scholarly journals Xer1-Mediated Site-Specific DNA Inversions and Excisions in Mycoplasma agalactiae

2010 ◽  
Vol 192 (17) ◽  
pp. 4462-4473 ◽  
Author(s):  
Stefan Czurda ◽  
Wolfgang Jechlinger ◽  
Renate Rosengarten ◽  
Rohini Chopra-Dewasthaly
Keyword(s):  
High Frequency ◽  
Phase Variation ◽  
Etiologic Agent ◽  
Reporter System ◽  
Recognition Sequence ◽  
Site Specific ◽  
Phase Switching ◽  
Site Specific Recombination ◽  
Mycoplasma Agalactiae ◽  
Variable Surface

ABSTRACT Surface antigen variation in Mycoplasma agalactiae, the etiologic agent of contagious agalactia in sheep and goats, is governed by site-specific recombination within the vpma multigene locus encoding the Vpma family of variable surface lipoproteins. This high-frequency Vpma phase switching was previously shown to be mediated by a Xer1 recombinase encoded adjacent to the vpma locus. In this study, it was demonstrated in Escherichia coli that the Xer1 recombinase is responsible for catalyzing vpma gene inversions between recombination sites (RS) located in the 5′-untranslated region (UTR) in all six vpma genes, causing cleavage and strand exchange within a 21-bp conserved region that serves as a recognition sequence. It was further shown that the outcome of the site-specific recombination event depends on the orientation of the two vpma RS, as direct or inverted repeats. While recombination between inverted vpma RS led to inversions, recombination between direct repeat vpma RS led to excisions. Using a newly developed excision assay based on the lacZ reporter system, we were able to successfully demonstrate under native conditions that such Xer1-mediated excisions can indeed also occur in the M. agalactiae type strain PG2, whereas they were not observed in the control xer1-disrupted VpmaY phase-locked mutant (PLMY), which lacks Xer1 recombinase. Unless there are specific regulatory mechanisms preventing such excisions, this might be the cost that the pathogen has to render at the population level for maintaining this high-frequency phase variation machinery.

scholarly journals Surface Diversity in Mycoplasma agalactiae Is Driven by Site-Specific DNA Inversions within the vpma Multigene Locus

2002 ◽  
Vol 184 (21) ◽  
pp. 5987-5998 ◽  
Author(s):  
Michelle D. Glew ◽  
Marc Marenda ◽  
Renate Rosengarten ◽  
Christine Citti
Keyword(s):  
Promoter Sequence ◽  
Untranslated Regions ◽  
Dna Rearrangements ◽  
Site Specific ◽  
Reading Frame ◽  
Mycoplasma Agalactiae ◽  
Variable Surface ◽  
A Site ◽  
Active Promoter ◽  
Minimal Region

ABSTRACT The ruminant pathogen Mycoplasma agalactiae possesses a family of abundantly expressed variable surface lipoproteins called Vpmas. Phenotypic switches between Vpma members have previously been correlated with DNA rearrangements within a locus of vpma genes and are proposed to play an important role in disease pathogenesis. In this study, six vpma genes were characterized in the M. agalactiae type strain PG2. All vpma genes clustered within an 8-kb region and shared highly conserved 5′ untranslated regions, lipoprotein signal sequences, and short N-terminal sequences. Analyses of the vpma loci from consecutive clonal isolates showed that vpma DNA rearrangements were site specific and that cleavage and strand exchange occurred within a minimal region of 21 bp located within the 5′ untranslated region of all vpma genes. This process controlled expression of vpma genes by effectively linking the open reading frame (ORF) of a silent gene to a unique active promoter sequence within the locus. An ORF (xer1) immediately adjacent to one end of the vpma locus did not undergo rearrangement and had significant homology to a distinct subset of genes belonging to the λ integrase family of site-specific xer recombinases. It is proposed that xer1 codes for a site-specific recombinase that is not involved in chromosome dimer resolution but rather is responsible for the observed vpma-specific recombination in M. agalactiae.

scholarly journals Control of ϕC31 integrase-mediated site-specific recombination by protein trans-splicing

2019 ◽  
Vol 47 (21) ◽  
pp. 11452-11460 ◽  
Author(s):  
Femi J Olorunniji ◽  
Makeba Lawson-Williams ◽  
Arlene L McPherson ◽  
Jane E Paget ◽  
W Marshall Stark ◽  
...  
Keyword(s):  
Genome Engineering ◽  
Genetic Circuits ◽  
Reporter System ◽  
Site Specific ◽  
Site Specific Recombination ◽  
Recombination System ◽  
Split Intein ◽  
Genetic Switches ◽  
Trans Splicing

Abstract Serine integrases are emerging as core tools in synthetic biology and have applications in biotechnology and genome engineering. We have designed a split-intein serine integrase-based system with potential for regulation of site-specific recombination events at the protein level in vivo. The ϕC31 integrase was split into two extein domains, and intein sequences (Npu DnaEN and Ssp DnaEC) were attached to the two termini to be fused. Expression of these two components followed by post-translational protein trans-splicing in Escherichia coli generated a fully functional ϕC31 integrase. We showed that protein splicing is necessary for recombination activity; deletion of intein domains or mutation of key intein residues inactivated recombination. We used an invertible promoter reporter system to demonstrate a potential application of the split intein-regulated site-specific recombination system in building reversible genetic switches. We used the same split inteins to control the reconstitution of a split Integrase-Recombination Directionality Factor fusion (Integrase-RDF) that efficiently catalysed the reverse attR x attL recombination. This demonstrates the potential for split-intein regulation of the forward and reverse reactions using the integrase and the integrase-RDF fusion, respectively. The split-intein integrase is a potentially versatile, regulatable component for building synthetic genetic circuits and devices.

scholarly journals Characterization of a Multigene Family Undergoing High-Frequency DNA Rearrangements and Coding for Abundant Variable Surface Proteins in Mycoplasma agalactiae

2000 ◽  
Vol 68 (8) ◽  
pp. 4539-4548 ◽  
Author(s):  
M. D. Glew ◽  
L. Papazisi ◽  
F. Poumarat ◽  
D. Bergonier ◽  
R. Rosengarten ◽  
...  
Keyword(s):  
Phase Variation ◽  
Gene Families ◽  
Surface Proteins ◽  
Dna Rearrangements ◽  
Reading Frame ◽  
Mycoplasma Agalactiae ◽  
Amino Terminal ◽  
Clonal Variant ◽  
Variable Surface

ABSTRACT A family of abundant surface proteins (Vpmas [variable proteins ofMycoplasma agalactiae]) undergoing phase variation inM. agalactiae has been characterized using monoclonal antibodies and specific polyclonal sera. Two expressed members of 39 kDa (Vpma39) and 34 kDa (Vpma34), which varied in expression between clones of a lineage, shared a common amino-terminal sequence but were immunologically distinct. An amino-terminal oligonucleotide probe identified multiple vpma genes which were clustered within a 14-kb ClaI genomic fragment. Rearrangements were found to have occurred within the vpma locus between clones which correlated with changes in their Vpma phenotype. Two neighboringvpma genes were cloned and sequenced from one M. agalactiae clonal variant expressing Vpma39. The two genes,vpmaX and vpmaY, were orientated divergently and shared highly homologous 5′ untranslated regions, 25-amino-acid (aa) lipoprotein leader sequences, and amino-terminal sequences. ThevpmaY gene coded for 346 aa and 84% of the open reading frame, comprised of 1.5 units of a large repeat of 186 aa. Although the sequence for an entire second vpmaY repeat was present, it was prematurely terminated by insertion of two nucleotides. ThevpmaX gene encoded 221 aa and possessed 102 aa of the 186-aa repeat of vpmaY. Many of the features in common between the vpma genes were also found to be shared by thevsp genes of M. bovis, which also undergo DNA rearrangements concomitant with phenotypic changes. Since M. bovis is the closest phylogenetic relative to M. agalactiae, the vpma and vsp gene families most probably represent homologous systems.

scholarly journals FREQUENCY AND DIRECTIONALITY OF GENE CONVERSION EVENTS INVOLVING THE CYC7-H3 MUTATION IN SACCHAROMYCES CEREVISIAE

Genetics ◽  
1986 ◽  
Vol 114 (2) ◽  
pp. 347-361
Author(s):  
Patricia J Pukkila ◽  
Michael D Stephens ◽  
David M Binninger ◽  
Beverly Errede
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Gene Conversion ◽  
High Frequency ◽  
Yeast Genome ◽  
Junction Region ◽  
Sequence Comparisons ◽  
Site Specific ◽  
Site Specific Recombination ◽  
Conversion Frequency ◽  
Aberrant Segregation

ABSTRACT The CYC7-H3 mutation is a 5-kb deletion that causes overproduction of iso-2 cytochrome c. Unlike most mutations in yeast, the CYC7-H3 mutation is preferentially lost when it is involved in a gene conversion event. We have shown that cloned copies of CYC7-H3 DNA that are inserted into the yeast genome are associated with a high frequency of recombination and aberrant segregation events. Since parity in conversion frequency was observed when the extensive insertion/deletion heterozygosity at this locus was eliminated, we conclude that the CYC7-H3 sequences are inherently capable of acting as donors or recipients in gene conversion events, although they are unlikely to act as donors when they are located opposite a large heterology. DNA sequence comparisons revealed similarities between the CYC7-H3 junction region and the 2-µm circle DNA region that is involved in site-specific recombination.

scholarly journals The IntP C-Terminal Segment Is Not Required for Excision of Bacteriophage Mx8 from the Myxococcus xanthus Chromosome

2003 ◽  
Vol 185 (7) ◽  
pp. 2187-2193 ◽  
Author(s):  
Nobuki Tojo ◽  
Teruya Komano
Keyword(s):  
Amino Acid ◽  
High Frequency ◽  
Myxococcus Xanthus ◽  
Terminal Segment ◽  
Site Specific ◽  
Site Specific Recombination ◽  
Coding Sequence ◽  
Phage Dna ◽  
New Protein

ABSTRACT During lysogenization of myxophage Mx8, phage DNA can be integrated into the attB site of the Myxococcus xanthus chromosome through site-specific recombination. We previously demonstrated that the Mx8 attP site is located within the coding sequence of the Mx8 intP gene. Hence, the integration of Mx8 into the M. xanthus chromosome results in the conversion of the 112-amino-acid C-terminal segment of the IntP protein into a 13-amino-acid C-terminal segment of a new protein, IntR. To examine whether IntR is active for Mx8 excision, we have constructed a series of plasmids carrying various lengths of the intP-attP or intR-attR regions as well as the lacZ gene. The integrated Mx8 was excised at a high frequency, indicating that IntR is active for the excision. For Mx8 excision, a gene designated xis was shown to be required in addition to intR.

scholarly journals High-frequency phase-switching of modB methylase is associated with phenotypic ceftriaxone susceptibility in Neisseria gonorrhoeae

2020 ◽  
Author(s):  
Ola B Brynildsrud ◽  
Magnus N Osnes ◽  
Kevin C Ma ◽  
Yonatan H Grad ◽  
Michael Koomey ◽  
...  
Keyword(s):  
High Frequency ◽  
Genetic Basis ◽  
Meta Analysis ◽  
Phase Variation ◽  
Phase Variable ◽  
Phase Switching ◽  
Expression Of Genes ◽  
Genome Wide ◽  
Restriction Modification ◽  
Frequency Phase

AbstractThe gonococcal adenine methylases modA and modB, belonging to separate Type III restriction modification systems, are phase variable and could thus enable rapid adaptation to changing environments. However, the frequency of phase variation across transmission chains and the phenotypic impact of phase variation are largely unknown.Here we show that the repeat tracts enabling phase variation expand and contract at high rates in both modA and modB. For modB, multiple ON/OFF transition events were identified over the course of a single outbreak.A mixed effects model using population samples from Norway and a global meta-analysis collection indicates that modB in the OFF state is predictive of moderately decreased ceftriaxone susceptibility. Our findings suggest that modB orchestration of genome-wide 6-methyladenine modification controls the expression of genes modulating ceftriaxone susceptibility.ImportanceDespite significant progress, our current understanding of the genetic basis of antibiotic susceptibility remains incomplete. The gonococcal methylase modB is phase variable, meaning that it can be switched ON or OFF via contraction or expansion of a repeat tract in the gene during replication. We find that transitions between the ON and OFF state occur at high frequency. Furthermore, isolates harbouring modB in a configuration predicted to be inactive had decreased susceptibility to ceftriaxone, an antibiotic used to treat gonorrhea. This finding improves understanding of the genetic underpinnings of antibiotic resistance, but further work is needed to elucidate the mechanics and broader phenotypic effects of epigenetic modifications and transcription.

scholarly journals Multiple regulatory mechanisms control the production of CmrRST, an atypical signal transduction system in Clostridioides difficile

2021 ◽  
Author(s):  
Elizabeth M Garrett ◽  
Anchal Mehra ◽  
Ognjen Sekulovic ◽  
Rita Tamayo
Keyword(s):  
Signal Transduction ◽  
Phase Variation ◽  
Phenotypic Characterization ◽  
Signal Transduction System ◽  
Site Specific ◽  
Environmental Signals ◽  
Site Specific Recombination ◽  
Multiple Promoters ◽  
Clostridioides Difficile ◽  
The Right

Clostridioides difficile, an intestinal pathogen and leading cause of nosocomial infection, exhibits extensive phenotypic heterogeneity through phase variation by site-specific recombination. The signal transduction system CmrRST, which encodes two response regulators (CmrR and CmrT) and a sensor kinase (CmrS), impacts C. difficile cell and colony morphology, surface and swimming motility, biofilm formation, and virulence in an animal model. CmrRST is subject to phase variation through site-specific recombination and reversible inversion of the ‘cmr switch’, and expression of cmrRST is also regulated by c-di-GMP through a riboswitch. The goal of this study was to determine how the cmr switch and c-di-GMP work together to regulate cmrRST expression. We generated “phase locked” strains by mutating key residues in the right inverted repeat flanking the cmr switch. Phenotypic characterization of these phase locked cmr-ON and -OFF strains demonstrates that they cannot switch between rough and smooth colony morphologies, respectively, or other CmrRST-associated phenotypes. Manipulation of c-di-GMP levels in these mutants showed that c-di-GMP promotes cmrRST expression and associated phenotypes independent of cmr switch orientation. We identified multiple promoters controlling cmrRST transcription, including one within the ON orientation of cmr switch and another that is positively autoregulated by CmrR. Overall, this work reveals a complex regulatory network that governs cmrRST expression and a unique intersection of phase variation and c-di-GMP signaling. These findings suggest that multiple environmental signals impact the production of this signaling transduction system.

scholarly journals Juxtaposition of an Active Promoter to vsp Genes via Site-Specific DNA Inversions Generates Antigenic Variation inMycoplasma bovis

2001 ◽  
Vol 183 (19) ◽  
pp. 5698-5708 ◽  
Author(s):  
Innesa Lysnyansky ◽  
Yael Ron ◽  
David Yogev
Keyword(s):  
High Frequency ◽  
Transcription Initiation ◽  
Northern Blot Analysis ◽  
Antigenic Variation ◽  
Regulatory Element ◽  
Phase Variation ◽  
Upstream Region ◽  
Site Specific ◽  
Active Promoter ◽  
Frequency Phase

ABSTRACT Mycoplasma bovis, the most important etiological agent of bovine mycoplasmosis, undergoes extensive antigenic variation of major and highly immunogenic surface lipoprotein antigens (Vsps). A family of 13 related but divergent vsp genes, which occur as single chromosomal copies, was recently found in the chromosome ofM. bovis. In the present study, the molecular mechanism mediating the high-frequency phase variation of two Vsps (VspA and VspC) as representatives of the Vsp family was investigated. Analysis of clonal isolates exhibiting phase transitions of VspA or of VspC (i.e., ON→OFF→ON) has shown that DNA inversions occur during Vsp phase variation. The upstream region of each vsp gene contains two sequence cassettes. The first (cassette no. 1), a 71-bp region upstream of the ATG initiation codon, exhibits 98% homology among all vsp genes, while the second (cassette no. 2), upstream of cassette no. 1, ranges in size from 50 to 180 bp and is more divergent. Examination of the ends of the inverted fragments during VspA or VspC phase variation revealed that in both cases, a change in the organization of vsp upstream cassettes involving three vsp genes had occurred. Primer extension and Northern blot analysis have shown that a specific cassette no. 2, designated A2, is an active promoter and that juxtaposition of this regulatory element to a silent vsp gene by DNA inversions allows transcription initiation of the recipient gene. Further genetic analysis revealed that phase variation of VspA or of VspC involves two site-specific DNA inversions occurring between inverted copies of a specific 35-bp sequence present within the conserved cassette no. 1. A model for the control of Vsp phase variation is proposed.

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