Recombinant Escherichia coli Inner membrane protein YbhL (ybhL)

Primary and Secondary Structure

Recombinant YbhL is produced as a full-length protein (1–234 amino acids) fused to an N-terminal hexahistidine (His) tag for purification . The amino acid sequence begins with MDRFPRSDSIVQPRAGLQTYMAQVYGWMTVGLLLTAFVAWYAANSAAVMELLFTNRVFLIGLIIAQLALVIVLSAMIQKLSAGVTTMLFMLYSALTGLTLSSIFIVYTAASIASTFVVTA GMFGAMSLYGYTTKRDLSGFGNMLFMALIGIVLASLVNFWLKSEALMWAVTYIGVIVFVGLTAYDTQKLKNMGEQIDTRDTSNLRKYSILGALTLYLDFINLFLMLLRIFGNRR, featuring hydrophobic domains characteristic of inner membrane proteins . Circular dichroism studies predict α-helical transmembrane segments, consistent with its localization in the inner membrane .

Table 1: Biophysical Properties of Recombinant YbhL

Role in Membrane Transport and Stress Response

YbhL is hypothesized to function as an acetate transporter, facilitating the movement of short-chain fatty acids across the inner membrane . This activity aligns with its interaction partners, including YbhM (a BAX Inhibitor-1 family paralog) and YdiF (a CoA transferase involved in fatty acid metabolism) . Genetic knockout studies suggest that YbhL contributes to E. coli’s adaptation to osmotic stress and nutrient limitation, possibly by modulating intracellular acetate levels .

Involvement in the SOS Response

YbhL is regulated by the LexA repressor, a key mediator of the SOS response to DNA damage . Under stress conditions, LexA cleavage derepresses ybhL transcription, enabling YbhL synthesis. This regulatory link positions YbhL within a network of stress-response proteins, including YdjM and YcgB, which collectively enhance bacterial survival under adverse conditions .

YjjQ-Mediated Repression of ybhL

The transcriptional regulator YjjQ directly represses ybhL by binding to a 15-bp palindromic motif (5′-TGTCCATCAGTTGAC-3′) overlapping the promoter region . Electrophoretic mobility shift assays (EMSAs) confirm that YjjQ binds this motif with high affinity (K_d = 12 nM), reducing ybhL expression by 4.5-fold in E. coli K-12 . Mutagenesis of the YjjQ-binding site abolishes repression, underscoring the motif’s necessity for regulatory control .

Table 2: YjjQ-Binding Motifs in ybhL and Other Targets

Cross-Regulation with RcsA-RcsB

At the flhDC promoter, YjjQ and the RcsA-RcsB two-component system independently repress transcription, highlighting overlapping regulatory mechanisms . While RcsA-RcsB responds to envelope stress, YjjQ-mediated repression is constitutive, suggesting distinct environmental triggers for ybhL regulation .

Role in Avian Pathogenic E. coli (APEC)

In APEC, YjjQ deletion mutants exhibit upregulated ybhL expression, correlating with enhanced motility and virulence in poultry . Comparative transcriptomics revealed that YbhL contributes to capsule synthesis and biofilm formation, critical for host colonization .

Impact on Uropathogenic E. coli (UPEC)

In UPEC strains CFT073 and 536, YbhL repression by YjjQ reduces bladder epithelial cell invasion by 40%, as shown in murine infection models . This phenotype is linked to YbhL’s modulation of YfiRNB, a cyclic di-GMP signaling system governing cellulose production and biofilm integrity .

Recombinant Expression Protocols

Recombinant YbhL is expressed in E. coli BL21(DE3) using a pET-28a(+) vector, yielding 15–20 mg/L culture after nickel-affinity chromatography . Optimal reconstitution requires deionized water supplemented with 50% glycerol to maintain stability during storage (−80°C) .

Table 3: Expression and Purification Parameters