JOURNAL OF BacTeRIOLocy, Dec. 1975, p. 1630-1634 Copyright © 1975 American Society for Microbiology Vol. 124, No. 3 Printed in U.S.A. Transfectability of Rough Strains of Salmonella typhimurium H. BURSZTYN,* V. SGARAMELLA,' O. CIFERRI,? anp J. LEDERBERG Department of Genetics, J. P. Kennedy, Jr., Laboratories for Molecular Medicine, Stanford University School of Medicine, Stanford, California 94305 Received for publication 19 June 1975 Cells of rough (but not smooth) strains of Salmonella typhimurium become competent for transfection by phage P22 deoxyribonucleic acid after treatment with 0.1 M CaCl,. The yield of infectious centers is about 10-* per genome equivalent of deoxyribonucleic acid. However, different sorts of rough strains vary in their ability to become competent in a fashion that can be correlated with the level of the genetic block in cell wall lipopolysaccharide synthesis. The most amenable strains are blocked by defects in the addition of galactose units I and II of the lipopolysaccharide by the inability to synthesize uridine 5'-diphosphate- galactose (galE point mutants and gal deletion mutants). Strains blocked only in the addition of galactose I, glucose I, or heptose II have low levels of transfectability, whereas strains with either more complete or more deficient lipopolysaccharide core are not competent for transfection. When normal lipopolysaccharide synthesis is restored either genetically or by furnishing exogenous galactose (galE point mutants that can still use it), the cells are no longer competent for transfection. Mandel and Higa (10), Cohen et al. (3), and Oishi and Cosloy (11) have described the transfectability of Escherichia coli cells treated with CaCl,. We report here the induction of competence to transfection by phage P22 deoxy- ribonucleic acid (DNA) as a result of exposure of Salmonella typhimurium cells to CaCl. Operationally, two classes of S. typhimurium strains can be defined (15): smooth strains able to adsorb P22 phage and rough strains unable to adsorb P22 phage. Wilkinson et al. (14) de- scribed various classes of rough mutants of S. typhimurium, all lacking the O antigenic speci- ficity of their smooth parent as a consequence of alterations in the polysaccharide of the somatic lipopolysaccharide (LPS). After a treatment with 0.1 M CaCl,, certain rough strains of S. typhimurium, but not smooth ones, can be transfected by the DNA extracted from the smooth-specific bacterio- phage P22, a temperature-sensitive C2 mutant (13). An overnight inoculum is diluted 200-fold into L (7) or Penassay (Difco antibiotic medium 3) broth and grown at 37 C with aeration to an optical density at 600 nm of 0.6 per centimeter (ca. 2 x 10° cells/ml). The cells are chilled, recovered by centrifugation at 12,000 x gat 4 C, and suspended to original volume in 0.1 M *Permanent address: Laboratorio di Genetica Biochimica ed Evoluzionistica del C.N.R., Pavia, Italv. ?Permanent address: Instituto di Microbiologia e Fisi- ologia Vegetale, Universita di Pavia, Pavia, Italy. MgCl,. The suspension is spun-down and the cells are resuspended in 0.5 of the original volume of 0.1 M CaCl, and kept at 0 C for 20 min. Finally, the cells are pelleted and resus- pended in Yo of the original volume of 0.1 M CaCl, at OC (‘‘treated” cells). DNA was pre- pared by phenol extraction of CsCl-purified phage particles (13). No obvious heterogeneity was noted by electron microscopy; less than one break per five molecules was detected by sedi- mentation in alkaline sucrose gradients. The transfection was performed by mixing native P22 DNA, diluted in 0.1 ml of SSC (0.15 M NaCl plus 0.015 M sodium citrate), with 0.2 to 0.4 ml of ‘‘treated”’ cells. A preincubation of 5 to 60 min at 0 C followed by a thermal treatment, 2 min at 42 C or 5 min at 37 C (0.3 to0.5 ml ina 10 by 100 nm tube was transferred to 42 or 37 C water bath and left there for 2 or 5 min, respectively), was required to bring about opti- mal induction of competence for transfection. It was found that no significant difference was observed by varying the length of the incuba- tion period at OC or that of the thermal treatment. Because these rough mutants do not adsorb P22 phage, the appearance of plaques requires the presence of smooth cells as indicators. At the end of the thermal treatment, 2 x 10° to5 x 10% stationary phase smooth indicator cells (proC90 [5] or SL1027 [12]) were added to the treatment mixture, 2.5 ml of soft agar was 1630 Vor. 124, 1975 added, and the suspension was plated on tryp- tone agar. The plaques were counted after overnight incubation at 37 C. Omission of the thermal treatment caused a 30-fold decrease in the number of plaques. If the thermal treatment was at 22C, the plaques produced were less than half of those obtained at 42 C. The DNA concentration dependence of the transfection process is reported in Fig. 1. Plaques are evidently increasing at DNA con- centrations higher than 0.1 ug/plate. A plateau appears to be reached at a concentration of approximately 1 yug/plate, but its level varied from one batch of cells to another. If the CaCl, treatment was omitted, no plaques were de- tected. In the DNA range 0.1 to 1.0 wg/plate, the efficiency of transfection equals 5 x 1078 plaques per genome equivalent. Recently, a high efficiency of transfection of spheroplasts 1631 NOTES 1,000 uu e < a a i 100 4 > q g PLUS CaCly 7 ¢ 1 a oe 4 a 4 MINUS CaCly 4 108 e eo—-e-e ~ 0.01 01 10 10 DNA (ug/PLATE] Fic. 1. Effect of DNA concentration on CaCl,- dependent transfection. Cells of S. typhimurium TA1659 were grown, harvested, and treated as re- ported in the text. In the control, the CaCl, solution was replaced by L broth. TABLE 1. Comparison of the transfectability of CaCl,-‘‘treated” cells Inferred No. of plaques Strain no. Description LPS per 10° cells character at lug of DNA 1. TA1659° LT2 chl-1013(Agal, bio, uurB) Re 700-3, 000 2. SL1694 TA1659 carrying F'8-gal+ + <1 3. SL1694 Gal- Gal- segregant of SL1694 Re 300-2,000 4. SL1657¢ LT2 galE496 Re 100-400 5. TA1701 LT2 hisC3076, chi-1038(Agal, bio, uurB, aroG, nic) Re 100-200 6. TA 1701-H*+ Hist spontaneous revertant of TA1701 Re 100-200 7. TA1657 LT2 chl-1011(Agal, bio, uurB) Re 100-400 8. TA1662 LT2 chl-1016(Agal, bio, uurB) Re 90-200 9. SL1181¢° rfaF511 Rd, 10-100 10. EL199 His mutant of $L4213 (2) galE496 Re 50-300 11. his-519 LT2 deletion of his and rfb clusters Ra 5 12. his-520 LT2 deletion of his and rfb clusters Ra 5 13. TA1674 LT2 chl-1028( Agal, bio, uurB, nic, dho, aroG) Re 200 14, SL1747 LT2 metE47, galE161 Re 40 15. $L1752 SL1747 carrying F’8-gal* +