Characterization of the
            <i>dnaG</i>
            Locus in
            <i>Mycobacterium smegmatis</i>
            Reveals Linkage of DNA Replication and Cell Division

Klann, Amy G.; Belanger, Aimee E.; Mello, Angelica Abanes-De; Lee, Janice Y.; Hatfull, Graham F.

doi:10.1128/jb.180.1.65-72.1998

Cited by 30 publications

(18 citation statements)

References 48 publications

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“…In contrast, cells overexpressing 6C sRNA or treated with CRISPRi both contained many more and smaller nucleoids along the cell filament (Figure 7E). These phenotypes are reminiscent of the growth defect observed in the dnaA and dnaG conditional deletion mutants of M. smegmatis (39,40), in which chromosomal replication is also disrupted at the initiation steps.…”

Section: Resultsmentioning

confidence: 86%

Mycobacterium tuberculosis 6C sRNA binds multiple mRNA targets via C-rich loops independent of RNA chaperones

Mai

Rao

Watt

et al. 2019

Nucleic Acids Research

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Bacterial small regulatory RNAs (sRNAs) are the most abundant class of post-transcriptional regulators and have been well studied in Gram-negative bacteria. Little is known about the functions and mechanisms of sRNAs in high GC Gram-positive bacteria including Mycobacterium and Streptomyces . Here, we performed an in-depth study of 6C sRNA of Mycobacterium tuberculosis , which is conserved among high GC Gram-positive bacteria. Forty-seven genes were identified as possible direct targets of 6C sRNA and 15 of them were validated using an in vivo translational lacZ fusion system. We found that 6C sRNA plays a pleotropic role and regulates genes involved in various cellular processes, including DNA replication and protein secretion. Mapping the interactions of 6C sRNA with mRNA targets panD and dnaB revealed that the C-rich loops of 6C sRNA act as direct binding sites to mRNA targets. Unlike in Gram-negative bacteria where RNA binding proteins Hfq and ProQ are required, the interactions of 6C sRNA with mRNAs appear to be independent of RNA chaperones. Our findings suggest that the multiple G–C pairings between single stranded regions are sufficient to establish stable interactions between 6C sRNA and mRNA targets, providing a mechanism for sRNAs in high GC Gram-positive bacteria.

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Section: Resultsmentioning

confidence: 86%

Mycobacterium tuberculosis 6C sRNA binds multiple mRNA targets via C-rich loops independent of RNA chaperones

Mai

Rao

Watt

et al. 2019

Nucleic Acids Research

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“…Liquid M. smegmatis cultures were grown in 7H9 expression medium (Middlebrook 7H9 medium (Fluka Analytical ® ) supplemented with 0.05% (v/v) tween 80, 0.2% (v/v) glycerol, 1.11 m M d ‐glucose and 100 µg/mL hygromycin B). The doubling time of M. smegmatis is around 3–4 h . Wild type M. smegmatis mc 2 155 was grown in 7H9 expression medium without antibiotics.…”

Section: Methodsmentioning

confidence: 99%

“…Mycobacterium smegmatis mc 2 155 was transformed with positive vector DNA by employing the electroporation method, 28 29 Wild type M. smegmatis mc 2 155 was grown in 7H9 expression medium without antibiotics.…”

Section: Strains and Mediamentioning

confidence: 99%

A versatile vector for mycobacterial protein production with a functional minimized acetamidase regulon

Vergara¹,

Kallenberg²,

Rogasch³

et al. 2017

Protein Science

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Recombinant protein expression is a prerequisite for diverse investigations of proteins at the molecular level. For targets from Mycobacterium tuberculosis it is favorable to use M. smegmatis as an expression host, a species from the same genus. In the respective shuttle vectors, target gene expression is controlled by the complex tetra-cistronic acetamidase regulon. As a result, the size of those vectors is large, rendering them of limited use, especially when the target proteins are expressed from multi-cistronic operons. Therefore, in the current work we present a versatile new expression vector in which the acetamidase regulon has been minimized by deleting the two genes amiD and amiS. We assessed the functional properties of the resulting vector pMyCA and compared it with those of the existing vector pMyNT that contains the full-length acetamidase regulon. We analyzed the growth features and protein expression patterns of M. smegmatis cultures transformed with both vectors. In addition, we created mCherry expression constructs to spectroscopically monitor the expression properties of both vectors. Our experiments showed that the minimized vector exhibited several advantages over the pMyNT vector. First, the overall yield of expressed protein is higher due to the higher yield of bacterial mass. Second, the heterologous expression was regulated more tightly, offering an expression tool for diverse target proteins. Third, it is suitable for large multi-protein complexes that are expressed from multi-cistronic operons. Additionally, our results propose a new understanding of the regulation mechanism of the acetamidase regulon with the potential to construct more optimized vectors in the future.

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“…To achieve this aim, we evolved a phage by serial passages under different conditions using M. smegmatis as model host. M. smegmatis is a non-pathogenic bacterium with a faster life cycle than other Mycobacterium species [17], thus offering a good system to explore and set up directed evolution protocols.…”

Section: Introductionmentioning

confidence: 99%

Directed Evolution of a Mycobacteriophage

2019

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Bacteriophages represent an alternative strategy to combat pathogenic bacteria. Currently, Mycobacterium tuberculosis infections constitute a major public health problem due to extensive antibiotic resistance in some strains. Using a non-pathogenic species of the same genus as an experimental model, Mycobacterium smegmatis, here we have set up a basic methodology for mycobacteriophage growth and we have explored directed evolution as a tool for increasing phage infectivity and lytic activity. We demonstrate mycobacteriophage adaptation to its host under different conditions. Directed evolution could be used for the development of future phage therapy applications against mycobacteria.

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Characterization of the dnaG Locus in Mycobacterium smegmatis Reveals Linkage of DNA Replication and Cell Division

Cited by 30 publications

References 48 publications

Mycobacterium tuberculosis 6C sRNA binds multiple mRNA targets via C-rich loops independent of RNA chaperones

Mycobacterium tuberculosis 6C sRNA binds multiple mRNA targets via C-rich loops independent of RNA chaperones

A versatile vector for mycobacterial protein production with a functional minimized acetamidase regulon

Directed Evolution of a Mycobacteriophage

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