Proton motive force-driven and ATP-dependent drug extrusion systems in multidrug-resistant Lactococcus lactis.

Three mutants of Lactococcus lactis subsp. lactis MG1363, termed EthR, DauR, and RhoR, were selected for resistance to high concentrations of ethidium bromide, daunomycin, and rhodamine 6G, respectively. These mutants were found to be cross resistant to a number of structurally and functionally unrelated drugs, among which were typical substrates of the mammalian multidrug transporter (P-glycoprotein) such as daunomycin, quinine, actinomycin D, gramicidin D, and rhodamine 6G. The three multidrug-resistant strains showed an increased rate of energy-dependent ethidium and daunomycin efflux compared with that of the wild-type strain. This suggests that resistance to these toxic compounds is at least partly due to active efflux. Efflux of ethidium from the EthR strain could occur against a 37-fold inwardly directed concentration gradient. In all strains, ethidium efflux was inhibited by reserpine, a well-known inhibitor of P-glycoprotein. Ionophores which selectively dissipate the membrane potential or the pH gradient across the membrane inhibited ethidium and daunomycin efflux in the wild-type strain, corresponding with a proton motive force-driven efflux system. The ethidium efflux system in the EthR strain, on the other hand, was inhibited by ortho-vanadate and not upon dissipation of the proton motive force, which suggests the involvement of ATP in the energization of transport. The partial inhibition of ethidium efflux by ortho-vanadate and nigericin in the DauR and RhoR strains suggest that a proton motive force-dependent and an ATP-dependent system are expressed simultaneously. This is the first report of an ATP-dependent transport system in prokaryotes which confers multidrug resistance to the organism.     

Multidrug transporters and antibiotic resistance in Lactococcus lactis

A novel bioactive lipid, cyclic phosphatidic acid (cPA), was isolated originally from myxoamoebae of a true slime mold, Physarum polycephalum, and has now been detected in a wide range of organisms from slime molds to humans. It has a cyclic phosphate at the sn-2 and sn-3 positions of the glycerol carbons, and this structure is absolutely necessary for its activities. This substance shows specific biological functions, including antimitogenic regulation of the cell cycle, regulation of actin stress fiber formation and rearrangement, inhibition of cancer cell invasion and metastasis, regulation of differentiation and viability of neuronal cells, and mobilization of intracellular calcium. Although the structure of cPA is similar to that of lysophosphatidic acid (LPA), its biological activities are apparently distinct from those of LPA. In the present review, we focus mainly on the enzymatic formation of cPA, the antimitogenic regulation of the cell cycle, the inhibition of cancer cell invasion and metastasis, and the neurotrophic effect of cPA.     

Cholate resistance in Lactococcus lactis is mediated by an ATP-dependent multispecific organic anion transporter

The cholate-resistant Lactococcus lactis strain C41-2, derived from wild-type L. lactis MG1363 through selection for growth on cholate-containing medium, displayed a reduced accumulation of cholate due to an enhanced active efflux. However, L. lactis C41-2 was not cross resistant to deoxycholate or cationic drugs, such as ethidium and rhodamine 6G, which are typical substrates of the multidrug transporters LmrP and LmrA in L. lactis MG1363. The cholate efflux activity in L. lactis C41-2 was not affected by the presence of valinomycin plus nigericin, which dissipated the proton motive force. In contrast, cholate efflux in L. lactis C41-2 was inhibited by ortho-vanadate, an inhibitor of P-type ATPases and ATP-binding cassette transporters. Besides ATP-dependent drug extrusion by LmrA, two other ATP-dependent efflux activities have previously been detected in L. lactis, one for the artificial pH probe 2',7'-bis-(2-carboxyethyl)-5(and 6)-carboxyfluorescein (BCECF) and the other for the artificial pH probe N-(fluorescein thio-ureanyl)-glutamate (FTUG). Surprisingly, the efflux rate of BCECF, but not that of FTUG, was significantly enhanced in L. lactis C41-2. Further experiments with L. lactis C41-2 cells and inside out membrane vesicles revealed that cholate and BCECF inhibit the transport of each other. These data demonstrate the role of an ATP-dependent multispecific organic anion transporter in cholate resistance in L. lactis.     

Ofloxacin-resistant Pseudomonas aeruginosa mutants with elevated drug extrusion across the inner membrane.

We selected the quinolone-resistant mutants from the protein F deficient Pseudomonas aeruginosa. The mutants showed cross-resistance to tetracycline, minocycline and chloramphenicol, but not to the beta-lactam antibiotics. The MIC of ofloxacin (OFLX) against the mutants, but not in the parent, became 2 to 4 times lower as the medium pH was raised from 6.5 to 8.5. The mutants accumulated about half as much OFLX as the parent. The OFLX accumulation in the mutants increased 6.5- to 8-fold in the presence of 100 mM carbonyl cyanide m-chlorophenylhydrazone, while that in the parent was 4-fold. The OFLX sensitivity of the mutant DNA-gyrase was comparable with that of the parent's enzyme. These results suggest that the resistance of these mutants to OFLX is associated with the membrane potential dependent drug efflux.     

Characterization of N-deoxyribosyltransferase from Lactococcus lactis subsp. lactis

A nucleoside N-deoxyribosyltransferase-homologous gene was detected by homological search in the genomic DNA of Lactococcus lactis subsp. lactis. The gene yejD is composed of 477 nucleotides encoding 159 amino acids with only 25% identity, which is low in comparison to the amino acid sequences of the N-deoxyribosyltransferases from other lactic acid bacteria, i.e. Lactobacillus leichmannii and Lactobacillus helveticus. The residues responsible for catalytic and substrate-binding sites in known enzymes are conserved at Gln49, Asp73, Asp93 (or Asp95), and Glu101, respectively. The recombinant YejD expressed in Escherichia coli shows a 2-deoxyribosyl transfer activity to and from both bases of purine and pyrimidine, showing that YejD should be categorized as a class II N-deoxyribosyltransferase. Interestingly, the base-exchange activity as well as the heat stability of YejD was enhanced by the presence of monovalent cations such as K(+), NH(4)(+), and Rb(+), indicating that the Lactococcus enzyme is a K(+)-activated Type II enzyme. However, divalent cations including Mg(2+) and Ca(2+) significantly inhibit the activity. Whether or not the yejD gene product actually participates in the nucleoside salvage pathway of Lc. lactis remains unclear, but the lactic acid bacterium possesses the gene coding for the nucleoside N-deoxyribosyltransferase activated by K(+) on its genome.     

Lactococcus lactis as host for overproduction of functional membrane proteins

Lactococcus lactis has many properties that are ideal for enhanced expression of membrane proteins. The organism is easy and inexpensive to culture, has a single membrane and relatively mild proteolytic activity. Methods for genetic manipulation are fully established and a tightly controlled promoter system is available, with which the level of expression can be varied with the inducer concentration. Here we describe our experiences with lactococcal expression of the mechanosensitive channel, the human KDEL receptor and transporters belonging to the ABC transporter family, the major facilitator superfamily, the mitochondrial carrier family and the peptide transporter family. Previously published expression studies only deal with the overexpression of prokaryotic membrane proteins, but in this paper, experimental data are presented for the overproduction of mitochondrial and hydrogenosomal carriers and the human KDEL receptor. These eukaryotic membrane proteins were expressed in a functional form and at levels amenable to structural work.     

Comparison of cell wall proteinases from Lactococcus lactis subsp. cremoris AC1 and Lactococcus lactis subsp. lactis NCDO 763

Summary Cell wall-associated proteinases were isolated from Lactococcus lactis subsp. cremoris AC1 and subsp. lactis NCDO 763 in order to compare their specificities towards different caseins. Two purification strategies were applied. Cells grown in casein-free M17 medium were a suitable starting material for purification, since electrophoretic purity could be achieved after one chromatographic step. Both enzymes has an apparent molecular mass of about 145000 daltons as judged by sodium dodecyl sulphate-polyacrylamide gel electrophoresis. Electrophoresis and reversed phase HPLC patterns of hydrolysates of _s1-, _s2-, -, and K-caseins indicated that both proteinases had a similar specificity. The enzyme of L. lactis subsp. lactis split _s1- and _s2-caseins more extensively than that of L. lactis subsp. cremoris.     

Comparison of cell wall proteinases from Lactococcus lactis subsp. cremoris AC1 and Lactococcus lactis subsp. lactis NCDO 763

Summary The cell wall proteinases of Lactococcus lactis subsp. lactis NCDO 763 and L. lactis subsp. cremoris AC1 hydrolyse -casein with a similar specificity even though some quantitative differences can be observed for a few degradation products analysed by reverse phase HPLC and sodium dodecyl sulphate-polyacrylamide gel electrophoresis. The main peptides soluble in 1.1% trifluoroacetic acid and liberated by the two proteinases were identified and have been found to be the same for the two enzymes. They are located in two areas of the -casein sequence (53–93 and the C-terminal part: 129–209) and they include bitter tasting or physiologically active fragments. No narrow specificity was observed for these proteinases. However, glutamine and serine residues are more frequently encountered in position P1 and P1 of the sensitive peptide bond and the close environment (position P2 to P4 and P2 to P4) of the cleaved bond is mainly hydrophobic.     

Genetic evidence for a defective xylan degradation pathway in Lactococcus lactis

Genetic and biochemical evidence for a defective xylan degradation pathway was found linked to the xylose operon in three lactococcal strains, Lactococcus lactis 210, L. lactis IO-1, and L. lactis NRRL B-4449. Immediately downstream of the xylulose kinase gene (xylB) (K. A. Erlandson, J.-H. Park, W. El Khal, H.-H. Kao, P. Basaran, S. Brydges, and C. A. Batt, Appl. Environ. Microbiol. 66:3974-3980, 1999) are two open reading frames encoding a mutarotase (xylM) and a xyloside transporter (xynT) and a partial open reading frame encoding a beta-xylosidase (xynB). These are functions previously unreported for lactococci or lactobacilli. The mutarotase activity of the putative xylM gene product was confirmed by overexpression of the L. lactis enzyme in Escherichia coli and purification of recombinant XylM. We hypothesize that the mutarotase links xylan degradation to xylose metabolism due to the anomeric preference of xylose isomerase. In addition, Northern hybridization experiments suggested that the xylM and xynTB genes are cotranscribed with the xylRAB genes, responsible for xylose metabolism. Although none of the three strains appeared to metabolize xylan or xylobiose, they exhibited xylosidase activity, and L. lactis IO-1 and L. lactis NRRL B-4449 had functional mutarotases.     

Exopolysaccharide expression in Lactococcus lactis subsp. cremoris Ropy352: evidence for novel gene organization

Lactococcus lactis subsp. cremoris Ropy352 produces two distinct heteropolysaccharides, phenotypically described as ropy and mucoid, when cultured in nonfat milk. One exopolysaccharide precipitated with 50% ethanol as a series of elongated threads and was composed of glucose and galactose in a molar ratio of 3:2. The second exopolysaccharide precipitated with 75% ethanol as a fine flocculant and consisted of galactose, glucose, and mannose with a molar ratio of 67:21:12. A mutant strain, L. lactis subsp. cremoris EK240, lacking the ropy phenotype did not produce the exopolysaccharide that precipitated with 50% ethanol; however, it produced the exopolysaccharide that precipitated with 75% ethanol, indicating that the former exopolysaccharide is essential for the ropy phenotype. Cultures of L. lactis subsp. cremoris Ropy352 in 10% nonfat milk reached a viscosity of 25 Pa-s after 24 h, while those of the nonropy L. lactis subsp. cremoris EK240 mutant did not change. A mutation abolishing ropy exopolysaccharide expression mapped to a region on a plasmid containing two open reading frames, epsM and epsN, encoding novel glycosyltransferases bordered by ISS1 elements oriented in the same direction. Sequencing of this plasmid revealed two other regions involved in exopolysaccharide expression, an operon located between partial IS981 and IS982 elements, and an independent gene, epsU. Two and possibly three of these regions are involved in L. lactis subsp. cremoris Ropy352 exopolysaccharide expression and are arranged in a novel fashion different from that of typical lactococcal exopolysaccharide loci, and this provides genetic evidence for exopolysaccharide gene reorganization and evolution in Lactococcus.     

Functional expression of eukaryotic membrane proteins in Lactococcus lactis

The overproduction of eukaryotic membrane proteins is a major impediment in their structural and functional characterization. Here we have used the nisin-inducible expression system of Lactococcus lactis for the overproduction of 11 mitochondrial transport proteins from yeast. They were expressed at high levels in a functional state in the cytoplasmic membrane. The results also show that the level of expression is influenced by the N-terminal regions of the transporters. Expression levels were improved >10-fold either by replacing or truncating these regions or by adding lactococcal signal peptides. The observed expression levels are now compatible with a realistic exploration of crystallization conditions. The lactococcal expression system may be used for the high-throughput functional characterization of eukaryotic membrane proteins and structural genomics.     

Identification of four phage resistance plasmids from Lactococcus lactis subsp. cremoris HO2

The bacteriophage-host sensitivity patterns of 16 strains of Lactococcus lactis originally isolated from a mixed strain Cheddar cheese starter culture were determined. Using phages obtained from cheese factory whey, four of the strains were found to be highly phage resistant. One of these isolates, Lactococcus lactis subsp. cremoris HO2, was studied in detail to determine the mechanisms responsible for the phage insensitivity phenotypes. Conjugal transfer of plasmid DNA from strain HO2 allowed a function to be assigned to four of its six plasmids. A 46-kb molecule, designated pCI646, was found to harbor the lactose utilization genes, while this and plasmids of 58 kb (pCI658), 42 kb (pCI642), and 4.5 kb (pCI605) were shown to be responsible for the phage resistance phenotypes observed against the small isometric-headed phage phi712 (936 phage species) and the prolate-headed phage phic2 (c2 species). pCI658 was found to mediate an adsorption-blocking mechanism and was also responsible for the fluffy pellet phenotype of cells containing the molecule. pCI642 and pCI605 were both shown to be required for the operation of a restriction-modification system.     

Isolation and characterization of nisin-producing Lactococcus lactis subsp. lactis from bean-sprouts

Bacterial isolates from bean-sprouts were screened for anti- Listeria monocytogenes bacteriocins using a well diffusion method. Thirty-four of 72 isolates inhibited the growth of L.monocytogenes Scott A. One, HPB 1688, which had the biggest inhibition zone against L.monocytogenes Scott A, was selected for subsequent analysis. Both ribotyping and DNAsequencing of 16S ribosomal RNA gene demonstrated that the isolate was Lactococcus lactis subsp. lactis . Polymerase chain reaction and nucleotide sequencing revealed that thegenomic DNA of the bean-sprout isolates contained a nisin Z structural gene. In MRS broth,bean-sprout isolate HPB 1688 survived at 3–4·5°C for at least 20 d, grew at 4°Cand produced anti-listerial compoundsat 5°C. When co-cultured with L. monocytogenes in MRS broth, the isolate inhibited thegrowth of L. monocytogenes at 4°C after 14d and at 10°C after 2 d. When co-inoculatedwith 10²cells g⁻¹ of L.monocytogenes on fresh-cut ready-to-eat Caesar salad, L. lactis subsp. lactis (10⁸cells g⁻¹) was able to reduce the number of L. monocytogenes by 1–1·4 logs after storage for 10 d at 7° and 10°C. A bacteriocin-producing Enterococcusfaecium was also able to reduce the numbers of L. monocytogenes onCaesar salad, butdid not act synergistically when co-inoculated with L. lactis subsp. lactis .     

Intracellular X-prolyl dipeptidyl peptidase from Lactococcus lactis spp. lactis: purification and properties

An X-prolyl dipeptidyl peptidase (EC 3.4.14.5) has been purified from a crude intracellular extract from Lactococcus lactis spp. lactis NCDO 763 by ion-exchange chromatography and gel filtration. One protein band was detected after electrophoresis of the purified enzyme in the presence or absence of sodium dodecyl sulphate. The enzyme is a 190 kDa dimer composed of identical subunits. Optimal activity occurs at pH 8.5 and 40–45°C and the enzyme is inhibited by reagents specific for serine proteases, such as diisopropylfluorophosphate. The enzyme hydrolyzes p -nitroanilide- or β-naphthylamide-substituted X-Pro dipeptides, as well as β-casomorphin.     

A membrane protein is required for bacteriophage c2 infection of Lactococcus lactis subsp. lactis C2.

Phage-resistant mutants, isolated from cultures of Lactococcus lactis subsp. lactis C2 infected with phage c2, did not form plaques but bound phage normally. The mutants were sensitive to another phage, sk1, although the number of plaques was reduced approximately 56% and the plaques were four times smaller. Binding to phage sk1 was reduced about 10%. Another group of phage-resistant mutants, isolated from cultures infected with phage sk1, bound normally to both phages c2 and sk1 but did not form plaques with either phage. Carbohydrate analyses by gas chromatography of the cell walls showed no significant differences in saccharide compositions between the wild-type and phage-resistant cells. However, a difference was observed in the interactions of the phage with the cytoplasmic membranes. Membranes from the wild-type cells, but not mutant cells, inactivated phage c2. Phage sk1 was not inactivated by membrane from either strain. Treatment of wild-type membranes with proteinase K eliminated the ability of the membrane to inactivate the phage, whereas treatment with mutanolysin had no effect. On the basis of this ability to inactivate the phage, a membrane protein was partially purified by gel filtration and ion-exchange chromatography. Under nondenaturing conditions, the phage-inactivating protein has an apparent Mr of approximately 350,000. The protein has an apparent subunit size of 32 kDa, which suggests that it normally exists as a multimer with 10 to 12 subunits or in association with other membrane components. It is proposed that this protein is required for phage c2 infection.     

Multidrug resistance: Versatile drug sensors of bacterial cells.

One way that bacteria resist antimicrobial agents involves 'drug sensors', proteins that bind a range of structurally unrelated antimicrobial drugs and induce expression of multidrug resistance pumps. The basis for drug recognition is now becoming clear from the recently determined crystal structure of a drug sensor.     

Mechanism and energetics of dipeptide transport in membrane vesicles of Lactococcus lactis.

Alanyl-alpha-glutamate transport has been studied in Lactococcus lactis ML3 cells and in membrane vesicles fused with liposomes containing beefheart cytochrome c oxidase as a proton-motive-force-generating system. The uptake of Ala-Glu observed in de-energized cells can be stimulated 26-fold upon addition of lactose. No intracellular dipeptide pool could be detected in intact cells. In fused membranes, a 40-fold accumulation of Ala-Glu was observed in response to a proton motive force. Addition of ionophores and uncouplers resulted in a rapid efflux of the accumulated dipeptide, indicating that Ala-Glu accumulation is directly coupled to the proton motive force as a driving force. Ala-Glu uptake is an electrogenic process and the dipeptide is transported in symport with two protons. In both fused membranes and intact cells the same affinity constant (0.70 mM) for Ala-Glu uptake was found. Accumulated Ala-Glu is exchangeable with externally added alanyl-glutamate, glutamyl-glutamate, and leucyl-leucine, while no exchange occurred upon addition of the amino acid glutamate or alanine. These results indicate that the Ala-Glu transport system has a broad substrate specificity.