JOURNAL OF BACTERIOLOGY, OCt. 1983, p. 450-4540021-9193/83/100450-05$02.00/0Copyright C 1983, American Society for Microbiology

Vol. 156, No. 1

Local Anesthetics Block Transient Expression of InducibleFunctions for Transformation in Streptococcus sanguis

JAWAHAR L. RAINAtDepartment ofBiology, The University of Chicago, Chicago, Illinois 60637

Received 11 March 1983/Accepted 7 August 1983

Procaine and tetracaine reversibly inhibit transformation by preventing thetransient expression of competence-specific, inducible functions, which areusually triggered in response to cellular.stimulation with competence protein.Affinity studies with "4C-labeled procaine showed that the anesthetic bound to cellsurface macromolecules specifically in the initiation phases of competence-specific events and blocked transfer of information imparted by cellular mem-brane receptor(s) upon interaction with competence protein.

In Streptococcus sanguis and Streptococcuspneumoniae, cell surface receptors (6, 19), afterinteraction with the ligand, competence protein(11, 19), impart information that transientlymodifies cellular physiology. This transientphysiological change, called competence, ischaracterized by the uptake, integration, andexpression of exogenous donor DNA by recipi-ent cells (7, 15). Competence results from the denovo synthesis of a set of at least 10 newpolypeptides (9, 17), ranging from 14,000 to51,000 in molecular weight. Two of these poly-peptides, a 16,000-dalton donor, single-strandedDNA binding protein (17) (19,500-dalton in S.pneumoniae [9]) and a 51,000-dalton recombina-tion-specific protein (13), have been implicatedin DNA-mediated gene transfer by transforma-tion. I used procaine and tetracaine to determinethe role of the cell surface receptor-ligand inter-action in triggering cellular competence in S.sanguis. In Escherichia coli, procaine preventsthe processing of precursor forms of some ex-ported proteins (3, 5, 8) but mostly acts byinhibiting the synthesis of specific outer mem-brane proteins (OmpF, OmpC, OmpB) at thetranscriptional level (10).

(These results were first presented at theInternational Conference on Streptococcal Ge-netics sponsored by the American Society forMicrobiology, 9-12 November 1981, Sarasota,Fla.)

Physiologically noncompetent cells, stimulat-ed with exogenous competence protein for 20min, were allowed to undergo the developmentof competence in the absence and presence of14C-labeled procaine (Fig. 1). There seems to bea direct correlation between the binding of the

t Present address: Pfizer Central Research, Groton, CT06340.

drug and the inhibition of transformation (Fig.1A); at 30 mM procaine, transformabiity wasvirtually eliminated, although binding increasedup to 50mM procaine. Fluorographic analysis ofcellular polypeptides in [3H]leucine pulse-la-beled cells before (Fig. 1B, lane a) and after (Fig.1B, lane b) competence protein stimulationshowed the appearance of two polypeptides,E16 and CipA+, 16,000 and 51,000 daltons,respectively, previously implicated in the proc-ess of transformation (13, 17). However, whenprocaine was present during the competenceprotein stimulation period of the cells, theexpression of these polypeptides decreased rath-er specifically and was completely inhibited at aprocaine concentration above 30 mM (Fig. 1B,lanes c through h).

Quantitative data (not shown) on the net rateof incorporation of radioactive precursors intoDNA, RNA, and protein and estimates of cellviability suggested that the physiological state ofcells remained unaffected after treatment of cellswith 30 mM procaine for 20 min at 37°C. Hence,this concentration of procaine was used to studyits effect on transformability of cells duringvarious phases of competence development.

Cells in the precompetence phase showedhigher binding affinity for, and extreme sensitiv-ity to, procaine as reflected in a virtual lack oftransformability during the first 60 min, whencompetence achieved a maximal level in controlcells (Fig. 2A). Procaine binding decreased sig-nificantly over 80 min (Fig. 2A), and procainebound had only a small effect on transforma-bility by 70 min. Postcompetence-phase cellsshowed minimal affinity for binding procaine,and its presence had no effect on the decay ofany residual competence at 80 min (Fig. 2A).Competence-specific, inducible polypeptidesCipA+ and E16 (Fig. 2B, arrowheads) were fully

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NOTES 451

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FIG. 1. Effect of procaine on exogenous competence protein-stimulated development of competence. (A)Recipient cells (2 x 108) were induced with exogenous competence protein in the absence and presence of 5 to 50mM 14C-labeled procaine (0.7 mCi/mmol; Amersham Corp., England) for 20 min at 37°C. Cells were thenwashed, transformed (streptomycin resistance), and the efficiency of transformation determined as previouslydescribed (17). Transformation efficiency of procaine-treated cells is expressed as a fraction of untreated controlcells (@) (100% = 1.1 x 10-2 transformants per CFU). "4C-labeled procaine bound is expressed as counts per

minute bound per milliliter of cells (0). (B) Recipient cells and conditions for competence induction were

identical to that in (A), except cells were stimulated in the absence (lane b) and presence of 5, 10, 20, 30, 40, or 50mM procaine (lanes c through h) washed, pulse-labeled with [3H]leucine (100 ,uCi/ml), and fluorographed as

previously described (17). Cellular polypeptides in uninduced cells (lane a) are shown for comparison.Arrowheads indicate the positions of competence-specific, inducible donor single-stranded DNA binding protein(E16) (17) and recombination-specific protein (CipA+) (13).

expressed within 10 to 20 min of the initiation ofthese events (Fig. 2B, lanes a and b) and contin-ued during the maximal transformability phaseof the cells (Fig. 2B, lanes c and d). Procaineuniquely inhibited the expression of compe-tence-specific, inducible polypeptides, specifi-cally during the precompetence (Fig. 2B, lanesa' and b') and maximal (Fig. 2B, lanes c' and d')competence phases, whereas no effect was ap-parent on their residual synthesis in postcompe-tence-phase cells (Fig. 2B, lanes e, f, e' and f').

Tetracaine, a more potent analog of procaine,inhibited transformation in a way similar to

procaine. Inhibition of transformation with pro-caine and tetracaine was reversible (Table 1) andoccurred without any additional stimulation withcompetence protein. Clearly, competence pro-tein stimulation-dependent information was im-parted independently of anesthetic binding tocells and was expressed, thereafter, upon with-drawal of the drugs. The transformability ofanesthetic-treated cells correlated with the re-

stored synthesis of competence-specific, induc-ible polypeptides, as revealed by fluorographicanalysis of [3H]leucine pulse-labeled cells (datanot shown).

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FIG. 2. Effect of procaine on endogenous competence protein-stimulated development of competence. (A)Recipient cells (2 x 108) sampled at 10-min intervals during growth in competence development medium wereincubated further without (0) and with (0) "C-labeled procaine (30 mM) for 20 min at 37°C. Cells were thenwashed, transformed, and the efficiency of transformation determined as previously described (17). Transforma-tion efficiency (transformants per CFU) of procaine-treated cells is expressed as a fraction of untreated controlcells (100% = 1.8 x 10-2 transformants per CFU). "C-labeled procaine bound is expressed as counts per minutebound per milliliter of cells (A). (B) Recipient cells and experimental conditions were identical to (A), exceptuntreated (lanes a through f) and procaine-treated (a' through f') cells were pulse-labeled with [3Hlleucine andfluorographed as previously described (17). Sampling times are: 30 min (lanes a and a'); 40 min (lanes b and b');50 min (lanes c and c'); 60 min (lanes d and d'); 70 min (lanes e and e'); and 80 min (lanes f and f'). Arrowheadsare as described in Fig. 1. Arrows show competence-specific, inducible polypeptides of undefined functions.

When physiologically noncompetent cellswere stimulated exogenously with competenceprotein in the presence of an increasing concen-tration of methyl-triphenyl-phosphonium bro-mide (MTB) (4), transformability decreased tothe limits of detectability at 3 mM MTB (Fig.3A). This loss in transformability was concomi-tant with the loss in the donor DNA bindingability of cells, as measured by the uptake of[3H]thymidine-labeled donor DNA in the DN-Ase I resistant form (data not shown). TheMTB-induced effect on protein synthesis duringthe development of competence was similar tothat of the anesthetics in that the inhibition wasselective for the expression of competence-spe-cific, inducible polypeptides CipA+ and E16(Fig. 3B). Additionally, other more subtle effects

were evident from the fluorographic analysis ofMTB-treated cells. First, the threshold concen-tration of MTB required for complete inhibitionof CipA+ and E16 polypeptides was greater thanthat of the other, less abundant competence-specific, inducible polypeptides (Fig. 3B, ar-rows). Second, the expression of some normalcellular polypeptides was clearly enhanced, par-ticularly at higher concentrations of MTB (Fig.3B, double arrows). Third, the expression of anew polypeptide (E16p), possibly a precursorform of the DNA binding protein (E16) (14, 16,17), was evident in MTB-treated cells (Fig. 3B,lanes d through g).What are the molecular mechanisms underly-

ing the action of local anesthetics on eucaryoticand procaryotic cells? In eucaryotic cells, the

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FIG. 3. Effect of MTB on exogenous competence protein-stimulated development of competence. (A)Exogenous competence protein stimulation was in the absence and presence of 0.5 to 3.0 mM MTB for 20 min at37°C. Recipient cells and experimental conditions were as described in the legend to Fig. 1. Cells weretransformed and transformation efficiency (transformants per CFU) were determined as previously described(17). (B) Competence protein-stimulated cells in the absence (lane b) and presence of 0.5 mM MTB (lane c), 1.0mM MTB (lane d), 1.5 mM MTB (lane e), 2.0 mM MTB (lane f), 2.5 mM MTB (lane g), and 3.0mM MTB (lane h)were pulse-labeled with [3H]leucine and fluorographed as previously described (17). Polypeptides in uninducedcells (lane a) are shown for comparison. Arrowheads and arrows are as described in Fig. 2. Double arrowsindicate the positions of normal cellular polypeptides whose synthesis was enhanced with MTB.

available data point much more plausibly toproteins rather than lipids as the primary targetsites on the cellular membrane (2). In procaryot-ic cells, local anesthetics interfere with mem-brane events, blocking the processing and trans-location of periplasmic proteins (1, 3, 5, 8, 10,12).The data presented here support the hypothe-

sis that local anesthetics act on S. sanguis bybinding directly to specific target sites on thecellular membrane. The fact that the inhibitory

effect of procaine and tetracaine corresponds totheir binding affinities which, by measure oftheir octanol-water partition coefficients (18),correlate to their hydrophobicity, suggest thatthese sites are amphiphilic in nature. MTB, acationic membrane-permeant agent, mimics theeffect of anesthetics at high concentrations, butat low concentrations it more likely acts byblocking the processing and translocation of the16,000-dalton outer membrane protein, E16. Ineither case, the molecular mechanism of their

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454 NOTES

TABLE 1. Reversibility of anesthetic effect ontransformation

Transformation efficiency (transformants/Incubation CFU) for the following anesthetics presenttime (min) during induction of competence'

Control Procaine Tetracaine

0 4.6 x 10-2 2.1 x 10-4 1.3 x 10-410 3.5 x 10-2 1.3 x 10-3 1.0 X 10-320 9.9 X 10-3 5.2 x 10-3 5.0 x 10-330 5.1 x 10-3 4.7 x 10-3 4.2 x 10-3

a Transformants per CFU. CFU increased fromabout 8 x 107/ml to 1.1 x 108/ml during 30 min ofincubation in control and 30 mM procaine- and 3 mMtetracaine-treated samples. Cells (5 x 108) were in-duced with exogenous competence protein without(control) and with anesthetics as indicated for 20 minat 37°C. Induced cells were washed twice in the coldand incubated for up to 30 min at 37°C in fresh mediumbefore exposure to donor DNA. Efficiency of transfor-mation was determined as previously described (17).Each number represents an average of three repli-cates. Variation between replicates was less than±10O.

action appears to be independent of the size,shape, and chemical nature but to be due ratherto their apolar and polar characteristics. Thespecificity of procaine binding to cells, uniquelyduring the early onset of the development ofcompetence, permits me to suggest the exis-tence of a new regulatory element in the bio-chemical cascade that leads to competence pro-tein-stimulated induction of competence-specific functions, with two novel features: itmediates information transfer imparted by cellu-lar membrane receptors upon interaction withthe ligand (competence protein) by a mechanismpossibly involving some proteolysis, and itstransient synthesis regulates the development ofcompetence for gene transfer by transformation.

I am grateful to Frederick Mansma for expert technicalassistance, to John Stratton for art work, and to DorotheaBochain for preparing the manuscript.

This work was supported by Public Health Service grantGM27059 from the National Institutes of Health.

LITERATURE CITED

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2. Franks, N. P., and W. R. Lieb. 1982. Molecular mecha-

nism to general anaesthesia. Nature (London) 300:487-493.

3. Gayda, R. C., G. W. Henderson, and A. Markovitz. 1979.Neuroactive drugs inhibit trypsin and outer membraneprotein processing in Escherichia coli K-12. Proc. Natl.Acad. Sci. U.S.A. 76:2138-2142.

4. Gfrinus, L. L., A. A. Jasaitls, Y. P. Kadzyankas, E. A.Lkberman, V. P. Skulachner, V. P. Topall, L. M. Tsofina,and M. A. Vladhlirova. 1970. Conversion of biomem-brane-produced energy into electrical form. Biochim.Biophys. Acta 216:1-12.

5. Granett, S., and M. J. VIllarejo. 1981. Selective inhibitionof carbohydrate transport by the local anesthetic procainein Escherichia coli. J. Bacteriol. 147:289-296.

6. Horn, D., S. Ploch, and A. Tomasz. 1977. Cell surfacecomponents implicated as attachment sites for the Pneu-mococcal competence activator, p. 11-34. In A. Potoles,R. Lopez, and M. Espinose (ed.), Modem trends inbacterial transformation and transfection. Elsevier/North-Holland Publishing Co., Amsterdam.

7. Lacks, S. 1962. Molecular fate ofDNA in genetic transfor-mation of Pneumococcus. J. Mol. Biol. 5:119-131.

8. Lannskl, C., D. Baty, and J. M. Pages. 1979. Procaine, alocal anaesthetic interacting with cell membrane, inhibitsthe processing of precursor forms of periplasmic proteinsin Escherichia coli. Eur. J. Biochem. 96:49-59.

9. Morrison, D. A., and M. F. Baker. 1979. Competence forgenetic transformation in Pneumococcus depends on thesynthesis of a small set of proteins. Nature (London)282:215-217.

10. Pages, J. M., and C. Lazdunskl. 1982. Transcriptionalregulation of ompF and lamB genetic expression by localanaesthetics. FEMS Lett. 15:153-157.

11. Pakula, R., and W. Walesek. 1963. On the nature ofcompetence of transformable Streptococci. J. Gen. Mi-crobiol. 31:125-133.

12. Pugsley, A. D., D. J. Conrad, C. A. Schnadtman, and T. I.Gregg. 1980. In vivo effects of local anaesthetics on theproduction of major outer membrane proteins by Esche-richia coli. Biochim. Biophys. Acta 599:1-12.

13. Raina, J. L., and F. L. Macrina. 1982. A competencespecific inducible protein promotes in vivo recombinationin Streptococcus sanguis. Mol. Gen. Genet. 185:21-29.

14. Raina, J. L., E. Metzer, and A. W. Ravin. 1979. Fate ofheterospecific transforming DNA bound to Streptococcussanguis. J. Bacteriol. 133:1223-1231.

15. RaIna, J. L., and A. W. Ravin. 1978. Fate of homospecifictransforming DNA bound to Streptococcus sanguis. J.Bacteriol. 133:1212-1223.

16. Radna, J. L., and A. W. Ravin. 1979. Presynaptic donorDNA-protein complexes in transformation of Streptococ-cus sanguis: identification of protein component. Bio-chem. Biophys. Res. Commun. 93:228-234.

17. Raina, J. L., and A. W. Ravln. 1980. Switches in macro-molecular synthesis during induction of competence fortransformation of Streptococcus sanguis. Proc. Natl.Acad. Sci. U.S.A. 77:6062-6066.

18. Seaman, P. 1972. The membrane action of ianaestheticsand tranquilizers. Pharm. Rev. 24:583-1113.

19. Ziegler, R., and A. Tomasz. 1970. Binding of the compe-tence factor to receptors in spheroplast membranes ofPneumococci. Biochem. Biophys. Res. Commun.41:1342-1349.

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