Mycobacterium Smegmatis Porin Research Articles

Precise Construction and Tuning of an Aerolysin Single-Biomolecule Interface for Single-Molecule Sensing

Precise Construction and Tuning of an Aerolysin Single-Biomolecule Interface for Single-Molecule Sensing

Increasing the accuracy of nanopore DNA sequencing using a time-varying cross membrane voltage.

Nanopore DNA sequencing is limited by low base calling accuracy. Improved base-calling accuracy has so far relied on specialized base-calling algorithms, different nanopores and motor enzymes, or biochemical methods to re-read DNA molecules. Two primary error modes hamper sequencing accuracy: enzyme mis-steps and sequences with indistinguishable signals. We vary the driving voltage across an MspA nanopore between 100 to 200 mV with a frequency of 200 Hz, changing how the DNA strand moves through the nanopore. As a DNA helicase moves the DNA through the nanopore in discrete steps, the variable voltage positions the DNA continuously between these steps. The resulting electronic signal can be analysed to overcome the primary error modes in base-calling. Single-passage de novo base-calling accuracy in our device increases from 62.7 ± 0.5% with a constant driving voltage to 79.3 ± 0.3% with a variable driving voltage. Our variable-voltage sequencing mode is complementary to other advances in nanopore sequencing and is amenable to incorporation into other enzyme-actuated nanopore sequencing devices.

A Single-molecule Mycobacterium Smegmatis Porin A Protein Nanopore Sensor for Host-Guest Chemistry

The mycobacterium smegmatis porin A (MspA) protein nanopore was prepared by E. coil prokaryotic expression system. The extraction of MspA nanopore using surfactants were carried out under the optimal conditions such as 0.5% extraction agent, 30 min of extraction and at 90 °C. The interactions of MspA nanopore with a variety of cyclodextrins were examined at single-molecule level. The interaction between MspA and Per-6-amino-β-cyclodextrin (am7-β-CD) was stronger and had consistent blocking platform. Its retention time in the MspA nanopore decreased with the increasing voltage, whereas the blocking current was not affected by the voltage. So, am7-β-CD was selected as a non-covalent adapter and lodged within single MspA nanopore to study the single-molecule host-guest chemistry. After adding am7-β-CD to cis side and displaying a first blocked platform, a low concentration of 50 nM for the amantadine hydrochloride could show a second blocked platform and its residence time decreased with the increasing voltage. This new protein nanopore sensor can be used as a single-molecule detector and identifier for small organic molecules, which will greatly broaden the application range of MspA nanopore.

Thermal Motion of DNA in an MspA Pore

We report on an experiment and calculations that determine the thermal motion of a voltage-clamped single-stranded DNA-NeutrAvidin complex in a Mycobacterium smegmatis porin A nanopore. The electric force and diffusion constant of DNA inside a Mycobacterium smegmatis porin A pore were determined to evaluate the thermal position fluctuations of DNA. We show that an out-of-equilibrium state returns to equilibrium so quickly that experiments usually measure a weighted average over the equilibrium position distribution. Averaging over the equilibrium position distribution is consistent with results of state-of-the-art nanopore sequencing experiments. It is shown how a reduction in thermal position fluctuations can be achieved by increasing the electrophoretic force used in nanopore sequencing devices.

High-throughput optical sensing of nucleic acids in a nanopore array.

Protein nanopores such as α-hemolysin and MspA can potentially be used to sequence long strands of DNA quickly and at low cost. In order to provide high-speed sequencing, large arrays of nanopores are required that allow the nanopores to individually addressed, but current nanopore sequencing methods rely on ionic current measurements and such methods are likely to prove difficult to scale up. Here, we show that, by optically encoding the ionic flux through protein nanopores, the discrimination of nucleic acid sequences and the detection of sequence-specific nucleic acid binding events can be parallelized. We make optical recordings at a density of ~104 nanopores per mm2 in a single droplet interface bilayer. Nanopore blockades can discriminate between DNAs with sub-pA equivalent resolution, and specific miRNA sequences can be identified by differences in unzipping kinetics. By creating an array of 2500 bilayers with a micro-patterned hydrogel chip, we are also able to load different samples into specific bilayers, suitable for high-throughput nanopore recording.

Tailored Polymeric Membranes for Mycobacterium Smegmatis Porin A (MspA) Based Biosensors.

Nanopores based on protein channels inserted into lipid membranes have paved the way towards a wide-range of inexpensive biosensors, especially for DNA sequencing. A key obstacle in using these biological ion channels as nanodevices is the poor stability of lipid bilayer membranes. Amphiphilic block copolymer membranes have emerged as a robust alternative to lipid membranes. While previous efforts have shown feasibility, we demonstrate for the first time the effect of polymer composition on MspA protein functionality. We show that membrane-protein interaction depends on the hydrophobic-hydrophilic ratio (f-ratio) of the block copolymer. These effects are particularly pronounced in asymmetric protein pores like MspA compared to the cylindrical α-Hemolysin pore. A key effect of membrane-protein interaction is the increased 1/fα noise. After first showing increases in 1/fα behaviour arise from increased substate activity, the noise power spectral density S(f) was used as a qualitative tool for understanding protein-membrane interactions in polymer membranes. Polymer compositions with f-ratios close to lipid membranes caused noise behaviour not observed in lipid membranes. However, by modifying the f-ratio using a modular synthetic approach, we were able to design a block copolymer exhibiting noise properties similar to a lipid membrane, albeit with better stability. Thus, by careful optimization, block copolymer membranes can emerge as a robust alternative for protein-pore based nano-biosensors.

Ultra-Precision Nanopore Tool to Study Enzymes at Work

The protein pore Mycobacterium smegmatis porin A (MspA) is enabling single-molecule nanopore DNA sequencing. In nanopore sequencing an electric field causes an ion current to flow through a single nano-meter sized pore embedded in a membrane. The electric field also draws single-stranded DNA through the pore. DNA bases inside the pore specifically modulate the measured ion current. We use molecular motors to regulate DNA's translocation speed through MspA. Here, we use this system to examine these motors by measuring the movement of DNA. With MspA, we can detect DNA motion as low as 35 picometers on sub millisecond timescales. To demonstrate the sensitivity of MspA we studied the detailed motions of DNA moved by a Super Family II helicase.

Nanopore Sequencing of “Alien” DNA Bases

Recently, it was shown for the first time that Escherichia coli can efficiently propagate a genetic alphabet expanded from the four canonical DNA nucleotides to include the synthetic “alien” bases d5SICS and dNaM. Traditional sequencing platforms cannot detect these alien bases. We tested whether nanopore sequencing with the protein pore Mycobacterium smegmatis porin A (MspA) is sensitive to d5SICS and dNaM. In nanopore sequencing, a nanometer scale pore provides the only electrical connection between two electrolyte solutions. An applied voltage causes a current to flow through the pore. Negatively-charged single stranded DNA is drawn through the pore, causing a nucleotide specific reduction in the measured current. Thus-far, MspA is the only nanopore shown to demonstrate single-nucleotide sensitivity to standard nucleotides as well as methyl cytosine and hydroxymethyl cytosine. We use the φ29 DNA polymerase to regulate DNA motion to single-nucleotide steps. Here, we demonstrate the direct detection d5SICS and dNaM with MspA-enabled nanopore sequencing.

DNA Sequence Detection using the Nanopore MspA

Nanopore DNA sequencing is a single molecule sequencing method in which DNA is pulled through a nanometer-sized pore, while the nucleotides modulate an ionic current. We use a protein pore based on Mycobacterium smegmatis porin A (MspA) that we developed to have sensitivity to single nucleotides in DNA. When using the phi29 DNA polymerase to move DNA in single-nucleotide steps through MspA, we observe a succession of well resolved ionic current levels. Each current level is primarily determined by the two nucleotides centered in MspA's narrowest constriction and is partially influenced by the flanking nucleotides. We present progress towards decoding algorithms that translate the observed ionic current patterns to DNA sequence.

Force Spectroscopy of Single Stranded DNA with Biological Nanopore MspA

Biological nanopores are single molecule sensors able to probe individual DNA molecules. In this method, a nanometer scale pore is inserted into a lipid bilayer in an electrolyte solution. Voltage is applied across the bilayer and the ionic current through the pore is measured. DNA nucleotides residing in the pore constriction modulate the resistance of the DNA-pore system. Our group previously introduced Mycobacterium smegmatis porin A (MspA) as a biological nanopore sensor with high signal to noise resolution of DNA nucleotides. In this work, we perform force spectroscopy on individual single-stranded DNA molecules. DNA molecules are attached to NeutrAvidin and driven into MspA until the NeutrAvidin comes to rest on MspA's entrance. Approximately 14 nucleotides span the distance to MspA's constriction. The nucleotides within the pore constriction experience a force proportional to the applied voltage. By varying the voltage applied across the pore, we detect stretching of the DNA with angstrom precision. These experiments validate MspA as a single molecule tool to study mechanical properties of DNA.

MspA Nanopores from Subunit Dimers

Mycobacterium smegmatis porin A (MspA) forms an octameric channel and represents the founding member of a new family of pore proteins. Control of subunit stoichiometry is important to tailor MspA for nanotechnological applications. In this study, two MspA monomers were connected by linkers ranging from 17 to 62 amino acids in length. The oligomeric pore proteins were purified from M. smegmatis and were shown to form functional channels in lipid bilayer experiments. These results indicated that the peptide linkers did not prohibit correct folding and localization of MspA. However, expression levels were reduced by 10-fold compared to wild-type MspA. MspA is ideal for nanopore sequencing due to its unique pore geometry and its robustness. To assess the usefulness of MspA made from dimeric subunits for DNA sequencing, we linked two M1-MspA monomers, whose constriction zones were modified to enable DNA translocation. Lipid bilayer experiments demonstrated that this construct also formed functional channels. Voltage gating of MspA pores made from M1 monomers and M1-M1 dimers was identical indicating similar structural and dynamic channel properties. Glucose uptake in M. smegmatis cells lacking porins was restored by expressing the dimeric mspA M1 gene indicating correct folding and localization of M1-M1 pores in their native membrane. Single-stranded DNA hairpins produced identical ionic current blockades in pores made from monomers and subunit dimers demonstrating that M1-M1 pores are suitable for DNA sequencing. This study provides the proof of principle that production of single-chain MspA pores in M. smegmatis is feasible and paves the way for generating MspA pores with altered stoichiometries. Subunit dimers enable better control of the chemical and physical properties of the constriction zone of MspA. This approach will be valuable both in understanding transport across the outer membrane in mycobacteria and in tailoring MspA for nanopore sequencing of DNA.

Mycobacterium smegmatis porin A (MspA) nanopore–based DNA sequencing using phage DNA polymerase and blocking oligomers

Mycobacterium smegmatis porin A (MspA) nanopore–based DNA sequencing using phage DNA polymerase and blocking oligomers

Reading DNA at single-nucleotide resolution with a mutant MspA nanopore and phi29 DNA polymerase

Nanopore technologies are being developed for fast and direct sequencing of single DNA molecules through detection of ionic current modulations as DNA passes through a pore's constriction. Here we demonstrate the ability to resolve changes in current that correspond to a known DNA sequence by combining the high sensitivity of a mutated form of the protein pore Mycobacterium smegmatis porin A (MspA) with phi29 DNA polymerase (DNAP), which controls the rate of DNA translocation through the pore. As phi29 DNAP synthesizes DNA and functions like a motor to pull a single-stranded template through MspA, we observe well-resolved and reproducible ionic current levels with median durations of ∼28 ms and ionic current differences of up to 40 pA. Using six different DNA sequences with readable regions 42-53 nucleotides long, we record current traces that map to the known DNA sequences. With single-nucleotide resolution and DNA translocation control, this system integrates solutions to two long-standing hurdles to nanopore sequencing.

Expression of the ompATb operon accelerates ammonia secretion and adaptation of Mycobacterium tuberculosis to acidic environments

Homeostasis of intracellular pH is a trait critical for survival of Mycobacterium tuberculosis in macrophages. However, mechanisms by which M. tuberculosis adapts to acidic environments are poorly understood. In this study, we analysed the physiological functions of OmpATb, a surface-accessible protein of M. tuberculosis. OmpATb did not complement the permeability defects of a Mycobacterium smegmatis porin mutant to glucose, serine and glycerol, in contrast to the porin MspA. Uptake rates of these solutes were unchanged in an ompATb operon mutant of M. tuberculosis indicating that OmpATb is not a general porin. Chemical analysis of low-pH culture filtrates showed that the proteins encoded by the ompATb operon are involved in generating a rapid ammonia burst, which neutralized medium pH and preceded exponential growth of M. tuberculosis. Addition of ammonia accelerated growth of the ompATb operon mutant demonstrating that ammonia secretion is indeed a mechanism by which M. tuberculosis neutralizes acidic environments. Infection experiments revealed that the ompATb operon was not required for full virulence in mice suggesting that M. tuberculosis has multiple mechanisms of resisting phagosomal acidification. Taken together, these results show that the ompATb operon is necessary for rapid ammonia secretion and adaptation of M. tuberculosis to acidic environments in vitro but not in mice.

Nanopore DNA sequencing with MspA.

Nanopore sequencing has the potential to become a direct, fast, and inexpensive DNA sequencing technology. The simplest form of nanopore DNA sequencing utilizes the hypothesis that individual nucleotides of single-stranded DNA passing through a nanopore will uniquely modulate an ionic current flowing through the pore, allowing the record of the current to yield the DNA sequence. We demonstrate that the ionic current through the engineered Mycobacterium smegmatis porin A, MspA, has the ability to distinguish all four DNA nucleotides and resolve single-nucleotides in single-stranded DNA when double-stranded DNA temporarily holds the nucleotides in the pore constriction. Passing DNA with a series of double-stranded sections through MspA provides proof of principle of a simple DNA sequencing method using a nanopore. These findings highlight the importance of MspA in the future of nanopore sequencing.

Functions of the Periplasmic Loop of the Porin MspA from Mycobacterium smegmatis

MspA is the major porin of Mycobacterium smegmatis and mediates diffusion of small and hydrophilic solutes across the outer membrane. The octameric structure of MspA, its sharply defined constriction zone, and a large periplasmic loop L6 represent novel structural features. L6 consists of 13 amino acids and is directly adjacent to the constriction zone. Deletion of 3, 5, 7, 9, and 11 amino acids of the L6 loop resulted in functional pores that restored glucose uptake and growth of a porin mutant of M. smegmatis. Lipid bilayer experiments revealed that all mutant channels were noisier than wild type (wt) MspA, indicating that L6 is required for pore stability in vitro. Voltage gating of the Escherichia coli porin OmpF was attributed to loops that collapse into the channel in response to a strong electrical field. Here, we show that deletion mutants Delta7, Delta9, and Delta11 had critical voltages similar to wt MspA. This demonstrated that the L6 loop is not the primary voltage-dependent gating mechanism of MspA. Surprisingly, large deletions in L6 resulted in 3-6-fold less extractable pores, whereas small deletions did not alter expression levels of MspA. Pores with large deletions in L6 were more permissive for glucose than smaller deletion mutants, whereas their single channel conductance was similar to that of wt MspA. These results indicate that translocation of ions through the MspA pore is governed by different mechanisms than that of neutral solutes. This is the first study identifying a molecular determinant of solute translocation in a mycobacterial porin.

Computational identification of beta-barrel outer-membrane proteins in Mycobacterium tuberculosis predicted proteomes as putative vaccine candidates

Mycobacterial porins and other beta-barrel outer-membrane proteins are represented by the structure of Mycobacterium smegmatis porin MspA. On the basis of existing knowledge of beta-barrel outer-membrane proteins, several state of the art prediction methods, as well as a new in-house program (PROB) were employed for the systematic exploration of Mycobacterium tuberculosis predicted proteomes for potential beta-barrel structures. PROB allowed parameter optimization while functioning with an adaptive algorithm for the detection of outer-membrane beta-barrel proteins in highly divergent proteomes. As a result of the predictions, 114 proteins in total were predicted to be beta-barrel structures; of these, 40 were PE-PPE proteins, 8 Mce proteins, 24 hypothetical, 11 probable membrane proteins, 10 transporters, 4 lipoproteins, and 14 classified as other. The congruence among three of the predictors, PROB, TMB-Hunt, and BOMP, was low with only three proteins (MT0318, MT0356, and MT2423) predicted by the three. Overall, 79 new proteins for which no previous experimental work has been performed are reported. At least 10 of these have high potential of being not only surface-exposed but also served as putative vaccine candidates as determined by in silico predictions of CD4T cell MHC-II restricted epitopes.

Topology of the Porin MspA in the Outer Membrane of Mycobacterium smegmatis

MspA is the major porin of Mycobacterium smegmatis mediating the exchange of hydrophilic solutes across the outer membrane (OM). It is the prototype of a new family of octameric porins with a single central channel of 9.6 nm in length and consists of two hydrophobic beta-barrels of 3.7 nm in length and a more hydrophilic, globular rim domain. The length of the hydrophobic domain of MspA does not match the thicknesses of mycobacterial OMs of 5-12 nm as derived from electron micrographs. Further, the membrane topology of MspA is unknown as it is for any other mycobacterial OM protein. We used MspA as a molecular ruler to define the boundaries of the OM of M. smegmatis by surface labeling of single cysteine mutants. Seventeen mutants covered the surface of the rim domain and were biotinylated with a membrane-impermeable reagent. The label efficiencies in vitro were remarkably similar to the predicted accessibilities of the cysteines. By contrast, six of these mutants were protected from biotinylation in M. smegmatis cells. Tryptophan 21 defines a horizontal plane that dissects the surface-exposed versus the membrane-protected residues of MspA. The 8 phenylalanines at position 99 form a ring at the periplasmic end of the hydrophobic beta-barrel domain. These results indicated that (i) the membrane boundaries of MspA are defined by aromatic girdles as in porins of Gram-negative bacteria and (ii) loops and a 3.4-nm long part of the hydrophilic rim domain are embedded into the OM of M. smegmatis. This is the first report suggesting that elements other than hydrophobic alpha-helices or beta-sheets are integrated into a lipid membrane.

The growth rate of Mycobacterium smegmatis depends on sufficient porin‐mediated influx of nutrients

Mycobacteria have a unique outer membrane (OM) that is thicker than any other known biological membrane. Nutrients cross this permeability barrier by diffusion through porins. MspA is the major porin of Mycobacterium smegmatis. In this study we showed that three paralogues of MspA, namely MspB, MspC and MspD are also porins. However, only the mspA and mspC genes were expressed in the wild-type strain. None of the single deletion mutants displayed a significant OM permeability defect except for the mspA mutant. Deletion of the mspA gene caused activation of transcription of mspB and/or mspD in three independent strains by unknown chromosomal mutations. It is concluded that mspB and mspD provide backup porins for M. smegmatis. This also indicated that a minimal porin-mediated OM permeability is essential for survival of M. smegmatis. Electron microscopy in combination with quantitative image analysis of protein gels revealed that the number of pores per cell dropped from 2400 to 800 and 150 for the DeltamspA and DeltamspA DeltamspC mutant (ML10) respectively. The very low number of pores correlated well with the at least 20-fold lower channel activity of detergent extracts of the ML10 strain and its 15- and 75-fold lower permeability to nutrient molecules such as serine and glucose respectively. The amount of Msp porin and the OM permeability of the triple porin mutant lacking mspA, mspC and mspD was not altered. The growth rate of M. smegmatis dropped drastically with its porin-mediated OM permeability in contrast to porin mutants of Escherichia coli. These results show that porin-mediated influx of nutrients is a major determinant of the growth rate of M. smegmatis.

Mycobacterial porins--new channel proteins in unique outer membranes.

Mycobacteria protect themselves with an outer lipid bilayer, which is the thickest biological membrane hitherto known and has an exceptionally low permeability rendering mycobacteria intrinsically resistant to many antibiotics. Pore proteins spanning the outer membrane mediate the diffusion of hydrophilic nutrients. Mycobacterium tuberculosis possesses at least two porins in addition to the low activity channel protein OmpATb. OmpATb is essential for adaptation of M. tuberculosis to low pH and survival in macrophages and mice. The channel activity of OmpATb is likely to play a major role in the defence of M. tuberculosis against acidification within the phagosome of macrophages. MspA is the main porin of Mycobacterium smegmatis. It forms a tetrameric complex with a single central pore of 10 nm length and a cone-like structure. This structure differs clearly from that of the trimeric porins of Gram-negative bacteria, which form one 4 nm long pore per monomer. The 45-fold lower number of porins compared to Gram-negative bacteria and the exceptional length of the pores are two major determinants of the low permeability of the outer membrane of M. smegmatis for hydrophilic solutes. The importance of the synergism between slow transport through the porins and drug efflux or inactivation for the development of drugs against M. tuberculosis is discussed.