication proceeds. Comparisons of various well-characterized bacteria suggest that the mechanisms of chromosome segregation are likely to be diverse, and that in many bacteria, multiple overlapping mechanisms may contribute to efficient segregation. One system in which the molecular mechanisms of chromosome segregation are beginning to be elucidated is that of sporulating cells of Bacillus subtilis. The key components of this system have been identified, and their functions are understood, in outline. Although this system appears to be specialized, most of the functions are conserved widely throughout the bacteria.

In bacteria, genes are often expressed from multiple promoters to allow for a greater spectrum of regulation. Transcription of rRNA genes in Escherichia coli uses two promoters, rrn P1 and rrn P2. Under the conditions examined previously, the P1 and P2 promoters were regulated in response to many of the same changes in nutritional conditions. We report here that rrn P2 promoters play unique roles in rRNA expression during transitional situations. rrn P2 promoters play a dominant role in rRNA synthesis as cells enter into and persist in stationary phase. rrn P2 promoters also play a role in the rapid increases in rRNA synthesis that occur during outgrowth from stationary phase and during the initial stages of rapid shifts to richer media. We demonstrate that rrnB P2 directly senses the concentrations of guanosine 5'-disphosphate 3'-diphosphate (ppGpp) and the initiating nucleoside triphosphate (iNTP), thereby accounting, at least in part, for the observed patterns of regulation. Our work significantly extends previous information about the regulators responsible for control of the rrn P2 promoters and the relationship between the tandem rRNA promoters.

The mechanosensitive (MS) channels MscS and MscL are essential for the survival of hypoosmotic shock by Escherichia coli cells. We demonstrate that MscS and MscL are induced by osmotic stress and by entry into stationary phase. Reduced levels of MS proteins and reduced expression of mscL- and mscS-LacZ fusions in an rpoS mutant strain suggested that the RNA polymerase holoenzyme containing sigmaS is responsible, at least in part, for regulating production of MS channel proteins. Consistent with the model that the effect of sigmaS is direct, the MscS and MscL promoters both use RNA polymerase containing sigmaS in vitro. Conversely, clpP or rssB mutations, which cause enhanced levels of sigmaS, show increased MS channel protein synthesis. RpoS null mutants are sensitive to hypoosmotic shock upon entry into stationary phase. These data suggest that MscS and MscL are components of the RpoS regulon and play an important role in ensuring structural integrity in stationary phase bacteria.

The control of ribosomal RNA transcription is one of the most enduring issues in molecular microbiology, having been subjected to intense scrutiny for over 50 years. Rapid changes in rRNA expression occur during transitions in the bacterial growth cycle and following nutritional shifts during exponential growth. Genetic approaches and measurements of initiating nucleoside triphosphate (iNTP) and guanosine 5'-diphosphate, 3'-diphosphate (ppGpp) concentrations and of rRNA promoter activities showed that rapid changes in the concentrations of iNTPs and ppGpp account for the rapid changes in rRNA expression. The two regulatory signals are nonredundant: changes in iNTP concentration dominate regulation during outgrowth from stationary phase, whereas changes in ppGpp concentration are responsible for regulation following upshifts and downshifts during exponential phase. The results suggest a molecular logic for the use of two homeostatic regulatory mechanisms to monitor different aspects of ribosome activity and provide general insights into the nature of overlapping regulatory circuits.

The seven rRNA operons in Escherichia coli each contain two promoters, rrn P1 and rrn P2. Most previous studies have focused on the rrn P1 promoters. Here we report a systematic analysis of the activity and regulation of the rrnB P2 promoter in order to define the intrinsic properties of rrn P2 promoters and to understand better their contributions to rRNA synthesis when they are in their natural setting downstream of rrn P1 promoters. In contrast to the conclusions reached in some previous studies, we find that rrnB P2 is regulated: it displays clear responses to amino acid availability (stringent control), rRNA gene dose (feedback control), and changes in growth rate (growth rate-dependent control). Stringent control of rrnB P2 requires the alarmone ppGpp, but growth rate-dependent control of rrnB P2 does not require ppGpp. The rrnB P2 core promoter sequence (-37 to +7) is sufficient to serve as the target for growth rate-dependent regulation.