In iron-limited medium, Azotobacter winelandii strain UW produces three catecholate siderophores : the tricatecholate protochelin, the dicatecholate azotochelin and the monocatecholate aminochelin. Each siderophore was found to bind Fe3+ preferentially to Fez+, in a 1igand:Fe ratio of 1 :1,3:2 and 3: 1 , respectively. Protochelin had the highest affinity for Fe3+, with a calculated proton-independent solubility coefficient of lWg, comparable to ferrioxamine B. Iron-limited wild-type strain UW grown under N,-fixing or nitrogen-sufficient conditions hyper-produced catecholate siderophores in response to oxidative stress caused by high aeration. In addition, superoxide dismutase activity was greatly diminished in iron-limited cells, whereas catalase activity was maintained. The ferredoxin I (Fdl)-negative A. vinelandii strain LM100 also hyper-produced catecholates, especially protochelin, under oxidative stress conditions, but had decreased activities of both superoxide dismutase and catalase, and was about 10 times more sensitive to paraquat than strain UW. Protochelin and azotochelin held Fe3+ firmly enough to prevent its reduction by -0; and did not promote the generation of hydroxyl radical by the Fenton reaction. Ferric-aminochelin was unable to resist reduction by -0; and was a Fenton catalyst. These data suggest that under iron-limited conditions, A. vinelandii suffers oxidative stress caused by .O;. The catecholate siderophores azotochelin, and especially protochelin, are hyper-produced to offer chemical protection from oxidative damage catalysed by -0; and Fe3+. The results are also consistent with Fdl being required for oxidative stress management in A. winelandii.

Both molybdate and iron are metals that are required by the obligately aerobic organism Azotobacter vinelandii to survive in the nutrient-limited conditions of its natural soil environment. Previous studies have shown that a high concentration of molybdate (1 mM) affects the formation of A. vinelandii siderophores such that the tricatecholate protochelin is formed to the exclusion of the other catecholate siderophores, azotochelin and aminochelin. It has been shown previously that molybdate combines readily with catecholates and interferes with siderophore function. In this study, we found that the manner in which each catecholate siderophore interacted with molybdate was consistent with the structure and binding potential of the siderophore. The affinity that each siderophore had for molybdate was high enough that stable molybdosiderophore complexes were formed but low enough that the complexes were readily destabilized by Fe 3؉ . Thus, competition between Fe 3؉ and molybdate did not appear to be the primary cause of protochelin accumulation; in addition, we determined that protochelin accumulated in the presence of vanadate, tungstate, Zn 2؉ , and Mn 2؉ . We found that all five of these metal ions partially inhibited uptake of 55 Fe-protochelin and 55 Feazotochelin complexes. Also, each of these metal ions partially inhibited the activity of ferric reductase, an enzyme important in the deferration of ferric siderophores. Our results suggest that protochelin accumulates in the presence of molybdate because protochelin uptake and conversion into its component parts, azotochelin and aminochelin, are inhibited by interference with ferric reductase.

Cultures of Vibrio cholerae 01, biotype El Tor, from the current epidemic of cholera in the Western Hemisphere, and of the new V. cholerae serogroup 0139, from the current outbreak in India and Bangladesh, revealed marked colonial heterogeneity when received by the authors. By comparison with reference colony types, using a stereoscope and transmitted oblique illumination, colonies of approximately 10 different degrees of opacity could be distinguished. In contrast, strains freshly isolated from patients and rapidly and carefully preserved were more homogeneous although still differentiable by this technique. These (and older) observations prompted the questions: (1) why is a V. cholerae colony opaque or translucent? and (2) what benefit is it to the vibrios to vary their colonial appearance? The observed changes in colonial opacity, which are reversible, are sometimes (rarely) accompanied by changes in virulence for infant rabbits and, more frequently, by other phenotypic variations including the ability to produce poly-p-hydroxybutyrate inclusion bodies on glycerol-containing medium, the degree of encapsulation in 0139, changes in outer-membrane proteins, alteration in lipopolysaccharide structure, changes in expression of glycolytic pathways, and differences in ability to survive under adverse conditions. Colonial variations in choleragenic vibrios are phenotypically multifactorial. The genetic mechanisms(s) underlying the observed phenotypic changes remain to be defined.Keywords : Vibrio cholerae, colonial variation, opacity, poly-P-hydroxybutyrate (PHB), capsules INTRODUCTIONCholera vibrios have long been known to be mercurial in their ability to exhibit variations in colonial morphology. Robert Koch was the first to observe that the colonial appearance of cholera vibrios was distinct from that of other bacterial species (Pollitzer, 1959, p. 125). Soon thereafter, Kolle and Gotschlich (Pollitzer, 1959, pp. 452-456), and subsequently others, noted that vibrio subcultures contained opaque (0) and translucent (T) colonial variants. Lankford (1960) observed that nearly all colonies of classical biotype Vibrio cholerae on primary culture from patients in Calcutta, India, in 1953, and in Bangkok, Thailand, in 1958, were of a single characteristic ' typically opaque and striated ' colony type, whereas most stock cultures of V. cholerae Abbreviations: LOS, lipooligosaccharide; MR, methyl red; OU, opacity unit; PHB, poly-P-hydroxybutyrate; VP, Voges-Proskauer.were mixtures of colony types or pure types differing from each other. Colonies which deviated from the ' typical ' colony type usually had reduced virulence for 13-d-old chicken embryos (Lankford, 1960 ;Gardner et al., 1964). The apparent homogeneity of fresh isolates has also been the author's (R. A. F.'s) experience during cholera epidemics of El Tor and classical biotypes in the Philippines, India, Vietnam and Thailand during the period from 1961to 1967. In 1966, it was noted (Finkelstein, 1966 that stock cultures of El Tor biotype V. cholerae sent f...

The csbX gene of Azotobacter vinelandii was regulated in an iron-repressible manner from a divergent promoter upstream of the catecholate siderophore biosynthesis (csb) operon and was predicted to encode an efflux pump of the major facilitator superfamily. Other proteins that were most similar to CsbX were encoded by genes found in the catecholate siderophore biosynthesis operons of Aeromonas hydrophila and Stigmatella aurantiaca. Inactivation of csbX resulted in 57-100% decrease in the amount of catecholates released when compared to the wild-type in iron-limited medium. CsbX was most important for the export of the high affinity chelator protochelin with the majority of the catecholates released by csbX mutants being the protochelin intermediates azotochelin and aminochelin.

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