title: A markerless protocol for genetic analysis of Aggregatibacter actinomycetemcomitans.
abstract: OBJECTIVE
  The genomes of different Aggregatibacter actinomycetemcomitans (A actinomycetemcomitans) strains contain many strain-specific genes and genomic islands (defined as DNA found in some but not all strains) of unknown functions. Genetic analysis for the functions of these islands will be constrained by the limited availability of genetic markers and vectors for A actinomycetemcomitans. In this study, we tested a novel genetic approach of gene deletion and restoration in a naturally competent A actinomycetemcomitans strain D7S-1.
  
  
  METHODS
  Specific genes' deletion mutants and mutants restored with the deleted genes were constructed by a markerless loxP/Cre system. In mutants with sequential deletion of multiple genes loxP with different spacer regions were used to avoid unwanted recombinations between loxP sites.
  
  
  RESULTS
  Eight single-gene deletion mutants, four multiple-gene deletion mutants, and two mutants with restored genes were constructed. No unintended non-specific deletion mutants were generated by this protocol. The protocol did not negatively affect the growth and biofilm formation of A actinomycetemcomitans.
  
  
  CONCLUSIONS
  The protocol described in this study is efficient and specific for genetic manipulation of A actinomycetemcomitans, and will be amenable for functional analysis of multiple genes in A actinomycetemcomitans.
authors: School of Dentistry and Graduate Institute of Clinical Dentistry, National Taiwan University, Taipei, Taiwan.
authors: Ya-An
authors: YA
authors: Cheng
authors: Division of Periodontology, Diagnostic Sciences and Dental Hygiene, Herman Ostrow School of Dentistry of the University of Southern California, Los Angeles, CA, USA.
authors: Jason
authors: J
authors: Jee
authors: Division of Periodontology, Diagnostic Sciences and Dental Hygiene, Herman Ostrow School of Dentistry of the University of Southern California, Los Angeles, CA, USA.
authors: Genie
authors: G
authors: Hsu
authors: Division of Periodontology, Diagnostic Sciences and Dental Hygiene, Herman Ostrow School of Dentistry of the University of Southern California, Los Angeles, CA, USA.
authors: Yanyan
authors: Y
authors: Huang
authors: Division of Periodontology, Diagnostic Sciences and Dental Hygiene, Herman Ostrow School of Dentistry of the University of Southern California, Los Angeles, CA, USA.
authors: Casey
authors: C
authors: Chen
authors: School of Dentistry and Graduate Institute of Clinical Dentistry, National Taiwan University, Taipei, Taiwan. Electronic address: pinlin@ntu.edu.tw.
authors: Chun-Pin
authors: CP
authors: Lin
mesh_terms: D016976:Aggregatibacter actinomycetemcomitans / Q000235:genetics*; D017353:Gene Deletion; D044404:Genomic Islands
keywords: aggressive periodontitis; gene deletion; genetic analysis; genomic islands