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Ag43 is an abundant outer membrane autotransporter adhesin present in most commensal and pathogenic  Escherichia coli . Expression of the  agn43  gene is characterized by a regulated reversible switch or phase variation between the  agn43  ON and  agn43  OFF states. Although the  agn43  regulatory switch leads to a heterogeneous population of ON and OFF bacteria, studies of Ag43 seldom consider potential biases associated with phase variation. We monitored  agn43  ON/OFF phase-variation status genetically and phenotypically and we show that the use of populations with random  agn43  ON or OFF status could result in misleading conclusions about Ag43 function or regulation. In particular, we demonstrate that Lrp and MqsR, previously identified as  agn43  regulators, do not regulate  agn43  expression or ON/OFF switch frequency. We also show that biofilm formation in dynamic flow conditions does not influence  agn43  ON/OFF switching but physically selects aggregating  agn43  ON cells. This indicates that misinterpretation is possible when studying gene expression within biofilms. Finally, we provide evidence that ignoring the initial  agn43  ON/OFF status of the  E. coli  populations studied is likely to bias analyses of phenotypes associated with other  E. coli  adhesins. This study therefore emphasizes the importance of monitoring Ag43 phase variation and indicates that caution is required when interpreting experiments using strains that are neither deleted for  agn43  nor carefully assessed for  agn43  ON/OFF status.

Did I Pick the Right Colony? Pitfalls in the Study of Regulation of the Phase Variable Antigen 43 Adhesin