Search

The dependence of native Bacillus subtilis proteins expressed from their chromosomal genes on SecA is still unclear. Hence, the consequences of the regulation of the SecA level on the secretion of levansucrase and alpha-amylase during the exponential phase of growth were compared. Findings revealed a great contrast in SecA dependency for the secretion of levansucrase and alpha-amylase. While the secretion of levansucrase gradually varies with the SecA levl in the cell, the secretion of alpha-amylase is influenced only under conditions of strong SecA depletion.

Protein export in Escherichia coli is controlled by translocase, a multisubunit membrane protein complex with SecA as the peripheral subunit and the SecY, SecE and SecG proteins as the integral membrane domain. SecA, SecY and SecE have been identified in Bacillus subtilis by genetic analysis. Sequence comparison of the Bacillus chromosome revealed a possible homologue of SecG, called YvaL. A chromosomal disruption of the yvaL gene caused a mild cold sensitivity and a beta-latamase secretion defect.

The capability of nisin, a pore-forming antimicrobial peptide, to induce transmembrane transfer of a fluorescent phospholipid in lipid vesicles was studied. Nisin-induced transfer of the fluorescent phospholipid from the inner leaflet to the outer leaflet of the membranes attained stable levels, which were dependent on the concentration of the nisin introduced. The rate constant of this nicin-induced transmembrane movement increased with the nisin concentration but was not dependent on temperature from 5 to 30 deg C.

A study was conducted to characterize two enantiomeric forms of plantaricin A-22 (PlnA-22), and evaluate them against a secondary structure. Lactobacillus plantarum C11 was utilized as the bacteriocin-producing strain while fluorescent probe was carried out to produce transmembrane electrical potential. Experimental results indicated that the two forms of PlnA-22 supported pheromone activities that were coupled in nature.

In Escherichia coli, SecA ATPase bound to SecYEG membrane protein complex mediates protein translocation, which depends on the presence of negatively charged lipids. A new study investigates the lipid accessability of SecA bound at the translocase, using a phospholipid with an acyl chain. The findings suggest that translocase-bound SecA is mostly shielded from the phospholipid acyl chain, suggesting that acidic phospholipids may be implicated in translocation through their action on the lipid-bound fraction of SecA.

Research was conducted to investigate the isolation of large quantities of homogeneously pure SecYEG protein utilizing IMV's derived from Escherichia coli cells that associates the complex with a hexahistidine-tagged SecY. Strains were grown aerobically in an incubator until the end of the logarithmic phase. Results showed that the SecYEG complex can be purified in large quantities in a functional state while retaining several of its catalytic and functional properties in micellar solution.

A study was conducted on lactococcin G, a bacteriocin whose activity is dependent on the complementary action of two peptides. Both peptides can bind independently to the target cell surface. However, activity requires the complementary peptide. Moreover, the findings indicate that lactococcin G does not only causes the release of potassium ions but has also broader specificity for monovalent cations.

Bifunctional photoactivatable nucleotide analogue 3'-arylazido-8-azidoadenosine 5'-triphosphate (diN3ATP) was utilized to probe the spatial arrangement of the nucleotide binding sites of the SecA dimer in Escherichia coli. Results have shown that crosslinking of the protein occurred at NBS2 located at the domain of SecA. UV irradiation resulted in the formation of crosslinked SecA dimers.

Researchers have sought to gain further information about the mechanisms by which nucleotide-induced conformational changes in SecA, the peripheral subunit of the preprotein translocase of Escherichia coli, are connected with preprotein translocation. This involved raising monoclonal antibodies (mAbs) against specific parts of SecA in order to gain more detail about the functions of the C- and N-domains. It was possible to isolate an mAb which recognizes an epitope situated in the carboxy-terminal 30-kDa membrane inserting fragment of SecA.

The role of the transmembrane hydrogen-ion concentration (pH) gradient in pore formation and the characteristics of nisin variants were analyzed in sensitive Lactococcus lactis cells and proteoliposomes. Analysis of the mechanisms of pore formation in Lactococcus lactis indicated the ability of the transmembrane pH gradient to activate lantibiotic nisin variants. Furthermore, the mechanisms of transmembrane pH gradient-induced pore formation correlated with the wedge-model of nisin-induced pore formation.

Resistance of Lactococcus lactis to cytotoxic compounds shares features with the multidrug resistance phenotype of mammalian tumor cells. Here, we report the gene cloning and functional characterization in Escherichia coli of LmrA, a lactococcal structural and functional homolog of the human multidrug resistance P-glycoprotein MDR1. LmrA is a 590-aa polypeptide that has a putative topology of six [Alpha]-helical transmembrane segments in the N-terminal hydrophobic domain, followed by a hydrophilic domain containing the ATP-binding site. LmrA is similar to each of the two halves of MDR1 and may function as a homodimer. The sequence conservation between LmrA and MDR1 includes particular regions in the transmembrane domains and connecting loops, which, in MDR1 and the MDR1 homologs in other mammalian species, have been implicated as determinants of drug recognition and binding. LmrA and MDR1 extrude a similar spectrum of amphiphilic cationic compounds, and the activity of both systems is reversed by reserpine and verapamil. As LmrA can be functionally expressed in E. coli, it offers a useful prokaryotic model for future studies on the molecular mechanism of MDR1-like multidrug transporters.

Tryptophan fluorescence spectroscopy, differential scanning calorimetry, tryptic digestion, and dynamic light scattering were performed to characterize the domain interactions of the homodimeric SecA protein. The protein, which promotes the translocation of the preprotein translocase across the bacterial cytoplasmic membrane, was examined when ADP or AMP-PNP are attached to one or both of its nucleotide binding sites.

Plantaricin C is a pore-forming bacteriocin that causes dissipation of the delta-p on plasma membrane and release of intracellular molecules. Plantaricin C dissipates the proton motive force and inhibits amino acid transport in sensitive cells, while it dissipates the transmembrane electrical potential in proteoliposomes. In liposomes, plantaricin C elicits the efflux of entrapped carboxyfluorescein. Plantaricin C molecules form multipeptide pore complexes by their combined action to allow the passage of large molecules.

Energy transduction in the anaerobic thermoalkaliphilic strain LBS3 occurs primarily by sodium cycling. Na+ stimulates the membrane-bound ATPase activity in invaginated membrane vesicles of the bacterium. The Na+ -dependent energy transduction instead of the H+ -dependent one in aerobic alkalophiles improves intracellular pH regulation due to the lower permeability of membranes to Na ions. But an antiporter mechanism other than the Na ion/H+ antiporter may be involved in pH homeostasis of this strain.

The basic amino acid permease (system VI) in the filamentous fungus Penicillium chrysogenum is responsible for transfer of basic amino acids in plasma membrane vesicles. Lysine and arginine collect in the membrane vesicles in the presence of reduced cytochrome c. In the external pH range of 5.5-7.5, the transmembrane electrical potential drives the uphill transport of arginine. The amino acid permease is an H+ ion symporter, functioning in a reversible manner. The H+ -arginine symport stoichiometry is 1:1.

The uptake of sulfate by Penicillium chrysogenum cells through the plasma membrane is regulated by the electroneutral transmembrane pH gradient rather than the transmembrane electrical potential. The transport mechanism has a 2:1 H+/sulfate stoichiometry as it involves the symport of one sulfate ion with two protons. Divalent ions, such as Ca2+, are not involved in the transport. The same pathway is used for sulfate transport during both sulfate-deficient and -sufficient conditions. The transport mechanism has a narrow substrate specificity.

Lactococcin G is an antibacterial agent produced by lactic acid bacteria. This novel lactococcal bacteriocin require two peptides, termed alpha and beta, to exert bactericidal activity. A study of the impact of lactococcin G on the energy-transducing properties of Lactococcus lactis revealed that lcatococcin G forms potassium-selective channels by the alpha and beta peptides in the target bacterial membrane.

The tryptophan residue 36 of SecB helps in the binding of precursor proteins. The intrinsic tryptophan fluorescence of proteins helps in collecting the information on the folding state of a protein while forming complexes with a molecular chaperon. The binding of SecB to bovine pancreas trypsin inhibitor (BPTI) increases the fluorescence of the surface-exposed tryptophan residues 36 of SecB. In vivo, SecB binds to nascent polypeptides. The binding of AR-BPTI to SecB shifts the emission maximum of acrylodan fluorescence.

The Penicillium chrysogenum, a fungus used to produce penicillin commercially, uses phenylacetic acid (PA) as a side chain precursor in the synthesis of penicillin G. The PA enters the fungal cell via the plasma membrane by passive diffusion of the protonated species. The process of PA passage is studied in mycelia and liposomes. The rate-limiting factor in the biosynthesis of penicillin G is not the influx of PA.

A study of the mechanism of L-glutamate uptake in Rhodobacter sphaeroides showed that the uptake is influenced by a high-affinity (K{sub t} of 1.2 micro meter) and shock-sensitive transport system which is inhibited by vanadate and dependent on the internal pH. The L-glutamate and L-glutamine is bound by the protein that limits concentrations of glutamine in the growth medium at a maximum expression level. This information suggests that in R. sphaeroides, the L-glutamate- and L-aspartate-binding proteins work with the same membrane transporter.

Experimental studies on the permeabilization of liposomes by cationic polycyclic bacteriocin nisin reveal that nisin functions as an anionic carrier in dioleoylphosphatidylcholine liposomes. The induction of a transmembrane electric potential causes nisin to permeate into the phospholipid membrane and to distort the structure of the vesicle. Several anionic fluorescent dyes such as 6-carboxyfluorescein are released by nisin. Electrostatic interactions between nisin molecules and the anionic lipids prevent the activity of nisin as an anion carrier.

The three mutants Eth(R), Dau(R) and Rho(R) of Lactococcus lactis subspecies lactis MG1363 are investigated to determine their resistive property toward ethidium bromide, daunomycin and rhodamine 6G, respectively. The three mutants resist several drugs including mammalian multidrug transporters. The resistance to these toxic compounds is attributed to the active efflux. The ethidium efflux is inhibited by reserpine in all bacterial strains and yields a proton motive force-driven efflux system. The proton motive force is the primary cause for the resistive property of the mutants.

SecA, the peripheral ATPase region of Escherichia coli precursor protein translocase was denatured in 6M guanidine hydrochloride. The protein is converted into a random-coli configuration. SecA refolds into its native, functional conformation as a homodimer, when the chaotropic agent is diluted. Homodimers derived from native and 8-azido-ATP-inactivated SecA were used to examine the half-of-the-sites activity. Heterodimers are incapable of enhancing translocation of the precursor protein proOmpA in an in vitro translocation system.

The activity of pediocin PA-1 was studied, and it was revealed that it reacts with the cytoplasmic membrane of target cells by forming voltage-independent pores. These pores cause an efflux of vital cellular metabolites. Pediocin PA-1 bacteriocin is synthesized by Pediococcus acidilactici PAC1.0. Pediocin PA-1 destroys Pediococcus cells that are sensitive, and reacts with the cytoplasmic membrane.

High intracellular pools of both proline and betaine in Lactococcus lactis subsp. lactis ML3 are formed as a result of specific transport systems, both substances being compatible solutes in L. lactis subsp. lactis ML3. Passive diffusion does not affect the proline uptake in L. lactis as it is mediated by an enzyme. The uptake is influenced by the pH and other factors, such as the intracellular concentration. Metabolic energy determines the uptake of both solutes, and the transport systems are ATP-driven.

Wild-type lac permease and lac permease mutated at each of the 8 cysteinyl residues in the molecule were solubilized from the membrane, purified and transformed into proteoliposomes. The lactose equilibrium exchange, and efflux actions of mutants with Ser in the place of Cys117, Cys176, Cys234, Cys333, Cys353 or Cys355 were the same as wild-type permease. The lack of the low Km pathway in the C148S permease is correlated with the lack of a high-affinity binding area for p-nitrophenyl alpha-D-galactopyranoside (NPG).

The generation of metabolic energy by histidine/histamine antiport in Lactobacillus buchneri was demonstrated. It was shown that L. buchneri generates metabolic energy from the decarboxylation of histidine and the electrogenic antiport of histidine and histamine. The results suggest that decarboxylation coupled to proton motive force generation is widespread among bacteria.

Research was conducted on the influence of bipolar tetraether lipids sourced from Sulfolobus acidocaldarius on the leucine transport system of Lactococcus lactis. Results showed that bipolar lipids possessed a monomolecular configuration at elevated Sulfolobus acdocaldarius lipid content. Based on the experimental results, the possibility of transport protein reconstitution into membranes containing membrane-spanning lipids was proposed.

A study was done on the kinetic and thermodynamic role of ATP and the protonmotive force in bacterial protein translocation. It was shown that ATP binding to SecA ATPase drives the insertion step which initiates translocation. On the other hand, protonmotive force stimulates the translocation process in the absence of ATP, allows small stable folded domains to translocate in the presence of ATP and specifies the direction of translocation by its polarity.