A family of removable cassettes designed to obtain antibiotic-resistance-free genomic modifications of Escherichia coli and other bacteria

Palmeros, Beatrı́z; Wild, Jadwiga; Szybalski, Waclaw; Borgne, Sylvie Le; Hernández-Chávez, Georgina; Gosset, Guillermo; Valle, Fernando; Bolívar, Francisco

doi:10.1016/s0378-1119(00)00075-5

Cited by 80 publications

(58 citation statements)

References 28 publications

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“…The Cre recombinase of bacteriophage P1 catalyzes site-specific recombination between lox sites and, in particular, the in vivo excision of DNA regions flanked by codirectional loxP recognition sites (19). Cre expression from plasmid pCM157 (18) in M. gryphiswaldense was verified by reverse transcription-PCR.…”

Section: Methodsmentioning

confidence: 99%

The Major Magnetosome Proteins MamGFDC Are Not Essential for Magnetite Biomineralization inMagnetospirillum gryphiswaldensebut Regulate the Size of Magnetosome Crystals

et al. 2008

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Magnetospirillum gryphiswaldense and related magnetotactic bacteria form magnetosomes, which are membrane-enclosed organelles containing crystals of magnetite (Fe 3 O 4 ) that cause the cells to orient in magnetic fields. The characteristic sizes, morphologies, and patterns of alignment of magnetite crystals are controlled by vesicles formed of the magnetosome membrane (MM), which contains a number of specific proteins whose precise roles in magnetosome formation have remained largely elusive. Here, we report on a functional analysis of the small hydrophobic MamGFDC proteins, which altogether account for nearly 35% of all proteins associated with the MM. Although their high levels of abundance and conservation among magnetotactic bacteria had suggested a major role in magnetosome formation, we found that the MamGFDC proteins are not essential for biomineralization, as the deletion of neither mamC, encoding the most abundant magnetosome protein, nor the entire mamGFDC operon abolished the formation of magnetite crystals. However, cells lacking mamGFDC produced crystals that were only 75% of the wild-type size and were less regular than wild-type crystals with respect to morphology and chain-like organization. The inhibition of crystal formation could not be eliminated by increased iron concentrations. The growth of mutant crystals apparently was not spatially constrained by the sizes of MM vesicles, as cells lacking mamGFDC formed vesicles with sizes and shapes nearly identical to those formed by wild-type cells. However, the formation of wild-type-size magnetite crystals could be gradually restored by in-trans complementation with one, two, and three genes of the mamGFDC operon, regardless of the combination, whereas the expression of all four genes resulted in crystals exceeding the wild-type size. Our data suggest that the MamGFDC proteins have partially redundant functions and, in a cumulative manner, control the growth of magnetite crystals by an as-yet-unknown mechanism.The ability of magnetotactic bacteria (MTB) to orient in the earth's magnetic field is based on specific organelles, the magnetosomes, which are membrane-enveloped crystals of a magnetic mineral that are arranged in chain-like structures within the cell (5). Magnetosome crystals display a variety of species-specific shapes and sizes, which for most MTB are between 35 and 120 nm (3, 4). In MTB of the genus Magnetospirillum, cubo-octahedral nanocrystals of the magnetic mineral magnetite (Fe 3 O 4 ) are synthesized within magnetosome membrane (MM) vesicles, which are roughly spherical and are formed by invagination from the cytoplasmic membrane prior to magnetite biomineralization (2, 15, 16). The MM is a phospholipid bilayer with a distinctive biochemical composition (8,29,35). In Magnetospirillum gryphiswaldense, a specific set of 20 MM proteins (MMPs) was identified by biochemical and proteomic approaches (9,10,22), and these proteins were speculated to be involved in magnetosome biomineralization (29). However, the individual functions of M...

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Section: Methodsmentioning

confidence: 99%

The Major Magnetosome Proteins MamGFDC Are Not Essential for Magnetite Biomineralization inMagnetospirillum gryphiswaldensebut Regulate the Size of Magnetosome Crystals

et al. 2008

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“…Colonies were selected on LB agar plates containing Cm, and successful gene replacement with the lox71-Cm R -lox66 cassette was confirmed by direct colony PCR. The Cm R marker was subsequently eliminated by a helper plasmid pJW168, which contains a temperaturesensitive replication origin and the IPTG-inducible Cre recombinase (Palmeros et al, 2000). The loss of Cm R was further verified by PCR.…”

Section: Deletion Of Chromosomal Genesmentioning

confidence: 99%

Metabolic engineering of Escherichia coli for the production of putrescine: A four carbon diamine

Qian

Xia

Lee

2009

Biotech & Bioengineering

227

178

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A four carbon linear chain diamine, putrescine (1,4-diaminobutane), is an important platform chemical having a wide range of applications in chemical industry. Biotechnological production of putrescine from renewable feedstock is a promising alternative to the chemical synthesis that originates from non-renewable petroleum. Here we report development of a metabolically engineered strain of Escherichia coli that produces putrescine at high titer in glucose mineral salts medium. First, a base strain was constructed by inactivating the putrescine degradation and utilization pathways, and deleting the ornithine carbamoyltransferase chain I gene argI to make more precursors available for putrescine synthesis. Next, ornithine decarboxylase, which converts ornithine to putrescine, was amplified by a combination of plasmid-based and chromosome-based overexpression of the coding genes under the strong tac or trc promoter. Furthermore, the ornithine biosynthetic genes (argC-E) were overexpressed from the trc promoter, which replaced the native promoter in the genome, to increase the ornithine pool. Finally, strain performance was further improved by the deletion of the stress responsive RNA polymerase sigma factor RpoS, a well-known global transcription regulator that controls the expression of ca. 10% of the E. coli genes. The final engineered E. coli strain was able to produce 1.68 g L À1 of putrescine with a yield of 0.168 g g À1 glucose. Furthermore, high cell density cultivation allowed production of 24.2 g L À1 of putrescine with a productivity of 0.75 g L À1 h À1 . The strategy reported here should be useful for the bio-based production of putrescine from renewable resources, and also for the development of strains capable of producing other diamines, which are important as nitrogen-containing platform chemicals.

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“…More recently, a Cre-loxP system has been adapted for use in S. mutans (5). The Cre-loxP approach has the advantage of circumventing the requirement for a mutant recipient strain and has been used to create unmarked gene deletions in a large variety of bacteria (5,8,21,28,30,36,39,42). However, the resulting mutant strains also retain the loxP site after the excision step (5,30,36), which could be problematic for the creation of various types of mutations, such as truncations, point mutations, fusion proteins, etc.…”

mentioning

confidence: 99%

Cloning-Independent and Counterselectable Markerless Mutagenesis System in Streptococcus mutans

Xie

Okinaga

et al. 2011

Appl Environ Microbiol

120

156

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Insertion duplication mutagenesis and allelic replacement mutagenesis are among the most commonly utilized approaches for targeted mutagenesis in bacteria. However, both techniques are limited by a variety of factors that can complicate mutant phenotypic studies. To circumvent these limitations, multiple markerless mutagenesis techniques have been developed that utilize either temperature-sensitive plasmids or counterselectable suicide vectors containing both positive-and negative-selection markers. For many species, these techniques are not especially useful due to difficulties of cloning with Escherichia coli and/or a lack of functional negative-selection markers. In this study, we describe the development of a novel approach for the creation of markerless mutations. This system employs a cloning-independent methodology and should be easily adaptable to a wide array of Gram-positive and Gram-negative bacterial species. The entire process of creating both the counterselection cassette and mutation constructs can be completed using overlapping PCR protocols, which allows extremely quick assembly and eliminates the requirement for either temperature-sensitive replicons or suicide vectors. As a proof of principle, we used Streptococcus mutans reference strain UA159 to create markerless in-frame deletions of 3 separate bacteriocin genes as well as triple mutants containing all 3 deletions. Using a panel of 5 separate wild-type S. mutans strains, we further demonstrated that the procedure is nearly 100% efficient at generating clones with the desired markerless mutation, which is a considerable improvement in yield compared to existing approaches.Streptococcus mutans is a Gram-positive bacterial species that resides within multispecies oral biofilms formed on human tooth surfaces. It is also considered to be one of the principal species associated with dental caries initiation (6,7,34,35,44, 47,52). S. mutans genetic research has benefited tremendously from the many genetic tools that have been adapted for use in studies of the organism (4,5,13,15,22,25,26,29,33,45,51). For genetic studies of S. mutans, defined mutations are usually engineered in either of two ways: insertion duplication mutagenesis via single-crossover homologous recombination or marked allelic replacement mutagenesis using double-crossover homologous recombination (25,27,41). Both approaches are highly reliable strategies for mutagenesis and are simple to engineer, but they also have the potential to create unwanted artifacts that could influence the outcome of a genetic study. For example, insertion duplication mutations often result in the production of truncated proteins. Rarely is it known with certainty whether the protein fragments actually influence mutant phenotypes. Furthermore, due to significant polar effects downstream of the mutation site, both insertion duplication mutagenesis and allelic replacement mutagenesis are of limited utility within operons. In some cases, these issues have been addressed by creating allelic replacement mut...

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A family of removable cassettes designed to obtain antibiotic-resistance-free genomic modifications of Escherichia coli and other bacteria

Cited by 80 publications

References 28 publications

The Major Magnetosome Proteins MamGFDC Are Not Essential for Magnetite Biomineralization inMagnetospirillum gryphiswaldensebut Regulate the Size of Magnetosome Crystals

The Major Magnetosome Proteins MamGFDC Are Not Essential for Magnetite Biomineralization inMagnetospirillum gryphiswaldensebut Regulate the Size of Magnetosome Crystals

Metabolic engineering of Escherichia coli for the production of putrescine: A four carbon diamine

Cloning-Independent and Counterselectable Markerless Mutagenesis System in Streptococcus mutans

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