Teach­ers Cor­ner: Who bears the load?

by Roberto

First, let me com­ment on the ti­tle I wrote. It should have read, what bears the load? Bet­ter yet, what part of the bac­te­r­ial cell en­ve­lope is the load-bear­ing com­po­nent? When­ever I taught bac­te­r­ial cell bi­ol­ogy I al­ways started by de­scrib­ing the com­po­nents of cell en­ve­lope, cy­to­plas­mic mem­brane, cell wall and, in the Gram-neg­a­tives, the outer mem­brane. Then I would in­vari­ably pro­nounce that the cell wall, with its re­mark­able mesh of co­va­lently linked gly­can strands and pep­tide crosslinks, was the rigid load-bear­ing en­ve­lope com­po­nent. Just look­ing at the struc­ture sug­gested it was a stiff mol­e­cule able to bear the me­chan­i­cal stresses that a bac­terium might en­counter in its chang­ing en­vi­ron­ments. Right? Well, not quite… Ap­par­ently, for Gram-neg­a­tives, the outer mem­brane is a very im­por­tant load-bear­ing com­po­nent of the cell en­ve­lope. That is the find­ing that was re­ported in 2018 in a pa­per whose first au­thor is En­rique Ro­jas, who worked with Julie The­riot and K.C. Huang at Stan­ford and is now in the fac­ulty at NYU. The pa­per presents the re­sults of a col­lab­o­ra­tion that in­volved not only those labs but also groups at UCSF and Madi­son. Over­all, I found the re­sults quite com­pelling and they cer­tainly changed the way I view the stiff­ness of the Gram-neg­a­tive cell en­ve­lope.

Fig­ure 1. De­ter­gent treat­ment af­ter plas­mo­ly­sis causes fur­ther con­trac­tion of the Gram-neg­a­tive cell wall. a, Model of the cell wall–ou­t­er mem­brane com­plex as par­al­lel lin. springs with spring con­stants k_cw, k_om, and rest lengths l_cw, l_om. b, E. coli cells (turgid, plas­mol­ysed (Plasm.), lysed) stained with WGA-488 and FM 4–64. The white ar­row in­di­cates resid­ual phase sig­nal af­ter ly­sis. Im­ages are rep­re­sen­ta­tive of 84 cells from 3 ex­per­iments. Outer mem., outer mem­brane. c, Left, cell-wall length ver­sus time dur­ing hyper­osmotic shock and treat­ment with deter­gent. Data are rep­resen­tative of 84 cells. The red ar­row indic­ates sharp swell­ing upon ly­sis. Right, a model of the turgid, plas­molysed and lysed cel­lular states. d–f, His­tograms of length con­traction upon plas­molysis (d, n = 79 cells), ly­sis (e, n = 56 cells) and in to­tal (f, n = 56 cells). Cir­cle and er­ror bars in­dicate the mean ± s.d. Source

The re­searchers were led to think­ing that the outer mem­brane was rather stiff based on prior ob­ser­va­tions in­di­cat­ing that the mo­tion of outer mem­brane pro­teins is con­strained. So, they asked, which is stiffer, the cell wall or the outer mem­brane? To ad­dress the ques­tion, they mea­sured the change in the length of the cell wall go­ing from a fully turgid cell (high tur­gor pres­sure due to high os­mo­lar­ity in­side the cell) to plas­molyzed cell (due to the hy­per­os­motic shock of be­ing placed in 3M sor­bitol) to a cell with­out outer mem­brane and lysed (through the ad­di­tion of 5% lau­royl sar­co­sine, a strong an­ionic de­ter­gent). Quite sur­pris­ingly, the cell wall con­tracted a to­tal of 25±8.6% (in some cases the cell wall con­tracted 50%!). By cal­cu­lat­ing the ex­pected length of cells lack­ing cell wall (di­gested with lysozyme) they found that the rest length was the same as the length of the fully turgid cell. From their mea­sure­ments they were also able to cal­cu­late the rel­a­tive stiff­ness of both the cell wall and the outer mem­brane. Here again, their re­sults were sur­pris­ing. Un­der their con­di­tions the outer mem­brane was stiffer than the cell wall! Us­ing both chem­i­cal and ge­netic means, they al­tered the com­po­si­tion of the outer mem­brane. They con­clude that both the lipolysac­cha­ride and outer mem­brane pro­teins con­tribute to the load-bear­ing ca­pac­ity of the outer mem­brane. All in all, I found these re­sults forced me into a new way of think­ing about the Gram-neg­a­tive cell en­ve­lope, a real eye-opener.