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.2023 Sep 29:14:1270665.
doi: 10.3389/fmicb.2023.1270665. eCollection 2023.

"Influence of plasmids, selection markers and auxotrophic mutations onHaloferax volcanii cell shape plasticity"

Affiliations

"Influence of plasmids, selection markers and auxotrophic mutations onHaloferax volcanii cell shape plasticity"

Megha Patro et al. Front Microbiol..

Abstract

Haloferax volcanii and other Haloarchaea can be pleomorphic, adopting different shapes, which vary with growth stages. Several studies have shown thatH. volcanii cell shape is sensitive to various external factors including growth media and physical environment. In addition, several studies have noticed that the presence of a recombinant plasmid in the cells is also a factor impactingH. volcanii cell shape, notably by favoring the development of rods in early stages of growth. Here we investigated the reasons for this phenomenon by first studying the impact of auxotrophic mutations on cell shape in strains that are commonly used as genetic backgrounds for selection during strain engineering (namely: H26, H53, H77, H98, and H729) and secondly, by studying the effect of the presence of different plasmids containing selection markers on the cell shape of these strains. Our study showed that most of these auxotrophic strains have variation in cell shape parameters including length, aspect ratio, area and circularity and that the plasmid presence is impacting these parameters too. Our results indicated that ΔhdrB strains andhdrB selection markers have the most influence onH. volcanii cell shape, in addition to the sole presence of a plasmid. Finally, we discuss limitations in studying cell shape inH. volcanii and make recommendations based on our results for improving reproducibility of such studies.

Keywords: HDRB; Haloferax volcanii; auxotrophy; cell shape; haloarchaea; plasmid.

Copyright © 2023 Patro, Duggin, Albers and Ithurbide.

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Conflict of interest statement

The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest. The author(s) declared that they were an editorial board member of Frontiers, at the time of submission. This had no impact on the peer review process and the final decision.

Figures

Figure 1
Figure 1
Analysis of the morphology ofH. volcanii DS2 at different stages of growth.(A) Relative frequency distribution of cell circularity (left) measured from phase contrast image analysis (right) of DS2 samples collected at OD600 0.01 to 0.2 (bottom to top). Cell types R (rods), I (intermediates) and P (plates) are determined depending on the cell circularity as described in the text and each cell shape percentage is indicated on the graphs. TheY-axis indicates the percentage of cells at each OD. Sum of the graph height per OD600 equals 100%. Scale bars on micrographs represent 4 μm.(B) Violin plot distribution of cells circularity at different OD600. Data set is the same as in(A).(C) Violin plot distribution of cells aspect ratio at different OD600(D) Violin plot distribution of cells area (μm2) at different OD600(E) Violin plot distribution of cells length (μm) at different OD600. The statistical analysis in(B–E) were performed using Kruskal–Wallis-test in GraphPad Prism and data represent more than 1,100 cells from three independent experiments. Black line indicates mean; bottom and top lines indicate the standard deviation. Additional results of Kruskal–Wallis-tests are represented in Supplementary Figure S5.
Figure 2
Figure 2
Morphological analysis of different auxotrophicH. volcanii strains at different stages of growth.(A) Phase contrast micrographs showing DS2, H26, H53, H77, H98 and H729 sampled at different growth stages from OD600 0.01 (bottom) to 0.2 (top). Scale bars represent 4 μm. The genotype of each strain (coloured) is indicated at the bottom of the micrographs.(B) Relative frequency distribution of cell circularity measured from micrographs in(A) at OD600 0.01 to 0.2 (bottom to top). Dashed vertical lines delimit the different cell types R (rods), I (intermediates) and P (plates) determined depending on the cell circularity as described in the text. TheY-axis indicates the percentage of cells at each OD. Sum of the graph height per OD600 equals 100%.(C) Bar graph indicating the percentage of each cell shape types at each sampled OD600 for each strain background. Colour representation—R (rods): black, I (intermediate): blue and P (plates): grey.(B,C) (N > 500 from three independent experiments). Data set for strain DS2 is the same as in Figure 1.
Figure 3
Figure 3
Morphological analysis of H26 with plasmids compared to H26 without plasmid at different stages of growth.(A) Left—phase contrast micrographs showing H26, H26 pTA1392, H26 pTA230 sampled at different growth stages from OD600 0.01 (bottom) to 0.2 (top). Scale bars represent 4 μm. The genotype of each strain (coloured) is indicated at the bottom of the micrographs. Right—Relative frequency distribution of cell circularity measured from micrographs in(A) at OD600 0.01 to 0.2 (bottom to top). Dashed vertical lines delimit the different cell types R (rods), I (intermediates) and P (plates) determined depending on the cell circularity as described in the text. TheY-axis indicates the percentage of cells at each OD. Sum of the graph height per OD600 equals 100%.(B) Bar graph indicating the percentage of each cell shape types at each sampled OD600 for each strain background. Colour representation—R (rods): black, I (intermediate): blue and P (plates): grey.(C) Violin plot distribution of cells area (μm2) at different OD600.(D) Violin plot distribution of cell length (μm) at different OD600.(E) Violin plot distribution of cells aspect ratio at different OD600. The statistical analysis in(C–E) were performed using Kruskal–Wallis-test in GraphPad Prism and data represent more than 1,300 cells from three independent experiments. Black line indicates mean; bottom and top lines indicate the standard deviation. Additional results of Kruskal–Wallis-tests are represented in Supplementary Figure S6. Data set for strain H26 is the same as in Figure 1.
Figure 4
Figure 4
Morphological analysis of H53 with plasmids compared to H53 without plasmid at different stages of growth.(A) Left—phase contrast micrographs showing H53, H53 pTA1392, H53 pTA230, H53 pTA231 sampled at different growth stages from OD600 0.01 (bottom) to 0.2 (top). Scale bars represent 4 μm. The genotype of each strain (coloured) is indicated at the bottom of the micrographs. Right—relative frequency distribution of cell circularity measured from micrographs in(A) at OD600 0.01 to 0.2 (bottom to top). Dashed vertical lines delimit the different cell types R (rods), I (intermediates) and P (plates) determined depending on the cell circularity as described in the text. TheY-axis indicates the percentage of cells at each OD. Sum of the graph height per OD600 equals 100%.(B) Bar graph indicating the percentage of each cell shape types at each sampled OD600 for each strain background. Colour representation—R (rods): black, I (intermediate): blue and P (plates): grey.(C) Violin plot distribution of cells area (μm2) at different OD600.(D) Violin plot distribution of cell length (μm) at different OD600.(E) Violin plot distribution of cells aspect ratio at different OD600. The statistical analysis in(C–E) were performed using Kruskal–Wallis-test in GraphPad Prism and data represent more than 500 cells from three independent experiments. Black line indicates mean; bottom and top lines indicate the standard deviation. Additional results of Kruskal–Wallis-tests are represented in Supplementary Figure S7. Data set for strain H53 is the same as in Figure 1.
Figure 5
Figure 5
Morphological analysis of H77 with plasmids compared to H77 without plasmid at different stages of growth.(A) Left—phase contrast micrographs showing H77, H77 pTA231 sampled at different growth stages from OD600 0.01 (bottom) to 0.2 (top). Scale bars represent 4 μm. The genotype of each strain (coloured) is indicated at the bottom of the micrographs. Right—relative frequency distribution of cell circularity measured from micrographs in(A) at OD600 0.01 to 0.2 (bottom to top). Dashed vertical lines delimit the different cell types R (rods), I (intermediates) and P (plates) determined depending on the cell circularity as described in the text. TheY-axis indicates the percentage of cells at each OD. Sum of the graph height per OD600 equals 100%.(B) Bar graph indicating the percentage of each cell shape types at each sampled OD600 for each strain background. Colour representation—R (rods): black, I (intermediate): blue and P (plates): grey.(C) Violin plot distribution of cells area (μm2) at different OD600.(D) Violin plot distribution of cells length (μm) at different OD600.(E) Violin plot distribution of cells aspect ratio at different OD600. The statistical analysis in(C–E) were performed using Kruskal–Wallis-test in GraphPad Prism and data represent more than 500 cells from three independent experiments. Black line indicates mean; bottom and top lines indicate the standard deviation. Additional results of Kruskal–Wallis-tests are represented in Supplementary Figure S8. Data set for strain H77 is the same as in Figure 1.
Figure 6
Figure 6
Morphological analysis of H729 with plasmids compared to H729 without plasmid at different stages of growth.(A) Left—phase contrast micrographs showing H729, H729 pTA1392, H729 pTA233 sampled at different growth stages from OD600 0.01 (bottom) to 0.2 (top). Scale bars represent 4 μm. The genotype of each strain (coloured) is indicated at the bottom of the micrographs. Right—relative frequency distribution of cell circularity measured from micrographs in(A) at OD600 0.01 to 0.2 (bottom to top). Dashed vertical lines delimit the different cell types R (rods), I (intermediates) and P (plates) determined depending on the cell circularity as described in the text. TheY-axis indicates the percentage of cells at each OD. Sum of the graph height per OD600 equals 100%.(B) Bar graph indicating the percentage of each cell shape types at each sampled OD600 for each strain background. Colour representation—R (rods): black, I (intermediate): blue and P (plates): grey.(C) Violin plot distribution of cells area (μm2) at different OD600.(D) Violin plot distribution of cell length (μm) at different OD600.(E) Violin plot distribution of cells aspect ratio at different OD600. The statistical analysis in(C–E) were performed using Kruskal–Wallis-test in GraphPad Prism and data represent more than 900 cells from three independent experiments. Black line indicates mean; bottom and top lines indicate the standard deviation. Additional results of Kruskal–Wallis-tests are represented in Supplementary Figure S9. Data set for strain H729 is the same as in Figure 1.
Figure 7
Figure 7
Morphological analysis of H98 with plasmids compared to H98 without plasmid at different stages of growth.(A) Left—phase contrast micrographs showing H98, H98 pTA1392, H98 pTA230, H98 pTA233 sampled at different growth stages from OD600 0.01 (bottom) to 0.2 (top). Scale bars represent 4 μm. The genotype of each strain (coloured) is indicated at the bottom of the micrographs. Right—relative frequency distribution of cell circularity measured from micrographs in(A) at OD600 0.01 to 0.2 (bottom to top). Dashed vertical lines delimit the different cell types R (rods), I (intermediates) and P (plates) determined depending on the cell circularity as described in the text. TheY-axis indicates the percentage of cells at each OD. Sum of the graph height per OD600 equals 100%.(B) Bar graph indicating the percentage of each cell shape types at each sampled OD600 for each strain background. Colour representation—R (rods): black, I (intermediate): blue and P (plates): grey.(C) Violin plot distribution of cells area (μm2) at different OD600.(D) Violin plot distribution of cell length (μm) at different OD600.(E) Violin plot distribution of cells aspect ratio at different OD600. The statistical analysis in(C–E) were performed using Kruskal–Wallis-test in GraphPad Prism and data represent more than 1,700 cells from three independent experiments. Black line indicates mean; bottom and top lines indicate the standard deviation. Additional results of Kruskal–Wallis-tests are represented in Supplementary Figure S10. Data set for strain H98 is the same as in Figure 1.
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Grants and funding

The author(s) declare financial support was received for the research, authorship, and/or publication of this article. SI was supported by a Momentum grant from the VW Science Foundation (grant number 94933) granted to S-VA. MP was supported by grant AL1206/4-3 by the German Science Foundation (DFG). ID was supported by the Australian Research Council (DP160101076 and FT160100010).

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