Surface-layer (S-layer) proteins sap and EA1 govern the binding of the S-layer-associated protein BslO at the cell septa of Bacillus anthracis

Abstract

The Gram-positive pathogen Bacillus anthracis contains 24 genes whose products harbor the structurally conserved surface-layer (S-layer) homology (SLH) domain. Proteins endowed with the SLH domain associate with the secondary cell wall polysaccharide (SCWP) following secretion. Two such proteins, Sap and EA1, have the unique ability to self-assemble into a paracrystalline layer on the surface of bacilli and form S layers. Other SLH domain proteins can also be found within the S layer and have been designated Bacillus S-layer-associated protein (BSLs). While both S-layer proteins and BSLs bind the same SCWP, their deposition on the cell surface is not random. For example, BslO is targeted to septal peptidoglycan zones, where it catalyzes the separation of daughter cells. Here we show that an insertional lesion in the sap structural gene results in elongated chains of bacilli, as observed with a bslO mutant. The chain length of the sap mutant can be reduced by the addition of purified BslO in the culture medium. This complementation in trans can be explained by an increased deposition of BslO onto the surface of sap mutant bacilli that extends beyond chain septa. Using fluorescence microscopy, we observed that the Sap S layer does not overlap the EA1 S layer and slowly yields to the EA1 S layer in a growth-phase-dependent manner. Although present all over bacilli, Sap S-layer patches are not observed at septa. Thus, we propose that the dynamic Sap/EA1 S-layer coverage of the envelope restricts the deposition of BslO to the SCWP at septal rings.

Fig 1

Genetic organization of the S-layer locus of B. anthracis. The chromosomal region of the S-layer locus encodes the predicted polysaccharide protein CsaA, the function of which is unknown; the pyruvyl transferase protein CsaB, which modifies the SCWP to permit SLH-domain-mediated binding; and the two surface-layer proteins Sap and EA1 (eag encoded). The open arrowhead indicates the position of the bursa aurealis transposon insertion. Sap is a two-domain protein comprising the helix-rich SLH domain repeats and a strand-rich passenger domain that is thought to promote monomer crystallization into S layers. Secondary-structure prediction was performed by using PSIPRED, version 2.0 (http://cmr.jcvi.org/cgi-bin/CMR/shared/GenePage.cgi?locus=NTL05BA0826) and by incorporating two feed-forward neural networks, which performed an analysis on the output obtained from PSI-BLAST (Position-Specific Iterated BLAST) (2). The amino acid (AA) and nucleotide (NT) numbers corresponding to the sequence of BAS0841 are displayed at the bottom.

Fig 2

B. anthracis sap variants exhibit a chaining phenotype. (A) Phase micrographs of bacillus chains at exponential and stationary growth phases. (B) Box-and-whisker plots of bacillus chain lengths measured from phase micrographs observed at hourly time intervals. Boxes bound the 25th and 75th percentiles, with the median values indicated by the black bar. Black dots represent first- and fourth-quartile observations. The median chain lengths were as follows for the 3- through 8-h time points: 42.30, 43.58, 28.61, 24.38, 17.92, and 17.17 μm, respectively, for Sterne and 70.91, 84.56, 53.30, 27.17, 25.07, and 22.78 μm, respectively, for the sap mutant (n = 100 data points for each time indicated). (C) Coomassie-stained PAGE gels of Sterne and sap extracts. Total protein extracts from whole cultures (T) or surface proteins (S) were separated by PAGE and stained with Coomassie. Extracts were prepared by using Sterne and the sap mutant with no plasmid (ø) or the sap mutant carrying the complementing plasmid (psap) or an empty vector control (pOS1). White arrowheads point to the band corresponding to Sap. MW, molecular weight (in thousands). (D and E) Phase micrographs and corresponding box-and-whisker plots of bacillus chain lengths. Bacterial cultures of Sterne, the sap mutant, and the sap mutant carrying the complementing plasmid psap or the empty vector control pOS1 were grown for 3 h. Measurements were performed as described above for panel B (n = 50 data points for each sample).

Fig 3

B. anthracis sap mutants resist BslO-catalyzed dechaining and fail to restrict BslO localization. (A) Total protein extracts from whole cultures (T) or surface proteins (S) were separated by PAGE, and proteins were transferred onto PVDF membranes for immunoblotting with anti-BslO polyclonal serum. Extracts were prepared by using strain Sterne and the sap mutant. (B) Box-and-whisker plots of bacillus chain lengths after incubation with rBslO at the indicated concentrations (0, 1, and up to 25 mg/ml). Data were collected by using phase micrographs and as described in the legend of Fig. 2B (n = 100 data points for each concentration). (C) Fluorescence and phase micrographs of Sterne and sap bacilli incubated with 20 mg/ml Alexa Fluor 488-conjugated BslO for 10 min at room temperature. Fluorescence intensities of the axial transects (yellow dashed line) are plotted in the line plots on the right.

Fig 4

Sap patterning excludes BslO and EA1 localization in the S layer of B. anthracis. (A) Phase and fluorescence micrographs of sap::sap-mcherry and eag::eag-mcherry strain variants in which the sap or eag gene was replaced with hybrid constructs, leading to the production of Sap::mCherry and EA1-mCherry, respectively. Both strains were incubated with 10 mg/ml Alexa Fluor 488-conjugated BslO for 10 min at room temperature. In the merged image, Sap- and EA1-mCherry are pseudocolored as red signals, and Alexa 488-BslO is pseudocolored as green signals. (B) Line plots of the normalized fluorescence intensity of each fluorophore as measured from a line scan tracing the perimeter of the bacillus chain from panel A. (C) Scatter plots and axial frequency histograms of mCherry and Alexa Fluor 488 fluorescence (FL) intensity signal pairs. Points represent pixels extracted from line scan traces of chain perimeters for a population of cells (n = 25 per sample).

Fig 5

Sap-mCherry and EA1-mCherry occupy distinct locations in the S layer of B. anthracis vegetative forms. (A) Phase and fluorescence (FL) micrographs of sap::sap-mcherry or eag::eag-mcherry strain variant chains imaged at late exponential (time [T] = 6 h and 9 h) and stationary growth (T = 12 h) phases. (B) Cartoon of representative distribution of Sap-mCherry (left) or EA1-mCherry (right) patch locations, shown in red, with respect to the placement on bacillus chains.

Fig 6

Sap-mCherry and EA1-mBanana S-layer patches in the envelope of B. anthracis exclude each other. (A) Phase and fluorescence micrographs of bacilli coproducing Sap-mCherry and EA1-mBanana. The merged image depicts mCherry and mBanana signals pseudocolored red and green, respectively. (B) Line plots of the normalized fluorescence (FL) intensity of each fluorophore, as measured from a line scan tracing the perimeter of the bacillus chain shown in panel A. (C) Scatter plots and axial frequency histograms of mCherry and mBanana fluorescence (FL) intensity signal pairs. Points represent pixels extracted from line scan traces of chain perimeters for a population of cells (n = 25 per sample).

Fig 7

Sap-mCherry arrays separate during cell wall elongation of B. anthracis vegetative forms. (A) Fluorescence frames from a time-lapse experiment imaging sap::sap-mcherry bacillus growth at the indicated time points. The white arrowhead points to a region of Sap-mCherry patch separation with chain elongation. The red line segment indicates a portion of the cell perimeter (transect) analyzed in panel B. The green-line segment spans the perimeter transect between the local fluorescence intensity maxima of two adjacent Sap-mCherry patches. Corresponding phase micrographs are shown in lower left insets. (B) Fluorescence intensity transects (length of the red line segment shown in panel A) of Sap-mCherry signals. Measurements in micrometers are the Euclidean distances between fluorescence intensity maxima between two adjacent Sap-mCherry patches, depicted by a green-line segment in panel A.