Tri-methylation of histone H3 lysine 9 is important for recruiting heterochromatin protein 1 (HP1) to discrete regions of the genome, thereby regulating gene expression, chromatin packaging and heterochromatin formation. Here we show that HP1alpha, -beta, and -gamma are released from chromatin during the M phase of the cell cycle, even though tri-methylation levels of histone H3 lysine 9 remain unchanged. However, the additional, transient modification of histone H3 by phosphorylation of serine 10 next to the more stable methyl-lysine 9 mark is sufficient to eject HP1 proteins from their binding sites. Inhibition or depletion of the mitotic kinase Aurora B, which phosphorylates serine 10 on histone H3, causes retention of HP1 proteins on mitotic chromosomes, suggesting that H3 serine 10 phosphorylation is necessary for the dissociation of HP1 from chromatin in M phase. These findings establish a regulatory mechanism of protein-protein interactions, through a combinatorial readout of two adjacent post-translational modifications: a stable methylation and a dynamic phosphorylation mark.

Biofilm formation is a complex, ordered process. In the opportunistic pathogen Pseudomonas aeruginosa, Psl and Pel exopolysaccharides and extracellular DNA (eDNA) serve as structural components of the biofilm matrix. Despite intensive study, Pel's chemical structure and spatial localization within mature biofilms remain unknown. Using specialized carbohydrate chemical analyses, we unexpectedly found that Pel is a positively charged exopolysaccharide composed of partially acetylated 1→4 glycosidic linkages of N-acetylgalactosamine and N-acetylglucosamine. Guided by the knowledge of Pel's sugar composition, we developed a tool for the direct visualization of Pel in biofilms by combining Pel-specific Wisteria floribunda lectin staining with confocal microscopy. The results indicate that Pel cross-links eDNA in the biofilm stalk via ionic interactions. Our data demonstrate that the cationic charge of Pel is distinct from that of other known P. aeruginosa exopolysaccharides and is instrumental in its ability to interact with other key biofilm matrix components.biofilms | exopolysaccharide | extracellular DNA | Pel | Psl

Allelic exchange is an efficient method of bacterial genome engineering. This protocol describes the use of this technique to make gene knockouts and knockins, as well as single nucleotide insertions, deletions and substitutions in Pseudomonas aeruginosa. Unlike other approaches to allelic exchange, this protocol does not require heterologous recombinases to insert or excise selective markers from the target chromosome. Rather, positive and negative selection are enabled solely by suicide vector-encoded functions and host cell proteins. Here, mutant alleles, which are flanked by regions of homology to the recipient chromosome, are synthesized in vitro and then cloned into allelic exchange vectors using standard procedures. These suicide vectors are then introduced into recipient cells by conjugation. Homologous recombination then results in antibiotic resistant single-crossover mutants in which the plasmid has integrated site-specifically into the chromosome. Subsequently, unmarked double-crossover mutants are isolated directly using sucrose-mediated counter-selection. This two-step process yields seamless mutations that are precise to a single base pair of DNA. The entire procedure requires ~2 weeks.

Bacterial biofilms are surface-associated, multicellular, morphologically complex microbial communities1-7. Biofilm-forming bacteria such as the opportunistic pathogen7-10 Pseudomonas aeruginosa are phenotypically distinct from their free-swimming, planktonic counterparts. Much work has focused on factors impacting surface adhesion and it is known that P. aeruginosa secretes the Psl exopolysaccharide, which promotes surface attachment by acting as a ‘molecular glue’11-15. However, how individual surface-attached bacteria self-organize into microcolonies, the first step in communal biofilm organization, is not well understood. Here, we identify a new role for Psl in early biofilm development using a massively parallel cell-tracking algorithm to extract the motility history of every cell on a newly colonized surface via a search-engine based approach16. By combining these techniques with fluorescent Psl staining and computer simulations, we show that P. aeruginosa deposits a trail of Psl as it moves on a surface, which influences the surface motility of subsequent cells that encounter these trails and thus generate positive feedback. Both experiments and simulations indicate that the web of secreted Psl controls the distribution of surface visit frequencies, which can be approximated by a power law. This Zipf's Law17 indicates that the bacterial community self-organizes in a manner analogous to a capitalist economic system18, a ‘rich-get-richer’ mechanism of Psl accumulation that results in a small number of ‘elite’ cells extremely enriched in communally produced Psl. Using engineered strains with inducible Psl production, we show that local Psl levels determine post-division cell fates and that high local Psl levels ultimately allow ‘elite’ cells to serve as the founding population for initial microcolony development.

SUMMARY Biofilm cells are less susceptible to antimicrobials than their planktonic counterparts. While this phenomenon is multifactorial, the ability of the biofilm matrix to reduce antibiotic penetration into the biofilm is thought to be of limited importance, as previous studies suggest that antibiotics move fairly rapidly through biofilms. In this study, we monitored the transport of two clinically relevant antibiotics, tobramycin and ciprofloxacin, into non-mucoid P. aeruginosa biofilms. To our surprise, we showed that the positively charged antibiotic tobramycin is sequestered to the biofilm periphery, while the neutral antibiotic ciprofloxacin readily penetrated. We provide evidence that tobramycin in the biofilm periphery both stimulated a localized stress response and killed bacteria in these regions, but not in the underlying biofilm. Although it is unclear which matrix component binds tobramycin, its penetration was increased by the addition of cations in a dose-dependent manner, which led to increased biofilm death. These data suggest that ionic interactions of tobramycin with the biofilm matrix limit its penetration. We propose that tobramycin sequestration at the biofilm periphery is an important mechanism in protecting metabolically active cells that lie just below the zone of sequestration.