Identification and characterization of PorH, a new cell wall channel of Corynebacterium glutamicum

The cell wall of Corynebacterium glutamicum contains the cation-selective channel (porin) PorA_C.glut and the anion-selective channel PorB_C.glut for the passage of hydrophilic solutes. Lipid bilayer experiments with organic solvent extracts of whole C. glutamicum cells cultivated in minimal medium suggested that also another cation-selective channel-forming protein, named PorH_C.glut, is present in C. glutamicum. The protein was purified to homogeneity by fast-protein liquid chromatography across a HiTrap-Q column. The pure protein had an apparent molecular mass of about 12 kDa on SDS-PAGE. Western blot analysis suggested that the cell wall channel is presumably formed by protein oligomers. The purified protein forms cation-selective channels with an average single-channel conductance of about 2.5 nS in 1 M KCl in the lipid bilayer assay. The PorH_C.glut protein was partially sequenced, and based on the resulting amino acid sequence, the corresponding gene, designated as porH_C.glut, was identified in the published genome sequence of C. glutamicum ATCC13032. PorH_C.glut contains only the inducer methionine but no N-terminal extension, which suggests that the export and assembly of the protein follow a yet unknown pathway. PorH_C.glut is coded in the bacterial chromosome by a gene that is localized in the vicinity of porA_C.glut, within a putative operon of 13 genes. RT-PCR revealed that both porins are cotranscribed. They coexist according to immunological detection experiments in the cell wall of C. glutamicum together with PorB_C.glut and PorC_C.glut.     

Identification and characterization of the channel-forming protein in the cell wall of Corynebacterium amycolatum

The mycolic-acid layer of certain gram-positive bacteria, the mycolata, represents an additional permeability barrier for the permeation of small water-soluble solutes. Consequently, it was shown in recent years that the mycolic acid layer of individual bacteria of the group mycolata contains pores, called porins, for the passage of hydrophilic solutes. Corynebacterium amycolatum, a pathogenic Corynebacterium species, belongs to the Corynebacteriaceae family but it lacks corynomycolic acids in its cell wall. Despite the absence of corynomycolic acids the cell wall of C. amycolatum contains a cation-selective cell wall channel, which may be responsible for the limited permeability of the cell wall of C. amycolatum. Based on partial sequencing of the protein responsible for channel formation derived from C. amycolatum ATCC 49368 we were able to identify the gene coram0001_1986 within the known genome sequence of C. amycolatum SK46 that codes for the cell wall channel. The corresponding gene of C. amycolatum ATCC 49368 was cloned into the plasmid pXHis for its expression in Corynebacterium glutamicum ?porA?porH. Biophysical characterization of the purified protein (PorA_coram) suggested that coram0001_1986 is indeed the gene coding for the pore-forming protein PorA_coram in C. amycolatum ATCC 49368. The protein belongs to the DUF (Domains of Unknown Function) 3068 superfamily of proteins, mainly found in bacteria from the family Corynebacteriaceae. The nearest relative to PorA_coram within this family is an ORF which codes for PorA_cres, which was also recognized in reconstitution experiments as a channel-forming protein in Corynebacterium resistens.     

The properties and contribution of the Corynebacterium glutamicum MscS variant to fine-tuning of osmotic adaptation

Based on sequence similarity, the mscCG gene product of Corynebacterium glutamicum belongs to the family of MscS-type mechanosensitive channels. In order to investigate the physiological significance of MscCG in response to osmotic shifts in detail, we studied its properties using both patch-clamp techniques and betaine efflux kinetics. After heterologous expression in an Escherichiacoli strain devoid of mechanosensitive channels, in patch-clamp analysis of giant E. coli spheroplasts MscCG showed the typical pressure dependent gating behavior of a stretch-activated channel with a current/voltage dependence indicating a strongly rectifying behavior. Apart from that, MscCG is characterized by significant functional differences with respect to conductance, ion selectivity and desensitation behavior as compared to MscS from E. coli. Deletion and complementation studies in C. glutamicum showed a significant contribution of MscCG to betaine efflux in response to hypoosmotic conditions. A detailed analysis of concomitant betaine uptake (by the betaine transporter BetP) and efflux (by MscCG) under hyperosmotic conditions indicates that MscCG may act in osmoregulation in C. glutamicum by fine-tuning the steady state concentration of compatible solutes in the cytoplasm which are accumulated in response to hyperosmotic stress.     

The FHA domain of OdhI interacts with the carboxyterminal 2-oxoglutarate dehydrogenase domain of OdhA in Corynebacterium glutamicum

In Corynebacterium glutamicum, the unphosphorylated 15-kDa OdhI protein inhibits the activity of the 2-oxoglutarate dehydrogenase complex (ODHc) by binding to OdhA, which in corynebacteria and mycobacteria is a large fusion protein with two major domains exhibiting structural features of E1o and E2 proteins. Using copurification and surface plasmon resonance experiments with different OdhI and OdhA length variants it was shown that the entire forkhead-associated (FHA) domain of OdhI and the C-terminal dehydrogenase domain of OdhA are required for interaction. The FHA domain was also sufficient for inhibition of ODHc activity. Phosphorylated OdhI was binding-incompetent and did not inhibit ODHc activity.

Structured summary

MINT-7713362:OdhI (uniprotkb:Q8NQJ3) binds (MI:0407) to OdhA (uniprotkb:Q8NRC3) by surface plasmon resonance (MI:0107)MINT-7713261:OdhI (uniprotkb:Q8NQJ3) physically interacts (MI:0915) with OdhA (uniprotkb:Q8NRC3) by pull down (MI:0096)     

Activity regulation of the betaine transporter BetP of Corynebacterium glutamicum in response to osmotic compensation

As a response to hyperosmotic stress bacterial cells accumulate compatible solutes by synthesis or by uptake. Beside the instant activation of uptake systems after an osmotic upshift, transport systems show also a second, equally important type of regulation. In order to adapt the pool size of compatible solutes in the cytoplasm to the actual extent of osmotic stress, cells down-regulate solute uptake when the initial osmotic stress is compensated. Here we describe the role of the betaine transporter BetP, the major uptake carrier for compatible solutes in Corynebacterium glutamicum, in this adaptation process. For this purpose, betP was expressed in cells (C. glutamicum and Escherichia coli), which lack all known uptake systems for compatible solutes. Betaine uptake mediated by BetP as well as by a truncated form of BetP, which is deregulated in its response to hyperosmotic stress, was dissected into the individual substrate fluxes of unidirectional uptake, unidirectional efflux and net uptake. We determined a strong decrease of unidirectional betaine uptake by BetP in the adaptation phase. The observed decrease in net uptake was thus mainly due to a decrease of V_max of BetP and not a consequence of the presence of separate efflux system(s). These results indicate that adaptation of BetP to osmotic compensation is different from activation by osmotic stress and also different from previously described adaptation mechanisms in other organisms. Cytoplasmic K⁺, which was shown to be responsible for activation of BetP upon osmotic stress, as well as a number of other factors was ruled out as triggers for the adaptation process. Our results thus indicate the presence of a second type of signal input in the adaptive regulation of osmoregulated carrier proteins.     

Corynebacterium heidelbergense sp. nov., isolated from the preen glands of Egyptian geese (Alopochen aegyptiacus)

Two strains (pedersoli^T and girotti) of a new species of bacteria were isolated from the preen glands of wild Egyptian geese (Alopochen aegyptiacus) from the river Neckar in southern Germany in two subsequent years. The strains were lipophilic, fastidious, Gram-positive rods and belonged to the genus Corynebacterium. Phylogenetically, the isolates were most closely related to Corynebacterium falsenii DSM 44353^T which has been found to be associated with birds before. 16S rRNA gene sequence similarity to all known Corynebacterium spp. was significantly <97%. Corresponding values of rpoB showed low levels of similarity <87% and ANIb was <73%. G + C content of the genomic DNA was 65.0 mol% for the type strain of the goose isolates, as opposed to 63.2 mol% in Corynebacterium falsenii. MALDI-TOF MS analysis of the whole-cell proteins revealed patterns clearly different from the related species, as did biochemical tests, and polar lipid profiles. We therefore conclude that the avian isolates constitute strains of a new species, for which the name Corynebacterium heidelbergense sp. nov. is proposed. The type strain is pedersoli^T (=DSM 104638^T = LMG 30044^T).     

Metabolic engineering of Escherichia coli and Corynebacterium glutamicum for the production of l-threonine

l-threonine is an essential amino acid for mammals and as such has a wide and expanding application in industry with a fast growing market demand. The major method of production of l-threonine is microbial fermentation. To optimize l-threonine production the fundamental solution is to develop robust microbial strains with high productivity and stability. Metabolic engineering provides an effective alternative to the random mutation for strain development. In this review, the updated information on genetics and molecular mechanisms for regulation of l-threonine pathways in Escherichia coli and Corynebacterium glutamicum are summarized, including l-threonine biosynthesis, intracellular consumption and trans-membrane export. Upon such knowledge, genetically defined l-threonine producing strains have been successfully constructed, some of which have already achieved the productivity of industrial producing strains. Furthermore, strategies for strain construction are proposed and potential problems are identified and discussed. Finally, the outlook for future strategies to construct industrially advantageous strains with respect to recent advances in biology has been considered.     

Two types of ion channel formation of tolaasin,a Pseudomonas peptide toxin

Tolaasin is a peptide toxin produced by Pseudomonas tolaasii and causes brown blotch disease of the cultivated mushrooms. Two types of ion channels were identified by the incorporation of tolaasin into lipid bilayer. The slope conductance of type 1 channel measured in the buffer containing 100 mM KCl was 150 pS with a linear current vs. voltage relationship. The type 2 tolaasin channel had two subconductance states of 300 and 500 pS. Both channels were inhibited by Zn(2+). Ion channel formations of tolaasin were concentration-dependent and single channel currents were successfully obtained at 0.6 unit tolaasin, 15.9 nM. The type 1 channel was obtained more frequently than the type 2 channel and the ratio of their appearance was approximately 4:1, respectively.     

Statistical properties of ion channel records. Part II: estimation from the macroscopic current

Macroscopic ion channel current is the summation of the stochastic records of individual channel currents and therefore relates to their statistical properties. As a consequence of this relationship, it may be possible to derive certain statistical properties of single channel records or even generate some estimates of the records themselves from the macroscopic current when the direct measurement of single channel currents is not applicable. We present a procedure for generating the single channel records of an ion channel from its macroscopic current when the stochastic process of channel gating has the following two properties: (I) the open duration is independent of the time of opening event and has a single exponential probability density function (pdf), (II) all the channels have the same probability to open at time t. The application of this procedure is considered for cases where direct measurement of single channel records is difficult or impossible. First, the probability density function (pdf) of opening events, a statistical property of single channel records, is derived from the normalized macroscopic current and mean channel open duration. Second, it is shown that under the conditions (I) and (II), a non-stationary Markov model can represent the stochastic process of channel gating. Third, the non-stationary Markov model is calibrated using the results of the first step. The non-stationary formulation increases the model ability to generate a variety of different single channel records compared to common stationary Markov models. The model is then used to generate single channel records and to obtain other statistical properties of the records. Experimental single channel records of inactivating BK potassium channels are used to evaluate how accurately this procedure reconstructs measured single channel sweeps.     

Production of poly(3-hydroxybutyrate-co-3-hydroxyvalerate) in recombinant Corynebacterium glutamicum using propionate as a precursor

Lipopolysaccharides free P[3-hydroxybutyrate (3HB)-co-3-hydroxyvalerate (3HV)] production was achieved using recombinant Corynebacterium glutamicum harboring polyhydroxyalkanoate (PHA) biosynthetic genes from Ralstonia eutropha. Cells grown on glucose with feeding of propionate as a precursor of 3HV unit accumulated 8-47 wt% of P(3HB-co-3HV). The 3HV fraction in the copolymer was varied from 0 to 28 mol% depending on the propionate concentrations.     

Role of polyphosphate in regulation of the Streptomyces lividans KcsA channel

We examine the hypotheses that the Streptomyces lividans potassium channel KcsA is gated at neutral pH by the electrochemical potential, and that its selectivity and conductance are governed at the cytoplasmic face by interactions between the KcsA polypeptides and a core molecule of inorganic polyphosphate (polyP). The four polypeptides of KcsA are postulated to surround the end unit of the polyP molecule with a collar of eight arginines, thereby modulating the negative charge of the polyP end unit and increasing its preference for binding monovalent cations. Here we show that KcsA channels can be activated in planar lipid bilayers at pH 7.4 by the chemical potential alone. Moreover, one or both of the C-terminal arginines are replaced with residues of progressively lower basicity-lysine, histidine, valine, asparagine-and the effects of these mutations on conductance and selectivity for K⁺ over Mg²⁺ is tested in planar bilayers as a function of Mg²⁺ concentration and pH. As the basicity of the C-terminal residues decreases, Mg²⁺ block increases, and Mg²⁺ becomes permeant when medium pH is greater than the pI of the C-terminal residues. The results uphold the premise that polyP and the C-terminal arginines are decisive elements in KcsA channel regulation.     

Comparison of the gramicidin a potassium/sodium permeability and single channel conductance ratio

If the ion concentration is low enough that most channels are unoccupied, then the ‘independence relations’ should be satisfied and the permeability ratio should equal the conductance ratio. It has been previously reported that for the gramicidin A channel these ratios for Na⁺ and K⁺ were not equal at concentrations as low as 10 mM. However, these ratios were not measured at the same applied potential, as is required by the theory. Instead, the conductance ratio was measured at 100 mV and corrected using calculated current-voltage relations. In this report the comparison between permeability and conductance ratios is reexamined using data obtained at the correct potential. There is no significant difference in the ratios at 10 mM when they are measured at the same voltage. This implies that most channels are not occupied by sodium or potassium ions at 10 mM.     

Temperature dependence of the Langmuir monolayer packing of mycolic acids from Mycobacterium tuberculosis

Phase diagrams of the Langmuir monolayer of dicyclopropyl alpha mycolic acid (α-MA), cyclopropyl methoxy mycolic acid (MeO-MA), and cyclopropyl ketomycolic acids (Keto-MA) from Mycobacterium tuberculosis were obtained by thermodynamic analysis of the surface pressure (π) vs. average molecular area (A) isotherms at temperatures in the range of 10-46 °C. The Langmuir monolayers of MAs were shown to exhibit various phases depending on the temperature (T) and the π values. In the Langmuir monolayer of Keto-MA, the carbonyl group in the meromycolate chain apparently touches the water surface to give the molecule a W-shape in all the temperatures and surface pressures studied. Keto-MA formed a rigid solid condensed film, with four hydrocarbon chains packing together, not observed in the others. In contrast, the monolayer films of α-and MeO-MAs having no such highly hydrophilic intra-chain groups in the meromycolate chain were mostly in liquid condensed phase. This novel insight into the packing of mycolic acids opens up new avenues for the study of the role of mycolic acids in the mycobacterial cell envelopes and pathogenic processes.     

Cloning and functional expression of Rh50-like glycoprotein, a putative ammonia channel, in Aedes albopictus mosquitoes

Evidence has shown that female mosquitoes can deaminate more than 80% of the ingested bloodmeal protein amino acids, and thus lead to a massive amount of ammonia production. Ammonia transport is a critical step for detoxifying ammonia in organisms. Here we characterized a putative ammonia channel gene, Rhesus (Rh) 50 glycoprotein, from Aedes albopictus (AalRh50) and determined the difference of its expression profile in different tissues at both message and protein levels as well as its response to a blood meal. We showed that AalRh50 shares a low identity with E. coli ammonia transporter (EcoAmtB), but higher identities with human RhBG and Drosophila Rh50 genes. The analysis of ammonia-conductance sites indicates that AalRh50 has residue substitutions of S237L (equivalent to S219 in AmtB) in the external vestibule, F127I (equivalent to F107 in AmtB) in the pore entrance, and S281N (equivalent to S263 in AmtB) in the internal vestibule, which could alter or reduce ammonia-conductance activity. The results from quantitative real-time-PCR and immunohistochemistry revealed that AalRh50 is expressed at significantly higher levels in the head, Malpighian tubules, and thorax of the non-blood-fed females, suggesting that AalRh50 might play roles in maintaining normal neurotransmitter metabolism, acid-base balance, and flight energy production in different tissues of mosquitoes at the non-blood-fed condition. A blood meal significantly increases AalRh50 expression in midgut, fat body, and Malpighian tubules from 3 or 6 to 24 h post feeding, indicating that AalRh50 plays an important role in detoxification of excess systemic ammonia of female adults during the gonotrophic cycle.     

Noise analysis of an ion channel in the cardiac myocyte--study based on a Markov model

Mechanical contraction of a cardiac muscle cell is related to the electric activation of the plasma membrane. As in the neuron cell, inflow of the Na(+) ions across the cell membrane causes electric activation with amplitude of about 100 mV. However, differently from the nerve cell, the action potential lasts a few hundred milliseconds before repolarization. Moreover, several types of K(+) channel such as the classical inward rectifier K(+) channel, the voltage dependent channel and others are responsible for the formation of the action potential. The mechanism of opening and closing the K(+) channels is not thoroughly elucidated. In the present paper, a four state Markov model with one open and three closed states is studied to obtain open and close probabilities of the gates constituting a specific ionic channel. The probability density functions of durations of opening and closing of the channel are also discussed.     

Heme-dependent autophosphorylation of a heme sensor kinase, ChrS, from Corynebacterium diphtheriae reconstituted in proteoliposomes

Corynebacterium diphteriae employs the response regulator, ChrA, and the sensor kinase, ChrS, of a two-component signal transduction system to utilize host heme iron. Although ChrS is predicted to encode a heme sensor, the sensing mechanism remains to be characterized. In this report, ChrS expressed in Eshcherichia coli membranes was solubilized and purified using decylmaltoside. ChrS protein incorporated into proteoliposomes catalyzed heme-dependent autophosphorylation by ATP. Other metalloporphyrins and iron did not stimulate kinase activity. The UV-Vis spectrum of hemin in the ChrS-proteoliposomes indicated that heme directly interacts with ChrS. This is the first functional reconstitution of a bacterial heme-sensing protein.     

The G314S KCNQ1 mutation exerts a dominant-negative effect on expression of KCNQ1 channels in oocytes

Congenital long QT syndrome is a cardiac disorder characterized by prolongation of QT interval on the surface ECG associated with syncopal attacks and a high risk of sudden death. Mutations in the voltage-gated potassium channel subunit KCNQ1 induce the most common form of long QT syndrome (LQT1). We previously identified a hot spot mutation G314S located within the pore region of the KCNQ1 ion channel in a Chinese family with long QT syndrome. In the present study, we used oocyte expression of the KCNQ1 polypeptide to study the effects of the G314S mutation on channel properties. The results of electrophysiological studies indicate G314S, co-expressed with KCNE1 was unable to assemble to form active channel. G314S, co-expressed with WT KCNQ1 and KCNE1, suppressed I_ks currents in a dominant-negative manner, which is consistent with long QT syndrome in the members of the Chinese family carrying G314S KCNQ1 mutation.