Tatlockia, a Genetically and Chemically Distinct Group of Bacteria. Proposal to Transfer Legionella maceachernii (Brenner et al.) to the Genus Tatlockia, as Tatlockia maceachernii comb. nov.

System. Appl. Microbiol. 14, 52-56 (1991) © Gustav Fischer Verlag, StuttgartlNew York

Tatlockia, a Genetically and Chemically Distinct Group of Bacteria. Proposal to Transfer Legionella maceachernii (Brenner et al.) to the Genus Tatlockia, as Tatlockia maceachernii comb. nov. K. F. FOX l ,3, A. BROWN l ,2,3,*, A. FOX\ and G. SCHNITZER l 1 Department 3 Research

of Microbiology and Immunology, and 2 Department of Medicine, University of South Carolina School of Medicine, and Service, W. J. B. Dorn Veterans Hospital, Columbia, SC 29201, USA

Received July 25, 1990

Summary In addition to the previously reported DNA hybridization and base composition data, comparison of the nucleotide sequence of 16S-ribosomal RNA fragments and the total cellular carbohydrate content of Tatlockia micdadei and Legionella pneumophila strains strongly support their separation into different genera. In the present study Legionella maceachernii was found to be very similar to T. micdadei, and like T. micdadei, it was easily distinguished from the other legionellae. Based on the accumulated phenotypic and genotypic data Legionella maceachernii should be transferred to the genus Tatlockia, as Tatlockia maceachernii comb. nov.

Key words: 16S-rRNA, Legionella - Tatlockia - Legionellaceae - Taxonomy

The family Legionellaceae consists of ubiquitous environmental organisms, some of which have been associated with human pneumonia (Legionnaires' disease) (Fraser et ai. 1977; McDade et aI., 1977) and a "fIulike" disease (Pontiac fever) (Fraser et aI., 1979; Glick et aI., 1978). The family contains at least 28 named species, with 45 serogroups (Benson et aI., 1989; Brenner et aI., 1984; Brenner et aI., 1985; Brenner et aI., 1979) and several proposed "subspecies" (Brenner et aI., 1988). Available phenotypic, immunologic, chemical and genetic data support the division of the family, Legionellaceae, into several genera (Chumakov et aI., 1986; Collins and Gilbart, 1983; Fox et aI., 1984; Fox et aI., 1990; Fox and Brown, 1989; Fox and Brown, 1989; Garrity et aI., 1980; Hebert, 1981; Joly and Kenny, 1982; Karr et aI., 1982; Lambert and Moss, 1989; Lema and Brown, 1983; Ludwig and Stackebrandt, 1983; Moss et aI., 1977; Selander et aI., 1985; Sonesson et aI., 1989; Vickers and Yu, 1984; Walla et ai. 1984). Objections to the division of this family have primarily been based on the reported inability to * To whom reprint requests should be addressed.

distinquish the proposed genera phenotypically and the practicality of placing phenotypically similar organisms in the same genus (Brenner et aI., 1985). However, with the rapidly expanding number of recognized species, the single genus, Legionella, is becoming increasingly cumbersome and difficult to justify. In addition, a simple battery of tests, which can distinguish the genera Legionella, Tatlockia, and Fluoribacter, has recently been reported (Fox and Brown, 1989). Carbohydrate analysis of whole bacterial cells by gaschromatography, can also distinquish the proposed genera (Fox et aI., 1984; Fox et aI., 1990, Walla et aI., 1984). Such analysis has shown T. micdadei and L. maceachernii contain large amounts of rhamnose and fucose, in addition to two unusual sugars, fucosamine (X2) and an unidentified O-methylated sugar (X3). L. pneumophila was found to contain small amounts of quinovosamine (Xl) ,(Fox et aI., 1984; Fox et aI., 1990; Sonesson et aI., 1989) and rhamnose but no fucose or XL Although Fluoribacter species had diverse carbohydrate profiles, most contained quinovosamine (Xl) and fucosamine (X2) (Fox et aI., 1984; Fox et aI., 1990) and had profiles which could be

Transfer of L. maceachernii to the Genus Tatlockia

easily distinguished from those of other legionellae. In this study, data from 16S-rRNA sequencing and carbohydrate analysis clearly demonstrated that T. micdadei and 1. maceachernii are very closely related but otherwise quite distinct froJ:Il other legionellae studied and should therefore be considered members of the same genus, Tatlockia.

Materials and Methods Bacterial strains. The bacterial strains used in this study and corresponding sequence accession numbers are listed in Table 1. Growth conditions were as previously described (Fox and Brown, 1989; Fox and Brown, 1989). Preparation of ribosomal RNA. Bacterial cells were grown to late log phase in buffered yeast extract broth (500 ml). They were then harvested, and washed twice. The total RNA was extracted as previously described (Fox and Brown, 1989; Fox and Brown, 1990). Sequencing of 16S-rRNA. The method described by Lane et al. (1985) was used with minor modifications (Deborde et aI., 1986; Fox and Brown, 1990). Terminal deoxynucleotidyl transferase was added to the chase mixture which was then incubated for 45 min at 3rc. Sequence analysis. For similarity calculations, sequence alignment was accomplished using the FSTNSCAN program (LipmanPearson algorithm), available as part of the PC/GENE program suite (Intellegenetics, Mountain View, Cal. Physiological tests. All biochemical tests were done as previously described (Fox and Brown, 1989). They included the following tests: catalase (Weaver- and Feeley, 1979), gelatinase (Garrity et aI., 1980), Voges-Prokauer using a modified media and API-20E strip (Fox and Brown, 1989), starch hydrolysis and browning of tyrosine-containing medium (Baine et aI., 1979; Garrity et aI., 1980; Vickers and Yu, 1984), hippurate hydrolysis (Hebert, 1981), bromocresol-purple-spot test (Garrity et aI., 1980), and cytochrome oxidase (Fox and Brown, 1989). Colony and medium fluorescence were determined on the starch! tyrosine-containing buffered yeast extract agar platt;s. Carbohydrate analysis. Carbohydrate profiles were obtained using the alditol acetate derivatization procedure as previously described (Fox et aI., 1989). Two to 10 mg of each sample were hydrolyzed in 2 N sulfuric acid. After extraction and derivitization, gas chromatographic-mass spectrometric analysis was carried out with an HP-5970 mass-selective detector (HewlettPackard USA) interfaced to an HP-5890 gas-chromatograph equipped with an automated sample injector (HP-7673A) and a SP2330 fused silica capillary column (Supelco, Bellefonte, PAl.

Results and Discussion The 16S-rRNA of twelve legionellae were partially sequenced using primers complementary to nucleotides #521-533, #913-928, and #1392-1407 of the Escherichia coli 16S-rRNA sequence. These sequences consisting of a total of 450 to 600 bases, which represented approximately 113 of the 16S-rRNA molecule, were also compared with the published sequences of E. coli, Proteus vulgaris, Pseudomonas testosteroni, Chlamydia psittaci, Rochalimaea quintana, Bacterioides fragilis, Anacystis nidulans and Agxobacterium tumefaciens. In addition to randomly located differences, characteristic differences were noted at nucleotides #834-,-836 and

53

843-849, which distinguished the T. micdadei and 1. maceachernii strains from the others tested. T. micdadei and 1. maceachernii sequences contained CC(U)U at bases 834--836 and UGAGG at bases 843-849, whereas 1. pneumophila and F. bozemanae sequences contained UUA and AAUAA at these sites, and those of F. dumoffii and F. gormanii contained AUA and AAUAU. The legionella strains used and the corresponding GENBANK accession numbers are listed in Table 1.

Table 1. Bacterial strains used for sequencing - Genbank accession numbers *

Legionella pneumophila Philadelphia-1 Chicago-2 Dallas-IE

M19444 M19450 M22266 M19442 M19448 M22264 M19447 M19452 M22265

Tatlockia micdadei Tatlock PGH-12 PPA-JC

M19449 M19443 M22261 M22262 M19446 M22263 M19451 M19445 M22260

Fluoribacter Bozemanae WIGA MI-15

M24620 M24645 M24646 M24618 M24643 M24649

Fluoribacter Dumoffii NY-23 Tex-KL

M24619 M24641 M24647 M24617 M24642 M24650

Fluoribacter gormanii LS-13

M24640 M24644 M24648

Legionella maceachernii Px-l-G2-E2

M34712 M34713 M34714

Sequence similarity values are shown in Table 2. T. micdadei 16S-rRNA fragments were 97.2% (+/-0.6%) similar to those of 1. maceachernii. In turn, the 1. maceachernii sequence was 92.3% (+/-0.8%) similar to 1. pneumophila strains and 92.5%, 93.3%, and 91.9% similar to F. bozemanae, F. dumoffii, and F. gormanii, respectively. The mean intergroup similarity among 1. pneumophila and Tatlockia strains was 92.2% (+/"':'1.3%), and among Tatlockia and Fluoribacter strains it was 92.4% (+/-1.0%). For comparison, the corresponding fragments of the reported 16S-rRNA sequences of E. coli and P. vulgaris, both members of the class Proteobacteria (Stackebrandt et al., 1988) and of the family Enterobacteriaceae, were 94.7% similar. The 16S-rRNA of legionella strains was 81.2 % (+ / -1.5 %) similar to that of E. coli, also a member of the gamma subdivision of Proteobacteria. Similarity to P. testosteroni, a member of the beta subdivision, and A. tumefaciens, a member of the alpha subdivision (Woese et al., 1985), was slightly less (77.9 +/-1.0% and 75.4 +/-1.5%, respectively). These data are consistent with previous reports (Chumakov et al., 1986; Ludwig and Stackebrandt, 1983). It is interesting to note that the sequences of the Legionella and Tatlockia strains showed less similarity to one another than

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K. F. Fox, A. Brown, A. Fox, and G. Schnitzer

Table 2. Percent 16S-ribosomal RNA similarity Strains T. micdadei Tatlock PGH-12 PPA-JC

Px-1-G"-E2

Px-l-G2-E2 Tatlock

97.7 97.5 98.0 100

,Proteus vulgaris!

100 96.6 96.6

80.9 79.9 80.9

97.7

83.0

L. pneumophila Philadelphia-l Chicago-2 "Dallas-IE"

91.4 92.8 92.8

91.8 92.5 91.8

81.0 83.0 81.2

F. bozemanae WIGA MI-15

91.3 93.7

90.3 93.7

83.3 82.4

F.dumoffii NY-23 Tex-KL

93.1 93.5

91.5 92.4

82.6 83.7

F.gormanii LS-13

91.9

91.5

83.2

Other Proteobacteria 2 P. vulgaris E. coli P. testosteroni A. tumefaciens R.quintana C. psittaci

83.0 82.7 78.6 76.9 78.2 78.2

80.9 80.5 77.1 74.1 73.9 75.4

100 94.7 81.9 79.1 78.8 79.2

Other Eubacteria! B. fragilis A. nidulans

71.9 71.5

69.6 68.9

73.7 71.1

From published sequences. The strain used is a CDC derived serogroup 5 L. pneumophila strain, but is apparently not the original Dallas-IE strain. 1

2

that seen between two distinct genera of the Enterobacteriaceae. These data are consistent with that obtained from DNA-DNA hybridization studies of the legionellae (Brenner et ai., 1985; Garrity et ai., 1980). All members of the family Legionellaceae contain a relatively large amount of lipid. In addition, 80% of the fatty acids are branched chain; with minimal amounts of hydroxy-fatty acids (Lambert and Moss, 1989; Moss et ai., 1977), characteristics which are unusual for Gramnegative organisms. Based on their fatty acid profiles, Lambert and Moss (1989) recently grouped the legionellae into three subdivisions. L. micdadei and L. maceachernii, which have indistinguishable profiles, were placed in group A15/16C. Members of this group contain C a1S :O and C i16 : 0 in approximately equal amounts. These two species were differentiated from other members of the A15/16C group by having more than 3% Ca17 :0 and by the presence of C 21 :0 . T. micdadei and L. maceachernii also have similar ubiquinone profiles, with no Q9 or Q10 (Collins and Gilbart, 1983; Lambert and Moss, 1989). The cellular carbohydrate content of T. micdadei was identical to that of L. maceachernii but both were dramat-

ically different from the other legionellae tested. L. micdadei strains and L. maceachernii contained large amounts of rhamnose and fucose. In addition, tWo unique sugars, X3, and fucosamine (Xl) were present, but quinovosamine (Xl, commonly found in other legionellae) was absent (Fox et al., 1990). Brenner justified the inclusion of all legionellae strains into one genus by stating, "Species reflects evolutionary divergence; a genus is a man-made division". In addition, he stated the inclusion of an organism into a genus "based on phenotype is preferable" if the genotypic and phenotypic data are not in agreement (Brenner et al., 1985). Ludwig and Stackebrandt, using oligonucleotide cataloging, evaluated four Legionella species; L. pneumophila, L. bozemanii (F. bozemanae), L. (T.) micdadei, and L. jordanis (Ludwig and Stackebrandt, 1983), each represented by a single strain. They concluded that by "comparing the data obtained on Legionella with those of the enterobacteria, each of the four Legiol1ella species investigated could be described as a new genus". They qualifed this, however, by pointing out that "the high degree of phylogenetic relatedness found among and within genera of Enterobacteriaceae is unique among eubacterial families." As a starting point for further evaluation and discussion, we would like to propose that, in addition to accepting DNA similarity values of 65-70% or more as indicating species level relatedness, the similarity of 16S-rRNA sequences (assuming adequate sample size) should be at least 94% for species of the same genus and at least 90% for genera of the same family . It should be noted that there appears to be considerable overlap in the 16S-rRNA similarity values for genus and species. Legionella (as represented by species other than L. maceachernii) and Tatlockia are distinct genera, with less than 20% of control reannealing by DNA-DNA hybridization and less than 93.8% (mean = 92.2%) 16S-rRNA sequence similarity. The Fluoribacter can be separated from T atlockia by the same criteria. On the other hand, L. maceachernii should be included within the genus Tatlockia based primarily on the striking 97.8% 16S-rRNA sequence similarity. Characteristics, capable of distinguishing T. micdadei and L. maceachernii from other legionellae, include their blue gray colonies on dye-containing media (Vickers et al., 1981), an inability to hydrolize hippurate, the absence of medium or colony fluoresecence, the absence of browning on tyrosine-containing media, a positive bromocresol purple spot test, and negative oxidase activity (Fox and Brown, 1989). T. micdadei can be distinquished from L. maceachernii by the positive Voges-Proskauer reaction of the former (Fox and Brown, 1989). Fatty acid content of T. micdadei and L. maceachernii are indistinguishable and can be separated from other legionellae by the presence of C a1S :O and C i16 :0 in approximately equal amounts (Lambert and Moss, 1989) . T. micdadei strains and L. maceachernii carbohy'drate profiles are distinctive, containing large amounts of rhamnose and fucose and the characteristic sugars X3 and fucosamine (X2) but no quinovosamine (Xl) (Fox et al., 1990). Finally, ubiquinone profiles are identical and distinct for these two species, which contain no Q9 or Q10

Transfer of L. maceachernii to the Genus Tatlockia

(Collins and Gilbart, 1983; Lambert and Moss, 1989). The genus Tatlockia should now be considered to consist of two species, T. micdadei and T. maceachernii, comb. nov. The type strain of the species, Tatlockia maceachernii, remains PX-1-G2-E2 (ATCC 35300). Acknowledgement. This work was in part supported by the V A Medical Research Service.

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Dr. A. Brown, Research Service, W.J. B. Dorn Veterans Hospital, Columbia, SC 29201, USA