Journal:

Article Title: Identification and Genetic Characterization of a Novel Proteinase, PrtR, from the Human Isolate Lactobacillus rhamnosus BGT10

doi: 10.1128/AEM.69.10.5802-5811.2003

Figure Lengend Snippet: Bacterial strains and plasmids used in this study

Article Snippet: The results showed that the smallest construct, pEB99, is active (Table ) and carries the minimal promoter region of the prtR gene situated on a DNA fragment with the 5′ end 44 bp upstream and the 3′-end 54 bp downstream from the prtR transcription start point (Fig. and ). fig ft0 fig mode=article f1 fig/graphic|fig/alternatives/graphic mode="anchored" m1 Open in a separate window FIG. 4. caption a7 Determination of the prtR transcription start point. (A) Primer extension analysis of the prtR promoter was performed with RNA from L. casei ATCC 393 T cells harboring pEB471. (B) L. casei ATCC 393 T cells harboring pEB640 (lane 1) and L. rhamnosus BGT10 (lane 2).

Techniques: Plasmid Preparation

Journal:

Article Title: Identification and Genetic Characterization of a Novel Proteinase, PrtR, from the Human Isolate Lactobacillus rhamnosus BGT10

doi: 10.1128/AEM.69.10.5802-5811.2003

Figure Lengend Snippet: Determination of the prtR transcription start point. (A) Primer extension analysis of the prtR promoter was performed with RNA from L. casei ATCC 393T cells harboring pEB471. (B) L. casei ATCC 393T cells harboring pEB640 (lane 1) and L. rhamnosus BGT10 (lane 2). The sequencing reactions (A, C, G, and T) were performed with the same primers as the corresponding primer extension reactions. The primer extension products are indicated with an arrow. (C) Interpretation of the primer extension results. The putative −35 and −10 prtR promoter haxamers (underlined), the transcription start point (+1, underlined), and the putative ribosome binding site (RBS) (boldface) are shown. The prtR coding sequence is in italic.

Article Snippet: The results showed that the smallest construct, pEB99, is active (Table ) and carries the minimal promoter region of the prtR gene situated on a DNA fragment with the 5′ end 44 bp upstream and the 3′-end 54 bp downstream from the prtR transcription start point (Fig. and ). fig ft0 fig mode=article f1 fig/graphic|fig/alternatives/graphic mode="anchored" m1 Open in a separate window FIG. 4. caption a7 Determination of the prtR transcription start point. (A) Primer extension analysis of the prtR promoter was performed with RNA from L. casei ATCC 393 T cells harboring pEB471. (B) L. casei ATCC 393 T cells harboring pEB640 (lane 1) and L. rhamnosus BGT10 (lane 2).

Techniques: Sequencing, Binding Assay

Journal:

Article Title: Identification and Genetic Characterization of a Novel Proteinase, PrtR, from the Human Isolate Lactobacillus rhamnosus BGT10

doi: 10.1128/AEM.69.10.5802-5811.2003

Figure Lengend Snippet: Expression of the prtR-gusA fusions in CDM supplemented with casitone and in MRS broth

Article Snippet: The results showed that the smallest construct, pEB99, is active (Table ) and carries the minimal promoter region of the prtR gene situated on a DNA fragment with the 5′ end 44 bp upstream and the 3′-end 54 bp downstream from the prtR transcription start point (Fig. and ). fig ft0 fig mode=article f1 fig/graphic|fig/alternatives/graphic mode="anchored" m1 Open in a separate window FIG. 4. caption a7 Determination of the prtR transcription start point. (A) Primer extension analysis of the prtR promoter was performed with RNA from L. casei ATCC 393 T cells harboring pEB471. (B) L. casei ATCC 393 T cells harboring pEB640 (lane 1) and L. rhamnosus BGT10 (lane 2).

Techniques: Expressing, Activity Assay

Journal:

Article Title: Biosynthesis of Lipoteichoic Acid in Lactobacillus rhamnosus : Role of DltD in d -Alanylation

doi:

Figure Lengend Snippet: Bacterial strains, phages, and plasmids used in this study a

Article Snippet: All bacterial strains, phages, and plasmids used in this study are listed in Table . table ft1 table-wrap mode="anchored" t5 TABLE 1 caption a7 Strain, plasmid, or phage Description Source or reference Strains L. rhamnosus 7469 Formerly named L. casei ATCC 7469 ATCC L. casei 102S L. casei ATCC 393 cured from plasmids B. Chassy L. casei 102S dltD :: erm Em r ; dltD interrupted by erm This study E. coli XL-1 Blue MRF′ Δ( mcrA ) 183 Δ ( mcrCB-hsdSMR-mrr ) 173end1A supE44thi-1 recA1 gyrA96 relA1lac [F′ proAB lac q Z ΔM15 Tn 10 (Tet r )] Stratagene E. coli BL21(DE3) pLysS F − ompT hsdS B (r B − m B − ) gal dcm (DE3)pLysS Novagene E. coli DH5α F − f80d lacZ ΔM15 Δ( lacZYA-argF ) U169 deoR recA1 endA1 hsdR17 (r k − m k + ) supE44 λ − thi-1 girA96 relA1 Gibco-BRL Phages (phagemids) λZAPII [Bluescript SK( − )] Ap r cloning vector Stratagene VCSM13 Helper phage Stratagene λZAPII(−)DE43 4.3 kb of L. rhamnosus DNA containing dltA , dltB , dltC , and partial dltD 10 λZAPII(−)A65 6.5 kb of L. rhamnosus DNA containing partial dltB , dltC , dltD , and downstream flanking region This study Plasmids pET-3d Expression vector Novagene pDltD Expression plasmid; dltD cloned into pET-3d This study pΔDltD Expression plasmid; Δ dltD cloned into pET-3d This study pVE6006 Em r Cm r Ts shuttle vector 30 pNZ123 Cm r shuttle vector 46 pΔDltD/ erm 1.2-kb Eco ICR erm fragment of pVE6006 joined to Bam HI blunt-ended pΔDltD in direct orientation to Δ dltD This study pNZ123/ dlt 4.895-kb fragment containing dlt operon cloned into Hin dIII of pNZ123 This study Open in a separate window a Em r , erythromycin resistant; Cm r , choramphenicol resistant; Ap r , ampicillin resistant; ATCC, American Type Culture Collection.

Techniques: Plasmid Preparation, Cloning, Expressing, Clone Assay

Journal:

Article Title: Biosynthesis of Lipoteichoic Acid in Lactobacillus rhamnosus : Role of DltD in d -Alanylation

doi:

Figure Lengend Snippet: Incorporation of d -[ 14 C]alanine into permeabilized cells and membranes from L. casei 102S ( dltD + ) and its dltD :: erm mutant

Article Snippet: All bacterial strains, phages, and plasmids used in this study are listed in Table . table ft1 table-wrap mode="anchored" t5 TABLE 1 caption a7 Strain, plasmid, or phage Description Source or reference Strains L. rhamnosus 7469 Formerly named L. casei ATCC 7469 ATCC L. casei 102S L. casei ATCC 393 cured from plasmids B. Chassy L. casei 102S dltD :: erm Em r ; dltD interrupted by erm This study E. coli XL-1 Blue MRF′ Δ( mcrA ) 183 Δ ( mcrCB-hsdSMR-mrr ) 173end1A supE44thi-1 recA1 gyrA96 relA1lac [F′ proAB lac q Z ΔM15 Tn 10 (Tet r )] Stratagene E. coli BL21(DE3) pLysS F − ompT hsdS B (r B − m B − ) gal dcm (DE3)pLysS Novagene E. coli DH5α F − f80d lacZ ΔM15 Δ( lacZYA-argF ) U169 deoR recA1 endA1 hsdR17 (r k − m k + ) supE44 λ − thi-1 girA96 relA1 Gibco-BRL Phages (phagemids) λZAPII [Bluescript SK( − )] Ap r cloning vector Stratagene VCSM13 Helper phage Stratagene λZAPII(−)DE43 4.3 kb of L. rhamnosus DNA containing dltA , dltB , dltC , and partial dltD 10 λZAPII(−)A65 6.5 kb of L. rhamnosus DNA containing partial dltB , dltC , dltD , and downstream flanking region This study Plasmids pET-3d Expression vector Novagene pDltD Expression plasmid; dltD cloned into pET-3d This study pΔDltD Expression plasmid; Δ dltD cloned into pET-3d This study pVE6006 Em r Cm r Ts shuttle vector 30 pNZ123 Cm r shuttle vector 46 pΔDltD/ erm 1.2-kb Eco ICR erm fragment of pVE6006 joined to Bam HI blunt-ended pΔDltD in direct orientation to Δ dltD This study pNZ123/ dlt 4.895-kb fragment containing dlt operon cloned into Hin dIII of pNZ123 This study Open in a separate window a Em r , erythromycin resistant; Cm r , choramphenicol resistant; Ap r , ampicillin resistant; ATCC, American Type Culture Collection.

Techniques:

Journal:

Article Title: Biosynthesis of Lipoteichoic Acid in Lactobacillus rhamnosus : Role of DltD in d -Alanylation

doi:

Figure Lengend Snippet: Formation of d-alanyl–Dcp and d-alanyl–ACP in the presence of DltD+, DltD−, and DltD−/DltD+ membranes. Nondenaturing PAGE (15%) was used to monitor the amount of either d-[14C]alanyl–Dcp or d-[14C]alanyl–ACP according to the method of Heaton and Neuhaus (19). The reactions were performed in 250-μl mixtures containing 8 U of Dcl, 5 nmol of either Dcp or ACP, 5 mM MgCl2, 5 mM DTT, 30 mM bis-Tris buffer (pH 6.5), 10 mM ATP, and 110 μM d[14C]alanine (46 mCi/mmol) in the absence of membranes (w/o) and in the presence of membranes (130 μg) from L. casei 102S (dltD+), L. casei 102S (dltD::erm), and the dltD mutant which had been complemented with pNZ123/dlt (dltD::erm/dltD+). All reaction mixtures were incubated for 1 h at 37°C.

Article Snippet: All bacterial strains, phages, and plasmids used in this study are listed in Table . table ft1 table-wrap mode="anchored" t5 TABLE 1 caption a7 Strain, plasmid, or phage Description Source or reference Strains L. rhamnosus 7469 Formerly named L. casei ATCC 7469 ATCC L. casei 102S L. casei ATCC 393 cured from plasmids B. Chassy L. casei 102S dltD :: erm Em r ; dltD interrupted by erm This study E. coli XL-1 Blue MRF′ Δ( mcrA ) 183 Δ ( mcrCB-hsdSMR-mrr ) 173end1A supE44thi-1 recA1 gyrA96 relA1lac [F′ proAB lac q Z ΔM15 Tn 10 (Tet r )] Stratagene E. coli BL21(DE3) pLysS F − ompT hsdS B (r B − m B − ) gal dcm (DE3)pLysS Novagene E. coli DH5α F − f80d lacZ ΔM15 Δ( lacZYA-argF ) U169 deoR recA1 endA1 hsdR17 (r k − m k + ) supE44 λ − thi-1 girA96 relA1 Gibco-BRL Phages (phagemids) λZAPII [Bluescript SK( − )] Ap r cloning vector Stratagene VCSM13 Helper phage Stratagene λZAPII(−)DE43 4.3 kb of L. rhamnosus DNA containing dltA , dltB , dltC , and partial dltD 10 λZAPII(−)A65 6.5 kb of L. rhamnosus DNA containing partial dltB , dltC , dltD , and downstream flanking region This study Plasmids pET-3d Expression vector Novagene pDltD Expression plasmid; dltD cloned into pET-3d This study pΔDltD Expression plasmid; Δ dltD cloned into pET-3d This study pVE6006 Em r Cm r Ts shuttle vector 30 pNZ123 Cm r shuttle vector 46 pΔDltD/ erm 1.2-kb Eco ICR erm fragment of pVE6006 joined to Bam HI blunt-ended pΔDltD in direct orientation to Δ dltD This study pNZ123/ dlt 4.895-kb fragment containing dlt operon cloned into Hin dIII of pNZ123 This study Open in a separate window a Em r , erythromycin resistant; Cm r , choramphenicol resistant; Ap r , ampicillin resistant; ATCC, American Type Culture Collection.

Techniques: Mutagenesis, Incubation

Journal:

Article Title: Phenotypic and Genotypic Characterization of Non-Starter Lactic Acid Bacteria in Mature Cheddar Cheese

doi:

Figure Lengend Snippet: (a) RAPD profiles of type and collection strains of L. paracasei subsp. paracasei (lanes 2 to 9, LMG 14766, LMG 11473, LMG 13087T, LMG 9193, LMG 11965, LMG 13722, LMG 13717, and LMG 13724a, respectively, and lanes 11 to 14, LMG 13724b, LMG 13732, LMG 12586, and LMG 9192T, respectively), L. paracasei subsp. tolerans (lanes 15 and 16, LMG 9191aT and LMG 9191bT, respectively), and L. casei (lane 17, LMG 6904T). Lanes 1, 10, and 18 contain 100-bp molecular size markers. (b) RAPD profiles of type and collection strains of L. rhamnosus (lanes 2 to 7, LMG 10769, LMG 10775, LMG 6400Ta, LMG 6400Tb, LMG 8153, and LMG 12166, respectively), L. plantarum (lanes 9 to 16, LMG 11475, LMG 14769a, LMG 14769b, LMG 6907T, LMG 1284, LMG 14760, LMG 9205, and LMG 11460, respectively), and L. pentosus (lane 17, LMG 10755T). Lanes 1, 8, and 18 contain 100-bp molecular size markers. (c) RAPD profiles of type and collection strains of L. curvatus (lanes 2 to 6, LMG 12009, LMG 12007, LMG 9198T, LMG 12008, and LMG 11479, respectively). Lanes 1 and 7 contain 100-bp molecular size markers.

Article Snippet: The type strains of L. paracasei subsp. paracasei (LMG 13087 T , cluster 30) and L. casei subsp. casei (LMG 6904 T [ATCC 393, reclassified as L. zeae ], cluster 69) and the proposed neotype strain of L. casei subsp. casei ATCC 334 (LMG 12586, cluster 57), in place of ATCC 393 ( 12 ), were all well separated (Fig. ).

Techniques: