CRISPR Interference for Rapid Knockdown of Essential Cell Cycle Genes in <italic toggle="yes">Lactobacillus plantarum</italic>

ABSTRACT Studies of essential genes in bacteria are often hampered by the lack of accessible genetic tools. This is also the case for Lactobacillus plantarum, a key species in food and health applications. Here, we develop a clustered regularly interspaced short palindromic repeat interference (CRIS...

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Autores principales:	Ine Storaker Myrbråten, Kamilla Wiull, Zhian Salehian, Leiv Sigve Håvarstein, Daniel Straume, Geir Mathiesen, Morten Kjos
Formato:	article
Lenguaje:	EN
Publicado:	American Society for Microbiology 2019
Materias:	CRISPRi Lactobacillus plantarum acm2 bacterial cell cycle cozE dnaA Microbiology QR1-502
Acceso en línea:	https://doaj.org/article/bc455616091f4e57899ab6b41c4ec1f3
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spelling	oai:doaj.org-article:bc455616091f4e57899ab6b41c4ec1f32021-11-15T15:22:22ZCRISPR Interference for Rapid Knockdown of Essential Cell Cycle Genes in <italic toggle="yes">Lactobacillus plantarum</italic>10.1128/mSphere.00007-192379-5042https://doaj.org/article/bc455616091f4e57899ab6b41c4ec1f32019-04-01T00:00:00Zhttps://journals.asm.org/doi/10.1128/mSphere.00007-19https://doaj.org/toc/2379-5042ABSTRACT Studies of essential genes in bacteria are often hampered by the lack of accessible genetic tools. This is also the case for Lactobacillus plantarum, a key species in food and health applications. Here, we develop a clustered regularly interspaced short palindromic repeat interference (CRISPRi) system for knockdown of gene expression in L. plantarum. The two-plasmid CRISPRi system, in which a nuclease-inactivated Cas9 (dCas9) and a gene-specific single guide RNA (sgRNA) are expressed on separate plasmids, allows efficient knockdown of expression of any gene of interest. We utilized the CRISPRi system to gain initial insights into the functions of key cell cycle genes in L. plantarum. As a proof of concept, we investigated the phenotypes resulting from knockdowns of the cell wall hydrolase-encoding acm2 gene and of the DNA replication initiator gene dnaA and of ezrA, which encodes an early cell division protein. Furthermore, we studied the phenotypes of three cell division genes which have recently been functionally characterized in ovococcal bacteria but whose functions have not yet been investigated in rod-shaped bacteria. We show that the transmembrane CozE proteins do not seem to play any major role in cell division in L. plantarum. On the other hand, RNA-binding proteins KhpA and EloR are critical for proper cell elongation in this bacterium. IMPORTANCE L. plantarum is an important bacterium for applications in food and health. Deep insights into the biology and physiology of this species are therefore necessary for further strain optimization and exploitation; however, the functions of essential genes in the bacterium are mainly unknown due to the lack of accessible genetic tools. The CRISPRi system developed here is ideal to quickly screen for phenotypes of both essential and nonessential genes. Our initial insights into the function of some key cell cycle genes represent the first step toward understanding the cell cycle in this bacterium.Ine Storaker MyrbråtenKamilla WiullZhian SalehianLeiv Sigve HåvarsteinDaniel StraumeGeir MathiesenMorten KjosAmerican Society for MicrobiologyarticleCRISPRiLactobacillus plantarumacm2bacterial cell cyclecozEdnaAMicrobiologyQR1-502ENmSphere, Vol 4, Iss 2 (2019)
institution	DOAJ
collection	DOAJ
language	EN
topic	CRISPRi Lactobacillus plantarum acm2 bacterial cell cycle cozE dnaA Microbiology QR1-502
spellingShingle	CRISPRi Lactobacillus plantarum acm2 bacterial cell cycle cozE dnaA Microbiology QR1-502 Ine Storaker Myrbråten Kamilla Wiull Zhian Salehian Leiv Sigve Håvarstein Daniel Straume Geir Mathiesen Morten Kjos CRISPR Interference for Rapid Knockdown of Essential Cell Cycle Genes in <italic toggle="yes">Lactobacillus plantarum</italic>
description	ABSTRACT Studies of essential genes in bacteria are often hampered by the lack of accessible genetic tools. This is also the case for Lactobacillus plantarum, a key species in food and health applications. Here, we develop a clustered regularly interspaced short palindromic repeat interference (CRISPRi) system for knockdown of gene expression in L. plantarum. The two-plasmid CRISPRi system, in which a nuclease-inactivated Cas9 (dCas9) and a gene-specific single guide RNA (sgRNA) are expressed on separate plasmids, allows efficient knockdown of expression of any gene of interest. We utilized the CRISPRi system to gain initial insights into the functions of key cell cycle genes in L. plantarum. As a proof of concept, we investigated the phenotypes resulting from knockdowns of the cell wall hydrolase-encoding acm2 gene and of the DNA replication initiator gene dnaA and of ezrA, which encodes an early cell division protein. Furthermore, we studied the phenotypes of three cell division genes which have recently been functionally characterized in ovococcal bacteria but whose functions have not yet been investigated in rod-shaped bacteria. We show that the transmembrane CozE proteins do not seem to play any major role in cell division in L. plantarum. On the other hand, RNA-binding proteins KhpA and EloR are critical for proper cell elongation in this bacterium. IMPORTANCE L. plantarum is an important bacterium for applications in food and health. Deep insights into the biology and physiology of this species are therefore necessary for further strain optimization and exploitation; however, the functions of essential genes in the bacterium are mainly unknown due to the lack of accessible genetic tools. The CRISPRi system developed here is ideal to quickly screen for phenotypes of both essential and nonessential genes. Our initial insights into the function of some key cell cycle genes represent the first step toward understanding the cell cycle in this bacterium.
format	article
author	Ine Storaker Myrbråten Kamilla Wiull Zhian Salehian Leiv Sigve Håvarstein Daniel Straume Geir Mathiesen Morten Kjos
author_facet	Ine Storaker Myrbråten Kamilla Wiull Zhian Salehian Leiv Sigve Håvarstein Daniel Straume Geir Mathiesen Morten Kjos
author_sort	Ine Storaker Myrbråten
title	CRISPR Interference for Rapid Knockdown of Essential Cell Cycle Genes in <italic toggle="yes">Lactobacillus plantarum</italic>
title_short	CRISPR Interference for Rapid Knockdown of Essential Cell Cycle Genes in <italic toggle="yes">Lactobacillus plantarum</italic>
title_full	CRISPR Interference for Rapid Knockdown of Essential Cell Cycle Genes in <italic toggle="yes">Lactobacillus plantarum</italic>
title_fullStr	CRISPR Interference for Rapid Knockdown of Essential Cell Cycle Genes in <italic toggle="yes">Lactobacillus plantarum</italic>
title_full_unstemmed	CRISPR Interference for Rapid Knockdown of Essential Cell Cycle Genes in <italic toggle="yes">Lactobacillus plantarum</italic>
title_sort	crispr interference for rapid knockdown of essential cell cycle genes in <italic toggle="yes">lactobacillus plantarum</italic>
publisher	American Society for Microbiology
publishDate	2019
url	https://doaj.org/article/bc455616091f4e57899ab6b41c4ec1f3
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CRISPR Interference for Rapid Knockdown of Essential Cell Cycle Genes in <italic toggle="yes">Lactobacillus plantarum</italic>

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