The distinct cell physiology of Bradyrhizobium at the population and cellular level

Abstract

The α-Proteobacteria belonging to Bradyrhizobium genus are microorganisms of extreme slow growth. Despite their extended use as inoculants in soybean production, their physiology remains poorly characterized. In this work, we produced quantitative data on four different isolates: B. diazoefficens USDA110, B. diazoefficiens USDA122, B. japonicum E109 and B. japonicum USDA6 which are representative of specific genomic profiles. Notably, we found conserved physiological traits conserved in all the studied isolates: (i) the lag and initial exponential growth phases display cell aggregation; (ii) the increase in specific nutrient concentration such as yeast extract and gluconate hinders growth; (iii) cell size does not correlate with culture age; and (iv) cell cycle presents polar growth. Meanwhile, fitness, cell size and in vitro growth widely vary across isolates correlating to ribosomal RNA operon number. In summary, this study provides novel empirical data that enriches the comprehension of the Bradyrhizobium (slow) growth dynamics and cell cycle.

Keywords: Bradyrhizobium; Bacterial physiology; Cell polarity; Growth rate.

Fig. 1

General features ofBradyrhizobium growth.(a) A representative growth curve manually performed by following OD_450nm along time in classical YEM medium of B. japonicum E109 (Blue), B. japonicum USDA6^T (Red), B. diazoefficiens USDA110 (green) and B. diazoefficiens USDA122 (purple). (b) Colony Forming Units per mL from a) of culture are plotted as a function of time. (c) Photograph of typical cell aggregates observed for the four isolates that spontaneously appear during the first 48hs accompanying the OD reduction (left). Microscopy observation of B. japonicum E109 aggregates using 1000X magnification (right). The photograph shows the cellular nature of aggregates. The black bar indicates 10 μm. (d) Generation times were calculated from several manual growth curves. The graph shows the box & whiskers plot minimum to maximum showing all the individual values. The central line indicates the median (n ≥ 6). Statistical significance was analyzed using Kruskal-Wallis non-parametric tests followed by Dunn’s multiple comparisons. *** means p < 0.001

Fig. 2

Increasing nutrient concentration inhibitsBradyrhizobium growth. Growth curves plotting OD_450nm as a function of time (in hours) of Bd1110 (shades of green) and, Bj109 (shades of blue) in manual growth curves. Strains were grown on increasing yeast extract concentration as indicated by darker colors. Upper panels show representative growth curves out of 3 performed that were used to calculate the GT values. The lower panels plot the mean value of generation times for both strains at the indicated YE concentration. Statistical significance was analyzed using One-way ANOVA and the Tukey test for multiple comparisons. Letters denote groups displaying statistically significant differences (P of at least < 0.05)

Fig. 3

Cell size dynamics along the growth curve. Bd110 (green) and BjE109 (blue) were grown on YEM medium. Samples were taken at different phases of the growth curve. Cells were photographed under the microscope and the OD_450nm of the culture was measured. (a) The distribution of individual cells lengths of Bd1110 (green, USDA110) and, Bj109 (Blue E109) was plotted along the experiment. The thick lines indicate the median for each timepoint. Dotted lines show the quartile range. Descriptive statistics can be found in Additional File 1, Table S2. (b) Each median of cell length (left axis) is plotted as a function of OD_450nm (right axis) is plotted as a function of time for each strain

Fig. 4

B. japonicum tend to display a fitness advantage over B. diazoefficiens during lag to early-exponential phase. Pairwise competition experiments between Bd110::gfp + and Bd122 (purple), Bd110 (green), Bj6 (red) and, Bj109 (blue) was performed as described in material and methods. For each time point OD_450nm was determined. (a) Relative fitness at early (3 days), late exponential (7 days) and stationary phase (10 days) of growth in co-culture. The Bd110 against the same strain expressing gfp was taken as reference for relativizing fitness (green dotted line). Points represent mean with SEM (n = 4). Statistical differences were computed using Two-way ANOVA and Holm-Sidak for multiple comparisons. * and ** mean p < 0.05 and p < 0.01 respectively. (b) A representative dataset where W_rel (left axis, filled dots, dotted connecting lines) and OD_450nm (right axis, solid connecting lines, empty dots) at each time point were plotted as a function of elapsed time since the beginning of the experiment

Fig. 5

Bradyrhizobium present an extreme asymmetry. Cells were stained with sBADA and photographed using CLSM as indicates un material and methods. A. fabrum C58 an asymmetric α-Proteobacterium was used as a control of an asymmetric cell and E.coli as a regular bacterium dividing by binary fission. The white bar at the central panel corresponds to 5 μm for all panels

Fig. 6

Bradyrhizobium displays stronger growth asymmetry than Agrobacterium fabrum C58. Using FIJI plugin MicrobeJ [48], we built an sBADA local maxima density heatmap in B. japonicum E109 (a) (n = 162), B. diazoefficiens USDA110 (b) (n = 86) and (A) fabrum C58 (c) (n = 90) cells. Grey dots represent events of local maxima detected within the cell. (d) Proportion histogram of local maxima events shown in (a), (b) and (c), along (B) japonicum E109, B. diazoefficiens USDA110 and A. fabrum C58 cells. Cell poles 1 and − 1 were defined by intensity of sBADA channel, being cell pole 1 the one with higher intensity. The vertical grey line denotes the cell center. We used the same threshold settings of intensity and Z-score for sBADA maxima detection in every experiment