Homodimerization and binding of specific domains to the target DNA are essential requirements for HlyU to regulate expression of the virulence gene rtxA1, encoding the repeat-in-toxin protein in the human pathogen Vibrio vulnificus.
The virulence gene rtxA1, encoding the repeat-in-toxin protein, plays an essential role in the pathogenesis of Vibrio vulnificus infections. Expression of this gene is controlled by the HlyU regulator by direct contact of the DNA upstream of the rtxA1 toxin operon acting as a derepressor of the H-NS protein. The crystal structure suggests that HlyU forms a homodimer in vitro. However, knowledge of the biological implications of these findings in vivo is limited. In this work, we endeavored to dissect, using genetic and biochemical approaches, the domains of this protein that are essential for homodimer formation and the interaction of HlyU with the target DNA. We identified that residues L18, N22, R25, S54, Q55, L57, W59, R61, K70, and Y77 are essential for the HlyU protein binding to the DNA and that amino acids L17 and L91 are important for HlyU dimerization. We also determined that HlyU homodimer formation is an essential requirement for binding to the upstream region of the rtxA1 operon and is the key feature in relieving the H-NS repression of rtxA1 transcription.
Department of Molecular Microbiology and Immunology, Oregon Health and Science University, Portland, OR 97239, USA.
Moqing
M
Liu
Michael
M
Rose
Jorge H
JH
Crosa
D000595:Amino Acid Sequence; D001426:Bacterial Proteins / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism*; D001427:Bacterial Toxins / Q000235:genetics* / Q000378:metabolism; D004269:DNA, Bacterial / Q000235:genetics / Q000378:metabolism; D019281:Dimerization; D015964:Gene Expression Regulation, Bacterial; D006801:Humans; D008969:Molecular Sequence Data; D009876:Operon; D011485:Protein Binding; D017434:Protein Structure, Tertiary; D016415:Sequence Alignment; D014157:Transcription Factors / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D014735:Vibrio Infections / Q000382:microbiology; D041261:Vibrio vulnificus / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D037521:Virulence Factors / Q000235:genetics* / Q000378:metabolism
D001426:Bacterial Proteins; D001427:Bacterial Toxins; D004269:DNA, Bacterial; D014157:Transcription Factors; D037521:Virulence Factors