How to grow shrubs

by Christoph
 

Exactly eight years ago today, we introduced you to the Hedgehogs In Your Garden. No, we had not turned our backs on the Small Things and became gardeners instead. Again today, it is not about gardening activities, namely, How to grow shrubs. Let me explain...
 

(click to enlarge)
Figure 1. Corncob structures formed in plaque. Co­ryne­bacterium cells (magenta) are visible as long fila­ments, with cocci (green) bound to the tips of the filaments. Partially disrupted plaque was hy­bri­dized with a probe for Co­ry­ne­bac­te­ri­um and a universal bacterial probe. Image was acquired using a Zeiss Axio­Imager 63/Plan-Apo­chro­mat 1.4 N.A. ob­jec­tive and Apo­tome struc­tured illu­mi­na­tion (Scale bar: 20 μm). Source. Frontispiece: C. matruchotii colony. Courtesy of © Scott Chi­mi­les­ki

When Jessica Mark Welch and her colleagues studied the multi-species consortia in dental plaque, they reproducibly found structures in these biofilms that looked like miniature hedge-hogs or corncobs to them. What was striking about both structures was that a thicket of cory­ne­bacteria had settled near the base with which the biofilm was attached to the tooth surfaces close to the gum (gingiva). This thicket was surrounded and interspersed with other oral bac­teria that looked attached like leaves to shrubs. Figure 1 shows a particularly revealing micro­scopic image; see a diagram here.
 

In this earlier study, the shrub-forming bacteria were identified as Corynebacterium matru­cho­tii and, in smaller numbers, as Corynebacterium durum. Corynebacteria and other members of the order Mycobac­teriales (class Actinomycetes) are not known for filamentous growth as, for example, Streptomyces spp. (class Actino­mycetes). This puzzle led Chimileski et al. (2024) to in­ves­tigate the growth and cell division of C. ma­truchotii  in more detail (Disclosure: Scott Chi­mileski co‑authored Life at the Edge of Sight with Roberto and designed the current STC logo).
 

By tracking individual cells as they developed into microcolonies, the authors were able to solve the puzzle, collecting a few more puzzles along the way (Figure 2).

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Figure 2. Microcolony development is driven by filament elongation and simultaneous multiple division (SMD). The phases of the C. matruchotii cell cycle highlighted by tracking a single cell as it develops into a micro­colony. In phase 1, the progenitor cell elongates and forms a filament. The outline of the progenitor cell at t=0 (purple) is overlaid on the 3.5 h image. Multiple septa then form along the filament (phase 2, arrows), and it divides into many daughter cells simultaneously, with some daughter cells transiently attached by a hinge-like structure (phase 3, arrow). After multiple divi­sion, new thinner filaments emerge from daughter cells (phase 4, arrows). After the first division event, con­tinued cycles of filament elongation, multiple divi­sion, and daughter filament outgrowth drive exponential growth of a microcolony (phase 5). Scale bars for Top panels and detail panels below are 20 and 5 μm, respectively. Source. Modification by the author: white arrowheads pointing to balloon-shaped cells (see text)

Figure 2.1. Unlike E. coli or B. sub­tilis which grow by adding new cell wall material along their sidewalls, bacteria of the class Actinomycetes grow by tip extension, that is, they incorporate new cell wall material subapical at both cell poles, but not always symmetrical (see here). In contrast, C. ma­truchotii single cells grow exclusively from one cell pole and elongate into a fi­lament that can reach lengths of 30 µm, even 70 µm, under lab conditions. It is unclear how the cells manage to grow from one cell pole only.

Figure 2.2. The filaments reach different lengths, but at one point septation occurs simulta­ne­ously at several sites in one segment of a filament and then spreads over its entire length. Septa are placed at regular distances only in the first septating segment (see Figure 3) and at more varying distances thereafter. Septation does not occur in a zipper-like fashion from one end as it is known for sporulating aerial hyphae of Streptomyces venezuelae (see here).

Figure 2.3. Shortly after septa­tion, the filaments disintegrate almost simultaneously into mul­tiple cells in a division process (SMD that corresponds to that of other Mycobacteria in one particular aspect: cells that separate from each other remain connected by a "hinge" for a short time. In Mycobacteria, this is known and referred to as "V‑snapping." It may be due to their complex cell wall, which requires an orderly separation of the M and AG layers in addi­tion to separation of the peptidoglycan (PG) layer (see here).

Figure 2.4. It appears that out­growth of dividing cells occurs already when they are still con­nected by a hinge, and always as narrow filament at one pole only (it is not possible or useful to distinguish between the old pole and the new pole in a filament that disintegrates into indi­vidual cells). The narrow filaments ap­pear to elongate at different "speeds," which leads to the impression that any remaining syn­chrony of growth and septation/division is lost at this stage. Rarely, tip growth during narrow fila­ment elongation happens so quickly that PG synthesis can hardly keep up, causing the cells to look temporarily like tiny balloons (white arrowheads in Figures 2.4+2.5).

Figure 2.5. Since outgrowing narrow filaments grow to different lengths before they gain in dia­meter again, septate and disintegrate by cell division, and since the outgrowing filaments grow in all directions and there is no parallel alignment of filaments, a richly disordered, "unkempt" shrub structure already results at the stage of a microcolony.

(click to enlarge)
Figure 3. New peptidoglycan is synthesized at one pole of elongating filaments and at the sites of multiple septa developing simultaneously within dividing cells. Maximum intensity projection images ac­quired from a two-color virtual time-lapse experiment, showing a di­viding cell grown in the pres­ence of the fluorescent D-amino acid HADA for 4 h, then washed and grown with NADA for 45 min. The Left panel is the transmitted light image. Bottom image panels are maximum in­ten­sity views of the X, Z plane showing 6 z-slices, representing 1.6 μm of depth. A fluorescence in­ten­sity profile of each FDAA along the cell length is also shown. Source

You can watch the snapshots in Figure 2 as a time-lapse video here, or download the video. What may be even more apparent when watching the movie repeatedly is that the initially nar­row filaments thicken asynchronously during elongation but before the largely simultaneous sep­tum formation and cell division. This successive "thickening" of the filaments may have to do with lagging completion of the complex cell wall of the Mycobacteria (see here).
 

It is interesting to compare the formation of the initially narrow, very fast-growing filaments of C. matru­chotii as observed by Chimileski et al. (2024) with a similar phenomenon found by Jones et al. (2017) for Streptomy­ces venezuelae when it was grown on YPD plates in the pre­sence of S. cerevisiae: "The rate of expansion of the leading edge of these rapidly migrating cells was ten-fold faster than the rate of hyphal tip extension of these fila­mentous bacte­ria! Wow! What's going on? Imaging of the cells using scanning electron microscopy revealed that the morpho­logy of the bacteria in the rapidly expanding areas was drastically changed from typical hyphae, their filaments were much thinner and they did not branch." (as Roberto said here in STC). Although it is not trivial to find realis­ti­cally comparable conditions for a "speed test," the tip ex­tension rate of S. venezuelae's "exploratory hyphae" (90 µm/h) is about five times faster than the tip extension of "narrow fila­ments" of C. ma­truchotii, for which Chimileski et al. (2024) measured rates of over 16 μm/h, which is still five times faster than that of any other Mycobacterium or Corynebacterium.
 

And despite their morphological similarity (narrow width, unbranched), there is a difference: S. venezuelae forms "exploratory hy­phae" when triggered by environmental cues, whereas the formation of narrow filaments by C. ma­tru­chotii occurs under all tested growth conditions and is apparently an integral part of its cell cycle. A further difference is that a number of Streptomyces spp. are capable of forming "explora­tory hy­­phae," whereas filamentous growth has not been observed in other Corynebac­terium spp. to date.
 

Whether the "accelerated growth" of both the thin "exploratory hy­phae" of S. venezuelae and the narrow C. matruchotii fila­ments should be de­scribed as a new, previously uncategorized form of bacterial motility remains to be seen. In any case, the rapid filamentous growth of these non-motile cells leads to a spatio-temporal range expansion that equals that of motile species in efficiency. And since the elongation of the filaments is asynchronous and their lengths vary considerably, the result in the case of Corynebacterium matruchotii is the rather unkempt, disheveled shrub you see in Figure 1.
 

Frontispiece. What looks like a spaghetti tangle at the edge of a colony of C. matruchotii (order Mycobacteriales; family Corynebacteriaceae) is not a result of the unusual cell division of this bacterium that you see in Figure 2 (different scale!). Back in 2019, our colleagues from the Contamination Club found a very similar spaghetti tangle on a leftover Petri dish and named the bacterium "Noodlococcus" (see here an image from their tweet). Sequencing revealed that "Noodlococcus" is another, distantly related actinobacterium, Kocuria rhizophila (or­der Micrococcales; family Micrococcaceae).
 

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