The lipoprotein DolP affects cell separation in Escherichia coli, but not as an upstream regulator of NlpD

Gabriela Boelter; Jack A Bryant; Hannah Doherty; Peter Wotherspoon; Dema Alodaini; Xuyu Ma; Micheal B Alao; Patrick J Moynihan; Danesh Moradigaravand; Monika Glinkowska; Timothy J Knowles; Ian R Henderson; Manuel Banzhaf

doi:10.1099/mic.0.001197

The lipoprotein DolP affects cell separation in Escherichia coli, but not as an upstream regulator of NlpD

Gabriela Boelter, Jack A Bryant, Hannah Doherty, Peter Wotherspoon, Dema Alodaini, Xuyu Ma, Micheal B Alao, Patrick J Moynihan, Danesh Moradigaravand, Monika Glinkowska, Timothy J Knowles, Ian R Henderson, Manuel Banzhaf

Research output: Contribution to journal › Article › peer-review

6 Downloads (Pure)

Abstract

Bacterial amidases are essential to split the shared envelope of adjunct daughter cells to allow cell separation. Their activity needs to be precisely controlled to prevent cell lysis. In Escherichia coli, amidase activity is controlled by three regulatory proteins NlpD, EnvC and ActS. However, recent studies linked the outer membrane lipoprotein DolP (formerly YraP) as a potential upstream regulator of NlpD. In this study we explored this link in further detail. To our surprise DolP did not modulate amidase activity in vitro and was unable to interact with NlpD in pull-down and MST (MicroScale Thermophoresis) assays. Next, we excluded the hypothesis that Δ dolP phenocopied Δ nlpD in a range of envelope stresses. However, morphological analysis of double deletion mutants of amidases (AmiA, AmiB AmiC) and amidase regulators with dolP revealed that Δ amiAΔ dolP and Δ envCΔ dolP mutants display longer chain length compared to their parental strains indicating a role for DolP in cell division. Overall, we present evidence that DolP does not affect NlpD function in vitro, implying that DolP is not an upstream regulator of NlpD. However, DolP may impact daughter cell separation by interacting directly with AmiA or AmiC, or by a yet undiscovered mechanism.

Original language	English
Article number	001197
Journal	Microbiology
Volume	168
Issue number	5
DOIs	https://doi.org/10.1099/mic.0.001197
Publication status	Published - 23 May 2022

Bibliographical note

Funding Information:
G.B. and M.A were supported by a PhD studentship from the Darwin Trust of Edinburgh. M.B was supported by a UKRI Future Leaders Fellowship [MR/V027204/1]. H.D. was supported by the Wellcome Trust AAMR DTP. M.G. is supported by the National Science Centre Poland [UMO-2017/27/B/ NZ2/00747].

Publisher Copyright:
© 2022 The Authors.

Keywords

Amidohydrolases/genetics
Bacterial Proteins/genetics
Cell Separation
Escherichia coli/metabolism
Escherichia coli Proteins/genetics
Lipoproteins/genetics
Peptidoglycan/metabolism
bacterial cell division
peptidoglycan
amidases
Gram-negative cell envelope
DolP

ASJC Scopus subject areas

Microbiology

Access to Document

10.1099/mic.0.001197Licence: Creative Commons: Attribution (CC BY)

Boelter2022_the_Lipoprotein_DolPFinal published version, 2.03 MBLicence: Creative Commons: Attribution (CC BY)

Cite this

Boelter, G., Bryant, J. A., Doherty, H., Wotherspoon, P., Alodaini, D., Ma, X., Alao, M. B., Moynihan, P. J., Moradigaravand, D., Glinkowska, M., Knowles, T. J., Henderson, I. R., & Banzhaf, M. (2022). The lipoprotein DolP affects cell separation in Escherichia coli, but not as an upstream regulator of NlpD. Microbiology, 168(5), [001197]. https://doi.org/10.1099/mic.0.001197

@article{404350ff7c444af1af8dffc54ccde1dc,

title = "The lipoprotein DolP affects cell separation in Escherichia coli, but not as an upstream regulator of NlpD",

abstract = "Bacterial amidases are essential to split the shared envelope of adjunct daughter cells to allow cell separation. Their activity needs to be precisely controlled to prevent cell lysis. In Escherichia coli, amidase activity is controlled by three regulatory proteins NlpD, EnvC and ActS. However, recent studies linked the outer membrane lipoprotein DolP (formerly YraP) as a potential upstream regulator of NlpD. In this study we explored this link in further detail. To our surprise DolP did not modulate amidase activity in vitro and was unable to interact with NlpD in pull-down and MST (MicroScale Thermophoresis) assays. Next, we excluded the hypothesis that Δ dolP phenocopied Δ nlpD in a range of envelope stresses. However, morphological analysis of double deletion mutants of amidases (AmiA, AmiB AmiC) and amidase regulators with dolP revealed that Δ amiAΔ dolP and Δ envCΔ dolP mutants display longer chain length compared to their parental strains indicating a role for DolP in cell division. Overall, we present evidence that DolP does not affect NlpD function in vitro, implying that DolP is not an upstream regulator of NlpD. However, DolP may impact daughter cell separation by interacting directly with AmiA or AmiC, or by a yet undiscovered mechanism. ",

keywords = "Amidohydrolases/genetics, Bacterial Proteins/genetics, Cell Separation, Escherichia coli/metabolism, Escherichia coli Proteins/genetics, Lipoproteins/genetics, Peptidoglycan/metabolism, bacterial cell division, peptidoglycan, amidases, Gram-negative cell envelope, DolP",

author = "Gabriela Boelter and Bryant, {Jack A} and Hannah Doherty and Peter Wotherspoon and Dema Alodaini and Xuyu Ma and Alao, {Micheal B} and Moynihan, {Patrick J} and Danesh Moradigaravand and Monika Glinkowska and Knowles, {Timothy J} and Henderson, {Ian R} and Manuel Banzhaf",

note = "Funding Information: G.B. and M.A were supported by a PhD studentship from the Darwin Trust of Edinburgh. M.B was supported by a UKRI Future Leaders Fellowship [MR/V027204/1]. H.D. was supported by the Wellcome Trust AAMR DTP. M.G. is supported by the National Science Centre Poland [UMO-2017/27/B/ NZ2/00747]. Publisher Copyright: {\textcopyright} 2022 The Authors.",

year = "2022",

month = may,

day = "23",

doi = "10.1099/mic.0.001197",

language = "English",

volume = "168",

journal = "Microbiology",

issn = "1350-0872",

publisher = "Society for General Microbiology",

number = "5",

}

TY - JOUR

T1 - The lipoprotein DolP affects cell separation in Escherichia coli, but not as an upstream regulator of NlpD

AU - Boelter, Gabriela

AU - Bryant, Jack A

AU - Doherty, Hannah

AU - Wotherspoon, Peter

AU - Alodaini, Dema

AU - Ma, Xuyu

AU - Alao, Micheal B

AU - Moynihan, Patrick J

AU - Moradigaravand, Danesh

AU - Glinkowska, Monika

AU - Knowles, Timothy J

AU - Henderson, Ian R

AU - Banzhaf, Manuel

N1 - Funding Information: G.B. and M.A were supported by a PhD studentship from the Darwin Trust of Edinburgh. M.B was supported by a UKRI Future Leaders Fellowship [MR/V027204/1]. H.D. was supported by the Wellcome Trust AAMR DTP. M.G. is supported by the National Science Centre Poland [UMO-2017/27/B/ NZ2/00747]. Publisher Copyright: © 2022 The Authors.

PY - 2022/5/23

Y1 - 2022/5/23

N2 - Bacterial amidases are essential to split the shared envelope of adjunct daughter cells to allow cell separation. Their activity needs to be precisely controlled to prevent cell lysis. In Escherichia coli, amidase activity is controlled by three regulatory proteins NlpD, EnvC and ActS. However, recent studies linked the outer membrane lipoprotein DolP (formerly YraP) as a potential upstream regulator of NlpD. In this study we explored this link in further detail. To our surprise DolP did not modulate amidase activity in vitro and was unable to interact with NlpD in pull-down and MST (MicroScale Thermophoresis) assays. Next, we excluded the hypothesis that Δ dolP phenocopied Δ nlpD in a range of envelope stresses. However, morphological analysis of double deletion mutants of amidases (AmiA, AmiB AmiC) and amidase regulators with dolP revealed that Δ amiAΔ dolP and Δ envCΔ dolP mutants display longer chain length compared to their parental strains indicating a role for DolP in cell division. Overall, we present evidence that DolP does not affect NlpD function in vitro, implying that DolP is not an upstream regulator of NlpD. However, DolP may impact daughter cell separation by interacting directly with AmiA or AmiC, or by a yet undiscovered mechanism.

AB - Bacterial amidases are essential to split the shared envelope of adjunct daughter cells to allow cell separation. Their activity needs to be precisely controlled to prevent cell lysis. In Escherichia coli, amidase activity is controlled by three regulatory proteins NlpD, EnvC and ActS. However, recent studies linked the outer membrane lipoprotein DolP (formerly YraP) as a potential upstream regulator of NlpD. In this study we explored this link in further detail. To our surprise DolP did not modulate amidase activity in vitro and was unable to interact with NlpD in pull-down and MST (MicroScale Thermophoresis) assays. Next, we excluded the hypothesis that Δ dolP phenocopied Δ nlpD in a range of envelope stresses. However, morphological analysis of double deletion mutants of amidases (AmiA, AmiB AmiC) and amidase regulators with dolP revealed that Δ amiAΔ dolP and Δ envCΔ dolP mutants display longer chain length compared to their parental strains indicating a role for DolP in cell division. Overall, we present evidence that DolP does not affect NlpD function in vitro, implying that DolP is not an upstream regulator of NlpD. However, DolP may impact daughter cell separation by interacting directly with AmiA or AmiC, or by a yet undiscovered mechanism.

KW - Amidohydrolases/genetics

KW - Bacterial Proteins/genetics

KW - Cell Separation

KW - Escherichia coli/metabolism

KW - Escherichia coli Proteins/genetics

KW - Lipoproteins/genetics

KW - Peptidoglycan/metabolism

KW - bacterial cell division

KW - peptidoglycan

KW - amidases

KW - Gram-negative cell envelope

KW - DolP

UR - http://www.scopus.com/inward/record.url?scp=85130480027&partnerID=8YFLogxK

U2 - 10.1099/mic.0.001197

DO - 10.1099/mic.0.001197

M3 - Article

C2 - 35604759

SN - 1350-0872

VL - 168

JO - Microbiology

JF - Microbiology

IS - 5

M1 - 001197

ER -

The lipoprotein DolP affects cell separation in Escherichia coli, but not as an upstream regulator of NlpD

Abstract

Bibliographical note

Keywords

ASJC Scopus subject areas

Access to Document

Fingerprint

Cite this