The soluble peptides of strains of Legionella pneumophila, Tatlockia micdadei, ..... Lane 1,Philadelphia 1 (serogroup 1); lane 2, Togus 1 (serogroup 2); lane 3, ...
Vol. 17, No. 6
JOURNAL OF CLINICAL MICROBIOLOGY, June 1983, p. 1132-1140 0095-1137/83/061132-09$02.00/0 Copyright © 1983, American Society for Microbiology
Electrophoretic Characterization of Soluble Protein Extracts of Legionella pneumophila and Other Members of the Family Legionellaceae MICHAEL LEMA AND ARNOLD BROWN* Research Service, Wm. Jennings Bryan Dorn Veterans Hospital, Columbia, South Carolina 29201 Received 17 November 1982/Accepted 31 January 1983
The soluble peptides of strains of Legionella pneumophila, Tatlockia micdadei, Fluoribacter bozemanae, Fluoribacter dumoffii, and Fluoribacter gormanii were studied by polyacrylamide gel electrophoresis. Characteristic patterns were seen for Legionella and Tatlockia strains, whereas the patterns for the Fluoribacter strains were variable as would be expected for this genetically heterogenous group. Grouping by peptide pattern was consistent with proposed taxons based on DNA-DNA homology. By using a new silver stain technique, the sensitivity and ease of pattern recognition were enhanced significantly. This technique is an easily applied general method for distinguishing between strains in epidemiological studies.
Legionella pneumophila and phenotypically similar organisms of the genera Legionella, Tatlockia, and Fluoribacter (8, 19) are recently recognized pathogens (12, 18, 21, 24, 33, 41) which are widely distributed in the environment (13, 16, 17, 22, 50). These organisms have been identified and characterized by antigen-antibody techniques (5, 12, 14, 20, 34, 53-55), fatty acid analysis (12, 20, 32, 38, 39), ubiquinone profiling (28), nucleic acid hybridization (5-8, 12, 19, 20, 24, 37, 46) and relatively few routine phenotypic tests (2, 3, 6, 8, 12, 19, 23, 24, 40, 44, 45, 51, 52). The most definitive test, that of nucleic acid hybridization, is complex and tedious. On the other hand, the standard phenotypic tests are not sufficiently discriminating (20); therefore, other techniques are needed to characterize these organisms. Electrophoresis of crude extracts of soluble protein has been used either alone or as a supplement to other characterization and identification techniques in the study of many bacterial groups (4, 10, 11, 15, 25, 26, 35, 36, 42, 43, 47-49). This analytic technique is simple to perform with large numbers of samples, and the amount of information obtained is great. This paper provides preliminary information about the protein patterns obtained with various strains of L. pneumophila and other members of the family Legionellaceae. MATERIALS AND METHODS Bacterial strains and media. Bacterial strains used in this study are listed in Table 1. The bacteria were grown on buffered charcoal-yeast extract agar (46) in air at 37°C for 3 days.
Preparations of cell-free extracts. Half of the confluent bacterial growth on a Petri plate (15 by 100 mm) was harvested and suspended in 2 ml of a solution of 0.1 M Tris-hydrochloride (pH 6.8)-15% glycerol-2 mM phenylmethylsulfonyl fluoride. To this, 10% sodium dodecyl sulfate (SDS) was added to yield a final concentration of 2.0%. The suspension was vigorously mixed and placed in a boiling water bath. After 5 min, the suspension was again vigorously mixed and then centrifuged at room temperature for 10 min at 10,000 rpm in a JA-20 rotor in a Beckman J21B centrifuge. The supernatant was removed, and the protein content was determined by the method of Lowry et al. (31), using bovine serum albumin as the standard. Extracts were stored at -20°C. SDS-polyacrylamide gel electrophoresis. Polyacrylamide gel electrophoresis was performed by a modification of the method of Laemmli (30). The gel slabs were 1.5 mm thick, 140 mm wide, and 100 mm long. The running gel, consisting of 8.5% polyacrylamide, 0.375 M Tris-hydrochloride, and 0.1% SDS (pH 8.8), was poured to within 1 cm from the bottom of the well former. The stacking gel, which filled the remaining area, was composed of 4.5% polyacrylamide, 0.125 M Tris-hydrochloride, and 0.1% SDS (pH 6.8). The running buffer was 0.025 M Tris-hydrochloride, 0.2 M glycine, and 0.1% SDS (pH 8.8). Equal volumes of sample and 2x sample buffer (10% glycerol, 4.0%
SDS, 0.02% bromthymol blue, 10% [vol/vol] P-mercaptoethanol, 0.125 M Tris-hydrochloride [pH 6.8]) were mixed and placed in a boiling water bath for 5 min. Samples contained approximately 150 to 200 ,ug of protein in Coomassie blue-stained gels. Gels which were to be stained with the silver stain contained approximately 10 ,ug of protein per sample well. Electrophoresis was performed at room temperature at 50 V (constant voltage) for 15 min and then at 100 V (constant voltage) until the tracking dye reached the bottom of the gel. The gels were fixed for 20 min in
1132
SDS-PAGE ANALYSIS OF LEGIONELLACEAE PEPTIDES
VOL. 17, 1983
1133
TABLE 1. Bacterial strains used in this study Isolate
L. pneumophila Philadelphia 1 Knoxville 1 Togus 1 Atlanta 1 Bloomington 2 Los Angeles 1 Dallas 1E 687 Chicago 2 Houston 2
1CMH 11EJ 10E36 1OE115 HWT3 11ES 7W FOS CAR KEL SMH1
Serogroup
1 1 2 2 3 4 5 S 6 6 1 1 1 1 1 1 1 1 1 1 1
Source
CDCb CDC CDC CDC CDC CDC CDC GSPHc CDC CDC
PVAd PVA PVA PVA PVA PVA PVA PVA PVA PVA
Isolated from:
Plasmida
Human Human Human Human Environment Human
62 Mdal
Environment Environment Human Human Environment Environment Environment Environment Environment Environment Environment Human
80 80 80 80 80
ND Mdal Mdal Mdal Mdal Mdal
SMHe
Human Human Human
CDC
Guinea pig
PUHf CDC/PVA
Human
Environmental
ND
PUH PUH PUH PVA PVA PVA
Human Human Human Human Human Human
ND ND ND
F. bozemanae WIGA MI-15
CDC PUH/CDC
Human Human
35, 40, 45, 80 Mdal 23 Mdal
F. dumoffii NY-23
PUH/CDC
Environment
25, 40, 55, 80 Mdal
F. gormanii LS-13
PUH/CDC
Environment
>80 Mdal
T. micdadei TATLOCK PPA-EK PPA-PGH-12 PPA-JC PPA-GL PPA-ML PPA-MCC PPA-MAC PPA-CAR
ND
None found; ND, not done. Centers for Disease Control, Atlanta, Ga. c R. Yee, Graduate School of Public Health, University of Pittsburgh, Pittsburgh, Pa. d Veterans Administration Medical Center, Pittsburgh, Pa. e F. S. Nolte, Strong Memorial Hospital, Rochester, N.Y. f A. W. Pasculle, Presbyterian-University Hospital, Pittsburgh, Pa. a
-,
b
12.5% (wt/vol) trichloroacetic acid and then were stained overnight with 0.018% (wt/vol) Coomassie blue in a methanol-water-acetic acid solution (5:5:1). Destaining was accomplished with several changes of 10% (vol/vol) acetic acid. Gels were also stained by a silver staining technique (Gelcode; The Upjohn Co., Kalamazoo, Mich.) according to the manufacturers protocol. Analysis of gels. Photographs of the gels were taken with a Mamiya camera (2/4 by 2/4 in. [ca. 5.72 by 5.72 cm]) equipped with an 80-mm lens on Kodalith film or
with a Polaroid MP4 camera and Polacolor type 58 sheet film (4 by 5 in. [ca. 10.16 by 12.70 cm]). In addition, densitometry tracings of Coomassie bluestained gels were obtained with an EC scanning densitometer. The tracings were compared by marking peaks, overlying the two tracings to be compared, and measuring the height on both tracings, at each designated peak area. Slight shifting of the tracing was done to line up obviously related peaks. Pearson productmoment correlation coefficients were obtained for each comparison (29). Numerical comparisons were
1134
LEMA AND BROWN
J. CLIN. MICROBIOL.
only performed for samples run in the same gel slab. Sample size ranged from 20 to 35 measurements.
strains seen in Fig. 2 is due to the presence of a dense low-molecular-weight band in the extracts of the five plasmid-containing environmental isolates (lanes 1 through 5). The extract in lane 1 contains an additional, slightly less dense band not seen in the other preparations. Other more subtle differences may be seen between the plasmid-containing and plasmid-less strains in several regions. However, except for the differences mentioned above, the major band patterns are otherwise almost identical. When the densitometer tracings of Coomassie blue-stained gels were compared, very high correlation coefficients were obtained for the following intragroup comparisons: (i) different preparations of the same strain of L. pneumophila (Philadelphia 1), r = 0.983 + 0.014 (n = 7); (ii) epidemiologically related plasmid-containing environmental L. pneumophila strains, r = 0.985 ± 0.004 (n = 7); and (iii) Tatlockia micdadei isolates (TATLOCK and four different clinical Pittsburgh pneumonia agent strains), r = 0.983 ± 0.010 (n = 5). When the patterns of plasmidcontaining environmental isolates were compared with plasmid-less environmental or clinical isolates from the Pittsburgh Veterans Administration Medical Center, a correlation coefficient of 0.710 ± 0.009 was obtained; however, when the most prominent plasmid-associated band was eliminated from the comparison, the correlation coefficient increased to 0.966 ± 0.005. The intergroup comparisons between L.
RESULTS
Figures 1 and 2 show the patterns obtained with Coomassie blue staining of soluble protein extracts from isolates representing each of the six serogroups of L. pneumophila. The patterns are visually very similar. Approximately five to eight major bands can be seen in all isolates as well as a number of minor bands. The most obvious differences among strains are seen in the region of the gel where the lower-molecularweight peptides are found. In Fig. 1 lanes 1, 3, 6, and 9, a prominent band can be seen 9/loths of the way through the gel; in addition, the extracts in lanes 5 and 6 appear to be missing the dense band near the end of the gel that is seen in the other lanes. Figure 2 shows the Coomassie blue staining pattern of extracts obtained from serogroup 1 L. pneumophila isolated at the Pittsburgh Veterans Administration Medical Center (9). The characteristics of these strains have been reported previously (9); they include environmental isolates containing a unique 80 megadalton (Mdal) plasmid (lanes 1 through 5), environmental isolates without a plasmid (lanes 6 and 7), and plasmid-less clinical isolates (lanes 8 and 9). With these strains the patterns are recognizably similar to those of the various strains seen in Fig. 1. The most obvious difference among 1
2
4J;,.
3
4
5
_ * _.o Ft* #-e!s;
.06%.WAN*.
qvmmlmop ANOW
..-.;
".
6
*
7
9
8
4 W
W
,
ATWI. .wW x. .-?,
-4000W--AM.
bkft-
-'s.
me .."me
FIG. 1. Coomassie blue-stained gel patterns of extracts of strains representing the six serogroups of L. pneumophila. Lane 1, Philadelphia 1 (serogroup 1); lane 2, Togus 1 (serogroup 2); lane 3, Atlanta 1 (serogroup 2); lane 4, Bloomington 2 (serogroup 3); lane 5, Los Angeles 1 (serogroup 4); lane 6, Dallas 1E (serogroup 5); lane 7, 687 (serogroup 5); lane 8, Chicago 2 (serogroup 6); lane 9, Houston 2 (serogroup 6). Dashed lines in the unnumbered lane indicate position of major L. pneumophila bands seen on silver-stained gels.
SDS-PAGE ANALYSIS OF LEGIONELLACEAE PEPTIDES
VOL. 17, 1983 l
2
3
4
5
6
7
8
1135
9
-.:~
_ _ _
Ant
_ _ _
FIG. 2. Coomassie blue-stained gel patterns of L. pneumophila strains from the Pittsburgh Veterans Administration Medical Center, which included plasmid-containing environmental isolates (lanes 1 through 5), plasmid-less environmental isolates (lanes 6 and 7), and plasmid-less clinical isolates (lanes 8 and 9). The specific isolates were as follows: lane 1, 1CMH; lane 2, 11EJ; lane 3, 10E36; lane 4, 10E115; lane 5, HWT3; lane 6, 11ES; lane 7, 7W; lane 8, FOS; lane 9, CAR. Dashed lines in the unnumbered lane indicate the position of major L. pneumophila bands seen on silver-stained gels.
pneumophila (Knoxville 1) and Fluoribacter bozemanae (WIGA) or T. micdadei (TATLOCK) yielded values of r = 0.593 and 0.869, respectively. The correlation coefficients obtained for comparisons between the pattern of T. micdadei (TATLOCK) and the various Fluoribacter species, represented by isolates WIGA, NY-23, and LS-13, were 0.825, 0.898, and 0.873, respectively. The values obtained for comparisons between the Fluoribacter species were: LS-13 and NY23, 0.934; WIGA and NY23, 0.913; WIGA and LS-13, 0.783; MI-15 and LS-13, 0.780; and WIGA and MI-15, 0.766. Probabilities based on z-transformation (29) increased the range between the comparisons slightly (data not shown). Photographs of gels stained by the silver staining technique are shown in Fig. 3a through d. More than 50 to 60 distinct bands of various colors can be seen in each lane. Even though 20fold less material (-10 ,ug of total protein as opposed to -200 ,ug in the Coomassie bluestained gels) was loaded in each lane of the gels stained with the silver stain, more bands can be seen, with sharper definition, than on gels stained with Coomassie blue. Figure 3a shows the patterns obtained with extracts of isolates representing the six serogroups of L. pneumophila. As in the Coomassie blue-stained gels (Fig. 1), the patterns are simi-
lar. The most striking characteristic bands appear at approximately 1.3, 3.8, 5.3, 10.2, 11.4, and 12.2 cm, with a light band, or relative void, at 6.4 cm. These bands appear at the same position in each lane and have approximately the same color. Differences can be seen in many of the minor bands, in terms of both position and color. The most striking difference is due to the presence of a light-yellow band which occurs at approximately 11.4 cm in lanes 1 and 6 through 9. Lanes 3, 7, and 9 contain extracts of strains which had been prepared approximately 1 month before electrophoretic analysis (stored at -20°C) and were compared with the corresponding freshly prepared extracts contained in lanes 2, 6, and 8. Upon visual inspection, there is a one-to-one correspondence between the pairs (lanes 2 and 3, 6 and 7, 8 and 9) in terms of band position and color. Figure 3b shows the patterns obtained with extracts of serogroup 1 L. pneumophila isolates, including plasmid-containing environmental isolates (lanes 7 through 9), environmental isolates without a plasmid (lanes 5 and 6), clinical isolates without a plasmid (lanes 2 through 4), and a clinical isolate from another institution (lane 1, plasmid content not tested). The patterns are similar to those seen in Fig. 3a. The major characteristic bands occur at
a
1
2
3
4
5 6
.:_-l.s2*Fif$'"--
;.^ i;_ 8
7
9
i-':>.!.*
.F:
..
*'
. .. i
.-.--.
°:-
KiS
3 .-
.i _ *.,
>
.h
.2
-.te!g
t0
u. r;ee,,,, ,_ __ .;
...
.S.;.
Es X ww
a M
!
w XF
s _ _
E F.#F'
:, '.1
s =
-!
i_
_r
1_
.
!
^&
.flt .>.o.
11
9-
_r
-
F
i
- - -
b_
1
2
34
56
. *!..
8
7
9
-
.....
I
W i'V
4
FIG. 3. (a) silver-stained gel patterns of extracts of strains representing the six serogroups of L. pneumophila. Lane 1, Philadelphia 1 (serogroup 1); lane 2, Togus 1 (serogroup 2, extracted 7 December); lane 3, Togus 1 (serogroup 2, extracted 8 August); lane 4, Bloomington 2 (serogroup 3); lane 5, Los Angeles 1 (serogroup 4); lane 6, Dallas 1E (serogroup 5, extracted 7 December); lane 7, Dallas 1E (serogroup 5, extracted 8 August); lane 8, Houston 2 (serogroup 6, extracted 7 December); lane 9, Houston 2 (serogroup 6, extracted 8 August). Unmarked lane: -4.2 cm, bovine serum albumin, 66 kilodaltons; -7.6 cm, ovalbumin, 45 kilodaltons; -12.5 cm, lysozyme, 14.3 kilodaltons. Lines in the margin indicate the positions of visually prominent bands present in extracts of all L. pneumophila isolates tested. (b) Silver-stained gel patterns of strains of serogroup 1 L. pneumophila strains from the Pittsburgh Veterans Administration Medical Center, which include plasmid-containing environmental isolates (lanes 7 through 9), plasmid-less environmental isolates (lanes 5 and 6), plasmid-less clinical isolates 1136
1
C
3
2
4
;
..=4
;, _ d t-
.4
A.1,
s79.i8
,6u
5
4
L
j.
4
-
.4
.
*;
..
_ ^_ a
6' -
-e
'.
_-
&
-.4 4~~~~~~~~~~~~~~~
F:
..1
.
..
f
t
SO
I _ew
_'a
I I
d
1
r
5
4
3
2
6
!
7r7p9
1.t Lr:
M.B.
.4. I
4~
0=0 ...-
4
...
a. .
..
Eb.-
I1
X -:
ca..
I
(lanes 2 through 4), and a clinical isolate with the plasmid content not determined (lane 1). Lane 1, SMH1; lane 2, KEL; lane 3, CAR; lane 4, FOS; lane 5, 7W; lane 6, liES; lane 7, 10E36; lane 8, llEJ; lane 9, 1CMH. Lines in the margin indicate the positions of visually prominent bands present in extracts of all L. pneumophila isolates tested. (c) Silver-stained gel patterns of strains of T. micdadei. Lane 1, TATLOCK; lane 2, PPA-EK; lane 3, PPA-PGH-12; lane 4, PPA-JC; lane 5, PPA-GL; lane 6, PPA-ML; lane 7, PPA-MCC; lane 8, PPA-MAC; lane 9, PPA-CAR. (d) Silver-stained gel patterns of extracts of L. pneumophila (lanes 1 through 3), T. micdadei (lanes 4 and 5), F. bozemanae (lanes 6 and 7), F. dumof i (lane 8), and F. gormanii (lane 9). Lane 1, Knoxville 1; lane 2, Togus 1; lane 3, 1CMH; lane 4, PPA-EK; lane 5, TATLOCK; lane 6, WIGA; lane 7, MI-15; lane 8, NY-23; lane 9, LS-13. 1137
1138
LEMA AND BROWN
approximately 1.2, 3.8, 5.0, 9.4, 10.7, and 11.4 cm, again with a lighter band, or void, seen at approximately 6.0 cm. The patterns may be visually grouped into environmental plasmidcontaining isolates (lanes 7 through 9), clinical and environmental plasmid-less isolates (lanes 2 through 6), and the "foreign" clinical isolate (lane 1). The patterns obtained with extracts of T. micdadei are shown in Fig. 3c. Lane 1 contains the strain TATLOCK and the remaining lanes contain Pittsburgh pneumonia agent strains isolated in Pittsburgh. Visually the patterns are very similar but they are different than the patterns obtained with L. pneumophila (Fig. 3a and b). The most striking characteristic bands are seen at approximately 1.0 (triple band), 2.0, 4.4, 4.7, 5.4, 5.7, 7.3, 7.9, 8.3, 8.5, 9.0, 9.6, 10, 10.4, 10.6, 11, and 11.5 cm. A comparison of extracts of istolates representing the three groups of organisms, L. pneumophila (lanes 1 through 3), T. micdadei (lanes 4 and 5), and several Fluoribacter species (lanes 6 through 9), is shown in Fig. 3d. Three general patterns corresponding to the three groups can be seen. The patterns seen with Tatlockia strains (also see Fig. 3c) are the most uniform in that almost all of the bands match visually. The Legionella strains appear to be the next most uniform (also see Fig. 3a and b), with the majority of the bands matching. The Fluoribacter strains show the greatest diversity. DISCUSSION Previously, workers have used polyacrylamide gel analysis of soluble bacterial peptides stained with Coomassie blue for the characterization or classification of various groups of organisms (4, 10, 11, 15, 25, 26, 29, 35, 36, 42, 43, 47-49). These patterns were analyzed by simple visual comparison (10, 36, 42) or by complex numerical analysis of a digitized densitometry scan (26, 29). In this study we examined the ability of SDS-polyacrylamide gel electrophoresis analysis of soluble bacterial proteins to characterize and distinguish between strains of L. pneumophila and several Legionella-like organisms. Two different staining procedures were used to visualize the protein bands, Coomassie blue staining and a new silver staining technique. On Coomassie blue-stained gels, characteristic and reproducible patterns could be seen for all L. pneumophila strains tested. Differences among the patterns for different strains were observed primarily in the minor band regions, and some might be a reflection of antigenic differences among these strains. However, on the whole the patterns were sufficiently similar to be useful for identification.
J. CLIN. MICROBIOL.
It is interesting to note the presence of a dense band in the low-molecular-weight region of the electropherogram of extracts of the plasmidbearing environmental isolates which is not seen in extracts of the clinical or environmental plasmid-less strains (Fig. 2 and 3b). However, although this finding is quite reproducible (unpublished observation), an examination of Fig. 1 and 3a shows that this band was also seen in some plasmid-less strains. It is possible that the presence of the plasmid does not code for but merely promotes the synthesis of this peptide or that the coincident peptides are, in fact, different proteins. Densitometry scans were digitized by measuring peak height at positions defined by the presence of a peak on one or both of the scans compared. The Pearson product-moment correlation coefficient was then determined for the paired data (29). Most intragroup comparisons resulted in correlation coefficients ranging from 0.983 to 0.985; however, it was found that the presence of a single plasmid-associated band was sufficient to change the correlation coefficient for comparisons between otherwise very similar strains from 0.966 to 0.710. Thus, a difference in one major band between two patterns resulted in a correlation coefficient which was as low, or even lower, than that found in a comparison of patterns which were not at all visually similar. Therefore, although this test could determine similarity it was not useful to distinguish between degrees of difference, at least as we performed the data analysis. With automated digitization of the scans and with the comparison of a larger number of smaller intervals the useful range of values would probably be greater. The data obtained from the Coomassie blue staining patterns are restricted to band position and density. Undoubtedly many of the bands contain several different proteins which happen to have the same electrophoretic mobility; in addition, a matching of bands does not necessarily mean that the proteins are identical. With the silver stain used in this study, another parameter, that of color, was added to that of position and density, thus significantly increasing the amount of available information. The clarity and distinctiveness of these patterns makes data analysis simple. Characteristic patterns were observed for the L. pneumophila and T. micdadei strains. Patterns seen with Tatlockia extracts were the least variable, whereas those seen with L. pneumophila extracts were slightly more variable. The Fluoribacter spp., not unexpectedly, being a genetically heterogeneous group, visually showed significant differences in peptide profiles. Other workers have found that there is a good correlation between similarities
VOL. 17, 1983
SDS-PAGE ANALYSIS OF LEGIONELLACEAE PEPTIDES
found by DNA homology studies and similarities found through the examination of protein electrophoretic patterns (4, 10, 49). A comparison of the peptide patterns photographically presented in this paper with the DNA homology studies of Garrity et al. (19) and Brown et al. (8) shows that both methods are in agreement with respect to the grouping of organisms tested. Other techniques, such as carbohydrate profiling, also appear to support these taxonomic distinctions (A. Fox, P. Y. Lau, A. Brown, S. L. Morgan, Z.-T. Zhu, and M. Lema, submitted for publication). In summary, these data support the proposed classification which separates this group of organisms into the genera Legionella, Tatlockia, and Fluoribacter (8, 19). The function and origin of the distinctive peptides found consistently in the plasmid-containing environmental isolates from Pittsburgh, but also seen in some plasmid-less isolates from other cities, remain to be elucidated. Because of its sensitivity and ability to discriminate between proteins with color differences, the silver staining technique appears to be a powerful tool for both taxonomic and epidemiological studies. Other techniques currently used for epidemiological characterization of strains have distinct drawbacks. For example, serotyping requires the availability of sera, which may vary in specificity unless monoclonal reagents are used, bacteriocin and phage typing schemes require viable indicator strains or phage, biotyping is subject to problems of standardization, and strain identification by antibiotic sensitivity patterns may be affected by mutation, plasmid transfer or loss, etc. Although we and others have used plasmid profiling to characterize Legionella strains (1, 9, 27), plasmid analysis may be of limited sensitivity since most strains, particularly those of clinical origin, are plasmid-less and because of the limited variety of L. pneumophila plasmids detected so far. Therefore, for epidemiological studies, no other technique may be comparable to peptide profiling in terms of simplicity, sensitivity, and, probably, universal applicability. ACKNOWLEDGMENTS This work was supported by the Veterans Administration Medical Research Service. We thank Dorothea Barwick for her excellent secretarial assistance. LITERATURE CITED 1. Aye, T., K. Wachsmuth, J. C. Feeley, R. J. Gibbon, and S. R. Johnson. 1981. Plasmid profiles of Legionella spe-
cies. Curr. Microbiol. 6:389-394. 2. Baine, W. B., and T. K. Rasheed. 1979. Aromatic substrate specificity of browning by cultures of the Legionnaires disease bacterium. Ann. Intern. Med. 90:619-620. 3. Berdal, B. P., 0. Hushovd, 0. Olsvik, 0. R. Odegard, and T. Bergan. 1982. Demonstration of extracellular proteolytic enzymes from Legionella species strains by using
4.
5.
6.
7.
8.
9.
10. 11.
12.
13.
1139
synthetic chromogenic peptide substrates. Acta Pathol. Microbiol. Immunol. Scand. Sect. B 90:119-123. Biavati, B., V. Scardovi, and W. E. C. Moore. 1982. Electrophoretic patterns of proteins in the genus Bifidobacterium and proposal of four new species. Int. J. Syst. Bacteriol. 32:358-373. Bibb, W. F., R. J. Sorg, B. M. Thomason, M. D. Hicklin, A. G. Steigerwalt, D. J. Brenner, and M. R. Wulf. 1981. Recognition of a second serogroup of Legionella longbeachae. J. Clin. Microbiol. 14:674-677. Brenner, D. J., A. G. Steigerwalt, G. W. Gorman, R. E. Weaver, J. C. Feeley, L. G. Cordes, H. W. Wilkinson, C. Patton, B. M. Thomason, and K. R. Lewallen-Sasseville. 1980. Legionella bozemanii species nova and Legionella dumoffi species nova: classification of two additional species of Legionella associated with human pneumonia. Curr. Microbiol. 4:111-116. Brenner, D. J., A. G. Steigerwalt, and J. E. McDade. 1979. Legionnaires' disease bacterium. Legionella pneumophila, genus novum, species nova, of the family Legionellaceae familia nova. Ann. Intern. Med. 90:656-658. Brown, A., G. M. Garrity, and R. M. Vickers. 1981. Fluoribacter dumoffi (Brenner et al.) comb. nov. and Fluoribacter gormanii (Morris et al.) comb. nov. Int. J. Syst. Bacteriol. 31:111-115. Brown, A., R. M. Vickers, E. M. Elder, M. Lema, and G. M. Garrity. 1982. Plasmid and surface antigen markers of endemic and epidemic Legionella pneumophila strains. J. Clin. Microbiol. 16:230-235. Cato, E. P., D. E. Hash, L. V. Holdeman, and W. E. C. Moore. 1982. Electrophoretic study of Clostridium species. J. Clin. Microbiol. 15:688-702. Cato, E. P., L. V. Holdeman, and W. E. C. Moore. 1982. Clostridium perenne and Clostridium paraperfringens: later subjective synonyms of Clostridium barati. Int. J. Syst. Bacteriol. 32:77-81. Cherry, W. B., G. W. Gorman, L. H. Orrison, C. W. Moss, A. G. Steigerwalt, H. W. Wilkinson, S. E. Johnson, R. M. McKinney, and D. J. Brenner. 1982. Legionella jordanis: a new species of Legionella isolated from water and sewage. J. Clin. Microbiol. 15:290-297. Cordes, L. G., A. M. Wiesenthal, G. W. Gorman, J. P. Phair, H. M. Sommers, A. Brown, W. L. Yu, M. H. Magnussen, R. D. Meyer, J. S. Wolf, K. N. Shands, and D. W. Fraser. 1981. Isolation of Legionella pneumophila from hospital shower heads. Ann. Intern. Med. 94:195-
197. 14. Cordes, L. G., H. W. Wilkinson, G. W. Gorman, B. J. Fikes, and D. W. Fraser. 1979. Atypical legionella-like organisms: fastidious water associated bacteria pathogenic for man. Lancet ii:927-930. 15. Daniels, M. J., and B. M. Meddins. 1973. Polyacrylamide gel electrophoresis of Mycoplasma proteins in sodium dodecyl sulfate. J. Gen. Microbiol. 76:239-242. 16. Fliermans, C. B., W. B. Cherry, L. H. Orrison, S. J. Smith, D. L. Tison, and D. H. Pope. 1981. Ecological distribution of Legionella pneumophila. Appl. Environ. Microbiol. 41:9-16. 17. Fliermans, C. B., W. B. Cherry, L. H. Orrison, and L. Thacker. 1979. Isolation of Legionella pneumophila from nonepidemic-related aquatic habitats. Appl. Environ. Microbiol. 37:1239-1242. 18. Fraser, D. W., T. R. Tsai, W. Orenstein, W. E. Parkin, H. J. Beecham, R. G. Sharrar, J. Harris, G. F. Mallison, S. M. Martin, J. E. McDade, C. C. Shepard, P. S. Brachman, and the Field Investigation Team. 1977. Legionnaires' disease: description of an epidemic of pneumonia. N. Engl. J. Med. 297:1197-1203. 19. Garrity, G. M., A. Brown, and R. M. Vickers. 1980. Tatlockia and Fluoribacter: two new genera of organisms resembling Legionella pneumophila. Int. J. Syst. Bacteriol. 30:609-614. 20. Garrity, G. M., E. M. Elder, B. Davis, R. M. Vickers, and A. Brown. 1982. Serological and genotypic diversity among serogroup 5-reacting environmental Legionella
1140
LEMA AND BROWN
isolates. J. Clin. Microbiol. 15:646-653. 21. Glick, T. H., M. B. Gregg, B. Berman, G. Mallison, W. W. Rhodes, and I. Kassanoff. 1978. Pontiac fever: an epidemic of unknown etiology in a health department I. Clinical and epidemiological aspects. Am. J. Epidemiol. 107:149-160. 22. Gorman, G. W., V. L. Yu, A. Brown, J. A. Hall, W. T. Martin, W. F. Bibb, G. K. Morris, M. H. Magnussen, and D. W. Fraser. 1980. Isolation of Pittsburgh pneumonia agent from nebulizers used in respiratory therapy. Ann. Intern. Med. 93:572-573. 23. Hebert, G. A. 1981. Hippurate hydrolysis by Legionella pneumophila. J. Clin. Microbiol. 13:240-242. 24. Hebert, G. A., A. G. Steigerwalt, and D. J. Brenner. 1980. Legionella micdadei species nova: classification of a third species of Legionella associated with human pneumonia. Curr. Microbiol. 3:225-257. 25. Izard, D., C. Ferragut, F. Gavini, K. Kersters, J. De Ley, and H. Leclerc. 1981. Klebsiella terrigena, a new species from soil and water. Int. J. Syst. Bacteriol. 31:116-127. 26. Jarvis, A. W., and J. M. Wolff. 1979. Grouping of lactic streptococci by gel electrophoresis of soluble cell extracts. Appl. Environ. Microbiol. 37:391-398. 27. Johnson, S. R., and W. 0. Schalla. 1982. Plasmids of serogroup 1 strains of Legionella pneumophila. Curr. Microbiol. 7:143-146. 28. Karr, D. E., W. F. Bibb, and C. W. Moss. 1982. Isoprenoid quinones of the genus Legionella. J. Clin. Microbiol. 15:1044-1048. 29. Kersters, K., and J. De Ley. 1975. Identification and grouping of bacteria by numerical analysis of their electrophoretic protein patterns. J. Gen. Microbiol. 87:333-342. 30. Laemmli, U. K. 1970. Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature (London) 227:680-685. 31. Lowry, 0. H., N. J. Rosebrough, A. L. Farr, and R. J. Randall. 1951. Protein measurement with the Folin phenol reagent. J. Biol. Chem. 193:265-275. 32. Mayberry, W. R. 1981. Dihydroxy and monohydroxy fatty acids in Legionella pneumophila. J. Bacteriol. 147:373-381. 33. McKinney, R. M., R. Porschen, P. H. Edelstein, M. L. Bissett, P. P. Harris, S. P. Bondell, A. G. Steigerwalt, R. E. Weaver, M. E. Ein, D. S. Linguist, R. S. Kops, and D. J. Brenner. 1981. Legionella longbeachae sp. nov., another etiologic agent of human pneumonia. Ann. Intern. Med. 94:739-743. 34. McKinney, R. M., H. W. Wilkinson, H. M. Sommers, B. J. Fikes, K. R. Sasseville, M. M. Yungbluth, and J. S. Wolf. 1980. Legionella pneumophila serogroup six: isolation from cases of legionellosis, identification by immunofluorescence staining, and immunological response to infection. J. Clin. Microbiol. 12:395-401. 35. Mocca, L. F., and C. E. Frasch. 1982. Sodium dodecyl sulfate-polyacrylamide gel typing system for characterization of Neisseria meningitidis isolates. J. Clin. Microbiol. 16:240-244. 36. Moore, W. E. C., D. E. Hash, L. V. Holdeman, and E. P. Cato. 1980. Polyacrylamide slab gel electrophoresis of soluble proteins for studies of bacterial floras. Appl. Environ. Microbiol. 39:900-907. 37. Morris, G. K., A. Steigerwalt, J. C. Feeley, E. S. Wong, W. T. Martin, C. M. Patton, and D. J. Brenner. 1980. Legionella gormanii sp. J. Clin. Microbiol. 12:718-721. 38. Moss, C. W., D. E. Karr, and S. B. Dees. 1981. Cellular fatty acid composition of Legionella longbeachae sp. nov. J. Clin. Microbiol. 14:692-694. 39. Moss, C. W., R. E. Weaver, S. B. Dees, and W. B. Cher-
J. CLIN. MICROBIOL.
40.
41.
42.
43. 44.
45.
46.
47.
48.
49. 50.
51.
52.
53.
ry. 1977. Cellular fatty acid composition of isolates from Legionnaires disease. J. Clin. Microbiol. 6:140-143. Muller, H. E. 1981. Enzymatic profile of Legionella pneumophila. J. Clin. Microbiol. 13:423-426. Myerowitz, R. L., A. W. Pasculle, J. N. Dowling, G. J. Pazin, M. Puerzer, R. B. Yee, C. R. Rinaldo, and T. R. Hakala. 1979. Opportunistic lung infection due to "Pittsburgh pneumonia agent." N. Engl. J. Med. 301:953-958. Nicolet, J., Ph. Parot, and M. Krawinkler. 1980. Polyacrylamide gel electrophoresis of whole-cell proteins of porcine strains of Haemophilus. Int. J. Syst. Bacteriol. 30:69-76. Noble, R. C., and S. C. Schell. 1978. Acrylamide gel electrophoresis of proteins of Neisseria gonorrhoeae as an epidemiological tool. Infect. Immun. 19:178-186. Nolte, F. S., G. E. Hollick, and R. G. Robertson. 1982. Enzymatic activities of Legionella pneumophila and Legionella-like organisms. J. Clin. Microbiol. 15:175-177. Orrison, L. H., W. B. Cherry, C. B. Fliermans, S. B. Dees, L. K. McDougal, and D. J. Dodd. 1981. Characteristics of environmental isolates of Legionella pneumophila. Appl. Environ. Microbiol. 42:109-115. Pasculle, A. W., J. C. Feeley, R. J. Gibson, L. G. Cordes, R. L. Myerowitz, C. M. Patton, G. W. Gorman, L. L. Carmack, J. W. Ezzell, and J. N. Dowling. 1980. Pittsburgh pneumonia agent: direct isolation from human lung tissue. J. Infect. Dis. 141:727-732. Rouatt, J. W., G. W. Skyring, V. Purkayastha, and C. Quadling. 1970. Soil bacteria: numerical analysis of electrophoretic protein patterns developed in acrylamide gels. Can. J. Microbiol. 16:202-205. Seiter, J. A., and J. M. Jay. 1980. Application of polyacrylamide gel electrophoresis to the characterization and identification of Arthrobacter species. Int. J. Syst. Bacteriol. 30:460-465. Swings, J., and J. De Ley. 1977. The biology of Zymomonas. Bacteriol. Rev. 41:1-46. Tobin, J. O., C. L. R. Bartlett, S. A. Waitkins, G. I. Baron, A. D. Macrae, A. G. Taylor, R. J. Fallon, and F. R. N. Lynch. 1981. Legionnaires' disease: further evidence to implicate water storage and distribution systems as sources. Br. Med. J. 282:573. Vickers, R. M., A. Brown, and G. M. Garrity. 1981. Dyecontaining buffered charcoal-yeast extract medium for the differentiation of members of the family Legionellaceae. J. Clin. Microbiol. 13:380-382. Weaver, R. E., and J. C. Feely. 1979. Cultural and biochemical characterization of the Legionnaires' disease bacterium, p. 20-25. In G. L. Jones and G. A. Hebert (ed.), Legionnaires: the disease, the bacterium and methodology. U.S. Department of Health, Education and Welfare, Public Health Service, Centers for Disease Control, Atlanta, Ga. Wilkinson, H. W., and B. J. Fikes. 1980. Slide agglutination test for serogrouping Legionella pneumophila and atypical Legionella-like organisms. J. Clin. Microbiol.
11:99-101. 54. Wilkinson, H. W., B. J. Fikes, and D. D. Cruce. 1979. Indirect immunofluorescent test for serodiagnosis of Legionnaires disease: evidence for serogroup diversity of Legionnaires disease bacterial antigens and for multiple specificity of human antibodies. J. Clin. Microbiol. 9:379383. 55. Wong, K. O., W. 0. Schalla, R. J. Arko, J. C. Bollard, and J. C. Feeley. 1979. Immunochemical, serologic, and immunologic properties of major antigens isolated from the Legionnaires' disease bacterium. Ann. Intern. Med. 90:634-638.
