Determination of the ribosome-binding sequence and spacer length between binding site and initiation codon for efficient protein expression in Bifidobacterium longum 105-A

He, Jianlong; Sakaguchi, Kouta; Suzuki, Tomohiko

doi:10.1016/j.jbiosc.2011.11.019

Cited by 18 publications

(15 citation statements)

References 20 publications

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“…Normalization of the PepI expression data with respect to the corresponding transcript data reveals that spacing is a factor affecting translation initiation efficiency for the canonical SD sequence, as described for other bacteria (50)(51)(52). A spacing effect can also be seen with regard to the Bacteroides ribosomal binding site (comparing pGH022 and pGH049).…”

Section: Discussionmentioning

confidence: 75%

Defining the Bacteroides Ribosomal Binding Site

Wegmann¹,

Horn²,

Carding

2013

Appl Environ Microbiol

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The human gastrointestinal tract, in particular the colon, hosts a vast number of commensal microorganisms. Representatives of the genus Bacteroides are among the most abundant bacterial species in the human colon. Bacteroidetes diverged from the common line of eubacterial descent before other eubacterial groups. As a result, they employ unique transcription initiation signals and, because of this uniqueness, they require specific genetic tools. Although some tools exist, they are not optimal for studying the roles and functions of these bacteria in the human gastrointestinal tract. Focusing on translation initiation signals in Bacteroides, we created a series of expression vectors allowing for different levels of protein expression in this genus, and we describe the use of pepI from Lactobacillus delbrueckii subsp. lactis as a novel reporter gene for Bacteroides. Furthermore, we report the identification of the 3= end of the 16S rRNA of Bacteroides ovatus and analyze in detail its ribosomal binding site, thus defining a core region necessary for efficient translation, which we have incorporated into the design of our expression vectors. Based on the sequence logo information from the 5= untranslated region of other Bacteroidales ribosomal protein genes, we conclude that our findings are relevant to all members of this order.

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Section: Discussionmentioning

confidence: 75%

Defining the Bacteroides Ribosomal Binding Site

Wegmann¹,

Horn²,

Carding

2013

Appl Environ Microbiol

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“…We also constructed a gene-knockout system using a temperature-sensitive plasmid [55], pyrE -dependent bidirectional selection marker [56], improved promoter [57] and oxygen tolerance [58]. By the combination of these molecular genetic tools, the construction of gene deletion in Bifidobacteria , using the double cross-over homologous recombination system, became available at the practical level [55, 56, 59].…”

Section: Introductionmentioning

confidence: 99%

Capsular polysaccharide inhibits adhesion of Bifidobacterium longum 105-A to enterocyte-like Caco-2 cells and phagocytosis by macrophages

et al. 2017

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Background Bifidobacterium longum 105-A produces markedly high amounts of capsular polysaccharides (CPS) and exopolysaccharides (EPS) that should play distinct roles in bacterial–host interactions. To identify the biological function of B. longum 105-A CPS/EPS, we carried out an informatics survey of the genome and identified the EPS-encoding genetic locus of B. longum 105-A that is responsible for the production of CPS/EPS. The role of CPS/EPS in the adaptation to gut tract environment and bacteria-gut cell interactions was investigated using the ΔcpsD mutant.ResultsA putative B. longum 105-A CPS/EPS gene cluster was shown to consist of 24 putative genes encoding a priming glycosyltransferase (cpsD), 7 glycosyltransferases, 4 CPS/EPS synthesis machinery proteins, and 3 dTDP-L-rhamnose synthesis enzymes. These enzymes should form a complex system that is involved in the biogenesis of CPS and/or EPS. To confirm this, we constructed a knockout mutant (ΔcpsD) by a double cross-over homologous recombination. Compared to wild-type, the ∆cpsD mutant showed a similar growth rate. However, it showed quicker sedimentation and formation of cell clusters in liquid culture. EPS was secreted by the ∆cpsD mutant, but had altered monosaccharide composition and molecular weight. Comparison of the morphology of B. longum 105-A wild-type and ∆cpsD by negative staining in light and electron microscopy revealed that the formation of fimbriae is drastically enhanced in the ∆cpsD mutant while the B. longum 105-A wild-type was coated by a thick capsule. The fimbriae expression in the ∆cpsD was closely associated with the disappearance of the CPS layer. The wild-type showed low pH tolerance, adaptation, and bile salt tolerance, but the ∆cpsD mutant had lost this survivability in gastric and duodenal environments. The ∆cpsD mutant was extensively able to bind to the human colon carcinoma Caco-2 cell line and was phagocytosed by murine macrophage RAW 264.7, whereas the wild-type did not bind to epithelial cells and totally resisted internalization by macrophages.ConclusionsOur results suggest that CPS/EPS production and fimbriae formation are negatively correlated and play key roles in the survival, attachment, and colonization of B. longum 105-A in the gut.Electronic supplementary materialThe online version of this article (doi:10.1186/s13099-017-0177-x) contains supplementary material, which is available to authorized users.

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“…We have tried to construct genetic tools for bifidobacteria, such as high efficiency transformation methods [4, 5], and to improve gene expression by tuning promoter and ribosomal binding sequences [27, 28]. In this study, we reported a new selection marker in Bifidobacterium.…”

Section: Resultsmentioning

confidence: 99%

The <i>pyrE</i> Gene as a Bidirectional Selection Marker in <i>Bifidobacterium Longum</i> 105-A

Sakaguchi

Funaoka

Tani

et al. 2013

Bioscience of Microbiota, Food and Health

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We constructed a deletion mutant of the pyrE gene in Bifidobacterium longum 105-A. A pyrE knockout cassette was cloned into pKKT427, a Bifidobacterium-Escherichia coli shuttle vector, and then introduced into B. longum 105-A by electroporation. The transformants were propagated and spread onto MRS plates containing 5-fluoroorotic acid (5-FOA) and uracil. 5-FOA-resistant mutants were obtained at a frequency of 4.7 × 10−5 integrations per cell. To perform pyrE gene complementation, the pyrE gene was amplified by PCR and used to construct a complementation plasmid (pKKT427-pyrE+). B. longum 105-A ∆pyrE harboring this plasmid could not grow on MRS plates containing 5-FOA, uracil and spectinomycin. We also developed a chemically defined medium (bifidobacterial minimal medium; BMM) containing inorganic salts, glucose, vitamins, isoleucine and tyrosine for positive selection of pyrE transformants. B. longum 105-A ∆pyrE could not grow on BMM agar, but the same strain harboring pKKT427-pyrE+ could. Thus, pyrE can be used as a counterselection marker in B. longum 105-A and potentially other Bifidobacterium species as well. We demonstrated the effectiveness of this system by constructing a knockout mutant of the xynF gene in B. longum 105-A by using the pyrE gene as a counterselection marker. This pyrE-based selection system will contribute to genetic studies of bifidobacteria.

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Determination of the ribosome-binding sequence and spacer length between binding site and initiation codon for efficient protein expression in Bifidobacterium longum 105-A

Cited by 18 publications

References 20 publications

Defining the Bacteroides Ribosomal Binding Site

Defining the Bacteroides Ribosomal Binding Site

Capsular polysaccharide inhibits adhesion of Bifidobacterium longum 105-A to enterocyte-like Caco-2 cells and phagocytosis by macrophages

The <i>pyrE</i> Gene as a Bidirectional Selection Marker in <i>Bifidobacterium Longum</i> 105-A

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