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DNA replication is tightly controlled to ensure accurate inheritance of genetic information. In all organisms, initiator proteins possessing AAA+ (ATPases associated with various cellular activities) domains bind replication origins to license new rounds of DNA synthesis (1). In bacteria the master initiator protein, DnaA, is highly conserved and has two crucial DNA binding activities (2). DnaA monomers recognize the replication origin (oriC) by binding double-stranded DNA sequences (DnaA-boxes); subsequently, DnaA filaments assemble and promote duplex unwinding by engaging and stretching a single DNA strand (3-5). While the specificity for duplex DnaA-boxes by DnaA has been appreciated for over 30 years, the sequence specificity for single-strand DNA binding has remained unknown. Here we identify a new indispensable bacterial replication origin element composed of a repeating trinucleotide motif that we term the DnaA-trio. We show that the function of the DnaA-trio is to stabilize DnaA filaments on a single DNA strand, thus providing essential precision to this binding mechanism. Bioinformatic analysis detects DnaA-trios in replication origins throughout the bacterial kingdom, indicating that this element is part of the core oriC structure. The discovery and characterization of the novel DnaA-trio extends our fundamental understanding of bacterial DNA replication initiation, and because of the conserved structure of AAA+ initiator proteins these findings raise the possibility of specific recognition motifs within replication origins of higher organisms., |\ The master bacterial DNA replication initiator, DnaA, is a highly conserved multifunctional protein that utilizes distinct domains to achieve its two key DNA binding activities. DnaA recognizes double-stranded (ds)DNA [...]

This paper presents, from the perspectives of both social scientists and microbiologists, a case study of the implementation and practice of Responsible Innovation (RI) in a UK-based synthetic biology project. We highlight the impact of interdisciplinary working and examine the benefits that arise from creating the time and space for shared reflection on research. Our discussions over the course of the project included concerns about the potential escape to the environment of laboratory-constructed genetic material and alternatives to the role that antibiotic resistance genes play in synthetic biology. As a result, the design of a key product of the project was altered in ways that go beyond normal institutional requirements. We highlight our view of the importance of continued interdisciplinary collaboration and the utility of the framework of Responsible Innovation in achieving this. We conclude with observations on the difficulties of sustaining such collaborations. [ABSTRACT FROM AUTHOR]

SMC complexes, loaded at ParB-parS sites, are key mediators of chromosome organization in bacteria. ParA/Soj proteins interact with ParB/Spo0J in a pathway involving adenosine triphosphate (ATP)-dependent dimerization and DNA binding, facilitating chromosome segregation in bacteria. In Bacillus subtilis, ParA/Soj also regulates DNA replication initiation and along with ParB/Spo0J is involved in cell cycle changes during endospore formation. The first morphological stage in sporulation is the formation of an elongated chromosome structure called an axial filament. Here, we show that a major redistribution of SMC complexes drives axial filament formation in a process regulated by ParA/Soj. Furthermore, and unexpectedly, this regulation is dependent on monomeric forms of ParA/Soj that cannot bind DNA or hydrolyze ATP. These results reveal additional roles for ParA/Soj proteins in the regulation of SMC dynamics in bacteria and yet further complexity in the web of interactions involving chromosome replication, segregation and organization, controlled by ParAB and SMC. [ABSTRACT FROM AUTHOR]

To link to full-text access for this article, visit this link: http://dx.doi.org/10.1016/j.cell.2008.07.044 Byline: Heath Murray (1), Jeff Errington (1) Keywords: CELLBIO; CELLCYCLE; MICROBIO Abstract: Regulation of DNA replication and segregation is essential for all cells. Orthologs of the plasmid partitioning genes parA, parB, and parS are present in bacterial genomes throughout the prokaryotic evolutionary tree and are required for accurate chromosome segregation. However, the mechanism(s) by which parABS genes ensure proper DNA segregation have remained unclear. Here we report that the ParA ortholog in B. subtilis (Soj) controls the activity of the DNA replication initiator protein DnaA. Subcellular localization of several Soj mutants indicates that Soj acts as a spatially regulated molecular switch, capable of either inhibiting or activating DnaA. We show that the classical effect of Soj inhibiting sporulation is an indirect consequence of its action on DnaA through activation of the Sda DNA replication checkpoint. These results suggest that the pleiotropy manifested by chromosomal parABS mutations could be the indirect effects of a primary activity regulating DNA replication initiation. Author Affiliation: (1) Centre for Bacterial Cell Biology, Institute for Cell and Molecular Biosciences, Newcastle University, Framlington Place, Newcastle Upon Tyne, NE2 4HH, UK Article History: Received 3 March 2008; Revised 25 May 2008; Accepted 31 July 2008 Article Note: (miscellaneous) Published: October 2, 2008

Author(s): Tomas T. Richardson; Omar Harran; Heath Murray Nature 534, 412416 (2016); doi:10.1038/nature17962 In Fig. 2c of this Letter, the rectangle at the bottom right corner was erroneously labelled ADP [...]

For bacteria to proliferate they must duplicate their genetic material so that it can be passed to their progeny. This requires that DNA replication is coordinated with cell growth and division. In the natural environment bacterial growth is dynamic and strongly influenced by changes in nutrient availability. Recent studies have found that bacteria utilize a range of regulatory systems, many of them species-specific, to coordinate DNA replication with cell growth. This variability likely reflects the diverse lifestyles of different bacterial types. [ABSTRACT FROM AUTHOR]

Bacterial genomes typically consist of a single chromosome and, optionally, one or more plasmids. But whole-genome sequencing reveals about ten per-cent of them to be multipartite, with additional replicons which by size and indispensability are considered secondary chromosomes. This raises the questions of how their replication and partition is managed without compromising genome stability and of how such genomes arose. Vibrio cholerae, with a 1 Mb replicon in addition to its 3 Mb chromosome, is the only species for which maintenance of a multipartite genome has been investigated. In this study we have explored the more complex genome of Burkholderia cenocepacia (strain J2315). It comprises an extra replicon (c2) of 3.21 Mb, comparable in size to the3.87Mb main chromosome (c1), another extra replicon(c3) of 0.87 Mb and a plasmid of 0.09 Mb. The replication origin of c1 is typically chromosomal and those of c2 and c3 are plasmid-like; all are replicated bidirectionally. Fluorescence microscopy of tagged origins indicates that all initiate replication at mid-cell and segregate towards the cell quarter positions sequentially, c1-c2-p1/c3. c2 segregation is as well-phased with the cell cycle as c1, implying that this plasmid-like origin has become subject to regulation not typical of plasmids; in contrast, c3 segregates more randomly through the cycle. Disruption of individual Par systems by deletion of parAB or by addition of parS sites showed each Par system to govern the positioning of its own replicon only. Inactivation of c1, c2 and c3 Par systems not only reduced growth rate, generated anucleate cells and compromised viability but influenced processes beyond replicon partition, notably regulation of replication, chromosome condensation and cell size determination. In particular, the absence of the c1 ParA protein altered replication of all three chromosomes, suggesting that the partition system of the main chromosome is a major participant in the choreography of the cell cycle. [ABSTRACT FROM AUTHOR]

The prokaryotic nucleoid-associated protein (NAP) HU is both highly conserved and ubiquitous. Deletion of HU causes pleiotropic phenotypes, making it difficult to uncover the critical functions of HU within a bacterial cell. In their recent work, Karaboja and Wang (J Bacteriol 204:e00119-22, 2022, https://doi.org/10.1128/JB.00119-22) show that one essential function of Bacillus subtilis HU (HBsu) is to drive the DnaA-dependent initiation of DNA replication at the chromosome origin. We discuss the possible roles of HBsu in replication initiation and other essential cellular functions. [ABSTRACT FROM AUTHOR]

In many bacteria the rate of DNA replication is linked with cellular physiology to ensure that genome duplication is coordinated with growth. Nutrient-mediated growth rate control of DNA replication initiation has been appreciated for decades, however the mechanism(s) that connects these cell cycle activities has eluded understanding. In order to help address this fundamental question we have investigated regulation of DNA replication in the model organism Bacillus subtilis. Contrary to the prevailing view we find that changes in DnaA protein level are not sufficient to account for nutrient-mediated growth rate control of DNA replication initiation, although this regulation does require both DnaA and the endogenous replication origin. We go on to report connections between DNA replication and several essential cellular activities required for rapid bacterial growth, including respiration, central carbon metabolism, fatty acid synthesis, phospholipid synthesis, and protein synthesis. Unexpectedly, the results indicate that multiple regulatory systems are involved in coordinating DNA replication with cell physiology, with some of the regulatory systems targeting oriC while others act in a oriC-independent manner. We propose that distinct regulatory systems are utilized to control DNA replication in response to diverse physiological and chemical changes. [ABSTRACT FROM AUTHOR]

Chromosome copy number in cells is controlled so that the frequency of initiation of DNA replication matches that of cell division. In bacteria, this is achieved through regulation of the interaction between the initiator protein DnaA and specific DNA elements arrayed at the origin of replication. DnaA assembles at the origin and promotes DNA unwinding and the assembly of a replication initiation complex. SirA is a DnaA-interacting protein that inhibits initiation of replication in diploid B acillus subtilis cells committed to the developmental pathway leading to formation of a dormant spore. Here we present the crystal structure of SirA in complex with the N-terminal domain of DnaA revealing a heterodimeric complex. The interacting surfaces of both proteins are α-helical with predominantly apolar side-chains packing in a hydrophobic interface. Site-directed mutagenesis experiments confirm the importance of this interface for the interaction of the two proteins in vitro and in vivo. Localization of GFP- SirA indicates that the protein accumulates at the replisome in sporulating cells, likely through a direct interaction with DnaA. The SirA interacting surface of DnaA corresponds closely to the HobA-interacting surface of DnaA from H elicobacter pylori even though HobA is an activator of DnaA and SirA is an inhibitor. [ABSTRACT FROM AUTHOR]

When starved, Bacillus subtilis cells can enter the developmental programme of endospore formation by activation of the master transcriptional regulator Spo0 A. Correct chromosome copy number is crucial for the production of mature and fully resistant spores. The production and maintenance of one chromosome for the mother cell and one copy for the forespore requires accurate co-ordination between DNA replication and initiation of sporulation. Here, we show that Spo0 A regulates chromosome copy number by directly binding to a number of Spo0 A binding sites that are present near the origin of replication ( oriC). We demonstrate that cells lacking three specific Spo0 A binding sites at oriC display increased chromosome copy numbers when sporulation is induced. Our data support the hypothesis that Spo0 A directly controls DNA replication during sporulation by binding to oriC. [ABSTRACT FROM AUTHOR]

Control of DNA replication initiation is essential for normal cell growth. A unifying characteristic of DNA replication initiator proteins across the kingdoms of life is their distinctive AAA+ nucleotide-binding domains. The bacterial initiator DnaA assembles into a right-handed helical oligomer built upon interactions between neighbouring AAA+ domains, that in vitro stretches DNA to promote replication origin opening. The Bacillus subtilis protein Soj/ParA has previously been shown to regulate DnaA-dependent DNA replication initiation; however, the mechanism underlying this control was unknown. Here, we report that Soj directly interacts with the AAA+ domain of DnaA and specifically regulates DnaA helix assembly. We also provide critical biochemical evidence indicating that DnaA assembles into a helical oligomer in vivo and that the frequency of replication initiation correlates with the extent of DnaA oligomer formation. This work defines a significant new regulatory mechanism for the control of DNA replication initiation in bacteria. [ABSTRACT FROM AUTHOR]

Phage φ29 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 φ29 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 φ29 genome. In addition, we demonstrate that Spo0A, the master regulator for initiation of sporulation, suppresses φ29 development by repressing the main early φ29 promoters via different and novel mechanisms and also by preventing activation of the single late φ29 promoter. [ABSTRACT FROM AUTHOR]

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. [Copyright &y& Elsevier]

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. [ABSTRACT FROM AUTHOR]