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Fifty-three strains of M. avium and related species all produced one or more exochelins, the extracellular iron-binding compounds of the mycobacteria, when grown iron deficiently. Only those strains which could grow without the addition of mycobactin (i.e., mycobactin independent) produced mycobactin, the intracellular iron-binding compound of the mycobacteria. Exochelins varied from 20 to 2,000 ,ug per g of cell dry weight; mycobactins were between 1 and 10 mg per g of cell dry weight. M. paratuberculosis (13 strains) and 13 strains of M. avium, both species dependent upon mycobactin for growth, failed to produce spectrophotometrically detectable amounts of mycobactin (<0.2 ,ug per g of cell dry weight), although mycobactin could be recognized in one strain of M. avium grown with an additional supply of salicylate and examined by a radiolabeling technique. On repeated subculture three of the mycobactin-dependent strains of M. avium, but none of those of M. paratuberculosis, lost their mycobactin dependence and on reexamination were found to produce their own mycobactin at 0.3 mg per g of cell dry weight. It is concluded that mycobactin biosynthesis is probably strongly repressed in the mycobactin-dependent strains rather than being a genetic deletion. The exochelins, when examined by high-pressure thin-layer chromatography were revealed as being multiples of similar compounds, with up to 20 individual iron-binding compounds being recognizable with some strains. It is argued that the exochelins represent the single most important means of iron acquisition in mycobacteria growing in vitro and in vivo, and their elaboration by the fastidious M. paratuberculosis and related species explains how these organisms are able to grow in vivo in the absence of an external supply of mycobactin. 1138 on July 6, 2020 by guest

Methods were devised to purify the cell-associated, iron-binding compounds known as mycobactins from the closely related species Mycobacterum avium, Mycobacterium intraceUulare, and Mycobacterium scrofulaceum (i.e., the MAIS complex of organisms). The mycobactins from these three species showed a structure that is common to the mycobactins from all the mycobacteria examined to date. However, these mycobactins were unique in that they had more than one alkyl chain. The M. scrofulaceum mycobactins differed from other MAIS mycobactins by a shift in the position of the double bond in the Rl alkyl chain. Traces of other mycobactin types were observed in ethanol extracts of the three species, and examination of the chromatographic properties of these mycobactins showed that each species produced five mycobactin types. Each mycobactin could be subdivided further by the lengthi of its Rl alkyl chain. No differences in the production of these novel mycobactins were observed among species. Mycobactins from three strains of Mycobactenium paratuberculosis and two wood pigeon strains of Mycobacterium avium which had lost their original growth requirements for mycobactin after repeated subculturing in laboratory growth media were examined by thin-layer chromatography and high-pressure liquid chromatography. Each organism produced a mycobactin with similar chromatographic properties to thope synthesized by MAIS organisms. M. paratuberculosis NADC 18 produced at least two components in our laboratory, and nuclear magnetic resonance analysis of the major component showed this mycobactdli to be identical to that produced by M. intraceUlure M12. However, a samn4le of mycobactin J isolated by Merkal and McCullougb (Curr. Microbiol. 7:333-335, 1982) from M. paratuberculosis NADC 18 was different from our isolates and appeared to correspond to a minor mycobactin component we had seen by thin-layer chromatography. No reason for this difference could be evinced. Our findings indicate that there is a close taxonomic relationship between M. paratuberculosis and the MAIS complex.

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Summary. Of 120 laboratory-maintained strains of Listeria rnonocytogenes and two of L. ivanovii examined for haemolytic and lipolytic activity, 62 exhibited haemolytic activity alone, 20 of these showed haemolytic and lipolytic activity and 40 had neither activity. The L. ivanovii strains showed both activities. The results indicated a relationship between haemolysin production and lipolytic activity which was not explained by the serotype of the organism. In addition, the following hydrolytic activities were detected in the cell-free growth media of strains L. rnonocytogenes Boldy and L. ivanovii (formerly L. rnonocytogenes) Type 5 (substrates acted upon are given in parentheses) : acid phosphatase (4-nitrophenylphosphate, naphthyl phosphate, glycerophosphate, phosphorylcholine and GTP); neutral phosphatase (4-nitrophenylphosphate, naphthyl phosphate, phosphorycholine, NADP and UDPG); phosphodiesterase (bis-4-nitrophenylphosphate, ATP and NADP) ; NADase (NAD) ; phospholipase C (4-nitrophenylphosphoryl-choline, phosphatidyl choline and ethanolamine, and sphingomyelin) ; and lipase and esterase (triacetin, tributyrin, triolein, naphthyl-laurate, -myristate, -caprylate, -palmitate and -oleate, 4-nitrophenyl-acetate -1aurate and Tween 80). The preparations also showed weak catalase activity. No evidence was found for the presence of RNAase, DNAase, peptidase/amidase, phosphoamidase, a-amylase, glucosidase, galactosidase, pyranosidase or glucose aminidase. IntroductionThe clinical features of the diseases caused by Listeria spp. suggest that toxins and enzymes produced by the bacteria contribute to their pathogenicity. The toxicity is associated partly with the cell envelope (Stanley, 1949 ;Patocka and Mara, 1973) but much of it is associated with the extracellular products. The discovery that L. monocytogenes produces a thiol-activated haemolysin (Harvey and Faber, 1941) helped to explain the nature of the extracellular toxic activities of this organism. However, in early attempts to differentiate the toxic activities of the haemolysin from those of the cell envelope, cell-free culture filtrates rather than purified haemolysin were used. Later attempts to ascribe an enzyme activity to the haemolysin showed that the culture filtrates also contained lipolytic (Girard et al., 1963) and nicotin-