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Growth under conditions of iron-restriction and the production of siderophores was examined in 21 typical and 14 atypical strains of Aeromonas salmonicida. With the exception of one atypical strain, all strains grew and multiplied in the presence of the high-affinity iron chelators ethylenediamine di(o-hydroxyphenylacetic acid), a, a'-dipyridyl or transferrin. Chrome azurol S agar was used to screen bacterial strains growing under these conditions for the production of siderophores. Siderophore production was detected only in the typical strains. Siderophores were also detected in the iron-restricted culture supernatants of typical strains, where they were associated with an iron-binding activity. The siderophore was extracted from iron-restricted culture supernatant of one strain by adsorption onto an XAD-7 resin; it behaved as a 2,3-diphenol-catechol in several colorimetric assays. The results indicate that although both typical and atypical strains of A. safmunicida grow and multiply under conditions of iron-restriction, they use different iron-uptake mechanisms, siderophore-mediated and siderophoreindependent, respectively. In cross-feeding assays, growth of typical strains was stimulated only by homologous iron-restricted supernatant, suggesting strain differences in the siderophore produced. However, one strain produced a culture supernatant with growth-stimulating activity for other typical and also atypical strains.

Abstract. The ability of A‐layer‐positi ve (A+) and A‐layer‐negative (A−) strains of Aeromonas salmonicida to utilize haem sources of iron under conditions of iron‐restriction was evaluated. In a plate bioassay, only A+ strains of A. salmonicida were able to utilize haem from a variety of sources including haem, haemin, myoglobin, haemoglobin, haemoglobin‐ haptoglobin and haem‐albumin complexes. Trypsin‐digestion of whole cells abolished haem‐ binding, indicating that binding was cell‐surface associated, involving a protein binding site or receptor. Competitive binding studies indicated that all haem compounds were bound by a common receptor, which was not iron‐regulated and was associated with the presence of the 49‐kDa A‐layer protein. The ability of both typical A+ (siderophore‐positive) and atypical A+ (siderophore‐negative) strains to utilize haem indicated that the mechanism of haem utilization was not siderophore‐mediated and that A. salmonicida possesses both siderophore‐dependent and siderophore‐independent mechanisms to overcome iron‐restricted conditions encountered in vivo.

The ability of typical and atypical strains of Aemmnas salmonicida to utilize non-haem sources of protein-bound iron was evaluated. (i) In a plate bioassay, the suppression of growth imposed on typical and atypical A. Ealmonicida by addition of the high-aff inity iron chelator ethylenediamine-di(ohydroxyphenylacetic acid) (EDDA) to the growth medium was reversed by the addition of 30% or 90% iron-saturated bovine or human transferrin (Tf) or lactoferrin (Lf) to the growth medium. (ii) The mechanism of obtaining iron from Tf was investigated by the addition of bovine Tf contained within a dialysis bag. The reversal of iron-restricted growth suppression differed between the strains in that the atypical strains were unable to utilize Tf contained within a dialysis bag while the typical strains were able to do so.This suggested a siderophore-mediated uptake of iron from Tf by the typical strains, which are known to produce siderophores while atypical strains do not. (iii) A solid-phase binding assay using horseradish-peroxidase-conjugated or biotinylated Tf or Lf failed to detect Tf/Lf-binding activity using whole typical or atypical cells. (iv) When atypical extracellular products (ECP) plus bovine Tf or salmon serum were enclosed in a dialysis bag, diffusible products were released which could reverse the EDDA-imposed growth suppression of an atypical strain. This reversal was negated by inhibition of the ECP metalloprotease with EDTA. (v) Purified 70 kDa serine protease of a typical strain was able to digest bovine Tf to low molecular mass fragments as observed in SDS-PAGE. These results indicate that typical and atypical strains of A. sslmonicida differ in their mechanism of utilization of non-haem proteinbound sources of iron. Typical strains utilize T f via a siderophore-mediated mechanism and are also able to digest Tf with the extracellular serine protease. Atypical strains utilize Tf by a siderophore-independent mechanism probably involving the proteolytic degradation of Tf by the extracellular metal loprotease.