Construction of a cloning site near one end of Tn917 into which foreign DNA may be inserted without affecting transposition in Bacillus subtilis or expression of the transposon-borne erm gene

Youngman, Philip; Perkins, John B.; Losick, Richard

doi:10.1016/0147-619x(84)90061-1

Cited by 381 publications

(225 citation statements)

References 29 publications

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“…B. subtilis strains were derivatives of the wildtype strain PY79 (27) and are listed in Table S1. All general methods were carried out as described (28).…”

Section: Methodsmentioning

confidence: 99%

Spatial organization of a replicating bacterial chromosome

Berlatzky

Rouvinski

Ben-Yehuda

2008

Proc. Natl. Acad. Sci. U.S.A.

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Emerging evidence indicates that the global organization of the bacterial chromosome is defined by its physical map. This architectural understanding has been gained mainly by observing the localization and dynamics of specific chromosomal loci. However, the spatial and temporal organization of the entire mass of newly synthesized DNA remains elusive. To visualize replicated DNA within living cells, we developed an experimental system in the bacterium Bacillus subtilis whereby fluorescently labeled nucleotides are incorporated into the chromosome as it is being replicated. Here, we present the first visualization of replication morphologies exhibited by the bacterial chromosome. At the start of replication, newly synthesized DNA is translocated via a helical structure from midcell toward the poles, where it accumulates. Next, additionally synthesized DNA forms a second, visually distinct helix that interweaves with the original one. In the final stage of replication, the space between the two helices is filled up with the very last synthesized DNA. This striking geometry provides insight into the three-dimensional conformation of the replicating chromosome.Bacillus subtilis ͉ bacterial cell biology ͉ DNA replication ͉ nucleoid ͉ replisome F aithful DNA replication and segregation are essential for all prokaryotic and eukaryotic cells to maintain their genomic integrity. The topological features of DNA, combined with its enormous length, raise the fundamental questions of how replication is organized spatially and what is the geometry of the replicating chromosome. These questions are even more crucial for bacteria, because they are capable of initiating a new round of DNA replication before the previous round has been completed, resulting in multifork replication (1). Moreover, bacteria lack a nucleus compartmentalizing their DNA; instead the bacterial DNA mass (nucleoid) occupies the majority of the cytoplasmic space. Nevertheless, the nucleoid appears as a well organized structure in which chromosome replication and partitioning are carried out with remarkable spatial and temporal regulation (2-9).Like many bacteria, Bacillus subtilis has a single circular chromosome (Ϸ4,200 kilobase pairs), where DNA replication is initiated from a single origin (oriC) and proceeds bidirectionally. Visualization of specific B. subtilis chromosomal loci has revealed that, in general, the oriC regions are duplicated at the cell center and then move toward opposite poles, whereas the unduplicated terminus region remains centrally located throughout the replication process (10-14). The replication machinery (replisome) is composed of two complexes, one for each replication fork, that tend to remain at midcell during chromosome duplication (15,16). It has been proposed that unduplicated DNA is pulled into the replisome and newly synthesized DNA is extruded bidirectionally from the complex (4, 16).The spatial and temporal organization of the replicating bacterial chromosome has been elucidated mainly by observing the localization a...

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“…B. subtilis strains were derivatives of the wildtype strain PY79 (27) and are listed in Table S1. All general methods were carried out as described (28).…”

Section: Methodsmentioning

confidence: 99%

Spatial organization of a replicating bacterial chromosome

Berlatzky

Rouvinski

Ben-Yehuda

2008

Proc. Natl. Acad. Sci. U.S.A.

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“…The B. subtilis prototroph PY79 (Youngman et al, 1984) was used as the wild-type strain and for the generation of all mutant derivatives. Mutant strains were constructed by transformation of PY79 with genomic DNA from the different strains carrying the genes of interest substituted with antibiotic-resistance markers.…”

Section: Methodsmentioning

confidence: 99%

Characterization of tRNACys processing in a conditional Bacillus subtilis CCase mutant reveals the participation of RNase R in its quality control

et al. 2010

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We generated a conditional CCase mutant of Bacillus subtilis to explore the participation in vivo of the tRNA nucleotidyltransferase (CCA transferase or CCase) in the maturation of the singlecopy tRNA Cys , which lacks an encoded CCA 39 end. We observed that shorter tRNA Cys species, presumably lacking CCA, only accumulated when the inducible Pspac : cca was introduced into an rnr mutant strain, but not in combination with pnp. We sequenced the tRNA 39 ends produced in the various mutant tRNA Cys species to detect maturation and decay intermediates and observed that decay of the tRNA Cys occurs through the addition of poly(A) or heteropolymeric tails. A few clones corresponding to full-size tRNAs contained either CCA or other C and/or A sequences, suggesting that these are substrates for repair and/or decay. We also observed editing of tRNA Cys at position 21, which seems to occur preferentially in mature tRNAs. Altogether, our results provide in vivo evidence for the participation of the B. subtilis cca gene product in the maturation of tRNAs lacking CCA. We also suggest that RNase R exoRNase in B. subtilis participates in the quality control of tRNA.

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“…All strains were derivatives of PY79 (Youngman et al 1984) and were built with the following constructs: spoIIE::erm (representing a replacement of most of the spoIIE-coding sequence with erm; laboratory stock); thr::spoIIQ-lacZ (Arigoni et al 1999 (Kenney and Moran 1987); ftsZ⍀pPL80 (pPL80 is a derivative of pAG58 generously provided by P. Levin, J. Quisel, and A. Grossman, pers. comm.…”

Section: Strain Constructionmentioning

confidence: 99%

Septation, dephosphorylation, and the activation of sigma F during sporulation in Bacillus subtilis

King

Dreesen²,

Stragier³

et al. 1999

Genes & Development

124

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Cell-specific activation of transcription factor F during sporulation in Bacillus subtilis requires the formation of the polar septum and the activity of a serine phosphatase (SpoIIE) located in the septum. The SpoIIE phosphatase indirectly activates F by dephosphorylating a protein (SpoIIAA-P) in the pathway that controls the activity of the transcription factor. By use of a SpoIIE-GFP fusion protein in time-course and time-lapse experiments and by direct visualization of septa in living cells, we show that SpoIIE is present in the predivisional sporangium, where it often localizes near both cell poles in structures known as E-rings. We also present evidence consistent with the view that SpoIIE is present in both progeny cells after polar division. These findings are incompatible with a model for the control of F activity in which the phosphatase is simply sequestered to one cell. Instead, we conclude that the function of SpoIIE is subject to regulation, and we present evidence that this occurs in two stages. The first stage, which involves the phosphatase function of SpoIIE, depends on the cell division protein FtsZ and could correspond to the FtsZ-dependent assembly of SpoIIE into E-rings. The second stage occurs after the dephosphorylation of SpoIIAA-P and is dependent on the later-acting, cell-division protein DivIC. Evidence based on the use of modified and mutant forms of the phosphatase protein indicates that SpoIIE blocks the capacity of unphosphorylated SpoIIAA to activate F until formation of the polar septum is completed.

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Construction of a cloning site near one end of Tn917 into which foreign DNA may be inserted without affecting transposition in Bacillus subtilis or expression of the transposon-borne erm gene

Cited by 381 publications

References 29 publications

Spatial organization of a replicating bacterial chromosome

Spatial organization of a replicating bacterial chromosome

Characterization of tRNACys processing in a conditional Bacillus subtilis CCase mutant reveals the participation of RNase R in its quality control

Septation, dephosphorylation, and the activation of sigma F during sporulation in Bacillus subtilis

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