title: Identification of genes essential for pellicle formation in Acinetobacter baumannii.
abstract: BACKGROUND
  Acinetobacter baumannii is an opportunistic pathogen, which has the ability to persist in the clinical environment, causing acute and chronic infections. A possible mechanism contributing to survival of A. baumannii is its ability to form a biofilm-like structure at the air/liquid interface, known as a pellicle. This study aimed to identify and characterise the molecular mechanisms required for pellicle formation in A. baumannii and to assess a broad range of clinical A. baumannii strains for their ability to form these multicellular structures.
  
  
  RESULTS
  Random transposon mutagenesis was undertaken on a previously identified hyper-motile variant of A. baumannii ATCC 17978 designated 17978hm. In total three genes critical for pellicle formation were identified; cpdA, a phosphodiesterase required for degradation of cyclic adenosine monophosphate (cAMP), and A1S_0112 and A1S_0115 which are involved in the production of a secondary metabolite. While motility of the A1S_0112::Tn and A1S_0115::Tn mutant strains was abolished, the cpdA::Tn mutant strain displayed a minor alteration in its motility pattern. Determination of cAMP levels in the cpdA::Tn strain revealed a ~24-fold increase in cellular cAMP, confirming the role CpdA plays in catabolising this secondary messenger molecule. Interestingly, transcriptional analysis of the cpdA::Tn strain showed significant down-regulation of the operon harboring the A1S_0112 and A1S_0115 genes, revealing a link between these three genes and pellicle formation. Examination of our collection of 54 clinical A. baumannii strains revealed that eight formed a measurable pellicle; all of these strains were motile.
  
  
  CONCLUSIONS
  This study shows that pellicle formation is a rare trait in A. baumannii and that a limited number of genes are essential for the expression of this phenotype. Additionally, an association between pellicle formation and motility was identified. The level of the signalling molecule cAMP was found to be controlled, in part, by the cpdA gene product, in addition to playing a critical role in pellicle formation, cellular hydrophobicity and motility. Furthermore, cAMP was identified as a novel regulator of the operon A1S_0112-0118.
authors.affiliation: School of Biological Sciences, Flinders University, GPO Box 2100, 5001, Adelaide, SA, Australia. sarah.giles@flinders.edu.au.
authors.forename: Sarah K
authors.initials: SK
authors.lastname: Giles
authors.affiliation: School of Biological Sciences, Flinders University, GPO Box 2100, 5001, Adelaide, SA, Australia. uwe.stroeher@flinders.edu.au.
authors.forename: Uwe H
authors.initials: UH
authors.lastname: Stroeher
authors.affiliation: School of Biological Sciences, Flinders University, GPO Box 2100, 5001, Adelaide, SA, Australia. bart.eijkelkamp@adelaide.edu.au.|Research Centre for Infectious Diseases, School of Biological Sciences University of Adelaide, Adelaide, Australia. bart.eijkelkamp@adelaide.edu.au.
authors.forename: Bart A
authors.initials: BA
authors.lastname: Eijkelkamp
authors.affiliation: School of Biological Sciences, Flinders University, GPO Box 2100, 5001, Adelaide, SA, Australia. melissa.brown@flinders.edu.au.
authors.forename: Melissa H
authors.initials: MH
authors.lastname: Brown
mesh_terms: D040981:Acinetobacter baumannii / Q000502:physiology*; D001426:Bacterial Proteins / Q000235:genetics* / Q000378:metabolism*; D018441:Biofilms / Q000254:growth & development*; D000242:Cyclic AMP / Q000378:metabolism; D020869:Gene Expression Profiling; D015964:Gene Expression Regulation, Bacterial; D020043:Genes, Essential; D057927:Hydrophobic and Hydrophilic Interactions; D016254:Mutagenesis, Insertional; D009876:Operon; D040641:Quantitative Trait Loci
chemical_list: D001426:Bacterial Proteins; D000242:Cyclic AMP