Journal: Journal of Bacteriology

Article Title: Functional Analysis of the Holin-Like Proteins of Mycobacteriophage Ms6

doi: 10.1128/jb.01519-10

Figure Lengend Snippet: FIG. 1. Holin-like proteins of mycobacteriophages Ms6 and D29. (A) CLUSTALW alignment of Ms6 Gp4 (AAG48320) with similar sequences of Pham 95 members included in subcluster F1: Llij Gp32 (ABD58248), Pacc40 Gp32 (YP002241616), PMC Gp32 (ABE67533), Che8 Gp34 (NP817372), Fruitloop Gp31 (YP002241716), Tweety Gp32 (YP001469265), Ramsey Gp35 (YP002241822), and Boomer Gp34 (YP002014250) (the primary accession numbers of the UniProtKB/TrEmbl database are given in parentheses). Identical (*) and highly similar (:) amino acids are indicated. Numbers refer to the amino acid positions. The two TMDs are indicated in a gray box. (B) Sequences of genes coding for the class II holin (gp4) and class III holin (gp5) of Ms6 and class II holin of D29 (gp11). Charged residues are indicated by a plus or minus sign. TMDs are indicated in gray. Amino acid residues in the SAR domain of Gp4 that are predicted to be weakly hydrophobic are shown in lowercase. Potential translation start codons and corresponding Shine-Dalgarno sequences are in bold and underlined. (C) Topological model for Ms6 Gp4 (N-in, C-in), Gp5 (N-out, C-in), and D29 Gp11 (Hol) (N-in, C-in).

Article Snippet: Strains, bacteriophages, and plasmids used in this studya Strain, bacteriophage, or plasmid Description Reference or source Strains E. coli JM109 endA1 recA1 gyrA96 thi-1 hsdR17(rK mK ) relA1 supE44 (lac-proAB) F traD36 proAB lacIqZ M15 Stratagene BL21 F ompT hsdSB(rB mB ) gal dcm Novagen BL21(DE3) F ompT hsdSB(rB mB ) gal dcm (DE3) Novagen M. smegmatis 31 mc2155 High-transformation-efficiency mutant of M. smegmatis ATCC 607 Bacteriophages D29 Lytic phage that infects both fast- and slow-growing mycobacterial species Institute Pasteur collection gt11 cIts857 Sam100 Stratagene Ms6wt Temperate bacteriophage from M. smegmatis 26 Ms6 gp1 213-bp in-frame deletion of the Ms6 gp1 gene 2 Ms6 gp4 210-bp in-frame deletion of the Ms6 gp4 gene This study Ms6 gp5 366-bp in-frame deletion of the Ms6 gp5 gene This study Ms6 gp1 gp4 213-bp and 210-bp in-frame deletions of the Ms6 gp1and gp4 genes, respectively This study Ms6 gp1 gp5 213-bp and 366-bp in-frame deletions of the Ms6 gp1and gp5 genes, respectively This study Plasmids pQE30 Expression vector, T5 promoter; Ampr lacIq Qiagen pET29a( ) Expression vector, T7 promoter; Kanr Novagen pJV53 Derivative of pLAM12 with Che9c 60 and 61 under control of the acetamidase promoter; Kanr 34 pMG231A lysA cloned into pQE30 4 pMP300 Ms6 lysA and gp4 cloned in pQE30 4 pMP310 Ms6 gp4 cloned in pQE30 4 pMJC21 Ms6 gp5 cloned in pQE30 This study pMJC22 Ms6 gp4 and gp5 cloned in pQE30 This study pMJC23 D29 gp11 cloned in pQE30 This study pMJC24 R and Ms6 gp4 cloned in pQE30 This study pMJC25 R and Ms6 gp5 cloned in pQE30 This study pMJC27 Ms6 gp5 cloned in pMG231A This study pMJC28 Ms6 gp4 and gp5 cloned in pMG231A This study pMJC29 D29 gp11 cloned in pMG231A This study pMJC30 Ms6 gp4 cloned in pET29a( ) This study pMJC31 Ms6 gp5 cloned in pET29a( ) This study pMJC32 Ms6 gp4 and gp5 cloned in pET29a( ) This study a The accession number for the Ms6 lysis genes is AF319619. on D ecem ber 23, 2012 by B row n U niversity Library http://jb.asm .org/ D ow nloaded from gene gp11 was amplified by PCR using D29 genomic DNA as a template with primers PholD29fwd/PholD29rv and cloned into SacI/HindIII sites of pQE30. pMJC24 and pMJC25 were constructed in two steps: the R gene was amplified using the genomic DNA of bacteriophage gt11 as a template with primers P Rfwd/P Rrv and cloned into BamHI/SacI sites of pQE30.

Techniques:

Journal: Journal of Bacteriology

Article Title: Functional Analysis of the Holin-Like Proteins of Mycobacteriophage Ms6

doi: 10.1128/jb.01519-10

Figure Lengend Snippet: FIG. 2. (A) Expression of the holin-like proteins from mycobacteriophages Ms6 and D29 in E. coli. E. coli JM109 cells carrying plasmid pQE30 containing no insert or cloned genes were grown in LB broth at 37°C to an OD600 of 0.2. At time zero, transcription of cloned lysis genes was induced by addition of 1 mM IPTG. (B) Release of -galactosidase from E. coli BL21 expressing Ms6 and D29 holin-like proteins. Activity was determined in the supernatants of induced cultures. Results are averages of triplicate experiments.

Article Snippet: Strains, bacteriophages, and plasmids used in this studya Strain, bacteriophage, or plasmid Description Reference or source Strains E. coli JM109 endA1 recA1 gyrA96 thi-1 hsdR17(rK mK ) relA1 supE44 (lac-proAB) F traD36 proAB lacIqZ M15 Stratagene BL21 F ompT hsdSB(rB mB ) gal dcm Novagen BL21(DE3) F ompT hsdSB(rB mB ) gal dcm (DE3) Novagen M. smegmatis 31 mc2155 High-transformation-efficiency mutant of M. smegmatis ATCC 607 Bacteriophages D29 Lytic phage that infects both fast- and slow-growing mycobacterial species Institute Pasteur collection gt11 cIts857 Sam100 Stratagene Ms6wt Temperate bacteriophage from M. smegmatis 26 Ms6 gp1 213-bp in-frame deletion of the Ms6 gp1 gene 2 Ms6 gp4 210-bp in-frame deletion of the Ms6 gp4 gene This study Ms6 gp5 366-bp in-frame deletion of the Ms6 gp5 gene This study Ms6 gp1 gp4 213-bp and 210-bp in-frame deletions of the Ms6 gp1and gp4 genes, respectively This study Ms6 gp1 gp5 213-bp and 366-bp in-frame deletions of the Ms6 gp1and gp5 genes, respectively This study Plasmids pQE30 Expression vector, T5 promoter; Ampr lacIq Qiagen pET29a( ) Expression vector, T7 promoter; Kanr Novagen pJV53 Derivative of pLAM12 with Che9c 60 and 61 under control of the acetamidase promoter; Kanr 34 pMG231A lysA cloned into pQE30 4 pMP300 Ms6 lysA and gp4 cloned in pQE30 4 pMP310 Ms6 gp4 cloned in pQE30 4 pMJC21 Ms6 gp5 cloned in pQE30 This study pMJC22 Ms6 gp4 and gp5 cloned in pQE30 This study pMJC23 D29 gp11 cloned in pQE30 This study pMJC24 R and Ms6 gp4 cloned in pQE30 This study pMJC25 R and Ms6 gp5 cloned in pQE30 This study pMJC27 Ms6 gp5 cloned in pMG231A This study pMJC28 Ms6 gp4 and gp5 cloned in pMG231A This study pMJC29 D29 gp11 cloned in pMG231A This study pMJC30 Ms6 gp4 cloned in pET29a( ) This study pMJC31 Ms6 gp5 cloned in pET29a( ) This study pMJC32 Ms6 gp4 and gp5 cloned in pET29a( ) This study a The accession number for the Ms6 lysis genes is AF319619. on D ecem ber 23, 2012 by B row n U niversity Library http://jb.asm .org/ D ow nloaded from gene gp11 was amplified by PCR using D29 genomic DNA as a template with primers PholD29fwd/PholD29rv and cloned into SacI/HindIII sites of pQE30. pMJC24 and pMJC25 were constructed in two steps: the R gene was amplified using the genomic DNA of bacteriophage gt11 as a template with primers P Rfwd/P Rrv and cloned into BamHI/SacI sites of pQE30.

Techniques: Expressing, Plasmid Preparation, Clone Assay, Lysis, Activity Assay