scholarly journals Protein-DNA Complexes in Mycobacteriophage L5 Integrative Recombination

1999 ◽  
Vol 181 (2) ◽  
pp. 454-461 ◽  
Author(s):  
Carol E. A. Peña ◽  
J. Michelle Kahlenberg ◽  
Graham F. Hatfull
Keyword(s):  
Mycobacterium Tuberculosis ◽  
Mycobacterium Bovis ◽  
Binding Sites ◽  
Mycobacterium Smegmatis ◽  
Recombination Event ◽  
Specific Protein ◽  
Integration Host Factor ◽  
Dna Complexes ◽  
Active Complex ◽  
Mycobacteriophage L5

ABSTRACT The temperate mycobacteriophage L5 integrates site specifically into the genomes of Mycobacterium smegmatis,Mycobacterium tuberculosis, and Mycobacterium bovis bacillus Calmette-Guérin. This integrative recombination event occurs between the phage L5 attP site and the mycobacterial attB site and requires the phage-encoded integrase and mycobacterial-encoded integration host factor mIHF. Here we show that attP, Int-L5, and mIHF assemble into a recombinationally active complex, the intasome, which is capable of attB capture and formation of products. The arm-type integrase binding sites within attP play specialized roles in the formation of specific protein-DNA architectures; the intasome is constructed by the formation of intramolecular integrase bridges between one pair of sites, P4-P5, and the attP core, while an additional pair of sites, P1-P2, is required for interaction with attB.

scholarly journals Multiple small RNAs identified in Mycobacterium bovis BCG are also expressed in Mycobacterium tuberculosis and Mycobacterium smegmatis

2010 ◽  
Vol 38 (12) ◽  
pp. 4067-4078 ◽  
Author(s):  
J. M. DiChiara ◽  
L. M. Contreras-Martinez ◽  
J. Livny ◽  
D. Smith ◽  
K. A. McDonough ◽  
...  
Keyword(s):  
Mycobacterium Tuberculosis ◽  
Mycobacterium Bovis ◽  
Mycobacterium Smegmatis ◽  
Small Rnas ◽  
Mycobacterium Bovis Bcg

The MspA porin promotes growth and increases antibiotic susceptibility of both Mycobacterium bovis BCG and Mycobacterium tuberculosis

Microbiology ◽  
2004 ◽  
Vol 150 (4) ◽  
pp. 853-864 ◽  
Author(s):  
Claudia Mailaender ◽  
Norbert Reiling ◽  
Harald Engelhardt ◽  
Stefan Bossmann ◽  
Stefan Ehlers ◽  
...  
Keyword(s):  
Mycobacterium Tuberculosis ◽  
Outer Membrane ◽  
Mycobacterium Bovis ◽  
Mycobacterium Smegmatis ◽  
Drug Susceptibility ◽  
Nucleic Acid Binding ◽  
Outer Membrane Permeability ◽  
Syto 9 ◽  
Slow Growing ◽  
Hydrophilic Solutes

Porins mediate the diffusion of hydrophilic solutes across the outer membrane of mycobacteria, but the efficiency of this pathway is very low compared to Gram-negative bacteria. To examine the importance of porins in slow-growing mycobacteria, the major porin MspA of Mycobacterium smegmatis was expressed in Mycobacterium tuberculosis and Mycobacterium bovis. Approximately 20 and 35 MspA molecules per μm2 cell wall were observed in M. tuberculosis and M. bovis BCG, respectively, by electron microscopy and quantitative immunoblot experiments. Surface accessibility of MspA in M. tuberculosis was demonstrated by flow cytometry. Glucose uptake was twofold faster, indicating that the outer membrane permeability of M. bovis BCG to small and hydrophilic solutes was increased by MspA. This significantly accelerated the growth of M. bovis BCG, identifying very slow nutrient uptake as one of the determinants of slow growth in mycobacteria. The susceptibility of both M. bovis BCG and M. tuberculosis to zwitterionic β-lactam antibiotics was substantially enhanced by MspA, decreasing the minimal inhibitory concentration up to 16-fold. Furthermore, M. tuberculosis became significantly more susceptible to isoniazid, ethambutol and streptomycin. Fluorescence with the nucleic acid binding dye SYTO 9 was 10-fold increased upon expression of mspA. These results indicated that MspA not only enhanced the efficiency of the porin pathway, but also that of pathways mediating access to large and/or hydrophobic agents. This study provides the first experimental evidence that porins are important for drug susceptibility of M. tuberculosis.

scholarly journals Comparison of the Construction of Unmarked Deletion Mutations in Mycobacterium smegmatis, Mycobacterium bovis Bacillus Calmette-Guérin, and Mycobacterium tuberculosis H37Rv by Allelic Exchange

1999 ◽  
Vol 181 (16) ◽  
pp. 4780-4789 ◽  
Author(s):  
Martin S. Pavelka ◽  
William R. Jacobs
Keyword(s):  
Mycobacterium Tuberculosis ◽  
Mycobacterium Bovis ◽  
Mycobacterium Smegmatis ◽  
Tween 80 ◽  
Defined Medium ◽  
Lysine Biosynthesis ◽  
Rich Medium ◽  
Deletion Mutations ◽  
Allelic Exchange ◽  
Slow Growing

ABSTRACT Until recently, genetic analysis of Mycobacterium tuberculosis, the causative agent of tuberculosis, was hindered by a lack of methods for gene disruptions and allelic exchange. Several groups have described different methods for disrupting genes marked with antibiotic resistance determinants in the slow-growing organismsMycobacterium bovis bacillus Calmette-Guérin (BCG) and M. tuberculosis. In this study, we described the first report of using a mycobacterial suicidal plasmid bearing the counterselectable marker sacB for the allelic exchange of unmarked deletion mutations in the chromosomes of two substrains ofM. bovis BCG and M. tuberculosis H37Rv. In addition, our comparison of the recombination frequencies in these two slow-growing species and that of the fast-growing organismMycobacterium smegmatis suggests that the homologous recombination machinery of the three species is equally efficient. The mutants constructed here have deletions in the lysA gene, encoding meso-diaminopimelate decarboxylase, an enzyme catalyzing the last step in lysine biosynthesis. We observed striking differences in the lysine auxotrophic phenotypes of these three species of mycobacteria. The M. smegmatis mutant can grow on lysine-supplemented defined medium or complex rich medium, while the BCG mutants grow only on lysine-supplemented defined medium and are unable to form colonies on complex rich medium. The M. tuberculosis lysine auxotroph requires 25-fold more lysine on defined medium than do the other mutants and is dependent upon the detergent Tween 80. The mutants described in this work are potential vaccine candidates and can also be used for studies of cell wall biosynthesis and amino acid metabolism.

scholarly journals Diferenciação de micobactérias por PCR multiplex

2009 ◽  
Vol 42 (6) ◽  
pp. 716-722 ◽  
Author(s):  
Diogo da Rocha Poroca ◽  
Andrea Santos Lima ◽  
Juliana Falcão de Araújo Lima ◽  
Heidi Lacerda Alves da Cruz ◽  
Rosana de Albuquerque Montenegro ◽  
...  
Keyword(s):  
Mycobacterium Tuberculosis ◽  
Mycobacterium Bovis ◽  
Mycobacterium Avium ◽  
Mycobacterium Smegmatis ◽  
Pcr Multiplex

O trabalho visou à otimização de um método baseado na reação em cadeia da polimerase multiplex - para diferenciação de micobactérias de interesse para a saúde pública. A PCR Multiplex baseou-se na amplificação simultânea do genehsp65, presente em todo gênero Mycobacterium, do gene dnaJ, presente apenas em Mycobacterium tuberculosis e Mycobacterium avium e da sequência de inserção IS6110 presente no complexo Mycobacterium tuberculosis, gerando amplicons de 165pb, 365pb e 541pb, respectivamente. O limite de detecção foi de 1fg para o alvo hsp65, 100pg para o dnaJ e 0,1fg para o IS6110. A PCR multiplex detectou até 100pg de DNA de Mycobacterium tuberculosis. O sistema demonstrou ser específico e sensível na detecção de Mycobacterium tuberculosis, Mycobacterium bovis, Mycobacterium avium e Mycobacterium smegmatis. Os resultados obtidos utilizando cepas de referência demonstraram que a PCR multiplex pode ser uma ferramenta rápida, sensível e específica na diferenciação de micobactérias.

scholarly journals Protein and DNA Effectors Control the TraI Conjugative Helicase of Plasmid R1

2009 ◽  
Vol 191 (22) ◽  
pp. 6888-6899 ◽  
Author(s):  
Marta V. Sut ◽  
Sanja Mihajlovic ◽  
Silvia Lang ◽  
Christian J. Gruber ◽  
Ellen L. Zechner
Keyword(s):  
Dna Binding ◽  
Binding Sites ◽  
Specific Protein ◽  
Integration Host Factor ◽  
Cooperative Interaction ◽  
Plasmid R1 ◽  
Ligand Interactions ◽  
In The Wild ◽  
Coupling Protein ◽  
Dna Strand

ABSTRACT The mechanisms controlling progression of conjugative DNA processing from a preinitiation stage of specific plasmid strand cleavage at the transfer origin to a stage competent for unwinding the DNA strand destined for transfer remain obscure. Linear heteroduplex substrates containing double-stranded DNA binding sites for plasmid R1 relaxosome proteins and various regions of open duplex for TraI helicase loading were constructed to model putative intermediate structures in the initiation pathway. The activity of TraI was compared in steady-state multiple turnover experiments that measured the net production of unwound DNA as well as transesterase-catalyzed cleavage at nic. Helicase efficiency was enhanced by the relaxosome components TraM and integration host factor. The magnitude of stimulation depended on the proximity of the specific protein binding sites to the position of open DNA. The cytoplasmic domain of the R1 coupling protein, TraDΔN130, stimulated helicase efficiency on all substrates in a manner consistent with cooperative interaction and sequence-independent DNA binding. Variation in the position of duplex opening also revealed an unsuspected autoinhibition of the unwinding reaction catalyzed by full-length TraI. The activity reduction was sequence dependent and was not observed with a truncated helicase, TraIΔN308, lacking the site-specific DNA binding transesterase domain. Given that transesterase and helicase domains are physically tethered in the wild-type protein, this observation suggests that an intramolecular switch controls helicase activation. The data support a model where protein-protein and DNA ligand interactions at the coupling protein interface coordinate the transition initiating production and uptake of the nucleoprotein secretion substrate.

scholarly journals Regulation Mechanism of thealdGene Encoding Alanine Dehydrogenase in Mycobacterium smegmatis and Mycobacterium tuberculosis by the Lrp/AsnC Family Regulator AldR

2015 ◽  
Vol 197 (19) ◽  
pp. 3142-3153 ◽  
Author(s):  
Ji-A Jeong ◽  
Jaekyung Hyun ◽  
Jeong-Il Oh
Keyword(s):  
Mycobacterium Tuberculosis ◽  
Binding Sites ◽  
Mycobacterium Smegmatis ◽  
Quaternary Structure ◽  
Consensus Sequence ◽  
Underlying Mechanism ◽  
Cooperative Binding ◽  
Regulation Mechanism ◽  
Content Type ◽  
Alanine Dehydrogenase

ABSTRACTIn the presence of alanine, AldR, which belongs to the Lrp/AsnC family of transcriptional regulators and regulatesaldencoding alanine dehydrogenase inMycobacterium smegmatis, changes its quaternary structure from a homodimer to an octamer with an open-ring conformation. Four AldR-binding sites (O2, O1, O4, and O3) with a consensus sequence of GA/T-N2-NWW/WWN-N2-A/TC were identified upstream of theM. smegmatisaldgene by means of DNase I footprinting analysis. O2, O1, and O4 are required for the induction ofaldexpression by alanine, while O3 is directly involved in the repression ofaldexpression. In addition to O3, both O1 and O4 are also necessary for full repression ofaldexpression in the absence of alanine, due to cooperative binding of AldR dimers to O1, O4, and O3. Binding of a molecule of the AldR octamer to thealdcontrol region was demonstrated to require two AldR-binding sites separated by three helical turns between their centers and one additional binding site that is in phase with the two AldR-binding sites. The cooperative binding of AldR dimers to DNA requires three AldR-binding sites that are aligned with a periodicity of three helical turns. ThealdRgene is negatively autoregulated independently of alanine. Comparative analysis ofaldexpression ofM. smegmatisandMycobacterium tuberculosisin conjunction with sequence analysis of bothaldcontrol regions led us to suggest that the expression of thealdgenes in both mycobacterial species is regulated by the same mechanism.IMPORTANCEIn mycobacteria, alanine dehydrogenase (Ald) is the enzyme required both to utilize alanine as a nitrogen source and to grow under hypoxic conditions by maintaining the redox state of the NADH/NAD+pool. Expression of thealdgene was reported to be regulated by the AldR regulator that belongs to the Lrp/AsnC (feast/famine) family, but the underlying mechanism was unknown. This study revealed the regulation mechanism ofaldinMycobacterium smegmatisandMycobacterium tuberculosis. Furthermore, a generalized arrangement pattern ofcis-acting regulatory sites for Lrp/AsnC (feast/famine) family regulators is suggested in this study.

scholarly journals Characterization of the mIHF Gene ofMycobacterium smegmatis

1998 ◽  
Vol 180 (20) ◽  
pp. 5473-5477 ◽  
Author(s):  
Marisa L. Pedulla ◽  
Graham F. Hatfull
Keyword(s):  
Dna Binding ◽  
Stationary Phase ◽  
Mycobacterium Smegmatis ◽  
Binding Proteins ◽  
Dna Binding Proteins ◽  
Integration Host Factor ◽  
Heat Stable ◽  
Mycobacteriophage L5

ABSTRACT Integration of mycobacteriophage L5 requires the mycobacterial integration host factor (mIHF) in vitro. mIHF is a 105-residue heat-stable polypeptide that is not obviously related to HU or any other small DNA-binding proteins. mIHF is most abundant just prior to entry into stationary phase and is essential for the viability ofMycobacterium smegmatis.

EVALUATION OF TISSUE STEROID BINDING IN VITRO

1971 ◽  
Vol 68 (1_Suppl) ◽  
pp. S223-S246 ◽  
Author(s):  
C. R. Wira ◽  
H. Rochefort ◽  
E. E. Baulieu
Keyword(s):  
Protein Binding ◽  
Binding Sites ◽  
Experimental System ◽  
Specific Protein ◽  
Coupling Mechanism ◽  
Target Tissue ◽  
Protein Binding Sites ◽  
Definition Of ◽  
Hormone Specificity

ABSTRACT The definition of a RECEPTOR* in terms of a receptive site, an executive site and a coupling mechanism, is followed by a general consideration of four binding criteria, which include hormone specificity, tissue specificity, high affinity and saturation, essential for distinguishing between specific and nonspecific binding. Experimental approaches are proposed for choosing an experimental system (either organized or soluble) and detecting the presence of protein binding sites. Techniques are then presented for evaluating the specific protein binding sites (receptors) in terms of the four criteria. This is followed by a brief consideration of how receptors may be located in cells and characterized when extracted. Finally various examples of oestrogen, androgen, progestagen, glucocorticoid and mineralocorticoid binding to their respective target tissues are presented, to illustrate how researchers have identified specific corticoid and mineralocorticoid binding in their respective target tissue receptors.

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