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Lactococcus lactis NZ9000 and its parent MG1363 are the most commonly used lactic acid bacteria for expression and physiological studies. We noted unexpected but significant differences in the growth behaviors of both strains. We sequenced the entire genomes of the original NZ9000 and MG1363 strains using an ultradeep sequencing strategy. The analysis of the L. lactis NZ9000 genome yielded 79 differences, mostly point mutations, with the annotated genome sequence of L. lactis MG1363. Resequencing of the MG1363 strain revealed that 73 out of the 79 differences were due to errors in the published sequence. Comparative transcriptomic studies revealed several differences in the regulation of genes involved in sugar fermentation, which can be explained by two specific mutations in a region of the ptcC promoter with a key role in the regulation of cellobiose and glucose uptake. doi: 10.1128/JB.00533-10

This study describes the characterization of the riboflavin transport protein RibU in the lactic acid bacterium Lactococcus lactis subsp, cremoris NZ9000. RibU is predicted to contain five membrane-spanning segments and is a member of a novel transport protein family, not described in the Transport Classification Database. Transcriptional analysis revealed that ribU transcription is downregulated in response to riboflavin and flavin mononucleotide (FMN), presumably by means of the structurally conserved RFN (riboflavin) element located between the transcription start site and the start codon. An L. lactis strain carrying a mutated ribU gene exhibits altered transcriptional control of the riboflavin biosynthesis operon ribGBAH in response to riboflavin and FMN and does not consume riboflavin from its growth medium. Furthermore, it was shown that radiolabeled riboflavin is not taken up by the ribU mutant strain, in contrast to the wild-type strain, directly demonstrating the involvement of RibU in riboflavin uptake. FMN and the toxic riboflavin analogue roseoflayin were shown to inhibit riboflavin uptake and are likely to be RibU substrates. FMN transport by RibU is consistent with the observed transcriptional regulation of the ribGBAH operon by external FMN. The presented transport data are consistent with a uniport mechanism for riboflavin translocation and provide the first detailed molecular and functional analysis of a bacterial protein involved in riboflavin transport.

The crystal structures of BtuCD and its cognate periplasmic binding protein BtuF are determined. It is suggested that binding protein may not dissociate from the transporter during the catalytic cycle that are relevant to the mechanisms of other ABC transporter systems under the tight binding of BtuF to BtuCD.

A LacS-IIA truncation mutant of LacS is constructed to determine whether unphosphorylated LacS-IIA inhibits, or the phosphorylated form stimulates lactose counterflow. The results show that unphosphorylated LacS-IIA does not functionally interact with the carrier domain, instead, interaction of the phosphorylated form of LacS-IIA with the carrier stimulates lactose counterflow transport.

The current view of the cytoplasm as a 'bustling and well-organized metropolitan city' raises the issue of how physicochemical forces control the macromolecular interactions and transport of metabolites and energy in the cell. Motivated by studies on bacterial osmosensors, we argue that charged cytoplasmic macromolecules are stabilized electrostatically by their ionic atmospheres. The high cytoplasmic crowding (25-50% of cell volume) shapes the remaining cell volume (50-75%) into transient networks of electrolyte pathways and pools. The predicted 'semi-conductivity' of the electrolyte pathways guides the flow of biochemical ions throughout the cytoplasm. This metabolic and signaling current is powered by variable electrochemical gradients between the pools. The electrochemical gradients are brought about by cellular biochemical reactions and by extracellular stimuli. The cellular metabolism is thus vectorial not only across the membrane but also throughout the cytoplasm.

To link to full-text access for this article, visit this link: http://dx.doi.org/10.1016/j.tibs.2005.08.002 Byline: Jan J. Spitzer (a), Bert Poolman (b) Abstract: The current view of the cytoplasm as a 'bustling and well-organized metropolitan city' raises the issue of how physicochemical forces control the macromolecular interactions and transport of metabolites and energy in the cell. Motivated by studies on bacterial osmosensors, we argue that charged cytoplasmic macromolecules are stabilized electrostatically by their ionic atmospheres. The high cytoplasmic crowding (25-50% of cell volume) shapes the remaining cell volume (50-75%) into transient networks of electrolyte pathways and pools. The predicted 'semi-conductivity' of the electrolyte pathways guides the flow of biochemical ions throughout the cytoplasm. This metabolic and signaling current is powered by variable electrochemical gradients between the pools. The electrochemical gradients are brought about by cellular biochemical reactions and by extracellular stimuli. The cellular metabolism is thus vectorial not only across the membrane but also throughout the cytoplasm. Author Affiliation: (a) IPM Emulsion Polymer Research, 6643 Lyndonville Drive, Charlotte, NC 28277-4616, USA (b) Department of Biochemistry, Groningen Biomolecular Sciences and Biotechnology Institute, and Materials Science Center.sup.plus, University of Groningen, Nijenborgh 4, 9747 AG Groningen, The Netherlands

Membranes are important sites for the regulation of metabolic functions because they contain transport molecules, which often catalyze the first step in a pathway, and signal-transduction components, which allow the cell to communicate with the environment. Given the catalytic importance of transport proteins and their role in membrane stability, it is possible that oligomerization is used to regulate their function. This review evaluates knowledge of the functions that are associated with the oligomeric organization of secondary transport proteins, which are a major class of solute-translocation systems in all living species.

Results show that the Lactococcus lactis peptide transport protein DtpT is specific for di- and tripeptides and show high affinity to hydrophobic amino acid residues. Data also suggest that bacterial transporter exhibit a restrictive substrate recognition as opposed to eukayotic homologue tranport proteins.

Research has been conducted on the di-tripeptide binding protein ABC transporter of Lactococcus lactis. The cloning of the gene that encodes this binding protein is described.

Research has been conducted on the reaction of microorganisms to the hyperosmotic stress. The use of the transport system by Lactococcus lactis for glycine betaine has been examined.

A Pho84p/cytochrome C oxidase proteoliposome can be a useful model for studying the nature of the P(sub)i transporter. Pho84p is an H(super)+/phosphate cotransporter in Saccharomyces cerevisiae.

Large peptides have slower dissociation rates than smaller ones when interacting with the oligopeptide binding protein of Lactococcus lactis. The slower rates are the rate-determining step in the translocation of peptides across the bacterial membrane.

Maleimide spin-label attached to the single cysteine mutation in K373C in the lactose transport protein (LacS) from Streptococcus thermophilus is used to determine the location of interhelix loop 10-11 with respect to the substrate binding center in the protein. Cross-polarization magic-angle spinning nuclear magnetic resonance spectroscopy is used to observe the substrate binding. Results show that the loop region is in close proximity to the substrate binding sites of LacS and is expected to be incorporated within the membrane-spanning region of the protein.

A study was conducted to analyze the use of detergents in the functional reconstitution of the secondary multidrug transporter LmrP in Lactococcus lactis. A unique protein expression system for cytotoxic proteins was utilized to carry out the analysis. Experimental results indicated that LmrP is capable of mediating drug transport without the need for accessory proteins or cytosolic components.

A study relating to research on lactic acid bacteria was conducted to probe the kinetics and specificity of the oligopeptide transport system Opp of Lactococcus lactis through the competitive inhibition of the uptake of a radiolabeled reporter peptide that is not degraded by the cell. The kinetics of uptake of distinct peptides were also inferred from HPLC analysis of extra and intracellular fractions. The results of the experiment are discussed.

A study was conducted to characterize a framework for the unidirectional integration of lactose transport protein (LacS) of Streptococcus thermophilus into liposomes influenced by Triton X-100 using cryotransmission electron microscopy. Results showed the existence of an equilibrium between Triton-X-100-saturated liposomes and mixed micelles. Findings also indicated that the orientation of LacS in proteoliposomes correlated with transport activity in the counterflow assay.

A study was conducted to analyze the influence of salt and nonelectrolyte stress on the growth of Lactobacillus plantarum. The R 3010 quadrupole mass spectrometer was utilized to record electrospray mass spectra while L. plantartum cells were determined on cellulose actetate filters. Results indicated that severe growth inhibition correlated with the inability of cells to accommodate K+ to levels needed to restore turgor.

A study was conducted on the regulation of betaine and L-carnitine accumulation in Listeria monocytogenes in response to changes in the osmolarity of the growth medium. The results indicate that betaine and L-carnitine are taken up by separate highly specific transport systems. There is also evidence to support a secondary transport mechanism for betaine uptake in L monocytogenes.

Membrane topology of the dipeptide and tripeptide transport protein of Lactococcus lactis was established through construction and analyses of the gene fusions of di- and tripeptide transport protein (dtpT) with alkaline phodsphatase (phoA). Results have shown that the last predicted transmembrane segment, which is composed of residues from 433-451 of DtpT, was not crossing the membrane despite the high hydrophobicity and high ampipathy of the region.

The inverse PCR method was utilized in an experiment to clone the gene that encodes the di- and tripeptide transport protein of Lactobacillus helveticus. In addition, the functional expression in an Escherichia coli protein was evaluated. Results show that the proton motive force affects peptide transport through DtpT in membrane vesicles. The specificity for di-and tripeptides is not applicable to tetrapeptides or amino acids.

The gene encoding the di- and tripeptide transport protein (DtpT) of Lactobacillus helveticus (DtpT(sub SH)) was cloned with the help of the inverse PCR method and employed to complement the dipeptide transport-deficient and proline-auxotrophic Escherichia coli E1772. The dtpT(sub SH) gene is composed of 1,491 bp, which translates into a 497-amino-acid polypeptide. DtpT(sub SH) has 34% similarity to the di- and tripeptide transport protein of Lactococcus lactis and is also homologous to various peptide transporters of eukaryotic origin.

A decreased capacity of multiple-peptidase mutants of Lactococcus lactis, to break down peptides to free amino acids, leads to a reduction in their ability to grow in milk. An increase in the number of peptidase gene mutants leads to a lesser growth rate in milk giving rise to smaller amounts of intracellular amino acids. Single deficiencies in the genes, however, PepX, PepO, PepC, and PepT, barring PepN produce little effect on the growth rates of the mutants in milk.

Lactobacillus plantarum ATCC 14917 respond to osmotic stress by accumulation of glycine-betaine in the cytoplasmic pools of potassium, proline, alanine and glycine. The synthesis of glycine-betaine, however, results in the decrease of these other important solutes. Atomic absorption spectrophotometry and high-performance liquid chromatography analysis showed that L. plantarum ATCC 14917 increased the uptake of proline and glycine-betaine under hyperosmotic conditions. Glutamate uptake was not affected by osmotic shock while uptake of alanine was low under both hypo- and hyper-osmolarity.

A transcriptional factor called glutamate uptake regulatory protein (Grp) has been identified in Zymomonas mobilis. The protein increased glutamate uptake in E. coli but inhibited the secondary H+

A third peptide transport system is indicated for Lactococcus lactis because hydrophobic di- and tripeptides continue to be transported even when the di-tripeptide carrier protein is eliminated by a specific mutation in some peptides. This transport system, DtpP, is specific for di- and tripeptides that are relatively hydrophobic. An ATP or related energy- rich, phosphorylated intermediate is necessary to provide the proton motive force to drive the peptide uptake.

Protein transport in Saccharomyces cerevisiae operate primarily through the extensive fluxing of lipids from intracellular organelles to the plasma membrane. The transport is facilitated by the hydrolysis of ATP molecules by the plasma membrane to generate a proton motive force that will fuel secondary transport processes. In S. cerevisiae, transport activity may proceed via uniport systems or via proton symport systems. Both are regulated at the transcription level.

Th accumulation of cyclic hydrocarbons such as terpenes, aromatics and cycloalkanes in the membrane lipid bilayer in different microorganisms produces toxic effects important to their microbial degradation. The membrane alters its functional properties and increases its permeability to protons and ions, thereby resulting to a pH imbalance. The increase in hydrocarbons in the membrane also affects proteins embedded in the membrane, resulting in the modification of the outer lipid polysaccharide and cell wall composition.

A study in Bacillus subtilis membrane vesicles revealed that L-glutamate and L-aspartate are transported by the proton motive force (delta p) electrogenically in symport with at least 2 protons. The gene gltP(sub Bsu), has been cloned and functionally expressed in Escherichia coli JC5412, and its nucleotide sequence has been determined. A polypeptide of 414 amino acid residues is encoded by the gene, which is homologous to a group of proteins that transport compounds such as glutamate and aspartate.

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.

Studies conducted on the kinetics and genetics of citrate transport in Leuconostoc oenos show that the metabolism and citrate uptake cause the formation of an electrochemical gradient of protons across the cytoplasmic membrane. The citrate transport is an electrogenic process where a uniport mechanism assists the translocation of H2cit-. Energy generation during citrate utilization occurs due to the uptake of citrate anions, which yield a membrane potential and form H+-consuming reactions that yield a pH gradient.

N,N,N',N'-tetramethyl-P-phenylenediamine, ascorbate and cytochrome C enhance maltose intake against the concentration gradient in Lactobacillus sanfrancisco cells. A proton symport mechanism transports maltose within the cell. Intracellular glucose formation increases when maltose is used as a substrate, leading to excessive glucose secretion through the glucose import system.

A nucleotide sequence is inserted in pVS8 which encodes an endopeptidase and a functional oligopeptide transport system, as revealed by the peptide intake experiments. While assessing the participation of genes encoding the oligopeptide binding proteins in the functioning of casein and oligopeptides, the genes were disturbed in the chromosome of L. lactis. Lactococcal proteolytic system shows that extracellular hydrolysis of casein by proteinase PrtP is possible and that di-, tri- and oligopeptides are generated to support the growth of lactococci in milk.

The genomic integration of plasmids by homologous recombination results in the formation of two multiple copies of homologous DNA across the integrated plasmid. The two multiple copies are structurally parallel to insertion sequence-mediated cointegrates which are multi-faceted mechanisms for genetic manipulation, inactivation, substitution or cohesion of genes and for genetic labelling of specific genes. Plasmid integration facilitates deletion or insertion of genes. A promoterless chloramphenicol acetyltransferase (cat) gene, when inserted between the chromosomal lacS and lacZ genes, becomes a functional part of the operon and is expressed and regulated with respect to the operon.

The transport of 4-toluene sulfonate (TS) in Comamonas testosteroni T-2 was investigated. The TS transport system is only active in cells grown in the presence of TS or 4-methylbenzoate. Uptake of TS is inhibited by uncouplers and by a few structurally related compounds. Under anaerobic conditions, TS was accumulated in induced cells upon imposition of anartificial transmembrane pH gradient. Taken together, the results provide evidence that the transport is mediated by an inducible secondary proton symport system.

The ability of some bacterias to extrude 2', 7' -bis-(2-carboxyethyl)-5 (and -6) -carboxyfluorescein (BCECF) was determined to be due to an ATP-driven transport mechanism and not with a proton driven force. This efflux of the BCECF was found to occur against a BCECF gradient. An examination of the transport process in Lactococcus lactis showed that the system was inhibited by vanadate and possessed similarities to eukaryotic Mdr P-glycoproteins.

The toxic action of the lipophilic compound tetralin on bacterial membranes was studied using a membrane model system composed of proteoliposomes with reconstituted beef heart cytochrome c oxidase. Tetralin was found to cause expansion of liposomal membranes, altered membrane permeability and inhibited respiratory enzymes. These changes result in impairment of metabolic functions and low growth rates of the bacteria.

The analysis of the metabolic energy conservation by the malolactic fermentation pathway in Leoconostoc oenos indicates the build up of a negative membrane potential upon the addition of L-malate to the membranes. The L-malate fermentation process led to the accumulation of leucine against the concentration gradient, however, leucine was eliminated when the membrane potential was dissipated by valimycin and nigericin.

The anaerobic, thermophilic bacterium Clostridium fervidus utilizes Na(+) as the coupling ion for the energy transduction process, with Na(+) pump (ATPase) producing the sodium motive forces essential for the process. At high temperatures, the sodium motive force is more useful than the proton motive force. It requires less maintenance energy than the proton force at high temperatures. The function of Na+ as the coupling ion for energy transduction enables the C. fervidus to adapt to environmental changes at low bioenergetic costs.

Research was conducted to examine the mechanism of osmotic activation of the quaternary ammonium compound transport system in Lactobacillus plantarum. To elucidate the contribution of the transport systems to the overall flux of glycine betaine, mutants defective in glycine betaine uptake were generated and characterized. Results indicate that a channel protein responding to membrane stretch or tension mediates the rapid efflux of glycine betaine upon osmotic downshock.