A Novel Repressor of the
ica
Locus Discovered in Clinically Isolated Super-Biofilm-Elaborating
Staphylococcus aureus
Authors: Yu Liansheng , Hisatsune Junzo , Hayashi Ikue , Tatsukawa Nobuyuki , Sato’o Yusuke , Mizumachi Emiri , Kato Fuminori , Hirakawa Hideki , Pier Gerald B. , Sugai Motoyuki 
Published: February 2017

Staphylococcus aureus
TF2758 is a clinical isolate from an atheroma and a super-biofilm-elaborating/polysaccharide intercellular adhesin (PIA)/poly-
N
-acetylglucosamine (PNAG)-overproducing strain (L. Shrestha et al., Microbiol Immunol 60:148–159, 2016,
https://doi.org/10.1111/1348-0421.12359
). A microarray analysis and DNA genome sequencing were performed to identify the mechanism underlying biofilm overproduction by TF2758. We found high transcriptional expression levels of a 7-gene cluster (
satf2580
to
satf2586
) and the
ica
operon in TF2758. Within the 7-gene cluster, a putative transcriptional regulator gene designated
rob
had a nonsense mutation that caused the truncation of the protein. The complementation of TF2758 with
rob
from FK300, an
rsbU
-repaired derivative of
S. aureus
strain NCTC8325-4, significantly decreased biofilm elaboration, suggesting a role for
rob
in this process. The deletion of
rob
in non-biofilm-producing FK300 significantly increased biofilm elaboration and PIA/PNAG production. In the search for a gene(s) in the 7-gene cluster for biofilm elaboration controlled by
rob
, we identified open reading frame (ORF) SAOUHSC_2898 (
satf2584
). Our results suggest that ORF SAOUHSC_2898 (
satf2584
) and
icaADBC
are required for enhanced biofilm elaboration and PIA/PNAG production in the
rob
deletion mutant. Rob bound to a palindromic sequence within its own promoter region. Furthermore, Rob recognized the TATTT motif within the
icaR-icaA
intergenic region and bound to a 25-bp DNA stretch containing this motif, which is a critically important short sequence regulating biofilm elaboration in
S. aureus
. Our results strongly suggest that Rob is a long-sought repressor that recognizes and binds to the TATTT motif and is an important regulator of biofilm elaboration through its control of SAOUHSC_2898 (SATF2584) and Ica protein expression in
S. aureus
.

IMPORTANCE
During the search for molecular mechanisms underlying biofilm overproduction of
Staphylococcus aureus
TF2758, we found a putative transcriptional regulator gene designated
rob
within a 7-gene cluster showing a high transcriptional expression level by microarray analysis. The deletion of
rob
in non-biofilm-producing FK300, an
rsbU
-repaired derivative of NCTC8325-4, significantly increased biofilm elaboration and PIA/PNAG production. The search for a gene(s) in the 7-gene cluster for biofilm elaboration controlled by
rob
identified ORF SAOUHSC_2898. Besides binding to its own promoter region to control ORF SAOUHSC_2898 expression, Rob recognized the TATTT motif within the
icaR-icaA
intergenic region and bound to a 25-bp DNA stretch containing this motif, which is a critically important short sequence regulating biofilm elaboration in
S. aureus
. Our results strongly suggest that Rob is a long-sought repressor that recognizes and binds to the TATTT motif and is a new important regulator of biofilm elaboration through its control of SAOUHSC_2898 and Ica protein expression in
S. aureus
.