The MG1363 and IL1403 laboratory strains of Lactococcus lactis and several dairy strains are diploid

Abstract

Bacteria are normally haploid, maintaining one copy of their genome in one circular chromosome. We have examined the cell cycle of laboratory strains of Lactococcus lactis, and, to our surprise, we found that some of these strains were born with two complete nonreplicating chromosomes. We determined the cellular content of DNA by flow cytometry and by radioactive labeling of the DNA. These strains thus fulfill the criterion of being diploid. Several dairy strains were also found to be diploid while a nondairy strain and several other dairy strains were haploid in slow-growing culture. The diploid and haploid strains differed in their sensitivity toward UV light, in their cell size, and in their D period, the period between termination of DNA replication and cell division.

FIG. 1.

Phase-contrast micrograph of the cells that were used for the flow cytometry experiment shown in Fig. 2.

FIG. 2.

Flow cytometric DNA histograms of L. lactis subsp. cremoris MG1363 grown in MM supplemented with glucose and lipoic acid at a generation time of 228 min and of E. coli grown with proline as a carbon source. The DNA histograms were resolved into B, C, and D periods. The two samples were recorded with the same settings. D′ is the D period minus the generation time.

FIG. 3.

(A) A flow cytometric DNA histogram of haploid L. lactis subsp. lactis NCDO2118 growing at a generation time of 139 min. The histogram has been resolved for cell cycle periods. The status of chromosomal replication is also indicated on this panel (from left to right): just initiated, replicating, nonreplicating, and just initiated. The cells to the right in the histogram are cells that stick together. These cells can be seen as a shadow in the upper part of the cytogram in panel C. (B) A flow cytometric DNA histogram of an E. coli strain growing on proline as a carbon energy source and recorded at the same settings as the histogram in panel A. The status of chromosomal replication is also indicated on this panel (from left to right): nonreplicating, just initiated, replicating, and nonreplicating. (C) A flow cytometric cytogram of DNA versus size of the NCDO2118 culture. The white line indicates the change in DNA content and growth of each cell in the culture.

FIG. 4.

Fluorescence micrograph of MG1363 and NCDO2118 from slow-growing cultures with generation times of 126 min and 161 min, respectively. Red arrows indicate cells about to divide with four (MG1363) or two (NCDO2118) nucleoids. Black arrows indicate new-born cells with two nucleoids (MG1363) or one (NCDO2118) nucleoid.

FIG. 5.

Flow cytometric cytograms of cells from slow-growing cultures of L. lactis subsp. cremoris MG1363 and L. lactis subsp. lactis NCDO2118 growing with 125-min and 161-min generation times, respectively. The cytograms were recorded with the same settings on the instrument. Numbers along the y axis indicate the number of genomes.

FIG. 6.

UV killing of MG1363 and NCDO2118. The strains were grown in MM supplemented with glucose and lipoic acid. The generation times for MG1363 and NCDO2118 were 264 min and 154 min, respectively. Half-lives for UV-exposed NCDO2118 and MG1363 were 19 and 21 s, respectively. Simulations in which one (NCDO2118) or two (MG1363) hits were needed for killing the bacteria, have been fitted to the curves. The number of survivors has been normalized. Actual starting numbers were 1.2 × 10⁷ and 2.2 ×10⁷ cells per ml for MG1363 and NCDO2118, respectively.

FIG. 7.

Cell cycle periods D and C of MG1363 and NCDO2118. The periods have been determined by resolving flow cytometric DNA histograms of cultures grown at different generation times. See text for discussion of D′ versus D periods.

FIG. 8.

The cell cycles of diploid (MG1363) and haploid (NCDO2118) L. lactis strains. The cell cycle from division (div) to division consists of a B period, a C period, and a D′ period. The chromosomes are shown as dark-gray open figures, with the replication start points (origins) represented as solid black circles. The diploid cell cycle of the MG1363 strain is shown to the left. The replication end points (termini) that are split at division in the bottom of the figure are those formed after replication of the terminus (denoted with T in the top cell), as indicated with the dotted line. The haploid cell cycle of NCDO2118 is shown to the right. Initiation of replication occurs simultaneously with cell division. Consequently, this strain has no B period.

FIG. 9.

Flow cytometric DNA histograms of L. lactis subsp. cremoris MG1363 grown at different generation times (in MM plus N, generation time is 143 min; in MM, generation time is 185 min panel) and of E. coli (with two identical histograms). E. coli was grown with proline as a carbon source and incubated with rifampin and cephalexin for 4 h; the histograms show cells with 1 and 2 chromosomes. Rifampin inhibits initiation of replication but not elongation. E. coli treated with rifampin thus terminates ongoing replication and ends with completely replicated chromosomes. Cephalexin inhibits cell division and thus prevents those cell divisions that otherwise would occur in the presence of rifampin. Arrows numbered 1 to 4 correspond to peaks representing 1 to 4 chromosomes, respectively.

FIG. 10.

Flow cytometric cytogram of the cells shown in Fig. 2. Concomitant increases in mass (light scatter) and DNA (fluorescence) can be seen.