Site-Specific Recombination in the Cyanobacterium Anabaena sp. Strain PCC 7120 Catalyzed by the Integrase of Coliphage HK022

ABSTRACT The integrase (Int) of the λ-like coliphage HK022 catalyzes the site-specific integration and excision of the phage DNA into and from the chromosome of its host, Escherichia coli. Int recognizes two different pairs of recombining sites attP × attB and attL × attR for integration and excision, respectively. This system was adapted to the cyanobacterium Anabaena sp. strain PCC 7120 as a potential tool for site-specific gene manipulations in the cyanobacterium. Two plasmids were consecutively cointroduced by conjugation into Anabaena cells, one plasmid that expresses HK022 Int recombinase and the other plasmid that carries the excision substrate PglnA -attL-T1/T2-attR-lacZ, where T1/T2 are the strong transcription terminators of rrnB, to prevent expression of the lacZ reporter under the constitutive promoter PglnA . The Int-catalyzed site-specific recombination reaction was monitored by the expression of lacZ emanating as a result of T1/T2 excision. Int catalyzed the site-specific excision reaction in Anabaena cells when its substrate was located either on the plasmid or on the chromosome with no need to supply an accessory protein, such as integration host factor and excisionase (Xis), which are indispensable for this reaction in its host, E. coli.

Download Full-text

Site-Specific Recombination in the Cyanobacterium Anabaena sp. Strain PCC 7120 Catalyzed by the Integrase of Coliphage HK022

Journal of Bacteriology ◽

10.1128/jb.00813-09 ◽

2009 ◽

Vol 191 (18) ◽

pp. 5879-5879 ◽

Cited By ~ 1

Author(s):

Olga Melnikov ◽

Arieh Zaritsky ◽

Aliza Zarka ◽

Sammy Boussiba ◽

Natalia Malchin ◽

...

Keyword(s):

Site Specific ◽

Site Specific Recombination ◽

Cyanobacterium Anabaena ◽

Anabaena Sp ◽

Pcc 7120

Download Full-text

Functional Analysis of the Acinetobacter baumannii XerC and XerD Site-Specific Recombinases: Potential Role in Dissemination of Resistance Genes

Antibiotics ◽

10.3390/antibiotics9070405 ◽

2020 ◽

Vol 9 (7) ◽

pp. 405

Author(s):

David L. Lin ◽

German M. Traglia ◽

Rachel Baker ◽

David J. Sherratt ◽

Maria Soledad Ramirez ◽

...

Keyword(s):

Escherichia Coli ◽

Functional Analysis ◽

Acinetobacter Baumannii ◽

Potential Role ◽

Covalent Attachment ◽

Recombination Reaction ◽

Site Specific ◽

Site Specific Recombination ◽

E Coli ◽

Resistant Genes

Modules composed of a resistance gene flanked by Xer site-specific recombination sites, the vast majority of which were found in Acinetobacter baumannii, are thought to behave as elements that facilitate horizontal dissemination. The A. baumannii xerC and xerD genes were cloned, and the recombinant clones used to complement the cognate Escherichia coli mutants. The complemented strains supported the resolution of plasmid dimers, and, as is the case with E. coli and Klebsiella pneumoniae plasmids, the activity was enhanced when the cells were grown in a low osmolarity growth medium. Binding experiments showed that the partially purified A. baumannii XerC and XerD proteins (XerCAb and XerDAb) bound synthetic Xer site-specific recombination sites, some of them with a nucleotide sequence deduced from existing A. baumannii plasmids. Incubation with suicide substrates resulted in the covalent attachment of DNA to a recombinase, probably XerCAb, indicating that the first step in the recombination reaction took place. The results described show that XerCAb and XerDAb are functional proteins and support the hypothesis that they participate in horizontal dissemination of resistant genes among bacteria.

Download Full-text

Osmoregulation of Dimer Resolution at the Plasmid pJHCMW1 mwr Locus by Escherichia coli XerCD Recombination

Journal of Bacteriology ◽

10.1128/jb.184.6.1607-1616.2002 ◽

2002 ◽

Vol 184 (6) ◽

pp. 1607-1616 ◽

Cited By ~ 12

Author(s):

Huong Pham ◽

Ken J. Dery ◽

David J. Sherratt ◽

Marcelo E. Tolmasky

Keyword(s):

Escherichia Coli ◽

Central Region ◽

High Salt ◽

Recombination Reaction ◽

Recombination Site ◽

Site Specific ◽

Site Specific Recombination ◽

E Coli ◽

Low Salt ◽

Ionic Effects

ABSTRACT Xer-mediated dimer resolution at the mwr site of plasmid pJHCMW1 is osmoregulated in Escherichia coli. Whereas under low-salt conditions, the site-specific recombination reaction is efficient, under high-salt conditions, it proceeds inefficiently. Regulation of dimer resolution is independent of H-NS and is mediated by changes in osmolarity rather than ionic effects. The low level of recombination at high salt concentrations can be overcome by high levels of PepA or by mutating the ARG box to a sequence closer to the E. coli ARG box consensus. The central region of the mwr core recombination site plays a role in regulation of site-specific recombination by the osmotic pressure of the medium.

Download Full-text

Functional Analysis of the Acinetobacter baumannii XerC and XerD Site-Specific Recombinases: Potential Role in Dissemination of Resistance Genes

10.20944/preprints202006.0163.v1 ◽

2020 ◽

Author(s):

David L. Lin ◽

German M. Traglia ◽

Rachel Baker ◽

David J. Sherratt ◽

Maria S. Ramirez ◽

...

Keyword(s):

Escherichia Coli ◽

Functional Analysis ◽

Acinetobacter Baumannii ◽

Potential Role ◽

Covalent Attachment ◽

Recombination Reaction ◽

Site Specific ◽

Site Specific Recombination ◽

E Coli ◽

Resistant Genes

Modules composed of a resistance gene flanked by Xer site-specific recombination sites, the vast majority of which were found in Acinetobacter baumannii, are thought to behave as elements that facilitate horizontal dissemination. The xerCAb and xerDAb genes were cloned, and the recombinant clones used to complement the cognate Escherichia coli mutants. The complemented strains supported resolution of plasmid dimers, and, as is the case with E. coli and Klebsiella pneumoniae plasmids, the activity was enhanced when cells were growing in low osmolarity growth medium. Binding experiments showed that partially purified A. baumannii XerC and XerD proteins (XerCAb and XerDAb) bound synthetic Xer site-specific recombination sites, some of them with a nucleotide sequence deduced from existing A. baumannii plasmids. Incubation with suicide substrates resulted in covalent attachment of DNA to a recombinase, probably XerCAb, indicating that the first step in the recombination reaction took place. The results described show that XerCAb and XerDAb are functional proteins that actively participate in horizontal dissemination of resistant genes among bacteria.

Download Full-text

Expanding the Natural Products Heterologous Expression Repertoire in the Model Cyanobacterium Anabaena sp. Strain PCC 7120: Production of Pendolmycin and Teleocidin B-4

10.26434/chemrxiv.11316098.v1 ◽

2019 ◽

Cited By ~ 1

Author(s):

Patrick Videau ◽

Kaitlyn Wells ◽

Arun Singh ◽

Jessie Eiting ◽

Philip Proteau ◽

...

Keyword(s):

Natural Products ◽

Genome Mining ◽

Gene Clusters ◽

Combinatorial Biosynthesis ◽

Test Case ◽

Biosynthetic Gene ◽

Biosynthetic Gene Clusters ◽

Cyanobacterium Anabaena ◽

Anabaena Sp ◽

Pcc 7120

Cyanobacteria are prolific producers of natural products and genome mining has shown that many orphan biosynthetic gene clusters can be found in sequenced cyanobacterial genomes. New tools and methodologies are required to investigate these biosynthetic gene clusters and here we present the use of <i>Anabaena </i>sp. strain PCC 7120 as a host for combinatorial biosynthesis of natural products using the indolactam natural products (lyngbyatoxin A, pendolmycin, and teleocidin B-4) as a test case. We were able to successfully produce all three compounds using codon optimized genes from Actinobacteria. We also introduce a new plasmid backbone based on the native <i>Anabaena</i>7120 plasmid pCC7120ζ and show that production of teleocidin B-4 can be accomplished using a two-plasmid system, which can be introduced by co-conjugation.

Download Full-text