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Genome duplication is essential for the proliferation of cellular life and this process is generally initiated by dedicated replication proteins at chromosome origins. In bacteria, DNA replication is initiated by the ubiquitous DnaA protein, which assembles into an oligomeric complex at the chromosome origin (oriC) that engages both double-stranded DNA (dsDNA) and single-stranded DNA (ssDNA) to promote DNA duplex opening. However, the mechanism of DnaA specifically opening a replication origin was unknown. Here we show that Bacillus subtilis DnaAATP assembles into a continuous oligomer at the site of DNA melting, extending from a dsDNA anchor to engage a single DNA strand. Within this complex, two nucleobases of each ssDNA binding motif (DnaA-trio) are captured within a dinucleotide binding pocket created by adjacent DnaA proteins. These results provide a molecular basis for DnaA specifically engaging the conserved sequence elements within the bacterial chromosome origin basal unwinding system (BUS). Here, the authors show that a DnaA:origin complex promotes specific nucleobase capture from a single DNA strand. It is proposed that this mechanism may play a key role stimulating opening of bacterial chromosome origins. [ABSTRACT FROM AUTHOR]

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]

The human microbiota, which plays an important role in health and disease, uses complex carbohydrates as a major source of nutrients. Utilization hierarchy indicates that the host glycosaminoglycans heparin (Hep) and heparan sulfate (HS) are high-priority carbohydrates for Bacteroides thetaiotaomicron, a prominent member of the human microbiota. The sulfation patterns of these glycosaminoglycans are highly variable, which presents a significant enzymatic challenge to the polysaccharide lyases and sulfatases that mediate degradation. It is possible that the bacterium recruits lyases with highly plastic specificities and expresses a repertoire of enzymes that target substructures of the glycosaminoglycans with variable sulfation or that the glycans are desulfated before cleavage by the lyases. To distinguish between these mechanisms, the components of the B. thetaiotaomicron Hep/HS degrading apparatus were analyzed. The data showed that the bacterium expressed a single-surface endo-acting lyase that cleaved HS, reflecting its higher molecular weight compared with Hep. Both Hep and HS oligosaccharides imported into the periplasm were degraded by a repertoire of lyases, with each enzyme displaying specificity for substructures within these glycosaminoglycans that display a different degree of sulfation. Furthermore, the crystal structures of a key surface glycan binding protein, which is able to bind both Hep and HS and periplasmic sulfatases reveal the major specificity determinants for these proteins. The locus described here is highly conserved within the human gut Bacteroides, indicating that the model developed is of generic relevance to this important microbial community. [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]

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]

Control of DNA replication initiation is essential for cell growth. A unifying characteristic of DNA replication initiator proteins 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 to form an active initiation complex. Recently we developed a unique cross-linking assay that specifically detects ATP-dependent DnaA helix assembly. Here we have utilized this assay to show that two DnaA regulatory proteins in Bacillus subtilis, YabA and DnaD, inhibit DnaA helix formation. These results, in combination with our previous finding that the regulatory factor Soj/ ParA also targets DnaA filament formation, highlight the critical importance of regulating DnaA helix formation during the initiation reaction. Moreover, these observations lead us to suggest that DnaA oligomerization may be the main regulatory step of the initiator assembly pathway in B. subtilis, in contrast to the prevailing model of bacterial DNA replication based on Escherichia coli DnaA where ATP binding appears to be the targeted activity. [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]

Control of DNA replication initiation is essential for bacterial cells to co-ordinate the faithful replication and segregation of their genetic material. The Bacillus subtilis ATPase Soj is a dynamic protein that regulates DNA replication initiation by either inhibiting or activating the DNA replication initiator protein DnaA. Here we report that the key event which switches Soj regulatory activity is a transition in its oligomeric state from a monomer to an ATP-dependent homodimer capable of DNA binding. We show that the DNA binding activity of the Soj dimer is required both for activation of DNA replication initiation and for interaction with Spo0J. Finally, we demonstrate that Spo0J inhibits Soj dimerization by stimulating Soj ATPase activity. The data provide a molecular explanation for the dichotomous regulatory activities of Soj, as well as assigning unique Soj conformations to distinct cellular localization patterns. We discuss how the regulation of Soj ATPase activity by Spo0J could be utilized to control the initiation of DNA replication during the cell cycle. [ABSTRACT FROM AUTHOR]

The phage shock protein (Psp) response in Gram-negative bacteria counteracts membrane stress. Transcription of the PspF regulon ( pspABCDE and pspG) in Escherichia coli is induced upon stresses that dissipate the proton motive force (pmf). Using GFP fusions we have visualized the subcellular localizations of PspA (a negative regulator and effector of Psp) and PspG (an effector of Psp). It has previously been proposed that PspA evenly coates the inner membrane of the cell. We now demonstrate that instead of uniformly covering the entire cell, PspA (and PspG) is highly organized into what appear to be distinct functional classes (complexes at the cell pole and the lateral cell wall). Real-time observations revealed lateral PspA and PspG complexes are highly mobile, but absent in cells lacking MreB. Without the MreB cytoskeleton, induction of the Psp response is still observed, yet these cells fail to maintain pmf under stress conditions. The two spatial subspecies therefore appear to be dynamically and functionally distinct with the polar clusters being associated with sensory function and the mobile complexes with maintenance of pmf. [ABSTRACT FROM AUTHOR]

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. [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]

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. [ABSTRACT FROM AUTHOR]

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 isuggested that the RNA polymerase holoenzyme containing σ[SUPs] is responsible, at least in part, for regulating production of MS channel proteins. Consistent with the model that the effect of σ[SUPs] is direct, the MscS and MscL promoters both use RNA polymerase containing ers in vitro. Conversely, clpP or rssB mutations, which cause enhanced levels of σ[SUPs] 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. [ABSTRACT FROM AUTHOR]

Genome duplication is essential for cell proliferation, and DNA synthesis is generally initiated by dedicated replication proteins at specific loci termed origins. In bacteria, the master initiator DnaA binds the chromosome origin (oriC) and unwinds the DNA duplex to permit helicase loading. However, despite decades of research it remained unclear how the information encoded within oriC guides DnaA‐dependent strand separation. To address this fundamental question, we took a systematic genetic approach in vivo and identified the core set of essential sequence elements within the Bacillus subtilis chromosome origin unwinding region. Using this information, we then show in vitro that the minimal replication origin sequence elements are necessary and sufficient to promote the mechanical functions of DNA duplex unwinding by DnaA. Because the basal DNA unwinding system characterized here appears to be conserved throughout the bacterial domain, this discovery provides a framework for understanding oriC architecture, activity, regulation and diversity. Synopsis: Bacterial replication origin sequences are complex and diverse. In vivo dissection of a basal bacterial chromosome unwinding mechanism in Bacillus subtilis provides a framework for understanding oriC architecture, function and diversity. A B. subtilis strain with a synthetic replication origin allows identification of a minimal set of essential sequences within B. subtilis oriC.Two DnaA‐boxes adjacent to repeating DnaA‐trios are required for DnaA‐dependent strand separation.DnaA‐dependent unwinding could be reconstituted in vitro using just these core origin sequences.DnaA filaments are guided from DnaA‐boxes onto adjacent DnaA‐trios where they stretch ssDNA to promote origin unwinding. [ABSTRACT FROM AUTHOR]