Most bacteria possess a peptidoglycan cell wall that determines their morphology and provides mechanical robustness during osmotic challenges. The biosynthesis of this structure is achieved by a large set of synthetic and lytic enzymes with varying substrate specificities. Although the biochemical functions of these proteins are conserved and well-investigated, the precise roles of individual factors and the regulatory mechanisms coordinating their activities in time and space remain incompletely understood. Here, we comprehensively analyze the autolytic machinery of the alphaproteobacterial model organism Caulobacter crescentus, with a specific focus on LytM-like endopeptidases, soluble transglycosylases, and amidases. Our data reveal a high degree of redundancy within each protein family but also specialized functions for individual family members under stress conditions. In addition, we identify two lytic transglycosylases and an amidase as new divisome components that are recruited to midcell at distinct stages of the cell cycle. The midcell localization of these proteins is affected by two LytM factors with degenerate catalytic domains, DipM and LdpF, which may serve as regulatory hubs coordinating the activities of multiple autolytic enzymes during cell constriction and fission, respectively. These findings set the stage for in-depth studies of the molecular mechanisms that control peptidoglycan remodeling in C. crescentus.This article is protected by copyright. All rights reserved.3

The bacterial plasma membrane is an important cellular compartment. In recent years it has become obvious that protein complexes and lipids are not uniformly distributed within membranes. Current hypotheses suggest that flotillin proteins are required for the formation of complexes of membrane proteins including cell-wall synthetic proteins. We show here that bacterial flotillins are important factors for membrane fluidity homeostasis. Loss of flotillins leads to a decrease in membrane fluidity that in turn leads to alterations in MreB dynamics and, as a consequence, in peptidoglycan synthesis. These alterations are reverted when membrane fluidity is restored by a chemical fluidizer. In vitro, the addition of a flotillin increases membrane fluidity of liposomes. Our data support a model in which flotillins are required for direct control of membrane fluidity rather than for the formation of protein complexes via direct protein-protein interactions.

Curcumin is the main constituent of turmeric, a seasoning popularized around the world with Indian cuisine. Among the benefits attributed to curcumin are anti-inflammatory, antimicrobial, antitumoral, and chemopreventive effects. Besides, curcumin inhibits the growth of the gram-positive bacterium Bacillus subtilis. The anti-B. subtilis action happens by interference with the division protein FtsZ, an ancestral tubulin widespread in Bacteria. FtsZ forms protofilaments in a GTP-dependent manner, with the concomitant recruitment of essential factors to operate cell division. By stimulating the GTPase activity of FtsZ, curcumin destabilizes its function. Recently, curcumin was shown to promote membrane permeabilization in B. subtilis. Here, we used molecular simplification to dissect the functionalities of curcumin. A simplified form, in which a monocarbonyl group substituted the β-diketone moiety, showed antibacterial action against gram-positive and gram-negative bacteria of clinical interest. The simplified curcumin also disrupted the divisional septum of B. subtilis; however, subsequent biochemical analysis did not support a direct action on FtsZ. Our results suggest that the simplified curcumin exerted its function mainly through membrane permeabilization, with disruption of the membrane potential necessary for FtsZ intra-cellular localization. Finally, we show here experimental evidence for the requirement of the β-diketone group of curcumin for its interaction with FtsZ.

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20The bacterial plasma membrane is an important cellular compartment. In recent years 21 it has become obvious that protein complexes and lipids are not uniformly distributed 22 within membranes. Current hypotheses suggest that flotillin proteins are required for the 23 formation of complexes of membrane proteins including cell-wall synthetic proteins. We 24show here that bacterial flotillins are important factors for membrane fluidity 25 homeostasis. Loss of flotillins leads to a decrease in membrane fluidity that in turn leads 26to alterations in MreB dynamics and, as a consequence, in peptidoglycan synthesis. These 27 alterations are reverted when membrane fluidity is restored by a chemical fluidizer. In 28 sufficient to convert spherical cells to a rod shape 8,9 . In Bacillus subtilis, the motion of MreB 54 along the membrane is associated with elongasome activity 10,11 and the velocity of MreB 55 patches is related to growth rate 12 , indicating that MreB motion can be used as a marker for 56 elongasome activity. Interestingly, MreB localizes to and organizes regions of increased 57 membrane fluidity (RIF) 13 , which in turn is linked to the presence of LipidII, which favours a 58 more fluid membrane and promotes local membrane disorder 14,15 . Inhibition of LipidII 59 synthesis by genetic or chemical means results in a dissolution of membrane structures 60 observed with the dye FM 4-64 and release of MreB from the membrane 10,11,16,17 . 61Next to RIFs, membrane regions of decreased fluidity have been identified in bacteria 7,18,19 . 62