Transcription of the trp operon in Lactococcus lactis is controlled by antitermination in the leader region

Frenkiel, HéléeGne; Bardowski, Jacek; Ehrlich, S. Dusko; Chopin, Alain

doi:10.1099/00221287-144-8-2103

Cited by 13 publications

(10 citation statements)

References 37 publications

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“…This is exemplified by the downregulation of the trpE-R-IVET clone by all rich medium components and by the subsequent demonstration that this effect is achieved specifically by the tryptophan present in these components. These findings are consistent with previous reports that describe the tryptophanmediated transcriptional control of the trpEFG locus in L. lactis (11). Analogously, we evaluated the effect of arginine on the promoter element of the arcA.…”

Section: Vol 74 2008 High-throughput Analysis Of In Vivo-expressed supporting

confidence: 82%

High-Throughput Identification and Validation of In Situ-Expressed Genes of Lactococcus lactis

Bachmann

Kleerebezem

Vlieg

2008

Appl Environ Microbiol

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Understanding the functional response of bacteria to their natural environment is one of the current challenges in microbiology. Over the past decades several techniques have been developed to study gene expression in complex natural habitats. Most of these methods, however, are laborious, and validation of results under in situ conditions is cumbersome. Here we report the improvement of the recombinase-based in vivo expression technology (R-IVET) by the implementation of two additional reporter genes. The first one is an ␣-galactosidase gene (melA), which facilitates the rapid identification of in vivo-induced genes. Second, the bacterial luciferase genes (luxAB) are transcriptionally coupled to the resolvase gene, which allows rapid validation and characterization of in vivo-induced genes. The system is implemented and validated in the industrially important lactic acid bacterium Lactococcus lactis. We demonstrate the applicability of the advanced R-IVET system by the identification and validation of lactococcal promoter elements that are induced in minimal medium compared to the commonly used rich laboratory medium M17. R-IVET screening led to the identification of 19 promoters that predominantly control expression of genes involved in amino acid and nucleotide metabolism and in transport functions. Furthermore, the luciferase allows high-resolution transcription analysis and enabled the identification of complex medium constituents and specific molecules involved in promoter control. Rapid target validation exemplifies the high-throughput potential of the extended R-IVET system. The system can be applied to other bacterial species, provided that the reporter genes used are functional in the organism of interest.Understanding the responses of microorganisms in their natural environments is one of the big challenges of the postgenomic era. Complex niches such as soil, decaying plant material, and the (mammalian) gastrointestinal tract show a rich bacterial diversity and complex microbial activity patterns. Sampling complexity and environmental dynamics hamper comprehensive transcriptional analysis of a specific microorganism residing in such a complex environment. To overcome these challenges, a number of tools to study in vivo gene expression were developed during the past decades (25). One of the most powerful technologies is termed in vivo expression technology (IVET) and is based on transcriptional fusions of genomic DNA fragments to a selectable reporter gene (32). Recombinase-based IVET (R-IVET) is an IVET variation that employs a resolvase as the primary reporter gene. Upon expression of the resolvase, a chromosomally located marker, situated between two recombination sites, is excised from the genome (1). This irreversible step "records" in situ promoter activity and acts as a promoter-trap system, allowing the accumulation of promoter activity detection over extended in situ incubation periods. Moreover, the system is able to detect very low and transient gene expression in complex environmental condi...

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Section: Vol 74 2008 High-throughput Analysis Of In Vivo-expressed supporting

confidence: 82%

High-Throughput Identification and Validation of In Situ-Expressed Genes of Lactococcus lactis

Bachmann

Kleerebezem

Vlieg

2008

Appl Environ Microbiol

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“…The T box mechanism is used to regulate expression of the trp operon of Lactococcus lactis. (14) This illustrates the regulatory diversity that exists in controlling expression of genes performing the same function in different organisms. The role of many of the elements conserved in leader RNAs in the T box family remains to be determined, but formation of a pocket to stabilize the tRNA-leader interaction is likely to require a complex set of structural constraints.…”

Section: Trna-mediated Transcription Antitermination By the T Box Mecmentioning

confidence: 97%

Regulation by transcription attenuation in bacteria: how RNA provides instructions for transcription termination/antitermination decisions

2002

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Regulation of gene expression by premature termination of transcription, or transcription attenuation, is a common regulatory strategy in bacteria. Various mechanisms of regulating transcription termination have been uncovered, each can be placed in either of two broad categories of termination events. Many mechanisms involve choosing between two alternative hairpin structures in an RNA transcript, with the decision dependent on interactions between ribosome and transcript, tRNA and transcript, or protein and transcript. In other examples, modification of the transcription elongation complex is the crucial event. This article will describe and compare several of these regulatory strategies, and will cite specific examples to illustrate the different mechanisms employed.

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“…The T-box pattern (Fig. 9) was constructed using a training set of known T-boxes (Henkin et al 1992Vander Horn and Zahler 1992;Grundy et al 1997a;Luo et al 1997Luo et al , 1998Frenkiel et al 1998;Pelchat and Lapointe 1999;Panina et al 2003;Rodionov et al 2003Rodionov et al , 2004Grundy and Henkin 2004;Gutierrez-Preciado et al 2005) and includes the most conserved structures in T-boxes, namely, the specifier hairpin and the alternative antiterminator/ terminator structures. The identified T-boxes were aligned using the sequence/structure alignment program MultAl (A.A. Mironov, unpubl.)…”

Section: Methodsmentioning

confidence: 99%

Comparative genomic analysis of T-box regulatory systems in bacteria

Vitreschak

Миронов²,

Lyubetsky³

et al. 2008

RNA

124

223

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T-box antitermination is one of the main mechanisms of regulation of genes involved in amino acid metabolism in Gram-positive bacteria. T-box regulatory sites consist of conserved sequence and RNA secondary structure elements. Using a set of known Tbox sites, we constructed the common pattern and used it to scan available bacterial genomes. New T-boxes were found in various Gram-positive bacteria, some Gram-negative bacteria (d-proteobacteria), and some other bacterial groups (Deinococcales/Thermales, Chloroflexi, Dictyoglomi). The majority of T-box-regulated genes encode aminoacyl-tRNA synthetases. Two other groups of T-box-regulated genes are amino acid biosynthetic genes and transporters, as well as genes with unknown function. Analysis of candidate T-box sites resulted in new functional annotations. We assigned the amino acid specificity to a large number of candidate amino acid transporters and a possible function to amino acid biosynthesis genes. We then studied the evolution of the T-boxes. Analysis of the constructed phylogenetic trees demonstrated that in addition to the normal evolution consistent with the evolution of regulated genes, T-boxes may be duplicated, transferred to other genes, and change specificity. We observed several cases of recent T-box regulon expansion following the loss of a previously existing regulatory system, in particular, arginine regulon in Clostridium difficile and methionine regulon in Lactobacillaceae. Finally, we described a new structural class of T-boxes containing duplicated terminator-antiterminator elements and unusual reduced T-boxes regulating initiation of translation in the Actinobacteria.

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Transcription of the trp operon in Lactococcus lactis is controlled by antitermination in the leader region

Cited by 13 publications

References 37 publications

High-Throughput Identification and Validation of In Situ-Expressed Genes of Lactococcus lactis

High-Throughput Identification and Validation of In Situ-Expressed Genes of Lactococcus lactis

Regulation by transcription attenuation in bacteria: how RNA provides instructions for transcription termination/antitermination decisions

Comparative genomic analysis of T-box regulatory systems in bacteria

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