ECA29 has integrated into the 5′-end of pflA. If this process led to a nonfunctional or absent PflA protein, further degeneration of the coding sequence may have occurred. Therefore, the PflA amino acid sequence was compared with the sequences of its homologues in other enteric species, showing that a full-length PflA is predicted (barring the five residue N-terminal

disruption caused by prophage integration), without any premature truncations or mutations that would obviously eliminate function (Supporting Information, Fig. S1). pflA codes for pyruvate formate lyase-activating enzyme. Once activated, pyruvate formate lyase is responsible for catalysing the first committed step of anaerobic glucose metabolism. We first attempted Selleck BYL719 to detect a pflA transcript by RT-PCR. Using primers to the 3′-end of the gene, a transcript was detected, suggesting that there is an outward-reading

promoter at the 3′-end of ECA29 (Fig. 2). Integration of phages in the 5′-end of genes can alter the expression of such genes by generating polar mutations or by providing alternative promoters. Streptococcus pyogenes provides a number of examples, where such transcription-altering prophages appear to be an important class of genetic regulatory elements (discussed by McShan & Ferretti, 2007). In this case, if the pflA transcript is translated, albeit without the five N-terminal residues, a functional protein may be produced. Therefore, anaerobic growth of wild-type AZD2281 in vivo Pa was compared with a strain carrying the full-length pflA gene in trans on plasmid pTE13 with glucose Interleukin-3 receptor as the sole carbon source. Serial dilutions of each strain were plated on MM plates in an anaerobic chamber. Viable counts of only 102 cells mL−1 were observed, and this was the same regardless of the presence or the absence of pTE13 (data not shown). This low cell count (109 cells mL−1 were observed when plates were grown aerobically) demonstrates that wild-type Pa cannot grow anaerobically on MM and neither is growth possible in the presence of the full-length pflA gene. We cannot rule out functionally important mutations either in this gene or in other genes essential for anaerobic

metabolism. Prophages often contain cargo genes that contribute to virulence. Analysis of the prophage sequences did not reveal the presence of any genes that obviously contribute to pathogenicity, such as toxins, although a number of uncharacterized, hypothetical genes are present. Interestingly, microarray studies have shown that ECA2598 and ECA2617 (present in ECA29) are upregulated in a quorum-sensing mutant at 12 h postinfection in the potato (Liu et al., 2008). These genes encode a putative exported protein and a phage lysis protein, respectively. Additionally, the protein products of uncharacterized genes ECA3710 and ECA3737 (present in ECA41) have been detected in an unrelated proteomics investigation of the Pa intracellular proteome (K.