Models to determine the kinetic mechanisms of ion-coupled transporters.

With high-resolution structures available for many ion-coupled (secondary active) transporters, a major challenge for the field is to determine how coupling is accomplished. Knowledge of the kinetic mechanism of the transport reaction, which defines the binding order of substrate and co-ions, together with the sequence with which all relevant states are visited by the transporter, will help to reveal this coupling mechanism. Here, we derived general mathematical models that can be used to analyze data from steady-state transport measurements and show how kinetic mechanisms can be derived. The models describe how the apparent maximal rate of substrate transport depends on the co-ion concentration, and vice versa, in different mechanisms. Similarly, they describe how the apparent affinity for the transported substrate is affected by the co-ion concentration and vice versa. Analyses of maximal rates and affinities permit deduction of the number of co-ions that bind before, together with, and after the substrate. Hill analysis is less informative, but in some mechanisms, it can reveal the total number of co-ions transported with the substrate. However, prior knowledge of the number of co-ions from other experimental approaches is preferred when deriving kinetic mechanisms, because the models are generally overparameterized. The models we present have wide applicability for the study of ion-coupled transporters.

Arginine and Citrulline Catabolic Pathways Encoded by the arc Gene Cluster of Lactobacillus brevis ATCC 367.

High concentrations of l-arginine or l-citrulline in the growth medium provided the wine bacterium Lactobacillus brevis with a significant growth advantage. The arginine deiminase pathway (ADI) arc gene cluster of Lactobacillus brevis contains three genes-arcD, arcE1, and arcE2-encoding putative l-arginine/l-ornithine exchangers. Uptake experiments with Lactococcus lactis cells expressing the genes showed that all three transported l-ornithine with affinities in the micromolar range. Similarly, ArcD and ArcE2 transported l-arginine, while ArcE1 transported l-citrulline, an intermediate of the ADI pathway. Chase experiments showed very efficient exchange of l-arginine and l-ornithine by ArcD and ArcE2 and of l-citrulline and l-ornithine by ArcE1. Low affinities (millimolar range) combined with low translocation rates were found for ArcD and ArcE2 with l-citrulline and for ArcE1 with l-arginine. Resting cells of Lactobacillus brevis grown in the presence of l-arginine and l-citrulline rapidly consumed l-arginine and l-citrulline, respectively, while producing ammonia and l-ornithine. About 10% of l-arginine degraded was excreted by the cells as l-citrulline. Degradation of l-arginine and l-citrulline was not subject to carbon catabolite repression by glucose in the medium. At a high medium pH, l-citrulline in the medium was required for induction of the l-citrulline degradation pathway. Pathways are proposed for the catabolic breakdown of l-arginine and l-citrulline that merge at the level of ornithine transcarbamylase in the ADI pathway. l-Arginine uptake is catalyzed by ArcD and/or ArcE2, l-citrulline by ArcE1. l-Citrulline excretion during l-arginine breakdown is proposed to be catalyzed by ArcD and/or ArcE2 through l-arginine/l-citrulline exchange.IMPORTANCELactobacillus brevis, a bacterium isolated from wine, as well as other food environments, expresses a catabolic pathway for the breakdown of l-citrulline in the medium that consists of the l-citrulline/l-ornithine exchanger ArcE1 and part of the catabolic arginine deiminase (ADI) pathway enzymes. The proposed pathways for l-arginine and l-citrulline breakdown provide a mechanism for l-citrulline accumulation in fermented food products that is the precursor of the carcinogen ethyl carbamate.

Convergent evolution of the arginine deiminase pathway: the ArcD and ArcE arginine/ornithine exchangers.

The arginine deiminase (ADI) pathway converts L-arginine into L-ornithine and yields 1 mol of ATP per mol of L-arginine consumed. The L-arginine/L-ornithine exchanger in the pathway takes up L-arginine and excretes L-ornithine from the cytoplasm. Analysis of the genomes of 1281 bacterial species revealed the presence of 124 arc gene clusters encoding the pathway. About half of the clusters contained the gene encoding the well-studied L-arginine/L-ornithine exchanger ArcD, while the other half contained a gene, termed here arcE, encoding a membrane protein that is not a homolog of ArcD. The arcE gene product of Streptococcus pneumoniae was shown to take up L-arginine and L-ornithine with affinities of 0.6 and 1 µmol/L, respectively, and to catalyze metabolic energy-independent, electroneutral exchange. ArcE of S. pneumoniae could replace ArcD in the ADI pathway of Lactococcus lactis and provided the cells with a growth advantage. In contrast to ArcD, ArcE catalyzed translocation of the pathway intermediate L-citrulline with high efficiency. A short version of the ADI pathway is proposed for L-citrulline catabolism and the presence of the evolutionary unrelated arcD and arcE genes in different organisms is discussed in the context of the evolution of the ADI pathway.

Structure and elevator mechanism of the Na+-citrate transporter CitS.

The recently determined crystal structure of the bacterial Na+-citrate symporter CitS provides unexpected structural and mechanistic insights. The protein has a fold that has not been seen in other proteins, but the oligomeric state, domain organization and proposed transport mechanism strongly resemble those of the sodium-dicarboxylate symporter vcINDY, and the putative exporters YdaH and MtrF, thus hinting at convergence in structure and function. CitS and the related proteins are predicted to translocate their substrates by an elevator-like mechanism, in which a compact transport domain slides up and down through the membrane while the dimerization domain is stably anchored. Here we review the large body of available biochemical data on CitS in the light of the new crystal structure. We show that the biochemical data are fully consistent with the proposed elevator mechanism, but also demonstrate that the current structural data cannot explain how strict coupling of citrate and Na+ transport is achieved. We propose a testable model for the coupling mechanism.

ArcD1 and ArcD2 Arginine/Ornithine Exchangers Encoded in the Arginine Deiminase Pathway Gene Cluster of Lactococcus lactis.

UNLABELLED: The arginine deiminase (ADI) pathway gene cluster in Lactococcus lactis contains two copies of a gene encoding an l-arginine/l-ornithine exchanger, the arcD1 and arcD2 genes. The physiological function of ArcD1 and ArcD2 was studied by deleting the two genes. Deletion of arcD1 resulted in loss of the growth advantage observed in the presence of high l-arginine in different growth media. Uptake of l-arginine and l-ornithine by resting cells was reduced to the low level observed for an ArcD1/ArcD2 double deletion mutant. Deletion of the arcD2 gene did not affect the growth enhancement, and uptake activities were slightly reduced. Nevertheless, recombinant expression of ArcD2 in the ArcD1/ArcD2 double mutant did recover the growth advantage. Kinetic characterization of ArcD1 and ArcD2 showed high affinities for both l-arginine and l-ornithine (Km in the micromolar range). A difference between the two transporters was the significantly lower affinity of ArcD2 for the cationic amino acids l-ornithine, l-lysine, and l-histidine. In contrast, the affinity of ArcD2 was higher for the neutral amino acid l-alanine. Moreover, ArcD2 efficiently translocated l-alanine, while ArcD1 did not. Both transporters revealed affinities in the mM range for agmatine, cadaverine, histamine, and putrescine. These amines bind but are not translocated. It is concluded that ArcD1 is the main l-arginine/l-ornithine exchanger in the ADI pathway and that ArcD2 is not functionally expressed in the media used. ArcD2 is proposed to function together with the arcT gene that encodes a putative transaminase and is found adjacent to the arcD2 gene. IMPORTANCE: The arginine deiminase (ADI) pathway gene cluster in Lactococcus lactis contains two copies of a gene encoding an l-arginine/l-ornithine exchanger, the arcD1 and arcD2 genes. The physiological function of ArcD1 and ArcD2 was studied by deleting the two genes. It is concluded that ArcD1 is the main l-arginine/l-ornithine exchanger in the ADI pathway. ArcD2 is proposed to function as a l-arginine/l-alanine exchanger in a pathway together with the arcT gene, which is found adjacent to the arcD2 gene in the ADI gene cluster.

The Hill analysis and co-ion-driven transporter kinetics.

Interaction of multiple ligands with a protein or protein complex is a widespread phenomenon that allows for cooperativity. Here, we review the use of the Hill equation, which is commonly used to analyze binding or kinetic data, to analyze the kinetics of ion-coupled transporters and show how the mechanism of transport affects the Hill coefficient. Importantly, the Hill analysis of ion-coupled transporters can provide the exact number of transported co-ions, regardless of the extent of the cooperativity in ion binding.

Physiology and substrate specificity of two closely related amino acid transporters, SerP1 and SerP2, of Lactococcus lactis.

The serP1 and serP2 genes found adjacently on the chromosome of Lactococcus lactis strains encode two members of the amino acid-polyamine-organocation (APC) superfamily of secondary transporters that share 61% sequence identity. SerP1 transports L-serine, L-threonine, and L-cysteine with high affinity. Affinity constants (Km) are in the 20 to 40 µM range. SerP2 is a DL-alanine/DL-serine/glycine transporter. The preferred substrate appears to be DL-alanine for which the affinities were found to be 38 and 20 µM for the D and L isomers, respectively. The common substrate L-serine is a high-affinity substrate of SerP1 and a low-affinity substrate of SerP2 with affinity constants of 18 and 356 µM, respectively. Growth experiments demonstrate that SerP1 is the main L-serine transporter responsible for optimal growth in media containing free amino acids as the sole source of amino acids. SerP2 is able to replace SerP1 in this role only in medium lacking the high-affinity substrates L-alanine and glycine. SerP2 plays an adverse role for the cell by being solely responsible for the uptake of toxic D-serine. The main function of SerP2 is in cell wall biosynthesis through the uptake of D-alanine, an essential precursor in peptidoglycan synthesis. SerP2 has overlapping substrate specificity and shares 42% sequence identity with CycA of Escherichia coli, a transporter whose involvement in peptidoglycan synthesis is well established. No evidence was obtained for a role of SerP1 and SerP2 in the excretion of excess amino acids during growth of L. lactis on protein/peptide-rich media.

Identification of CDP-archaeol synthase, a missing link of ether lipid biosynthesis in Archaea.

Archaeal membrane lipid composition is distinct from Bacteria and Eukarya, consisting of isoprenoid chains etherified to the glycerol carbons. Biosynthesis of these lipids is poorly understood. Here we identify and characterize the archaeal membrane protein CDP-archaeol synthase (CarS) that catalyzes the transfer of the nucleotide to its specific archaeal lipid substrate, leading to the formation of a CDP-activated precursor (CDP-archaeol) to which polar head groups are attached. The discovery of CarS enabled reconstitution of the entire archaeal lipid biosynthesis pathway in vitro, starting from simple isoprenoid building blocks and using a set of five purified enzymes. The cell free synthetic strategy for archaeal lipids we describe opens opportunity for studies of archaeal lipid biochemistry. Additionally, insights into archaeal lipid biosynthesis reported here allow addressing the evolutionary hypothesis of the lipid divide between Archaea and Bacteria.

Ca2+-citrate uptake and metabolism in Lactobacillus casei ATCC 334.

The putative citrate metabolic pathway in Lactobacillus casei ATCC 334 consists of the transporter CitH, a proton symporter of the citrate-divalent metal ion family of transporters CitMHS, citrate lyase, and the membrane-bound oxaloacetate decarboxylase complex OAD-ABDH. Resting cells of Lactobacillus casei ATCC 334 metabolized citrate in complex with Ca(2+) and not as free citrate or the Mg(2+)-citrate complex, thereby identifying Ca(2+)-citrate as the substrate of the transporter CitH. The pathway was induced in the presence of Ca(2+) and citrate during growth and repressed by the presence of glucose and of galactose, most likely by a carbon catabolite repression mechanism. The end products of Ca(2+)-citrate metabolism by resting cells of Lb. casei were pyruvate, acetate, and acetoin, demonstrating the activity of the membrane-bound oxaloacetate decarboxylase complex OAD-ABDH. Following pyruvate, the pathway splits into two branches. One branch is the classical citrate fermentation pathway producing acetoin by α-acetolactate synthase and α-acetolactate decarboxylase. The other branch yields acetate, for which the route is still obscure. Ca(2+)-citrate metabolism in a modified MRS medium lacking a carbohydrate did not significantly affect the growth characteristics, and generation of metabolic energy in the form of proton motive force (PMF) was not observed in resting cells. In contrast, carbohydrate/Ca(2+)-citrate cometabolism resulted in a higher biomass yield in batch culture. However, also with these cells, no generation of PMF was associated with Ca(2+)-citrate metabolism. It is concluded that citrate metabolism in Lb. casei is beneficial when it counteracts acidification by carbohydrate metabolism in later growth stages.

Biochemical and genetic characterization of the Enterococcus faecalis oxaloacetate decarboxylase complex.

Enterococcus faecalis encodes a biotin-dependent oxaloacetate decarboxylase (OAD), which is constituted by four subunits: E. faecalis carboxyltransferase subunit OadA (termed Ef-A), membrane pump Ef-B, biotin acceptor protein Ef-D, and the novel subunit Ef-H. Our results show that in E. faecalis, subunits Ef-A, Ef-D, and Ef-H form a cytoplasmic soluble complex (termed Ef-AHD) which is also associated with the membrane. In order to characterize the role of the novel Ef-H subunit, coexpression of oad genes was performed in Escherichia coli, showing that this subunit is vital for Ef-A and Ef-D interaction. Diminished growth of the oadA and oadD single deletion mutants in citrate-supplemented medium indicated that the activity of the complex is essential for citrate utilization. Remarkably, the oadB-deficient strain was still capable of growing to wild-type levels but with a delay during the citrate-consuming phase, suggesting that the soluble Ef-AHD complex is functional in E. faecalis. These results suggest that the Ef-AHD complex is active in its soluble form, and that it is capable of interacting in a dynamic way with the membrane-bound Ef-B subunit to achieve its maximal alkalinization capacity during citrate fermentation.

Genome Sequence of Lactobacillus saerimneri 30a (Formerly Lactobacillus sp. Strain 30a), a Reference Lactic Acid Bacterium Strain Producing Biogenic Amines.

Lactobacillus sp. strain 30a (Lactobacillus saerimneri) produces the biogenic amines histamine, putrescine, and cadaverine by decarboxylating their amino acid precursors. We report its draft genome sequence (1,634,278 bases, 42.6% G+C content) and the principal findings from its annotation, which might shed light onto the enzymatic machineries that are involved in its production of biogenic amines.

Three-component lysine/ornithine decarboxylation system in Lactobacillus saerimneri 30a.

Lactic acid bacteria play a pivotal role in many food fermentations and sometimes represent a health threat due to the ability of some strains to produce biogenic amines that accumulate in foods and cause trouble following ingestion. These strains carry specific enzymatic systems catalyzing the uptake of amino acid precursors (e.g., ornithine and lysine), the decarboxylation inside the cell, and the release of the resulting biogenic amines (e.g., putrescine and cadaverine). This study aimed to identify the system involved in production of cadaverine from lysine, which has not been described to date for lactic acid bacteria. Strain Lactobacillus saerimneri 30a (formerly called Lactobacillus sp. 30a) produces both putrescine and cadaverine. The sequencing of its genome showed that the previously described ornithine decarboxylase gene was not associated with the gene encoding an ornithine/putrescine exchanger as in other bacteria. A new hypothetical decarboxylation system was detected in the proximity of the ornithine decarboxylase gene. It consisted of two genes encoding a putative decarboxylase sharing sequence similarities with ornithine decarboxylases and a putative amino acid transporter resembling the ornithine/putrescine exchangers. The two decarboxylases were produced in Escherichia coli, purified, and characterized in vitro, whereas the transporter was heterologously expressed in Lactococcus lactis and functionally characterized in vivo. The overall data led to the conclusion that the two decarboxylases and the transporter form a three-component decarboxylation system, with the new decarboxylase being a specific lysine decarboxylase and the transporter catalyzing both lysine/cadaverine and ornithine/putrescine exchange. To our knowledge, this is an unprecedented observation of a bacterial three-component decarboxylation system.

Cloning, expression, and functional characterization of secondary amino acid transporters of Lactococcus lactis.

Fourteen genes encoding putative secondary amino acid transporters were identified in the genomes of Lactococcus lactis subsp. cremoris strains MG1363 and SK11 and L. lactis subsp. lactis strains IL1403 and KF147, 12 of which were common to all four strains. Amino acid uptake in L. lactis cells overexpressing the genes revealed transporters specific for histidine, lysine, arginine, agmatine, putrescine, aromatic amino acids, acidic amino acids, serine, and branched-chain amino acids. Substrate specificities were demonstrated by inhibition profiles determined in the presence of excesses of the other amino acids. Four knockout mutants, lacking the lysine transporter LysP, the histidine transporter HisP (formerly LysQ), the acidic amino acid transporter AcaP (YlcA), or the aromatic amino acid transporter FywP (YsjA), were constructed. The LysP, HisP, and FywP deletion mutants showed drastically decreased rates of uptake of the corresponding substrates at low concentrations. The same was observed for the AcaP mutant with aspartate but not with glutamate. In rich M17 medium, the deletion of none of the transporters affected growth. In contrast, the deletion of the HisP, AcaP, and FywP transporters did affect growth in a defined medium with free amino acids as the sole amino acid source. HisP was essential at low histidine concentrations, and AcaP was essential in the absence of glutamine. FywP appeared to play a role in retaining intracellularly synthesized aromatic amino acids when these were not added to the medium. Finally, HisP, AcaP, and FywP did not play a role in the excretion of accumulated histidine, glutamate, or phenylalanine, respectively, indicating the involvement of other transporters.

Uptake of α-ketoglutarate by citrate transporter CitP drives transamination in Lactococcus lactis.

Transamination is the first step in the conversion of amino acids into aroma compounds by lactic acid bacteria (LAB) used in food fermentations. The process is limited by the availability of α-ketoglutarate, which is the best α-keto donor for transaminases in LAB. Here, uptake of α-ketoglutarate by the citrate transporter CitP is reported. Cells of Lactococcus lactis IL1403 expressing CitP showed significant levels of transamination activity in the presence of α-ketoglutarate and one of the amino acids Ile, Leu, Val, Phe, or Met, while the same cells lacking CitP showed transamination activity only after permeabilization of the cell membrane. Moreover, the transamination activity of the cells followed the levels of CitP in a controlled expression system. The involvement of CitP in the uptake of the α-keto donor was further demonstrated by the increased consumption rate in the presence of L-lactate, which drives CitP in the fast exchange mode of transport. Transamination is the only active pathway for the conversion of α-ketoglutarate in IL1403; a stoichiometric conversion to glutamate and the corresponding α-keto acid from the amino acids was observed. The transamination activity by both the cells and the cytoplasmic fraction showed a remarkably flat pH profile over the range from pH 5 to pH 8, especially with the branched-chain amino acids. Further metabolism of the produced α-keto acids into α-hydroxy acids and other flavor compounds required the coupling of transamination to glycolysis. The results suggest a much broader role of the citrate transporter CitP in LAB than citrate uptake in the citrate fermentation pathway alone.

Genomic distribution of the small multidrug resistance protein EmrE over 29 Escherichia coli strains reveals two forms of the protein.

Analysis of the genomes of 29 Escherichia coli strains revealed two different versions of the EmrE protein, a member of the small multidrug resistance family. The versions are different in length and contain 110 residues (EMRE(110)) and 165 residues (EMRE(165)). The N-terminal extension found in the longer sequence has the properties of a signal sequence, i.e. contains at the extreme N-terminus a hydrophobic region followed by a predicted cleavage site. Analysis of the genetic context of the genes in the different strains showed that all of the genes encoding EMRE(165) had the same context, whereas the genes encoding EMRE(110) were distributed over four different, but similar, contexts. The different genetic contexts corresponded to the branching of the phylogenetic tree of the emrE genes. Membrane topology studies using translational fusions with the two reporter proteins alkaline phosphatase and green fluorescent protein showed the well-described dual topology mode of insertion of EMRE(110). In contrast, but in line with the presence of the signal sequence, EMRE(165) was inserted in a single orientation into the membrane, with the C-terminus in the periplasm. The N-terminal region was removed from the protein after insertion into the membrane. In contrast to cells expressing EMRE(110), cells expressing only mature EMRE(165) were not able to grow on plates containing ethidium bromide. The reults suggest that if dimers were formed from EMRE(165) monomers with the same orientation in the membrane, they would not be active in drug extrusion.

Role of individual positive charges in the membrane orientation and activity of transporters of the small multidrug resistance family.

The effect of individual positively charged residues on the orientation in the membrane was analyzed in three dual-topology transporters of the small multidrug resistance (SMR) family: AAVE4701aave of Acidovorax avenae, EMREecol of Escherichia coli, and RRUA0272rrub of Rhodospirillum rubrum. It is shown that (i) individual positive charges have different impacts on the orientation, (ii) positive charges that are conserved in the three different proteins do not have the same impact on the orientation, (iii) positive charges in odd- and even-numbered loops have different impacts, (iv) for some, but not all, the impact depends on the presence of other positive charges, and (v) proteins from which all positive charges are removed in some cases are dual-topology proteins and in other cases have a single orientation. A small number of positive charges placed in the loops of the latter proteins results in the violation of the so-called positive-inside rule that has been reported previously [Kolbusz, M. A., et al. (2010) J. Mol. Biol. 402, 127-138]. We conclude that each positive charge shifts the distribution between the two orientations toward the state that has the positive charge in the cytoplasm but that intrinsic factors other than positive charges determine the orientation as well. The ability of the mutants of AAVE4701aave and EMREecol to confer resistance against ethidium bromide revealed an essential role in catalysis for a conserved pair of positive charges in the second loop. No significant relation between activity and the relative orientation of the monomeric subunits in the dimer could be demonstrated.

Rerouting citrate metabolism in Lactococcus lactis to citrate-driven transamination.

Oxaloacetate is an intermediate of the citrate fermentation pathway that accumulates in the cytoplasm of Lactococcus lactis ILCitM(pFL3) at a high concentration due to the inactivation of oxaloacetate decarboxylase. An excess of toxic oxaloacetate is excreted into the medium in exchange for citrate by the citrate transporter CitP (A. M. Pudlik and J. S. Lolkema, J. Bacteriol. 193:4049-4056, 2011). In this study, transamination of amino acids with oxaloacetate as the keto donor is described as an additional mechanism to relieve toxic stress. Redirection of the citrate metabolic pathway into the transamination route in the presence of the branched-chain amino acids Ile, Leu, and Val; the aromatic amino acids Phe, Trp, and Tyr; and Met resulted in the formation of aspartate and the corresponding α-keto acids. Cells grown in the presence of citrate showed 3.5 to 7 times higher transaminase activity in the cytoplasm than cells grown in the absence of citrate. The study demonstrates that transaminases of L. lactis accept oxaloacetate as a keto donor. A significant fraction of 2-keto-4-methylthiobutyrate formed from methionine by citrate-driven transamination in vivo was further metabolized, yielding the cheese aroma compounds 2-hydroxy-4-methylthiobutyrate and methyl-3-methylthiopropionate. Reducing equivalents required for the former compound were produced in the citrate fermentation pathway as NADH. Similarly, phenylpyruvate, the transamination product of phenylalanine, was reduced to phenyllactate, while the dehydrogenase activity was not observed for the branched-chain keto acids. Both α-keto acids and α-hydroxy acids are known substrates of CitP and may be excreted from the cell in exchange for citrate or oxaloacetate.

Substrate specificity of the citrate transporter CitP of Lactococcus lactis.

The citrate transporter CitP of lactic acid bacteria catalyzes electrogenic precursor-product exchange of citrate versus L-lactate during citrate-glucose cometabolism. In the absence of sugar, L-lactate is replaced by the metabolic intermediates/end products pyruvate, α-acetolactate, and acetate. In this study, the binding and translocation properties of CitP were analyzed systematically for a wide variety of mono- and dicarboxylates of the form X-CR(2)-COO(-), where X represents OH (2-hydroxy acid), O (2-keto acid), or H (acid) and R groups differ in size, hydrophobicity, and composition. It follows that CitP is a very promiscuous carboxylate transporter. A carboxylate group is both essential and sufficient for recognition by the transporter. A C-2 atom is not essential, formate is a substrate, and C-2 may be part of a ring structure, as in benzoate. The R group may be as bulky as an indole ring structure. For all monocarboxylates of the form X-CHR-COO(-), the hydroxy (X = OH) analogs were the preferred substrates. The preference for keto (X = O) or acid (X = H) analogs was dependent on the bulkiness of the R group, such that the acid was preferred for small R groups and the 2-ketoacid was preferred for more bulky R groups. The C(4) to C(6) dicarboxylates succinate, glutarate, and adipate were also substrates of CitP. The broad substrate specificity is discussed in the context of a model of the binding site of CitP. Many of the substrates of CitP are intermediates or products of amino acid metabolism, suggesting that CitP may have a broader physiological function than its role in citrate fermentation alone.

Improved acid stress survival of Lactococcus lactis expressing the histidine decarboxylation pathway of Streptococcus thermophilus CHCC1524.

Degradative amino acid decarboxylation pathways in bacteria generate secondary metabolic energy and provide resistance against acid stress. The histidine decarboxylation pathway of Streptococcus thermophilus CHCC1524 was functionally expressed in the heterologous host Lactococcus lactis NZ9000, and the benefits of the newly acquired pathway for the host were analyzed. During growth in M17 medium in the pH range of 5-6.5, a small positive effect was observed on the biomass yield in batch culture, whereas no growth rate enhancement was evident. In contrast, a strong benefit for the engineered L. lactis strain was observed in acid stress survival. In the presence of histidine, the pathway enabled cells to survive at pH values as low as 3 for at least 2 h, conditions under which the host cells were rapidly dying. The flux through the histidine decarboxylation pathway in cells grown at physiological pH was under strict control of the electrochemical proton gradient (pmf) across the membrane. Ionophores that dissipated the membrane potential (ΔΨ) and/or the pH gradient (ΔpH) strongly increased the flux, whereas the presence of glucose almost completely inhibited the flux. Control of the pmf over the flux was exerted by both ΔΨ and ΔpH and was distributed over the transporter HdcP and the decarboxylase HdcA. The control allowed for a synergistic effect between the histidine decarboxylation and glycolytic pathways in acid stress survival. In a narrow pH range around 2.5 the synergism resulted in a 10-fold higher survival rate.

Evidence of two functionally distinct ornithine decarboxylation systems in lactic acid bacteria.

Biogenic amines are low-molecular-weight organic bases whose presence in food can result in health problems. The biosynthesis of biogenic amines in fermented foods mostly proceeds through amino acid decarboxylation carried out by lactic acid bacteria (LAB), but not all systems leading to biogenic amine production by LAB have been thoroughly characterized. Here, putative ornithine decarboxylation pathways consisting of a putative ornithine decarboxylase and an amino acid transporter were identified in LAB by strain collection screening and database searches. The decarboxylases were produced in heterologous hosts and purified and characterized in vitro, whereas transporters were heterologously expressed in Lactococcus lactis and functionally characterized in vivo. Amino acid decarboxylation by whole cells of the original hosts was determined as well. We concluded that two distinct types of ornithine decarboxylation systems exist in LAB. One is composed of an ornithine decarboxylase coupled to an ornithine/putrescine transmembrane exchanger. Their combined activities results in the extracellular release of putrescine. This typical amino acid decarboxylation system is present in only a few LAB strains and may contribute to metabolic energy production and/or pH homeostasis. The second system is widespread among LAB. It is composed of a decarboxylase active on ornithine and l-2,4-diaminobutyric acid (DABA) and a transporter that mediates unidirectional transport of ornithine into the cytoplasm. Diamines that result from this second system are retained within the cytosol.