scholarly journals Gene Disruption through Homologous Recombination inSpiroplasma citri: an scm1-Disrupted Motility Mutant Is Pathogenic

1999 ◽  
Vol 181 (24) ◽  
pp. 7449-7456 ◽  
Author(s):  
Sybille Duret ◽  
Jean-Luc Danet ◽  
Monique Garnier ◽  
Joël Renaudin
Keyword(s):  
Homologous Recombination ◽  
Gene Disruption ◽  
Reca Protein ◽  
Specific Gene ◽  
Gene Fragment ◽  
Wild Type ◽  
Spiroplasma Citri ◽  
E Coli ◽  
Circulifer Haematoceps ◽  
Motility Mutant

ABSTRACT To determine whether homologous recombination could be used to inactivate selected genes in Spiroplasma citri, plasmid constructs were designed to disrupt the motility gene scm1. An internal scm1 gene fragment was inserted into plasmid pKT1, which replicates in Escherichia coli but not inS. citri, and into the S. citri oriC plasmid pBOT1, which replicates in spiroplasma cells as well as in E. coli. Electrotransformation of S. citri with the nonreplicative, recombinant plasmid pKTM1 yielded no transformants. In contrast, spiroplasmal transformants were obtained with the replicative, pBOT1-derived plasmid pCJ32. During passaging of the transformants, the plasmid was found to integrate into the chromosome by homologous recombination either at the oriC region or at the scm1 gene. In the latter case, plasmid integration by a single crossover between the scm1 gene fragment carried by the plasmid and the full-length scm1 gene carried by the chromosome led to a nonmotile phenotype. Transmission of thescm1-disrupted mutant to periwinkle (Catharanthus roseus) plants through injection into the leafhopper vector (Circulifer haematoceps) showed that the motility mutant multiplied in the insects and was efficiently transmitted to plants, in which it induced symptoms similarly to the wild-type S. citri strain. These results suggest that the spiroplasmal motility may not be essential for pathogenicity and that, more broadly, the S. citri oriC plasmids can be considered promising tools for specific gene disruption by promoting homologous recombination in S. citri, a mollicute which probably lacks a functional RecA protein.

Ordered Intracellular Reca-Dna Assemblies

2000 ◽  
Vol 6 (S2) ◽  
pp. 860-861
Author(s):  
S. G. Wolf ◽  
S. Levin-Zaidman ◽  
D. Frenkiel-Krispin ◽  
E. Shimoni ◽  
I. Sabanay ◽  
...  
Keyword(s):  
Dna Damage ◽  
Dna Repair ◽  
Electron Micrographs ◽  
Reca Protein ◽  
Sos Response ◽  
Structural Features ◽  
Wild Type ◽  
E Coli ◽  
Dna Damaging Agents

The inducible SOS response increases the ability of bacteria to cope with DNA damage through various DNA repair processes in which the RecA protein plays a central role. We find that induction of the SOS system in wild-type E. coli bacteria results in fast and massive intracellular coaggregation of RecA and DNA into lateral assemblies, which comprise substantial portions of both the cellular RecA and the DNA complement. The structural features of the coaggregates and their relation to in-vitro RecA-DNA are consistent with the possibility that the intracellular assemblies represent a functional entity in which RecA-mediated DNA repair and protection activities occur.Bacterial chromatin is demarcated in electron micrographs of metabolically active cells as amorphous ribosome-free spaces that are irregularly spread over the cytoplasm (Fig. A). Wild-type E. coli cells exposed to DNA-damaging agents that induce the SOS response reveal a strikingly different morphology (Fig. B).

scholarly journals Bacterial RecA Protein Promotes Adenoviral Recombination duringIn VitroInfection

mSphere ◽  
2018 ◽  
Vol 3 (3) ◽  
Author(s):  
Jeong Yoon Lee ◽  
Ji Sun Lee ◽  
Emma C. Materne ◽  
Rahul Rajala ◽  
Ashrafali M. Ismail ◽  
...  
Keyword(s):  
Homologous Recombination ◽  
Hot Spots ◽  
Genetic Recombination ◽  
Reca Protein ◽  
Human Adenovirus ◽  
Penton Base ◽  
Content Type ◽  
E Coli ◽  
Recombination Hot Spots ◽  
Loop 2

ABSTRACTAdenovirus infections in humans are common and sometimes lethal. Adenovirus-derived vectors are also commonly chosen for gene therapy in human clinical trials. We have shown in previous work that homologous recombination between adenoviral genomes of human adenovirus species D (HAdV-D), the largest and fastest growing HAdV species, is responsible for the rapid evolution of this species. Because adenovirus infection initiates in mucosal epithelia, particularly at the gastrointestinal, respiratory, genitourinary, and ocular surfaces, we sought to determine a possible role for mucosal microbiota in adenovirus genome diversity. By analysis of known recombination hot spots across 38 human adenovirus genomes in species D (HAdV-D), we identified nucleotide sequence motifs similar to bacterial Chi sequences, which facilitate homologous recombination in the presence of bacterial Rec enzymes. These motifs, referred to here as ChiAD, were identified immediately 5′ to the sequence encoding penton base hypervariable loop 2, which expresses the arginine-glycine-aspartate moiety critical to adenoviral cellular entry. Coinfection with two HAdV-Ds in the presence of anEscherichia colilysate increased recombination; this was blocked in a RecA mutant strain,E. coliDH5α, or upon RecA depletion. Recombination increased in the presence ofE. colilysate despite a general reduction in viral replication. RecA colocalized with viral DNA in HAdV-D-infected cell nuclei and was shown to bind specifically to ChiADsequences. These results indicate that adenoviruses may repurpose bacterial recombination machinery, a sharing of evolutionary mechanisms across a diverse microbiota, and unique example of viral commensalism.IMPORTANCEAdenoviruses are common human mucosal pathogens of the gastrointestinal, respiratory, and genitourinary tracts and ocular surface. Here, we report finding Chi-like sequences in adenovirus recombination hot spots. Adenovirus coinfection in the presence of bacterial RecA protein facilitated homologous recombination between viruses. Genetic recombination led to evolution of an important external feature on the adenoviral capsid, namely, the penton base protein hypervariable loop 2, which contains the arginine-glycine-aspartic acid motif critical to viral internalization. We speculate that free Rec proteins present in gastrointestinal secretions upon bacterial cell death facilitate the evolution of human adenoviruses through homologous recombination, an example of viral commensalism and the complexity of virus-host interactions, including regional microbiota.

scholarly journals Reduced Infectivity of a Leishmania donovani Biopterin Transporter Genetic Mutant and Its Use as an Attenuated Strain for Vaccination

2002 ◽  
Vol 70 (1) ◽  
pp. 62-68 ◽  
Author(s):  
Barbara Papadopoulou ◽  
Gaétan Roy ◽  
Marie Breton ◽  
Christoph Kündig ◽  
Carole Dumas ◽  
...  
Keyword(s):  
Homologous Recombination ◽  
Recent Work ◽  
Interferon Gamma ◽  
Protective Immunity ◽  
Gene Disruption ◽  
Leishmania Donovani ◽  
Vaccination Strategy ◽  
Enzyme Linked Immunosorbent Assay ◽  
Null Mutant ◽  
Wild Type

ABSTRACT Pterins are essential for the growth of Leishmania species, and recent work has led to the isolation of the biopterin transporter BT1. In this study, we inactivated the Leishmania donovani biopterin transporter BT1 by gene disruption mediated by homologous recombination. No transport of biopterin was detected in this mutant. The L. donovani BT1 null mutant showed a much lesser capacity for inducing infection in mice than wild-type parasites and could elicit protective immunity in mice susceptible to infection against a L. donovani challenge. Splenocytes isolated from mice immunized with the BT1 null mutant parasites produced significant amounts of interferon gamma following stimulation with L. donovani promastigotes as measured by enzyme-linked immunosorbent assay and enzyme-linked immunospot assays. Overall, these results show that by genetically manipulating the pterin transport in L. donovani, it is possible to generate an attenuated organism that could be part of a vaccination strategy.

scholarly journals Mutagenesis by Insertion of Tn4001 into the Genome of Spiroplasma citri: Characterization of Mutants Affected in Plant Pathogenicity and Transmission to the Plant by the Leafhopper Vector Circulifer haematoceps

1997 ◽  
Vol 10 (4) ◽  
pp. 454-461 ◽  
Author(s):  
X. Foissac ◽  
J. L. Danet ◽  
C. Saillard ◽  
P. Gaurivaud ◽  
F. Laigret ◽  
...  
Keyword(s):  
Type Strain ◽  
Culture Medium ◽  
Wild Type Strain ◽  
Single Copy ◽  
Wild Type ◽  
Spiroplasma Citri ◽  
Leafhopper Vector ◽  
Circulifer Haematoceps ◽  
Plant Pathogenicity

Two hundred and fifty-seven transposon Tn4001 mutants of Spiroplasma citri strain GII3 were used for transmission assays by the leafhopper vector Circulifer haematoceps into periwinkle (Catharanthus roseus) plants. Multiplication of the mutants in the two hosts, the leafhopper and the plant, as well as the symptom expression in the plant were studied. Two mutants, GMT 470 and GMT 553, caused no symptoms on plants. Tn4001 is inserted as a single copy in the genome of these mutants. Mutant GMT 470 did not multiply, or multiplied only poorly, in the leaf-hopper and was not transmitted by the insect to the plant, nor to culture medium through Parafilm membrane. The growth rate of GMT 470 in SP4 medium was twice as slow as that of wild-type strain GII3. Mutant GMT 553 multiplied in the leafhopper as well as the wild-type spiro-plasma, and was transmitted by the leafhoppers into the plants, where it reached the same titers as the wild-type strain but in approximately twice as much time. The plants containing high titers of mutant GMT 553 remained symptomless for several weeks. However, symptoms began to develop at a time when revertants that had lost the transposon were detected.

Molecular characterization ofGluconobacter oxydans recAgene and its inhibitory effect on the function of the host wild-typerecAgene

1998 ◽  
Vol 44 (2) ◽  
pp. 149-156 ◽  
Author(s):  
Yu-Tien Liu ◽  
Der-Chiang Chao ◽  
Fan Lee ◽  
Chia-Geun Chen ◽  
Dar-Der Ji
Keyword(s):  
Pseudomonas Aeruginosa ◽  
Amino Acid ◽  
Nucleotide Sequence ◽  
Amino Acid Sequence ◽  
Reca Protein ◽  
Gluconobacter Oxydans ◽  
Inhibitory Effect ◽  
Reca Gene ◽  
Wild Type ◽  
E Coli

A DNA fragment containing the recA gene of Gluconobacter oxydans was isolated and further characterized for its nucleotide sequence and ability to functionally complement various recA mutations. When expressed in an Escherichia coli recA host, the G. oxydans recA protein could efficiently function in homologous recombination and DNA damage repair. The recA gene's nucleotide sequence analysis revealed a protein of 344 amino acids with a molecular mass of 38 kDa. We observed an E. coli-like LexA repressor-binding site in the G. oxydans recA gene promoter region, suggesting that a LexA-like mediated response system may exist in G. oxydans. The expression of G. oxydans recA in E. coli RR1, a recA+strain, surprisingly caused a remarkable reduction of the host wild-type recA gene function, whereas the expression of both Serratia marcescens recA and Pseudomonas aeruginosa recA gene caused only a slight inhibitory effect on function of the host wild-typerecA gene product. Compared with the E. coli RecA protein, the identity of the amino acid sequence of G. oxydans RecA protein is much lower than those RecA proteins of both S. marcescens and Pseudomonas aeruginosa. This result suggests that the expression of another wild-type RecA could interfere with host wild-type recA gene's function, and the extent of such an interference is possibly correlated to the identity of the amino acid sequence between the two classes of RecA protein.Key words: Gluconobacter oxydans, recA gene, recombination, SOS function, interference.

scholarly journals Genetic Functions Promoting Homologous Recombination in Escherichia coli: A Study of Inversions in Phage Λ

Genetics ◽  
1987 ◽  
Vol 115 (1) ◽  
pp. 11-24
Author(s):  
Don G Ennis ◽  
Susan K Amundsen ◽  
Gerald R Smith
Keyword(s):  
Escherichia Coli ◽  
Homologous Recombination ◽  
Molecular Model ◽  
Reca Protein ◽  
Dna Polymerase I ◽  
E Coli ◽  
The Right ◽  
Polymerase I ◽  
Recbcd Enzyme ◽  
A Current

ABSTRACT We have studied homologous recombination in a derivative of phage λ containing two 1.4-kb repeats in inverted orientation. Inversion of the intervening 2.5-kb segment occurred efficiently by the Escherichia coli RecBC pathway but markedly less efficiently by the λ Red pathway or the E. coli RecE or RecF pathways. Inversion by the RecBCD pathway was stimulated by Chi sites located to the right of the invertible segment; this stimulation decreased exponentially by a factor of about 2 for each 2.2 kb between the invertible segment and the Chi site. In addition to RecA protein and RecBCD enzyme, inversion by the RecBC pathway required single-stranded DNA binding protein, DNA gyrase, DNA polymerase I and DNA ligase. Inversion appeared to occur either intra- or intermolecularly. These results are discussed in the framework of a current molecular model for the RecBC pathway of homologous recombination.

scholarly journals Spiralin Is Not Essential for Helicity, Motility, or Pathogenicity but Is Required for Efficient Transmission of Spiroplasma citri by Its Leafhopper Vector Circulifer haematoceps

2003 ◽  
Vol 69 (10) ◽  
pp. 6225-6234 ◽  
Author(s):  
Sybille Duret ◽  
Nathalie Berho ◽  
Jean-Luc Danet ◽  
Monique Garnier ◽  
Joël Renaudin
Keyword(s):  
Fusion Protein ◽  
Type Strain ◽  
Wild Type Strain ◽  
Insect Vector ◽  
Wild Type ◽  
Spiroplasma Citri ◽  
Leafhopper Vector ◽  
Gfp Fusion Protein ◽  
Circulifer Haematoceps ◽  
Efficient Transmission

ABSTRACT Spiralin is the most abundant protein at the surface of the plant pathogenic mollicute Spiroplasma citri and hence might play a role in the interactions of the spiroplasma with its host plant and/or its insect vector. To study spiralin function, mutants were produced by inactivating the spiralin gene through homologous recombination. A spiralin-green fluorescent protein (GFP) translational fusion was engineered and introduced into S. citri by using an oriC-based targeting vector. According to the strategy used, integration of the plasmid by a single-crossover recombination at the spiralin gene resulted in the expression of the spiralin-GFP fusion protein. Two distinct mutants were isolated. Western and colony immunoblot analyses showed that one mutant (GII3-9a5) did produce the spiralin-GFP fusion protein, which was found not to fluoresce, whereas the other (GII3-9a2) produced neither the fusion protein nor the wild-type spiralin. Both mutants displayed helical morphology and motility, similarly to the wild-type strain GII-3. Genomic DNA analyses revealed that GII3-9a5 was unstable and that GII3-9a2 was probably derived from GII3-9a5 by a double-crossover recombination between plasmid sequences integrated into the GII3-9a5 chromosome and free plasmid. When injected into the leafhopper vector Circulifer haematoceps, the spiralinless mutant GII3-9a2 multiplied to high titers in the insects (1.1 × 106 to 2.8 × 106 CFU/insect) but was transmitted to the host plant 100 times less efficiently than the wild-type strain. As a result, not all plants were infected, and symptom production in these plants was delayed for 2 to 4 weeks compared to that in the wild-type strain. In the infected plants however, the mutant multiplied to high titers (1.2 × 106 to 1.4 × 107 CFU/g of midribs) and produced the typical symptoms of the disease. These results indicate that spiralin is not essential for pathogenicity but is required for efficient transmission of S. citri by its insect vector.

scholarly journals Enhancement of Targeted Homologous Recombination in Mycoplasma mycoides subsp. capri by Inclusion of Heterologous recA

2010 ◽  
Vol 76 (20) ◽  
pp. 6951-6954 ◽  
Author(s):  
Ayman B. Allam ◽  
Leticia Reyes ◽  
Nacyra Assad-Garcia ◽  
John I. Glass ◽  
Mary B. Brown
Keyword(s):  
Escherichia Coli ◽  
Homologous Recombination ◽  
Gene Disruption ◽  
Selection Marker ◽  
Suicide Plasmid ◽  
E Coli ◽  
Targeted Gene Disruption ◽  
Knockout Mutants ◽  
Mycoplasma Mycoides

ABSTRACT A suicide plasmid, pExp1-ctpA::tetM-recAec, employing recA from Escherichia coli and tetM as a selection marker, was used to generate ctpA knockout mutants in Mycoplasma mycoides subsp. capri through targeted gene disruption. Inclusion of E. coli recA greatly enhanced both the consistency and the recovery of mutants generated by homologous recombination.

scholarly journals Disruption of a Gene Predicted To Encode a Solute Binding Protein of an ABC Transporter Reduces Transmission of Spiroplasma citri by the Leafhopper Circulifer haematoceps

2004 ◽  
Vol 70 (7) ◽  
pp. 3960-3967 ◽  
Author(s):  
A. Boutareaud ◽  
J. L. Danet ◽  
M. Garnier ◽  
C. Saillard
Keyword(s):  
Abc Transporter ◽  
Binding Protein ◽  
Functional Complementation ◽  
Wild Type ◽  
Gene Encoding ◽  
Spiroplasma Citri ◽  
Leafhopper Vector ◽  
Circulifer Haematoceps ◽  
The Family ◽  
Phloem Feeding

ABSTRACT Spiroplasma citri is transmitted from plant to plant by phloem-feeding leafhoppers. In an attempt to identify mechanisms involved in transmission, mutants of S. citri affected in their transmission must be available. For this purpose, transposon (Tn4001) mutagenesis was used to produce mutants which have been screened for their ability to be transmitted by the leafhopper vector Circulifer haematoceps to periwinkle plants. With one mutant (G76) which multiplied in leafhoppers as efficiently as S. citri wild-type (wt) strain GII-3, the plants showed symptoms 4 to 5 weeks later than those infected with wt GII-3. Thirty to fifty percent of plants exposed to leafhoppers injected with G76 remained symptomless, whereas for wt GII-3, all plants exposed to the transmission showed severe symptoms. This suggests that the mutant G76 was injected into plants by the leafhoppers less efficiently than wt GII-3. To check this possibility, the number of spiroplasma cells injected by a leafhopper through a Parafilm membrane into SP4 medium was determined. Thirty times less mutant G76 than wt GII-3 was transmitted through the membrane. These results suggest that mutant G76 was affected either in its capacity to penetrate the salivary glands and/or to multiply within them. In mutant G76, transposon Tn4001 was shown to be inserted into a gene encoding a putative lipoprotein (Sc76) In the ABCdb database Sc76 protein was noted as a solute binding protein of an ABC transporter of the family S1_b. Functional complementation of the G76 mutant with the Sc76 gene restored the wild phenotype, showing that Sc76 protein is involved in S. citri transmission by the leafhopper vector C. haematoceps.

Sequence analysis of theGluconobacter oxydansRecA protein and construction of arecA-deficient mutant

1999 ◽  
Vol 45 (4) ◽  
pp. 347-351 ◽  
Author(s):  
Yu-Tien Liu ◽  
Chia-Geun Chen ◽  
Der-Chiang Chao ◽  
Fan Lee ◽  
Ching-Len Liao ◽  
...  
Keyword(s):  
Amino Acid ◽  
Uv Irradiation ◽  
Reca Protein ◽  
Kanamycin Resistance ◽  
Reca Gene ◽  
Amino Acid Residues ◽  
Deficient Mutant ◽  
Wild Type ◽  
E Coli ◽  
Nucleotide Sequence Alignment

The deduced amino acid sequence of Gluconobacter oxydans RecA protein shows 75.2, 69.4, and 66.2% homology with those from Aquaspirillum magnetotacticum, Escherichia coli, andPseudomonas aeruginosa, respectively. The amino acid residues essential for function of the recombinase, protease, and ATPase in E. coli recA protein are conserved in G. oxydans. Of 24 amino acid residues believed to be the ATP binding domain of E. coli RecA, 17 are found to be identical in G. oxydans RecA. Interestingly, nucleotide sequence alignment between the SOS box of G. orphans recA gene and those from different microorganisms revealed that all the DNA sequences examined have dyad symmetry that can form a stem-loop structure. A G. oxydans recA-deficient mutant (LCC96) was created by allelic exchange using the cloned recA gene that had been insertionally inactivated by a kanamycin-resistance cassette. Such replacement of the wild-type recA with a kanamycin resistance gene in the chromosome was further verified by Southern hybridization. Phenotypically, the recA-deficient mutant is significantly more sensitive to UV irradiation than the wild-type strain, suggesting that the recA gene of G. oxydans ATCC9324 plays a role in repairing DNA damage caused by UV irradiation. Moreover, the mutant strain is much more plasmid transformable than its parent strain, illustrating that G. oxydans LCC96 could be used as a host to take up the recombinant plasmid for gene manipulation.Key words: Gluconobacter orphans, recA gene, DNA repair, recA mutant, SOS box.

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