CRISPR, cas genes, Phage and Biofilms

To better understand host genes involved in DMS3-mediated biofilm inhibition, we performed transposon mutagenesis of a P. aeruginosa DMS3 lysogen and identified mutants in which biofilm formation and swarming were restored. This approach led us to discover that certain mutations of the bacterial CRISPR region resulted in intact biofilm formation and swarming in DMS3 lysogens. The CRISPR region consists of two CRISPRs separated by 6 CRISPR associated (cas) genes with sequence and gene organization similar to the CRISPR region of Yersina pestis.

CRISPR, or Clustered Regularly Interspaced Short Palindromic Repeats, are found in most Archaea and many Bacteria, and are typically organized as 20 to 30 base pair (bp) identical repeats interspersed with similarly sized non-repeating spacer sequences often identical to bacteriophage sequences and other mobile genetic elements. Notably our analysis revealed thatthe entire second 32 bp spacer sequence of CRISPR-2 is identical to a region of the phage gene DMS3-24. The CRISPR region of P. aeruginosa strain PA14 is shown below.

CRISPR region of P. aeruginosa PA14.  Shown in the center of the figure is the chromosomal organization of the CRISPR genomic region, including the two CRISPRs and the CRISPR associated (cas) genes.  The PA14 gene designation is listed above each gene and the corresponding homolog from Y. pestis is shown below each gene. The size of each open reading frame is as follows: PA14_33300 (504 bp), PA14_33310 (1029 bp), PA14_33320 (984 bp), PA14_33330 (1305 bp), PA14_33340 (3231bp) and PA14_33350 (975 bp).  CRISPR-2 is annotated as PA14_33360 in the PA14 genome and is 1395 bp. The organization of the CRISPRs is also shown. Black boxes indicate repeats and gray boxes indicate spacers containing identical or partial nucleotide sequence matches from the indicated bacteriophages. The appropriate spacer number is listed above each spacer with known sequence identity to a phage, as is the last spacer number indicating the total number of spacers in each CRISPR. From Zegans, J. Bacteriol, 2009.

We are currently exploring how the interactions between phage and the CRISPR region impact group behaviors of P. aeruginosa.  A link to a recent study from our lab is below:

Zegans ME, Wagner JC, Cady KC, Murphy DM, Hammond JH, O'Toole GA. 2009. Interaction between bacteriophage DMS3 and host CRISPR region inhibits group behaviors of Pseudomonas aeruginosa.  J Bacteriol. 2009 191:210-9.