Genetic Manipulation of Lactococcus lactis by Using Targeted Group II Introns: Generation of Stable Insertions without Selection

ABSTRACT Despite their commercial importance, there are relatively few facile methods for genomic manipulation of the lactic acid bacteria. Here, the lactococcal group II intron, Ll.ltrB, was targeted to insert efficiently into genes encoding malate decarboxylase (mleS) and tetracycline resistance (tetM) within the Lactococcus lactis genome. Integrants were readily identified and maintained in the absence of a selectable marker. Since splicing of the Ll.ltrB intron depends on the intron-encoded protein, targeted invasion with an intron lacking the intron open reading frame disrupted TetM and MleS function, and MleS activity could be partially restored by expressing the intron-encoded protein in trans. Restoration of splicing from intron variants lacking the intron-encoded protein illustrates how targeted group II introns could be used for conditional expression of any gene. Furthermore, the modified Ll.ltrB intron was used to separately deliver a phage resistance gene (abiD) and a tetracycline resistance marker (tetM) into mleS, without the need for selection to drive the integration or to maintain the integrant. Our findings demonstrate the utility of targeted group II introns as a potential food-grade mechanism for delivery of industrially important traits into the genomes of lactococci.

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The carB Gene Encoding the Large Subunit of Carbamoylphosphate Synthetase from Lactococcus lactis Is Transcribed Monocistronically

Journal of Bacteriology ◽

10.1128/jb.180.17.4380-4386.1998 ◽

1998 ◽

Vol 180 (17) ◽

pp. 4380-4386 ◽

Cited By ~ 27

Author(s):

Jan Martinussen ◽

Karin Hammer

Keyword(s):

Lactococcus Lactis ◽

Large Subunit ◽

Arginine Deiminase ◽

Gene Encoding ◽

Pyrimidine Nucleotides ◽

Reading Frame ◽

Glutathione Peroxidases ◽

Carbamoylphosphate Synthetase ◽

Genes Encoding ◽

High Degree

ABSTRACT The biosynthesis of carbamoylphosphate is catalyzed by the heterodimeric enzyme carbamoylphosphate synthetase. The genes encoding the two subunits of this enzyme in procaryotes are normally transcribed as an operon, but the gene encoding the large subunit (carB) in Lactococcus lactis is shown to be transcribed as an isolated unit. Carbamoylphosphate is a precursor in the biosynthesis of both pyrimidine nucleotides and arginine. By mutant analysis,L. lactis is shown to possess only onecarB gene; the same gene product is thus required for both biosynthetic pathways. Furthermore, arginine may satisfy the requirement for carbamoylphosphate in pyrimidine biosynthesis through degradation by means of the arginine deiminase pathway. The expression of the carB gene is subject to regulation at the level of transcription by pyrimidines, most probably by an attenuator mechanism. Upstream of the carB gene, an open reading frame showing a high degree of similarity to those of glutathione peroxidases from other organisms was identified.

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An intronic open reading frame was released from one of group II introns in the mitochondrial genome of the haptophyteChrysochromulinasp. NIES-1333

Mobile Genetic Elements ◽

10.4161/mge.29384 ◽

2014 ◽

Vol 4 (3) ◽

pp. e29384 ◽

Cited By ~ 2

Author(s):

Yuki Nishimura ◽

Ryoma Kamikawa ◽

Tetsuo Hashimoto ◽

Yuji Inagaki

Keyword(s):

Mitochondrial Genome ◽

Open Reading Frame ◽

Group Ii Introns ◽

Reading Frame ◽

Group Ii

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Group II introns as controllable gene targeting vectors for genetic manipulation of bacteria

Nature Biotechnology ◽

10.1038/nbt1201-1162 ◽

2001 ◽

Vol 19 (12) ◽

pp. 1162-1167 ◽

Cited By ~ 139

Author(s):

Michael Karberg ◽

Huatao Guo ◽

Jin Zhong ◽

Robert Coon ◽

Jiri Perutka ◽

...

Keyword(s):

Gene Targeting ◽

Genetic Manipulation ◽

Group Ii Introns ◽

Group Ii

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Mobile group II introns of yeast mitochondrial DNA are novel site-specific retroelements.

Molecular and Cellular Biology ◽

10.1128/mcb.15.5.2828 ◽

1995 ◽

Vol 15 (5) ◽

pp. 2828-2838 ◽

Cited By ~ 73

Author(s):

J V Moran ◽

S Zimmerly ◽

R Eskes ◽

J C Kennell ◽

A M Lambowitz ◽

...

Keyword(s):

Group Ii Intron ◽

Group Ii Introns ◽

Dna Endonuclease ◽

Site Specific ◽

Reading Frame ◽

Defective Mutant ◽

Sequences Analysis ◽

Group Ii ◽

Precursor Rna ◽

Yeast Mitochondrial Dna

Group II introns aI1 and aI2 of the yeast mitochondrial COXI gene are mobile elements that encode an intron-specific reverse transcriptase (RT) activity. We show here that the introns of Saccharomyces cerevisiae ID41-6/161 insert site specifically into intronless alleles. The mobility is accompanied by efficient, but highly asymmetric, coconversion of nearby flanking exon sequences. Analysis of mutants shows that the aI2 protein is required for the mobility of both aI1 and aI2. Efficient mobility is dependent on both the RT activity of the aI2-encoded protein and a separate function, a putative DNA endonuclease, that is associated with the Zn2+ finger-like region of the intron reading frame. Surprisingly, there appear to be two mobility modes: the major one involves cDNAs reverse transcribed from unspliced precursor RNA; the minor one, observed in two mutants lacking detectable RT activity, appears to involve DNA level recombination. A cis-dominant splicing-defective mutant of aI2 continues to synthesize cDNAs containing the introns but is completely defective in both mobility modes, indicating that the splicing or the structure of the intron is required. Our results demonstrate that the yeast group II intron aI2 is a retroelement that uses novel mobility mechanisms.

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Faculty Opinions recommendation of Use of computer-designed group II introns to disrupt Escherichia coli DExH/D-box protein and DNA helicase genes.

Faculty Opinions – Post-Publication Peer Review of the Biomedical Literature ◽

10.3410/f.1017227.202274 ◽

2004 ◽

Author(s):

Barry Stoddard

Keyword(s):

Escherichia Coli ◽

Dna Helicase ◽

Group Ii Introns ◽

Group Ii

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Organellar Introns in Fungi, Algae, and Plants

Cells ◽

10.3390/cells10082001 ◽

2021 ◽

Vol 10 (8) ◽

pp. 2001

Author(s):

Jigeesha Mukhopadhyay ◽

Georg Hausner

Keyword(s):

Gene Expression ◽

Ribosomal Proteins ◽

Heterologous Gene Expression ◽

Group I Introns ◽

Group Ii Introns ◽

Homing Endonucleases ◽

Organellar Genomes ◽

Chloroplast Genomes ◽

Group I ◽

Group Ii

Introns are ubiquitous in eukaryotic genomes and have long been considered as ‘junk RNA’ but the huge energy expenditure in their transcription, removal, and degradation indicate that they may have functional significance and can offer evolutionary advantages. In fungi, plants and algae introns make a significant contribution to the size of the organellar genomes. Organellar introns are classified as catalytic self-splicing introns that can be categorized as either Group I or Group II introns. There are some biases, with Group I introns being more frequently encountered in fungal mitochondrial genomes, whereas among plants Group II introns dominate within the mitochondrial and chloroplast genomes. Organellar introns can encode a variety of proteins, such as maturases, homing endonucleases, reverse transcriptases, and, in some cases, ribosomal proteins, along with other novel open reading frames. Although organellar introns are viewed to be ribozymes, they do interact with various intron- or nuclear genome-encoded protein factors that assist in the intron RNA to fold into competent splicing structures, or facilitate the turn-over of intron RNAs to prevent reverse splicing. Organellar introns are also known to be involved in non-canonical splicing, such as backsplicing and trans-splicing which can result in novel splicing products or, in some instances, compensate for the fragmentation of genes by recombination events. In organellar genomes, Group I and II introns may exist in nested intronic arrangements, such as introns within introns, referred to as twintrons, where splicing of the external intron may be dependent on splicing of the internal intron. These nested or complex introns, with two or three-component intron modules, are being explored as platforms for alternative splicing and their possible function as molecular switches for modulating gene expression which could be potentially applied towards heterologous gene expression. This review explores recent findings on organellar Group I and II introns, focusing on splicing and mobility mechanisms aided by associated intron/nuclear encoded proteins and their potential roles in organellar gene expression and cross talk between nuclear and organellar genomes. Potential application for these types of elements in biotechnology are also discussed.

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