Transcription‐ and translation‐dependent changes in membrane dynamics in bacteria: testing the transertion model for domain formation

Abstract

SummaryCell cycle events have been proposed to be triggered by the formation of membrane domains in the process of coupled transcription, translation and insertion ('transertion') of nascent membrane and exported proteins. Disruption of domain structure should lead to changes in membrane dynamics. Membrane viscosity of Escherichia coli and Bacillus subtilis decreased after inhibition of protein synthesis by chloramphenicol or puromycin, or of RNA initiation by rifampicin, but not after inhibition of RNA elongation by streptolydigin or amino acid starvation of a stringent strain. The decrease caused by inhibitors of protein synthesis was prevented by streptolydigin if added simultaneously, but was not reversed if added later. The drug-induced decrease in membrane viscosity is energy dependent: it did not happen in KCN-treated cells. All treatments decreasing membrane viscosity also induced nucleoid compaction and fusion. Inhibition of macromolecular synthesis without membrane perturbation caused nucleoids to expand. Changes in membrane dynamics were also displayed during a nutritional shift-down transition that causes imbalance in macromolecular syntheses. The results are consistent with the transertion model, predicting dissipation of membrane domains by termination of protein synthesis or detachment of polysomes from DNA; domain structure is conserved if the transertion process is 'frozen'.

Compaction of the Escherichia coli nucleoid caused by Cyt1Aa

“…This run-out releases DNA from the membrane resulting in nucleoid compaction, as observed under treatment with CAM (Binenbaum et al, 1999). This suggestion is consistent with nucleoid expansion in cells treated with protamin (Fig.…”

“…Apparent membrane viscosity and dynamics of cells were inferred from measurements of fluorescence anisotropy of 1,3-diphenyl-1,3,5-hexatriene (DPH), following the general procedure described previously (Zaritsky et al, 1985;Parola et al, 1990;Binenbaum et al, 1999). Steady-state values were measured at 37 uC by spectrofluorometry (LS50B; Perkin Elmer) using excitation and emission wavelengths of 360 and 430 nm, and 2?5 and 7?0 nm slits, respectively, in the 'Read' mode, with a 3 s integration time.…”

“…Coupled transcription, translation and insertion ('transertion') of the nascent membrane and exported proteins are considered to expand the nucleoid (Norris, 1995;Woldringh et al, 1995). A compact nucleoid in the cell's centre and a reduced number of membrane attachment sites induced by rifampicin or CAM (Dworsky & Schaechter, 1973;Morgan et al, 1967;Van Helvoort et al, 1998;Zusman et al, 1973) have been explained by disrupting this coupling and causing an imbalance favouring the compaction forces (Binenbaum et al, 1999). Hence, nucleoid compaction by Cyt1Aa can occur by disrupting the DNA-membrane interactions.…”

“…Compaction of nucleoids caused by amino acid starvation of the relA1 mutant that does not occur in its isogenic stringent strain was explained (Binenbaum et al, 1999) by run-out of mRNA releasing the transertion elements rather than freezing them, as in the wild-type. Amino acid starvation would thus result in leakage of amino acids through the membrane pores formed by Cyt1Aa.…”

The bacterial nucleoid: A highly organized and dynamic structure

“…Its movement along the template strand, therefore, transiently generates domains of positive and negative supercoiling, respectively, ahead and behind the transcriptional bubble [Wu et al, 1988;Rahmouni and Wells, 1992;Moulin et al, 2005]. These topological changes are significantly stabilized if the transcript encodes a membrane protein [Liu and Wang, 1987;Lodge et al, 1989;Lynch and Wang, 1993], because transcription and co-transcriptional translation are coupled with the insertion of nascent polypeptides into the membrane in bacteria, thereby tethering RNA polymerase to the site of protein translocation [Binenbaum et al, 1999]. This makes the transcription complex an efficient barrier to the diffusion of supercoils between the DNA regions upstream and downstream of the transcriptional bubble.…”