Ion channels from the <i>Bacillus subtilis</i> plasma membrane incorporated into planar lipid bilayers

Alcayaga, C; Venegas, Rodrigo; Carrasco, Alicia; Wolff, Daniel

doi:10.1016/0014-5793(92)81112-y

Cited by 10 publications

(5 citation statements)

References 22 publications

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“…Channel Activity in Planar Bilayers-High conductance, low selectivity channels were routinely observed upon fusion of B. subtilis vesicles with planar bilayers, in agreement with the observations by Alcayaga et al (41). Incorporation of a vesicle invariably led to the appearance of several conductances, ranging from about 0.1 to 1.5 nS (100 mM KCl), some of which clearly represented substates of others.…”

Section: Resultssupporting

confidence: 77%

“…300 l were withdrawn from the cis chamber and replaced with 300 l of 3 M KCl to favor fusion and to establish a transmembrane salt concentration gradient. Mostly inside-out B. subtilis membrane vesicles, prepared as described (41), containing approximately 5-20 g of protein were then added to the cis side, and the chambers were stirred at intervals until incorporation occurred. Activity was monitored at a few voltages for a short time.…”

Section: Methodsmentioning

confidence: 99%

“…Over the past decade, the application of patch clamp has led to the discovery and characterization of non-porin, stretchactivated, voltage-dependent channels in the cytoplasmic membrane of Gram-negative (25)(26)(27)(28)(29)(30)(31)(32)(33)(34)(35)(36) and Gram-positive (37)(38)(39)(40)(41) bacteria. No information is yet available on the proteins forming these pores, with the exception of one E. coli channel that has been sequenced and cloned (35).…”

mentioning

confidence: 99%

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DNA Translocation Across Planar Bilayers Containing Bacillus subtilis Ion Channels

Szabó

Báthori²,

Tombola

et al. 1997

Journal of Biological Chemistry

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The mechanisms by which genetic material crosses prokaryotic membranes are incompletely understood. We have developed a new methodology to study the translocation of genetic material via pores in a reconstituted system, using techniques from electrophysiology and molecular biology. We report here that planar bilayer membranes become permeable to double-stranded DNA (kilobase range) if Bacillus subtilis membrane vesicles containing high conductance channels have been fused into them. The translocation is an electrophoretic process, since it does not occur if a transmembrane electrical field opposing the movement of DNA, a polyanion, is applied. It is not an aspecific permeation through the phospholipid bilayer, since it does not take place if no proteins have been incorporated into the membrane. The transport is also not due simply to the presence of polypeptides in the membrane, since it does not occur if the latter contains gramicidin A or a eukaryotic, multiprotein vesicle fraction exhibiting 30-picosiemens anion-selective channel activity. The presence of DNA alters the behavior of the bacterial channels, indicating that it interacts with the pores and may travel through their lumen. These results support the idea that DNA translocation may take place through proteic pores and suggest that some of the high conductance bacterial channels observed in electrophysiological experiments may be constituents of the DNA translocating machinery in these organisms.

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Section: Resultssupporting

confidence: 77%

Section: Methodsmentioning

confidence: 99%

mentioning

confidence: 99%

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DNA Translocation Across Planar Bilayers Containing Bacillus subtilis Ion Channels

Szabó

Báthori²,

Tombola

et al. 1997

Journal of Biological Chemistry

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“…Evidence for the presence of ion channels in the B. subtilis plasma membrane was also obtained by Alcayaga et al (1) in electrophysiological studies using fusions of plasma membrane vesicles with planar lipid bilayers. The channels detected in these studies showed a wide range of conductances and kinetic behaviors, suggesting that more than one channel-forming protein operates in B. subtilis.…”

supporting

confidence: 56%

Responses of Bacillus subtilis to Hypotonic Challenges: Physiological Contributions of Mechanosensitive Channels to Cellular Survival

Hoffmann

Boiangiu

Moses

et al. 2008

Appl Environ Microbiol

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Mechanosensitive channels are thought to function as safety valves for the release of cytoplasmic solutes from cells that have to manage a rapid transition from high-to low-osmolarity environments. Subsequent to an osmotic down-shock of cells grown at high osmolarity, Bacillus subtilis rapidly releases the previously accumulated compatible solute glycine betaine in accordance with the degree of the osmotic downshift. Database searches suggest that B. subtilis possesses one copy of a gene for a mechanosensitive channel of large conductance (mscL) and three copies of genes encoding proteins that putatively form mechanosensitive channels of small conductance (yhdY, yfkC, and ykuT). Detailed mutational analysis of all potential channelforming genes revealed that a quadruple mutant (mscL yhdY yfkC ykuT) has no growth disadvantage in high-osmolarity media in comparison to the wild type. Osmotic down-shock experiments demonstrated that the MscL channel is the principal solute release system of B. subtilis, and strains with a gene disruption in mscL exhibited a severe survival defect upon an osmotic down-shock. We also detected a minor contribution of the SigB-controlled putative MscS-type channel-forming protein YkuT to cellular survival in an mscL mutant. Taken together, our data revealed that mechanosensitive channels of both the MscL and MscS types play pivotal roles in managing the transition of B. subtilis from hyper-to hypo-osmotic environments.The gram-positive soil bacterium Bacillus subtilis has to frequently manage the transition from high-to low-osmolarity surroundings due to drying and flooding of the upper layers of the soil (7, 35). To fend off the negative effects of high osmolarity on cellular water content and cell physiology, B. subtilis accumulates compatible solutes through either synthesis or uptake from the environment (7,20). Compatible solutes are highly soluble organic osmolytes (e.g., proline and glycine betaine) that can be amassed by the cell to exceedingly high levels without interfering with cell physiology (12). In this way, the water content of the cell is balanced with the prevalent osmolarity of the environment and cell turgor is stabilized. Consequently, cell growth can occur under osmotically unfavorable conditions.The massive accumulation of compatible solutes permits cell survival under hypertonic circumstances. However, the very same compounds become a threat to the integrity of the cell when B. subtilis is suddenly exposed to low-osmolarity surroundings via rainfall or washout into fresh water sources (8,35). The compatible solutes accumulated by the cell increase the osmotic potential of the cytoplasm and thereby instigate an osmotically controlled water influx (36). This water influx drives up turgor to nonphysiological high values and in extreme cases can lead to cell lysis (4, 6). Various researchers have observed that a number of cytoplasmic solutes, including proline, glycine betaine, potassium, glutamate, trehalose, and ATP, are rapidly released from osmotically downshifte...

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“…lowing an osmotic downshock Levina et al, 1999 . In B. subtilis, the presence of ion-conducting pores that respond to mechanical forces has been demonstrated through electrophysiological studies. These channels display conductances ranging from a few hundred pS to more than 3 nS and most exhibit complex gating kinetics, suggesting that at least some are composed of subunits that Ž function cooperatively Alcayaga et al, 1992;Sz-. abo et al, 1992 .…”

Section: Expulsion Of Compatible Solutes: Protection Against Extreme mentioning

confidence: 99%

Osmosensing and osmoregulatory compatible solute accumulation by bacteria

Wood

Bremer

Csonka

et al. 2001

Comparative Biochemistry and Physiology Part A: Molecular &

412

352

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Osmosensing and osmoregulatory compatible solute accumulation by bacteria Wood, J.M.; Bremer, Erhard; Csonka, L.N.; Kraemer, R; Poolman, B.; van der Heide, Tiemen; Smith, L.T. Take-down policy If you believe that this document breaches copyright please contact us providing details, and we will remove access to the work immediately and investigate your claim.Downloaded from the University of Groningen/UMCG research database (Pure): http://www.rug.nl/research/portal. For technical reasons the number of authors shown on this cover page is limited to 10 maximum. AbstractBacteria inhabit natural and artificial environments with diverse and fluctuating osmolalities, salinities and temperatures. Many maintain cytoplasmic hydration, growth and survival most effectively by accumulating kosmotropic organic Ž . Ž . solutes compatible solutes when medium osmolality is high or temperature is low above freezing . They release these solutes into their environment when the medium osmolality drops. Solutes accumulate either by synthesis or by transport from the extracellular medium. Responses to growth in high osmolality medium, including biosynthetic accumulation of trehalose, also protect Salmonella typhimurium from heat shock. Osmotically regulated transporters and mechanosensitive channels modulate cytoplasmic solute levels in Bacillus subtilis, Corynebacterium glutamicum, Escherichia coli, Lactobacillus plantarum, Lactococcus lactis, Listeria monocytogenes and Salmonella typhimurium. Each organism harbours multiple osmoregulatory transporters with overlapping substrate specificities. Membrane proteins Ž that can act as both osmosensors and osmoregulatory transporters have been identified secondary transporters ProP of . E. coli and BetP of C. glutamicum as well as ABC transporter OpuA of L. lactis . The molecular bases for the modulation of gene expression and transport activity by temperature and medium osmolality are under intensive investigation with emphasis on the role of the membrane as an antenna for osmo-andror thermosensors. ᮊ 2001 Elsevier Science Inc. All rights reserved. Wood et al. r Comparati¨e Biochemistry and Physiology Part A 130 2001 437᎐460 438

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Ion channels from the Bacillus subtilis plasma membrane incorporated into planar lipid bilayers

Cited by 10 publications

References 22 publications

DNA Translocation Across Planar Bilayers Containing Bacillus subtilis Ion Channels

DNA Translocation Across Planar Bilayers Containing Bacillus subtilis Ion Channels

Responses of Bacillus subtilis to Hypotonic Challenges: Physiological Contributions of Mechanosensitive Channels to Cellular Survival

Osmosensing and osmoregulatory compatible solute accumulation by bacteria

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