Bacillus subtilis operon encoding a membrane receptor for bacteriophage SPP1

Abstract

The results reported here have identified yueB as the essential gene involved in irreversible binding of bacteriophage SPP1 to Bacillus subtilis. First, a deletion in an SPP1-resistant (pha-2) strain, covering most of the yueB gene, could be complemented by a xylose-inducible copy of yueB inserted at amyE. Second, disruption of yueB by insertion of a pMutin4 derivative resulted in a phage resistance phenotype regardless of the presence or absence of IPTG (isopropyl-beta-D-thiogalactopyranoside). YueB homologues are widely distributed in gram-positive bacteria. The protein Pip, which also serves as a phage receptor in Lactococcus lactis, belongs to the same family. yueB encodes a membrane protein of approximately 120 kDa, detected in immunoblots together with smaller forms that may be processed products arising from cleavage of its long extracellular domain. Insertional inactivation of yueB and the surrounding genes indicated that yueB is part of an operon which includes at least the upstream genes yukE, yukD, yukC, and yukBA. Disruption of each of the genes in the operon allowed efficient irreversible adsorption, provided that yueB expression was retained. Under these conditions, however, smaller plaques were produced, a phenotype which was particularly noticeable in yukE mutant strains. Interestingly, such reduction in plaque size was not correlated with a decreased adsorption rate. Overall, these results provide the first demonstration of a membrane-bound protein acting as a phage receptor in B. subtilis and suggest an additional involvement of the yukE operon in a step subsequent to irreversible adsorption.

FIG. 1.

(A) Gene organization of the B. subtilis chromosome region containing yueB. In databases, yukBA (superscript 1) appears to be divided into two different genes (yukB and yukA) due to a sequencing error (see the text). Inverted repeats with the potential to form stem-loop-type structures (ΔG ≤ −10.5) are indicated by circles. Bs-2 and Bs-9 represent primers used for screening deletion events in pha-2 strains. The region covered by the deletion Δ6 found in strain CSJ1 is boxed. The KpnI restriction sites in boldface were used to clone a PCR product harboring the Δ6 deletion boundaries. The numbers of predicted TMDs of putative membrane products are indicated. (B) Predicted membrane topology of Pip and YueB proteins. Putative TMDs are depicted as black rectangles. Coordinates of the amino acid (aa) residues that are expected to face the cell wall are indicated in parentheses.

FIG. 2.

Complementation of the Δ6 mutation. (A) Efficiencies of plating (E.O.P) of phage SPP1 in strain CSJ4 (Δ6) and in its derivative, CSJ6 (carrying a xylose-inducible yueB copy in the amyE locus), under repressed (0.1% glucose [gluc]) or induced (0.5% xylose [xyl]) conditions. Wt, wild-type. (B) Phage plaque morphologies in the different strains. (C) Kinetics of SPP1 irreversible adsorption to the wild-type strain (▪), strain CSJ4 (□), and strain CSJ6 under repressed (▴) or induced (▵) conditions. The adsorption constant values (min⁻¹ · U_OD⁻¹) obtained from each curve are indicated, except for strain CSJ4, for which no significant SPP1 adsorption could be measured.

FIG. 3.

K_ads values, efficiencies of plating (E.O.P.), and β-galactosidase activities measured in the wild-type strain (L16601) and in the different integration mutants in the absence or presence of 1 mM IPTG. A schematic representation of the relevant DNA structure of each integrant is provided (genes not drawn to scale). Disrupted ORFs are represented by interrupted white arrows, while intact genes are depicted as grey arrows. pMutin4-derived elements (spoVG-lacZ, transcriptional terminators, and P_spac promoter) are in black. Putative hairpin-like secondary structures are also indicated (open circles). The indicated K_ads and β-galactosidase activity values are the averages from at least three independent experiments ± standard deviations of the mean, except under conditions where no accurate K_ads measurements (≤0.01) could be performed. E.O.P. values are expressed as the ratio between the phage titer obtained in each mutant strain and that obtained in the wild-type strain (L16601). (*), minute phage plaques could be observed, suggesting an E.O.P almost identical to that observed in the presence (+) of IPTG, but their reduced size did not allow accurate counting.

FIG. 4.

Northern blot analysis of RNA extracted from B. subtilis strains. Lanes: 1, L16601 (wild-type strain); 2, CSJ2 (pha-2 strain); 3, CSJ1 (pha-2 Δ6 strain). Radiolabeled probes correspond to internal PCR products of the indicated genes. The yueB probe is included in the DNA segment deleted in Δ6.

FIG. 5.

SPP1 plaque morphology when plated in the wild-type strain and in the different integration mutants in the absence (−) or presence (+) of 1 mM IPTG.

FIG. 6.

SPP1 irreversible-adsorption constants measured with strain CSJ3 (which carries an IPTG-inducible yueB) as a function of IPTG concentration.

FIG. 7.

Western blot analysis of YueB polypeptides in membrane-enriched protein extracts (see Materials and Methods) obtained from different B. subtilis strains. Lanes: 1, CSJ1 (Δ6); 2, L16601 (wild-type strain); 3, CSJ3 (no IPTG); 4, CSJ3 (1 mM IPTG); 5, CSJ6 (0.1% glucose); 6, CSJ6 (0.5% xylose); 7, overexposed lane 2. The polyclonal serum anti-YueB780 was used at a 1:30,000 dilution.

FIG. 8.

Sequence alignment of a conserved region in YueB-like proteins (bacterial origin provided on the left). A group of 30 sequences, representing the primary structure diversity of this protein family, were compared using the CLUSTALW tool (a representative alignment with only 12 sequences is shown). The positions showing a conservation level of >70% in the 30 sequences analyzed are boxed, while positions with identical, strongly similar, and weakly similar residues are indicated by asterisks, colons, and periods, respectively. The numbers in parentheses refer to the coordinates of each of the regions relative to the whole protein sequence. Predicted secondary structures (α-helices and β-strands) of sequence segments are indicated above the alignment. Protein accession numbers: 1, NP_388897.1; 2, NP_243012.1; 3, NP_267826.1; 4, NP_469996.1; 5, NP_276964.1; 6, NP_781183.1; 7, NP_469397.1; 8, NP_831846.1; 9, NP_692233.1; 10, NP_391064.1; 11, NP_293801.1; 12, NP_765785.1. Bacteria: 1, Bacillus subtilis; 2, Bacillus halodurans; 3, Lactococcus lactis; 4, Listeria innocua; 5, Methanothermobacter thermautotrophicus; 6, Clostridium tetani; 7, L. innocua; 8, Bacillus cereus; 9, Oceanobacillus iheyensis; 10, B. subtilis; 11, Deinococcus radiodurans; 12, Staphylococcus epidermidis.