Figure 2. Lack of BacA leads to uncontrolled growth of the stalk and bud compartments.

(A) Immunoblot showing the levels of BacA in strain EC41 (ΔbacA P_Cu-bacA) over the course of BacA depletion. Cells were grown in copper-containing medium, washed, and transferred to inducer-free medium. At the indicated time points, samples were withdrawn and subjected to immunoblot analysis. Strains LE670 (wild-type) and EC28 (ΔbacA) were included as controls. The position of BacA is indicated. (B) Morphological defects induced by BacA depletion. Cells of strains LE670 (wild-type) and EC41 (ΔbacA P_Cu-bacA) were grown in medium containing 0.5 mM CuSO₄, washed, and incubated for 6 hr in inducer-free medium before they were transferred onto ASS-agarose pads lacking inducer and imaged at the indicated time points. Shown are representative images. Bar: 2 µm. (C) Changes in the growth pattern of H. neptunium in the absence of bactofilins. Cells of strains LE670 (wild-type), and EC33 (ΔbacAD) were stained with the fluorescent D-amino acid HADA prior to analysis by fluorescence microscopy. Shown are representative images of cells at different developmental stages. Bars: 2 µm.

Figure 2—figure supplement 1. Growth of ΔbacAD cells in a microfluidic flow cell.

Cells of strain EC33 (ΔbacAD) were flushed into a microfluidic flow cell and imaged at the indicated time points (in hours). Bar: 3 µm.

Figure 2—figure supplement 2. Muropeptide profiles of different H. neptunium strains.

Cell walls of strains LE670 (wild-type), EC28 (ΔbacA), EC23 (ΔbacD), and EC33 (ΔbacAD) were digested with cellosyl to release muropeptides, which were reduced and separated by HPLC. The identities of major muropeptide species, assigned based on the previously reported retention times (Cserti et al., 2017; Glauner, 1988), are given above the corresponding peaks. Tri, Tetra, and Penta stand for N-acetylglucosamine-N-acetylmuramitol tripeptide, tetrapeptide and pentapeptide, respectively. ‘Anh’ indicates muropeptides containing 1,6-anhydromuramic acid. Shown are representative HPLC profiles from two independent experiments. A quantification of the different muropeptide species is given in Supplementary file 1.

Figure 2—video 1. Unconstrained growth of a bactofilin-deficient H. neptunium mutant.

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Cells of strain EC33 (ΔbadAD) were grown in a microfluidic flow cell and imaged by DIC microsopy at 15 min intervals. Bar: 2 µm.

Figure 2—video 2. Normal growth of the H. neptunium wild-type strain.

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Cells of strain LE670 (wild-type) were grown in a microfluidic flow cell and imaged by DIC microscopy at 5 min intervals. Bar: 2 µm.