Temperature-dependent regulation of the Escherichia coli lpxT gene

Sciandrone, Barbara; Forti, Francesca; Perego, S.; Falchi, Federica A.; Briani, Federica

doi:10.1016/j.bbagrm.2019.06.010

Cited by 6 publications

(13 citation statements)

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“…Finally, we observed that tris -phosphorylated lipid A was not enhanced in lpxT overexpressing cells, as shown by MALDI-TOF MS, probably because of feedback regulation mechanisms that limit LpxT activity when the lpxT gene is induced [ 17 , 18 , 21 ]. This result rules against the hypothesis that LpxT toxicity may be due to hyperphosphorylation of lipid A. Contextually, an LpxT H190A variant devoid of catalytic activity [ 19 ] is even more detrimental for growth, at least in solid medium, than the wt protein.…”

Section: Discussionmentioning

confidence: 91%

“…To evaluate the effect of lpxT overexpression, we cloned the lpxT gene into plasmid pGM930 [ 25 ] under the transcriptional control of the araBp promoter, inducible with arabinose (plasmid pLPXT, Figure 2 A) [ 17 ], and analyzed the growth of E. coli BW25113/pLPXT upon lpxT induction.…”

Section: Resultsmentioning

confidence: 99%

“…To estimate the amount of LpxT that inhibited growth, we exploited a pLPXT derivative with an lpxT -sfGFP translational fusion under the araBp promoter (plasmid pLPXT-GFP; Figure 2 A) [ 17 ], which allowed us to quantitate the LpxT sfGFP fusion protein as fluorescence intensity. LpxT sfGFP was previously shown to be correctly targeted to the inner membrane [ 17 ] (see also Figure 2 F). The induction of lpxT -GFP, like that of lpxT , caused growth inhibition between 30 and 60 min p.i.…”

Section: Resultsmentioning

confidence: 99%

“…Total protein extracts were prepared from bacterial cultures as described [ 17 ]. Extraction from culture media was performed by withdrawing 1.5 OD 600 culture samples and removing the intact cells by centrifugation.…”

Section: Methodsmentioning

confidence: 99%

“…Indeed, lpxT transcription initiation is downregulated at 37–42 °C vs. 30 °C, in stationary phase and at high Mg 2+ concentration [ 15 , 17 , 18 ]. The lpxT mRNA stability is also modulated, as the mRNA is quickly destabilized after a temperature upshift from 30 to 37 °C [ 17 ]. The physiological meaning of lpxT regulation in response to temperature changes remains to be established.…”

Section: Introductionmentioning

confidence: 99%

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Overexpression of lpxT Gene in Escherichia coli Inhibits Cell Division and Causes Envelope Defects without Changing the Overall Phosphorylation Level of Lipid A

et al. 2020

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LpxT is an inner membrane protein that transfers a phosphate group from the essential lipid undecaprenyl pyrophosphate (C-55PP) to the lipid A moiety of lipopolysaccharide, generating a lipid A tris-phosphorylated species. The protein is encoded by the non-essential lpxT gene, which is conserved in distantly related Gram-negative bacteria. In this work, we investigated the phenotypic effect of lpxT ectopic expression from a plasmid in Escherichia coli. We found that lpxT induction inhibited cell division and led to the formation of elongated cells, mostly with absent or altered septa. Moreover, the cells became sensitive to detergents and to hypo-osmotic shock, indicating that they had cell envelope defects. These effects were not due to lipid A hyperphosphorylation or C-55PP sequestering, but most likely to defective lipopolysaccharide transport. Indeed, lpxT overexpression in mutants lacking the L,D-transpeptidase LdtD and LdtE, which protect cells with outer membrane defects from osmotic lysis, caused cell envelope defects. Moreover, we found that pyrophosphorylated lipid A was also produced in a lpxT deletion mutant, indicating that LpxT is not the only protein able to perform such lipid A modification in E. coli.

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Section: Discussionmentioning

confidence: 91%

Section: Resultsmentioning

confidence: 99%

Section: Resultsmentioning

confidence: 99%

Section: Methodsmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

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Overexpression of lpxT Gene in Escherichia coli Inhibits Cell Division and Causes Envelope Defects without Changing the Overall Phosphorylation Level of Lipid A

et al. 2020

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Human PNPase causes RNA stabilization and accumulation of R-loops in the Escherichia coli model system

Falchi,

Forti,

Carnelli

et al. 2023

Sci Rep

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Polyribonucleotide phosphorylase (PNPase) is a phosphorolytic RNA exonuclease highly conserved throughout evolution. In Escherichia coli, PNPase controls complex phenotypic traits like biofilm formation and growth at low temperature. In human cells, PNPase is located in mitochondria, where it is implicated in the RNA import from the cytoplasm, the mitochondrial RNA degradation and the processing of R-loops, namely stable RNA–DNA hybrids displacing a DNA strand. In this work, we show that the human PNPase (hPNPase) expressed in E. coli causes oxidative stress, SOS response activation and R-loops accumulation. Hundreds of E. coli RNAs are stabilized in presence of hPNPase, whereas only few transcripts are destabilized. Moreover, phenotypic traits typical of E. coli strains lacking PNPase are strengthened in presence of the human enzyme. We discuss the hypothesis that hPNPase expressed in E. coli may bind, but not degrade, the RNA, in agreement with previous in vitro data showing that phosphate concentrations in the range of those found in the bacterial cytoplasm and, more relevant, in the mitochondria, inhibit its activity.

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Role of the lipid bilayer in outer membrane protein folding in Gram-negative bacteria

Horne

Brockwell

Radford

2020

Journal of Biological Chemistry

115

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β-barrel outer membrane proteins (OMPs) represent the major proteinaceous component of the outer membrane (OM) of Gram-negative bacteria. These proteins perform key roles in cell structure and morphology, nutrient acquisition, colonisation and invasion, and protection against external toxic threats such as antibiotics. To become functional, OMPs must fold and insert into a crowded and asymmetric OM that lacks much freely accessible lipid. This feat is accomplished in the absence of an external energy source and is thought to be driven by the high thermodynamic stability of folded OMPs in the OM. With such a stable fold, the challenge that bacteria face in assembling OMPs into the OM is how to overcome the initial energy barrier of membrane insertion. In this review, we highlight the roles of the lipid environment and the OM in modulating the OMP folding landscape and discuss the factors that guide folding in vitro and in vivo. We particularly focus on the composition, architecture and physical properties of the OM and how an understanding of the folding properties of OMPs in vitro can help explain the challenges they encounter during folding in vivo. Current models of OMP biogenesis in the cellular environment are still in flux, but the stakes for improving the accuracy of these models are high. Since OMP folding is an essential process in all Gram-negative bacteria, and considering the looming crisis of widespread microbial drug resistance, to bring down the powerful, OMP-supported barrier against antibiotics, we must first understand how bacterial cells build it.

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Temperature-dependent regulation of the Escherichia coli lpxT gene

Cited by 6 publications

References 46 publications

Overexpression of lpxT Gene in Escherichia coli Inhibits Cell Division and Causes Envelope Defects without Changing the Overall Phosphorylation Level of Lipid A

Overexpression of lpxT Gene in Escherichia coli Inhibits Cell Division and Causes Envelope Defects without Changing the Overall Phosphorylation Level of Lipid A

Human PNPase causes RNA stabilization and accumulation of R-loops in the Escherichia coli model system

Role of the lipid bilayer in outer membrane protein folding in Gram-negative bacteria

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