title: A novel strategy for the construction of genomic mutants of the Antarctic bacterium Pseudoalteromonas haloplanktis TAC125.
abstract: The sequencing and the annotation of the marine Antarctic Pseudoalteromonas haloplanktis TAC125 genome has paved the way to investigate on the molecular mechanisms involved in adaptation to cold conditions. The growing interest in this unique bacterium prompted the developing of several genetic tools for studying it at the molecular level. To allow a deeper understanding of the PhTAC125 physiology a genetic system for the reverse genetics in this bacterium was developed. In the present work, we describe a practical technique for allelic exchange and/or gene inactivation by in-frame deletion and the use of a counterselectable marker in P. haloplanktis. The construction of suitable non-replicating plasmid and methods used to carry out a two-step integration-segregation strategy in this bacterium are reported in detail.Furthermore two examples, in which the developed methodology was applied to find out gene function or to construct genetically engineered bacterial strains, were described.
authors.affiliation: Department of Organic Chemistry and Biochemistry, Università degli studi di Napoli Federico II, Naples, Italy.
authors.forename: Maria
authors.initials: M
authors.lastname: Giuliani
authors.forename: Ermenegilda
authors.initials: E
authors.lastname: Parrilli
authors.forename: Cinzia
authors.initials: C
authors.lastname: Pezzella
authors.forename: Valentina
authors.initials: V
authors.lastname: Rippa
authors.forename: Angela
authors.initials: A
authors.lastname: Duilio
authors.forename: Gennaro
authors.initials: G
authors.lastname: Marino
authors.forename: Maria Luisa
authors.initials: ML
authors.lastname: Tutino
mesh_terms: D000220:Adaptation, Biological / Q000235:genetics*; D000864:Antarctic Regions; D060846:Cell Engineering / Q000379:methods*; D003080:Cold Temperature; D003470:Culture Media / Q000737:chemistry; D017931:DNA Primers / Q000235:genetics; D015964:Gene Expression Regulation, Bacterial / Q000235:genetics*; D020868:Gene Silencing; D005818:Genetic Engineering / Q000379:methods*; D016296:Mutagenesis; D010957:Plasmids / Q000235:genetics; D031161:Pseudoalteromonas / Q000235:genetics*; D017384:Sequence Deletion / Q000235:genetics; D054793:Truncated Hemoglobins / Q000235:genetics / Q000378:metabolism
chemical_list: D003470:Culture Media; D017931:DNA Primers; D054793:Truncated Hemoglobins