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The 16s rRNAs from nine rapidly growing Mycobacterium species were partially sequenced by using the dideoxynucleotide-terminated, primer extension method with cDNA generated by reverse transcriptase. The sequences were aligned with 47 16s rRNA or DNA sequences that represented 30 previously described and 5 undescribed species of the genus Mycobacterium, and a dendrogram was constructed by using equally weighted distance values. Our results confirmed the phylogenetic separation of the rapidly and slowly growing mycobacteria and showed that the majority of the slowly growing members of the genus represent the most recently evolved organisms. The 24 strains which represented 21 rapidly growing species constituted several sublines, which were defined by the following taxa: (i) Mycobacterium neoaumm and M. diernhoferi, (ii) M. gudium, (iii) the M. chubuense cluster, (iv) the M. fortuitum cluster, (v) M. kommossense, (vi) M. sphagni, (vii) M. fallax and M. chitae, (viii) M. aurum and M. vaccae, (ix) the M. fravescens cluster, and (x) M. chelonae subsp. abscessus. Our phylogenetic analysis confirmed the validity of the phenotypically defined species mentioned above, but our conclusions disagree with most of the conclusions about intrageneric relationships derived from numerical phenetic analyses.In the early 1980s, using 16s rRNA cataloging methods, workers determined that Mycobacterium phlei, one of the fast-growing Mycobacterium species, is a member of the phylogenetically defined order Actinomycetales (39). Together with other mycolic acid-containing organisms (i.e., members of the genera Rhodococcus and Nocardia), the members of the genus Mycobacterium form one of the several sublines of descent within the radiation of the actinomycetes. Later, the results of reverse transcriptase sequencing of 16s rRNAs from a larger number of strains confirmed the phylogenetic coherency of this subline, which was extended by the inclusion of the genera Corynebacterium (35), Gordona (36), and Tsukamurella (5). Within the genus Mycobacterium, species fall into one of two large groups which by and large correspond to the traditional groups containing the slowly growing and rapidly growing members of the genus (35).With the introduction of even faster analytical methods (i.e., analysis of polymerase chain reaction-mediated DNA stretches [4,28]), the number of actinomycetes analyzed and consequently the knowledge about the natural relationships of these organisms have increased considerably during the last 3 years. From a phylogenetic point of view, the genus Mycobacterium is one of the most thoroughly investigated bacterial taxa. There are 55 or so validly described species in this genus (51), and 45 strains of 34 species have been subjected to either 16s rRNA analysis (31,35,40), 16s rDNA analysis (4, 28), or 23s rDNA analysis (21). The bifurcation within the genus Mycobactenurn has been confirmed, with the majority of the slowly growing strains forming a phylogenetically very shallow (i.e., recent) group. In addition to the phyloge...

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Strains of a new type of slowly growing scotochromogenic mycobacterium were isolated repeatedly from sphagnum vegetation and surface water of moors in New Zealand. These strains grew at 31 and 22°C but not at 37°C and possessed catalase, acid phosphatase, and arylsulfatase activities. They did not split amides, and most of them were susceptible to antituberculotic drugs. Furthermore, they did not tolerate 0.1% NaOH, and 0.2% picric acid and did not grow on compounds used as single carbon sources and single nitrogen and carbon sources. The internal similarity of the strains as determined by numerical taxonomy methods was 96.6% & 3.09%. The whole-mycolate pattern is unique in that it has not been found previously in 23 species of slowly growing mycobacteria. Evaluation of long-reverse-transcriptase-generated stretches of the primary structure of the 16s rRNA confirmed that these organisms belong to the genus Mycobacterium. The phylogenetic position of these bacteria is unique; they are situated between slowly growing pathogenic and rapidly growing saprophytic species. The strains are not pathogenic for mice, guinea pigs, and rabbits, but they provoke a nonspecific hypersensitivity reaction to bovine tuberculin. Hence, they are considered members of a new species of nonpathogenic, slowly growing mycobacteria, for which the name Mycobacterium cookii is proposed. Strain NZ2 is the type strain; a culture of this strain has been deposited in the American Type Culture Collection as strain ATCC 49103. During 1984 and 1985, 17 strains belonging to the genus Mycobacterium and having homogeneous properties were isolated from sphagnum vegetation and pond water obtained from sites throughout the North and South Islands of New Zealand (4, 11). These strains were not pathogenic for experimental animals, they stimulated nonspecific hypersensitivity to bovine tuberculin in sensitized guinea pigs (ll), and they were regarded as a source of nonspecific tuberculin reactions in cattle in some regions of New Zealand (4). The properties of these strains indicate that they belong to a new species. In this paper we describe the results of a taxonomic study of these unusual strains. MATERIALS AND METHODSBacterial strains. The designations and sources of 17 previously undescribed strains of mycobacteria (referred to below as the NZ strains) are shown in Table 1.Methods. Colony morphology and the ability to grow at various temperatures (22, 31, 37, 42, and 45°C) were determined after 4 weeks of incubation on Lowenstein-Jensen slants and Middlebrook 7H10 agar inoculated with mg of cells per plate. Pigment production in the dark and photoactivity after 6 weeks of incubation were determined as previously described (8, 24). The following tests were also performed as described previously: nitrate reduction and catalase activity (2); Tween hydrolysis (14); acid phosphatase production (10); arylsulfatase activity after 3 and 10 days (12); production of acidamidase, benzamidase, urease, isonicotinamidase , nicotinamidase , p yrazinamidase , salicy...