scholarly journals Characterization of the Chromosome Dimer Resolution Site in Caulobacter crescentus

2019 ◽  
Vol 201 (24) ◽  
Author(s):  
Ali Farrokhi ◽  
Hua Liu ◽  
George Szatmari
Keyword(s):  
Cell Division ◽  
Caulobacter Crescentus ◽  
Consensus Sequence ◽  
Markov Modeling ◽  
Control Of Gene Expression ◽  
Site Specific ◽  
Content Type ◽  
Site Specific Recombination ◽  
Resolution System

ABSTRACT Chromosome dimers occur in bacterial cells as a result of the recombinational repair of DNA. In most bacteria, chromosome dimers are resolved by XerCD site-specific recombination at the dif (deletion-induced filamentation) site located in the terminus region of the chromosome. Caulobacter crescentus, a Gram-negative oligotrophic bacterium, also possesses Xer recombinases, called CcXerC and CcXerD, which have been shown to interact with the Escherichia coli dif site in vitro. Previous studies on Caulobacter have suggested the presence of a dif site (referred to in this paper as dif1CC), but no in vitro data have shown any association with this site and the CcXer proteins. Using recursive hidden Markov modeling, another group has proposed a second dif site, which we call dif2CC, which shows more similarity to the dif consensus sequence. Here, by using a combination of in vitro experiments, we compare the affinities and the cleavage abilities of CcXerCD recombinases for both dif sites. Our results show that dif2CC displays a higher affinity for CcXerC and CcXerD and is bound cooperatively by these proteins, which is not the case for the original dif1CC site. Furthermore, dif2CC nicked substrates are more efficiently cleaved by CcXerCD, and deletion of the site results in about 5 to 10% of cells showing an altered cellular morphology. IMPORTANCE Bacteria utilize site-specific recombination for a variety of purposes, including the control of gene expression, acquisition of genetic elements, and the resolution of dimeric chromosomes. Failure to resolve dimeric chromosomes can lead to cell division defects in a percentage of the cell population. The work presented here shows the existence of a chromosomal resolution system in C. crescentus. Defects in this resolution system result in the formation of chains of cells. Further understanding of how these cells remain linked together will help in the understanding of how chromosome segregation and cell division are linked in C. crescentus.

scholarly journals SpyAD, a Moonlighting Protein of Group A Streptococcus Contributing to Bacterial Division and Host Cell Adhesion

2014 ◽  
Vol 82 (7) ◽  
pp. 2890-2901 ◽  
Author(s):  
Marilena Gallotta ◽  
Giovanni Gancitano ◽  
Giampiero Pietrocola ◽  
Marirosa Mora ◽  
Alfredo Pezzicoli ◽  
...  
Keyword(s):  
Cell Division ◽  
Mammalian Cells ◽  
Group A Streptococcus ◽  
Host Cells ◽  
Knockout Mutant ◽  
Content Type ◽  
Moonlighting Protein ◽  
Group A

ABSTRACTGroup A streptococcus (GAS) is a human pathogen causing a wide repertoire of mild and severe diseases for which no vaccine is yet available. We recently reported the identification of three protein antigens that in combination conferred wide protection against GAS infection in mice. Here we focused our attention on the characterization of one of these three antigens, Spy0269, a highly conserved, surface-exposed, and immunogenic protein of unknown function. Deletion of thespy0269gene in a GAS M1 isolate resulted in very long bacterial chains, which is indicative of an impaired capacity of the knockout mutant to properly divide. Confocal microscopy and immunoprecipitation experiments demonstrated that the protein was mainly localized at the cell septum and could interactin vitrowith the cell division protein FtsZ, leading us to hypothesize that Spy0269 is a member of the GAS divisome machinery. Predicted structural domains and sequence homologies with known streptococcal adhesins suggested that this antigen could also play a role in mediating GAS interaction with host cells. This hypothesis was confirmed by showing that recombinant Spy0269 could bind to mammalian epithelial cellsin vitroand thatLactococcus lactisexpressing Spy0269 on its cell surface could adhere to mammalian cellsin vitroand to mice nasal mucosain vivo. On the basis of these data, we believe that Spy0269 is involved both in bacterial cell division and in adhesion to host cells and we propose to rename this multifunctional moonlighting protein as SpyAD (StreptococcuspyogenesAdhesion andDivision protein).

Site-Specific Recombination Promoted in Vitro by the FLP Protein of the Yeast Two-Micron Plasmid

1986 ◽  
pp. 397-405
Author(s):  
Julie F. Senecoff ◽  
Robert C. Bruckner ◽  
Leslie Meyer-Leon ◽  
Cynthia A. Gates ◽  
Elizabeth Wood ◽  
...  
Keyword(s):  
Site Specific ◽  
Site Specific Recombination

Site-specific DNA binding of nuclear factor I: analyses of cellular binding sites

1985 ◽  
Vol 5 (5) ◽  
pp. 964-971
Author(s):  
R M Gronostajski ◽  
S Adhya ◽  
K Nagata ◽  
R A Guggenheimer ◽  
J Hurwitz
Keyword(s):  
Dna Binding ◽  
Hela Cell ◽  
Dna Sequences ◽  
Binding Sites ◽  
Consensus Sequence ◽  
Nuclear Factor ◽  
Site Specific ◽  
Factor I ◽  
Nuclear Factor I

Nuclear factor I is a cellular site-specific DNA-binding protein required for the efficient in vitro replication of adenovirus DNA. We have characterized human DNA sequences to which nuclear factor I binds. Three nuclear factor I binding sites (FIB sites), isolated from HeLa cell DNA, each contain the sequence TGG(N)6-7GCCAA. Comparison with other known and putative FIB sites suggests that this sequence is important for the binding of nuclear factor I. Nuclear factor I protects a 25- to 30-base-pair region surrounding this sequence from digestion by DNase I. Methylation protection studies suggest that nuclear factor I interacts with guanine residues within the TGG(N)6-7GCCAA consensus sequence. One binding site (FIB-2) contained a restriction endonuclease HaeIII cleavage site (GGCC) at the 5' end of the GCCAA motif. Digestion of FIB-2 with HaeIII abolished the binding of nuclear factor I. Southern blot analyses indicate that the cellular FIB sites described here are present within single-copy DNA in the HeLa cell genome.

scholarly journals Cell Division Protein FtsZ Is Unfolded for N-Terminal Degradation by Antibiotic-Activated ClpP

mBio ◽  
2020 ◽  
Vol 11 (3) ◽  
Author(s):  
Nadine Silber ◽  
Stefan Pan ◽  
Sina Schäkermann ◽  
Christian Mayer ◽  
Heike Brötz-Oesterhelt ◽  
...  
Keyword(s):  
Cell Division ◽  
Protein Unfolding ◽  
Multidrug Resistant ◽  
Unexpected Finding ◽  
Bacterial Cells ◽  
Clp Protease ◽  
Content Type ◽  
C Terminus ◽  
Cell Division Protein

ABSTRACT Antibiotic acyldepsipeptides (ADEPs) deregulate ClpP, the proteolytic core of the bacterial Clp protease, thereby inhibiting its native functions and concomitantly activating it for uncontrolled proteolysis of nonnative substrates. Importantly, although ADEP-activated ClpP is assumed to target multiple polypeptide and protein substrates in the bacterial cell, not all proteins seem equally susceptible. In Bacillus subtilis, the cell division protein FtsZ emerged to be particularly sensitive to degradation by ADEP-activated ClpP at low inhibitory ADEP concentrations. In fact, FtsZ is the only bacterial protein that has been confirmed to be degraded in vitro as well as within bacterial cells so far. However, the molecular reason for this preferred degradation remained elusive. Here, we report the unexpected finding that ADEP-activated ClpP alone, in the absence of any Clp-ATPase, leads to an unfolding and subsequent degradation of the N-terminal domain of FtsZ, which can be prevented by the stabilization of the FtsZ fold via nucleotide binding. At elevated antibiotic concentrations, importantly, the C terminus of FtsZ is notably targeted for degradation in addition to the N terminus. Our results show that different target structures are more or less accessible to ClpP, depending on the ADEP level present. Moreover, our data assign a Clp-ATPase-independent protein unfolding capability to the ClpP core of the bacterial Clp protease and suggest that the protein fold of FtsZ may be more flexible than previously anticipated. IMPORTANCE Acyldepsipeptide (ADEP) antibiotics effectively kill multidrug-resistant Gram-positive pathogens, including vancomycin-resistant enterococcus, penicillin-resistant Streptococcus pneumoniae (PRSP), and methicillin-resistant Staphylococcus aureus (MRSA). The antibacterial activity of ADEP depends on a new mechanism of action, i.e., the deregulation of bacterial protease ClpP that leads to bacterial self-digestion. Our data allow new insights into the mode of ADEP action by providing a molecular explanation for the distinct bacterial phenotypes observed at low versus high ADEP concentrations. In addition, we show that ClpP alone, in the absence of any unfoldase or energy-consuming system, and only activated by the small molecule antibiotic ADEP, leads to the unfolding of the cell division protein FtsZ.

scholarly journals Xer-mediated site-specific recombination in vitro.

1996 ◽  
Vol 15 (5) ◽  
pp. 1172-1181 ◽  
Author(s):  
S. D. Colloms ◽  
R. McCulloch ◽  
K. Grant ◽  
L. Neilson ◽  
D. J. Sherratt
Keyword(s):  
Site Specific ◽  
Site Specific Recombination

scholarly journals Gin-mediated site-specific recombination in bacteriophage Mu DNA: overproduction of the protein and inversion in vitro

1984 ◽  
Vol 3 (10) ◽  
pp. 2415-2421 ◽  
Author(s):  
Gabriele Mertens ◽  
Andrea Hoffmann ◽  
Helmut Blöcker ◽  
Ronald Frank ◽  
Regine Kahmann
Keyword(s):  
Bacteriophage Mu ◽  
Site Specific ◽  
Site Specific Recombination ◽  
And Inversion

scholarly journals Genomic Adaptations to the Loss of a Conserved Bacterial DNA Methyltransferase

mBio ◽  
2015 ◽  
Vol 6 (4) ◽  
Author(s):  
Diego Gonzalez ◽  
Justine Collier
Keyword(s):  
Cell Cycle ◽  
Cell Division ◽  
Experimental Evolution ◽  
Dna Methyltransferase ◽  
Metabolic Regulation ◽  
Caulobacter Crescentus ◽  
Point Mutations ◽  
Rich Medium ◽  
Content Type ◽  
Evolution Approach

ABSTRACTCcrM is an orphan DNA methyltransferase nearly universally conserved in a vast group ofAlphaproteobacteria.InCaulobacter crescentus, it controls the expression of key genes involved in the regulation of the cell cycle and cell division. Here, we demonstrate, using an experimental evolution approach, thatC. crescentuscan significantly compensate, through easily accessible genetic changes like point mutations, the severe loss in fitness due to the absence of CcrM, quickly improving its growth rate and cell morphology in rich medium. By analyzing the compensatory mutations genome-wide in 12 clones sampled from independent ΔccrMpopulations evolved for ~300 generations, we demonstrated that each of the twelve clones carried at least one mutation that potentially stimulatedftsZexpression, suggesting that the low intracellular levels of FtsZ are the major burden of ΔccrMmutants. In addition, we demonstrate that the phosphoenolpyruvate-carbohydrate phosphotransfer system (PTS) actually modulatesftsZandmipZtranscription, uncovering a previously unsuspected link between metabolic regulation and cell division inAlphaproteobacteria. We present evidence that point mutations found in genes encoding proteins of the PTS provide the strongest fitness advantage to ΔccrMcells cultivated in rich medium despite being disadvantageous in minimal medium. This environmental sign epistasis might prevent such mutations from getting fixed under changing natural conditions, adding a plausible explanation for the broad conservation of CcrM.IMPORTANCEIn bacteria, DNA methylation has a variety of functions, including the control of DNA replication and/or gene expression. The cell cycle-regulated DNA methyltransferase CcrM modulates the transcription of many genes and is critical for fitness inCaulobacter crescentus. Here, we used an original experimental evolution approach to determine which of its many targets make CcrM so important physiologically. We show that populations lacking CcrM evolve quickly, accumulating an excess of mutations affecting, directly or indirectly, the expression of theftsZcell division gene. This finding suggests that the most critical function of CcrM inC. crescentusis to promote cell division by enhancing FtsZ intracellular levels. During this work, we also discovered an unexpected link between metabolic regulation and cell division that might extend to otherAlphaproteobacteria.

scholarly journals The Nucleoid-Associated Protein GapR Uses Conserved Structural Elements To Oligomerize and Bind DNA

mBio ◽  
2020 ◽  
Vol 11 (3) ◽  
Author(s):  
Rogério F. Lourenço ◽  
Saumya Saurabh ◽  
Jonathan Herrmann ◽  
Soichi Wakatsuki ◽  
Lucy Shapiro
Keyword(s):  
Dna Binding ◽  
Caulobacter Crescentus ◽  
Genetic Material ◽  
Bacterial Cells ◽  
Structural Elements ◽  
Content Type ◽  
Time Dynamic ◽  
Associated Proteins ◽  
And Function

ABSTRACT Nucleoid-associated proteins (NAPs) are DNA binding proteins critical for the organization and function of the bacterial chromosome. A newly discovered NAP in Caulobacter crescentus, GapR, is thought to facilitate the movement of the replication and transcription machines along the chromosome by stimulating type II topoisomerases to remove positive supercoiling. Here, utilizing genetic, biochemical, and biophysical studies of GapR in light of a recently published DNA-bound crystal structure of GapR, we identified the structural elements involved in oligomerization and DNA binding. Moreover, we show that GapR is maintained as a tetramer upon its dissociation from DNA and that tetrameric GapR is capable of binding DNA molecules in vitro. Analysis of protein chimeras revealed that two helices of GapR are functionally conserved in H-NS, demonstrating that two evolutionarily distant NAPs with distinct mechanisms of action utilize conserved structural elements to oligomerize and bind DNA. IMPORTANCE Bacteria organize their genetic material in a structure called the nucleoid, which needs to be compact to fit inside the cell and, at the same time, dynamic to allow high rates of replication and transcription. Nucleoid-associated proteins (NAPs) play a pivotal role in this process, so their detailed characterization is crucial for our understanding of DNA organization into bacterial cells. Even though NAPs affect DNA-related processes differently, all of them have to oligomerize and bind DNA for their function. The significance of this study is the identification of structural elements involved in the oligomerization and DNA binding of a newly discovered NAP in C. crescentus and the demonstration that structural elements are conserved in evolutionarily distant and functionally distinct NAPs.

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