Regulator LdhR and
d
-Lactate Dehydrogenase LdhA of Burkholderia multivorans Play Roles in Carbon Overflow and in Planktonic Cellular Aggregate Formation
Authors: Silva Inês N. , Ramires Marcelo J. , Azevedo Lisa A. , Guerreiro Ana R. , Tavares Andreia C. , Becker Jörg D. , Moreira Leonilde M. 
Published: July 2017

LysR-type transcriptional regulators (LTTRs) are the most commonly found regulators in
Burkholderia cepacia
complex, comprising opportunistic pathogens causing chronic respiratory infections in cystic fibrosis (CF) patients. Despite LTTRs being global regulators of pathogenicity in several types of bacteria, few have been characterized in
Burkholderia
. Here, we show that gene
ldhR
of
B. multivorans
encoding an LTTR is cotranscribed with
ldhA
encoding a
d
-lactate dehydrogenase and evaluate their implication in virulence traits such as exopolysaccharide (EPS) synthesis and biofilm formation. A comparison of the wild type (WT) and its isogenic Δ
ldhR
mutant grown in medium with 2%
d
-glucose revealed a negative impact on EPS biosynthesis and on cell viability in the presence of LdhR. The loss of viability in WT cells was caused by intracellular acidification as a consequence of the cumulative secretion of organic acids, including
d
-lactate, which was absent from the Δ
ldhR
mutant supernatant. Furthermore, LdhR is implicated in the formation of planktonic cellular aggregates. WT cell aggregates reached 1,000 μm in size after 24 h in liquid cultures, in contrast to Δ
ldhR
mutant aggregates that never grew more than 60 μm. The overexpression of
d
-lactate dehydrogenase LdhA in the Δ
ldhR
mutant partially restored the formed aggregate size, suggesting a role for fermentation inside aggregates. Similar results were obtained for surface-attached biofilms, with WT cells producing more biofilm. A systematic evaluation of planktonic aggregates in
Burkholderia
CF clinical isolates showed aggregates in 40 of 74. As CF patients' lung environments are microaerophilic and bacteria are found as free aggregates/biofilms, LdhR and LdhA might have central roles in adapting to this environment.

IMPORTANCE
Cystic fibrosis patients often suffer from chronic respiratory infections caused by several types of microorganisms. Among them are the
Burkholderia cepacia
complex bacteria, which cause progressive deterioration of lung function that, in some patients, might develop into fatal necrotizing pneumoniae with bacteremia, known as “cepacia syndrome.”
Burkholderia
pathogenesis is multifactorial as they express several virulence factors, form biofilms, and are highly resistant to antimicrobial compounds, making their eradication from the CF patients' airways very difficult. As
Burkholderia
is commonly found in CF lungs in the form of cell aggregates and biofilms, the need to investigate the mechanisms of cellular aggregation is obvious. In this study, we demonstrate the importance of a
d
-lactate dehydrogenase and a regulator in regulating carbon overflow, cellular aggregates, and surface-attached biofilm formation. This not only enhances our understanding of
Burkholderia
pathogenesis but can also lead to the development of drugs against these proteins to circumvent biofilm formation.