- Email: [email protected]
The surface active properties and anti-bacterial activity of the compounds, trans-[Rh(L)4Cl2]Cl·nH2O
The Surface Active Properties and Antibacterial Activity of the Compounds,
trans-[Rh(L),Cl,]Cl -
nH,O
Thomas R. Jack Scarborough
Cortege. Ontario
ABSTRACT
The antibacterial activity and surfactant activity of the compounds tmns-[Rh(L)qClz] Cl-n&O increase in the order L = pyridine<4-methylpyridine<4ethylpyridine<4+tpropyIpyridine. As surfactants. the compounds are far more effective at reducing the interfacial tension, n-hexadecanelH20. than the surface tension. HpO/air. The most effective and efficient surfactant in this series. trans-[Rh(4+z-propylpyridine)&lp]CI-HZO, can cause the leakage of intracellular manganese ions from the gram-positive bacteria, R&iIur brevis ATCC 9999, at a concentration of 130 ppm but there is no observable effect on the retention of intracellular manganese ions at the minimum concentration required to prevent growth of this organism (-0.6 ppm at 23°C in nutrient broth). At 130 ppm, trans-[Rh(4+t-propyipyridine)qCt2]Cl-H~0 does not cause the loss of intracellular manganese ions from the gram-negative bacteria, EscherichCz coli JS-1. In this case, a concentration of at least 63 ppm of this rhodium compound is required to prevent the growth of this organism in M9TUH medium at 35’C. On the basis of these results, it is suggested that gross membrane disruption effects caused by the surfactants fmns-[Rh(L)4C12]C1-nH20 are not directly responsible for their observed antibacterial action.
INTRODUCTION In 1969, complexes of the type trans-[F&(L)4X2] Y*nH,O (where ‘L‘= a substituted pyridine, X = chloride or bromide, and Y = halide, nitrate, or perchlorate) were reported to be potent antibacterial agents capable of preventing the growth of a range of bacteria at very low concentrations [l] _Selected gram-positive bacteria were found to be far more sensitive to these compounds than selected gram-negative microbes [l-4] . For instance, trans-[Rh(4+z-propylpyridine)4C12] NO3 prevented the growth of Address reprint requests to: Thomas R. Jack, BC Research Council, 3650 Wcsbrook Mall, Vancouver, BC,
Canada, V6S 2L2.
Journal of Inorganic Biochemistry 12,187-199 (1980) 0 Elsevier North Holland, Inc., 1980 52 Vanderbilt Ave., New York, New York 10017
187 0162-0134/80/030187-13302.25
Surface Active Properties and Antibacterial
Activity of the Compounds
189
1H nrru in CDCla/TMS on a Varian T-60 and found to give spectra characteristic of the trans isomer and the presence of waters of crystallization in each case. Detailed work up procedures for the two compounds not specifically reported by Gillard et al. [14] are given below.
Prepared by the general method of Gillard et al. [14]. In this case, the product did not precipitate directly from the reaction mixture. Unreacted RhCIa-nHa0 was filtered off and the filtrate was taken to dryness under vacuum on a rotary evaporator. The product was precipitated from the resulting viscous orange solution by the addition of water plus a few drops of concentrated hydrochloric acid. The yellow solid was isolated and dried over P,Os in vacua overnight. Yield (as trans-[Rh(C,H,N),CI,JCl2H,O) was 37%. The product on recrystallization from hot aqueous ethanol gave a “gelatinous” precipitate which was induced to form a more granular solid by the addition of a few drops of hydrochloric acid. The pale yellow solid was isolated and dried over P205 iu vacua overnight. [Rh(C,H&)4C12]C1-2Ha0 requires: C, 49.90; H, 598. Found: C, 49.91, H, 5.96.
rransdichIorotetra(4n-propylpyridiue)rhodium(III)
chloride-Ha0
Prepared by the general method of Gillard et al. [14]_ The reaction mixture was filtered to remove a few orange prisms formed during the reaction. On concentrating the filtrate under vacuum on a rotary evaporator, creamy yellow, very fme needles precipitated. The needles were collected and oven dried at 60°C. Yield (as trans@&(CsH11N)4C12 ] CI=H,O), 43%. m-p_ 186”-188°C The very fme needles, recrystallized from hot H,O, were dried over P,O, in vacua overnight and crumbled during the drying process. [Rh(C8HllN)4C12 JCI-Ha0 requires: C, 53.98: H, 6.51. Found: C, 53.89; H, 656. Surface and interfacial tension measurements were carried out on a Fisher Autotensiomat that employs the de Nouy ring method [lS] _ The tensiometer was calibrated before and after each measurement using the standard l-g weight provided_ The surface tension of distilled deionized water measured on this instrument was found to be 72.6 f 0.8 dyn/cm at 22°C in good agreement with the literature due [16] _Distilled water deionized by a Millipore Mill&Q Reagent Grade Water System was used throughout these measurements. Interfacial tensions were measured by first standardizing the instrument with the ring immersed in an aqueous solution of the rhodium compound to be tested. Thenhexadecane (Aldrich) was then layered gently onto the aqueous phase and the measurement made immediately. The interfacial tension water/n-hexadecane was measured to be 48.1 dyn/cm by this method. Bacillus brevis ATCC 9999 obtained from the American Type Culture Collection and Escherichia coli W-l, an awotroph incapable of synthesizing histidiue, uracil or thymine, obtained from the Scarborough College collection of Dr. J. Silver were mairrtained on nutrient agar shurts. Culture purity was frequently checked on nutrient agar streak plates throughout the study. In all cases, cell numbers were determined using a Petroff-Hausser counting chamber.
Thomas R. Jack
XIRFACE TENSION (dynes/cm)
200 100 [Rh Compound]
300
’
(PPm)
FIGUREl_ The effect of the compound tmns-[Rh(L)4CI2JCI.nH20 on the surface tension in
aqueous so!ution; L = pyridine (X). L = 4-methylpyridine(01, L =4ethylpyridine (~1, and L = 4+propylpyridine (*I).
Growth studies for E. coli JS-1 were carried out at 37OC in a chemically defmed medium, M-9 salts plus glucose [17] , suppIemented with thymine (40 &ml), L(-)histidine (40 &ml), and uracil (10 pg/ml). Stock solutions of the rhodium compounds were prepared in this medium (M9TUH) and filtered steriIized. A series of tubes (compatible with use in a Bausch and Lomb Spectronic 20) were aseptically prepared having various concentrations of each rhodium compound in the M9TUH medium. Each tube was inoculated with E. coli JS-1 in exponential growth phase at 3S°C to give an initial ceil density of 3.7 X 107 cells/ml_ Growth was then followed as a function of time at 35°C by measuringthe changes in absorbance at A = 660 nm. Jn a similar fashion, a culture of B. brevis ATCC 9999 in exponential growth in nutrient broth (D&o) at 35OC was used to inoculate a series of aseptically prepared tubes containing various concentrations of rhodium compound in nutrient broth at 35OC. The initial cell density in each tube after inoculation was 4.1 X 107 ceRs/ml. In this case the occurrence of growth was checked by inspection after incubating the tubes at 35OCovernight. s4Mn was obtained as carrier-free 54Mn2+ in 0.5 M HCI (New England Nuclear Canada) Although the actual specific activity was somewhat less than the theoretical value (7.7 -X lo3 mCi/mg) due to “burn up” of the desired isotope to r%ln during synthesis 1181, the amount of manganese added as the label is negligible compared to
Surface Active Properties and Antibacterial Activity of the Compounds
191
1
cn
3u
I NTERFACIAL TENSION
iTa_ (dynes/cm)
f
#-
50 [Rh Cornpod!
I!50 (wm)
trans-[Rh(L)*CIZ]CI-nH20 on interfacial tension for n-hexadecane/HzO; L = pyridine (X), L = 4-methylpyridine (O), L = 4ethyIpyridine (A), and L = 4-n-propyIpyridine (*)_ FIGURE
2. The effect of the compound
the concentration of manganese present in the medium at the time of addition_ A 0.05 pCi/mJ solution is 1.2 X 1O-1o M in 54Mn. The M9TUH and nutrient broth media have been analyzed by radio-frequencyinduced argon plasma emission spectroscopy (Barringer Research, Rexdale, Ontario) and found to be C200 and 70 nM in manganese, respectively. In the uptake study exponential growth phase bacterial cells grown at 35°C in the appropriate medium (M9TUH for I?_ coli JS-1 and nutrient broth for B_ bra& ATCC 9999) were chilled to 4°C to stop growth and aliquots were transferred to 25 ml Erienmeyer flasks. These aliquots were then warmed to the temperature desired and 5*Mna+ was added (zero time in the accumulation study). Aliquots (1 ml) of the cell suspension were filtered periodically using a Millipore 3025 sampling manifold with 0.45 lun Sartor&s filters, which had been presoaked for several hours in the medium being used (either M9TUH or nutrient broth). The cells were washed twice with medium (3 ml total) to remove extraneous isotope. Partway through the uptake sequence, toluene or rhodium compound (as required) was added to selected flasks, and sampling continued as before. At the end of the samphng period, the filters were dried in a 60°C oven for 10 min and transferred to numbered scintillation viaIscontaining 10 ml of a standard scintillation cocktail consisting of PPO, POPOP, toluene, and Triton X-100_ After standing overnight to allow the filters to dissolve, the samples were counted on a Beckman J.,S250 liquid scintillation counter. Using the aH + l*C + 32P module, a counting efficiency of 53% was obtained. Detailed procedures for counting this isotope have been discriied elsewhere [19 ] _
Wi
Cl
0 0
1 1
2 2 between0.07 ond0.007
between0.16 and0.016
between2.2 and0.22
between32 and 3,2
B. brevlsa
0,16 2s
0008
1.25
5
B, subrilisb
0.16 0.08
0.32
0,63
I,25
B. anthrachb
Microorganism
63
92
120
>170
* Presentwork,35“C,nutrientbroth(B. breufs)or M9TUH(E, coli). b Reference[1],37’C,nutrientbroth. c Recrystallization anddryingconditionin the preparationof thiscompoundarenot idcnticolto thosewhichgavethe pen&hydrate[14].
NOa
Br Cl
Br Cl
4.n-propylpyridine Br
Br Cl
Br Cl
4-ethylpyridine
5 2
Cl
Cl
Cl
13
Cl
Cl Cl
II
Y
X
4.methylpyridlne
pyzidine
L
rrans-[Rh(L)dXg] Y*nH20
TABLE 1.Concentrations of trans-[Rh(L)4Xz]YenW20(ppm) Required to Prevent BacterialGrowth
80 40
80
10
90
B
6, collb
Surface Active Properties and Antibacterial
Activity of the Compounds
193
CONTROL
/
&
/
25ppn 37ppm
49 PPm 74PPm
*>BPpm
FIGURE 3. The inhibition of growth rate for Escherichfncoli JS-I in M9TUH at 35°C by various concentrations of trum-[Rh(4-methylpyridiie)&l~]Cl-2H20_
RESULTS AND DISCUSSION Surface Active Properties of the Compounds frons-[Rh(L)*Cl~]CI-nH,O From figures 1 and 2 it is evident that these compounds are surfactants of a special type. The effect ofthe seriestrans-[Rh(L),Cla] Cl*nHaO where L = pyridine, 4-methylpyridine, 4ethyIpyridine and 4-n-propylpyridine on surface tension (water/air) or interfacial tension (water/n-hexadecane) increases with the size of the alkyl substituent on the pyridine ligand. This is also the order of lipophilicity previously noted [l4] _ However, the reduction in surface tension observed for the most effective surfactant in the series, trans-[Rh(4+r-propylpyridine)4Cl,] Cl-H,O, is only comparable to that observed for routine culture media most of which have surface tensions from 57 to 63 dyn/cm. [8]. in contrast, the interfacial tension (water/n-hexadecane) is very appreciably reduced by these compounds and in considering the effect of surfactants on membrane functions a reduction in this interfacial tension may well be a more pertinent indication of surfactant activity than surface tension effects. Within a homologous series, there is a tendency for the optimally antibacterial compound to be close in the series to the one which has maximal effect on surface tension [9]. From the surface active data (Figures 1 and 2) and from the previous work of Gillard et al. [l-4] trans-[Rh(4+propyIpyridine)ine)4a,] Cl*HsO is expected to be the most effective antibacterial agent in this series of rhodium compounds.
194
Thomas R. Jack
IXPONENTIAL GROWTH
FIGURE 4. The inhIbitionof growth rate for E. coli JS-1 in M9TUH at 35°C by variousconcentrations of the compoundstmns-[Rh(dR pyridine)&12] Cl+zH,O: R = hydrogen(0). R = methyl (01, R = ethyl (I), and R = n-propyl(3.
Concentrations
Required to Prevent Bacterial Growth
The concentrations of the various rhodium compounds in the series trans-[RH(L)*Cl,] Cl which prevent growth of B_ brevis ATCC 9999 are in good agreement with data for reIated compounds and bacteria previously reported Cl] (Table 1). Within the homologous series reported herein, the effectiveness of a compound can be seen to clearly depend upon the size of the alkyl substituent on the pyridine ligand. The inhibition of the growth rate of E. cull JS-1 was monitored in M9TUH medium at 3S°C for various concentrations of each rhodium compound (the data for transCRh(Pme~y~pyridine)4C121Cl*2H,O only are presented in Figure 3). The data for the four compounds in the series expressed as a percentage of the control growth rate (no rhodium coinpound added) are summarized in fgure 4. The minimum concentrations of the compounds required to prevent microbial growth, obtained from the
Surface Active Properties and Antibacterial
Activity of the Compounds
195
X-intercept of this figure, are presented in Table 1, along with comparable data from the literature [l] . Again the dependence of the antibacterial activity on the size of the pyridine alkyl substituent‘ is particularly clear in the holomogous series presented herein. It had been previously reported that these rhodium compounds were capable of inducing filamentation in E. coli strain B at concentrations much lower than those required to prevent growth of this organism [1] and it had been originally planned to investigate the rates of macromolecular synthesis in the fiiamenting cells to see if these rhodium compounds substantially affect nucleic acid or protein synthesis. Hence the auxotrophic strain E. coZi JS-1 and a chemically defmed medium were chosen for this study. From Table 1, the order of antibacterial activity is seen to be the order of surface activity of these compounds (Figures 1 and ‘2) The most effective compound is trans[Bh(4+propylpyridine)~Cla] Cl-Ha0 which prevents the growth of the gram-positive bacterium, B. brevis ATCC 9999, at 0.07 ppm and that of the gram-negative microbe, E. Coli JS-1, at 63 ppm.
EFFECT
OF RHODIUM COMPOUNDS ON CELL MEMBRANE
PERMEABILITY
For surfactants which act as bacteriocides, a close correlation exists between the minimum antibacterial concentration of a compound and the concentration of that compound which causes an alteration in cell permeability [7,9] _In this study, the impact of rmns-~(4+r-propylpyride)4C1,] Cl-Ha0 has been assessed by monitoring its effect on the uptake and retention of saMna+ by the bacterial cells. In E_ coli and members of the genus Bacihs there exists a transport system specific for the divalent manganese ion that is temperature dependent and susceptible to chemical agents that poison energydependent processes [12,13] _Manganese can also be accumulated by alternate transport systems in these organisms. For example, 8. subtiii3W23 has a citrate inducible citrate-Mga+ or Mna+ system and E. coli B or K-12 can accumulate manganese as a low affinity substrate for the magnesium transport system [12,13]. The accumulated intracellular manganese is largely in a labile form_ The addition of toluene to the cell suspension results in the loss of 8% of the accumulated intracellular a4Mn from E. coli [12,20] and >90% from B. subtih (in exponential growth phase [21]) without any visible cell lysis occurring in either case. In f%ure 5, the uptake of s+ln by early log phase cells of B. brevis ATCC 9999 in nutrient broth at 23OC is evident (control). In contrast cells at 4°C retain only a low level of label absorbed to the cell surface, Mn2+ not being effectively transported into the cells at this temperature. (Silver et al. have reported that the uptake of Mn2+ by cells of B. subtih W23 was -5% inhibited at this temperature 1213 _) The effect of adding toluene or solid trans-[Rh(4+z-propylpyridine)4C12] Cl-H20 to the cell suspension results in the rapid loss of the accumulated 54Mn (Figure 5). In this case the rhodium compound is added in excess of its solubility and a saturated solution results at about 130 ppm. In an analogous uptake study, 54Mn2+ accumulation in the presence of 1 ppm or 0.01 ppm of rmns-CRh(4+r-propylpyridine)4C12]Cl~H20 was found to be indistinguishable from the control in rate and extent. Thus, although this rhodium compound
196
Thomas R Jack
CPM3
UPTAKE OF MANGANESE-54
Xlt13 /
CONTROL
lo 8 6
“NO
\y
//
4 2
I
+TOLUE NE
0 _--_-_-_---_-_--__--
4
8
0
12
---&$JC
16 20 24 Set X 10s2
I
FIGURE 5. The uptake of %fn2+ (0.032 &i/ml) by 2.04 X lo7 cells/ml of &xi&s bra& ATCC 9999 in nutrientbroth at 23°C. A-the additionof toluene(0.2 ml to 6 ml of cell suspension.B-the addition cf rrans-[Rh(~propylpyridine)qC12]CI-H20 (2 mg to 6 ml of cell suspension).100% uptake of isotope would correspondto 70,380 cpm.
is capable of inducing ‘1eakiness”in B. bra@& ATCC 9999 cells, this effect is not apparent at the minimum concentration required for complete suppression of microbial
growth (between 0.007 ppm and 0.07 ppm at 35°C and 4.6 ppm at 23OC). 23OC was chosen for the uptake study in order to minimize microbial growth during the experiment and to facilitate the direct comparison of the data obtained with that from previous work [12,13,20,21] _ Assuming that the nutrient broth medium contains the maximum possiile concentration of manganese (70 n&4), then the data for the control in figure 5 yield an overall accumulationrate of - 12 nmol/gdry weight-min and an ix&race&&r manganeseconcentration of >ioO run/mlcell water (assuming that the cell is 80% by weight water). Comparable values for the manganesetransport system in I?. subfiZ&W23 rate, SO nmol/gdry weight -min and intracellularmanganeseconcentration40 run/mlcell water 121J .
Surface-Active Properties and Antibacterial Activity of the Compounds
197
UPTAKE OF MANGANESE - 5%
+ RI-KIDIUM
I 8
76
24
32
40 set
Xld
’
FIGURE 6- The uptake of WUn2+ (0.032 &i/ml) by 3.4 x 108 cells/ml ofEscherichia cwli JS-1 in MSTUH at 23°C. A-the addition of toluene 0.2 ml to 6 ml of cell suspension. B-the addition of 2 mg of trans-fRh(4-n-propylppyridine)qC12] Cl-H20 to 6 ml of cell suspension.
FIGURE 7. The release and exchange of intracelhdar 54~~3 from Eschmihrh coli xi-i, 5.0 x 109 celfs/ml suspendedin M9TUH at 3?C. A-the addition of 0.2 ml of toluene to 9 ml of cell suspension.B-the addition of 2 mg of trans-[Rh(4_n_propyIpyridine)aci2] Cl-H20 to 9 ml of cell suspension.C-the additionof cold Mn*+ to the cell suspensionto a concentrationof 2 x IL! M_
MANGANESE-% +2Xl@M
2d
4
8
12
16
20
Mn=
24
28
E% set
/
Thomas R. Jack
198
The addition - mspeasion of E the accumulated appear, however,
of excess soIid trans-[Rh(an-propylpyridine)4ClzlCI~H20 to a cell coli JS-I ,in MBTUH at 23% does not result in a sign&ant Ioss of s4Mn2+ from the. celIs in an uptake study shown in figure 6. It does that the accumulation of the ion is curtailed about 900 set after the
addition of the rhodium compound. This observation is consistent with either a blockage of the active uptake process by either direct or indirect means or with a decrease in the net intraceU&ar pool of manganese_ To test this latter possibility, cells of E. coli
J!$1 were preloaded with 54Mn2+ by overnight growth in the presence of the isotope. These preloaded cells were then washed and resuspended in fresh M9TUH at 37°C. In this case, a concentration of rhodium compound (-130 ppm) comparable to that which can prevent growth (63 ppm, Table 1) neither induces leakage of the intraceWar manganese as does toIuene nor prevents the exchange of the labelled intra&War manganese with cold manganese added to the medium (Figure 75 CONCLUSIONS Compounds of the type rrans- [F&(4-~-a&ylpyridine)~Clz] Cl-H20 are surfactants which are particularly effective at reducing interfacial tension (n-hexadecane/HzO) with increasing effectiveness and efficiency in the order of increasing size for the alkyl substituent, (alkyl = H < Me < Et < Pr). This order matches the order of lipophilicity and antiiacterial activity. In a gram-positive bacterium,& brevis ATCC 9999, trans-[Rh(4+z-propyipyridine)4CI,]Cf-ff,O, the most effective surfactant in the series investigated, can cause leakage of intracelhdar ions such as manganese but only at concentrations more than two orders of magnitude greater than the minimum concentration
required to prevent
bacterial growth. For a gram-negative organism, E coli JS-1, a concentration similar to that which prevents growth does curtail the active uptake of manganese by the cells but does not cause leakage of the intracellular manganese and does not prevent exchange of the Iabile intracellular pool with added extracellular manganese. It may be concluded that the primary mechanism of the antibacterial action noted
of the type trans-[Rho4X,]Y~~H,O does not arise directIy from their ability to function as cationic surfactants. In further work on this problem, however, researchers should be aware that these compounds are capable of altering the permeability of bacterial cells under appropriate conditions_ for compounds
T%iswork wascarried out with the support of the National Research Council through grant A0835
REFERENCES l_ R. J. Bromfiehl, R. H_ Dainty, R. D. Gillard,and B. T. Heaton, Growth of Microorganismsin the Presenceof TransitionMetal Complexes: AntibacterialActivity of trans-Halogenotetrapy&inerhodium(Ill). Nature 735 (1967). 2. R. D. Gillard, Complexes of Pyridine Bases with Rhodium. P&inum Metab Rev. 14, SO-53 (1970). 3. R. D. GiRard,Femkomplexekes a Bakteriumok.KemMKozZemenyek 48,107-l 18 (1977). 4. R. D. GiiZad, unpublishedresultsby privatecommunication.
Surface-Active
5.
6. 7. 8. 9. 10.
11.
12. 13.
Properties and Antibacterial
Activity of the Compounds
199
R. A. Howard, E. Sherwood, A. Erck. A. P. Kimball, and J. L. Bear, Hydrophobicity of Several Rhodium(H) Carboxylates Correlated with Their Biological Activity. J. MeC. Chem. 20, 943-946 (1977). B. Rosenberg, Some Biological Effects of Platinum Compounds, Platinum Metals Rev. 15,4251 (1971). B. A_ Newton, The Mechanism of the Bacteriological Action of Surface Active Compounds: A Summary. L AppL Batter. 23,34S-349 <1960)_ A. J. Salle, Fundamental PrincipZes of Bacteriology. 7th ed. McGraw-Hill, Toronto, 1973. R. D_ Hot&kiss, The Nature of the Bacteriocidal Action of Surface Active Agents, Annals NY Acad_ Sci. 479-493 (1949). T. R. Jack and J. E. Zajic, The Immobilization of Whole Cells, in Advances in BiochemicaZ Engineering, T. K. Ghose. A. Fiechter and N. Blakebrough. Eds. Springer-Veriag. New York, 1977, pp_ 125-14s. T. R. Jack and J. E. Zajic, The Enzymatic Conversion of L-Histidine to Urccanic Acid by Whole Cells of Micrococcus luteus Immobilized on Carbodiimide Activated Carboxymethylcellulose. Biotechnology and Bioengineerbzg 19,631-648 (1977). S. Silver and P_ Jasper, Manganese Transport in Microorganisms, in Microorganirms and Miner&. E. D. Weinberg, Ed. Marcel Dekker, New York, 1977, p_ 105. S. Silver. Cations and Anions, in Bacterial Trampor?. B. P. Rosen, Ed. Marcel Dekker, New
York, 1978, Chap. 6. 14.
A. W. Addison, K. Dawson, R. D. Gillard, B. T. Heaton and H. Shaw, Synthesis and Characterization of Rhodium(II1) Complexes Containing Nitrogen Heterocyclic Ligands, J. Chem. Sot. Dalton 1972,589-596. 15. W. J. Moore, Physicrrl chemistry. 3rd ed. Prentice-Hall, Englewood Cliffs, NJ, 1962, p_ 731. 16. HandbookofChemisttyand Physics, 47th ed. Chemical Rubber Co., Cleveland, 1966, p_ F-27_ 17. Experiments in Microb@ Genetjcs, R. C. Clowes and W. Hayes, Eds. Blackwell Scientific Publications, Oxford and Edinburgh, 1968, p_ 187. 18. New England Nuclear, private communication. 19. S. Silver and P. Bhattacharyya, Cations, Antibiotics, and Membranes, in Methods in Enzymology. Academic Press, New York, 1974, Vol. 32, pp_ 881-893. 20. S. Silver and M. L. Kralovic, Manganese Accumulation by Escherichia coZi: Evidence for a Specitic Transport System, Biochem. Biophys Res Commun. 34,640-645 (1969). 21. E. Eisenthal. S. Fisher, Chi-Lui Der. and S. Silver, Manganese Transport in Bccillus s&t&% W23 During Growth and Sporulation. J. BQC 113.1363-1372 (1973). Received June 18, I9 79; revised August 30, 1979.
trans-[Rh(L),Cl,]Cl -
nH,O
Thomas R. Jack Scarborough
Cortege. Ontario
ABSTRACT
The antibacterial activity and surfactant activity of the compounds tmns-[Rh(L)qClz] Cl-n&O increase in the order L = pyridine<4-methylpyridine<4ethylpyridine<4+tpropyIpyridine. As surfactants. the compounds are far more effective at reducing the interfacial tension, n-hexadecanelH20. than the surface tension. HpO/air. The most effective and efficient surfactant in this series. trans-[Rh(4+z-propylpyridine)&lp]CI-HZO, can cause the leakage of intracellular manganese ions from the gram-positive bacteria, R&iIur brevis ATCC 9999, at a concentration of 130 ppm but there is no observable effect on the retention of intracellular manganese ions at the minimum concentration required to prevent growth of this organism (-0.6 ppm at 23°C in nutrient broth). At 130 ppm, trans-[Rh(4+t-propyipyridine)qCt2]Cl-H~0 does not cause the loss of intracellular manganese ions from the gram-negative bacteria, EscherichCz coli JS-1. In this case, a concentration of at least 63 ppm of this rhodium compound is required to prevent the growth of this organism in M9TUH medium at 35’C. On the basis of these results, it is suggested that gross membrane disruption effects caused by the surfactants fmns-[Rh(L)4C12]C1-nH20 are not directly responsible for their observed antibacterial action.
INTRODUCTION In 1969, complexes of the type trans-[F&(L)4X2] Y*nH,O (where ‘L‘= a substituted pyridine, X = chloride or bromide, and Y = halide, nitrate, or perchlorate) were reported to be potent antibacterial agents capable of preventing the growth of a range of bacteria at very low concentrations [l] _Selected gram-positive bacteria were found to be far more sensitive to these compounds than selected gram-negative microbes [l-4] . For instance, trans-[Rh(4+z-propylpyridine)4C12] NO3 prevented the growth of Address reprint requests to: Thomas R. Jack, BC Research Council, 3650 Wcsbrook Mall, Vancouver, BC,
Canada, V6S 2L2.
Journal of Inorganic Biochemistry 12,187-199 (1980) 0 Elsevier North Holland, Inc., 1980 52 Vanderbilt Ave., New York, New York 10017
187 0162-0134/80/030187-13302.25
Surface Active Properties and Antibacterial
Activity of the Compounds
189
1H nrru in CDCla/TMS on a Varian T-60 and found to give spectra characteristic of the trans isomer and the presence of waters of crystallization in each case. Detailed work up procedures for the two compounds not specifically reported by Gillard et al. [14] are given below.
Prepared by the general method of Gillard et al. [14]. In this case, the product did not precipitate directly from the reaction mixture. Unreacted RhCIa-nHa0 was filtered off and the filtrate was taken to dryness under vacuum on a rotary evaporator. The product was precipitated from the resulting viscous orange solution by the addition of water plus a few drops of concentrated hydrochloric acid. The yellow solid was isolated and dried over P,Os in vacua overnight. Yield (as trans-[Rh(C,H,N),CI,JCl2H,O) was 37%. The product on recrystallization from hot aqueous ethanol gave a “gelatinous” precipitate which was induced to form a more granular solid by the addition of a few drops of hydrochloric acid. The pale yellow solid was isolated and dried over P205 iu vacua overnight. [Rh(C,H&)4C12]C1-2Ha0 requires: C, 49.90; H, 598. Found: C, 49.91, H, 5.96.
rransdichIorotetra(4n-propylpyridiue)rhodium(III)
chloride-Ha0
Prepared by the general method of Gillard et al. [14]_ The reaction mixture was filtered to remove a few orange prisms formed during the reaction. On concentrating the filtrate under vacuum on a rotary evaporator, creamy yellow, very fme needles precipitated. The needles were collected and oven dried at 60°C. Yield (as trans@&(CsH11N)4C12 ] CI=H,O), 43%. m-p_ 186”-188°C The very fme needles, recrystallized from hot H,O, were dried over P,O, in vacua overnight and crumbled during the drying process. [Rh(C8HllN)4C12 JCI-Ha0 requires: C, 53.98: H, 6.51. Found: C, 53.89; H, 656. Surface and interfacial tension measurements were carried out on a Fisher Autotensiomat that employs the de Nouy ring method [lS] _ The tensiometer was calibrated before and after each measurement using the standard l-g weight provided_ The surface tension of distilled deionized water measured on this instrument was found to be 72.6 f 0.8 dyn/cm at 22°C in good agreement with the literature due [16] _Distilled water deionized by a Millipore Mill&Q Reagent Grade Water System was used throughout these measurements. Interfacial tensions were measured by first standardizing the instrument with the ring immersed in an aqueous solution of the rhodium compound to be tested. Thenhexadecane (Aldrich) was then layered gently onto the aqueous phase and the measurement made immediately. The interfacial tension water/n-hexadecane was measured to be 48.1 dyn/cm by this method. Bacillus brevis ATCC 9999 obtained from the American Type Culture Collection and Escherichia coli W-l, an awotroph incapable of synthesizing histidiue, uracil or thymine, obtained from the Scarborough College collection of Dr. J. Silver were mairrtained on nutrient agar shurts. Culture purity was frequently checked on nutrient agar streak plates throughout the study. In all cases, cell numbers were determined using a Petroff-Hausser counting chamber.
Thomas R. Jack
XIRFACE TENSION (dynes/cm)
200 100 [Rh Compound]
300
’
(PPm)
FIGUREl_ The effect of the compound tmns-[Rh(L)4CI2JCI.nH20 on the surface tension in
aqueous so!ution; L = pyridine (X). L = 4-methylpyridine(01, L =4ethylpyridine (~1, and L = 4+propylpyridine (*I).
Growth studies for E. coli JS-1 were carried out at 37OC in a chemically defmed medium, M-9 salts plus glucose [17] , suppIemented with thymine (40 &ml), L(-)histidine (40 &ml), and uracil (10 pg/ml). Stock solutions of the rhodium compounds were prepared in this medium (M9TUH) and filtered steriIized. A series of tubes (compatible with use in a Bausch and Lomb Spectronic 20) were aseptically prepared having various concentrations of each rhodium compound in the M9TUH medium. Each tube was inoculated with E. coli JS-1 in exponential growth phase at 3S°C to give an initial ceil density of 3.7 X 107 cells/ml_ Growth was then followed as a function of time at 35°C by measuringthe changes in absorbance at A = 660 nm. Jn a similar fashion, a culture of B. brevis ATCC 9999 in exponential growth in nutrient broth (D&o) at 35OC was used to inoculate a series of aseptically prepared tubes containing various concentrations of rhodium compound in nutrient broth at 35OC. The initial cell density in each tube after inoculation was 4.1 X 107 ceRs/ml. In this case the occurrence of growth was checked by inspection after incubating the tubes at 35OCovernight. s4Mn was obtained as carrier-free 54Mn2+ in 0.5 M HCI (New England Nuclear Canada) Although the actual specific activity was somewhat less than the theoretical value (7.7 -X lo3 mCi/mg) due to “burn up” of the desired isotope to r%ln during synthesis 1181, the amount of manganese added as the label is negligible compared to
Surface Active Properties and Antibacterial Activity of the Compounds
191
1
cn
3u
I NTERFACIAL TENSION
iTa_ (dynes/cm)
f
#-
50 [Rh Cornpod!
I!50 (wm)
trans-[Rh(L)*CIZ]CI-nH20 on interfacial tension for n-hexadecane/HzO; L = pyridine (X), L = 4-methylpyridine (O), L = 4ethyIpyridine (A), and L = 4-n-propyIpyridine (*)_ FIGURE
2. The effect of the compound
the concentration of manganese present in the medium at the time of addition_ A 0.05 pCi/mJ solution is 1.2 X 1O-1o M in 54Mn. The M9TUH and nutrient broth media have been analyzed by radio-frequencyinduced argon plasma emission spectroscopy (Barringer Research, Rexdale, Ontario) and found to be C200 and 70 nM in manganese, respectively. In the uptake study exponential growth phase bacterial cells grown at 35°C in the appropriate medium (M9TUH for I?_ coli JS-1 and nutrient broth for B_ bra& ATCC 9999) were chilled to 4°C to stop growth and aliquots were transferred to 25 ml Erienmeyer flasks. These aliquots were then warmed to the temperature desired and 5*Mna+ was added (zero time in the accumulation study). Aliquots (1 ml) of the cell suspension were filtered periodically using a Millipore 3025 sampling manifold with 0.45 lun Sartor&s filters, which had been presoaked for several hours in the medium being used (either M9TUH or nutrient broth). The cells were washed twice with medium (3 ml total) to remove extraneous isotope. Partway through the uptake sequence, toluene or rhodium compound (as required) was added to selected flasks, and sampling continued as before. At the end of the samphng period, the filters were dried in a 60°C oven for 10 min and transferred to numbered scintillation viaIscontaining 10 ml of a standard scintillation cocktail consisting of PPO, POPOP, toluene, and Triton X-100_ After standing overnight to allow the filters to dissolve, the samples were counted on a Beckman J.,S250 liquid scintillation counter. Using the aH + l*C + 32P module, a counting efficiency of 53% was obtained. Detailed procedures for counting this isotope have been discriied elsewhere [19 ] _
Wi
Cl
0 0
1 1
2 2 between0.07 ond0.007
between0.16 and0.016
between2.2 and0.22
between32 and 3,2
B. brevlsa
0,16 2s
0008
1.25
5
B, subrilisb
0.16 0.08
0.32
0,63
I,25
B. anthrachb
Microorganism
63
92
120
>170
* Presentwork,35“C,nutrientbroth(B. breufs)or M9TUH(E, coli). b Reference[1],37’C,nutrientbroth. c Recrystallization anddryingconditionin the preparationof thiscompoundarenot idcnticolto thosewhichgavethe pen&hydrate[14].
NOa
Br Cl
Br Cl
4.n-propylpyridine Br
Br Cl
Br Cl
4-ethylpyridine
5 2
Cl
Cl
Cl
13
Cl
Cl Cl
II
Y
X
4.methylpyridlne
pyzidine
L
rrans-[Rh(L)dXg] Y*nH20
TABLE 1.Concentrations of trans-[Rh(L)4Xz]YenW20(ppm) Required to Prevent BacterialGrowth
80 40
80
10
90
B
6, collb
Surface Active Properties and Antibacterial
Activity of the Compounds
193
CONTROL
/
&
/
25ppn 37ppm
49 PPm 74PPm
*>BPpm
FIGURE 3. The inhibition of growth rate for Escherichfncoli JS-I in M9TUH at 35°C by various concentrations of trum-[Rh(4-methylpyridiie)&l~]Cl-2H20_
RESULTS AND DISCUSSION Surface Active Properties of the Compounds frons-[Rh(L)*Cl~]CI-nH,O From figures 1 and 2 it is evident that these compounds are surfactants of a special type. The effect ofthe seriestrans-[Rh(L),Cla] Cl*nHaO where L = pyridine, 4-methylpyridine, 4ethyIpyridine and 4-n-propylpyridine on surface tension (water/air) or interfacial tension (water/n-hexadecane) increases with the size of the alkyl substituent on the pyridine ligand. This is also the order of lipophilicity previously noted [l4] _ However, the reduction in surface tension observed for the most effective surfactant in the series, trans-[Rh(4+r-propylpyridine)4Cl,] Cl-H,O, is only comparable to that observed for routine culture media most of which have surface tensions from 57 to 63 dyn/cm. [8]. in contrast, the interfacial tension (water/n-hexadecane) is very appreciably reduced by these compounds and in considering the effect of surfactants on membrane functions a reduction in this interfacial tension may well be a more pertinent indication of surfactant activity than surface tension effects. Within a homologous series, there is a tendency for the optimally antibacterial compound to be close in the series to the one which has maximal effect on surface tension [9]. From the surface active data (Figures 1 and 2) and from the previous work of Gillard et al. [l-4] trans-[Rh(4+propyIpyridine)ine)4a,] Cl*HsO is expected to be the most effective antibacterial agent in this series of rhodium compounds.
194
Thomas R. Jack
IXPONENTIAL GROWTH
FIGURE 4. The inhIbitionof growth rate for E. coli JS-1 in M9TUH at 35°C by variousconcentrations of the compoundstmns-[Rh(dR pyridine)&12] Cl+zH,O: R = hydrogen(0). R = methyl (01, R = ethyl (I), and R = n-propyl(3.
Concentrations
Required to Prevent Bacterial Growth
The concentrations of the various rhodium compounds in the series trans-[RH(L)*Cl,] Cl which prevent growth of B_ brevis ATCC 9999 are in good agreement with data for reIated compounds and bacteria previously reported Cl] (Table 1). Within the homologous series reported herein, the effectiveness of a compound can be seen to clearly depend upon the size of the alkyl substituent on the pyridine ligand. The inhibition of the growth rate of E. cull JS-1 was monitored in M9TUH medium at 3S°C for various concentrations of each rhodium compound (the data for transCRh(Pme~y~pyridine)4C121Cl*2H,O only are presented in Figure 3). The data for the four compounds in the series expressed as a percentage of the control growth rate (no rhodium coinpound added) are summarized in fgure 4. The minimum concentrations of the compounds required to prevent microbial growth, obtained from the
Surface Active Properties and Antibacterial
Activity of the Compounds
195
X-intercept of this figure, are presented in Table 1, along with comparable data from the literature [l] . Again the dependence of the antibacterial activity on the size of the pyridine alkyl substituent‘ is particularly clear in the holomogous series presented herein. It had been previously reported that these rhodium compounds were capable of inducing filamentation in E. coli strain B at concentrations much lower than those required to prevent growth of this organism [1] and it had been originally planned to investigate the rates of macromolecular synthesis in the fiiamenting cells to see if these rhodium compounds substantially affect nucleic acid or protein synthesis. Hence the auxotrophic strain E. coZi JS-1 and a chemically defmed medium were chosen for this study. From Table 1, the order of antibacterial activity is seen to be the order of surface activity of these compounds (Figures 1 and ‘2) The most effective compound is trans[Bh(4+propylpyridine)~Cla] Cl-Ha0 which prevents the growth of the gram-positive bacterium, B. brevis ATCC 9999, at 0.07 ppm and that of the gram-negative microbe, E. Coli JS-1, at 63 ppm.
EFFECT
OF RHODIUM COMPOUNDS ON CELL MEMBRANE
PERMEABILITY
For surfactants which act as bacteriocides, a close correlation exists between the minimum antibacterial concentration of a compound and the concentration of that compound which causes an alteration in cell permeability [7,9] _In this study, the impact of rmns-~(4+r-propylpyride)4C1,] Cl-Ha0 has been assessed by monitoring its effect on the uptake and retention of saMna+ by the bacterial cells. In E_ coli and members of the genus Bacihs there exists a transport system specific for the divalent manganese ion that is temperature dependent and susceptible to chemical agents that poison energydependent processes [12,13] _Manganese can also be accumulated by alternate transport systems in these organisms. For example, 8. subtiii3W23 has a citrate inducible citrate-Mga+ or Mna+ system and E. coli B or K-12 can accumulate manganese as a low affinity substrate for the magnesium transport system [12,13]. The accumulated intracellular manganese is largely in a labile form_ The addition of toluene to the cell suspension results in the loss of 8% of the accumulated intracellular a4Mn from E. coli [12,20] and >90% from B. subtih (in exponential growth phase [21]) without any visible cell lysis occurring in either case. In f%ure 5, the uptake of s+ln by early log phase cells of B. brevis ATCC 9999 in nutrient broth at 23OC is evident (control). In contrast cells at 4°C retain only a low level of label absorbed to the cell surface, Mn2+ not being effectively transported into the cells at this temperature. (Silver et al. have reported that the uptake of Mn2+ by cells of B. subtih W23 was -5% inhibited at this temperature 1213 _) The effect of adding toluene or solid trans-[Rh(4+z-propylpyridine)4C12] Cl-H20 to the cell suspension results in the rapid loss of the accumulated 54Mn (Figure 5). In this case the rhodium compound is added in excess of its solubility and a saturated solution results at about 130 ppm. In an analogous uptake study, 54Mn2+ accumulation in the presence of 1 ppm or 0.01 ppm of rmns-CRh(4+r-propylpyridine)4C12]Cl~H20 was found to be indistinguishable from the control in rate and extent. Thus, although this rhodium compound
196
Thomas R Jack
CPM3
UPTAKE OF MANGANESE-54
Xlt13 /
CONTROL
lo 8 6
“NO
\y
//
4 2
I
+TOLUE NE
0 _--_-_-_---_-_--__--
4
8
0
12
---&$JC
16 20 24 Set X 10s2
I
FIGURE 5. The uptake of %fn2+ (0.032 &i/ml) by 2.04 X lo7 cells/ml of &xi&s bra& ATCC 9999 in nutrientbroth at 23°C. A-the additionof toluene(0.2 ml to 6 ml of cell suspension.B-the addition cf rrans-[Rh(~propylpyridine)qC12]CI-H20 (2 mg to 6 ml of cell suspension).100% uptake of isotope would correspondto 70,380 cpm.
is capable of inducing ‘1eakiness”in B. bra@& ATCC 9999 cells, this effect is not apparent at the minimum concentration required for complete suppression of microbial
growth (between 0.007 ppm and 0.07 ppm at 35°C and 4.6 ppm at 23OC). 23OC was chosen for the uptake study in order to minimize microbial growth during the experiment and to facilitate the direct comparison of the data obtained with that from previous work [12,13,20,21] _ Assuming that the nutrient broth medium contains the maximum possiile concentration of manganese (70 n&4), then the data for the control in figure 5 yield an overall accumulationrate of - 12 nmol/gdry weight-min and an ix&race&&r manganeseconcentration of >ioO run/mlcell water (assuming that the cell is 80% by weight water). Comparable values for the manganesetransport system in I?. subfiZ&W23 rate, SO nmol/gdry weight -min and intracellularmanganeseconcentration40 run/mlcell water 121J .
Surface-Active Properties and Antibacterial Activity of the Compounds
197
UPTAKE OF MANGANESE - 5%
+ RI-KIDIUM
I 8
76
24
32
40 set
Xld
’
FIGURE 6- The uptake of WUn2+ (0.032 &i/ml) by 3.4 x 108 cells/ml ofEscherichia cwli JS-1 in MSTUH at 23°C. A-the addition of toluene 0.2 ml to 6 ml of cell suspension. B-the addition of 2 mg of trans-fRh(4-n-propylppyridine)qC12] Cl-H20 to 6 ml of cell suspension.
FIGURE 7. The release and exchange of intracelhdar 54~~3 from Eschmihrh coli xi-i, 5.0 x 109 celfs/ml suspendedin M9TUH at 3?C. A-the addition of 0.2 ml of toluene to 9 ml of cell suspension.B-the addition of 2 mg of trans-[Rh(4_n_propyIpyridine)aci2] Cl-H20 to 9 ml of cell suspension.C-the additionof cold Mn*+ to the cell suspensionto a concentrationof 2 x IL! M_
MANGANESE-% +2Xl@M
2d
4
8
12
16
20
Mn=
24
28
E% set
/
Thomas R. Jack
198
The addition - mspeasion of E the accumulated appear, however,
of excess soIid trans-[Rh(an-propylpyridine)4ClzlCI~H20 to a cell coli JS-I ,in MBTUH at 23% does not result in a sign&ant Ioss of s4Mn2+ from the. celIs in an uptake study shown in figure 6. It does that the accumulation of the ion is curtailed about 900 set after the
addition of the rhodium compound. This observation is consistent with either a blockage of the active uptake process by either direct or indirect means or with a decrease in the net intraceU&ar pool of manganese_ To test this latter possibility, cells of E. coli
J!$1 were preloaded with 54Mn2+ by overnight growth in the presence of the isotope. These preloaded cells were then washed and resuspended in fresh M9TUH at 37°C. In this case, a concentration of rhodium compound (-130 ppm) comparable to that which can prevent growth (63 ppm, Table 1) neither induces leakage of the intraceWar manganese as does toIuene nor prevents the exchange of the labelled intra&War manganese with cold manganese added to the medium (Figure 75 CONCLUSIONS Compounds of the type rrans- [F&(4-~-a&ylpyridine)~Clz] Cl-H20 are surfactants which are particularly effective at reducing interfacial tension (n-hexadecane/HzO) with increasing effectiveness and efficiency in the order of increasing size for the alkyl substituent, (alkyl = H < Me < Et < Pr). This order matches the order of lipophilicity and antiiacterial activity. In a gram-positive bacterium,& brevis ATCC 9999, trans-[Rh(4+z-propyipyridine)4CI,]Cf-ff,O, the most effective surfactant in the series investigated, can cause leakage of intracelhdar ions such as manganese but only at concentrations more than two orders of magnitude greater than the minimum concentration
required to prevent
bacterial growth. For a gram-negative organism, E coli JS-1, a concentration similar to that which prevents growth does curtail the active uptake of manganese by the cells but does not cause leakage of the intracellular manganese and does not prevent exchange of the Iabile intracellular pool with added extracellular manganese. It may be concluded that the primary mechanism of the antibacterial action noted
of the type trans-[Rho4X,]Y~~H,O does not arise directIy from their ability to function as cationic surfactants. In further work on this problem, however, researchers should be aware that these compounds are capable of altering the permeability of bacterial cells under appropriate conditions_ for compounds
T%iswork wascarried out with the support of the National Research Council through grant A0835
REFERENCES l_ R. J. Bromfiehl, R. H_ Dainty, R. D. Gillard,and B. T. Heaton, Growth of Microorganismsin the Presenceof TransitionMetal Complexes: AntibacterialActivity of trans-Halogenotetrapy&inerhodium(Ill). Nature 735 (1967). 2. R. D. Gillard, Complexes of Pyridine Bases with Rhodium. P&inum Metab Rev. 14, SO-53 (1970). 3. R. D. GiRard,Femkomplexekes a Bakteriumok.KemMKozZemenyek 48,107-l 18 (1977). 4. R. D. GiiZad, unpublishedresultsby privatecommunication.
Surface-Active
5.
6. 7. 8. 9. 10.
11.
12. 13.
Properties and Antibacterial
Activity of the Compounds
199
R. A. Howard, E. Sherwood, A. Erck. A. P. Kimball, and J. L. Bear, Hydrophobicity of Several Rhodium(H) Carboxylates Correlated with Their Biological Activity. J. MeC. Chem. 20, 943-946 (1977). B. Rosenberg, Some Biological Effects of Platinum Compounds, Platinum Metals Rev. 15,4251 (1971). B. A_ Newton, The Mechanism of the Bacteriological Action of Surface Active Compounds: A Summary. L AppL Batter. 23,34S-349 <1960)_ A. J. Salle, Fundamental PrincipZes of Bacteriology. 7th ed. McGraw-Hill, Toronto, 1973. R. D_ Hot&kiss, The Nature of the Bacteriocidal Action of Surface Active Agents, Annals NY Acad_ Sci. 479-493 (1949). T. R. Jack and J. E. Zajic, The Immobilization of Whole Cells, in Advances in BiochemicaZ Engineering, T. K. Ghose. A. Fiechter and N. Blakebrough. Eds. Springer-Veriag. New York, 1977, pp_ 125-14s. T. R. Jack and J. E. Zajic, The Enzymatic Conversion of L-Histidine to Urccanic Acid by Whole Cells of Micrococcus luteus Immobilized on Carbodiimide Activated Carboxymethylcellulose. Biotechnology and Bioengineerbzg 19,631-648 (1977). S. Silver and P_ Jasper, Manganese Transport in Microorganisms, in Microorganirms and Miner&. E. D. Weinberg, Ed. Marcel Dekker, New York, 1977, p_ 105. S. Silver. Cations and Anions, in Bacterial Trampor?. B. P. Rosen, Ed. Marcel Dekker, New
York, 1978, Chap. 6. 14.
A. W. Addison, K. Dawson, R. D. Gillard, B. T. Heaton and H. Shaw, Synthesis and Characterization of Rhodium(II1) Complexes Containing Nitrogen Heterocyclic Ligands, J. Chem. Sot. Dalton 1972,589-596. 15. W. J. Moore, Physicrrl chemistry. 3rd ed. Prentice-Hall, Englewood Cliffs, NJ, 1962, p_ 731. 16. HandbookofChemisttyand Physics, 47th ed. Chemical Rubber Co., Cleveland, 1966, p_ F-27_ 17. Experiments in Microb@ Genetjcs, R. C. Clowes and W. Hayes, Eds. Blackwell Scientific Publications, Oxford and Edinburgh, 1968, p_ 187. 18. New England Nuclear, private communication. 19. S. Silver and P. Bhattacharyya, Cations, Antibiotics, and Membranes, in Methods in Enzymology. Academic Press, New York, 1974, Vol. 32, pp_ 881-893. 20. S. Silver and M. L. Kralovic, Manganese Accumulation by Escherichia coZi: Evidence for a Specitic Transport System, Biochem. Biophys Res Commun. 34,640-645 (1969). 21. E. Eisenthal. S. Fisher, Chi-Lui Der. and S. Silver, Manganese Transport in Bccillus s&t&% W23 During Growth and Sporulation. J. BQC 113.1363-1372 (1973). Received June 18, I9 79; revised August 30, 1979.