2 research outputs found
Secreted citrate serves as iron carrier for the marine pathogen photobacterium damselae subsp damselae
Photobacterium damselae subsp damselae (Pdd) is a Vibrionaceae that has a wide
pathogenic potential against many marine animals and also against humans. Some
strains of this bacterium acquire iron through the siderophore vibrioferrin. However, there
are virulent strains that do not produce vibrioferrin, but they still give a strong positive
reaction in the CAS test for siderophore production. In an in silico search on the genome
sequences of this type of strains we could not find any ORF which could be related
to a siderophore system. To identify genes that could encode a siderophore-mediated
iron acquisition system we used a mini-Tn10 transposon random mutagenesis approach.
From more than 1,400 mutants examined, we could isolate a mutant (BP53) that showed
a strong CAS reaction independently of the iron levels of the medium. In this mutant the
transposon was inserted into the idh gene, which encodes an isocitrate dehydrogenase
that participates in the tricarboxylic acid cycle. The mutant did not show any growth
impairment in rich or minimal media, but it accumulated a noticeable amount of citrate
(around 7 mM) in the culture medium, irrespective of the iron levels. The parental strain
accumulated citrate, but in an iron-regulated fashion, being citrate levels 5–6 times higher
under iron restricted conditions. In addition, a null mutant deficient in citrate synthase
showed an impairment for growth at high concentrations of iron chelators, and showed
almost no reaction in the CAS test. Chemical analysis by liquid chromatography of the
iron-restricted culture supernatants resulted in a CAS-positive fraction with biological
activity as siderophore. HPLC purification of that fraction yielded a pure compound which
was identified as citrate from its MS and NMR spectral data. Although the production
of another citrate-based compound with siderophore activity cannot be ruled out, our
results suggest that Pdd secretes endogenous citrate and use it for iron scavenging
from the cell environmentThis work was supported by grants AGL2012-39274-C02-01/02
and AGL2015-63740-C2-1/2-R (AEI/FEDER, EU) from the State
Agency for Research (AEI) of Spain, and co-funded by the
FEDER Programme from the European Union. The support
of Xunta de Galicia (Spain) with grant GRC-2014/007 is also
acknowledgedS