Molecular Genetics of Mycobacteriophages

Hatfull, Graham F.

doi:10.1128/9781555818845.ch5

Cited by 57 publications

(47 citation statements)

References 210 publications

(223 reference statements)

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“…Middlebrook 7H11 agar (Difco) supplemented with 0.5% glycerol and BBL TM Middlebrook oleic acid-albumin-dextrose-catalase (OADC) enrichment (BD) was used for growth on solid medium (referred to as 7H11). M. tuberculosis was transformed as described previously (21). E. coli strains used for cloning and expression were grown in LB-Miller broth (Difco) at 37°C with aeration on a shaker or on LB agar.…”

Section: Methodsmentioning

confidence: 99%

Characterization of Guided Entry of Tail-Anchored Proteins 3 Homologues in Mycobacterium tuberculosis

Jordan

Zhang

et al. 2019

J Bacteriol

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We characterized an operon in Mycobacterium tuberculosis, Rv3679-Rv3680, in which each open reading frame is annotated to encode “anion transporter ATPase” homologues. Using structure prediction modeling, we found that Rv3679 and Rv3680 more closely resemble the guided entry of tail-anchored proteins 3 (Get3) chaperone in eukaryotes. Get3 delivers proteins into the membranes of the endoplasmic reticulum and is essential for the normal growth and physiology of some eukaryotes. We sought to characterize the structures of Rv3679 and Rv3680 and test if they have a role in M. tuberculosis pathogenesis. We solved crystal structures of the nucleotide-bound Rv3679-Rv3680 complex at 2.5 to 3.2 Å and show that while it has some similarities to Get3 and ArsA, there are notable differences, including that these proteins are unlikely to be involved in anion transport. Deletion of both genes did not reveal any conspicuous growth defects in vitro or in mice. Collectively, we identified a new class of proteins in bacteria with similarity to Get3 complexes, the functions of which remain to be determined. IMPORTANCE Numerous bacterial species encode proteins predicted to have similarity with Get3- and ArsA-type anion transporters. Our studies provide evidence that these proteins, which we named BagA and BagB, are unlikely to be involved in anion transport. In addition, BagA and BagB are conserved in all mycobacterial species, including the causative agent of leprosy, which has a highly decayed genome. This conservation suggests that BagAB constitutes a part of the core mycobacterial genome and is needed for some yet-to-be-determined part of the life cycle of these organisms.

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Section: Methodsmentioning

confidence: 99%

Characterization of Guided Entry of Tail-Anchored Proteins 3 Homologues in Mycobacterium tuberculosis

Jordan

Zhang

et al. 2019

J Bacteriol

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“…All plasmids made by PCR cloning were sequenced by GENEWIZ, Inc. to ensure the veracity of the cloned sequence. Plasmids were transformed into M. tuberculosis by electroporation as previously described (56). Single-colony transformants were isolated on 7H11 agar with antibiotic selection.…”

Section: Methodsmentioning

confidence: 99%

Mycobacterium tuberculosisRv0991c is a redox-regulated molecular chaperone

Becker

Ulrich

Dhabaria

et al. 2020

Preprint

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21The bacterial pathogen Mycobacterium (M.) tuberculosis is the leading cause of death by 22 an infectious disease among humans. Here, we describe a previously uncharacterized 23 M. tuberculosis protein, Rv0991c, as a molecular chaperone that is activated by oxidation. 24Rv0991c has homologues in most bacterial lineages and appears to function analogously 25 to the well-characterized Escherichia coli redox-regulated chaperone Hsp33, despite a 26 dissimilar protein sequence. Rv0991c is transcriptionally co-regulated with hsp60 and 27 hsp70 chaperone genes in M. tuberculosis, suggesting that Rv0991c functions with these 28 chaperones in maintaining protein quality control. Supporting this hypothesis, we found 29 that, like oxidized Hsp33, oxidized Rv0991c prevents the aggregation of a model unfolded 30 protein in vitro, and promotes its refolding by the M. tuberculosis Hsp70 chaperone 31 system. Furthermore, Rv0991c interacts with DnaK and associates with many other M. 32 tuberculosis proteins. Importantly, we found Rv0991c is required for the full virulence of 33 M. tuberculosis in mice. We therefore propose that Rv0991c, which we named "Ruc" 34 (redox-regulated protein with unstructured C-terminus), represents a founding member of 35 a new chaperone family that protects M. tuberculosis and other species from 36 proteotoxicity during oxidative stress. 37 38 IMPORTANCE 39 M. tuberculosis infections are responsible for more than one million human deaths per 40year. Developing effective strategies to combat this disease requires a greater 41 understanding of M. tuberculosis biology. As in all cells, protein quality control is essential 42 for the viability of M. tuberculosis, which likely faces proteome stress within a host. Here, 43we identify an M. tuberculosis protein, Ruc, that gains chaperone activity upon oxidation. 44Ruc represents a previously unrecognized family of redox-regulated chaperones found 45 throughout the bacterial super-kingdom. In addition to elucidating the activity of this 46 chaperone, we found that Ruc was required for full M. tuberculosis virulence in mice. This 47 6 discovery that the uncharacterized M. tuberculosis gene Rv0991c encodes a chaperone, 97 which we named Ruc (redox-regulated chaperone with unstructured C-terminus). Ruc 98 belongs to a previously unacknowledged, but evolutionarily widespread family of bacterial 99 proteins with little predicted structural similarity to other chaperones. Upon oxidation, Ruc 100 is capable of inhibiting protein aggregation and delivering unfolded proteins to DnaKJE 101 for refolding. Ruc was also found to interact with DnaK in M. tuberculosis and, while 102 dispensable for in vitro growth, was required for full M. tuberculosis virulence in mice. 103These observations ultimately suggest that Ruc is important for the ability of M. 104 tuberculosis, and potentially many other bacterial species, to withstand oxidation-105 associated proteotoxicity. 106 107 RESULTS 108

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“…7H11 agar (Difco) 392 supplemented with 0.5% glycerol and BBL TM Middlebrook OADC enrichment (BD) was 393 used for growth on solid medium ("7H11"). M. tuberculosis was transformed as 394 described (Hatfull & Jacobs, 2000). E. coli strains used for cloning and expression were 395 grown in LB-Miller broth (Difco) at 37 °C with aeration on a shaker or on LB agar.…”

Section: Materials and Methods 385mentioning

confidence: 99%

Cytokinin signaling inMycobacterium tuberculosis

Samanovic

Hsu

Jones

et al. 2017

Preprint

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SummaryIt was reported the human-exclusive pathogen Mycobacterium (M.) tuberculosis secretes cytokinins, which previously had only been known as plant hormones. Cytokinins are adenine-based signaling molecules in plants that have never been shown to participate in signal transduction in other kingdoms of life. Here, we show that cytokinins induce the strong expression of the M. tuberculosis gene, Rv0077c. We found that a TetR-like transcriptional regulator, Rv0078, directly repressed expression of the Rv0077c gene. Strikingly, cytokinin-induced expression of Rv0077c resulted in a loss of acid-fast staining of M. tuberculosis. Although acid-fast staining is thought to be associated with changes in the bacterial cell envelope and virulence, Rv0077c-induced loss of acid-fastness did not affect antibiotic susceptibility or attenuate bacterial growth in mice. Collectively, these findings show cytokinins signal transcriptional changes that affect the M. tuberculosis cell envelope, and that cytokinin signaling is no longer limited to the kingdom plantae.

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Molecular Genetics of Mycobacteriophages

Cited by 57 publications

References 210 publications

Characterization of Guided Entry of Tail-Anchored Proteins 3 Homologues in Mycobacterium tuberculosis

Characterization of Guided Entry of Tail-Anchored Proteins 3 Homologues in Mycobacterium tuberculosis

Mycobacterium tuberculosisRv0991c is a redox-regulated molecular chaperone

Cytokinin signaling inMycobacterium tuberculosis

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