Dividing cells secrete a factor that stimulates growth resumption

During our studies on growth resumption heterogeneity10 we speculated that growing cells produce a signal that stimulates the growth resumption of non-growing cells. To directly test this hypothesis we prepared a conditioned medium and tested its effect on cells that resume growth from stationary phase. Stationary phase cells were washed and resuspended in fresh medium. Half of the culture was immediately centrifuged and the supernatant was sterilized by filtration for use as a control medium. Prepared in this way, the control medium is almost like fresh medium, but also contains possible residual metabolites carried over by cells from stationary phase. The other half were grown until the middle exponential phase (OD (at 600 nm) about 0.5, while stationary phase OD is 1.0) and used for the preparation of conditioned medium. This conditioned medium should still have enough substrates to support growth and also contain factors secreted, if any, by growing cells.

We then took cells from stationary phase and compared their growth resumption in fresh, control, and conditioned media. At first we used our single-cell growth resumption assay10 where cells, carrying two plasmids encoding for fluorescent proteins GFP and Crimson, are grown into stationary phase with Crimson expression induced by arabinose (Fig. 1a). After that the arabinose is removed and a fresh carbon source is added together with IPTG to induce GFP. Cells that resume growth, begin active protein synthesis and initially become GFP-positive and later dilute their Crimson levels by cell division. GFP-positive cells clearly accumulate quicker in conditioned medium (Fig. 1b). The same effect is evident when comparing the optical density of different cultures – the lag phase is shorter in conditioned medium (Fig. 1c).

Figure 1 Conditioned medium stimulates growth resumption of stationary phase E. coli. (a) A scheme for single-cell growth resumption assay on flow cytometer. (b) Stationary phase cells were resuspended either in conditioned medium or control medium and GFP expression was induced. Growth resuming cells are GFP-positive and dividing cells have reduced Crimson content due to dilution by cell division. (c) Stationary phase cells were diluted into fresh medium, and fresh medium mixed with conditioned medium or control medium. Cells were grown in 96-well plate and OD was measured every 15 minutes. Cells exposed to conditioned medium resume growth earlier than cells in fresh medium. (d) Quantification of results in panel b. Δt is the time difference (in minutes) of reaching OD 0.06 (actual reading for 100 μl culture) between test medium and fresh medium. Dilution factor indicates how many times the tested medium was diluted in fresh medium. The average of three independent experiments are shown, error bars indicate standard error of the mean. Full size image

Compared to fresh medium, growth resumption is slightly inhibited in control medium. This is probably due to some inhibitory compounds carried over by stationary phase cells (see the preparation method above). At the same time the growth rate in exponential phase was the same for all cultures, thus indicating that only the growth resumption was affected. For quantification we compared the time it took for different cultures to reach OD 0.06 (measured OD value for 100 μl culture on 96-well plate at 600 nm, chosen to be approximately in the middle of the exponential phase) and calculated the difference between conditioned (or control) medium and the culture grown in fresh medium (Δt). The growth stimulatory effect of the conditioned medium was concentration dependent and still clearly detectable when diluted several times (Fig. 1d). These results demonstrate that conditioned medium has growth resumption promoting activity.

Cell wall derived muropeptides stimulate growth resumption

We tried to purify this activity from conditioned supernatant, but failed to get enough pure material for identification. However, during this process we learned that the molecule that facilitates growth has a relatively low molecular weight, is hydrophilic, and not strongly charged. Because cell wall derived muropeptides (MPs) fit this description and can induce spore germination in Bacillus18, we went on to test if MPs can also stimulate growth resumption in E. coli.

For this, we purified peptidoglycan (PG) from growing E. coli cells using an established method18 and digested it with mutanolysin to obtain individual MPs that consist of a disaccharide (N-acetyl-glucosamine linked to N-acetyl muramic acid) and a short peptide bound to muramic acid (Fig. 2a). When added to fresh medium, these solubilized MPs convey growth resumption stimulating activity whereas undigested PG does not (Fig. 2b).

Figure 2 Muropeptides stimulate growth resumption of E. coli. (a) Schematic representation of peptidoglycan and muropeptide. NAG – N-acetyl-glucosamine, NAM – N-acetyl-muramic acid. (b) Muropeptides (MPs) but not peptidoglycan (PG) can stimulate growth resumption of stationary phase cells in fresh medium. Different amounts of MP or PG were added to recovering cells and the Δt was calculated. The average and standard error of the mean of four independent experiments are shown. (c) Using mass-spectrometry, different MP variants can be detected from conditioned medium, but not from fresh medium. M4N – anhydromuro-tetrapeptide, M3N – anhydromuro-tripeptide, M5N – anhydromuro-pentapeptide, N.D. – not detected. The values shown are an average of two technical replicates. Full size image

In order to allow cell enlargement, peptidoglycan hydrolases cleave the PG sacculus during cell growth to generate PG fragments. The MPs released are usually recovered – both Gram-positive and Gram-negative bacteria have MP recycling systems that transport PG fragments back to their cytoplasm where they can be re-used19,20. However, some MPs escape the transport system and diffuse throughout the growth environment. We analyzed if there are MPs in our conditioned medium. For this, fresh and conditioned medium was concentrated and subjected to UPLC-MSe analysis. We detected anhydro disaccharide with tri-, tetra-, or pentapeptides in the conditioned medium, but not in fresh medium (Fig. 2c). This is in line with previous results21 and further supports our hypothesis that MPs act as a growth resumption signal in the conditioned medium.

It is necessary to observe that the effect of MPs is evident only when the lag phase is long enough. A longer stationary phase, good aeration during the stationary phase, and the use of “non-optimal” carbon sources in the growth resumption medium all prolong the lag phase and expose the effect of MPs. When stationary phase cells are resuspended in favorable media (LB or MOPS glucose), all cells resume growth quickly and the effect of MPs is not detectable on this background.

Bacterial PG is quite well conserved across different taxa and its basic structure is the same in most species22. It is thus reasonable to assume that E. coli growth resumption could also be stimulated by MPs from other species. One noticeable difference between the PG composition from Gram negatives and some Gram positives is the amino acid present at the third position of the peptide stem: Gram negatives, including E. coli, usually have meso-diaminopimelic acid (mDAP) in that position, while most Gram positives contain L-lysine (Lys)22. We prepared MPs from Gram positive bacteria Enterococcus faecalis (contain Lys) and Bacillus subtilis (contain mDAP) and also from Gram negative Pseudomonas aeruginosa (contain mDAP). It turns out that soluble MPs, but not PG, from all of these species can stimulate growth resumption of E. coli cells (Fig. 3a). Furthermore, all of these MPs were also able to stimulate the growth resumption of P. aeruginosa (Fig. 3b). This, together with the fact that conditioned medium from E. coli can induce B. subtilis spore germination18, indicates that MPs are likely to be universal stimulators of growth resumption across several bacterial species.

Figure 3 Cross-species recognition of muropeptides as growth resumption stimulators. (a) Muropeptides from different species stimulate growth resumption of E. coli cells. (b) Muropeptides from different species stimulate growth resumption of P. aeruginosa cells. Representative result of at least three independent experiments is shown. Full size image

The sugar-peptide bond within muropeptides is required to stimulate growth resumption

Digesting PG with mutanolysin results in a mixture of non-crosslinked (monomers) and crosslinked (e.g. dimers and trimers) MPs that can vary in their peptide stem length and composition23. In addition, such a preparation may contain remnants of lipids and proteins associated with PG. To get a better understanding of the activity of different MP variants we purified well-defined structures from the MP mixture and tested their growth resumption properties.

Several of these structures are active in our growth resumption assay (Fig. 4). Disaccharides with peptides that contain 4, 3 or 2 amino acids are all capable of stimulating growth resumption and anhydro forms tend to be more active than their hydrogenated counterparts. Even monosaccharide N-acetyl-muramic acid attached to 4 amino acid peptides (anhydro-muramyl-tetrapeptide) can stimulate growth resumption. In contrast, N-acetyl-muramic acid and tripeptide as separate molecules do not convey growth resumption stimulating activity. This indicates that the linkage between the sugar and peptide is crucial for the growth resumption stimulation activity and, when present, cells can respond to several MP variants.

Figure 4 Sugar – peptide bond is crucial for the MP growth resumption stimulation activity. Different MP variants were purified and tested in a growth resumption assay. Structures with intact sugar-peptide bonds can stimulate growth resumption, but NAM and its tripeptide together as separate molecules cannot. The average of three independent biological replicates is shown, error bars indicate standard error of the mean. Full size image

Muropeptide detection by the cells

E. coli has a well-described MP recycling system that imports, degrades, and recycles anhydromuropeptides released during cell growth19. The genes responsible include ampG (permease), ampD (amidase), and nagZ (N-acetylglucosaminidase). We expected this system to be responsible also for facilitating MP signaling during growth resumption. However, none of the single knockout strains of genes mentioned above had any phenotype in the growth resumption assay and all responded to MP stimulation like wt (Supplementary Fig. S1). This suggests that the primary MP signaling receptor is located either on the cell surface or in the periplasmic space.

In principle, MPs could act as an additional carbon source and stimulate growth resumption by simply providing more food for bacteria. There are two enzymes that allow intermediates of the MP recycling pathway to enter the central metabolism in E. coli: NagB converts glucosamine-6-phosphate to fructose-6-phosphate and MpaA cleaves off mDAP and releases Ala-Glu dipeptide that can be further metabolized19. We tested the knockout strains for both of these genes and found that they respond to MP stimulation like wt (Supplementary Fig. S2). We also found that adding NAM and tripeptide as separate entities does not stimulate growth resumption at all (Fig. 4) and MPs as a lone carbon source cannot support growth (Supplementary Fig. S1).

MPs as a carbon source should also increase growth rate, but this is not the case. Addition of pure MP (M3N) does not affect the doubling time and PG derived MPs actually increase it, probably due to some impurities (Supplementary Fig. S3). These observations, taken together, suggest that MPs do not exert their growth resumption stimulation effect by simply being an extra carbon source.

In the case of Bacillus spores, MPs are detected through eukaryotic-like protein kinase PrkC18. This gene family is, however, only present in Gram positive bacteria24,25 and absent in E. coli. It is thus clear that some new pathway for MP detection must be involved in Gram negative species.

In order to identify a putative signaling pathway we carried out a genetic screen to find mutants that resume growth relatively slowly in the presence of MPs, but with normal speed in the absence of MPs (see material and methods for details). As a result we identified a clone (BW1.3) whose response to MPs has changed (Fig. 5a). BW1.3 cells are more sensitive to MPs at lower concentrations and display a non-monotonous concentration dependency. We sequenced the genome of BW1.3 and identified 2 mutations that result in amino acid changes in two different proteins. In the rpoA gene, which encodes the RNA polymerase alpha subunit, the amino acid valine was changed to alanine at position 287 (V287A) and in the yggW gene, which encodes putative oxidoreductase, the aspartate in position 108 was changed to glutamate (D108E).

Figure 5 Point mutations in rpoA and yggW genes alter MP sensitivity. (a) The strain BW1.3 has an altered sensitivity to MPs. (b) Point mutations in rpoA and yggW genes are responsible for the change in MP sensitivity. The average of three biological replicates is shown, error bars indicate standard error of the mean. Full size image

To validate the role of these mutations we re-introduced these changes to the wt background using the CRMAGE technique26. Mutations were made both as single changes and in combination and the resulting strains were tested in our growth resumption assay. The BW-V287A strain is more sensitive to lower MP concentrations (compared to wt), but the MP effect is dampened at higher concentrations (Fig. 5b). BW-D108E is more similar to wt and the double mutant strain combines the two phenotypes displaying an MP effect with dual maxima. V287A and D108E mutations can recapitulate the BW1.3 phenotype and are thus the cause of the change in MP sensitivity.

We also tested a yggW knockout strain and found it to be very similar to wt in its response to MPs (Supplementary Fig. S2).

While the point mutations in rpoA and yggW genes changed the response to MPs, they did not have a big effect on the growth rate. We measured maximum growth rates for these strains in different media and only the rpoA V287A strain grew a bit slower in MOPS glycerol and MOPS gluconate media (Supplementary Fig. S3).