Abstract
The Acinetobacter baumannii BlsA photoreceptor has an N-terminal (NT) BLUF domain and a C-terminal (CT) amino acid sequence with no significant homology to characterized bacterial proteins. In this study, we tested the biological role of specific residues located in these BlsA regions. Site-directed mutagenesis, surface motility assays at 24°C and protein overexpression showed that residues Y7, Q51 and W92 are essential for not only light-regulated motility, but also BlsA’s solubility when overexpressed in a heterologous host. In contrast, residues A29 and F32, the latter representing a difference when compared with other BLUF-containing photoreceptors, do not play a major role in BlsA’s biological functions. Analysis of the CT region showed that the deletion of the last five BlsA residues has no significant effect on the protein’s light-sensing and motility regulatory functions, but the deletion of the last 14 residues as well as K144E and K145E substitutions significantly alter light-regulated motility responses. In contrast to the NT mutants, these CT derivatives were overexpressed and purified to homogeneity to demonstrate that although these mutations do not significantly affect flavin binding and photocycling, they do affect BlsA’s photodynamic properties. Notably, these mutations map within a potential fifth α-helical component that could play a role in predicted interactions between regulatory partners and BlsA, which could function as a monomer according to gel filtration data. All these observations indicate that although BlsA shares common structural and functional properties with unrelated photoreceptors, it also exhibits unique features that make it a distinct BLUF photoreceptor.
Highlights
Acinetobacter baumannii is a Gram-negative opportunistic pathogen and the causative agent of both nosocomial- and community-acquired infections [1]
These features reflect the general observation that light sensing by short photosensors is mediated by the interaction of the flavin adenine dinucleotide (FAD) chromophore with critically conserved as well as semi-conserved blue light sensing using FAD (BLUF) domain residues, while signal transduction is mediated by CT terminal residues and amino acid sequences that are not conserved and do not show significant similarity to known proteins, respectively [17]
This model shows the typical β1α1β2β3α2β4β5α3α4 structure described in BLUF proteins, the comparative analysis with Tll0078 and the light- and dark-adapted AppA BLUF domain shows that their predicted structures superimpose with the exception of the additional α helix (α5) formed by the blue-light-sensing protein A (BlsA) KKVGMVN residues, which are located between positions 144–150 (Figs 1 and 2C)
Summary
Acinetobacter baumannii is a Gram-negative opportunistic pathogen and the causative agent of both nosocomial- and community-acquired infections [1]. A. baumannii is commonly associated with hospital infections, it has been isolated from a wide range of environmental sources and samples including water and aquaculture environments [2], soil [3], varied food sources [4], animals [5, 6] and insects [7], all of which could be reservoirs for this. A. baumannii BlsA protein (NIGMS) (https://www.nigms.nih.gov), Miami University (http://miamioh.edu)
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