In vivo monitoring of the potassium channel KcsA in Streptomyces lividans hyphae using immuno-electron microscopy and energy-filtering transmission electron microscopy
The previous discovery of the Streptomyces lividans kcsA gene and its overexpression followed by the functional reconstitution of the purified gene product has resulted in new strategies to explore this channel protein in vitro. KcsA has evolved as a general model to investigate the structure/function relationship of ion channel proteins. Using specific antibodies raised against a domain of KcsA lacking membrane-spanning regions, KcsA has now been localized within numerous separated clusters between the outer face of the cytoplasm and the cell envelope in substrate hyphae of the S. lividans wild-type strain but not in a designed chromosomal disruption mutant ΔK, lacking a functional kcsA gene. Previous findings had revealed that caesium ions led to a block of KcsA channel activity within S. lividans protoplasts fused to giant vesicles. As caesium can be scored by electron energy loss spectroscopy better than potassium, this technique was applied to hyphae that had been briefly exposed to caesium instead of potassium ions. Caesium was found preferentially at the cell envelope. Compared to the ΔK mutant, the relative level of caesium was ≈30 % enhanced in the wild-type. This is attributed to the presence of KcsA channels. Additional visualization by electron spectroscopic imaging supported this conclusion. The data presented are believed to represent the first demonstration of in vivo monitoring of KcsA in its original host.
Blondelet-RouaultM. H, WeiserJ, LebrihiA, BrannyP, PernodetJ. L.
1997; Antibiotic resistance gene cassettes derived from the omega interposon for use in E. coli and Streptomyces . Gene 190:315–317[CrossRef]
BuckingH, BeckmannS, HeyserW, KottkeI.
1998; Elemental contents in vacuolar granules of extomycorrhizal fungi measured by EELS and EDXS. A comparison of different methods and preparation techniques. Micron 29:53–61[CrossRef]
ChavezF, JerezC. A,
LünsdorfH. 2004; Growth of polychlorinated-biphenyl-degrading bacteria in the presence of biphenyl and chlorobiphenyls generates oxidative stress and massive accumulation of inorganic polyphosphate. Appl Environ Microbiol 70:3064–3072[CrossRef]
CruzA. F.
2004; Element storage in spores of Gigaspora margarita Becker & Hall measured by electron energy loss spectroscopy (EELS). Acta Bot Bras 18:473–480[CrossRef]
DoyleD. A, CabralJ. M, PfuetznerR. A, KuoA, GulbisJ. M, CohenS. L, ChaitB. T, MacKinnonR.
1998; The structure of the potassium channel: molecular basis of K[sup]+[/sup] conduction and selectivity. Science 280:69–77[CrossRef]
GopingG, PollardH. B, SrivastavaM, LeapmanR.
2003; Mapping protein expression in mouse pancreatic islets by immunolabeling and electron energy loss spectrum-imaging. Microsc Res Tech 61:448–456[CrossRef]
HeinrichU. R, MaurerJ, MannW.
1998; Possible Ca[sup]2+[/sup]-dependent mechanism of apical outer hair cell modulation within the cochlea of the guinea pig. Cell Tissue Res 292:57–65[CrossRef]
KharkovetsT, HardelinJ. P, SafieddineS, SchweizerM, El AmraouiA, PetitC, JentschT. J.
2000; KCNQ4, a K[sup]+[/sup] channel mutated in a form of dominant deafness, is expressed in the inner ear and the central auditory pathway. Proc Natl Acad Sci U S A 97:4333–4338[CrossRef]
KottkeI.
1991; Electron energy loss spectroscopy and imaging technique for subcellular localization of elements in mycorrhiza. Methods Microbiol 23:369–382
KutznerH. J.
1981; The family Streptomycetaceae. In The Prokaryotes: a Handbook on Habitats, Isolation and Identification of Bacteria pp 2028–2090 Edited by
StarrM. P.,
StolpH.,
SchlegelH.,
TrüperH. G.,
BalowsA.
Berlin: Springer;
LiuD, KottkeI.
2003; Subcellular localization of chromium and nickel in root cells of Allium cepa by EELS and ESI. Cell Biol Toxicol 19:299–311[CrossRef]
MeuserD, SplittH, WagnerR, SchrempfH.
1999; Exploring the open pore of the K[sup]+[/sup] channel KcsA from Streptomyces lividans . FEBS Lett 462:447–452[CrossRef]
MeuserD, SplittH, WagnerR, SchrempfH.
2001; Mutations stabilizing an open conformation within the external region of the permeation pathway of the potassium channel KcsA. Eur Biophys J 30:385–391[CrossRef]
MolinaM. L, BarreraF. N, FernandezA. M, PovedaJ. A, RenartM. L, EncinarJ. A, RiquelmeG, Gonzalez-RosJ. M.
2006; Clustering and coupled gating modulate the activity in KcsA, a potassium channel model. J Biol Chemhttp://www.jbc.org/cgi/doi/10.1074/jbc.M600342200
MuthG, FarrM, HartmannV, WohllebenW.
1995; Streptomyces ghanaensis plasmid pSG5: nucleotide sequence analysis of the self-transmissible minimal replicon and characterization of the replication mode. Plasmid 33:113–126[CrossRef]
RasmussenH. B, MollerM, KnausH. G, JensenB. S, OlesenS. P, JorgensenN. K.
2004; Subcellular localization of the delayed rectifier K[sup]+[/sup] channels KCNQ1 and ERG1 in the rat heart. Am J Physiol Heart Circ Physiol 286:H1300–H1309
RocchettaH. L, LamJ. S.
1997; Identification and functional characterization of an ABC transport system involved in polysaccharide export of A-band lipopolysaccharide in Pseudomonas aeruginosa . J Bacteriol 179:4713–4724
SchlochtermeierA, NiemeyerF, SchrempfH.
1992; Biochemical and electron microscopic studies of the Streptomyces reticuli cellulase (Avicelase) in its mycelium-associated and extracellular forms. Appl Environ Microbiol 58:3240–3248
SchrempfH.
1999; Investigations of streptomycetes using tools of recombinant DNA technology. In Manual of Industrial Microbiology and Biotechnology, 2nd edn. pp 501–510 Edited by
DemainA. L., DaviesJ. E.
Washington, DC: American Society for Microbiology;
SchrempfH.
2005; Deciphering the Streptomyces lividans KcsA as a channel model. In Bacterial Ion Channels and Their Eukaryotic Homologs pp 14–67 Edited by
KubalskiA., MartinacB.
Washington, DC: American Society for Microbiology;
SplittH, MeuserD, BorovokI, BetzlerM, SchrempfH.
2000; Pore mutations affecting tetrameric assembly and functioning of the potassium channel KcsA from Streptomyces lividans . FEBS Lett 472:83–87[CrossRef]
van DalenA,
SchrempfH, KillianJ. A, de KruijffB. 2000; Efficient membrane assembly of the KcsA potassium channel in Escherichia coli requires the protonmotive force. EMBO Rep 1:340–346[CrossRef]
XiaoX, WangF, SaitoA, MajkaJ, SchrempfH,
SchlösserA. 2002; The novel Streptomyces olivaceoviridis ABC transporter Ngc mediates uptake of N -acetylglucosamine and N , N ′-diacetylchitobiose. Mol Genet Genomics 267:429–439[CrossRef]
ZakharianE, ReuschR. N.
2004; Functional evidence for a supramolecular structure for the Streptomyces lividans potassium channel KcsA. Biochem Biophys Res Commun 322:1059–1065[CrossRef]
In vivo monitoring of the potassium channel KcsA in Streptomyces lividans hyphae using immuno-electron microscopy and energy-filtering transmission electron microscopy