Results Deletion of cassettes reduces growth on some carbon sources To investigate how cassette genes may influence adaptation in their bacterial host, AICAR solubility dmso deletions of cassettes were created in the integron cassette array of Vibrio rotiferianus DAT722. Two cassette

deletion mutants within the 116-gene cassette array of Vibrio rotiferianus DAT722 were created (See Methods and Figure 1). These mutants removed cassettes 8-60 (designated d8-60a) in one case and cassettes 16-60 (d16-60) in the other. The ability PD-1/PD-L1 Inhibitor 3 mouse of these mutants to grow in various media were tested and compared to the wild type parent (Figure 2). It was found that both mutants were able to grow normally CA4P concentration in a complete medium (LB20) albeit with a slightly extended lag phase for d8-60a (Figure 2A). The two mutants were also examined for growth in marine minimal medium (2M salts, a medium that mimics marine seawater [20]) with glucose (Figure 2B) or pyruvate (Figure 2C) as a sole carbon source. The growth of both mutants was normal compared to wild type (Vibrio rotiferianus DAT722)

in 2M + glucose as was also the case for d16-60 in pyruvate. In contrast, d8-60a grew very poorly with pyruvate as sole carbon source. Growth of this mutant however could be restored on pyruvate with the addition of glycine-betaine, a known osmoprotectant (Figure 3). Glucose is also known to be a better osmoprotectant than pyruvate and we therefore tentatively conclude that the poor growth of d8-60a in pyruvate is a result of intolerance to osmotic changes and not a failure to extract carbon from this molecule. Further growth Decitabine nmr experiments supported this hypothesis with growth on other carbon sources that osmoprotect (eg trehalose) compared to failure to grow on other non-osmoprotectants (aspartic acid, glutamic acid, succinate and fumarate) (data not shown). These data suggested that this cassette array may include encoded proteins that integrate into and influence cellular processes more broadly in contrast to possessing proteins involved in single step

secondary metabolism. Specifically, in DAT722, at least one cassette product appears to influence normal growth under nutrient conditions analogous to those found in seawater, the natural free-living environment for Vibrio rotiferianus. Figure 1 Creation of deletions in cassette array of Vibrio rotiferianus DAT722. Genetic features of the integron are labelled in the figure (A). The numbers below each cassette indicates its position in the array relative to attI. The white (group 1) and grey cassettes (group 2) indicate the two groups of paralogous cassettes described in the Materials and Methods section. Not all cassettes are shown with gaps of missing cassettes marked with a ~ symbol.