199 FEMS Microbiology Letters 35 (1986) 199-204

Published by Elsevier

FEM 02465

Rifampin-resistant RNA polymerase in spirochetes **

( Leptospira bijlexa; Leptospira illini; Spirochaeta aurantia; Spirochaeta stenostrepta; Treponema denticola)

S.B. Leschine and E. Canale-Parola *

Department of Microbiology, University of Massachusetts, Amherst, MA 01003, U.S.A.

Received 7 March 1986

Accepted 13 March 1986

1. SUMMARY

Various free-living and host-associated spiro- chetes isolated by methods not involving rifampin were resistant to relatively high concentrations of

this antibiotic. The lowest concentrations of rifampin that were inhibitory for the spirochetes

ranged from 50 to more than 200 pg/ml, depend- ing on the species. The spirochete strains ex- amined were at least lo-fold more resistant to rifampin than Escherichia coli and lOOOO-fold more resistant than Staphylococcus aureus. The results support the conclusion that rifampin resis- tance is a general characteristic of spirochetes. Resistance of Spirochaeta aurantia to rifampin was not the result of detoxification of the antibio- tic in the culture medium. The activity of spirochete DNA-dependent RNA polymerase in vitro was completely resistant to 10 pg of rifampin per ml, a concentration that totally inhibited E. coli RNA polymerase. Higher concentrations de- creased the spirochetal activity. Thus, rifampin resistance may be due to a low affinity of spirochete RNA polymerase for the antibiotic.

2. INTRODUCTION

The antibiotic rifampin has been used as a selective agent for the isolation of spirochetes from the bovine rumen [1,2], the human gingival crevice [3], salt-marsh mud [4], deep-sea water [5], and thermal springs [6]. The concentrations of rifampin (l-10 pg/ml) in the media used in these isolations allowed growth of spirochetes, whereas the growth of many other bacteria was inhibited. These studies showed that spirochetes from a variety of natural environments are resistant to relatively high concentrations of rifampin. How-

ever, the above-mentioned studies did not de- termine whether resistance to rifampin is a prop- erty that is widespread among different genera of the order Spirochaetales, and did not provide in- formation on the physiological characteristics of spirochetes that allow these bacteria to be re- sistant to rifampin.

In the studies described in this article we ex-

amined the growth response of different spiro- chetes to relatively high concentrations of rifam- pin, and we investigated the mechanism of rifam- pin resistance in Spirochaeta aurantia.

* To whom correspondence should be addressed.

** A preliminary report of part of this work has been pre-

sented; S.B. Leschine and E. Canale-Parola, Abstr. Annu.

Meet. Am. Sot. Microbial. 1980, 120, p. 87.

037%1097/86/$03.50 0 1986 Federation of European Microbiological Societies

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3.4. Partial purification of RNA polymerase

RNA polymerases from Sp. aurantia and E. coli were partially purified by the method of Gross et al. [15] with modifications. Cells were grown in GTY broth in flasks incubated at 30°C in air. One

liter of GTY broth was inoculated with 50 ml of a

late logarithmic-phase culture of Sp. aurantia and incubated without shaking. An overnight culture of E. coli (20 ml) was used to inoculate 400 ml of

GTY broth and the culture was incubated on a rotary shaker (Model G76, New Brunswick Scien-

tific Co., New Brunswick, NJ) at 160 rpm. Sp.

aurantia cells were harvested after 22 h of incuba-

tion (final culture density, 2.6 X 10’ cells/ml) and E. coli cells after 3 h of incubation (final culture

density, 6.6 X 10’). The cultures were rapidly

chilled to 4°C in a dry ice-ethanol bath and the cells were harvested by centrifugation for 20 min at 5000 X g at 4°C. Each cell pellet was resus-

pended in 5 ml of lysis buffer (Tris-HCl, pH 7.9, 0.034 M; NaCl, 0.54 M; EDTA, 0.02 M; di- thiothreitol (added just prior to use), 0.01 M). Cells were disrupted with a French pressure cell at 10000 lb/in* (7 X lo6 kg/m*). RNA polymerase in cell extracts was precipitated with recrystallized polyethylene glycol [16] and extracted to obtain the ‘step (IV) extract’ as described by Gross et al. [15]. All extraction steps were carried out at 4°C. The protein content of cell extracts was de- termined using the Bio-Rad Protein Assay (Bio- Rad Laboratories, Richmond, CA).

3.5. Assay of RNA polymerase activity RNA polymerase was assayed in reaction mix-

tures (final volume, 0.2 ml) containing: Tris-HCl (pH 7.9) 25 mM; MgCl,, 10 mM; K,HPO, (pH 7.0) 1 mM; dithiothreitol, 1 mM; EDTA, 1 mM; bovine serum albumin, 100 pg; ATP, 0.2 mM; CTP, 0.2 mM; GTP, 0.2 mM; UTP, 0.025 mM; polydeoxyadenylic-thymidylic acid (poly-(dA-T)), 0.5 units; [14C]UTP (tetrasodium salt), 0.1 PCi; and Sp. aurantia extract protein, 36-45 pg or E.

coli extract protein, 40 pg. The final NaCl con- centration was adjusted to 200 mM in each assay mixture. Some reaction mixtures contained ri- fampin (final concentrations (pgg/ml): 20, 50, 100, and 200). Reactions were initiated by the addition of cell extract and incubated at 30°C. Radioactiv-

ity incorporated into trichloroacetic acid-precipi- table material was determined after 10, 20, and 30 min by pipetting samples (0.05 ml) of each reac- tion mixture onto Whatman 3MM filter paper circles (2.3 cm diameter). Filter papers were placed

in 5% (w/v) trichloroacetic acid and subsequently treated by the method of Bollum [17]. Radioactiv- ity of the dried filter papers was determined in 5

ml of scintillation fluid (Aquasol-2; New England Nuclear, Boston, MA) using a Beckman LS-100

Liquid Scintillation Counter. Poly(dA-T)-depen- dent RNA polymerase activity was determined by subtracting the radioactivity in control mixtures

lacking poly(dA-T) from radioactivity in complete

reaction mixtures.

3.7. Chemicals

All chemicals used were reagent grade. [U- 14C]UTP and [i4C]uracil were purchased from

New England Nuclear. Rifampin (rifampicin), bovine serum albumin (fraction V), ATP, CTP, GTP, UTP, and poly(dA-T) were from Sigma Chemical Co., St. Louis, MO.

4. RESULTS AND DISCUSSION

4.1. Natural resistance of spirochetes to rifampin

6 strains of Leptospira, Spirochaeta, and Treponema isolated by methods not involving rifampin were resistant to relatively high con- centrations of rifampin as compared to other bacteria tested (Table 1). The levels of rifampin sensitivity that we observed in species other than spirochetes were similar to those reported previ- ously [18]. Rifampin sensitivity in spirochetes varied considerably but even the most sensitive strains were approximately lo-fold more resistant than E. coli and lOOOO-fold more than S. aureus

(Table 1). L. illini, an obligately aerobic species and Sp. stenostrepta, an obligately anaerobic species, were the most resistant spirochetes tested. These results support the notion that resistance to relatively high concentrations of rifampin is a general characteristic of spirochetes. Furthermore, these results provide an explanation for the ef- fectiveness of rifampin as a selective agent for the isolation of spirochetes from a variety of environ-

202

Table 1

Rifampin sensitivity of spirochetes and other bacteria.

Organism

Leptospira illini 3055

Culture condition MIC

of rifampin

(kwmli a Aerobic >ZOOb

Spirochaeta stenostrepta Zl Anaerobic r2OOb

Leptospira bijlexa Bl6 Aerobic 200

Spirochaeta aurantia Ml Aerobic

or anaerobic 200

Spirochaeta aurantia J4T Anaerobic 200

Spirochaeta aurantia J4T Aerobic 100

Treponema denticola 10 Anaerobic 50

Escherichia coli K12 Aerobic

or anaerobic 5 Staphylococcus aweus Aerobic 0.01 Staphylococcus aureus Anaerobic 0.005

a Minimum Inhibitory Concentration: lowest concentration of

rifampin tested that reduced colony counts by more than 95%.

b Highest concentration tested (200 pg/ml) did not signifi-

cantly reduce viable counts.

ments. Indeed, when media containing 100 pg of rifampin per ml were inoculated with material from the gingival crevise often only spirochete

colonies were observed (data not shown). Some strains of Sp. auruntia were reported to

be sensitive to rifampin when tested using antibio- tic sensitivity discs [S]. However, the antibiotic disc procedure may have measured a reduced rate of growth in the presence of rifampin instead of growth inhibition.

To the best of our knowledge, no other group of eubacteria has been reported to be naturally resistant to high concentrations of rifampin. The growth of certain clostridia is relatively unaffected by rifampin [19]. However, this property is not widely distributed among the clostridia [19]. In- asmuch as the spirochetes comprise a phylogeneti- tally very deep [20,21], and therefore ancient group of eubacteria, apparently their resistance to ri- fampin is a highly conserved trait. Furthermore, evidence for a common mechanism of resistance would support the hypothesis that all existing spirochetes evolved through extensive physiologi- cal differentiation from a common ancestral pro- tospirochete [22].

4.2. Activity of rifampin in the growth medium of

Spirochaeta aurantia In order to determine whether rifampin was

inactivated in the culture medium during growth of Sp. aurantia, the inhibitory activity of rifampin

in spent spirochete growth medium was de-

termined using S. aureus viability as a bioassay.

The data in Table 2 show that rifampin was not inactivated significantly during the growth of Sp.

auruntia. The MIC of rifampin for S. aureus (0.01

pgg/ml) was the same whether the source of rifampin in the test medium was spent spirochete

medium or uninoculated spirochete medium. The growth of S. aureus was not inhibited on growth media prepared with spent spirochete medium that did not contain rifampin. These results indicate that the resistance of Sp. auruntia to rifampin was not due to detoxification of the antibiotic in the

growth medium in a manner analogous to the inactivation of penicillin by P-lactamase in the

growth medium of certain penicillin-resistant strains [23].

4.3. Effect of rifampin on Spirochaeta aurantia RNA polymerase activity

Rifampin inhibits the growth of sensitive

bacteria by binding to RNA polymerase and thus

Table 2

Effect of Sp. aurantia growth on inhibitory activity of rifam- pin ’

Rifampin Source of rifampin concentration

(ng/ml) b Spent Uninoculated

spirochete spirochete

medium

(W inhibition ‘)

medium

(W inhibition ‘)

0.001 10 15 0.002 3 7

0.005 68 14 0.01 > 99 z 99 0.02 > 99 z 99

a Determined by measuring the effect of spent Sp. aurantia rifampin-containing growth medium on viability of S. aureas.

b In agar medium in which S. aareas was grown.

’ Determined relative to viable count of S. aureus on agar

medium that did not contain spirochete spent medium ( = 7.2 x lO’cells/ml).

inhibiting RNA synthesis [24,25]. In contrast, we found that Sp. aurantia DNA-dependent RNA polymerase activity in vitro was unaffected by 10 pg rifampin per ml, a concentration that com-

pletely inhibited E. coli RNA polymerase (Fig. 1). Higher concentrations of rifampin (50 pg/ml or higher) decreased spirochetal RNA polymerase ac-

tivity (Table 3). These results suggest that rifam- pin resistance is due to a relatively low affinity of the spirochete RNA polymerase for the antibiotic.

Thus, unlike the RNA polymerases from most other bacteria, the spirochete enzyme presumably

forms an unstable complex with rifampin. Rifampin apparently interacts with the p subunit of RNA polymerases of bacteria that are inhibited

since the polymerases of rifampin-resistant mutants have been shown to contain an altered j3

2 - A S. aurantia

0 IO 20 Time (min)

0

Fig. 1. Effect of rifampin on RNA polymerase activity. Poly- (dA-T)-dependent RNA polymerase activity present in par- tially purified enzyme preparations of Sp. aurantia (A) and E.

coli (B) determined in the presence (solid circles) and absence (open circles) of rifampin (10 pg/ml, final concentration). Activity was calculated by subtracting the amount of radioac- tivity in reaction mixtures lacking poly(dA-T) from the amount of radioactivity in complete reaction mixtures.

203

subunit [26,27]. Recently, it was found that the fi subunit of Sp. aurantia RNA polymerase is smaller than that of E. cofi [31], an observation which suggests that the low affinity of spirochetal RNA

polymerase for rifampin is due to a fi subunit that

is substantially different from that of other

eubacteria. A naturally occurring rifampin-resistant RNA

polymerase has been identified in another eubac- terium, Clostridium acidi-urici [19]. However, this characteristic is not widespread among the

clostridia [19]. Thus, spirochetes are apparently a unique group of eubacteria not only in terms of their morphology [22] but also with regard to their

resistance to rifampin. Although 24% of Sp. aurantia RNA polymerase

activity in vitro was inhibited by 100 pg rifampin per ml (Table 3), a significant inhibition of colony formation occurred only when the rifampin con- centration in the medium was greater than 100 pgg/ml (Table 3). An analogous result has been obtained with other Gram-negative bacteria. For

example, it has been reported that 50% of the activity of E. coli RNA polymerase was inhillited by 0.02 pg rifampin per ml [28,29]. In contrast, E. coli viability was significantly reduced only when

the concentration of rifampin in the growth medium was greater than 2 pg/ml. The outer

Table 3

Effect of rifampin on RNA polymerase activity and cell viabil- ity of Sp. aurantia

Rifampin RNA polymerase activity a Viable count ’ concn.

pg/ml sp. act. b % Inhibition lO’cells/ml % Inhi-

bition

0 18.7 0 21 0 20 20.0 0 22 0 50 16.8 10 21 0

100 14.2 24 19 10 200 12.2 35 i 0.01 z== 99

a Poly(dA-T)-dependent RNA polymerase activity of Sp. auranria cell extract. RNA polymerase assayed as described in MATERIALS AND METHODS.

b Specific activity expressed as cpm.min-‘.pg-’ cell extract protein.

’ Viable counts determined on plates of GTY agar media in- cubated aerobically and containing the indicated concentrations of rifampin.

204

membrane of this bacterium apparently restricted the entry of rifampin into the cell inasmuch as it has been found that treating cells with EDTA increased the sensitivity of E. coli to rifampin [30]. We found that when Sp. aurantia cells were treated with EDTA, RNA synthesis was inhibited by rifampin to a greater extent than when cells were not treated with EDTA (data not shown). This suggests that the outer sheath of the spirochete restricted the uptake of rifampin in a manner analogous to the outer membrane of other Gram- negative bacteria and may explain why a higher concentration of rifampin is required to inhibit cell growth than to inhibit RNA polymerase activ- ity (Table 3).

ACKNOWLEDGEMENT

This research was supported by Public Health Service grant AI-17737 from the National In- stitutes of Health.

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