Acquisition of VanB-type vancomycin resistance by Bacillus subtilis: the impact on gene expression, cell wall composition and morphology

The vancomycin resistance operons from Enterococci, Staphylococci and Actinomycetes encode a VanRS two-component signal transduction system (TCS) and a suite of enzymes to modify the peptidoglycan biosynthetic precursor lipid II and to eliminate the D-Ala-D-Ala from the cell. Commingling of these regulatory and enzymatic activities with host functions has the potential to significantly impact host gene expression and cell wall metabolism. Here we report the effects of individually expressing the VanR(B) S(B) TCS and the VanY(B) WH(B) BX(B) resistance proteins in Bacillus subtilis. VanY(B) WH(B) BX(B) expression confers resistance to 2 μg ml(-1) of vancomycin with concomitant reduced Van-FL staining and leads to a cell division defect. In contrast to E. faecalis and S. aureus, VanS(B) is active in B. subtilis without vancomycin addition. Individual expression of the VanR(B) S(B) TCS and the VanY(B) WH(B) BX(B) resistance proteins repress and increase, respectively, expression of PhoPR regulon genes in the phosphate-limited state. When vancomycin-resistant cells are exposed to elevated vancomycin levels, mutant strains with increased resistance to vancomycin and a growth dependency on vanY(B) WH(B) BX(B) expression frequently arise. Mutation of the endogenous Ddl ligase is the necessary and sufficient cause of both phenotypes. We discuss how these effects may influence establishment of van operons in new host bacteria.     

AlgR-binding sites within the algD promoter make up a set of inverted repeats separated by a large intervening segment of DNA.

Activation of algD by AlgR is essential for mucoidy, a virulence factor expressed by Pseudomonas aeruginosa in cystic fibrosis. Two AlgR-binding sites, RB1 and RB2, located far upstream from the algD mRNA start site, are essential for the high-level activity of algD. However, the removal of RB1 and RB2 does not completely abolish inducibility of algD in response to environmental signals. In this work, a third binding site for AlgR, termed RB3, near the algD mRNA start site was characterized. Deletion of RB3 abrogated both the AlgR-binding ability and the residual inducibility of the algD promoter. DNase I footprinting analysis of RB3 resulted in a protection pattern spanning nucleotides -50 to -30. Eight of 10 residues encompassing a continuous region of protection within RB3 (positions -45 to -36) matched in the inverted orientation the conserved core sequence (ACCGTTCGTC) of RB1 and RB2. Quantitative binding measurements of AlgR association with RB1, RB2, and RB3 indicated that AlgR had significantly lower affinity for RB3 than for RB1 and RB2, with differences in the free energy of binding of 1.05 and 0.93 kcal/mol (4.39 and 3.89 kJ/mmol), respectively. Altering the core of RB2 to match the core of RB3 significantly reduced AlgR binding. Conversely, changing the core of RB3 to perfectly match the core of RB2 (mutant site termed RB3*) improved AlgR binding, approximating the affinity of RB2. RB3*, in the absence of the far upstream sites, showed an increase in activity, approaching the levels observed with the full-size algD promoter. Changing 4 nucleotides in two different combinations within the core of RB3 abolished the binding of AlgR to this site and resulted in a significant reduction of promoter activity in the presence of the far upstream sites. Thus, (i) the core sequence is essential for AlgR binding; (ii) the three binding sites, RB1, RB2, and RB3, are organized as an uneven palindrome with symmetrical sequences separated by 341 and 417 bp; and (iii) all three sites participate in algD activation.     

Co-ordinated regulation of amino sugar biosynthesis and degradation: the NagC repressor acts as both an activator and a repressor for the transcription of the glmUS operon and requires two separated NagC binding sites.

The NagC repressor controls the expression of the divergently transcribed nagE-nagBACD operons involved in the uptake and degradation of the amino sugars, N-acetyl-D-glucosamine (GlcNAc) and glucosamine (GlcN). The glmUS operon, encoding proteins necessary for the synthesis of GlcN (glmS) and the formation of UDP-GlcNAc (glmU), is transcribed from two promoters located upstream of glmU. In the absence of amino sugars both promoters are active. However, in the presence of GlcNAc, the glmU proximal promoter, P1, is inactive while the upstream promoter, P2, is subject to weak induction. Two binding sites for the NagC repressor are located at -200 and -47 bp upstream of P1. Mutations which prevent NagC binding to either of these sites eliminate expression from the P1 promoter. This shows that binding of NagC is necessary for expression of the glmU P1 promoter and implies that NagC is playing the role of activator for this promoter. Moreover, the location of the distal NagC site suggests that this site is behaving like an upstream activating sequence (UAS).