Restructured Lactococcus lactis strains with emergent properties constructed by a novel highly efficient screening system

Abstract Background After 2.83% genome reduction in Lactococcus lactis NZ9000, a good candidate host for proteins production was obtained in our previous work. However, the gene deletion process was time consuming and laborious. Here, we proposed a convenient gene deletion method suitable for large-scale genome reduction in L. lactis NZ9000. Results Plasmid pNZ5417 containing a visually selectable marker PnisZ-lacZ was constructed, which allowed more efficient and convenient screening of gene deletion mutants. Using this plasmid, two large nonessential DNA regions, L-4A and L-5A, accounting for 1.25% of the chromosome were deleted stepwise in L. lactis 9k-3. When compared with the parent strain, the mutant L. lactis 9k-5A showed better growth characteristics, transformability, carbon metabolic capacity, and amino acids biosynthesis. Conclusions Thus, this study provides a convenient and efficient system for large-scale genome deletion in L. lactis through application of visually selectable marker, which could be helpful for rapid genome streamlining and generation of restructured L. lactis strains that can be used as cell factories.

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Strains and Strategies for Large-Scale Gene Deletion Studies of the Diploid Human Fungal Pathogen Candida albicans

Eukaryotic Cell ◽

10.1128/ec.4.2.298-309.2005 ◽

2005 ◽

Vol 4 (2) ◽

pp. 298-309 ◽

Cited By ~ 389

Author(s):

Suzanne M. Noble ◽

Alexander D. Johnson

Keyword(s):

Candida Albicans ◽

Fungal Pathogen ◽

Large Scale ◽

Gene Deletion ◽

Selectable Marker ◽

Wild Type ◽

Position Effects ◽

Human Fungal Pathogen ◽

Chromosome Position ◽

Knockout Mutants

ABSTRACT Candida albicans is the most common human fungal pathogen and causes significant morbidity and mortality worldwide. Nevertheless, the basic principles of C. albicans pathogenesis remain poorly understood. Of central importance to the study of this organism is the ability to generate homozygous knockout mutants and to analyze them in a mammalian model of pathogenesis. C. albicans is diploid, and current strategies for gene deletion typically involve repeated use of the URA3 selectable marker. These procedures are often time-consuming and inefficient. Moreover, URA3 expression levels—which are susceptible to chromosome position effects—can themselves affect virulence, thereby complicating analysis of strains constructed with URA3 as a selectable marker. Here, we describe a set of newly developed reference strains (leu2Δ/leu2Δ, his1Δ/his1Δ; arg4Δ/arg4Δ, his1Δ/his1Δ; and arg4Δ/arg4Δ, leu2Δ/leu2Δ, his1Δ/his1Δ) that exhibit wild-type or nearly wild-type virulence in a mouse model. We also describe new disruption marker cassettes and a fusion PCR protocol that permit rapid and highly efficient generation of homozygous knockout mutations in the new C. albicans strains. We demonstrate these procedures for two well-studied genes, TUP1 and EFG1, as well as a novel gene, RBD1. These tools should permit large-scale genetic analysis of this important human pathogen.

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One-plasmid based CRISPR-Cas9 editing for gene deletion in Lactococcus lactis

10.21203/rs.3.rs-306012/v1 ◽

2021 ◽

Author(s):

José Miguel Miquelão Santos ◽

Gabriel António Amaro Monteiro ◽

Duarte Miguel de França Teixeira dos Prazeres ◽

Sofia de Oliveira Dias Duarte

Keyword(s):

Genome Editing ◽

Lactococcus Lactis ◽

Plasmid Dna ◽

Genome Engineering ◽

Gene Deletion ◽

Deletion Mutants ◽

Cell Factories ◽

Yield And Quality ◽

Delivery Vehicles

Abstract Lactococcus lactis strains are promising cell factories and delivery vehicles of plasmid DNA and recombinant protein for therapeutic applications. However, the limited yields of recombinant molecules obtained with these bacteria limits their wide applicability. Genome engineering of this host may solve the problem. However, the current genome editing toolbox available for L. lactis is either too laborious or incapable of large edits, limiting the scope of strain editing experiments. In this work, the basis for a one-plasmid CRISPR-Cas9 based genome editing plasmid was developed and tested. The new plasmid (pTCas9dO) adapted from the pKCcas9dO plasmid was used to delete 657 bp of the lactococcal nuclease nth of L. lactis subsp. lactis LMG19460, with the aim of improving yield and quality of plasmid DNA replicated in this strain. Although deletion mutants were successfully generated, plasmid curing was unsuccessful. Thus, further modifications are required before the plasmid is truly applicable for genome editing experiments. Unexpectedly, the generated deletion mutants generated a roughly 40% decrease in plasmid yield alongside with a decrease in the quality of produced pDNA.

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Adaptation of a parasitic lifestyle by Cuscuta gronovii Willd. ex Roem. & Schult.: large scale gene deletion, conserved gene orders, and low intraspecific divergence

Mitochondrial DNA Part B ◽

10.1080/23802359.2021.1911702 ◽

2021 ◽

Vol 6 (4) ◽

pp. 1475-1482

Author(s):

Yang Ni ◽

Mei Jiang ◽

Haimei Chen ◽

Linfang Huang ◽

Pinghua Chen ◽

...

Keyword(s):

Large Scale ◽

Gene Deletion ◽

Intraspecific Divergence ◽

Parasitic Lifestyle ◽

Cuscuta Gronovii ◽

Conserved Gene

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Plasmid Replicons for the Production of Pharmaceutical-Grade pDNA, Proteins and Antigens by Lactococcus lactis Cell Factories

International Journal of Molecular Sciences ◽

10.3390/ijms22031379 ◽

2021 ◽

Vol 22 (3) ◽

pp. 1379

Author(s):

Sofia O.D. Duarte ◽

Gabriel A. Monteiro

Keyword(s):

Lactococcus Lactis ◽

Recombinant Proteins ◽

Plasmid Copy Number ◽

Fermented Food ◽

Added Value ◽

Regulatory Sequences ◽

Plasmid Copy ◽

Mucosal Vaccination ◽

Cell Factories ◽

Plasmid Replicons

The Lactococcus lactis bacterium found in different natural environments is traditionally associated with the fermented food industry. But recently, its applications have been spreading to the pharmaceutical industry, which has exploited its probiotic characteristics and is moving towards its use as cell factories for the production of added-value recombinant proteins and plasmid DNA (pDNA) for DNA vaccination, as a safer and industrially profitable alternative to the traditional Escherichia coli host. Additionally, due to its food-grade and generally recognized safe status, there have been an increasing number of studies about its use in live mucosal vaccination. In this review, we critically systematize the plasmid replicons available for the production of pharmaceutical-grade pDNA and recombinant proteins by L. lactis. A plasmid vector is an easily customized component when the goal is to engineer bacteria in order to produce a heterologous compound in industrially significant amounts, as an alternative to genomic DNA modifications. The additional burden to the cell depends on plasmid copy number and on the expression level, targeting location and type of protein expressed. For live mucosal vaccination applications, besides the presence of the necessary regulatory sequences, it is imperative that cells produce the antigen of interest in sufficient yields. The cell wall anchored antigens had shown more promising results in live mucosal vaccination studies, when compared with intracellular or secreted antigens. On the other side, engineering L. lactis to express membrane proteins, especially if they have a eukaryotic background, increases the overall cellular burden. The different alternative replicons for live mucosal vaccination, using L. lactis as the DNA vaccine carrier or the antigen producer, are critically reviewed, as a starting platform to choose or engineer the best vector for each application.

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Bacterial Multicellularity: The Biology of Escherichia coli Building Large-Scale Biofilm Communities

Annual Review of Microbiology ◽

10.1146/annurev-micro-031921-055801 ◽

2021 ◽

Vol 75 (1) ◽

Author(s):

Diego O. Serra ◽

Regine Hengge

Keyword(s):

Escherichia Coli ◽

Large Scale ◽

Second Messengers ◽

Emergent Properties ◽

Annual Review ◽

Publication Date ◽

Growth And Survival ◽

Chemical Gradients ◽

Life Itself ◽

Scale Matrix

Biofilms are a widespread multicellular form of bacterial life. The spatial structure and emergent properties of these communities depend on a polymeric extracellular matrix architecture that is orders of magnitude larger than the cells that build it. Using as a model the wrinkly macrocolony biofilms of Escherichia coli, which contain amyloid curli fibers and phosphoethanolamine (pEtN)-modified cellulose as matrix components, we summarize here the structure, building, and function of this large-scale matrix architecture. Based on different sigma and other transcription factors as well as second messengers, the underlying regulatory network reflects the fundamental trade-off between growth and survival. It controls matrix production spatially in response to long-range chemical gradients, but it also generates distinct patterns of short-range matrix heterogeneity that are crucial for tissue-like elasticity and macroscopic morphogenesis. Overall, these biofilms confer protection and a potential for homeostasis, thereby reducing maintenance energy, which makes multicellularity an emergent property of life itself. Expected final online publication date for the Annual Review of Microbiology, Volume 75 is October 2021. Please see http://www.annualreviews.org/page/journal/pubdates for revised estimates.

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Scarless gene deletion in methylotrophic Hansenula polymorpha by using mazF as counter-selectable marker

Analytical Biochemistry ◽

10.1016/j.ab.2014.09.006 ◽

2015 ◽

Vol 468 ◽

pp. 66-74 ◽

Cited By ~ 5

Author(s):

Panpan Song ◽

Sha Liu ◽

Xuena Guo ◽

Xuejing Bai ◽

Xiuping He ◽

...

Keyword(s):

Gene Deletion ◽

Selectable Marker ◽

Hansenula Polymorpha

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Large-scale Phenotypic Profiling of Gene Deletion Mutants in Candida glabrata

BIO-PROTOCOL ◽

10.21769/bioprotoc.1530 ◽

2015 ◽

Vol 5 (14) ◽

Author(s):

Fabian Istel ◽

Tobias Schwarzm�ller ◽

Michael Tscherner ◽

Karl Kuchler

Keyword(s):

Candida Glabrata ◽

Large Scale ◽

Gene Deletion ◽

Deletion Mutants

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DNA Melting Analysis for Detection of Single Nucleotide Polymorphisms

Clinical Chemistry ◽

10.1093/clinchem/47.4.635 ◽

2001 ◽

Vol 47 (4) ◽

pp. 635-644 ◽

Cited By ~ 68

Author(s):

Robert H Lipsky ◽

Chiara M Mazzanti ◽

Joseph G Rudolph ◽

Ke Xu ◽

Gopal Vyas ◽

...

Keyword(s):

Single Nucleotide Polymorphisms ◽

Large Scale ◽

Denaturing Gradient Gel Electrophoresis ◽

Dna Melting ◽

Nucleotide Polymorphisms ◽

Single Nucleotide ◽

Efficient System ◽

Gradient Gel Electrophoresis ◽

Melting Analysis ◽

Scale Detection

Abstract Background: Several methods for detection of single nucleotide polymorphisms (SNPs; e.g., denaturing gradient gel electrophoresis and denaturing HPLC) are indirectly based on the principle of differential melting of heteroduplex DNA. We present a method for detecting SNPs that is directly based on this principle. Methods: We used a double-stranded DNA-specific fluorescent dye, SYBR Green I (SYBR) in an efficient system (PE 7700 Sequence Detector) in which DNA melting was controlled and monitored in a 96-well plate format. We measured the decrease in fluorescence intensity that accompanied DNA duplex denaturation, evaluating the effects of fragment length, dye concentration, DNA concentration, and sequence context using four naturally occurring polymorphisms (three SNPs and a single-base deletion/insertion). Results: DNA melting analysis (DM) was used successfully for variant detection, and we also discovered two previously unknown SNPs by this approach. Concentrations of DNA amplicons were readily monitored by SYBR fluorescence, and DNA amplicon concentrations were highly reproducible, with a CV of 2.6%. We readily detected differences in the melting temperature between homoduplex and heteroduplex fragments 15–167 bp in length and differing by only a single nucleotide substitution. Conclusions: The efficiency and sensitivity of DMA make it highly suitable for the large-scale detection of sequence variants.

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Microbial Interactions in Oral Communities Mediate Emergent Biofilm Properties

Journal of Dental Research ◽

10.1177/0022034519880157 ◽

2019 ◽

Vol 99 (1) ◽

pp. 18-25 ◽

Cited By ~ 10

Author(s):

P.I. Diaz ◽

A.M. Valm

Keyword(s):

Large Scale ◽

Taxonomic Composition ◽

Cell Interactions ◽

Microbial Interactions ◽

Spatial Proximity ◽

Individual Species ◽

Emergent Properties ◽

Oral Diseases ◽

Metabolic Cooperation ◽

Cell Cell

Oral microbial communities are extraordinarily complex in taxonomic composition and comprise interdependent biological systems. The bacteria, archaea, fungi, and viruses that thrive within these communities engage in extensive cell-cell interactions, which are both beneficial and antagonistic. Direct physical interactions among individual cells mediate large-scale architectural biofilm arrangements and provide spatial proximity for chemical communication and metabolic cooperation. In this review, we summarize recent work in identifying specific molecular components that mediate cell-cell interactions and describe metabolic interactions, such as cross-feeding and exchange of electron acceptors and small molecules, that modify the growth and virulence of individual species. We argue, however, that although pairwise interaction models have provided useful information, complex community-like systems are needed to study the properties of oral communities. The networks of multiple synergistic and antagonistic interactions within oral biofilms give rise to the emergent properties of persistence, stability, and long-range spatial structure, with these properties mediating the dysbiotic transitions from health to oral diseases. A better understanding of the fundamental properties of interspecies networks will lead to the development of effective strategies to manipulate oral communities.

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