Transcriptomic Response ofListeria monocytogenesto Iron Limitation andfurMutation

Abstract

Iron is required by almost all bacteria, but concentrations above physiological levels are toxic. In bacteria, intracellular iron is regulated mostly by the ferric uptake regulator, Fur, or a similar functional protein. Iron limitation results in the regulation of a number of genes, especially those involved in iron uptake. A subset of these genes is the Fur regulon under the control of Fur. In the present study, we have identified Fur-and iron-regulated genes in Listeria monocytogenes by DNA microarray analysis using a fur mutant and its isogenic parent. To identify genes regulated exclusively in response to iron limitation, the whole-genome transcriptional responses to the iron limitation of a fur mutant and its isogenic parent were compared. Fur-regulated genes were identified by comparing the transcriptional profile of the parent with the transcriptional profile of the isogenic fur mutant. Our studies have identified genes regulated exclusively in response to iron and those that are negatively regulated by Fur. We have identified at least 14 genes that were negatively regulated directly by Fur. Under iron-limited conditions, these genes were upregulated, while the expression of fur was found to be downregulated. To further investigate the regulation of fur in response to iron, an ectopic fur promoter-lacZ transcriptional fusion strain was constructed, and its isogenic fur and perR mutant derivatives were generated in L. monocytogenes 10403S. Analysis of the iron limitation of the perR mutant indicated that the regulation of genes under the negative control of Fur was significantly inhibited. Our results indicate that Fur and PerR proteins negatively regulate fur and that under iron-limited conditions, PerR is required for the negative regulation of genes controlled by Fur.

Role of the twin-arginine translocase (tat) system in iron uptake in Listeria monocytogenes

“…To determine the role of TatAC and FepCAB, the tatC and fepB genes were mutated by insertional disruption using a temperature-sensitive vector, pAUL-A (Chakraborty et al, 1992), as described by Ledala et al (2010). An internal 280 bp portion of tatC was PCR amplified using a forward primer (PtatCint-F) with a BamHI restriction site and a reverse primer (PtatCint-R) with an EcoRI restriction site.…”

“…Accumulation of iron above physiological levels causes the production of toxic free radicals by the Fenton reaction and is deleterious for cells. The fepB gene in L. monocytogenes has been shown to be derepressed fourfold under iron-limiting conditions (Ledala et al, 2010). If fepB encodes the peroxidase then it should be theoretically repressed rather than de-repressed under iron-limiting conditions and induced under excess iron and oxidative stress due to iron toxicity (Bagg & Neilands, 1987).…”

“…In contrast to Bacillus subtilis, where some paralogues of tatC and tatA are present, only one copy of each of these genes exists in L. monocytogenes (Jongbloed et al, 2002(Jongbloed et al, , 2004. In silico analysis of the listerial genome revealed the presence of tatA (lmo0362) and tatC (lmo0361) genes as a tatAC operon with a Fur-repressor binding sequence, the Fur box, in its promoter (Ledala et al, 2010). The Fur-repressor plays a central role in iron homeostasis by regulating furregulons in response to iron availability.…”

“…Transcriptomic analysis of a L. monocytogenes fur mutant revealed that the tatC, tatA and lmo0367 genes are highly expressed in response to a fur mutation or upon iron limitation (Ledala et al, 2010). lmo0367 is homologous to the iron-dependent peroxidase (FepB) in Staphylococcus aureus, which is translocated by the Tat-pathway in this organism (Biswas et al, 2009).…”

“…Several studies have shown that the concentration of intracellular iron influences the transcription rates of over 200 genes in bacteria (Ochsner et al, 2002;Camejo et al, 2009;Ledala et al, 2010). Due to the low availability of iron in the host, bacteria have evolved various iron acquisition systems such as citrateinducible ferric uptake (Adams et al, 1990;Andrews et al, 2003), a cell surface transferring binding protein (Hartford et al, 1993), an ABC transporter for HN/Hb, ferric hydroxamates (fhu), haemin/haemoglobin (hup) (Brown & Holden 2002;Faraldo-Gó mez et al, 2003;Jin et al, 2006;Weinberg, 2009) and extracellular and/or surface-associated iron reductases (McLaughlin et al, 2011).…”