Journal:

Article Title: Role of Porins in the Susceptibility of Mycobacterium smegmatis and Mycobacterium chelonae to Aldehyde-Based Disinfectants and Drugs ▿

doi: 10.1128/AAC.00590-09

Figure Lengend Snippet: GTA and OPA susceptibility of defined isogenic mutants of M. smegmatis mc2155. Results are expressed as CFU counts upon exposure of the test organisms to the indicated concentrations of disinfectants for 0 to 15 min. (A) mc2155ΔembA (closed circles), mc2155ΔembB (closed triangles), and mc2155ΔMSMEG4245 (closed diamonds) mutants are slightly more sensitive to GTA than is their WT parent, mc2155 (open triangle). GTA (B) and OPA (C) sensitivity of the porin mutants MN01 (closed rectangles, solid line) and ML10 (closed triangles, solid line); the complemented porin mutants MN01/pMN013 (open rectangles, dashed line) and ML10/pMN013 (open triangles, dotted line); and their WT parent, SMR5 (closed circles, solid line).

Article Snippet: We wish to thank M. Daffé (CNRS-IPBS, Toulouse, France), J.-M. Reyrat (INSERM U570, Paris, France), J. Dahl (Washington State University, Pullman, WA), Y. Av-Gay (University of British Columbia, Vancouver, British Columbia, Canada), and D. Chatterjee and D. Kaur (CSU, Fort Collins, CO) for providing the M. smegmatis isogenic mutants described in Table and F. Ripoll for sequence deposition.

Techniques:

Journal:

Article Title: Role of Porins in the Susceptibility of Mycobacterium smegmatis and Mycobacterium chelonae to Aldehyde-Based Disinfectants and Drugs ▿

doi: 10.1128/AAC.00590-09

Figure Lengend Snippet: Susceptibility of the M. chelonae strains ATCC 35752 and 9917 to GTA and OPA, and effect of expressing the mspA porin gene from M. smegmatis. Results are expressed as CFU counts upon exposure of the test organisms (M. chelonae ATCC 35752 [closed rectangles, solid line]; M. chelonae 9917 [open triangles, solid line]; M. chelonae ATCC 35752/pZS01 [closed rectangles, dotted line]; 9917/pZS01 [open triangles, dotted line]) to the indicated concentrations of GTA (A and B) or OPA (C) for 0 to 30 min. The increased susceptibility of 9917/pZS01 to GTA was consistently visible at the highest concentration of disinfectant (B) after 5 min of exposure, indicative of a more rapid killing of the M. chelonae recombinant isolate expressing the mspA porin gene.

Article Snippet: We wish to thank M. Daffé (CNRS-IPBS, Toulouse, France), J.-M. Reyrat (INSERM U570, Paris, France), J. Dahl (Washington State University, Pullman, WA), Y. Av-Gay (University of British Columbia, Vancouver, British Columbia, Canada), and D. Chatterjee and D. Kaur (CSU, Fort Collins, CO) for providing the M. smegmatis isogenic mutants described in Table and F. Ripoll for sequence deposition.

Techniques: Expressing, Concentration Assay, Recombinant

Journal: International Journal of Molecular Sciences

Article Title: Cloning and Partial Characterization of an Endo-α-(1→6)- d -Mannanase Gene from Bacillus circulans

doi: 10.3390/ijms20246244

Figure Lengend Snippet: Silver-stained SDS PAGE showing the GH-emn digestion products of native M. smegmatis LM and LAM. MWM: Molecular weight marker.

Article Snippet: Purified M. tuberculosis H37Rv phosphatidyl- myo -inositol hexamannosides (PIM 6 ) and M. smegmatis LAM were received from BEI resources.

Techniques: Staining, SDS Page, Molecular Weight, Marker

Journal: International Journal of Molecular Sciences

Article Title: Cloning and Partial Characterization of an Endo-α-(1→6)- d -Mannanase Gene from Bacillus circulans

doi: 10.3390/ijms20246244

Figure Lengend Snippet: Negative ion mass spectra of deacylated LM from M. smegmatis before and after digestion with GH-emn. ( A ) Mass spectrum of undigested d-LM. The spectrum shows the mass distribution of the mannan backbone of d-LM which contains up to 40 mannosyl residues (differing by the mass of one hexose). Inset: The isotopic pattern for quadruply-charged d-LM with 40 mannosyl residues indicates the sensitivity of the mass spectrometer for high-molecular weight compounds. ( B ) Mass spectrum of d-LM after digestion by GH-emn. The mass spectra for three different retention times (RT) are shown. At all retention times, the spectra are dominated by singly-charged PIM 2 and PIM 3 . RT 2.6 and 2.8 min show the presence of ions for Man17 to Man27 [M-3H] and Man9 to Man17 [M-2H]. RT 3.0 min shows low-molecular weight oligomannosides (Man 2 to Man 7 ) as singly charged and doubly charged chlorine adducts.

Article Snippet: Purified M. tuberculosis H37Rv phosphatidyl- myo -inositol hexamannosides (PIM 6 ) and M. smegmatis LAM were received from BEI resources.

Techniques: Mass Spectrometry, High Molecular Weight, Molecular Weight

Journal: Frontiers in Microbiology

Article Title: Rv1717 Is a Cell Wall - Associated β-Galactosidase of Mycobacterium tuberculosis That Is Involved in Biofilm Dispersion

doi: 10.3389/fmicb.2020.611122

Figure Lengend Snippet: Mycobacterium smegmatis expressing Rv1717 shows altered colony morphology and cell wall (CW) associated properties. (A) On MB 7H10 agar, M. smegmatis harboring empty vector (Msm pMV261 ) formed rough colonies with irregular margins, while M. smegmatis expressing Rv1717 (Msm Rv1717 ) formed smoother colonies with more regular margins. (B) Accumulation of EtBr and (C) Nile red by Msm pMV261 and Msm Rv1717 (D) Msm Rv1717 and Msm pMV261 were left untreated or treated with 0.05 or 0.1% (w/v) Sodium dodecyl sulfate (SDS) for 3 and 6 h. Viable cells in all samples were estimated by CFU assay. Statistical significance of data wherever applicable is indicated by ns: p > 0.05; *** p < 0.001. Data plotted are mean ± SD of three independent experiments.

Article Snippet: The membrane was blocked with 5% skimmed milk in 1 × PBS at room temperature for 1 h. The membrane was further incubated with primary antibodies [anti-6 × His-tag mouse monoclonal antibody (1: 5000) (Santa Cruz Biotechnology, Dallas, TX, United States; anti-mycobacterial Hsp65 mouse monoclonal antibody (Santa Cruz Biotechnology, Dallas, TX, United States; 1: 1000), and anti- M. smegmatis LAM monoclonal antibody NR-13798 (BEI Resources, Manassas, VA; 1: 200)], washed with PBS containing tween 20 (0.05%) and incubated with anti-mouse secondary antibody-HRP (1:5000; Santa Cruz Biotechnology, Dallas, TX, United States) in 5% BSA in PBST for 1–2 h. Membrane was washed with PBST before incubating with the Luminol Reagent (Santa Cruz Biotechnology, Dallas, TX).

Techniques: Expressing, Plasmid Preparation, Colony-forming Unit Assay

Journal: Frontiers in Microbiology

Article Title: Rv1717 Is a Cell Wall - Associated β-Galactosidase of Mycobacterium tuberculosis That Is Involved in Biofilm Dispersion

doi: 10.3389/fmicb.2020.611122

Figure Lengend Snippet: Exogenous Rv1717 protein inhibits biofilm formation and degrades biofilms. (A) Wild type M. smegmatis was allowed to form biofilm in Sauton’s medium containing different concentrations (0.01–5 μM) of purified Rv1717 protein in sodium phosphate buffer/buffer only/neither (untreated). On day 3, the biofilm biomass was quantified by crystal violet assay. (B) Fifth day air-liquid interface pellicle of M. smegmatis culture in Sauton’s medium in presence of 1 μM Rv1717. (C) Mycobacterium smegmatis biofilms were treated with 5 μM Rv1717 protein in sodium phosphate buffer pH 8.0 or buffer only. The biofilm biomass was quantified by crystal violet assay. Statistical significance of data wherever applicable is indicated by *** p < 0.001. Data plotted are mean ± SD of three independent experiments.

Article Snippet: The membrane was blocked with 5% skimmed milk in 1 × PBS at room temperature for 1 h. The membrane was further incubated with primary antibodies [anti-6 × His-tag mouse monoclonal antibody (1: 5000) (Santa Cruz Biotechnology, Dallas, TX, United States; anti-mycobacterial Hsp65 mouse monoclonal antibody (Santa Cruz Biotechnology, Dallas, TX, United States; 1: 1000), and anti- M. smegmatis LAM monoclonal antibody NR-13798 (BEI Resources, Manassas, VA; 1: 200)], washed with PBS containing tween 20 (0.05%) and incubated with anti-mouse secondary antibody-HRP (1:5000; Santa Cruz Biotechnology, Dallas, TX, United States) in 5% BSA in PBST for 1–2 h. Membrane was washed with PBST before incubating with the Luminol Reagent (Santa Cruz Biotechnology, Dallas, TX).

Techniques: Purification, Crystal Violet Assay

Journal: Frontiers in Microbiology

Article Title: Rv1717 Is a Cell Wall - Associated β-Galactosidase of Mycobacterium tuberculosis That Is Involved in Biofilm Dispersion

doi: 10.3389/fmicb.2020.611122

Figure Lengend Snippet: Rv1717 reduces the binding of WFL, a galactose-specific lectin to mycobacterial extracellular polymeric substance (EPS). (A) Mycobacterium smegmatis recombinant strains expressing RFP fused to Rv1717 (Msm Rv1717-rfp or RFP alone (Msm rfp ) were grown in Sauton’s medium to form biofilms on coverslips. On day 3, 4, and 5, the coverslips were stained with Fluorescein-tagged Wisteria floribunda lectin (WFL) and subjected to confocal laser scanning microscopy. Imaging was done with a 100× water immersion objective laser line. The representative images show WFL as green and bacteria as red entities. Absence of fluorescence mixing (orange) indicates that WFL stains the EPS and not the bacteria per se . WFL binding was seen on all days and was progressive in case of both strains. Bacteria are overlaid with the EPS on fifth day and hence are barely visible. (B) Fluorescein-WFL stained biofilms (upper panel) and planktonic culture (lower panel) of Mtb H37Ra. (C) Exopolysaccharides were extracted and purified from M. smegmatis biofilms, coated on 96-well ELISA plates and treated with Rv1717 protein (12 μM) in buffer or buffer without the protein for 1 h at 37°C. Residual EPS after thorough washing was stained with Fluorescein-tagged WFL and quantified by fluorescence measurement. Data plotted are mean ± SD of three independent experiments. Statistical significance of data wherever applicable is indicated by ** p < 0.01; *** p < 0.001.

Article Snippet: The membrane was blocked with 5% skimmed milk in 1 × PBS at room temperature for 1 h. The membrane was further incubated with primary antibodies [anti-6 × His-tag mouse monoclonal antibody (1: 5000) (Santa Cruz Biotechnology, Dallas, TX, United States; anti-mycobacterial Hsp65 mouse monoclonal antibody (Santa Cruz Biotechnology, Dallas, TX, United States; 1: 1000), and anti- M. smegmatis LAM monoclonal antibody NR-13798 (BEI Resources, Manassas, VA; 1: 200)], washed with PBS containing tween 20 (0.05%) and incubated with anti-mouse secondary antibody-HRP (1:5000; Santa Cruz Biotechnology, Dallas, TX, United States) in 5% BSA in PBST for 1–2 h. Membrane was washed with PBST before incubating with the Luminol Reagent (Santa Cruz Biotechnology, Dallas, TX).

Techniques: Binding Assay, Recombinant, Expressing, Staining, Confocal Laser Scanning Microscopy, Imaging, Bacteria, Fluorescence, Purification, Enzyme-linked Immunosorbent Assay

Journal: PLoS Genetics

Article Title: Leaderless Transcripts and Small Proteins Are Common Features of the Mycobacterial Translational Landscape

doi: 10.1371/journal.pgen.1005641

Figure Lengend Snippet: Candidate codons were tested in leadered and leaderless contexts for their activity in initiating translation of the adjacent lacZ gene. Putative positive (ATG) and negative (ATC) controls provided reference activities. The level of β-galactosidase expression in M . smegmatis from leaderless transcripts (upper right green bars beginning with an ATG or GTG codon) was similar to that from leadered transcripts (upper left black bars). CTG and TTG candidate codons were ineffective initiators at the leaderless position. Differences in β -galactosidase activities in M . smegmatis leaderless constructs were not due to effects of the +1 nucleotide (indicated by black background) on transcript abundance, as corresponding leadered constructs were not comparably affected.

Article Snippet: The mc 2 155 strain of M . smegmatis was grown in Middlebrook 7H9 broth supplemented with ADC and 0.05% Tween 80 with shaking at 230 RPM at 37°C to an OD 600 of ~1.0.

Techniques: Activity Assay, Expressing, Construct

Journal: PLoS Genetics

Article Title: Leaderless Transcripts and Small Proteins Are Common Features of the Mycobacterial Translational Landscape

doi: 10.1371/journal.pgen.1005641

Figure Lengend Snippet: (A) Libraries of leader sequences were generated using two overlapping oligonucleotides, each with a single randomized codon positioned either at the leaderless position (+1) or the leadered position (+30), in-frame with the zeocin-resistance ( zeo r ) gene. Self-primed heterodimers were inserted between the promoter and the zeo r gene and transformed into E . coli . The library was electroporated into M . smegmatis . Hygromycin selection allowed maintenance of the complete library, while zeocin selection required translation initiation at either one of the randomized codon sites. Following selection in zeocin, plasmids were recovered and the leader regions amplified for Ion-Torrent sequencing. Deep sequencing of amplicon libraries allowed the unbiased identification and estimation of relative efficiency of initiation codons. (B) A Shine-Dalgarno site was omitted to facilitate direct comparison between leaderless and leadered architectures. Read counts were compiled for each of the 64 possible codons at the leaderless position (columns) and leadered position (rows). Heat map indicates read counts of each combinatorial leaderless/leadered codon pair, from 10 0 (blue) through 10 4 (yellow). Only ATG or GTG at the leaderless position were capable of initiating translation of zeo r . At the leadered codon position, no enrichment indicated that translation initiation did not occur at any of the possible codons. A further reduction of the expected stop codons suggested that they prevented read through of leaderless ribosomes into the zeo r ORF. (C) A Shine-Dalgarno sequence enables efficient use of diverse leadered initiation codons. A consensus Shine-Dalgarno (SD) element was placed upstream of the randomized leadered codon position. Zeocin-resistant pools showed a complex pattern of active translation initiation codons at both the leaderless and leadered positions. The presence of a Shine-Dalgarno supported translation initiation activity of ATG and GTG triplets in the leadered position, as well as the less common TTG and ATT triplets.

Article Snippet: The mc 2 155 strain of M . smegmatis was grown in Middlebrook 7H9 broth supplemented with ADC and 0.05% Tween 80 with shaking at 230 RPM at 37°C to an OD 600 of ~1.0.

Techniques: Generated, Transformation Assay, Selection, Amplification, Sequencing, Comparison, Activity Assay

Journal: PLoS Genetics

Article Title: Leaderless Transcripts and Small Proteins Are Common Features of the Mycobacterial Translational Landscape

doi: 10.1371/journal.pgen.1005641

Figure Lengend Snippet: (A) Zeo-seq viability reporter libraries were generated to determine the sequence context preferences for a SD upstream of a leadered initiation codon. Randomized nucleotides were positioned upstream of a leadered initiation codon, and zeocin selection enriched for Shine-Dalgarno-like sequences, indicating that mycobacteria adhere to this canonical translation criterion. (B) Leaderless translation initiation exhibits no sequence preference in the adjacent mRNA. A block of 6 nt was randomized immediately downstream of a leaderless initiation zeocin reporter construct. Sequences in the recovered pools of zeocin-resistant M . smegmatis were not enriched in composition or motifs in this region. The absence of any detectable enrichment in the randomized region for the leaderless pool indicates that there are no nucleotide preferences for efficient leaderless initiation in mycobacteria downstream of the RTG codon.

Article Snippet: The mc 2 155 strain of M . smegmatis was grown in Middlebrook 7H9 broth supplemented with ADC and 0.05% Tween 80 with shaking at 230 RPM at 37°C to an OD 600 of ~1.0.

Techniques: Generated, Sequencing, Selection, Blocking Assay, Construct

Journal: PLoS Genetics

Article Title: Leaderless Transcripts and Small Proteins Are Common Features of the Mycobacterial Translational Landscape

doi: 10.1371/journal.pgen.1005641

Figure Lengend Snippet: (A) M . tuberculosis leaderless transcripts initiate unannotated small protein ORFs that terminate at the start of the annotated gene downstream more often than expected. All small protein ORF stop codons within 100 nucleotides of an annotated gene start are shown relative to that start codon (0 = coupled RTGA overlap). Three structural classes are identified: uORFs (the small ORF terminates upstream of the annotated start), coupled ORFs (linked by an RTGA tetramer), and overlapping ORFs. The y-axis shows the fraction of small ORFs that terminate a specified distance (x-axis) from the annotated start codon of the downstream gene. (B) One example of a coupled small protein in M . tuberculosis and M . smegmatis , upstream of orthologous genes. The primary sequence of the encoded small protein is not conserved, but the leaderless initiation and coupled linkage is maintained.

Article Snippet: The mc 2 155 strain of M . smegmatis was grown in Middlebrook 7H9 broth supplemented with ADC and 0.05% Tween 80 with shaking at 230 RPM at 37°C to an OD 600 of ~1.0.

Techniques: Sequencing

Journal: PLoS Genetics

Article Title: Leaderless Transcripts and Small Proteins Are Common Features of the Mycobacterial Translational Landscape

doi: 10.1371/journal.pgen.1005641

Figure Lengend Snippet: Small ORFs were identified upstream of annotated orthologous genes (A) cysA2 /Msmeg_5788, (B) Rv0485/Msmeg_0932, and (C) nirA /Msmeg_4527, in the M . tuberculosis and M . smegmatis genomes. Schematic representation of loci in M . tuberculosis (above) or M . smegmatis (below) that encode small proteins (yellow) upstream of genes conserved between these species. The deduced amino acid sequence of each small protein is shown, with the conserved amino acids in gray shaded boxes. The genes downstream in black block arrows are putative members of the same mRNA, and the gene designated by the gray arrow upstream is transcriptionally independent, but shown for context. The amino acid identity with the protein encoded by the corresponding M . tuberculosis gene is indicated below the respective M . smegmatis gene. Below are screen shots of RNA-seq and ribosome profiling profiles in M . smegmatis , and annotated gene predictions from two different annotation algorithms JCVI (black) and PATRIC (blue). Small proteins encoded throughout genomes are poorly annotated; note here that pipeline annotation algorithms predicted none of the small proteins, and in some cases predicted longer proteins on the opposite strand for which we see no transcriptional or translational evidence.

Article Snippet: The mc 2 155 strain of M . smegmatis was grown in Middlebrook 7H9 broth supplemented with ADC and 0.05% Tween 80 with shaking at 230 RPM at 37°C to an OD 600 of ~1.0.

Techniques: Sequencing, Blocking Assay, RNA Sequencing