FEMS Microbiology Letters 97 (1992) 95-100O 1992 Federation of European Microbiological Societies 03'78-1097 /92/505.00Published by Elsevier

FEMSLE 05071

Mercuric reductase in environmental Gram-positive bacteriasensitive to mercury

Elena S. Bogdanova ", Sofia Z. Mindlin o, Eva Pakrov5 b, M. Kocur b and Duncan A. Rouch '

" Institute of Molecular Genetics, Russian Academy of Sciences, Moscow, Russia, t' Czechoslot;ak CoLlection ol Microorganisms,Masaryk UniL;ersity, Brno, CzechosloL;akia, and ' School of Biological Sciences, Birminghant Unirersity, Birmingham, UK

Received 1 July 1992

Accepted 3 July 1992

Key words: Mercuric reductase; Mercury resistance; Cryptic mer operor,Environmental bacteria collections

1. SUMMARY

According to existing data, mercury resistanceoperons (mer operons) are in general thought tobe rare in bacteria, other than those from mer-cury-contaminated sites. We have found that a

high proportion of strains in environmental iso-lates of Gram-positive bacteria express mercuricreductase (MerA protein): the majority of thesestrains are apparently sensitive to mercury. Theexpression of MerA was also inducible in allcases. These results imply the presence of pheno-typically cryptic zer resistance operons, with boththe merA (mercuric reductase) and merR (regu-latory) genes still present, but the possible ab-sence of the transport function required to com-plete the resistance mechanism. This indicatesthat mer operons or parts thereof are more widelyspread in nature than is suggested by the fre-quency of mercury-resistant bacteria.

Correspondence to: E.S. Bogdanova, Institute of MolecularGenetics, Russian Academy of Sciences, Kurchatov Sq. 46,

Moscow 123182, Russia.

2. INTRODUCTION

A common mechanism for bacterial resistanceto non-organic mercury is mer operon-encodedenzymatic detoxification. mer operons from dif-ferent sources appear to encode a number ofcommon functions, which together confer resis-tance, viz. a regulatory protein (MerR), a mercurytransport system (MerT) and/or other proteins)and the enzyme mercuric reductase (MerA) [1].In the presence of mercuric ions, the regulatoryprotein promotes expression of the structuralproteins, whereupon mercuric ion is taken intothe cell by the transport system and subsequentlyreduced by MerA to non-toxic metallic mercury,which then volatilizes from the cell. Among bac-teria isolated from different environments mer-cury-resistant strains are rare (0.001 - 1 - 47a) t2- 51.

In studies of environmentally isolated strainsof Gram-positive bacteria from culture collec-tions we have occasionally found the presence ofinducible mercuric reductase in strains that areapparently sensitive to mercury. This suggestedthat mer determinants, or parts of these, aremore widely spread in nature than can be in-

96

ferred from the frequency or mercury-resistantbacteria. To test the validity of this notion a

number of strains of Gram-positive bacteria wereexamined for both the presence of MerA andresistance to mercury.

3. MATERIALS AND METHODS

3.1. Bacteriol strainsThe organisms uscd wcre te n Micrococcus

strains (M. lttteus B110 and B1045, M. t'arians825, 8485 and 81232, M. roseus 81236, Micro-coccus sp. 8490, 81108, 81233 and 81042), twostrains of Rhodococctts ( R. lentifragmentusAcl161 and R. ery-thropolis Ac11,50), two strainsof Arthrobacter (A. globiformis Ac1109 and A.oxydans Acll14) and one Mycobacterium strait(Mb. cyaneum Acll55) from All-Union Collec-tion of Microorganisms (VKM); one strain ofB re t' ib a c t e rium fla L' ttm B-42 (derive d ATCC 1 406 7)

from A1l-Union Collection of Industrial Microor-ganisms (VKIM) and 54 strains of seven Micro-coccus species from the Czechoslovak Collectionof Microorganisms (CCM) (Table 1).

The previously described mercury-resistantstrain of Arthrobacter sp. IMG TC28-1 and itsmercury-sensitive derivative were used as controlst6l.

Stock culturcs were maintained at 4-8'C onLB-agar [6].

3.2. MediaThc following nutrient media werc used: Luria

broth (LB) [6]; AP-broth [6]; PY-broth of thcfollowing composition: Bactopeptone (Difco), 1%,yeast extract (Dif'co), 0.5%, NaCl, 1'lo; PY-brothsotidificd with 1.57o agar Difco (PY-agar t); PY-broth solidificd with l.8c/o agar Ferak (PY-agar2).

3.3. MerA inductiortOvernight cultures of bacteria grown at 30"C

in LB were supplemented with 1/7 vcllumc ofAP-broth, 0.1% glucose and 0.15-0.3 pC/mlHgCl,. After 30 min of incubation,0.35-0.7pC/ml HgCl, was addcd to bacterial suspensions

and after 1-1.5 h of further incubation the cellswere harvested by centrifugation.

3.1. Determination of MerA in cell extractsMerA activity was dctermined in extracts by

spectrophotometry at 340 nm of the mercury-de-pendent oxidation of NADPH [7]. One unit ofMcrA catalysed HgClr-dependent oxidation of1.0 prmol of NADPH for 30 min at 30'C.

Electrophoresis of total bacterial cxtract pro-teins followed by immunoblotting with Typc IIIserum (obtained against the mycobacterial MerA)was carried out as in [8]. In all the experiments,proteins of both induced and not induced cul-tures were analyscd simultaneously.

3.5. Mercury susceptibility, testing3.5.1. The disk diffu.sion method. PY-agar 1

containing I pg/ml HgCl, (25 ml per plate) wasoverlaid with 5 ml of soft PY-agar (0.5o/o agar)containing 0.5 ml of induced culture with ,4onn -2.4 (a I cm wide cuvette). 6-mm diameter filterpaper disks loaded with 10 pg of HgCl, wereplaced on the plates. The growth inhibition zoncswere measured after incubation at 30"C for 20 h.

3.5.2. Determination of minimal inhibition con-centration L,alues (MIC). A. Bacterial suspensionsinduced as in section 3.3. were diluted to 10,,,,:0.15; 50 irl of diluted suspensions were spreadover half of the plate with PY agar 2 supple-mented with HgCl, at concentrations over therange ol 2-50 pg/ml.

B. 5 ir,l of induced bacterial cells with Atoo:0.1 were placed in drops on plates with PY-agar I

supplemented with I-10 pg/ml of HgClr.All the assays in section 3.5. were repeated

3-7 times for each strain, with mean values de-termined.

3.6. Antihiotic st,tt.sitit ity tt'stirtgBacteria to be tested wcrc grown for 36 h on

PY-agar I and individual colonies wcre rcplicatedto the same medium supplcmcntcd with antibi-otics at the final cclncentrations (in pg/ml): peni-cillin, 50; ampicillin, 10; streptomycin, 5 and 20;

chloramphenicol, 5; tetracyclin, 5; kanamycin, 5;

erythromycin, 5 and 50.

Table 1

Mercuric reductase activity, mercury and antibiotic resistance of bacterial strains

Bacterialstrains

Mercuric reductase HgCI, resistance Antibioticresi st anceActivity

(U /mgprote in )

Presenceof MerA(immunoblot)

Inhibitionzone(mm) "

MIC Ab(ps/ml)

MICBh1pg/ml).

tvt. tyt0e

RWHI lSLI66

CCM 2694

CCM 2696

TW226CCM 2693

KHYI2

M. luteu.t

CCM 144

CCM 1048

CCM 169

CCM 622

CCM 559

CCM 410

CCM 33ICCM 2494CCM 1335

M. t'ariansCCM 825

CCM2492

CCM 2431

CCM 552CCM 2490

CCM 547

M. nishinomiyaensisCCM 2670JL79CCM 2672

CCM 2I4OCCM 2671

M. kristinueCCM 2690

JM8

SM237SM332

M. sedentariusCCM 2691CCM 314CCM 2699

S

S

S

S

S

S

S

S

S

S

Pen

l52

NT3

2

2

NT

4

NT4

2

2

3

NT

t616

r'7

16

12

5

4

5

2

NT

8

6

NT3

NT

NTNT

3

4

NTNTNT

3

4

l319"t6

18

1B

5

6

20"

1.09

0.61

0.00

l.lti0.tt-5

0.59 .

0.040. l30.000.000.00

0.230.31

0.520.66 'NT0.020.060.03 '0.15

0.090.15

0.00

0.090.18 '0.020.03 '0.03

0.03 '0.000.000.00

0.800.700.(x)

0.000.00

0.200.21

0.220.25 '0.000.00

5

12'l

l3t720"20"21 '

24

l520

1g "

10

11

12',|

13 "17"/-)

l8

12l-2

l2

2

2

1.,21.-2

5

6

2

12I

2

1-2

2

2

1

PenS

PenPenPen

+

+

+

+

+

+

+

+

+

+

+

+

+

+

S

S

S

S

Pen, Amp

4

2

S

PenPen

S

Pen

S

Str, Erm

Pen, AmpPenPen, Amp

98

Table 1 (continucd)

Bacteria I

strai n s

Mcrcrrric reductase I-lgCl, resistance Antibioticre si st a nce

Activity(U/mgprotein)

Presenccof MerA( immu no-blot)

Inhibitionzone(mm) "

MICAb( p"g/ml)

MICBb( pg/ml)

Rfu t d ot o c c u.s I e n t iJiu gme r tt tts

VKM Acl167

B rc t' i but' I c riurn J lu t urtt

B-42

Arthrobacter sp.IMG TC28-1 ilg-rIMG TCl2l3-l Hg-s

0.790.26 '

0.49

0.270.139 '

l0 NT

NT

+ >102

Abbreviations: Pen, penicillin: Amp, ampicillin; Str, strcptomycin; Erm, erythromycin; NT' not tested.

" Inhibition zone diameter minus diameter of the disk.I' See MerlnreLs nNo MnrsoDS 3.5.2. A and B. respectively.

' Results of 2 3 experiments are shown.d In 50% of experiments bacteria formed a lawn only when double turbid suspension was used (1600:4.8).

' There were two growth inhibition zones around the disks: clear and turbid. The sum total of the two zone diameters is given.

The presence of MerA ancl antibiotic resistance were tested in an additional 20 strains of Micrococcus from CCM. MerA has

been found in immunoblot rn M. tytae RM32,l (S), M. roseus CCM 679 (S), CCM 570 (S), CCM 560 (S), CCM 837 (S), CCM 618 (S),

CCM 1679 (S), M. kristinae CCM 2691 (S), MK322 (Pen). MerA has not been found in M. lylae MK312 (S), M. tarians CCM 884

(S), CCM 2139 (S), M. roseus CCM 839 (S), CCM 691 (S), CCM 1145 (S), M. nLshinomiyaensis CCM 2699 (Pen), PM297 (Pen), M.

sedentarius CCM 2698 (Pen), CCM 3947 (Pen), CCM 4055 (Pen, Str): antibiotic resistance data are shown in parentheses.

NTNT

40

5

2

l00.91

0.00

4. RESULTS AND DISCUSSION

The prevalence of MerA in randomly chosenGram-positive bacteria was first revealed in in-vestigation of Micrococcus, Rhodococcus,Arthrobacter, Mycobocterium and Breuibacteriumstrains from VKM and VKIM (see naargntelsAND METHODs). MerA was observed in six out of16 strains under study, M. t:arians B25 and 8485,M. luteus 81045, M.sp.8490, R. lentifragmentusAC1167 and B. flaL:trm B-42 (data not shown;Table 1). This indicated the presence in thesebacteria of the merA gene. Furthermore, theexpression of MerA was inducible by mercuricion, implying the additional presence of the regu-lator protein gene, merR [1]. To examine furtherthe frequency of mer operons in environmentalGram-positive bacteria, the presence of MerAand mercury resistance was studied in 54 strains

of seven M. species from CCM.

Table 1 shows that MerA can be found in allMicrococcus species studied with high frequencyof occurrence: from 297o for M. nishinomiyaensisto 897o for M. luteus. The values could possibly

be underestimates, if MerA was not detected insome cases due to non-optimal induction condi-tions and the MerA was different enough not tobe detected by the heterologous serum (against

mycobacterial MerA). However, the proportionof undetected MerA-encoding mer operonsshould not be significant as MerA was clearlyseen on immunoblots in all cases in which MerAactivity could be determined in extracts (Fig. 1

and Table 1).

A high frequency of mercury resistance duc tomer operon-encoded enzymatic detoxification has

been described only for microorganisms isolatedfrom mercury-polluted areas 12-4,91 and clinicalspecimens [10-12]. Mercury is unlikely to occurin high concentrations in the habitats of micro-

KDA

m. zlarianss/d. ztgt a4g2 62l- riz a4go

--- Y-l--r

;-"l- +l- +l- +l- +l- + I

Fig. 1. Immunoblot of mercuric reductases of strains de-

scribed in Table 1. Strain designation is reported in the upperpart. ( ) and (+) denote the non-induced and inducedcultures. Std lanes show the molecular masses of mercuricreductases of strains: Arthrobacter sp. IMG TC43-4, My-cobacterium sp. IMG CHM22-8, IMG CHM19-3 and IMGCHM21-1 [6] used as molecular mass standards. Extract pro-

tein quantities in each lane are. M. luteus CCM 559, 40 pg;CCM 331, 30 pg: CCM 1048, 7 pg; CCM 169, I pg; CCM2494, 15 pg; CCM 410. 60 pg; M. Iylae RWHll, 2.5 pe;SL166, 0.5 pg: CCM 2696, 30 pg; CCM 2691, 60 pg; B.

llaaun B-42, 20 pe; M. r'arran-r CCM 2431, 7 pg; CCM 2492,

13 rrg; CCM 825, 6 pg; CCM 552, '10 pg; CCM 2490,40 pg.

cocci, since the normal environments of thesebacteria are soil, water, and human and animalskin [13]. Micrococci are also unlikely to have

been exposed to thc same kind of selective pres-

sures as clinical bacteria. For these, thewidespread occurrence of mercury resistanceseems to be maintained by selection for geneti-cally linked antibiotic resistance genes. Thus, as a

rule these mercury-resistant clinical isolates aresimultaneously resistant to at least one and com-monly a number of antibiotics [10,11,14]. This is

not so for Micrococcas in this study: Table 1

shows the members of M. lylae, M. luteus, M.

uarians and M. roseus to be mainly sensitive toantibiotics: the strains of the M. nishinomiyaensis,M. kristinae and M. sedentorius species, though

99

resistant to penicillin, show no correlation bc-tween the presence of the nrcr operon and antibi-otic resistance.

Thc majority of Mic:roc:occLts, BreL'ibacteriurnand possibly Rhodocr,tcc:tts studied are apparentlyscnsitivc to HgCl, (Table l), in accordance withthe usual criteria [2]. Only M. lylae RWHI I andArthrobactet' sp. IMG TC2tt-I Hg-r, as positivccontrol, could be classcd as HgCl ,-rcsistant. Thcmercury-sensitivc phcnotypc of thcse strains sug-gests the possible absence of a functional trans-port systcm, which is needed in addition to MerAto confer resistance [15]. Consequently, thescbacteria appear to contain phenotypically crypticmer operon derivatives, with thc rcgion that en-codes the transport component possibly beinginactive or deleted to some dcgree. The value forthe frequency of cryptic ffc',' ()perons suggestedby this study could be an underestimate bccausecryptic mer operons that failed to express MerAwould not have been detected.

In contrast with the proposed loss of transportfunction in most examples of cryptic mer operonsseen in this study, in several cases a different oradditional mer operon defect may occur. In M.

lylae CCM 2694, M. luteus CCM 559 and M.uarians CCld 2492 and 2437 approximately nor-mal quantities of MerA protein werc observed(Fig. 1), the corresponding enzymatic activitieswere inducible but were cxtremely low (Table 1).

These low activities were found not to result fromthe presence of MerA inhibitors, tested by exam-ining the effect of the extracts on MerA activityof M. kristinae JM8 (data not shown). A possible

explanation for the data is a defect in the MerAof these strains.

Thc phenotypically cryptic nc,' opcrons of Mi-crococcus, BreL'ibacteritrm and possibly Rhodococ-cus may be analogous to othcr cryptic genes andoperons, for example, the determinants for uti-lization of B-glucosides of E,nterobacteriaceae.Arbutin-spccific phospho-B-glucosidase activityhas been found in many wild-type Escherichia colistrains. but these strains cannot use arbutin bc-cause they do not express the transport and phos-phorylation genes (arbT or bgl operon) nccessaryfor arbutin utilization. -lhe arbT gcne or bgloperon can be activatcd by a variety of genetic

VZ. e uteusuru

sldpls_-_JJL __1e4a_{4g _t2lgg__4/o ,57\ *56- ffit2- .rde -- **a .q

i-+l*+l-+r-+t-+l- +t

m. eqtae 8.ffavumstd RtqHfl sL.t65 2696 26s4 sld *ret

---_-:a &

q d. @

r-+l-+l-+l-f1l-+l

1(X)

events. wlrich then allows growth of E. coli onarbutin [16 l9]. This suggested the possibilitythat thc cryptic r/r(,r' opcrons coulcl be reacl.ivatedby one or two rrutational cvcnts. On thc othcrhand. these cr1'ptic rircr opcrons ntay havc clc-

volvcd t<l a dcgrcc that prccludcs rcactivation bya snlrll nurnbcr o1' nrutations. Tl.rcsc altcrnutivcscoulcl bc tcstccl cxpcr.imcntirlll,. frragmcnts of nt(ropcrons nrav lravc bccn lirund in Stupltylocot:r'us[20] and (iram-ncgativc bactcria [2] 231.

Thc discovcr-y of a high frcqucncy of phcnotyp-ically cryptic nrcl' opcrons shows tlrat diffcrcnttypcs ol /ircr opcrons arc morc widcsprcad innaturc than can bc supposccl l}om the quantity ofmercury-rcsistant bactcria. A corollary to this no-tion is that tlie dctection of nrrr-related DNAseqLrences, for example by Southern blotting orPCR analysis, docs not ncccssarily indicatc thatthe source bacteria are resistant to mercury. Aprevalence of cryptic nrcr determinants may bethe case for at least the G + C-rich branch ofGram-positive bacteria. which is represented bythe Mic:rocr.tccus. Bret:ibacteriLtm and Rhodococ-cus strains examined here. However. this may notbc so for a iow G * C group of Gram-positivebacteria since only one strain (Bc. cereus 684,derived ATCC 10102) out of 26 Bc. cereus strainsfrom VKM studied was found to contain MerAand this wers also mercury-resistant (data notshown). Whethcr cryptic rilc'l opcrons occur at asignificant frequency among Gram-negative bac-teriu is un ()pcn question.

AC]KNOWLEDGEMEN-I'S

Wc thank Dr. V.C. Nikifbrov for disctrssingthe rcsults. and E.l. Molchanova and M.O. Var-nakova firr tcchniciil assistancc. D.A.R. was sup-portcd by a Mcdical Rcscarch Council, UK, GrantG. 902-5236C8 to Nigcl L. Brown.

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