γ-PGA Hydrolases of Phage Origin in Bacillus subtilis and Other Microbial Genomes

Abstract

Poly-γ-glutamate (γ-PGA) is an industrially interesting polymer secreted mainly by members of the class Bacilli which forms a shield able to protect bacteria from phagocytosis and phages. Few enzymes are known to degrade γ-PGA; among them is a phage-encoded γ-PGA hydrolase, PghP. The supposed role of PghP in phages is to ensure access to the surface of bacterial cells by dismantling the γ-PGA barrier. We identified four unannotated B. subtilis genes through similarity of their encoded products to PghP; in fact these genes reside in prophage elements of B. subtilis genome. The recombinant products of two of them demonstrate efficient polymer degradation, confirming that sequence similarity reflects functional homology. Genes encoding similar γ-PGA hydrolases were identified in phages specific for the order Bacillales and in numerous microbial genomes, not only belonging to that order. The distribution of the γ-PGA biosynthesis operon was also investigated with a bioinformatics approach; it was found that the list of organisms endowed with γ-PGA biosynthetic functions is larger than expected and includes several pathogenic species. Moreover in non-Bacillales bacteria the predicted γ-PGA hydrolase genes are preferentially found in species that do not have the genetic asset for polymer production. Our findings suggest that γ-PGA hydrolase genes might have spread across microbial genomes via horizontal exchanges rather than via phage infection. We hypothesize that, in natural habitats rich in γ-PGA supplied by producer organisms, the availability of hydrolases that release glutamate oligomers from γ-PGA might be a beneficial trait under positive selection.

Fig 1. Clustal alignment of B. subtilis gene products showing similarity to Bacillus phage ΦNIT1 PghP.

Black triangles below the sequences point to residues involved in Zn coordination according to the PghP structure [31, 32]. The valine residues enclosed in a rectangle in YoqZ and YndL sequences were transformed in the initial methionine in the recombinant proteins. An * (asterisk) in the clustal consensus line indicates positions which have a single, fully conserved residue. A: (colon) indicates conservation between groups of amino acids with strongly similar properties. A. (period) indicates conservation between groups of amino acids with weakly similar properties.

Fig 2. Purification of YndL and YoqZ.

His-tagged YndL and YoqZ were purified from the insoluble E. coli lysate fraction using Ni-NTA agarose beads under denaturing conditions as described in Material and Methods. Lanes 1 and 6: 12 μL flow-through fractions; lanes 2 and 5: 1 μL beads; lanes 3 and 4: 6 μL eluted proteins. Lanes 1–3 refer to YndL purification; lanes 4–6 refer to YoqZ purification. Molecular weight markers are indicated on the left.

Fig 3. Degradation of B. subtilis γ-DL-PGA by YndL and YoqZ.

A. B. subtilis γ-DL-PGA incubated with 0.045 μg YndL (lanes 2–5) or 0.009 μg YoqZ (lanes 7–10) at 37°C before separation on an agarose gel. Reactions were stopped after 1’ (lanes 2 and 7), 5’ (lanes 3 and 8), 10’ (lanes 4 and 9) and 20’ (lanes 5 and 10) by heating at 95°C for 3 min. Control reactions in lanes 1 and 6 were incubated for 20’ in the same conditions without enzyme. B. B. subtilis γ-DL-PGA was incubated at 37°C for 60’ with 0.5 μg YndL (lanes 2–5) or 0.5 μg YoqZ (lanes 6–9) with the addition of 10 mM (lanes 2 and 6), 50 mM (lanes 3 and 7), 100 mM (lanes 4 and 8) and 200 mM (lanes 5 and 9) EDTA. No enzyme was added in the control reaction in lane 1.

Fig 4. Resistance to enzymatic treatment of the B. anthracis capsule.

B. anthracis cells were grown under conditions that allowed (B) or did not allow (A) capsule production. Encapsulated cells were either incubated for 10 min at 37°C with YoqZ in digestion buffer (C) or with buffer alone (D) before staining (original magnification 100x).

Fig 5. B. anthracis γ-D-PGA is not a substrate for YndL and YoqZ.

B. subtilis γ-DL-PGA (2 μL in lanes 1–3), B. anthracis γ-D-PGA (2.5 μL lanes 4–6) or a mixture of both (2+2.5 μL in lanes 7–9) were incubated at 37°C for 60’ in the absence (lanes 1, 4, 7) or in the presence of 0.5 μg YndL (lanes 2, 5, 8) or 0.5 μg YoqZ (lanes 3, 6, 9). Enzymatic activity was stopped by heating at 95°C for 3 min before gel separation.

Fig 6. Relative distribution of pghP in bacteria and archaea genomes with respect to pgsB.

In the Venn diagram green circles represent species that contain at least one pghP-like gene; violet circles represent species that contain pgsB. Numbers inside circles refer to the number of species containing either pghP or pgsB. Species that contain both genes are represented by the overlapping region in black (number inside). For each group (namely Archaea, total Bacteria, Bacillales, non-Bacillales) the size of circles and their overlapping region is drawn to scale. The raw data are available in the worksheet “Table of PghP and PgsB” contained in the S1 Table.