Mechanism of transcription regulation by Acinetobacter baumannii HpaR in the catabolism of p‐hydroxyphenylacetate

Abstract

HpaR is a transcription regulator in the MarR family that controls the expression of the gene cluster responsible for conversion of p‐hydroxyphenylacetate to pyruvate and succinate for cellular metabolism. Here, we report the biochemical and structural characterization of Acinetobacter baumannii HpaR (AbHpaR) and its complex with cognate DNA. Our study revealed that AbHpaR binds upstream of the divergently transcribed hpaA gene and the meta‐cleavage operon, as well as the hpaR gene, thereby repressing their transcription by blocking access of RNA polymerase. Structural analysis of AbHpaR‐DNA complex revealed that the DNA binding specificity can be achieved via a combination of both direct and indirect DNA sequence readouts. DNA binding of AbHpaR is weakened by 3,4‐dihydroxyphenylacetate (DHPA), which is the substrate of the meta‐cleavage reactions; this likely leads to expression of the target genes. Based on our findings, we propose a model for how A. baumannii controls transcription of HPA‐metabolizing genes, which highlights the independence of global catabolite repression and could be beneficial for metabolic engineering toward bioremediation applications. Databases The structural data that support these findings are openly available in the wwPDB at https://doi.org/10.2210/pdb7EL2/pdb and https://doi.org/10.2210/pdb7EL3/pdb for AbHpaR and AbHpaR‐DNA complex, respectively.

Regulation of indole‐3‐acetic acid biosynthesis and consequences of auxin production deficiency in Serratia plymuthica

“…This cluster is homologous to the hpa gene cluster in Escherichia coli strain W (Díaz et al, 2001; Prieto et al, 1996). The hpaBC upper operon encodes the enzymes required to convert 4HPA to 3,4‐dihydroxyphenylacetic acid (3,4HPA), whereas the hpaGEDFHI meta operon encodes enzymes for the cleavage of the aromatic ring of 3,4HPA (Díaz et al, 2001; Permsirivisarn et al, 2022). It also encodes the AraC/XylS family transcriptional regulator HpaA and the MarR‐type regulator HpaR (Figure S2), which act as an activator and a repressor, respectively, of the upper and meta operons (Díaz et al, 2001; Galan et al, 2003; Prieto & García, 1997).…”

“…It also encodes the AraC/XylS family transcriptional regulator HpaA and the MarR‐type regulator HpaR (Figure S2), which act as an activator and a repressor, respectively, of the upper and meta operons (Díaz et al, 2001; Galan et al, 2003; Prieto & García, 1997). Different genetic and biochemical approaches suggest that 4HPA and 3,4HPA are ligands of HpaR that de‐repress expression of hpaGEDFHI (Galan et al, 2003; Permsirivisarn et al, 2022), whereas HpaA may preferentially sense 4HPA (Prieto & García, 1997). However, despite previous attempts (Permsirivisarn et al, 2022), there is, to our knowledge, no information available on the binding parameters of these regulators with their potential cognate ligands.…”

“…Different genetic and biochemical approaches suggest that 4HPA and 3,4HPA are ligands of HpaR that de‐repress expression of hpaGEDFHI (Galan et al, 2003; Permsirivisarn et al, 2022), whereas HpaA may preferentially sense 4HPA (Prieto & García, 1997). However, despite previous attempts (Permsirivisarn et al, 2022), there is, to our knowledge, no information available on the binding parameters of these regulators with their potential cognate ligands. To address this issue, we purified both HpaA and HpaR from A153, which share 62.0% and 66.4% identity, respectively, with homologous proteins in E. coli , and conducted isothermal titration calorimetry (ITC) studies.…”