Novel effects of a transposon insertion in the Vibrio fischeri glnD gene: defects in iron uptake and symbiotic persistence in addition to nitrogen utilization

Abstract

Vibrio fischeri is the sole species colonizing the light‐emitting organ of the Hawaiian squid, Euprymna scolopes. Upon entering the nascent light organ of a newly hatched juvenile squid, the bacteria undergo morphological and physiological changes that include the loss of flagellation and the induction of bioluminescence. These and other events reveal a pattern of genetic regulation that is a response to the colonization of host tissue. In this study, we isolated and characterized a glnD::mTn5Cm mutant of V. fischeri. In addition to the predicted defects in the efficiency of nitrogen utilization, this glnD mutant had an unexpected reduction in the ability to produce siderophore and grow under iron‐limiting conditions. Although the glnD mutant could colonize juvenile squid normally over the first 24 h, it was subsequently unable to persist in the light organ to the usual extent. This persistence phenotype was more severe if the mutant was pregrown under iron‐limiting conditions before inoculation, but could be ameliorated by the presence of excess iron. These results indicate that the ability to respond to iron limitation may be an important requirement in the developing symbiosis. Supplying the glnD gene in trans restored normal efficiency of nitrogen use, iron sequestration and colonization phenotypes to the glnD::mTn5Cm mutant; thus, there appears to be a genetic and/or metabolic linkage between nitrogen sensing, siderophore synthesis and symbiosis competence in V. fischeri that involves the glnD gene.

H-NOX–mediated nitric oxide sensing modulates symbiotic colonization by Vibrio fischeri

“…There was no significant loss in the viability of mutant or wildtype cultures when exposed up to 500 μM of the NO generator DEA-NONOate (Cayman Chemical) (Fig. S2); 10 mM N-acetyl-D-glucosamine (MP Biomedicals) served as the carbon and nitrogen source, and when desired, the iron in the medium was depleted by the addition of 50 μM of the deferrated iron chelator EDDHA (24).…”

“…During the onset of colonization by V. fischeri, the light organ is an iron-limited growth environment that requires the bacterium to scavenge this nutrient (24). However, although iron is essential for growth, a high concentration of free intracellular iron in the reducing environment of the cell can be toxic because of its role in generating hydroxyl radicals through the Fenton reaction.…”

GlnD Is Essential for NifA Activation, NtrB/NtrC-Regulated Gene Expression, and Posttranslational Regulation of Nitrogenase Activity in the Photosynthetic, Nitrogen-Fixing Bacterium Rhodospirillum rubrum

“…The glnD homolog in Rhizobium tropici appears to be involved in the induction of chlorosis on plants (42). In Vibrio fischeri, a mutant with a glnD transposon insertion displayed defects in iron sequestration, colonization of the light-producing organ of the squid Euprymna scolopes, and nitrogen utilization (15). The fact that GlnB and GlnJ appear to be involved in the light-dark regulation of the DRAT-DRAG system of R. rubrum also falls into this category.…”

Vibrio Iron Transport: Evolutionary Adaptation to Life in Multiple Environments

“…These molecules often have catechols or hydroxamates as part of the iron-binding moieties, although other structures have been identified. In some species, such as the squid symbiont V. fischeri, the structure of the siderophore is unknown (38), but a number of vibrio siderophores have been isolated and characterized, and there is considerable diversity in their structure and chemical properties. As described in more detail in the discussion of individual siderophores below, this diversity is likely a reflection of vibrios evolving to grow in different environments and to produce siderophores that can outcompete those produced by other members of the community when iron is scarce.…”

“…Another example of a link between metabolism and iron transport has been identified in V. fischeri, which couples nitrogen metabolism to iron acquisition. A mutation in glnD, which acts as a nitrogen sensor, not only caused a defect in nitrogen metabolism but also reduced siderophore synthesis by these bacteria (38). The glnD mutant was unable to persist in the squid light organ, but excess iron restored persistence to wild-type levels, indicating that the colonization defect was a result of the effect of the glnD mutation on iron acquisition.…”