Peptidoglycan Production by an Insect-Bacterial Mosaic

Abstract

Peptidoglycan (PG) is a defining feature of bacteria, involved in cell division, shape, and integrity. We previously reported that several genes related to PG biosynthesis were horizontally transferred from bacteria to the nuclear genome of mealybugs. Mealybugs are notable for containing a nested bacteria-within-bacterium endosymbiotic structure in specialized insect cells, where one bacterium, Moranella, lives in the cytoplasm of another bacterium, Tremblaya. Here we show that horizontally transferred genes on the mealybug genome work together with genes retained on the Moranella genome to produce a PG layer exclusively at the Moranella cell periphery. Furthermore, we show that an insect protein encoded by a horizontally transferred gene of bacterial origin is transported into the Moranella cytoplasm. These results provide a striking parallel to the genetic and biochemical mosaicism found in organelles, and prove that multiple horizontally transferred genes can become integrated into a functional pathway distributed between animal and bacterial endosymbiont genomes.

Graphical abstract

Figure 1

A Complete PG Biosynthesis Pathway Is Predicted by Genomics (A) Schematic representation of a single P. citri bacteriocyte (blue), where Moranella cells and their two lipid bilayers (green) reside inside of triple-membrane-bound Tremblaya cells (yellow). (B) Adapted from Typas et al., 2011. A pictorial representation of the genes present and expressed on the genomes of P. citri (blue represents native eukaryotic genes, brown represents HGTs of Gammaproteobacteria origin, orange represents HGTs of Bacteroidetes origin, and yellow represents Alphaproteobacteria origin) and Moranella (green) that are involved in PG production. The locations of the small colored cell schematics labeled with gene names are based on the predicted location of the protein product of that gene. See also Table S1.

Figure 2

PG Constituent Parts Are Present in Whole Insect Preparations (A) MS/MS spectrum showing fragments of the tetrapeptide disaccharide shown in (B) as annotated by Byonic (Bern et al., 2017). The label HexNAc indicates that GlcNAc cannot be formally distinguished from the stereochemically identical molecule N-acetyl-galactosamine. Pep+ 1+ represents the bare tetrapeptide L-Ala-D-Glu-mDap-D-Ala fragmented from PG glycans, and C₆H₈NO₂ and C₇H₈NO₂ represent rearrangements of HexNAc after neutral losses such as H₂O. (B) Schematic of the reduced β-1-4 linked GlcNAc-MurNAc^R disaccharide attached to a tetrapeptide stem made of L-Ala, D-Glu, mDAP, and D-Ala.

Figure S1

Representative Setup for Rearing and Feeding Experiments Involving P. citri, Related to Figures 2, 3, 4, and 5 Mealybugs (yellow arrows) were allowed to feed and reproduce on sprouted potatoes. Where treatments were applied, potatoes (often focusing on the sprouts) were covered and/or injected with 1 mL of the given treatment (modified D- or L-ala, cefsulodin, or water). Mealybugs were carefully placed on the potato with a paintbrush and allowed to eat ad libitum. Treatments were repeated each day for one week.

Figure 3

The PG-Specific Molecule D-Ala Is Specifically Localized at the Moranella Periphery (A) FISH imaging of a sectioned bacteriome from a mealybug treated with ¹⁵N D-Ala. Tremblaya cells are green, Moranella cells are yellow-red, and the insect nucleus is blue. Scale bar, 10 μm. (B) Reconstructed montage of a bacteriocyte from multiple nanoSIMS images shown as a heatmap of the fractional abundance of ¹⁵N over ¹⁴N [¹⁵N/(¹⁵N + ¹⁴N)] from the same mealybug section depicted in (A); ¹⁵N enrichment is observed as yellow rings around the edges of Moranella cells. (C) Close-up of a single Moranella cell highlighted in the red box in (B) shows enrichment of ¹⁵N D-Ala along Moranella’s periphery. (D) Three-dimensional representation of ¹⁵N D-Ala enrichment of the Moranella cell shown in (C). (E) A cross section through a portion of the bacteriocyte (black rectangle in B) reveals less ¹⁵N enrichment in either Tremblaya (green) or the insect tissue (blue) as compared with Moranella (orange). The row labeled ¹⁵F is the fractional abundance of ¹⁵N over ¹⁴N [¹⁵N/(¹⁵N + ¹⁴N)], the ¹⁴N/¹²C row depicts the ratio of the abundant natural isotope ¹⁴N over the common isotope ¹²C, and the bottom row shows this section of tissue in FISH microscopy. (F) FISH imaging of a sectioned bacteriome from a control mealybug treated with ¹⁵N L-Ala. Tremblaya cells are red, Moranella cells are green, and insect nuclei are blue. (G) The fractional abundance of ¹⁵N L-Ala as detected by nanoSIMS from the portion of (F) outlined in the pink box. The signal of ¹⁵N L-Ala is nearly uniform throughout the three organisms represented in this tissue, as expected for a molecule that is uniformly incorporated into protein. (H) Representative confocal image of Cu-catalyzed click-chemistry to a D-Ala variant showing enrichment at the Moranella periphery (green). Insect nuclei are stained with DAPI (blue). Image is comprised of four merged slices from a z stack. Scale bar, 10 μm.

Figure 4

A PG-Targeting Antibiotic Specifically Affects the Moranella Cell Envelope (A) Quantification of the distance between the inner- and outer-most membranes of Tremblaya (n = 200) and Moranella (n = 400) and the inner two membranes of Tremblaya (n = 200) from control and cefsulodin-treated insects; mean ± SEM with a jitter plot of all data points. All data points were collected from random sections from two independent biological replicates. There is a significant difference only in the periplasmic space of Moranella, 11.5 versus 26.1 nm (Student’s t test) with an effect size of 2.3. The effect sizes for Tremblaya’s inner-only and inner-to-outer measurements were 0.0016 and 0.034, respectively. (B and C) Representative TEM images of control (B) and 100 μg/mL cefsulodin-treated (C) insects with Tremblaya (blue arrow) and Moranella (red arrow) membranes visible. See also Table S3.

Figure 5

MurF, a PG-Related HGT of Alphaproteobacterial Origin, Is Localized to the Moranella Cytoplasm Representative confocal image of a sectioned bacteriome stained with an anti-MurF antibody (red). Insect nuclei are stained with Hoechst (blue). Signal is detected inside of the Moranella cells and insect tissue, but not Tremblaya. Scale bar, 10 μm.