Cloning of the <i>mspA</i> gene encoding a porin from <i>Mycobacterium smegmatis</i>

Niederweis, Michael; Ehrt, Sabine; Heinz, Christian; Klöcker, Uta; Karosi, Stefanie; Swiderek, Kristine M.; Riley, Lee W.; Benz, Roland

doi:10.1046/j.1365-2958.1999.01472.x

Cited by 141 publications

(202 citation statements)

References 46 publications

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“…For example, among those with higher fluorescence, we found insertions in an operon that encodes two putative efflux pumps ( msmeg5660-5659 ) and enzymes required for the modification of mycolic acids that constitute a major portion of the cell wall ( umaA ). Likewise, we found that insertions in mspA , a gene that encodes a well-characterized porin known to permit entry of molecules through the Msm cell wall resulted in lower median fluorescence 8 . We constructed targeted deletions 9 for several genes (Supplementary Table 2) and found that all had the predicted phenotypes (Fig.…”

Section: Main Textmentioning

confidence: 70%

Deletion of a mycobacterial divisome factor collapses single-cell phenotypic heterogeneity

Rego

Audette

Rubín

2017

Nature

158

177

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Summary Paragraph While microorganisms are often studied as populations, the behavior of single, individual cells can have profound consequences. For example, tuberculosis, caused by the bacterial pathogen Mycobacterium tuberculosis, requires months of antibiotic therapy even though the bulk of the bacterial population rapidly dies. Shorter courses lead to high rates of relapse because subpopulations of bacilli can survive despite being genetically identical to those that are easily killed 1. In fact, mycobacteria create variability every time a cell divides, producing daughter cells with different sizes and growth rates 2, 3. The mechanism(s) that underlie this high-frequency variation and how variability relates to survival of the population are unknown. Here we show that mycobacteria actively create heterogeneity. Using a fluorescent reporter and a FACS-based transposon screen, we find that deletion of lamA, a gene of previously unknown function, decreases the amount of heterogeneity in the population by decreasing asymmetric polar growth. LamA has no known homologs in other organisms, but is highly conserved across mycobacterial species. We find that LamA is a member of the mycobacterial division complex (“the divisome”). It inhibits growth at nascent new poles, creating asymmetry in polar growth. The kinetics of killing individual cells that lack lamA are more uniform and more rapid with rifampicin and certain drugs that target the cell wall. Our results show that mycobacteria encode a non-conserved protein that controls the pattern of cell growth, resulting in a population that is both heterogeneous and better able to survive antibiotic pressure.

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Section: Main Textmentioning

confidence: 70%

Deletion of a mycobacterial divisome factor collapses single-cell phenotypic heterogeneity

Rego

Audette

Rubín

2017

Nature

158

177

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“…CpnT does not appear to have a classical Sec signal sequence, as do porins of Gram-negative bacteria (45) and MspA, the only known mycobacterial porin (46). In addition, CpnT is unusually large for a porin and seems to have at least two domains.…”

Section: Discussionmentioning

confidence: 94%

An outer membrane channel protein of Mycobacterium tuberculosis with exotoxin activity

Danilchanka¹,

Sun²,

Pavlenok³

et al. 2014

Proc. Natl. Acad. Sci. U.S.A.

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The ability to control the timing and mode of host cell death plays a pivotal role in microbial infections. Many bacteria use toxins to kill host cells and evade immune responses. Such toxins are unknown in Mycobacterium tuberculosis. Virulent M. tuberculosis strains induce necrotic cell death in macrophages by an obscure molecular mechanism. Here we show that the M. tuberculosis protein Rv3903c (channel protein with necrosis-inducing toxin, CpnT) consists of an N-terminal channel domain that is used for uptake of nutrients across the outer membrane and a secreted toxic C-terminal domain. Infection experiments revealed that CpnT is required for survival and cytotoxicity of M. tuberculosis in macrophages. Furthermore, we demonstrate that the C-terminal domain of CpnT causes necrotic cell death in eukaryotic cells. Thus, CpnT has a dual function in uptake of nutrients and induction of host cell death by M. tuberculosis.transport | pore | secretion

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“…It was noted earlier that MspA, which was purified by preparative gel electrophoresis, did not dissociate in the presence of denaturing agents such as urea or after boiling in detergents (10). Selective extraction of functional MspA by extended heating of entire cells of M. smegmatis in the presence of nonionic detergents to 100°C indicated that MspA is indeed a very stable channel-forming protein (16).…”

mentioning

confidence: 91%

“…OmpATb of M. tuberculosis has low channel activity in vitro (8) and is important for the adaptation of M. tuberculosis to low pH and for survival in macrophages and mice (9). MspA was identified as a channel-forming protein in chloroform/methanol extracts of Mycobacterium smegmatis (10). Recently, it was shown that MspA is the major porin of M. smegmatis.…”

mentioning

confidence: 99%

The Core of the Tetrameric Mycobacterial Porin MspA Is an Extremely Stable β-Sheet Domain

Heinz

Engelhardt

Niederweis

2003

Journal of Biological Chemistry

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MspA is the major porin of Mycobacterium smegmatis mediating the exchange of hydrophilic solutes across the cell wall and is the prototype of a new family of tetrameric porins with a single central pore of 10 nm in length. Infrared and circular dichroism spectroscopy revealed that MspA consists mainly of antiparallel ␤-strands organized in a coherent domain. Heating to 92 and 112°C was required to dissociate the MspA tetramer and to unfold the ␤-sheet domain in the monomer, respectively. The stability of the MspA tetramer exceeded the remarkable stability of the porins of Gram-negative bacteria for every condition tested and was not reduced in the presence of 2% SDS and at any pH from 2 to 14. These results indicated that the interactions between the MspA subunits are different from those in the porins of Gram-negative bacteria and are discussed in the light of a channel-forming ␤-barrel as a core structure of MspA. Surprisingly, the channel activity of MspA in 2% SDS and 7.6 M urea at 50°C was reduced 13-and 30-fold, respectively, although the MspA tetramer and the ␤-sheet domain were stable under those conditions. Channel closure by conformational changes of extracellular loops under those conditions is discussed to explain these observations. This study presents the first experimental evidence that outer membrane proteins not only from Gram-negative bacteria but also from mycobacteria are ␤-sheet proteins and demonstrates that MspA constitutes the most stable transmembrane channel protein known so far. Thus, MspA may be of special interest for biotechnological applications.

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Cloning of the mspA gene encoding a porin from Mycobacterium smegmatis

Cited by 141 publications

References 46 publications

Deletion of a mycobacterial divisome factor collapses single-cell phenotypic heterogeneity

Deletion of a mycobacterial divisome factor collapses single-cell phenotypic heterogeneity

An outer membrane channel protein of Mycobacterium tuberculosis with exotoxin activity

The Core of the Tetrameric Mycobacterial Porin MspA Is an Extremely Stable β-Sheet Domain

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