MV production is quantitatively altered in bacteria lacking major MV proteins. Dry weight of MVs harvested from WT Salmonella and mutant strains was quantified (mean +/- standard error). *p<0.01 **p<0.001

(A) Size distribution of WT Salmonella MVs; x-axis represents MV size ranges (100 represents 1-100nm²). Compared to WT MVs, size distribution of (B) ompF MVs and (C) ompA MVs is similar (p>0.05; also ompC, nmpC, and ompX MVs, data not shown), while (D) tolA MV size is significantly increased (p<0.001; also pal and tolB MVs, data not shown). (E) WT MVs are released at constricted division septa and (F) along the cell body. (G) MVs are released along the cell body in the absence of OmpA (shown) and LppAB (), and (H) MV release occurs at division septa in tolA (shown), pal (), and tolB (data not shown) mutant strains. (I) Major MV proteins classified by envelope interconnections: Integral OM proteins OmpC, OmpF, OmpX, and NmpC lack extensive connectivity to envelope components, Lpp and OmpA bind PG (OM-PG linked), and Pal, TolB, and TolA form membrane-spanning protein complexes (OM-PG-IM linked). Dark shading represents N-termini, straight lines represent non-covalent interactions, and zig-zag line denotes covalent interaction. Bars = 200nm (except F inset, bar = 100nm), n = number of individual vesicles examined.

(A) MV release along cell body in the lppAB strain is (B) complemented with full length Lpp_1-58, but (C) expression of abundant mutated Lpp_1-57 unable to bind PG retains lppAB mutant MV production. (D) MV release at division septa in pal strain is (E) restored to WT MV release upon expression of full length Pal_1-153, but (F) cannot be complemented by expression of truncated Pal_1-123 unable to bind TolA. Bar = 200nm

Qualitative (A,B) and quantitative (C,D) analyses of membrane integrity. (A,C) WT Salmonella and mutants lacking integral OM proteins (OmpC, OmpF) are resistant to deoxycholate (DOC). OmpA-PG is dispensable for DOC-resistance, whereas loss of Lpp-PG and OM-PG-IM complexes (Pal, TolB, TolA) induces DOC-sensitivity. (B,D) DOC-sensitivity is dependent upon envelope linkages. Expression of full length, but not truncated, proteins complement deletions (complementation with pTolA is partial but significant compared to the tolA mutant; p<0.0001). For (A,B), three 10-fold dilutions shown from left to right. For (C,D), growth on DOC was quantified and adjusted to growth on LB alone; mean % growth +/- standard error is reported from at least three replicate experiments per strain. * p<0.001, ** p<0.0001

(A) Septal-derived MVs, harvested from septate filamentous cells, are significantly larger than WT MVs (p=1.1×10^-92), and (B) non septal-derived MVs, harvested from non-septate filamentous cells, are significantly smaller than WT MVs (p=1.3×10^-49). (C) Large MVs are released at constricted septa (Bar = 5um), and (D) small MVs are cell body derived (Bar=200nm). Arrowheads highlight constricted septa and arrows highlight MV release; n = number of individual vesicles measured.

(A) Whole-cell view, (B) close-up membrane view. Middle panel: Major MV proteins in the WT envelope: Integral OM proteins (dark gray), OM-PG linked proteins (light gray) and OM-PG-IM complex proteins (black). Left panel: Localized envelope remodeling induces release of small MVs at regions of lower density OM-PG connections along cell body. Right panel: Active invagination of IM and PG during cell division causes temporary disruption of septal OM-PG-IM complexes. OM release (MVs) occurs circumferentially at the septum due to lower density OM-PG-IM protein interconnections.