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The labelling of microorganisms with [35S]methionine, thioATP or inorganic sulphate for taxonomic purposes
Journal of Microbiological Methods 15 (1992) 31 - 40
31
© i992 Elsevier Science Publishers B.V. All rights reserved 0i67 - 7012/92/$ 5.00 MIMET 00477
The labelling o f microorganisms with [35 S] meth!on!ne, " " thi o A T P nr innreranlc, l]lnhat
fo
t a x o n o m i c purposes V. Furst, I. Smith and J. H o l t o n Department of Microbiology, University College and Middlesex School of Medicine, London, UK (Received 2 April 1991; accepted 18 November 1991)
Summary The aim of this study was to develop a rapid method of typing, widely applicable to bacteria and other microorganisms. A medium was devised in which all organisms tested would multiply. A standard method for labelling these organisms with [35Slmethionine, thioATP or inorganic sulphate was used. The labelled macromolecules present in the supernatent and the pelleted organisms separately, or the total cell lysate were treated with sodium dodecyl sulphate and dithiothreitol, and electrophoresed by one dimensional polyacrylamide gel electrophoresis. The gels were dried, and scanned in an Ambis radioscanner. In general, growth time per experiment was reduced to one tenth or less of what was previously necessary. The data from the radioscanner will be discussed in a subsequent paper. Methionine was taken up by all organisms examined and the washed pellets yielded patterns suitable for taxonomic studies. Not all organisms secreted labelled compounds but, when these were found, the patterns were simpler and equally adequate for taxonomy. The [35S]thioATP and inorganic sulphate were not always taken up and so were of less general use. However, as less complex patterns were seen, their use may be preferred in some instances.
Key words: Electrophoresis; [35S]lnorganic sulfate; [35S]Methionine; Radiolabelled bacterial protein pattern; Taxonomy; [35SlThioATP
Introduction Every cell is characterized genetically by its DNA. This DNA produces a characteristic set of RNAs which, in turn, translate to form an equally characteristic set of proteins. Thus DNA, RNA or protein can be used for taxonomic purposes. These three essential cellular components can be examined in different ways. DNA and RNA can be restricted and electrophoresed Ill and examined directly or be probed
Correspondence to: V. Furst, Department of Microbiology, University College and Middlesex School of Medicine, Riding House Street, London WIP 7DN, UK.
32
after electrophoresis as in human DNA fingerprinting [2]. It is, however, p~oteir. electrophoresis which has been used most frequently for the investigation of microorganisms because it is quicker and simpler. Gels have been examined by Coomassie Blue or silver staining. However, radiolabelling with [35S]methionine is often displacing the original method beca~se it is quicker, more sensitive and labels actively metabolizing cells only [3]. When microorganisms are grown in an appropriate medium frce of methionine but with the addition of [35S]methionine, the newly synthesized proteins are radiolabelled. On electrophoresis, a fingerprint of the separated, labelled proteins is obtained and this is characteristic of the particular cell examined. The incubation with [35S]methionine occurs in micro volumes. Thus the method lends itself to being used for rapid mass-screening of bacteria. Radiolabelling is a much more widely applicable method than Coomassie Blue or silver staining as a range of
TABLE SPECIES
1 AND GENERA
OF MICROORGANISMS
Name
EXAMINED Number o f strains
S o u r c e o f 35S
examined
Met
28
+
2 2
+ +
Streptococcus agaiactiae Enterococcus faecalis Bacillus sp. Corynebacterium jeikeium
1 1
+ +
1
+
1
+
Corynebacterium
1
+
2 3 1 1
+ + + +
10 4
+ +
1
+
sp.
33 1 1
+ + +
HaJnia sp. Pasteurella sp. Proteus mirabilis
1 1
+ +
1
+
Citrobacter sp.
1 1 1 1
+ + + +
9 8 11
+ + +
Staphylococcus ( C N S ) Staphylococcus aureus
Coagulase-negative
Methicillin-sensitive Methicillin-resistant
Mycobacterium Mycobacterium Mycobacterium Mycobacterium Mycobacterium
Candida
D2
vaccae gilvum flavescens duvali tuberculosis
sp.
Escherichia coli
Pseudomonas Shigella sp.
Salmonella
sp.
Klebsiella sp.
Morganella sp. Enterobacter sp. Helicobacter pylori
Campylobacter jejeni Spirochetes + , l a b e l l e d proteins; N D , n o t d o n e .
SO 4
ThioATP
-
+ + +
-
+
+
+
+ +
+ +
+ + + + +
+ + + + +
t +
+ +
+ + + + + + + + + + +
+ + + + + + + + + + +
+ ND
ND ND
33 precursor sources of 35S can be used to investigate subgroups of proteins. Hitherto much of the published work has examined the protein fingerprints of total cell lysates only. It is well known that many bacteria secrete extracellular proteins. Patterns derived from these cxtracellular proteins were likely to be simpler than those from total cell proteins, because there would be fewer p~otein bands, therefore we examined supernatants and pellets separately in man,, instances when labelling with methionine. Our aim was to develop a rapid, general micromethod for subspeciation and taxonomy of ,.,,,.,,..,.,h~"t'~r;~which u,,,,,ln...,u,.,,.,~.,ot~"be ~,r,~,,,,.,,u,,.°""t;"°r'~,.,~., " any microorganism. Materials and Methods
Bacteria The bacteria used were all clinical isolates, identified by standard microbiological techniques and retrieved from stock cultures (Table 1). Media All bacteria were grown initially on either 5% horse blood agar, CLED medium (Oxoid), Lowenstein Jensen medium or in nutrient broth (Oxoid). The constituents of the three media used for labelling are given in Tables 2 and 3. Labelling Pure isolates of each bacterium were taken from agar plates, or Lowenstein Jensen slopes in the case of mycobacteria. Coagulase-negative Staphylococcus (CNS) and Staphylococcus aureus (SA) were first grown in 1 ml of nutrient broth supplement with glycine (1 m g . m l - 1) overnight at 37 °C, then pelleted, washed with sterile saline and resuspended in an aliquot of 50 #l of the required labelling medium containing either l0 uCi of [35S]thioATP (NEN-Dupont), methionine (Amersham) or inorganic sulphate (NEN-Dupont). For all other organisms, a 1-/~l loopful of each was innoculated into the appropriate medium described above. With methionine, an incubation time of 2 h at 37 °C was used for all Gram-positive and -negative organisms. With thioATP and SO 4 the organisms were incubated for 2 h - 3 days in the medium at 37 °C. The mycobacteria were incubated for 4 - 7 days at 32 °C. L ysing o f bacteria Labelling was terminated, by the addition of 50/~l of lysing buffer (Ambis, San Diego). If the whole cells were to be examined, an equal volume of lysing buffer was added. Where pellet and supernatant were examined separately, the sample was spun in a bench centrifuge for i0 min. The supernatant was pipetted off and filtered through Millex-GV4 filters (Millipore). Supernatant volumes were estimated and an equivalent volume of lysing buffer added to each. The pellet was washed twice with 0.5 ml sterile saline and resuspended in 50/tl of saline and 50/~l lysing buffer. Pellets of Gram-positive bacteria were resuspended in 50/~l of lysostaphin (5 U-ml -~) for SA and CNS and lysozyme (5000 U.ml -l) for all the remaining Gram-positive bacterial for 2 h at 37°C, before the addition of lysing buffer. All samples were
34 TABLE 2 CONSTITUANTS OF SIOCK SOLUTIONS Amino acids at rag. 50 ml-I A: R.HCI, 50; H.HCI-H20, 10; I, 26; L, 26; K, 20; F, 16; T, 23; W, 5; Y, 20; V, 23; AI = A+C, 12; A2 = A alone; A3 = A+C, 12+M, 8; These need a small amount of NaOH IM and dissolve overnight. B: P, 10; N, 20; E, 12; A, 10; S, 10; G, 10; D: Bulk salts at rag. 50 ml-1 NaCl, 6800; KCI, 400; CaCl 2 •2H20, 265 (prepared from standard l M soln, BDH); NH4CI, 50; phenol red, 17; NaH2PO4, 158; glucose, 1000; for D1 and D3, add MgCIE.6H20, 150 and for D2 add MgSO4.7H20, 200; E: Trace elements at rag.50 ml -l CuAc 2, 3.3; KI, 1; FeCl 3 20; MnCl 2, 35; NH 4 molybdate, 20; ZnCl2, 33; HaBO 3, 5; F: Vitamins at mg. 50 ml-l biotin, Ca pantothenate, Choline. HCI, folic acid, nicotinamide, nicotinic acid, pyridoxal, pyridoxine, pyridoximine, thyamine. HCI and ascorbic acid all at 10; inositol, 20; riboflavin, l; PABA, 5; G: Hepes buffer at 4.76 g-50 ml-! H: Q, 292; J: Nucleosides all at l0 rag.10 ml -l A, T, G. C, U, deoxy A, C and G. Note: readily hydrolysed at room temperature by dilute acid. K- Ra¢~c
all ~t
1N m , - ~ . q r i ~,-~1-1
A, G, U, X NB. One-letter coding for amino acids, nucleosides and bases.
boiled in a water bath for 2 min and stored at - 2 0 °C until electrophoresis.
Electrophoresis Samples were centrifuged for 5 min at high speed in a bench centrifuge to pellet cell debris. Aliquots (6 #l of each) were loaded into each of 15 wells on the sample applicator. 14C molecular weight marker (NEN-Dupont) were added to the remaining three wells. Electrophoresis was carried out on the Ambis system (4) using the preprepared 12.5070 SDS - polyacrylamide gels maintained at 8 °C, with electrophoresis buffer, pH 8.3 (Ambis). Each isolate was electrophoresed in duplicate. Control of electrophoresis conditions was by computer and the gels were run at 20 W for 20 min, then 60 V~~ For the remaining time which was ~ 2 h. Runs were automatically terminated when the primary ion front reached the end-of-run detector at a mean run length of 12.4 cm. Gels were dried for 1 h in the Ambis vertical drier.
35 TABLE 3 PROPORTIONS OF STOCK SOLUTIONS TO PREPARE MED I A Mixture for ~:'.cubation with
Met
AI A2 A3 B DI D2 D3 E
+
SO 4
ATP
for 100 ml 5 5 5 5 5 5 5 50
+ + +
+ +
+ +
F G H J K H20 NaOH, l M to pH 7.5 and 1 5 - 2 0 ° C = 7.3 and 37 °C H20
+ + + + +
m
500 ul 5 ~I 5 i 5 ~ 70
#1 #1 ~1
#1
to 100 ml
M1 2 3 4 5 6 7 8 M
~_~
+ + + + + +
+ + + + +
ml ml ml mi ml ml ml #1
91011121314M
15M1617
~m~
E J
i ~:¸i
:i;!~i
.~i:il
~
0
..~
,.,
J
Fig. 1. Total cell proteins of Gram-positive and Gram-negative microorganisms (M) molecular weight markers: 1, Morganeila; 2, Pseudomonas sp.; 3, Citrobacter sp.; 4, Salmonella sp.; 5, Corynebacteria sp.; 6 - 7, S. epidermidis; 8, Staphylococcus aureus; 9 - 10, S. aureus, MRSA; l I - 12, Staphylococcus haemolyticus; 13, Streptococcus agalacteae; 14, Enterococcus faecalis; 15, Mycobacterium vaccae; 16, M. flavescens; 17, M. gilvum; labelled with [35S]methionine.
36 M1 2 3 4 5 6 7 8 M 9 1 0 1 1 12 3 4 M
v O
e
Fig. 2. Total cell proteins of Gram-negative organisms labelled with [35S]thioATP (M) molecular weight markers: 1, Klebsieila sp.; 2, Citrobacter sp.; 3, Proteus sp.; 4, Salmonella sp.; 5, Morganella; 6, Pasteurella sp.; 7: Pseudomonas sp.; 8, E. coil, 9, Hafnia sp.; 10, Shigeila sp.; 11, Enterobacter sp.
M 123
~.~,~3 ~ ,
o
4 5 6 78M
9105942
7M
.... , , ~ ....
.i
U
D
m
,
.~
~.
.
. . . .
..
. .
.
0
Fig. 3. Total cell proteins of Gram-positive organisms labelled with [35S]thoiATP (M) n~olecular weight markers: 1,5, Corynebacteria sp.; 2,9 S. aureus; 3,6, S. epidermidis; 4,7, S. aureus MRSA; 8,10, S. haemolyticus.
"1"1 ,J!
M1 2 3 4 5 6 7 8 M 9 1 0 1 1 1 2 3 4 M
i
m
i
~ •
iiii:!!
.....:.i!!
m R
|
|
m
e
l r ~
t ° ~-~
Fig. 4. Total cell proteins of Gram-negative organisms labelled with [35S]inorganic sulphate (M) molecular weight markers: l, Enterobacter sp.; 2, Morganella sp.; 3, Salmonella sp.; 4, Shigella sp.; 5, Hafnia sp.; 6, Pasreureiia sp.; 7, Kiebsieiia sp.; 8, Citrobacter sp.; 9, Proteus sp.; 10, E. coil; l 1, Pseudomonas sp.
Scanning The dried gels were scanned in an Ambis Mk 2 radioanalytic imaging system for periods varying from 3 - 24 h, to obtain a high-resolution computer image of the labelled proteins in the gel. Gels were also autoradiographed. Results All organisms gave patterns in the defined medium after 2 h with [35S]methionine, except for mycobacteria which required 4 - 7 days (Fig. 1). The labelling of Gramnegative organisms with [35S]thioATP was also satisfactory after 2 h except for Salmonella which gave weak patterns (Fig. 2). However, Gram-positive organisms needed 3 days incubation (Fig. 3). The use of [35S]inorganic sulphate gave more variable results. All of the Gramnegative organisms gave satisfactory patterns with the longer incubation of 3 days (Fig. 4), except for Hafnia sp. and Alorsanella sp. which gave very weak patterns. Of the Gram-positive organisms, only Corynebacteria, Mycobacteria, yeasts and Bacil-
38
M1 2 3 4 5 6 7 8
M 9 1911121314 M
151617 M
D
a
-_
a
_
.t W
!.......
|
-
_-
o
I i
I
Fig. 5.
Supernatant patterns from Gram-negative and Gram-positive organisms labelled with [35Slmethionine. Same order as Fig. I.
lus gave any labelled proteins after incubation. These results are summarised in Table 1. Many organisms, in particular, Gram-positive ones secreted sufficient proteins to give useful patterns from the supernate (Fig. 5). Discussion
Three basic enrict.:ed media were designed to provide salts, amino acids, trace elements, vitamins, r~ucleosides, bases, glutamine and buffer. These were identical but for the removal oi" the unlabelled equivalent of the labelled compound to be used. For example, nonradioactive SO 4 in the form of MgSO 4 was replaced by Cl in the form of MgCl 2, when [35S]SO4 was to be used as the label. We found that our enriched medium supported the growth of all the organisms we tried and that [35S]methionine was taken up by all the organisms to produce a set of labelled proteins. Little is known generally about the uptake and use of inorganic sulphate although there is an increasing use of it for labelling specific proteins and in the study of sulphate utilization. An early and thorough study of sulphate uptake in E. coli [5] showed that both cysteine and methionine in the medium inhibited its uptake from 50 to 100070 and for this reason we omitted both amino acids from the appropriate medium. Because sulphate is converted to methionine, we b~,~icved it would be quite possible that protein patterns would be similar to tbos!: ~rof, '" ' !i:'~S]methionine. However, there are several reasons why patterns may ":Je di',::.;erer,'../~with these two radioactive c o m -
39 pounds. For example, sulphate might produce labelled enzyme,; for the sulphate to methionine pathway which would not be found with methionine labelling. Also posttranslational modification would Gccur to form sulphated proteins [6] and polysaccharides. Such posttranslation modification may require longer times fiar labelling to occur and this may explain why 3 days were required for satisfactory patterns to be obtained when using SO 4. Alternative explanations are that sulphate is not taken up at all rapidly or that a sufficiency of nonlabelled sulp,hate is already present within the organism and this dilutes the uptake of labelled sulphate, or the appreciably longer incubation times needed for sulphate might simply be an indication of the far slower turnover and metabolism of SO 4 compared to methionine. The lack of patterns with SO 4 may be explained by our omission of cysteine and methionine, as they may be essential amino acids in these bacteria and the results obtained with E. coli may not be generally applicable. Many of the factors affecting sulphate labelling may apply to thioATP. It is also possible there are less sulphate or phosphate residues per total protein than methionine molecules and therefore more generations would be necessary with ATP and SO 4 than with methionine to increase the concentrations of aSS-labelled phosphoproteins or sulphoproteins. The patterns obtained were not as sharp as with the shorter incubation times which may be due to a lower ratio of SO 4 or PO 4 residues to total protein leading to a relative overloading of the lanes and smearing of the patterns. We found that the most generally useful of the three labels was methionine. However, the value of using the different labels will depend on the information required. If posphoproteins or sulphoproteins are of prime interest then [3zS]thioATP and SG 4 should be of use. In instances where translation is not occurring, i.e. in dormant or extremely slnw-ernwine nr~:ani~m~ n n ~ t t r n n ~ l ~ t l n n ~ l mndlflc'ntlnn ie~adlntr tn [35S]phosphoproteins or [35S]sulphoproteins may be useful for taxonomic purposes.
Secreted proteins Many organisms, in particular Gram-positive ones, secreted proteins labelled with [35S]methionine. Because of the simplicity of the patterns in many instances this method would be ideal for rapid typing of species, especially where computer analysis is not available. As many organisms did not give satisfactory secreted protein patterns, this method would not be useful generally. However, for typing within a species that is known to secrete proteins it could be useful. Where labelling with SO 4 and ATP is observed, secreted protein patterns may be of use, especially as secretion of sulphoproteins and phosphoproteins is now known to be common. The advantage of the [35S]-labelling method over the Coomassie Blue or silver staining method is particularly obvious in the case of mycobacteria. Here, patterns are obtained in 4 - 7 days rather than 7 - 1 0 wk necessary for culturing sufficient organisms to be examined by these chemical dyes.
40
Acknowledgements Thanks are due to the DHSS for financial support.
References 1 Clemens, K.V., Shankland, G.S., Richardson, M. D. and Stevens, D.A. (1991)Epidemiological study by DNA typing of a Candida albicans ombreak in heroin addicts. J. Clin. Microbiol. 29, 205 - 207. 2 Fox, B.C., Mobley, E. L. T. and Wade, J. C. (1989) The use of a DNA probe for epidemiological studies in candidiasis in immunocompromised hosts. J. Infect: Dis. 159, 488-493. 3 Tabaqchali, S., Silman, R. and Holland, D. (1987) Automation in clinical microbiology: a new approach to identifying micro-organisms by automated pattern matching of protein, labelled with 35Smethionine J. Clin. Pathol. 40, 1070- 1087. 4 Smith, I. (1985) The A,,nbis Beta scanning system. (1985) BioEssays 3,225 - 229. 5 Roberts, R.B., Cowie, D.B., Abelson, P.H., Bolton, E.T. and Britten, R.J. (1~,55) Studies of biosynthesis in E. coli, Chapter 18. Kirby, Washington. 6 Green, L.S. and Grossman, A.R. (1988) Changes in sulphate transport characteristics and protein composition of Anacystis nidulans R2 during sulphur deprivation. J. Bact. 170, 583 - 587.
31
© i992 Elsevier Science Publishers B.V. All rights reserved 0i67 - 7012/92/$ 5.00 MIMET 00477
The labelling o f microorganisms with [35 S] meth!on!ne, " " thi o A T P nr innreranlc, l]lnhat
fo
t a x o n o m i c purposes V. Furst, I. Smith and J. H o l t o n Department of Microbiology, University College and Middlesex School of Medicine, London, UK (Received 2 April 1991; accepted 18 November 1991)
Summary The aim of this study was to develop a rapid method of typing, widely applicable to bacteria and other microorganisms. A medium was devised in which all organisms tested would multiply. A standard method for labelling these organisms with [35Slmethionine, thioATP or inorganic sulphate was used. The labelled macromolecules present in the supernatent and the pelleted organisms separately, or the total cell lysate were treated with sodium dodecyl sulphate and dithiothreitol, and electrophoresed by one dimensional polyacrylamide gel electrophoresis. The gels were dried, and scanned in an Ambis radioscanner. In general, growth time per experiment was reduced to one tenth or less of what was previously necessary. The data from the radioscanner will be discussed in a subsequent paper. Methionine was taken up by all organisms examined and the washed pellets yielded patterns suitable for taxonomic studies. Not all organisms secreted labelled compounds but, when these were found, the patterns were simpler and equally adequate for taxonomy. The [35S]thioATP and inorganic sulphate were not always taken up and so were of less general use. However, as less complex patterns were seen, their use may be preferred in some instances.
Key words: Electrophoresis; [35S]lnorganic sulfate; [35S]Methionine; Radiolabelled bacterial protein pattern; Taxonomy; [35SlThioATP
Introduction Every cell is characterized genetically by its DNA. This DNA produces a characteristic set of RNAs which, in turn, translate to form an equally characteristic set of proteins. Thus DNA, RNA or protein can be used for taxonomic purposes. These three essential cellular components can be examined in different ways. DNA and RNA can be restricted and electrophoresed Ill and examined directly or be probed
Correspondence to: V. Furst, Department of Microbiology, University College and Middlesex School of Medicine, Riding House Street, London WIP 7DN, UK.
32
after electrophoresis as in human DNA fingerprinting [2]. It is, however, p~oteir. electrophoresis which has been used most frequently for the investigation of microorganisms because it is quicker and simpler. Gels have been examined by Coomassie Blue or silver staining. However, radiolabelling with [35S]methionine is often displacing the original method beca~se it is quicker, more sensitive and labels actively metabolizing cells only [3]. When microorganisms are grown in an appropriate medium frce of methionine but with the addition of [35S]methionine, the newly synthesized proteins are radiolabelled. On electrophoresis, a fingerprint of the separated, labelled proteins is obtained and this is characteristic of the particular cell examined. The incubation with [35S]methionine occurs in micro volumes. Thus the method lends itself to being used for rapid mass-screening of bacteria. Radiolabelling is a much more widely applicable method than Coomassie Blue or silver staining as a range of
TABLE SPECIES
1 AND GENERA
OF MICROORGANISMS
Name
EXAMINED Number o f strains
S o u r c e o f 35S
examined
Met
28
+
2 2
+ +
Streptococcus agaiactiae Enterococcus faecalis Bacillus sp. Corynebacterium jeikeium
1 1
+ +
1
+
1
+
Corynebacterium
1
+
2 3 1 1
+ + + +
10 4
+ +
1
+
sp.
33 1 1
+ + +
HaJnia sp. Pasteurella sp. Proteus mirabilis
1 1
+ +
1
+
Citrobacter sp.
1 1 1 1
+ + + +
9 8 11
+ + +
Staphylococcus ( C N S ) Staphylococcus aureus
Coagulase-negative
Methicillin-sensitive Methicillin-resistant
Mycobacterium Mycobacterium Mycobacterium Mycobacterium Mycobacterium
Candida
D2
vaccae gilvum flavescens duvali tuberculosis
sp.
Escherichia coli
Pseudomonas Shigella sp.
Salmonella
sp.
Klebsiella sp.
Morganella sp. Enterobacter sp. Helicobacter pylori
Campylobacter jejeni Spirochetes + , l a b e l l e d proteins; N D , n o t d o n e .
SO 4
ThioATP
-
+ + +
-
+
+
+
+ +
+ +
+ + + + +
+ + + + +
t +
+ +
+ + + + + + + + + + +
+ + + + + + + + + + +
+ ND
ND ND
33 precursor sources of 35S can be used to investigate subgroups of proteins. Hitherto much of the published work has examined the protein fingerprints of total cell lysates only. It is well known that many bacteria secrete extracellular proteins. Patterns derived from these cxtracellular proteins were likely to be simpler than those from total cell proteins, because there would be fewer p~otein bands, therefore we examined supernatants and pellets separately in man,, instances when labelling with methionine. Our aim was to develop a rapid, general micromethod for subspeciation and taxonomy of ,.,,,.,,..,.,h~"t'~r;~which u,,,,,ln...,u,.,,.,~.,ot~"be ~,r,~,,,,.,,u,,.°""t;"°r'~,.,~., " any microorganism. Materials and Methods
Bacteria The bacteria used were all clinical isolates, identified by standard microbiological techniques and retrieved from stock cultures (Table 1). Media All bacteria were grown initially on either 5% horse blood agar, CLED medium (Oxoid), Lowenstein Jensen medium or in nutrient broth (Oxoid). The constituents of the three media used for labelling are given in Tables 2 and 3. Labelling Pure isolates of each bacterium were taken from agar plates, or Lowenstein Jensen slopes in the case of mycobacteria. Coagulase-negative Staphylococcus (CNS) and Staphylococcus aureus (SA) were first grown in 1 ml of nutrient broth supplement with glycine (1 m g . m l - 1) overnight at 37 °C, then pelleted, washed with sterile saline and resuspended in an aliquot of 50 #l of the required labelling medium containing either l0 uCi of [35S]thioATP (NEN-Dupont), methionine (Amersham) or inorganic sulphate (NEN-Dupont). For all other organisms, a 1-/~l loopful of each was innoculated into the appropriate medium described above. With methionine, an incubation time of 2 h at 37 °C was used for all Gram-positive and -negative organisms. With thioATP and SO 4 the organisms were incubated for 2 h - 3 days in the medium at 37 °C. The mycobacteria were incubated for 4 - 7 days at 32 °C. L ysing o f bacteria Labelling was terminated, by the addition of 50/~l of lysing buffer (Ambis, San Diego). If the whole cells were to be examined, an equal volume of lysing buffer was added. Where pellet and supernatant were examined separately, the sample was spun in a bench centrifuge for i0 min. The supernatant was pipetted off and filtered through Millex-GV4 filters (Millipore). Supernatant volumes were estimated and an equivalent volume of lysing buffer added to each. The pellet was washed twice with 0.5 ml sterile saline and resuspended in 50/tl of saline and 50/~l lysing buffer. Pellets of Gram-positive bacteria were resuspended in 50/~l of lysostaphin (5 U-ml -~) for SA and CNS and lysozyme (5000 U.ml -l) for all the remaining Gram-positive bacterial for 2 h at 37°C, before the addition of lysing buffer. All samples were
34 TABLE 2 CONSTITUANTS OF SIOCK SOLUTIONS Amino acids at rag. 50 ml-I A: R.HCI, 50; H.HCI-H20, 10; I, 26; L, 26; K, 20; F, 16; T, 23; W, 5; Y, 20; V, 23; AI = A+C, 12; A2 = A alone; A3 = A+C, 12+M, 8; These need a small amount of NaOH IM and dissolve overnight. B: P, 10; N, 20; E, 12; A, 10; S, 10; G, 10; D: Bulk salts at rag. 50 ml-1 NaCl, 6800; KCI, 400; CaCl 2 •2H20, 265 (prepared from standard l M soln, BDH); NH4CI, 50; phenol red, 17; NaH2PO4, 158; glucose, 1000; for D1 and D3, add MgCIE.6H20, 150 and for D2 add MgSO4.7H20, 200; E: Trace elements at rag.50 ml -l CuAc 2, 3.3; KI, 1; FeCl 3 20; MnCl 2, 35; NH 4 molybdate, 20; ZnCl2, 33; HaBO 3, 5; F: Vitamins at mg. 50 ml-l biotin, Ca pantothenate, Choline. HCI, folic acid, nicotinamide, nicotinic acid, pyridoxal, pyridoxine, pyridoximine, thyamine. HCI and ascorbic acid all at 10; inositol, 20; riboflavin, l; PABA, 5; G: Hepes buffer at 4.76 g-50 ml-! H: Q, 292; J: Nucleosides all at l0 rag.10 ml -l A, T, G. C, U, deoxy A, C and G. Note: readily hydrolysed at room temperature by dilute acid. K- Ra¢~c
all ~t
1N m , - ~ . q r i ~,-~1-1
A, G, U, X NB. One-letter coding for amino acids, nucleosides and bases.
boiled in a water bath for 2 min and stored at - 2 0 °C until electrophoresis.
Electrophoresis Samples were centrifuged for 5 min at high speed in a bench centrifuge to pellet cell debris. Aliquots (6 #l of each) were loaded into each of 15 wells on the sample applicator. 14C molecular weight marker (NEN-Dupont) were added to the remaining three wells. Electrophoresis was carried out on the Ambis system (4) using the preprepared 12.5070 SDS - polyacrylamide gels maintained at 8 °C, with electrophoresis buffer, pH 8.3 (Ambis). Each isolate was electrophoresed in duplicate. Control of electrophoresis conditions was by computer and the gels were run at 20 W for 20 min, then 60 V~~ For the remaining time which was ~ 2 h. Runs were automatically terminated when the primary ion front reached the end-of-run detector at a mean run length of 12.4 cm. Gels were dried for 1 h in the Ambis vertical drier.
35 TABLE 3 PROPORTIONS OF STOCK SOLUTIONS TO PREPARE MED I A Mixture for ~:'.cubation with
Met
AI A2 A3 B DI D2 D3 E
+
SO 4
ATP
for 100 ml 5 5 5 5 5 5 5 50
+ + +
+ +
+ +
F G H J K H20 NaOH, l M to pH 7.5 and 1 5 - 2 0 ° C = 7.3 and 37 °C H20
+ + + + +
m
500 ul 5 ~I 5 i 5 ~ 70
#1 #1 ~1
#1
to 100 ml
M1 2 3 4 5 6 7 8 M
~_~
+ + + + + +
+ + + + +
ml ml ml mi ml ml ml #1
91011121314M
15M1617
~m~
E J
i ~:¸i
:i;!~i
.~i:il
~
0
..~
,.,
J
Fig. 1. Total cell proteins of Gram-positive and Gram-negative microorganisms (M) molecular weight markers: 1, Morganeila; 2, Pseudomonas sp.; 3, Citrobacter sp.; 4, Salmonella sp.; 5, Corynebacteria sp.; 6 - 7, S. epidermidis; 8, Staphylococcus aureus; 9 - 10, S. aureus, MRSA; l I - 12, Staphylococcus haemolyticus; 13, Streptococcus agalacteae; 14, Enterococcus faecalis; 15, Mycobacterium vaccae; 16, M. flavescens; 17, M. gilvum; labelled with [35S]methionine.
36 M1 2 3 4 5 6 7 8 M 9 1 0 1 1 12 3 4 M
v O
e
Fig. 2. Total cell proteins of Gram-negative organisms labelled with [35S]thioATP (M) molecular weight markers: 1, Klebsieila sp.; 2, Citrobacter sp.; 3, Proteus sp.; 4, Salmonella sp.; 5, Morganella; 6, Pasteurella sp.; 7: Pseudomonas sp.; 8, E. coil, 9, Hafnia sp.; 10, Shigeila sp.; 11, Enterobacter sp.
M 123
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4 5 6 78M
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Fig. 3. Total cell proteins of Gram-positive organisms labelled with [35S]thoiATP (M) n~olecular weight markers: 1,5, Corynebacteria sp.; 2,9 S. aureus; 3,6, S. epidermidis; 4,7, S. aureus MRSA; 8,10, S. haemolyticus.
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M1 2 3 4 5 6 7 8 M 9 1 0 1 1 1 2 3 4 M
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Fig. 4. Total cell proteins of Gram-negative organisms labelled with [35S]inorganic sulphate (M) molecular weight markers: l, Enterobacter sp.; 2, Morganella sp.; 3, Salmonella sp.; 4, Shigella sp.; 5, Hafnia sp.; 6, Pasreureiia sp.; 7, Kiebsieiia sp.; 8, Citrobacter sp.; 9, Proteus sp.; 10, E. coil; l 1, Pseudomonas sp.
Scanning The dried gels were scanned in an Ambis Mk 2 radioanalytic imaging system for periods varying from 3 - 24 h, to obtain a high-resolution computer image of the labelled proteins in the gel. Gels were also autoradiographed. Results All organisms gave patterns in the defined medium after 2 h with [35S]methionine, except for mycobacteria which required 4 - 7 days (Fig. 1). The labelling of Gramnegative organisms with [35S]thioATP was also satisfactory after 2 h except for Salmonella which gave weak patterns (Fig. 2). However, Gram-positive organisms needed 3 days incubation (Fig. 3). The use of [35S]inorganic sulphate gave more variable results. All of the Gramnegative organisms gave satisfactory patterns with the longer incubation of 3 days (Fig. 4), except for Hafnia sp. and Alorsanella sp. which gave very weak patterns. Of the Gram-positive organisms, only Corynebacteria, Mycobacteria, yeasts and Bacil-
38
M1 2 3 4 5 6 7 8
M 9 1911121314 M
151617 M
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Fig. 5.
Supernatant patterns from Gram-negative and Gram-positive organisms labelled with [35Slmethionine. Same order as Fig. I.
lus gave any labelled proteins after incubation. These results are summarised in Table 1. Many organisms, in particular, Gram-positive ones secreted sufficient proteins to give useful patterns from the supernate (Fig. 5). Discussion
Three basic enrict.:ed media were designed to provide salts, amino acids, trace elements, vitamins, r~ucleosides, bases, glutamine and buffer. These were identical but for the removal oi" the unlabelled equivalent of the labelled compound to be used. For example, nonradioactive SO 4 in the form of MgSO 4 was replaced by Cl in the form of MgCl 2, when [35S]SO4 was to be used as the label. We found that our enriched medium supported the growth of all the organisms we tried and that [35S]methionine was taken up by all the organisms to produce a set of labelled proteins. Little is known generally about the uptake and use of inorganic sulphate although there is an increasing use of it for labelling specific proteins and in the study of sulphate utilization. An early and thorough study of sulphate uptake in E. coli [5] showed that both cysteine and methionine in the medium inhibited its uptake from 50 to 100070 and for this reason we omitted both amino acids from the appropriate medium. Because sulphate is converted to methionine, we b~,~icved it would be quite possible that protein patterns would be similar to tbos!: ~rof, '" ' !i:'~S]methionine. However, there are several reasons why patterns may ":Je di',::.;erer,'../~with these two radioactive c o m -
39 pounds. For example, sulphate might produce labelled enzyme,; for the sulphate to methionine pathway which would not be found with methionine labelling. Also posttranslational modification would Gccur to form sulphated proteins [6] and polysaccharides. Such posttranslation modification may require longer times fiar labelling to occur and this may explain why 3 days were required for satisfactory patterns to be obtained when using SO 4. Alternative explanations are that sulphate is not taken up at all rapidly or that a sufficiency of nonlabelled sulp,hate is already present within the organism and this dilutes the uptake of labelled sulphate, or the appreciably longer incubation times needed for sulphate might simply be an indication of the far slower turnover and metabolism of SO 4 compared to methionine. The lack of patterns with SO 4 may be explained by our omission of cysteine and methionine, as they may be essential amino acids in these bacteria and the results obtained with E. coli may not be generally applicable. Many of the factors affecting sulphate labelling may apply to thioATP. It is also possible there are less sulphate or phosphate residues per total protein than methionine molecules and therefore more generations would be necessary with ATP and SO 4 than with methionine to increase the concentrations of aSS-labelled phosphoproteins or sulphoproteins. The patterns obtained were not as sharp as with the shorter incubation times which may be due to a lower ratio of SO 4 or PO 4 residues to total protein leading to a relative overloading of the lanes and smearing of the patterns. We found that the most generally useful of the three labels was methionine. However, the value of using the different labels will depend on the information required. If posphoproteins or sulphoproteins are of prime interest then [3zS]thioATP and SG 4 should be of use. In instances where translation is not occurring, i.e. in dormant or extremely slnw-ernwine nr~:ani~m~ n n ~ t t r n n ~ l ~ t l n n ~ l mndlflc'ntlnn ie~adlntr tn [35S]phosphoproteins or [35S]sulphoproteins may be useful for taxonomic purposes.
Secreted proteins Many organisms, in particular Gram-positive ones, secreted proteins labelled with [35S]methionine. Because of the simplicity of the patterns in many instances this method would be ideal for rapid typing of species, especially where computer analysis is not available. As many organisms did not give satisfactory secreted protein patterns, this method would not be useful generally. However, for typing within a species that is known to secrete proteins it could be useful. Where labelling with SO 4 and ATP is observed, secreted protein patterns may be of use, especially as secretion of sulphoproteins and phosphoproteins is now known to be common. The advantage of the [35S]-labelling method over the Coomassie Blue or silver staining method is particularly obvious in the case of mycobacteria. Here, patterns are obtained in 4 - 7 days rather than 7 - 1 0 wk necessary for culturing sufficient organisms to be examined by these chemical dyes.
40
Acknowledgements Thanks are due to the DHSS for financial support.
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