Acid tolerance of Streptococcus macedonicus as assessed by flow cytometry and single-cell sorting

Abstract

An in situ flow cytometric viability assay employing carboxyfluorescein diacetate and propidium iodide was used to identify Streptococcus macedonicus acid tolerance phenotypes. The logarithmic-phase acid tolerance response (L-ATR) was evident when cells were (i) left to autoacidify unbuffered medium, (ii) transiently exposed to nonlethal acidic pH, or (iii) systematically grown under suboptimal acidic conditions (acid habituation). Stationary-phase ATR was also detected; this phenotype was gradually degenerated while cells resided at this phase. Single-cell analysis of S. macedonicus during induction of L-ATR revealed heterogeneity in both the ability and the rate of tolerance acquisition within clonal populations. L-ATR was found to be partially dependent on de novo protein synthesis and compositional changes of the cell envelope. Interestingly, acid-habituated cells were interlaced in lengthier chains and exhibited an irregular pattern of active peptidoglycan biosynthesis sites when probed with BODIPY FL vancomycin. L-ATR caused cells to retain their membrane potential after lethal challenge, as judged by ratiometric analysis with oxonol [DiBAC(4)(3)]. Furthermore, F-ATPase was important during the induction of L-ATR, but in the case of a fully launched response, inhibition of F-ATPase affected acid resistance only partially. Activities of both F-ATPase and the glucose-specific phosphoenolpyruvate-dependent phosphotransferase system were increased after L-ATR induction, distinguishing S. macedonicus from oral streptococci. Finally, the in situ viability assessment was compared to medium-based recovery after single-cell sorting, revealing that the culturability of subpopulations with identical fluorescence characteristics is dependent on the treatments imposed to the cells prior to acid challenge.

FIG. 1.

Experimental design for the study of the S. macedonicus acid tolerance response. The sequence of subcultures and incubation conditions that led to nonadapted cells, autoacidified cells, acid-adapted cells (by transient exposure to nonlethal acidic pH), acid-habituated cells, cells reversed from acid habituation, and stationary-phase cells is presented schematically. o/n, overnight.

FIG. 2.

(A) Flow cytometric analysis of S. macedonicus acid tolerance at mid-log phase. Nonadapted (a) or acid-adapted (b to f) cells, after exposure to pH 3.5 for 15 min, were labeled with cFDA and PI and analyzed by flow cytometry. Prior to acid challenge, bacteria were adapted either by autoacidification (b), transient exposure to pH 6.0 and 5.5 (c and d), or acid habituation at pH 6.0 (e). Acid habituation was also reversed after regrowth of habituated cells in MRS-MOPS (f). As additional controls, unstressed (g) and heat-killed (h) mid-log-phase cells were analyzed in parallel. (B) Percentages (means ± standard deviations) of fluorescent subpopulations of the samples presented in panel A. (C) Percentages (means plus standard deviations) of fluorescent subpopulations of samples challenged at pH 3.5 for 30 min in the same order as that presented in panel A.

FIG. 3.

Kinetics of acid adaptation of S. macedonicus. Mid-log-phase cells were adapted to pH 5.5 for 0 to 60 min and exposed to lethal challenge for 15 or 30 min (A and B, respectively). Percentages (means ± standard deviations) of fluorescent subpopulations, as determined by flow cytometry after cFDA-PI labeling, are presented.

FIG. 4.

Effects of inhibitors on the induction of acid tolerance in S. macedonicus. Prior to acidic lethal challenge at pH 3.5 for 15 min, mid-log-phase cells were either left unadapted (control) or acid adapted at pH 5.5 for 30 min in the presence of chloramphenicol (Chl), actinomycin D (Act D), cerulenin (Cer), vancomycin (Van), or penicillin (Pen) or in the absence of any inhibitor (adapted). Percentages (means ± standard deviations) of fluorescent subpopulations, as determined by flow cytometry after cFDA-PI labeling, are presented.

FIG. 5.

(A) Flow cytometric analysis of active cell wall biosynthesis sites of nonadapted (a), acid-adapted (at pH 5.5 for 1 h) (b), acid-habituated (c), and stationary-phase (d) S. macedonicus cells labeled with BODIPY FL vancomycin. Results are presented as density plots of green fluorescence versus side scatter. Region R1 was drawn to enclose the core of nonadapted cells' distribution. (B) CLSM photographs of cells treated as described above (a to c). It should be noted that stationary-phase cells were negative for BODIPY FL vancomycin labeling under CLSM.

FIG. 6.

Ratiometric analysis of membrane potential changes in S. macedonicus cells after acidic lethal challenge with DiBAC₄(3). Untreated (a), depolarized (b), and heat-killed (c) mid-log-phase S. macedonicus cells served as controls. Nonadapted (d) or acid-adapted (at pH 5.5 for 30 min) (e) mid-log-phase cells were exposed to pH 3.5 for 15 min. All samples were labeled with DiBAC₄(3) and analyzed by flow cytometry. Results are presented as single-parameter histograms of the log ratios of green fluorescence to side scatter. M1, M2, and M3 were defined by the untreated, depolarized, and heat-killed cells' distributions, respectively.

FIG. 7.

Effect of DCCD on the induction of acid tolerance in S. macedonicus. Prior to lethal challenge, cells were nonadapted (a), acid adapted at pH 5.5 for 60 min, with DCCD being added either 20 min prior to and during the 60 min of acid adaptation (b) or only during the last 40 min of the treatment (c), and acid adapted under the same conditions in the absence of DCCD (d). Percentages (means ± standard deviations) of fluorescent subpopulations, as determined by flow cytometry after cFDA-PI labeling, are presented.

FIG. 8.

F-ATPase (A) and glucose-specific PEP-PTS (B) activities of S. macedonicus mid-log-phase cells that were nonadapted (a), autoacidified (b), acid adapted at pH 5.5 for 30 min in the presence of chloramphenicol (c) or cerulenin (d) or in the absence of inhibitors (e), and acid habituated (f).

FIG. 9.

Percentages of recovery of sorted cFDA⁺ PI⁻ and cFDA⁺ PI⁺ cells derived from nonadapted and acid-adapted populations after acid challenge at pH 3.5 for 15 min. Medium-based recovery of sorted cells was examined on MRS-MOPS, MRS (pH 6.0 and 5.5), and MRS-MOPS-1% NaCl agar plates. It should be noted that cFDA⁻ PI⁺ cells exhibited no recovery in all cases (data not shown).