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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.

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

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.

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.

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.

Investigates the mechanism and energetics of citrate transport in Leuconostoc oenos. Uniport of anionic citrate and proton consumption; Generation of proton motive force; Uptake in membrane vesicles; Kinetic analysis and specificity; Measurement of membrane potential and pH gradient; Transport assays.

Escherichia coli adapts to changing environmental osmolality to survive and maintain growth. It has been shown that the diffusion of green fluorescent protein (GFP) in cells adapted to osmotic upshifts is higher than expected from the increase in biopolymer volume fraction. To better understand the physicochemical state of the cytoplasm in adapted cells, we now follow the macromolecular crowding during adaptation with fluorescence resonance energy transfer (FRET)-based sensors. We apply an osmotic upshift and find that after an initial increase, the apparent crowding decreases over the course of hours to arrive at a value lower than that before the osmotic upshift. Crowding relates to cell volume until cell division ensues, after which a transition in the biochemical organization occurs. Analysis of single cells by microfluidics shows that changes in cell volume, elongation, and division are most likely not the cause for the transition in organization. We further show that the decrease in apparent crowding upon adaptation is similar to the apparent crowding in energy-depleted cells. Based on our findings in combination with literature data, we suggest that adapted cells have indeed an altered biochemical organization of the cytoplasm, possibly due to different effective particle size distributions and concomitant nanoscale heterogeneity. This could potentially be a general response to accommodate higher biopolymer fractions yet retaining crowding homeostasis, and it could apply to other species or conditions as well. [ABSTRACT FROM AUTHOR]

Sortases are transpeptidases that couple surface proteins to the peptidoglycan of Gram-positive bacteria, and several sortase-dependent proteins (SDPs) have been demonstrated to be crucial for the interactions of pathogenic and nonpathogenic bacteria with their hosts. Here, we studied the role of sortase A (SrtA) in Lactobacillus plantarum WCFS1, a model Lactobacillus for probiotic organisms. An isogenic srtA deletion derivative was constructed which did not show residual SrtA activity. DNA microarray-based transcriptome analysis revealed that the srtA deletion had only minor impact on the full-genome transcriptome of L plantarum, while the expression of SDP-encoding genes remained completely unaffected. Mass spectrometry analysis of the bacterial cell surface proteome, which was assessed by trypsinization of intact bacterial cells and by LiC1 protein extraction, revealed that SrtA is required for the appropriate subcellular location of specific SDPs and for their covalent coupling to the cell envelope, respectively. We further found that SrtA deficiency did not affect the persistence and/or survival of L. plantarum in the gastrointestinal tract of mice. In addition, an in vitro immature dendritic cell (iDC) assay revealed that the removal of surface proteins by LiCl strongly affected the proinflammatory signaling properties of the SrtA-deficient strain but not of the wild type, which suggests a role of SDPs in host immune response modulation. [ABSTRACT FROM AUTHOR]

Unlabelled: The GlnPQ transporter from Lactococcus lactis has the remarkable feature of having two substrate-binding domains (SBDs) fused to the N terminus of the transmembrane domain (TMD), and thus four SBDs are present in the homodimeric complex. Although X-ray structures and ligand binding data are available for both SBDs, little is known of how different amino acids compete with each other for transport via GlnPQ. Here we show GlnPQ has a broader substrate specificity than previously thought, with the ability to take up asparagine, glutamine, and glutamic acid, albeit via different routes and with different affinities. Asparagine and glutamine compete with each other at the level of binding to SBD1 and SBD2 (with differences in dissociation constant), but at the same time SBD1 and SBD2 compete with each other at the level of interaction with the translocator domain (with differences in affinity constant and rate of transport). Although glutamine transport via SBD1 is outcompeted by physiological concentrations of asparagine, SBD2 ensures high rates of import of the essential amino acid glutamine. Taken together, this study demonstrates that even in the presence of competing asparagine concentrations, GlnPQ has a high capacity to transport glutamine, which matches the high needs of the cell for glutamine and glutamate., Importance: GlnPQ is an ATP-binding cassette (ABC) transporter for glutamine, glutamic acid, and asparagine. The system is essential in various Gram-positive bacteria, including L. lactis and several pathogens. Here we show how the amino acids compete with each other for binding to the multiple SBDs of GlnPQ and how these SBDs compete with each other for substrate delivery to the transporter. Overall, our results show that GlnPQ has evolved to transport diverse substrates via different paths and to optimally acquire the abundant and essential amino acid glutamine., (Copyright © 2016, American Society for Microbiology. All Rights Reserved.)