YeeJ is an inverse autotransporter from Escherichia coli that binds to peptidoglycan and promotes biofilm formation.

Escherichia coli is a commensal or pathogenic bacterium that can survive in diverse environments. Adhesion to surfaces is essential for E. coli colonization, and thus it is important to understand the molecular mechanisms that promote this process in different niches. Autotransporter proteins are a class of cell-surface factor used by E. coli for adherence. Here we characterized the regulation and function of YeeJ, a poorly studied but widespread representative from an emerging class of autotransporter proteins, the inverse autotransporters (IAT). We showed that the yeeJ gene is present in ~40% of 96 completely sequenced E. coli genomes and that YeeJ exists as two length variants, albeit with no detectable functional differences. We demonstrated that YeeJ promotes biofilm formation in different settings through exposition at the cell-surface. We also showed that YeeJ contains a LysM domain that interacts with peptidoglycan and thus assists its localization into the outer membrane. Additionally, we identified the Polynucleotide Phosphorylase PNPase as a repressor of yeeJ transcription. Overall, our work provides new insight into YeeJ as a member of the recently defined IAT class, and contributes to our understanding of how commensal and pathogenic E. coli colonise their environments.

The dynamics and pH-dependence of Ag43 adhesins' self-association probed by atomic force spectroscopy.

Self-associating auto-transporter (SAAT) adhesins are two-domain cell surface proteins involved in bacteria auto-aggregation and biofilm formation. Antigen 43 (Ag43) is a SAAT adhesin commonly found in Escherichia coli whose variant Ag43a has been shown to promote persistence of uropathogenic E. coli within the bladder. The recent resolution of the tri-dimensional structure of the 499 amino-acids' ß-domain in Ag43a has shed light on the possible mechanism governing the self-recognition of SAAT adhesins, in particular the importance of trans-interactions between the L shaped ß-helical scaffold of two α-domains of neighboring adhesins. In this study, we use single-molecule force spectroscopy (SMFS) and dynamic force spectroscopy (DFS) to unravel the dynamics of Ag43-self association under various pH and molecular elongation rate conditions that mimic the situations encountered by E. coli in its natural environment. Results evidenced an important stretchability of Ag43α with unfolding of sub-domains leading to molecular extension as long as 150 nm. Nanomechanical analysis of molecular stretching data suggested that self-association of Ag43 can lead to the formation of dimers and tetramers driven by rapid and weak cis- as well as slow but strong trans-interaction forces with a magnitude as large as 100-250 pN. The dynamics of cis- and trans-interactions were demonstrated to be strongly influenced by pH and applied shear force, thus suggesting that environmental conditions can modulate Ag43-mediated aggregation of bacteria at the molecular level.

Did I pick the right colony? Pitfalls in the study of regulation of the phase variable antigen 43 adhesin.

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.