Effect of Cadmium on the Infection of Lactobacillus lactis by Bacteriophage LL-H

Abstract

SUMMARYThe infectivity of Lactobacillus lactis bacteriophage LL-H was shown to be calciumdependent. Of 10 different divalent cations screened, cadmium specifically decreased the infectivity of LL-H in the presence of calcium. At 1 to 2 mM, CdClz resulted in a decrease of the burst size of about 2.5-to 4-fold. Cd z+ was shown to reduce specifically the level of total phage DNA synthesis, resulting in a reduced progeny phage yield. Moreover, Cd 2+ had the most profound irreversible effect on progeny phage production between 20 and 60 rain after LL-H infection. This paralleled the beginning of phage DNA synthesis. Possible modes of action of Cd z+ on phage DNA replication are discussed.It is well documented, especially among bacteriophages active against Bacillus or Lactobacillus and other lactic acid-producing bacterial strains, that a Ca2+-dependent step(s) is frequently involved in the infection cycle (Potter & Nelson, 1953;Sozzi, 1972;Watanabe & Takesue, 1972;Steensma & Blok, 1979;Landry & Zsigray, 1980). This holds true also in the case of the Lactobacillus lactis bacteriophage LL-H. Of 10 different divalent cations screened, only Ca 2+ and Mg 2+ were capable of significantly promoting the infectivity of LL-H. Moreover, among these cations, cadmium was unique in being able to decrease the infectivity of LL-H in the presence of Ca 2+. The basis of this special effect of Cd 2+ on LL-H multiplication is investigated in this communication.Procedures for the growth of L. lactis and phage LL-H have been reported previously (Alatossava & Pyhtil/i, 1980). All plating experiments were carried out by the agar overlay technique described by Adams (1959). Soft and hard agar contained the same concentrations of divalent cations and chloramphenicol (Boehringer Mannheim) used as supplements in MRS medium (Difco). Standard procedures were used for one-step growth curve and burst size experiments.To reveal the roles of various elements on phage LL-H infectivity, the following cations were considered: Na +, K +, Ca 2+, Mg 2+, Ba z+, Sr 2+, Mn 2+, Zn 2+, Cu 2+, Fe 2+, Hg 2+, Cd 2+, and in addition spermine. Of these, only Ca 2+ in particular and Mg 2+ were able to increase the efficiency of plating of LL-H. The optimal concentrations for Ca 2+ and Mg z+ were between 20 and 40 mM. Supplementing MRS medium with 20 mM-CaC12 did not affect the rate of growth or DNA and protein syntheses of the host, L. lactis (results not shown). Of the cations studied, cadmium was the only one able to decrease the infectivity of LL-H in the presence of Ca 2+.To determine the Cd2+-sensitive step or steps in the lytic cycle of LL-H, the phage adsorption kinetics was first studied by determining the proportion of free phage in samples using Nuclepore membrane filters (pore size 0.4 lam). These filtration experiments showed that Cd 2+ did not inhibit phage infection at this step nor did LL-H adsorb to the Nuclepore filter. Accordingly, the CdZ+-sensitive step(s) must occur after the phage had adsorbed. Analysis of phage LL-H DNA replication showed this...

Transmembrane Cation Movements during Infection of Lactobacillus lactis by Bacteriophage LL-H

“…The divalent cations Ca 2÷ or Mg 2÷ are essential for LL-H infection (Alatossava et al, 1983). Ca 2÷ and Mg 2÷ are required for phage adsorption, but they also accumulate in the cell immediately after infection in amounts depending on their external concentrations and the m.o.i.…”

“…Thus, much higher intracellular Cd 2+ concentrations and consequently more drastic CdZ+-mediated inhibitory effects [e.g. lysis inhibition and lowered e.o.p, of LL-H as described by Alatossava et al (1983)] would be expected.…”

“…It occurred at external Cd 2÷ concentrations only slightly inhibitory for uninfected L. lactis (Alatossava et al, 1983). In order to investigate whether the sensitivity of LL-H-infected L. lactis to cadmium was associated with a phage-mediated Cd 2÷ movement, we measured by AAS the cellular Cd 2÷ contents after infection.…”

“…Mg 2÷ is known to substitute for Ca 2÷ during LL-H infection (Alatossava et at., 1983). We infected L. lactis with LL°H at various multiplicities in the presence of 20 mt, l-MgCl2 and followed the cellular K ÷, Na + and Mg 2+ contents by AAS.…”

“…This requirement could be associated with different steps of the infection cycle such as phage adsorption, penetration of the nucleic acid and intracellular phage development (Rountree, 1955;Watanabe & Takesue, 1972;Snipes et al, 1974;Steensma & Blok, 1979;Karnik & Gopinathan, 1980;Landry & Zsigray, 1980;Nagaraja & Gopinathan, 1980). Phage LL-H also shows a strict requirement for external divalent cations for successful infection of its Gram-positive host, Lactobacillus lactis (Alatossava et al, 1983). The molecular basis of this requirement is not yet understood.…”