Abstract
The BAM is a macromolecular machine responsible for the folding and the insertion of integral proteins into the outer membrane of diderm Gram-negative bacteria. In Escherichia coli, it consists of a transmembrane β-barrel subunit, BamA, and four outer membrane lipoproteins (BamB-E). Using BAM-specific antibodies, in E. coli cells, the complex is shown to localize in the lateral wall in foci. The machinery was shown to be enriched at midcell with specific cell cycle timing. The inhibition of septation by aztreonam did not alter the BAM midcell localization substantially. Furthermore, the absence of late cell division proteins at midcell did not impact BAM timing or localization. These results imply that the BAM enrichment at the site of constriction does not require an active cell division machinery. Expression of the Tre1 toxin, which impairs the FtsZ filamentation and therefore midcell localization, resulted in the complete loss of BAM midcell enrichment. A similar effect was observed for YidC, which is involved in the membrane insertion of cell division proteins in the inner membrane. The presence of the Z-ring is needed for preseptal peptidoglycan (PG) synthesis. As BAM was shown to be embedded in the PG layer, it is possible that BAM is inserted preferentially simultaneously with de novo PG synthesis to facilitate the insertion of OMPs in the newly synthesized outer membrane.
Highlights
The cell envelope of Gram-negative bacteria is a tripartite structure that offers protection and resistance against external insults, such as detergents, toxins, and antibiotics [1]
We revealed the spatiotemporal topography of the barrel assembly machinery (BAM) complex in E. coli cells, and its dependence on the presence of the Z-ring
The BAM machinery was immunolabelled with antibodies generated against the isolated BAM complex or with antibodies against its single subunits and found to exhibit a striking enrichment at the division site
Summary
The cell envelope of Gram-negative bacteria is a tripartite structure that offers protection and resistance against external insults, such as detergents, toxins, and antibiotics [1] This selective barrier is composed of an inner membrane (IM), a peptidoglycan (PG) layer, and an outer membrane (OM) [2]. BamA is the essential core of the machinery It is a β-barrel itself that forms the protein–lipid interface, and it contains a periplasmic domain composed of five extensible POTRA (polypeptide transport-associated) repeats. The BAM is still present at midcell in the absence of the late cell division proteins FtsQ, PBP3 and FtsN, at the constriction site. 2021, 22, 12101 similar cell division timing as BAM, and its midcell localization is dependent on the presence of FtsZ. Secapleobtearnitsieaqlucaal vtoe2aμt mth. at this midcell occurrence is erroneous, due to the putative higher membrane content during constriction, the BAM signal was compared to a generaTl omadedmrebsrsatnhee psotateinntiinagl c. aTvehaet twhaitldth-tisypmeidscterllaoinccuwrraesncgerioswernrontoeosutse,adduye tsotathtee, fixed, immptuounatoagtliaevbneeehrllaiegldhmewermmitbhermaαnbBeraAsntMaeinc,oiannngte.dnTsthtdeauiwnrieinlddg-twcyopinteshtsrttirhcateiionlniwp, tiahdsegdBryoAewMBn ostiodgnisptaeyla-wdCya1ss2tca.otBmeo,pfitahxreetddh,e BAM and timhemBuondoliapbye-lCled12wsiitghnαaBlAsMho, wanda sctoaninsetdanwtitchonthceelniptridatdioyne Boofdfilpuyo-rCe1s2c.eBnocteh athseaBfAuMnction of the caenldl tdhievBisoidoinpyc-yCc1l2esiaggneal (sFhiogwuraeco3nAst)a. nHt coownceevnetrra, tiwonheonf fltuhoereBscAenMce aasnadfuthnectiloinpid dye fluoroefsctheencelladvievriasigoencceylcllpe raogfeil(eFsigwuerere3Abi)n. nHeodwienve1r0, wdihfefenrtehnetBaAgeMcalansdsethse(1li0p–id10d0y%e), BAM showflsucolreeascremnciedacveelrlalgoecacelilzl aptrioofinle,sthwaetrebebcinonmedesinv1is0idbilfefearreonut angde 4cl0a%sseosf(t1h0–e1c0e0l%l d),iBvAisMion cycle age, swhohwilse ctlheaerBmoiddcipelyl -loCc1a2lizdaotioens,nthoatt(bFeicgoumrees3vBis)i.bTlehairsoruensdu4l0t %coonfftihremcseltlhdeivsispioencicfyicciltey of the αBAaαMgBeAm, wMidhmicleeidltlchefellulBfloourdoeiprsecyse-cCne1nt2tssidgiognenasalln..oIItnn(cFcoiognnucclruleus3isoBin)o.,nTin,hiiEns.rEceos.luic,loitmlic,ominmufinrmomluasbnteholelliasnbpgeeccloliifinncfigirtymcoosfnthtfhiaretms that all BAallMBAsuMbsuunbiutsniatsccauccmumuulalateteaatttthhee cceenntteerroof fthtehceeclledlludriungrisnegptsaetipotna,tpioronb,apbrlyobreaflbelcytirnegflecting midcmelildlcoelclaloliczaalitzioatnioonfotfhteheeennttiirree aasssseemmbbleldedBABMAMcomcopmlexp.lex
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