Temperature serves as a cue to regulate gene expression in Escherichia coli and other bacteria. Using DNA microarrays, we identified 297 genes whose expression is increased at 23 6C compared to 37 6C in E. coli K-12. Of these genes, 122 are RpoS-controlled, confirming genome-wide the model that low temperature serves as a primary cue to trigger the general stress response. Several genes expressed at 23 6C overlap with the cold-shock response, suggesting that strategies used to adapt to sudden shifts in temperature also mediate long-term growth at 23 6C. Another category of genes more highly expressed at 23 6C are associated with biofilm development, implicating temperature as an important cue influencing this developmental pathway. In a candidate set of genes tested, the biofilm genes (adrA, bolA, mlrA, nhaR, csgA, yceP/bssS) and cold-shock genes (otsA, yceP/bssS) were found to be RpoS-and DsrAdependent for their transcription at 23 6C. In contrast, transcription of three genes (ycgZ, dps and ymgB) was either partially or fully independent of these regulators, signifying there is an alternative thermoregulatory mechanism(s) that increases gene expression at 23 6C. Increased expression at 23 6C compared to 37 6C is retained in various media tested for most of the genes, supporting the relative importance of this cue in adaptation to changing environments. Both the RpoS-dependent gene otsA and the RpoS-independent gene ymgB demonstrated increased expression levels within 1 h after a shift from 37 to 23 6C, indicating a rapid response to this environmental cue. Despite changes in gene expression for many RpoS-dependent genes, experiments assessing growth rate at 23 6C and viability at 4 6C did not demonstrate significant impairment in rpoS : : Tn10 or dsrA : : cat mutant strains in comparison to the wild-type strain. Biofilm formation was favoured at low temperature and is moderately impaired in both the rpoS : : Tn10 and dsrA : : cat mutants at 23 6C, suggesting genes controlled by these regulators play a role necessary for optimal biofilm formation at 23 6C. Taken together, our data demonstrate that a large number of genes are increased in expression at 23 6C to globally respond to this environmental change and that at least two thermoregulatory pathways are involved in co-ordinating this response -the RpoS/DsrA pathway and an alternative thermoregulatory pathway, independent of these regulators.

Bacterial pathogens require multiple iron-specific acquisition systems for survival within the iron-limiting environment of the host. Francisella tularensis is a virulent intracellular pathogen that can replicate in multiple cell-types. To study the interrelationship of iron acquisition capability and virulence potential of this organism, we generated single and double deletion mutants within the ferrous iron (feo) and ferric-siderophore (fsl) uptake systems of the live vaccine strain (LVS). The Feo system was disrupted by a partial deletion of the feoB gene (ΔfeoB′), which led to a growth defect on iron-limited modified Muller Hinton agar plates. 55Fe uptake assays verified that the ΔfeoB′ mutant had lost the capacity for ferrous iron uptake but was still competent for 55Fe-siderophore-mediated ferric iron acquisition. Neither the ΔfeoB′ nor the siderophore-deficient ΔfslA mutant was defective for replication within J774A.1 murine macrophage-like cells, thus demonstrating the ability of LVS to survive using either ferrous or ferric sources of intracellular iron. A LVS ΔfslA ΔfeoB′ mutant defective for both ferrous iron uptake and siderophore production was isolated in the presence of exogenous F. tularensis siderophore. In contrast to the single deletion mutants, the ΔfslA ΔfeoB′ mutant was unable to replicate within J774A.1 cells and was attenuated in virulence following intraperitoneal infection of C57BL/6 mice. These studies demonstrate that the siderophore and feoB-mediated ferrous uptake systems are the only significant iron acquisition systems in LVS and that they operate independently. While one system can compensate for loss of the other, both are required for optimal growth and virulence.

Iron acquisition mechanisms in Francisella tularensis, the causative agent of tularemia, include the Francisella siderophore locus (fsl) siderophore operon and a ferrous iron–transport system comprising outer‐membrane protein FupA and inner‐membrane transporter FeoB. To characterize these mechanisms and to identify any additional iron uptake systems in the virulent subspecies tularensis, single and double deletions were generated in the fsl and feo iron acquisition systems of the strain Schu S4. Deletion of the entire fsl operon caused loss of siderophore production that could be restored by complementation with the biosynthetic genes fslA and fslC and Major Facilitator Superfamily (MFS) transporter gene fslB. 55Fe‐transport assays demonstrated that siderophore‐iron uptake required the receptor FslE and MFS transporter FslD. A ΔfeoB′ mutation resulted in loss of ability to transport ferrous iron (55Fe2+). A ΔfeoB′ ΔfslA mutant that required added exogenous siderophore for growth in vitro was unable to grow within tissue culture cells and was avirulent in mice, indicating that no compensatory cryptic iron uptake systems were induced in vivo. These studies demonstrate that the fsl and feo pathways function independently and operate in parallel to effectively support virulence of F. tularensis.

The Gram-negative bacterium Francisella tularensis secretes the siderophore rhizoferrin to scavenge necessary iron from the environment. Rhizoferrin, also produced by a variety of fungi and bacteria, comprises two citrate molecules linked by amide bonds to a central putrescine (diaminobutane) moiety. Genetic analysis has determined that rhizoferrin production in F. tularensis requires two enzymes: FslA, a siderophore synthetase of the nonribosomal peptide synthetase-independent siderophore synthetase (NIS) family, and FslC, a pyridoxal-phosphate-dependent decarboxylase. To discern the steps in the biosynthetic pathway, we tested F. tularensis strain LVS and its ΔfslA and ΔfslC mutants for the ability to incorporate potential precursors into rhizoferrin. Unlike putrescine supplementation, supplementation with ornithine greatly enhanced siderophore production by LVS. Radioactivity from L-[U-14C] ornithine, but not from L-[1-14C] ornithine, was efficiently incorporated into rhizoferrin by LVS. Although neither the ΔfslA nor the ΔfslC mutant produced rhizoferrin, a putative siderophore intermediate labeled by both [U-14C] ornithine and [1-14C] ornithine was secreted by the ΔfslC mutant. Rhizoferrin was identified by liquid chromatography and mass spectrometry in LVS culture supernatants, while citryl-ornithine was detected as the siderophore intermediate in the culture supernatant of the ΔfslC mutant. Our findings support a three-step pathway for rhizoferrin production in Francisella; unlike the fungus Rhizopus delemar, where putrescine functions as a primary precursor for rhizoferrin, biosynthesis in Francisella preferentially starts with ornithine as the substrate for FslA-mediated condensation with citrate. Decarboxylation of this citryl ornithine intermediate by FslC is necessary for a second condensation reaction with citrate to produce rhizoferrin.

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Francisella tularensis is a Gram-negative intracellular bacterium and the causative agent of the disease tularemia. F. tularensis is designated by the CDC as a Tier One Select Agent, due to its low infection dose, aerosol transmissibility, lack of a licensed vaccine, and for its potential use as an agent of biological warfare. Virulent Schu S4 and the attenuated live vaccine strain (LVS) are model strains used to study F. tularensis pathogenesis in tissue culture and in the mouse model of disease. Despite the genetic similarities between LVS and Schu S4, these strains have differences in iron uptake capability and virulence. Within the host, iron is tightly controlled and sequestered by iron binding proteins. Pathogenic bacteria, include F. tularensis, have devised methods for acquiring available ferrous and ferric iron from their hosts and they are frequently associated with virulence. A common method for ferric acquisition is the production of siderophores, which are low molecular weight iron binding chelators that are secreted into the environment and have high affinities for ferric iron. F. tularensis strains LVS and Schu S4 secrete an identical siderophore and encode genes required for siderophore biosynthesis and transport (fslABCDEF also referred to as figABCDEF). An in-frame deletion of fslC was generated in the LVS background (LVS ΔfslC), and similar to the previously published fslA mutant (LVS ΔfslA), this mutant was defective for siderophore production as determined by the liquid Chrome-Azurol S (CAS) assay. A fslABCDEF locus deletion mutant, LVS Δfsl, was complemented with plasmids containing wild-type copies of the fsl genes

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