Lactococci

Appl Environ Microbiol, 2005 Jan, 71(1), 303 - 11
Identification, Cloning, and Characterization of a Lactococcus lactis Branched-Chain {alpha}-Keto Acid Decarboxylase Involved in Flavor Formation; Smit BA et al.; The biochemical pathway for formation of branched-chain aldehydes, which are important flavor compounds derived from proteins in fermented dairy products, consists of a protease, peptidases, a transaminase, and a branched-chain alpha-keto acid decarboxylase (KdcA) . The activity of the latter enzyme has been found only in a limited number of Lactococcus lactis strains . By using a random mutagenesis approach, the gene encoding KdcA in L . lactis B1157 was identified . The gene for this enzyme is highly homologous to the gene annotated ipd, which encodes a putative indole pyruvate decarboxylase, in L . lactis IL1403 . Strain IL1403 does not produce KdcA, which could be explained by a 270-nucleotide deletion at the 3' terminus of the ipd gene encoding a truncated nonfunctional decarboxylase . The kdcA gene was overexpressed in L . lactis for further characterization of the decarboxylase enzyme . Of all of the potential substrates tested, the highest activity was observed with branched-chain alpha-keto acids . Moreover, the enzyme activity was hardly affected by high salinity, and optimal activity was found at pH 6.3, indicating that the enzyme might be active under cheese ripening conditions.

Microb Cell Fact . 2005 Jan 4;4(1):2 {Epub ahead of print}
Protein secretion in Lactococcus lactis: an efficient way to increase the overall heterologous protein production; Le Loir Y et al.; Lactococcus lactis, the model lactic acid bacterium (LAB), is a food grade and well-characterized Gram positive bacterium . It is a good candidate for heterologous protein delivery in foodstuff or in the digestive tract . L . lactis can also be used as a protein producer in fermentor . Many heterologous proteins have already been produced in L . lactis but only few reports allow comparing production yields for a given protein either produced intracellularly or secreted in the medium . Here, we review several works evaluating the influence of the localization on the production yields of several heterologous proteins produced in L . lactis . The questions of size limits, conformation, and proteolysis are addressed and discussed with regard to protein yields . These data show that i) secretion is preferable to cytoplasmic production; ii) secretion enhancement (by signal peptide and propeptide optimization) results in increased production yield; iii) protein conformation rather than protein size can impair secretion and thus alter production yields; and iv) fusion of a stable protein can stabilize labile proteins . The role of intracellular proteolysis on heterologous cytoplasmic proteins and precursors is discussed . The new challenges now are the development of food grade systems and the identification and optimization of host factors affecting heterologous protein production not only in L . lactis, but also in other LAB species.

Biofactors, 2004, 22(1-4), 119 - 22
Cytoplasmic fraction of Lactococcus lactis ssp . lactis induces apoptosis in SNU-1 stomach adenocarcinoma cells; Kim SY et al.; Lactic acid bacteria are known to have antitumor activity, but the underlying mechanisms remain unclear . Recently we showed that a cytoplasmic fraction - but not peptidoglycan - of Lactococcus lactis ssp . lactis (L.lac CF) had strong antiproliferative activity on SNU-1 human stomach adenocarcinoma cells . The present study investigated whether the antiproliferative activity of L.lac CF on SNU-1 is linked to the induction of apoptosis . Treatment of L.lac CF inhibited the proliferation of SNU-1 cells in a dose- and time-dependent manner . Furthermore, treatment of the cells with 50 microg/ml and 100 microg/ml L.lac CF resulted in DNA fragmentation and chromatin condensation, respectively . The results indicate that the inhibitory effect of L.lac CF on SNU-1 cell growth is mainly attributable to the induction of apoptosis.

J Bacteriol, 2005 Jan, 187(2), 601 - 10
Roles of Thioredoxin Reductase during the Aerobic Life of Lactococcus lactis; Vido K et al.; Thiol-disulfide bond balance is generally maintained in bacteria by thioredoxin reductase-thioredoxin and/or glutathione-glutaredoxin systems . Some gram-positive bacteria, including Lactococcus lactis, do not produce glutathione, and the thioredoxin system is presumed to be essential . We constructed an L . lactis trxB1 mutant . The mutant was obtained under anaerobic conditions in the presence of dithiothreitol (DTT) . Unexpectedly, the trxB1 mutant was viable without DTT and under aerated static conditions, thus disproving the essentiality of this system . Aerobic growth of the trxB1 mutant did not require glutathione, also ruling out the need for this redox maintenance system . Proteomic analyses showed that known oxidative stress defense proteins are induced in the trxB1 mutant . Two additional effects of trxB1 were not previously reported in other bacteria: (i) induction of proteins involved in fatty acid or menaquinone biosynthesis, indicating that membrane synthesis is part of the cellular response to a redox imbalance, and (ii) alteration of the isoforms of the glycolytic enzyme glyceraldehyde-3-phosphate dehydrogenase (GapB) . We determined that the two GapB isoforms in L . lactis differed by the oxidation state of catalytic-site cysteine C(152) . Unexpectedly, a decrease specific to the oxidized, inactive form was observed in the trxB1 mutant, possibly because of proteolysis of oxidized GapB . This study showed that thioredoxin reductase is not essential in L . lactis and that its inactivation triggers induction of several mechanisms acting at the membrane and metabolic levels . The existence of a novel redox function that compensates for trxB1 deficiency is suggested.

J Bacteriol, 2005 Jan, 187(2), 512 - 21
Probing Direct Interactions between CodY and the oppD Promoter of Lactococcus lactis; den Hengst CD et al.; CodY of Lactococcus lactis MG1363 is a transcriptional regulator that represses the expression of several genes encoding proteins of the proteolytic system . These genes include pepN, pepC, opp-pepO1, and probably prtPM, pepX, and pepDA2, since the expression of the latter three genes relative to nitrogen availability is similar to that of the former . By means of in vitro DNA binding assays and DNase I footprinting techniques, we demonstrate that L . lactis CodY interacts directly with a region upstream of the promoter of its major target known so far, the opp system . Our results indicate that multiple molecules of CodY interact with this promoter and that the amount of bound CodY molecules is affected by the presence of branched-chain amino acids and not by GTP . Addition of these amino acids strongly affects the extent of the region protected by CodY in DNase I footprints . Random and site-directed mutagenesis of the upstream region of oppD yielded variants that were derepressed in a medium with an excess of nitrogen sources . Binding studies revealed the importance of specific bases in the promoter region required for recognition by CodY.

Radiat Res, 2004 Nov, 162(5), 566 - 71
Radiation affects binding of Fpg repair protein to an abasic site containing DNA; Gillard N et al.; During the base excision repair of certain DNA lesions, the formamidopyrimidine-DNA glycosylase (Fpg) binds specifically to the DNA region containing an abasic (AP) site . Is this step affected by exposure to ionizing radiation? To answer this question, we studied a complex between a DNA duplex containing an analogue of an abasic site (the 1,3-propanediol site, Pr) and a mutated Lactococcus lactis Fpg (P1G-LlFpg) lacking strand cleavage activity . Upon irradiation of the complex, the ratio of bound/free partners decreased . When the partners were irradiated separately, the irradiated DNA still bound the unirradiated protein, whereas irradiated Fpg no longer bound unirradiated DNA . Thus irradiation hinders Fpg-DNA binding because of the damage to the protein . Using our radiolytic attack simulation procedure RADACK (Begusova et al., J . Biomol . Struct . Dyn . 19, 141-157, 2001), we reveal the potential hot spots for damage in the irradiated protein . Most of them are essential for the interaction of Fpg with DNA, which explains the radiation-induced loss of binding ability of Fpg . The doses necessary to destroy the complex are higher than those inactivating Fpg irradiated separately . As confirmed by our calculations, this can be explained by the partial protection of the protein by the bound DNA.

Int J Food Microbiol, 2005 Jan 15, 98(1), 89 - 105
A fuzzy logic-based model for the multistage high-pressure inactivation of Lactococcus lactis ssp . cremoris MG 1363; Kilimann KV et al.; The high-pressure inactivation (200 to 600 MPa) of Lactococcus lactis ssp . cremoris MG 1363 suspended in milk buffer was investigated with both experimental and theoretical methods . The inactivation kinetics were characterised by the determination of the viable cell counts, cell counts of undamaged cells, LmrP activity, membrane integrity, and metabolic activity . Pressures between 200 and 600 MPa were applied, and pressure holding times were varied between 0 and 120 min . Experiments were carried out in milk buffer at pH values ranging between 4.0 and 6.5, and the effect of the addition of molar concentrations of NaCl and sucrose was furthermore determined . The inactivation curves of L . lactis, as characterised by viable cell counts, exhibited typical sigmoid asymmetric shapes . Generally, inactivation of the membrane transport system LmrP was the most sensitive indicator of pressure-induced sublethal injury . Furthermore, the metabolic activity was inactivated concomitant with or prior to the loss of viability . Membrane integrity was lost concomitant with or later than cell death . For example, treatments at 200 MPa for 60 min in milk buffer did not inactivate L . lactis, but fully inactivated LmrP activity and reduced the metabolic activity by 50% . The membrane integrity was unaffected . Thus, the assay systems chosen are suitable to dissect the multistep high-pressure inactivation of L . lactis ssp . cremoris MG 1363 . A fuzzy logic model accounting for the specific knowledge on the multistep pressure inactivation and allowing the prediction of the quantities of sublethally damaged cells was formulated . Furthermore, the fuzzy model could be used to accurately predict pressure inactivation of L . lactis using conditions not taken into account in model generation . It consists of 160 rules accounting for several dependent and independent variables . The rules were generated automatically with fuzzy clustering methods and rule-oriented statistical analysis . The set is open for the integration of further knowledge-based rules . A very good overall agreement between measured and predicted values was obtained . Single, deviating results have been identified and can be explained to be measurement errors or model intrinsic deficiencies.

J Vet Sci, 2004 Dec, 5(4), 387 - 90
Experimental evaluation of pathogenicity of Lactococcus garvieae in black rockfish (Sebastes schlegeli); Kang SH et al.; Black rockfish (Sebastes schlegeli) is an important mariculture species in Korea . The production of this fish is drastically declined due to bacterial diseases, particularly streptococcosis caused by Lactococcus garvieae . The bacterial surface characteristics of SJ7 and TY6 were found to have capsule but not NB13 and YS18 . The experiential evaluation of L . garvieae pathogenicity, the capsular isolates showed high cumulative mortality i.e . SJ7 (100%) and TY6 (60%) compared to non-capsular isolates . Based on this result the capsular isolates L . garvieae were highly suspected as the causative agent of streptococcosis in rockfish.

J Biol Chem . 2004 Dec 21; {Epub ahead of print}
Lactococcus lactis uses MscL as its principal mechanosensitive channel; Folgering JH et al.; The functions of the mechanosensitive channels from Lactococcus lactis were determined by biochemical, physiological and electrophysiological methods . Patch-clamp studies showed that the genes yncB and mscL encode MscS and MscL-like channels, respectively, when expressed in E . coli or if the gene products were purified and reconstituted in proteoliposomes . However, unless yncB was expressed in trans, wild-type membranes of Lactococcus lactis displayed only MscL activity . Membranes prepared from a mscL disruption mutant did not show any mechanosensitive channel activity, irrespective of whether the cells had been grown on low or high osmolarity medium . In osmotic downshift assays, wild-type cells survived and retained 20% of the glycine betaine internalized under external high salt conditions . On the other hand, the mscL disruption mutant retained 40% of internalized glycine betaine and was significantly compromised in its survival upon osmotic downshifts . The data strongly suggest that Lactococcus lactis uses MscL as the main mechanosensitive solute-release system to protect the cells under conditions of osmotic downshift.

Lett Appl Microbiol, 2005, 40(1), 44 - 9
Heterologous expression of the plant coumarate : CoA ligase in Lactococcus lactis; Martinez-Cuesta MC et al.; Abstract m.c . martinez-cuesta, m.j . gasson and a . narbad . 2004.Aims: To demonstrate the expression of coumarate : CoA ligase of Arabidopsis thaliana in Lactococcus lactis as a first step of cloning the vanillin pathway . Methods and Results: The 4CL gene was amplified from a cDNA library of A . thaliana by PCR and subcloned into a multicopy lactococcal vector where the expression is under the nisA promoter . The maximum yield of the protein in the recombinant strain of L . lactis was obtained 3 h after induction with 10 ng ml(-1) of nisin . However, these levels were only fraction of those detected in cell extracts of Pseudomonas fluorescens AN103 strain which naturally expresses its own enzyme when grown in the presence of ferulic acid as a carbon source . Among different substrates examined, the enzyme was most active against coumaric acid . Conclusions: The gene encoding coumarate : CoA ligase in A . thaliana was isolated, sequenced, cloned and expressed in L . lactis . Significance and Impact of the Study: This study represents the first of the two steps for genetic engineering of the vanillin pathway in the GRAS (generally recognized as safe) organism L . lactis.

J Biol Chem . 2004 Dec 16; {Epub ahead of print}
A nudix enzyme removes pyrophosphate from dihyroneopterin triphosphate in the folate synthesis pathway of bacteria and plants; Klaus SM et al.; Removal of pyrophosphate from dihydroneopterin triphosphate (DHNTP) is the second step in the pterin branch of the folate synthesis pathway . There has been controversy over whether this reaction requires a specific pyrophosphohydrolase or is a metal ion-dependent chemical process . The genome of Lactococcus lactis has a multicistronic folate synthesis operon that includes an open reading frame (ylgG) specifying a putative Nudix hydrolase . Since many Nudix enzymes are pyrophosphohydrolases, YlgG was expressed in Escherichia coli and characterized . The recombinant protein showed high DHNTP pyrophosphohydrolase activity with a K(m) value of 2 microM, had no detectable activity against deoxynucleoside triphosphates or other typical Nudix hydrolase substrates, required a physiological level (~1 mM) of Mg(2+), and was active as a monomer . Essentially no reaction occurred without enzyme at 1 mM Mg(2+) . Inactivation of ylgG in L . lactis resulted in DHNTP accumulation and folate depletion, confirming that YlgG functions in folate biosynthesis . We therefore propose that ylgG be redesignated as folQ . The closest Arabidopsis homolog of YlgG (encoded by Nudix gene At1g68760) was expressed in E . coli and shown to have Mg(2+)-dependent DHNTP pyrophosphohydrolase activity . This protein (AtNUDT1) was previously reported to have NADH pyrophosphatase activity in the presence of 5 mM Mn(2+) (Dobrzanska et al., J . Biol . Chem., 277: 50482-50486, 2002) . However, we found that this activity is negligible at physiological levels of Mn(2+) and that, with 1 mM Mg(2+), AtNUDT1 prefers DHNTP and (deoxy)nucleoside triphosphates.

J Appl Microbiol, 2005, 98(1), 127 - 35
Development of food-grade cloning and expression vectors for Lactococcus lactis; Liu CQ et al.; Abstract c.-q . liu, p . su, n . khunajakr, y.-m . deng, s . sumual, w.s . kim, j.e . tandianus and n.w . dunn . 2004.Aims: To develop food-grade cloning and expression vectors for use in genetic modification of Lactococcus lactis . Methods and Results: Two plasmid replicons and three dominant selection markers were isolated from L . lactis and used to construct five food-grade cloning vectors . These vectors were composed of DNA only from L . lactis and contained no antibiotic resistance markers . Three of the vectors (pND632, pND648 and pND969) were based on the same plasmid replicon and carried, either alone or in combination, the three different selectable markers encoding resistance to nisin, cadmium and/or copper . The other two (pND965DJ and pND965RS) were derived from a cadmium resistance plasmid, and carried a constitutive promoter and a copper-inducible promoter, respectively, immediately upstream of a multicloning site . All vectors were stable in L . lactis LM0230 for at least 40 generations without selection pressure . The two groups of vectors were compatible in L . lactis LM0230 . The vectors pND648 and pND965RS, as representatives of the two groups, were transferred successfully by electroporation into and maintained in an industrial strain of L . lactis . The usefulness of the vectors was further demonstrated by expressing a phage resistance gene (abiI) in another industrial strain of L . lactis . Conclusions: The five food-grade vectors constructed are potentially useful for industrial strains of L . lactis . Significance and Impact of the Study: These vectors represent a new set of molecular tools useful for food-grade modifications of L . lactis.

Nucleic Acids Res, 2005 Jan 1, 33 Database Issue, D583 - 7
DynaProt 2D: an advanced proteomic database for dynamic online access to proteomes and two-dimensional electrophoresis gels; Drews O et al.; DynaProt 2D presents an advanced online database for dynamic access to proteomes and two-dimensional (2D) gels . The database was designed to administer complete in silico proteomes and links them with experimental proteomic data in the manner of 2D electrophoresis gels (IPG-Dalt) . The 2D gels serve as reference maps in 2D gel analysis as well as tools for navigation of the database to switch between experimental and predicted data . Therefore, all identified spots in the gels are clickable and linked with summarized protein information . The protein information tables contain calculated characteristics, which are often used in proteomics, such as the molecular weight, isoelectric point, codon adaptation index, grand average of hydropathicity, etc . The design of the database permits online extension of gel data and protein attributes without knowledge of any software language . Besides navigation via 2D gels, the clear graphical user interface permits quick and intuitive searching throughout complete proteomes and supports, e.g . the search for proteins with isoelectric points within pH ranges of interest or protein classes (e.g . ribosomal proteins or transporters) . The first organism implemented in the database is Lactococcus lactis . The database is available at www.wzw.tum.de/proteomik/lactis.

Biotechnol Lett, 2004 Nov, 26(22), 1713 - 6
Improvement of nisin production in pH feed-back controlled, fed-batch culture by Lactococcus lactis subsp . lactis; Lv W et al.; Nisin production by Lactococcus lactis subsp . lactis in fed-batch culture was doubled by using a pH feed-back controlled method . Sucrose concentration was controlled at 10 g l(-1) giving 5010 IU nisin ml(-1) compared to 2660 IU nisin ml(-1) in batch culture.

Biotechnol Lett, 2004 Sep, 26(17), 1341 - 5
Simple one-step purification of nisin Z from unclarified culture broth of Lactococcus lactis subsp . lactis A164 using expanded bed ion exchange chromatography; Cheigh CI et al.; A simple one-step purification method, using expanded bed, ion-exchange chromatography, for the fractionation of nisin Z produced by Lactococcus lactis subsp . lactis A164 was developed . The highest dynamic binding capacity (0.92) of the adsorbent was obtained at a superficial velocity of 367 cm h(-1), resulting in approx . 2.7-fold bed expansion . The range of pH for the maximum adsorption was 3-4 . The isocratic elution with 0.15 M NaCl led to approx . >90% recovery . Single-step purification of nisin Z from unclarified A164 culture broth resulted in 31-fold purification with a 90% yield.

J Bacteriol, 2005 Jan, 187(1), 114 - 24
Autolysis of Lactococcus lactis is increased upon D-alanine depletion of peptidoglycan and lipoteichoic acids; Steen A et al.; Mutations in the genes encoding enzymes responsible for the incorporation of D-Ala into the cell wall of Lactococcus lactis affect autolysis . An L . lactis alanine racemase (alr) mutant is strictly dependent on an external supply of D-Ala to be able to synthesize peptidoglycan and to incorporate D-Ala in the lipoteichoic acids (LTA) . The mutant lyses rapidly when D-Ala is removed at mid-exponential growth . AcmA, the major lactococcal autolysin, is partially involved in the increased lysis since an alr acmA double mutant still lyses, albeit to a lesser extent . To investigate the role of D-Ala on LTA in the increased cell lysis, a dltD mutant of L . lactis was investigated, since this mutant is only affected in the D-alanylation of LTA and not the synthesis of peptidoglycan . Mutation of dltD results in increased lysis, showing that D-alanylation of LTA also influences autolysis . Since a dltD acmA double mutant does not lyse, the lysis of the dltD mutant is totally AcmA dependent . Zymographic analysis shows that no degradation of AcmA takes place in the dltD mutant, whereas AcmA is degraded by the extracellular protease HtrA in the wild-type strain . In L . lactis, LTA has been shown to be involved in controlled (directed) binding of AcmA . LTA lacking D-Ala has been reported in other bacterial species to have an improved capacity for autolysin binding . Mutation of dltD in L . lactis, however, does not affect peptidoglycan binding of AcmA; neither the amount of AcmA binding to the cells nor the binding to specific loci is altered . In conclusion, D-Ala depletion of the cell wall causes lysis by two distinct mechanisms . First, it results in an altered peptidoglycan that is more susceptible to lysis by AcmA and also by other factors, e.g., one or more of the other (putative) cell wall hydrolases expressed by L . lactis . Second, reduced amounts of D-Ala on LTA result in decreased degradation of AcmA by HtrA, which results in increased lytic activity.

J Chromatogr A, 2004 Nov 12, 1056(1-2), 43 - 8
Isolation of genomic DNA using magnetic cobalt ferrite and silica particles; Prodelalova J et al.; Adsorption separation techniques as an alternative to laborious traditional methods (e.g., based on phenol extraction procedure) have been applied for DNA purification . In this work we used two types of particles: silica and cobalt ferrite (unmodified or modified with a reagent containing weakly basic aminoethyl groups, aminophenyl groups, or alginic acid) . DNA from chicken erythrocytes and DNA isolated from bacteria Lactococcus lactis were used for testing of adsorption/desorption properties of particles . The cobalt ferrite particles modified with different reagents were used for isolation of PCR-ready bacterial DNA from different dairy products.

Genetics, 2004 Nov, 168(3), 1145 - 57
Insertion-sequence-mediated mutations isolated during adaptation to growth and starvation in Lactococcus lactis; de Visser JA et al.; We studied the activity of three multicopy insertion sequence (IS) elements in 12 populations of Lactococcus lactis IL1403 that evolved in the laboratory for 1000 generations under various environmental conditions (growth or starvation and shaken or stationary) . Using RFLP analysis of single-clone representatives of each population, nine IS-mediated mutations were detected across all environmental conditions and all involving IS981 . When it was assumed that these mutations were neutral, their frequency was higher under shaken than under stationary conditions, possibly due to oxygen stress . We characterized seven of the nine mutations at the molecular level and studied their population dynamics where possible . Two were simple insertions into new positions and the other five were recombinational deletions (of <1->10 kb) among existing and new copies of IS981; in all but one case these mutations disrupted gene functions . The best candidate beneficial mutations were two deletions of which similar versions were detected in two populations each . One of these two parallel deletions, affecting a gene involved in bacteriophage resistance, showed intermediate rearrangements and may also have resulted from increased local transposition rates.

J Bacteriol, 2004 Dec, 186(24), 8337 - 46
Bacillus subtilis operon encoding a membrane receptor for bacteriophage SPP1; Sao-Jose C et al.; The results reported here have identified yueB as the essential gene involved in irreversible binding of bacteriophage SPP1 to Bacillus subtilis . First, a deletion in an SPP1-resistant (pha-2) strain, covering most of the yueB gene, could be complemented by a xylose-inducible copy of yueB inserted at amyE . Second, disruption of yueB by insertion of a pMutin4 derivative resulted in a phage resistance phenotype regardless of the presence or absence of IPTG (isopropyl-beta-D-thiogalactopyranoside) . YueB homologues are widely distributed in gram-positive bacteria . The protein Pip, which also serves as a phage receptor in Lactococcus lactis, belongs to the same family . yueB encodes a membrane protein of approximately 120 kDa, detected in immunoblots together with smaller forms that may be processed products arising from cleavage of its long extracellular domain . Insertional inactivation of yueB and the surrounding genes indicated that yueB is part of an operon which includes at least the upstream genes yukE, yukD, yukC, and yukBA . Disruption of each of the genes in the operon allowed efficient irreversible adsorption, provided that yueB expression was retained . Under these conditions, however, smaller plaques were produced, a phenotype which was particularly noticeable in yukE mutant strains . Interestingly, such reduction in plaque size was not correlated with a decreased adsorption rate . Overall, these results provide the first demonstration of a membrane-bound protein acting as a phage receptor in B . subtilis and suggest an additional involvement of the yukE operon in a step subsequent to irreversible adsorption.

J Fish Dis, 2004 Dec, 27(12), 679 - 86
Lancefield group C Streptococcus dysgalactiae infection responsible for fish mortalities in Japan; Nomoto R et al.; A Lancefield serological group C Streptococcus sp . was isolated from cultured amberjack, Seriola dumerili Risso, and yellowtail, Seriola quinqueradiata Temminck and Schlegel, immunized with Lactococcus garvieae commercial vaccines in Japan . The isolated bacteria were Gram-positive cocci, auto-aggregating in saline, morphologically long chains in growth medium, catalase negative and alpha-haemolytic on blood agar . An almost complete gene sequence of the 16S rDNA of two isolates was determined and compared with that of bacterial strains in the database . The isolates were identified as Streptococcus dysgalactiae based on the results of the 16S rDNA sequence, the bacteriological properties and the Lancefield serological grouping . Oligonucleotide primers specifically designed for the 16S-23S rDNA intergenic spacer region of S . dysgalactiae amplified a gene from all the fish isolates, as well as the type strains alpha-haemolytic S . dysgalactiae subsp . dysgalactiae ATCC430738 and beta-haemolytic S . dysgalactiae subsp . equisimilis ATCC35666, but not those of S . equi ATCC33398, Lactococcus garvieae ATCC43921 and L . garvieae KG9408 . The severe necrotic lesions of the caudal peduncle seen in experimentally infected fish were similar to those seen in naturally infected fish.

Appl Environ Microbiol, 2004 Dec, 70(12), 7365 - 71
Milk contamination and resistance to processing conditions determine the fate of Lactococcus lactis bacteriophages in dairies; Madera C et al.; Milk contamination by phages, the susceptibility of the phages to pasteurization, and the high levels of resistance to phage infection of starter strains condition the evolution dynamics of phage populations in dairy environments . Approximately 10% (83 of 900) of raw milk samples contained phages of the quasi-species c2 (72%), 936 (24%), and P335 (4%) . However, 936 phages were isolated from 20 of 24 (85%) whey samples, while c2 was detected in only one (4%) of these samples . This switch may have been due to the higher susceptibility of c2 to pasteurization (936-like phages were found to be approximately 35 times more resistant than c2 strains to treatment of contaminated milk in a plate heat exchanger at 72 degrees C for 15 s) . The restriction patterns of 936-like phages isolated from milk and whey were different, indicating that survival to pasteurization does not result in direct contamination of the dairy environment . The main alternative source of phages (commercial bacterial starters) does not appear to significantly contribute to phage contamination . Twenty-four strains isolated from nine starter formulations were generally resistant to phage infection, and very small progeny were generated upon induction of the lytic cycle of resident prophages . Thus, we postulate that a continuous supply of contaminated milk, followed by pasteurization, creates a factory environment rich in diverse 936 phage strains . This equilibrium would be broken if a particular starter strain turned out to be susceptible to infection by one of these 936-like phages, which, as a consequence, became prevalent.

RNA, 2005 Jan, 11(1), 14 - 28 Epub 2004 Dec 01.
Domain structure and three-dimensional model of a group II intron-encoded reverse transcriptase; Blocker FJ et al.; Group II intron-encoded proteins (IEPs) have both reverse transcriptase (RT) activity, which functions in intron mobility, and maturase activity, which promotes RNA splicing by stabilizing the catalytically active RNA structure . The LtrA protein encoded by the Lactococcus lactis Ll.LtrB group II intron contains an N-terminal RT domain, with conserved sequence motifs RT1 to 7 found in the fingers and palm of retroviral RTs; domain X, associated with maturase activity; and C-terminal DNA-binding and DNA endonuclease domains . Here, partial proteolysis of LtrA with trypsin and Arg-C shows major cleavage sites in RT1, and between the RT and X domains . Group II intron and related non-LTR retroelement RTs contain an N-terminal extension and several insertions relative to retroviral RTs, some with conserved features implying functional importance . Sequence alignments, secondary-structure predictions, and hydrophobicity profiles suggest that domain X is related structurally to the thumb of retroviral RTs . Three-dimensional models of LtrA constructed by "threading" the aligned sequence on X-ray crystal structures of HIV-1 RT (1) account for the proteolytic cleavage sites; (2) suggest a template-primer binding track analogous to that of HIV-1 RT; and (3) show that conserved regions in splicing-competent LtrA variants include regions of the RT and X (thumb) domains in and around the template-primer binding track, distal regions of the fingers, and patches on the protein's back surface . These regions potentially comprise an extended RNA-binding surface that interacts with different regions of the intron for RNA splicing and reverse transcription.

Eur J Pharm Biopharm, 2005 Jan, 59(1), 9 - 15
Evaluation of extrusion/spheronisation, layering and compaction for the preparation of an oral, multi-particulate formulation of viable, hIL-10 producing Lactococcus lactis; Huyghebaert N et al.; Three formulation techniques were compared in order to develop a multi-particulate formulation of viable, interleukin-10 producing Lactococcus lactis Thy12 . First, freeze-dried L . lactis was compacted into mini-tablets . Next, liquid L . lactis culture was used as the granulation fluid for the production of pellets by extrusion/spheronisation . Finally, liquid L . lactis culture was layered on inert pellets as an alternative technique for the production of pellets . L . lactis viability and interleukin-10 production was evaluated . Viability dropped to 15.7% after compaction of freeze-dried L . lactis and to 1.0% after pelletisation of liquid L . lactis by extrusion/spheronisation . The viability in the mini-tablets and pellets, stored for 1 week at RT and 10% RH was reduced to 23 and 0.5% of initial viability, respectively . Storage for 1 week at RT and 60% RH resulted in complete loss of viability . Layering of L . lactis on inert pellets resulted in low viability (4.86%), but 1 week after storage at RT and 10% RH, 68% of initial viability was maintained . Increasing product temperature and cell density of L . lactis in the layering suspension did not significantly change viability after layering and storage . Interleukin-10 production capacity of L . lactis Thy12 was maintained after layering.

Res Microbiol, 2004 Dec, 155(10), 847 - 54
Lactovum miscens gen . nov., sp . nov., an aerotolerant, psychrotolerant, mixed-fermentative anaerobe from acidic forest soil; Matthies C et al.; An aerotolerant, psychrotolerant anaerobe, anNAG3, was isolated from an acidic forest floor solution (in situ pH of 4.5) . Cells of anNAG3 stained Gram-positive did not form spores, and were not motile . Cells were ovoid, approximately 1 microm long and 0.7 microm wide, mostly in pairs, and contained a multi-layered cell wall and intracytoplasmic membranes . Growth was observed at pH 3.5-7.5 and 0-35 degrees C . Glucose, galactose, fructose, mannitol, glucosamine, N-acetylglucosamine, cellobiose, and maltose supported growth . Lactate, ethanol, formate, and acetate were end products . H(2) and CH(4) were not detected, and only very minor amounts of CO(2) were produced . The relative amount of a particular product was dependent on the substrate utilized, and product profiles indicated that (i) sugars were initially metabolized to pyruvate via glycolysis, and (ii) lactate dehydrogenase and pyruvate-formate lyase were responsible for the subsequent metabolism of pyruvate . O(2) was not significantly utilized and was not toxic to growth . anNAG3 did not contain detectable membranous or cytoplasmic cytochromes . Nitrate, sulfate, and Fe(III) were not dissimilated . Thus, anNAG3 was characterized as an aerotolerant, non-acetogenic chemoorganotroph with a mixed-fermentative metabolism . The G + C content of the DNA was 37.6 mol% . The similarity of the 16S rRNA gene sequence of anNAG3 to that of its closest phylogenetic relatives (which were in the genera Lactococcus and Streptococcus) approximated 88-89%, indicating that anNAG3 constitutes the type species of a new genus . Based on the collective properties of anNAG3, it is proposed that anNAG3 be termed Lactovum miscens.

Appl Microbiol Biotechnol . 2004 Nov 24; {Epub ahead of print}
Effect of Sorghum vulgare phosphoenolpyruvate carboxylase and Lactococcus lactis pyruvate carboxylase coexpression on succinate production in mutant strains of Escherichia coli; Lin H et al.; Sorghum vulgare phosphoenolpyruvate carboxylase (PEPC) and Lactococcus lactis pyruvate carboxylase (PYC) were overexpressed in Escherichia coli concurrently to improve the production of succinate, a valuable industrial specialty chemical . This coexpression system was also applied to E . coli mutant strains strategically designed by inactivating the competing pathways of succinate formation . The highest level of succinate production was observed in E . coli strains coexpressing both PEPC and PYC when compared with E . coli strains individually overexpressing either PEPC or PYC . Lactate production was also significantly reduced with PEPC and PYC coexpression . Lactate and acetate pathways were inactivated to eliminate the competing pathways of succinate formation . Results showed that inactivation of both the lactate and acetate pathways with the coexpression of PEPC and PYC was most effective in improving succinate production . Inactivating the lactate or acetate pathway alone only caused a majority of the carbon flux to shift to other metabolites rather than succinate . Coexpression of PEPC and PYC was also applied to an E . coli mutant strain deficient in lactate dehydrogenase and pyruvate:formate lyase that accumulated a substantial amount of the intermediate metabolite pyruvate during growth . Results showed that PEPC and PYC coexpression was effective in depleting pyruvate accumulation and increasing the production of metabolites.

Eur J Biochem, 2004 Nov, 271(22), 4392 - 400
Natural-abundance isotope ratio mass spectrometry as a means of evaluating carbon redistribution during glucose-citrate cofermentation by Lactococcus lactis; Mahmoud M et al.; The cometabolism of citrate and glucose by growing Lactococcus lactis ssp . lactis bv . diacetylactis was studied using a natural-abundance stable isotope technique . By a judicious choice of substrates differing slightly in their 13C/12C ratios, the simultaneous metabolism of citrate and glucose to a range of compounds was analysed . These end-products include lactate, acetate, formate, diacetyl and acetoin . All these products have pyruvate as a common intermediate . With the objective of estimating the degree to which glucose and citrate metabolism through pyruvate may be differentially regulated, the delta13C values of the products accumulated over a wide range of concentrations of citrate and glucose were compared . It was found that, whereas the relative accumulation of different products responds to both the substrate concentration and the ratio between the substrates, the delta13C values of the products primarily reflect the availability of the two substrates over the entire range examined . It can be concluded that in actively growing L . lactis the maintenance of pyruvate homeostasis takes precedence over the redox status of the cells as a regulatory factor.

J Food Prot, 2004 Nov, 67(11), 2521 - 9
Sequencing of the tyrosine decarboxylase cluster of Lactococcus lactis IPLA 655 and the development of a PCR method for detecting tyrosine decarboxylating lactic acid bacteria; Fernandez M et al.; The enzymatic decarboxylation of tyrosine produces tyramine, the most abundant biogenic amine in dairy products-especially in cheeses . The screening of lactic acid bacteria isolated from different artisanal cheeses and a number of microbial collections identified 22 tyramine-producing strains belonging to different genera . The Lactococcus lactis strain IPLA 655 was selected, and the genes encoding a putative tyrosyl tRNA synthetase, a tyrosine decarboxylase (tdcA), and a tyrosine-tyramine antiporter, found together as a cluster, were sequenced . The disruption of tdcA yielded a strain unable to produce tyramine . Comparison of the L . lactis IPLA 655 tdcA gene with database tdcA sequences led to the design of two primers for use in a PCR method that identified potential tyramine-producing strains . The proposed method can use purified DNA, isolated colonies, milk, curd, and even cheese as a template . Molecular tools for the rapid detection of tyramine-producing bacteria at any time during the fermentation process could help prevent tyramine accumulation in fermented foods . The proposed technique could be of great use to the food industry.

J Bacteriol, 2004 Dec, 186(23), 8010 - 7
Essentiality of the early transcript in the replication origin of the lactococcal prolate phage c2; Schiemann AH et al.; The genome of the prolate-headed lytic lactococcal bacteriophage c2 is organized into two divergently oriented blocks consisting of the early genes and the late genes . These blocks are separated by the noncoding origin of DNA replication . We examined the functional role of transcription of the origin in a plasmid model system . Deletion of the early promoter P(E)1 abolished origin function . Introduction of mutations into P(E)1 which did not eliminate promoter activity or replacement of P(E)1 with an unrelated but functional promoter did not abolish replication . The A-T-rich region upstream of P(E)1, which is conserved in prolate phages, was not required for plasmid replication . Replacement of the P(E)1 transcript template sequence with an unrelated sequence with a similar G+C content abolished replication, showing that the sequence encoding the transcript is essential for origin function . Truncated transcript and internal deletion constructs did not support replication except when the deletion was at the very 3' end of the DNA sequence coding for the transcript . The P(E)1 transcript could be detected for all replication-proficient constructs . Recloning in a plasmid vector allowed detection of P(E)1 transcripts from some fragments that did not support replication, indicating that stability of the transcript alone was not sufficient for replication . The data suggest that production of a transcript of a specific length and with a specific sequence or structure is essential for the function of the phage c2 origin in this model system.

Proteomics, 2004 Dec, 4(12), 3881 - 98
Acidic proteome of growing and resting Lactococcus lactis metabolizing maltose; Palmfeldt J et al.; The acidic proteome of Lactococcus lactis grown anaerobically was compared for three different growth conditions: cells growing on maltose, resting cells metabolizing maltose, and cells growing on glucose . In maltose metabolizing cells several proteins were up-regulated compared with glucose metabolizing cells, however only some of the up-regulated proteins had apparent relation to maltose metabolism . Cells growing on maltose produced formate, acetate and ethanol in addition to lactate, whereas resting cells metabolizing maltose and cells growing on glucose produced only lactate . Increased levels of alcohol-acetaldehyde dehydrogenase (ADH) and phosphate acetyltransferase (PTA) in maltose-growing cells compared with glucose-growing cells coincided with formation of mixed acids in maltose-growing cells . The resting cells did not grow due to lack of an amino acid source and fermented maltose with lactate as the sole product, although ADH and PTA were present at high levels . The maltose consumption rate was approximately three times lower in resting cells than in exponentially growing cells . However, the enzyme levels in resting and growing cells metabolizing maltose were similar, which indicates that the difference in product formation in this case is due to regulation at the enzyme level . The levels of 30S ribosomal proteins S1 and S2 increased with increasing growth rate for resting cells metabolizing maltose, maltose-growing cells and glucose-growing cells . A modified form of HPr was synthesized under amino acid starvation . This is suggested to be due to alanine misincorporation for valine, which L . lactis is auxotrophic for . L . lactis conserves the protein profile to a high extent, even after prolonged amino acid starvation, so that the protein expression profile of the bacterium remains almost invariant.

Microbiology, 2004 Nov, 150(Pt 11), 3831 - 41
Characterization of the fibrinogen-binding surface protein Fbl of Staphylococcus lugdunensis; Mitchell J et al.; The fbl gene of Staphylococcus lugdunensis encodes a protein Fbl that is 58 % identical to the clumping factor A (ClfA) of Staphylococcus aureus . The fbl gene was present in eight clinical isolates of S . lugdunensis . When Fbl was expressed on the surface of Lactococcus lactis it promoted adherence to immobilized fibrinogen and cell clumping in a fibrinogen solution . Purified recombinant Fbl region A bound to immobilized fibrinogen in a dose-dependent manner and inhibited the adherence of both Fbl-expressing and ClfA-expressing strains of L . lactis to fibrinogen . Adherence of S . lugdunensis and L . lactis Fbl(+) to immobilized fibrinogen was also inhibited by rabbit anti-Fbl region A antibodies and rabbit anti-ClfA region A antibodies, as well as by human immunoglobulin with a high level of anti-ClfA antibodies . Alignment of the A domains of CflA and Fbl revealed that all of the ClfA residues implicated in binding to the gamma-chain of fibrinogen are conserved in Fbl . Nevertheless Fbl had a tenfold lower affinity for fibrinogen, suggesting that sequence differences that occur elsewhere in the protein, possibly in beta-strand E of domain N2, affect ligand binding.

Appl Environ Microbiol, 2004 Nov, 70(11), 6738 - 47
DNA Macroarray profiling of Lactococcus lactis subsp . lactis IL1403 gene expression during environmental stresses; Xie Y et al.; This report describes the use of an oligonucleotide macroarray to profile the expression of 375 genes in Lactococcus lactis subsp . lactis IL1403 during heat, acid, and osmotic stress . A set of known stress-associated genes in IL1403 was used as the internal control on the array . Every stress response was accurately detected using the macroarray, compared to data from previous reports . As a group, the expression patterns of the investigated metabolic genes were significantly altered by heat, acid, and osmotic stresses . Specifically, 13 to 18% of the investigated genes were differentially expressed in each of the environmental stress treatments . Interestingly, the methionine biosynthesis pathway genes (metA-metB1 and metB2-cysK) were induced during heat shock, but methionine utilization genes, such as metK, were induced during acid stress . These data provide a possible explanation for the differences between acid tolerance mechanisms of L . lactis strains IL1403 and MG1363 reported previously . Several groups of transcriptional responses were common among the stress treatments, such as repression of peptide transporter genes, including the opt operon (also known as dpp) and dtpT . Reduction of peptide transport due to environmental stress will have important implications in the cheese ripening process . Although stress responses in lactococci were extensively studied during the last decade, additional information about this bacterium was gained from the use of this metabolic array.

Int J Food Microbiol, 2004 Dec 1, 97(1), 9 - 15
Two homologous oligopeptide binding protein genes (oppA) in Lactococcus lactis MG1363; Sanz Y et al.; In previous studies, it has been shown that inactivation of opp or even oppA abolishes the capacity of Lactococcus lactis to utilize oligopeptides . We now show that the opp operon has been duplicated in L . lactis MG1363 . The nucleotide sequence of the oppA and oppC homologues (appA and appC) and most of the oppB homologue (appB) indicate that the corresponding protein sequences are 83%, 92% and 91% identical, respectively . Inactivation of appA, via homologous recombination, as well as complementation studies were carried out to determine the possible function of appA in peptide utilization . As anticipated from studies with an oppA knock-out, peptide utilization was not impaired in an appA disruption mutant . Importantly, AppA expressed from a plasmid could restore the ability of oppA deletion mutants to utilize Leu-enkephalin, albeit with a lower efficiency than OppA . The differences in the ability to utilize this pentapeptide were not due to differences in expression levels but most likely reflect a different catalytic efficiency in oligopeptide utilization when AppA is used as ligand receptor.

Cell Mol Life Sci, 2004 Oct, 61(19-20), 2646 - 57
Proton motive force mediates a reorientation of the cytosolic domains of the multidrug transporter LmrP; Gbaguidi B et al.; LmrP from Lactococcus lactis is a 45-kDa membrane protein that confers resistance to a wide variety of lipophilic compounds by acting as a proton motive force-driven efflux pump . This study shows that both the proton motive force and ligand interaction alter the accessibility of cytosolic tryptophan residues to a hydrophilic quencher . The proton motive force mediates an increase of LmrP accessibility toward the external medium and results in higher drug binding . Residues Asp128 and Asp68, from cytosolic loops, are involved in the proton motive force-mediated accessibility change . Ligand binding does not modify the protein accessibility, but the proton motive force-mediated restructuring is prerequisite for a subsequent accessibility change mediated by ligand binding . Asp142 cooperates with other membrane-embedded carboxylic residues to promote a conformational change that increases LmrP accessibility toward the hydrophilic quencher . This drug binding-mediated reorganization may be related to the transition between the high- and low-affinity drug-binding sites and is crucial for drug release in the extracellular medium.

Infect Immun, 2004 Nov, 72(11), 6197 - 205
The fibrinogen receptor FbsA promotes adherence of Streptococcus agalactiae to human epithelial cells; Schubert A et al.; Streptococcus agalactiae is a major cause of bacterial pneumonia, sepsis, and meningitis in human neonates . During the course of infection, S . agalactiae adheres to a variety of epithelial cells but the underlying mechanisms are only poorly understood . The present report demonstrates the importance of the fibrinogen receptor FbsA for the streptococcal adherence and invasion of epithelial cells . Deletion of the fbsA gene in various S . agalactiae strains substantially reduced their binding of soluble fibrinogen and their adherence to and invasion of epithelial cells, indicating a role of FbsA in these different processes . The adherence and invasiveness of an fbsA deletion mutant were partially restored by reintroducing the fbsA gene on an expression vector . Heterologous expression of fbsA in Lactococcus lactis enabled this bacterium to adhere to but not to invade epithelial cells, suggesting that FbsA is a streptococcal adhesin . Flow cytometry experiments revealed a dose-dependent binding of FbsA to the surface of epithelial cells . Furthermore, tissue culture experiments exhibited an intimate contact of FbsA-coated latex beads with the surfaces of human epithelial cells . Finally, host cell adherence and invasion were significantly blocked in competition experiments with either purified FbsA protein or a monoclonal antibody directed against the fibrinogen-binding epitope of FbsA . Taken together, our studies demonstrate that FbsA promotes the adherence of S . agalactiae to epithelial cells but that FbsA does not mediate the bacterial invasion into host cells . Our results also indicate that fibrinogen-binding epitopes within FbsA are involved in the adherence of S . agalactiae to epithelial cells.

Bioorg Med Chem Lett, 2004 Dec 6, 14(23), 5823 - 6
Homology model of the multidrug transporter LmrA from Lactococcus lactis; Pleban K et al.; LmrA is an ATP dependent multidrug transporter from Lactococcus lactis conferring antibiotic resistance to 17 out of 21 most frequently administered antibiotics . Starting from the dimeric crystal structure of Vc-MsbA, we built two homology models, with NBD:NBD interfaces reflecting the nonenergized and energized state, respectively . The TMD:TMD topology of the dimer is consistent with the previously obtained substrate photoaffinity labeling pattern suggesting binding of substrates at the TMD:TMD interface involving helix 3 of one monomer and helices 5 and 6 of the other monomer.

Int J Pharm, 2004 Nov 22, 286(1-2), 117 - 24
Normal flora: living vehicles for non-invasive protein drug delivery; Shao J et al.; Feasibility to use probiotic bacteria as a living protein delivery system through oral route was assessed in vitro . Lactococcus lactis transformed with a plasmid to express and secret beta-lactamase was used to deliver beta-lactamase through Caco-2 monolayer, an intestine epithelium . Transport of beta-lactamase through Caco-2 monolayer was carried out in the transwells . The viability and integrity of the cell monolayers co-cultured with L . lactis was examined by trypan blue exclusion method and by measuring the transport of mannitol and propranolol as well as the transepithelial electrical resistance (TEER) . Results show that it is feasible to use cell culture technique to evaluate the drug delivery by normal flora . The transport rate of beta-lactamase when delivered by L . lactis was 2.0 +/- 0.1 x 10(-2)h(-1) (n = 9) and through free solution form was 1.0 +/- 0.1 x 10(-2)h-1 . When co-cultured with L . lactis, Caco-2 cell viability decreased to 98, 96, and 94% at 6, 8, and 10h, respectively . Transport of mannitol through Caco-2 cell monolayer was significantly increased and the transport of propranolol through Caco-2 cell monolayer was significantly decreased in the presence of L . lactis . Increase in the amount of protein delivered is probably due to the concentrate of the protein by L . lactis on the monolayer (absorption surface) and the opening of the tight junction of Caco-2 monolayer by L . lactis . copyright 2004 Elsevier B.V.

Appl Microbiol Biotechnol . 2004 Oct 13; {Epub ahead of print}
Using Lactococcus lactis for glutathione overproduction; Li Y et al.; Glutathione and gamma-glutamylcysteine were produced in Lactococcus lactis using a controlled expression system and the genes gshA and gshB from Escherichia coli encoding the enzymes gamma-glutamylcysteine synthetase and glutathione synthetase . High levels of gamma-glutamylcysteine were found in strains growing on chemically defined medium and expressing either gshA alone or both gshA and gshB . As anticipated, glutathione was found in a strain expressing gshA and gshB . The level of glutathione production could be increased by addition of the precursor amino acid cysteine to the medium . The addition of cysteine led to an increased activity of glutathione synthetase, which is remarkable because the amino acid is not a substrate of this enzyme . The final intracellular glutathione concentration attained was 358 nmol mg(-1) protein, which is the highest concentration reported for a bacterium, demonstrating the suitability of engineered L . lactis for fine-chemical production and as a model for studies of the impact of glutathione on flavour formation and other properties of food.

Appl Microbiol Biotechnol . 2004 Oct 12; {Epub ahead of print}
Glutathione: a review on biotechnological production; Li Y et al.; This Mini-Review summarizes the historic developments and technological achievements in the biotechnological production of glutathione in the past 30 years . Glutathione is the most abundant non-protein thiol compound present in living organisms . It is used as a pharmaceutical compound and can be used in food additives and the cosmetic industries . Glutathione can be produced using enzymatic methods in the presence of ATP and its three precursor amino acids ( l-glutamic acid, l-cysteine, glycine) . Alternatively, glutathione can be produced by direct fermentative methods using sugar as a starting material . In the latter method, Saccharomyces cerevisiae and Candida utilis are currently used to produce glutathione on an industrial scale . At the molecular level, the genes gshA and gshB, which encode the enzymes gamma-glutamylcysteine synthetase and glutathione synthetase, respectively, have been cloned from Escherichia coli and over-expressed in E . coli, S . cerevisiae, and Lactococcus lactis . It is anticipated that, with the design and/or discovery of novel producers, the biotechnological production of glutathione will be further improved to expand the application range of this physiologically and medically important tripeptide.

FEMS Microbiol Lett, 2004 Oct 15, 239(2), 205 - 12
A xylose-inducible expression system for Lactococcus lactis; Miyoshi A et al.; A new controlled production system to target heterologous proteins to cytoplasm or extracellular medium is described for Lactococcus lactis NCDO2118 . It is based on the use of a xylose-inducible lactococcal promoter, P(xylT) . The capacities of this system to produce cytoplasmic and secreted proteins were tested using the Staphylococcus aureus nuclease gene (nuc) fused or not to the lactococcal Usp45 signal peptide . Xylose-inducible nuc expression is tightly controlled and resulted in high-level and long-term protein production, and correct targeting either to the cytoplasm or to the extracellular medium . Furthermore, this expression system is versatile and can be switched on or off easily by adding either xylose or glucose, respectively . These results confirm the potential of this expression system as an alternative and useful tool for the production of proteins of interest in L . lactis.

Virology, 2004 Nov 10, 329(1), 40 - 52
Molecular and transcriptional analysis of the temperate lactococcal bacteriophage Tuc2009; Seegers JF et al.; The genome of bacteriophage Tuc2009 consists of 38347 base pairs on which 57 open reading frames (ORFs) were identified, divided in two oppositely transcribed regions . The leftward-transcribed region harbors three ORFs, two of which are involved in the establishment of lysogeny . The rightward-transcribed region contains 54 ORFs, which are assumed to be required for the lytic life cycle . An exception to the above organization is ORF 10, of unknown function, located within the rightward-transcribed region that has an orientation opposite to the ORFs surrounding it . Transcriptional analysis of the Tuc2009 genome following infection of a sensitive host revealed that most ORFs are transcribed in a sequential manner . ORFs that are presumed to form (part of) the genetic switch along with the superinfection exclusion-encoding gene are transcribed immediately after infection, followed by transcription of the presumed replication region . Subsequent to this, several small transcripts could be identified followed by a single 24-kb transcript . This latter transcript was shown to specify most of the identified structural proteins as well as two proteins required for host lysis . Interestingly, the 24-kb mRNA was shown to undergo splicing through the activity of a type I intron whose removal from the mRNA resulted in the formation of an ORF specifying a major structural protein . Primer extension analysis was employed to identify the 5' ends of mRNA transcripts and the genome and transcriptional data are discussed in relation to other lactococcal bacteriophages.

Appl Environ Microbiol, 2004 Oct, 70(10), 5825 - 32
Identification of Lactococcus lactis genes required for bacteriophage adsorption; Dupont K et al.; The aim of this work was to identify genes in Lactococcus lactis subsp . lactis IL1403 and Lactococcus lactis subsp . cremoris Wg2 important for adsorption of the 936-species phages bIL170 and phi 645, respectively . Random insertional mutagenesis of the two L . lactis strains was carried out with the vector pGh9:ISS1, and integrants that were resistant to phage infection and showed reduced phage adsorption were selected . In L . lactis IL1403 integration was obtained in the ycaG and rgpE genes, whereas in L . lactis Wg2 integration was obtained in two genes homologous to ycbC and ycbB of L . lactis IL1403 . rgpE and ycbB encode putative glycosyltransferases, whereas ycaG and ycbC encode putative membrane-spanning proteins with unknown functions . Interestingly, ycaG, rgpE, ycbC, and ycbB are all part of the same operon in L . lactis IL1403 . This operon is probably involved in biosynthesis and transport of cell wall polysaccharides (WPS) . Binding and infection studies showed that phi645 binds to and infects L . lactis Wg2, L . lactis IL1403, and L . lactis IL1403 strains with pGh9:ISS1 integration in ycaG and rgpE, whereas bIL170 binds to and infects only L . lactis IL1403 and cannot infect Wg2 . These results indicate that phi 645 binds to a WPS structure present in both L . lactis IL1403 and L . lactis Wg2, whereas bIL170 binds to another WPS structure not present in L . lactis Wg2 . Binding of bIL170 and phi 645 to different WPS structures was supported by alignment of the receptor-binding proteins of bIL170 and phi 645 that showed no homology in the C-terminal part.

Appl Environ Microbiol, 2004 Oct, 70(10), 5818 - 24
Identification of the receptor-binding protein in 936-species lactococcal bacteriophages; Dupont K et al.; The aim of this work was to identify genes responsible for host recognition in the lactococcal phages sk1 and bIL170 belonging to species 936 . These phages have a high level of DNA identity but different host ranges . Bioinformatic analysis indicated that homologous genes, orf18 in sk1 and orf20 in bIL170, could be the receptor-binding protein (RBP) genes, since the resulting proteins were unrelated in the C-terminal part and showed homology to different groups of proteins hypothetically involved in host recognition . Consequently, chimeric bIL170 phages carrying orf18 from sk1 were generated . The recombinant phages were able to form plaques on the sk1 host Lactococcus lactis MG1614, and recombination was verified by PCR analysis directly with the plaques . A polyclonal antiserum raised against the C-terminal part of phage sk1 ORF18 was used in immunogold electron microscopy to demonstrate that ORF18 is located at the tip of the tail . Sequence analysis of corresponding proteins from other lactococcal phages belonging to species 936 showed that the N-terminal parts of the RBPs were very similar, while the C-terminal parts varied, suggesting that the C-terminal part plays a role in receptor binding . The phages investigated could be grouped into sk1-like phages (p2, fd13, jj50, and phi 7) and bIL170-like phages (P008, P113G, P272, and bIL66) on the basis of the homology of their RBPs to the C-terminal part of ORF18 in sk1 and ORF20 in bIL170, respectively . Interestingly, sk1-like phages bind to and infect a defined group of L . lactis subsp . cremoris strains, while bIL170-like phages bind to and infect a defined group of L . lactis subsp . lactis strains.

Appl Environ Microbiol, 2004 Oct, 70(10), 5769 - 77
Riboflavin production in Lactococcus lactis: potential for in situ production of vitamin-enriched foods; Burgess C et al.; This study describes the genetic analysis of the riboflavin (vitamin B(2)) biosynthetic (rib) operon in the lactic acid bacterium Lactococcus lactis subsp . cremoris strain NZ9000 . Functional analysis of the genes of the L . lactis rib operon was performed by using complementation studies, as well as by deletion analysis . In addition, gene-specific genetic engineering was used to examine which genes of the rib operon need to be overexpressed in order to effect riboflavin overproduction . Transcriptional regulation of the L . lactis riboflavin biosynthetic process was investigated by using Northern hybridization and primer extension, as well as the analysis of roseoflavin-induced riboflavin-overproducing L . lactis isolates . The latter analysis revealed the presence of both nucleotide replacements and deletions in the regulatory region of the rib operon . The results presented here are an important step toward the development of fermented foods containing increased levels of riboflavin, produced in situ, thus negating the need for vitamin fortification.

Biochim Biophys Acta, 2004 Oct 4, 1658(3), 252 - 61
Energetics of wild-type and mutant multidrug resistance secondary transporter LmrP of Lactococcus lactis; Mazurkiewicz P et al.; LmrP, a proton/multidrug antiporter of Lactococcus lactis, transports a variety of cationic substrates . Previously, two membrane-embedded acidic residues, Asp142 and Glu327, have been reported to be important for multidrug transport activity of LmrP . Here we show that neither Glu327 nor Asp142 is essential for ethidium binding but that Glu327 is a critical residue for the high affinity binding of Hoechst 33342 . Substitution of these two residues, however, negatively influences the transport activity . The energetics of transport was studied of two closely related cationic substrates ethidium and propidium that carry one and two positive charges, respectively . Extrusion of monovalent ethidium is dependent on both the electrical membrane potential (Deltapsi) and transmembrane proton gradient (DeltapH), while extrusion of propidium predominantly depends on the DeltapH only . The LmrP mutants D142C and E327C, however, mediate electroneutral ethidium extrusion, but are unable to mediate DeltapH-dependent extrusion of propidium . These data indicate that Asp142 and Glu327 are involved in proton translocation.

Mol Microbiol, 2004 Sep, 53(5), 1331 - 42
Respiration metabolism reduces oxidative and acid stress to improve long-term survival of Lactococcus lactis; Rezaiki L et al.; The impact of oxygen on a cell is strongly dependent on its metabolic state: survival in oxygen of free-living Lactococcus lactis, best known as a fermenting, acidifying bacterium, is generally poor . In contrast, if haem is present, L . lactis uses oxygen to switch from fermentation to respiration metabolism late in growth, resulting in spectacularly improved long-term survival . Oxygen is thus beneficial rather than detrimental for survival if haem is provided . We examined the effects of respiration on oxygen toxicity by comparing integrity of stationary phase cells after aerated growth without and with added haem . Aeration (no haem) growth caused considerable cellular protein and chromosomal DNA damage, increased spontaneous mutation frequencies and poor survival of recA mutants . These phenotypes were greatly diminished when haem was present, indicating that respiration constitutes an efficient barrier against oxidative stress . Using the green fluorescent protein as an indicator of intracellular oxidation state, we showed that aeration growth provokes significantly greater oxidation than respiration growth . Iron was identified as a main contributor to mortality and DNA degradation in aeration growth . Our results point to two features of respiration growth in lactococci that are responsible for maintaining low oxidative damage: One is a more reduced intracellular state, which is because of efficient oxygen elimination by respiration . The other is a higher pH resulting from the shift from acid-forming fermentation to respiration metabolism . These results have relevance to other bacteria whose respiration capacity depends on addition of exogenous haem .

Blood . 2004 Sep 21; {Epub ahead of print}
FbsA, a fibrinogen-binding protein from Streptococcus agalactiae, mediates platelet aggregation; Pietrocola G et al.; The bacterium Streptococcus agalactiae is an etiological agent in the pathogenesis of endocarditis in humans . FbsA, a fibrinogen-binding protein produced by this pathogen, is considered to be an important virulence factor . In this present study we provide evidence that S . agalactiae clinical isolates bearing FbsA attach to fibrinogen and elicit a fibrinogen-dependent aggregation of platelets . Mutants of S . agalactiae lacking the fbsA gene lost the ability to attach to fibrinogen and to aggregate platelets . Plasmid-mediated expression of fbsA restored the capability for fibrinogen binding and platelet aggregation in S . agalactiae fbsA mutants, and allowed Lactococcus lactis to interact with fibrinogen and to aggregate human platelets . Moreover, a monoclonal anti-FbsA antibody inhibited bacterial adherence to fibrinogen and S . agalactiae-induced platelet aggregation . Platelet aggregation was inhibited by aspirin, PGE1, the peptide RGDS and the antibody abciximab demonstrating the specificity of platelet aggregation by S . agalactiae and indicating an involvement of integrin GPIIb/IIIa in the induction of platelet aggregation . Aggregation was also dependent on anti-FbsA IgG and could be inhibited by an antibody against the platelet FcgammaRIIA receptor . These findings indicate that FbsA is a crucial factor in S . agalactiae induced-platelet aggregation and may therefore play an important role in S . agalactiae-induced endocarditis.

Biochem Biophys Res Commun, 2004 Oct 22, 323(3), 884 - 90
Homologous and heterologous expression of RNase III from Lactococcus lactis; Amblar M et al.; The endoribonuclease III (RNase III), encoded by the rnc gene, is an important enzyme for RNA metabolism . In this report a chromosomal fragment containing the rnc gene from Lactococcus lactis was cloned and its expression was analyzed . Complementation assays performed in Escherichia coli demonstrate that the lactococcal RNase III (Lac-RNase III) is able to process rRNAs and to regulate the levels of polynucleotide phosphorylase (PNPase) . These results demonstrate that the lactococcal enzyme is able to substitute the Ec-RNase III not only in the rRNA processing, but also in the processing of mRNAs . The amount of lactococcal rnc transcript in an E . coli Deltarnc strain was 3.3-fold higher than in the wild type strain, suggesting that the E . coli RNase III triggers the degradation of the heterologous rnc mRNA . Lac-RNase III is able to cleave an in vitro synthesized mRNA substrate specific for the Bacillus subtilis homolog . Using this substrate, we standardized an enzymatic assay which allows the specific detection of the endonucleolytic activity of Lac-RNase III in L . lactis and E . coli crude extracts.

J Bacteriol, 2004 Oct, 186(19), 6492 - 500
A multifunction ABC transporter (Opt) contributes to diversity of peptide uptake specificity within the genus Lactococcus; Lamarque M et al.; Growth of Lactococcus lactis in milk depends on the utilization of extracellular peptides . Up to now, oligopeptide uptake was thought to be due only to the ABC transporter Opp . Nevertheless, analysis of several Opp-deficient L . lactis strains revealed the implication of a second oligopeptide ABC transporter, the so-called Opt system . Both transporters are expressed in wild-type strains such as L . lactis SK11 and Wg2, whereas the plasmid-free strains MG1363 and IL-1403 synthesize only Opp and Opt, respectively . The Opt system displays significant differences from the lactococcal Opp system, which made Opt much more closely related to the oligopeptide transporters of streptococci than to the lactococcal Opp system: (i) genetic organization, (ii) peptide uptake specificity, and (iii) presence of two oligopeptide-binding proteins, OptS and OptA . The fact that only OptA is required for nutrition calls into question the function of the second oligopeptide binding protein (Opts) . Sequence analysis of oligopeptide-binding proteins from different bacteria prompted us to propose a classification of these proteins in three distinct groups, differentiated by the presence (or not) of precisely located extensions.

Peptides, 2004 Sep, 25(9), 1405 - 14
Quorum sensing control of lantibiotic production; nisin and subtilin autoregulate their own biosynthesis; Kleerebezem M; Lantibiotics are produced by a variety of Gram-positive bacteria . The production of these peptides appears to be regulated at the transcriptional level in a cell-density-dependent manner in various bacteria . This phenomenon has been studied in detail for the production of nisin by Lactococcus lactis, and the production of the structurally similar subtilin by Bacillus subtilis . In this paper, the molecular mechanism underlying regulation of nisin and subtilin production is reviewed . This quorum sensing, autoregulatory module includes the lantibiotics themselves as peptide pheromones, the signal transduction by the corresponding two-component regulatory systems, and the lantibiotic-responsive promoter elements in the biosynthesis gene clusters . Finally, the exploitation of these regulatory characteristics for the development of highly effective controlled gene expression systems in Gram-positive bacteria is discussed.

Syst Appl Microbiol, 2004 Aug, 27(4), 414 - 22
Phylogenetic analysis of Lactococcus lactis subspecies based on decoding the sequence of the pepT tripeptidase gene, the pepV dipeptidase gene and 16S rRNA; Mori S et al.; Tripeptidase (PepT) and dipeptidase (PepV), the enzymes located in the final stage of the intracellular proteolytic system, were demonstrated to be distributed widely in lactic acid bacteria, especially in lactococci . Both the tripeptidase genes (pepT) and dipeptidase genes (pepV) of 15 lactococcal strains consisting of the type and domestic strains were cloned and sequenced using normal and TAIL PCR methods . Amino acid sequences of these enzymes were highly conserved among strains . Evolutionary distance trees based on the sequence of 1239 nucleotides of pepT and 1416 nucleotide of pepV showed a similar cluster as that obtained from the 1499 fragment of the 16S rRNA . Based on this profile, the species Lactococcus lactis is reasonably divided into three subspecies groups, subsp . lactis, cremoris, and hordniae, as in the current classification . Figure of trees from pepT and pepV were essentially identical to each other and slightly more intricate than that from 16S rRNA . The K nuc values obtained from pepT and pepV genes were approximately ten times as high as that from 16S rRNA . Considering these results, phylogenetic analysis based on pepT and pepV genes may aid in a more precise index of classification of L . lactis subspecies . PepT and PepV seem to have evolved in similar directions in lactococci.

Comp Biochem Physiol B Biochem Mol Biol, 2004 Sep, 139(1), 11 - 25
Purification and characterization of lysozyme from plasma of the eastern oyster (Crassostrea virginica); Xue QG et al.; Lysozyme was purified from the plasma of eastern oysters (Crassostrea virginica) using a combination of ion exchange and gel filtration chromatographies . The molecular mass of purified lysozyme was estimated at 18.4 kDa by SDS-PAGE, and its isoelectric point was greater than 10 . Mass spectrometric analysis of the purified enzyme revealed a high-sequence homology with i-type lysozymes . No similarity was found however between the N-terminal sequence of oyster plasma lysozyme and N-terminal sequences of other i-type lysozymes, suggesting that the N-terminal sequences of the i-type lysozymes may vary to a greater extent between species than reported in earlier studies . The optimal ionic strength, pH, cation concentrations, sea salt concentrations, and temperature for activity of the purified lysozyme were determined, as well as its temperature and pH stability . Purified oyster plasma lysozyme inhibited the growth of Gram-positive bacteria (e.g., Lactococcus garvieae, Enterococcus sp.) and Gram-negative bacteria (e.g., Escherichia coli, Vibrio vulnificus) . This is a first report of a lysozyme purified from an oyster species and from the plasma of a bivalve mollusc.

Biochem J, 2005 Jan 15, 385(Pt 2), 419 - 26
Arginine-482 is not essential for transport of antibiotics, primary bile acids and unconjugated sterols by the human breast cancer resistance protein (ABCG2); Janvilisri T et al.; The human BCRP (breast cancer resistance protein, also known as ABCG2) is an ABC (ATP-binding cassette) transporter that extrudes various anticancer drugs from cells, causing multidrug resistance . To study the molecular determinants of drug specificity of BCRP in more detail, we have expressed wild-type BCRP (BCRP-R) and the drug-selected cancer cell line-associated R482G (Arg482-->Gly) mutant BCRP (BCRP-G) in Lactococcus lactis . Drug resistance and the rate of drug efflux in BCRP-expressing cells were proportional to the expression level of the protein and affected by the R482G mutation, pointing to a direct role of BCRP in drug transport in L . lactis . In agreement with observations in mammalian cells, the BCRP-R-mediated transport of the cationic substrates rhodamine 123 and tetramethylrosamine was significantly decreased compared with the activity of BCRP-G . In addition, BCRP-R showed an enhanced interaction with the anionic anticancer drug methotrexate when compared with BCRP-G, suggesting that structure/substrate specificity relationships in BCRP, as observed in eukaryotic expression systems, are maintained in prokaryotic L . lactis . Interestingly, BCRP-R exhibited a previously unestablished ability to transport antibiotics, unconjugated sterols and primary bile acids in L . lactis, for which the R482G mutation was not critical . Since Arg482 is predicted to be present in the intracellular domain of BCRP, close to transmembrane segment 3, our results point to a role of this residue in electrostatic interactions with charged substrates including rhodamine 123 and methotrexate . Since unconjugated sterols are neutral molecules and bile acids and many antibiotics are engaged in protonation/deprotonation equilibria at physiological pH, our observations may point either to a lack of interaction between Arg482 and neutral or neutralized moieties in these substrates during transport or to the interaction of these substrates with regions in BCRP not including Arg482.

Biochemistry, 2004 Sep 21, 43(37), 11782 - 9
Only one of the two annotated Lactococcus lactis fabG genes encodes a functional beta-ketoacyl-acyl carrier protein reductase; Wang H et al.; The small genome of the Gram-positive bacterium Lactococcus lactis ssp . lactis IL1403 contains two genes that encode proteins annotated as homologues of Escherichia coli beta-hydroxyacyl-acyl carrier protein (ACP) reductase . E . coli fabG encodes beta-ketoacyl-acyl carrier protein (ACP) reductase, the enzyme responsible for the first reductive step of the fatty acid synthetic cycle . Both of the L . lactis genes are adjacent to (and predicted to be cotranscribed with) other genes that encode proteins having homology to known fatty acid synthetic enzymes . Such relationships have often been used to strengthen annotations based on sequence alignments . Annotation in the case of beta-ketoacyl-ACP reductase is particularly problematic because the protein is a member of a vast protein family, the short-chain alcohol dehydrogenase/reductase (SDR) family . The recent isolation of an E . coli fabG mutant strain encoding a conditionally active beta-ketoacyl-ACP reductase allowed physiological and biochemical testing of the putative L . lactishomologues . We report that expression of only one of the two L . lactis proteins (that annotated as FabG1) allows growth of the E . coli fabG strain under nonpermissive conditions and restores in vitro fatty acid synthetic ability to extracts of the mutant strain . Therefore, like E . coli, L . lactis has a single beta-ketoacyl-ACP reductase active with substrates of all fatty acid chain lengths . The second protein (annotated as FabG2), although inactive in fatty acid synthesis both in vivo and in vitro, was highly active in reduction of the model substrate, beta-ketobutyryl-CoA . As expected from work on the E . coli enzyme, the FabG1 beta-ketobutyryl-CoA reductase activity was inhibited by ACP (which blocks access to the active site) whereas the activity of FabG2 was unaffected by the presence of ACP . These results seem to be an example of a gene duplication event followed by divergence of one copy of the gene to encode a protein having a new function.

FEMS Microbiol Lett, 2004 Sep 15, 238(2), 367 - 74
Biochemical and molecular characterization of alpha-ketoisovalerate decarboxylase, an enzyme involved in the formation of aldehydes from amino acids by Lactococcus lactis; de la Plaza M et al.; In this paper, we report for the first time on the identification, purification, and characterization of the alpha-ketoisovalerate decarboxylase from Lactococcus lactis, a novel enzyme responsible for the decarboxylation into aldehydes of alpha-keto acids derived from amino acid transamination . The kivd gene consisted of a 1647 bp open reading frame encoding a putative peptide of 61 kDa . Analysis of the deduced amino acid sequence indicated that the enzyme is a non-oxidative thiamin diphosphate (ThDP)-dependent alpha-keto acid decarboxylase included in the pyruvate decarboxylase group of enzymes . The active enzyme is a homo-tetramer that showed optimum activity at 45 degrees C and at pH 6.5 and exhibited an inhibition pattern typical for metal-dependant enzymes . In addition to Mg(2+), activity was observed in presence of other divalent cations such as Ca(2+), Co(2+) and Mn(2+) . The enzyme showed the highest specific activity (80.7 Umg(-1)) for alpha-ketoisovalerate, an intermediate metabolite in valine and leucine biosynthesis . On the other side, decarboxylation of indole-3-pyruvate and pyruvate only could be detected by a 100-fold increase in the enzyme concentration present in the reaction.

Appl Environ Microbiol, 2004 Sep, 70(9), 5546 - 56
Lactococcal plasmid pNP40 encodes a novel, temperature-sensitive restriction-modification system; O'Driscoll J et al.; A novel restriction-modification system, designated LlaJI, was identified on pNP40, a naturally occurring 65-kb plasmid from Lactococcus lactis . The system comprises four adjacent similarly oriented genes that are predicted to encode two m(5)C methylases and two restriction endonucleases . The LlaJI system, when cloned into a low-copy-number vector, was shown to confer resistance against representatives of the three most common lactococcal phage species . This phage resistance phenotype was found to be strongly temperature dependent, being most effective at 19 degrees C . A functional analysis confirmed that the predicted methylase-encoding genes, llaJIM1 and llaJIM2, were both required to mediate complete methylation, while the assumed restriction enzymes, specified by llaJIR1 and llaJIR2, were both necessary for the complete restriction phenotype . A Northern blot analysis revealed that the four LlaJI genes are part of a 6-kb operon and that the relative abundance of the LlaJI-specific mRNA in the cells does not appear to contribute to the observed temperature-sensitive profile . This was substantiated by use of a LlaJI promoter-lacZ fusion, which further revealed that the LlaJI operon appears to be subject to transcriptional regulation by an as yet unidentified element(s) encoded by pNP40.

Appl Environ Microbiol, 2004 Sep, 70(9), 5477 - 84
The pool of ADP and ATP regulates anaerobic product formation in resting cells of Lactococcus lactis; Palmfeldt J et al.; Lactococcus lactis grows homofermentatively on glucose, while its growth on maltose under anaerobic conditions results in mixed acid product formation in which formate, acetate, and ethanol are formed in addition to lactate . Maltose was used as a carbon source to study mixed acid product formation as a function of the growth rate . In batch and nitrogen-limited chemostat cultures mixed acid product formation was shown to be linked to the growth rate, and homolactic fermentation occurred only in resting cells . Two of the four lactococcal strains investigated with maltose, L . lactis 65.1 and MG1363, showed more pronounced mixed acid product formation during growth than L . lactis ATCC 19435 or IL-1403 . In resting cell experiments all four strains exhibited homolactic fermentation . In resting cells the intracellular concentrations of ADP, ATP, and fructose 1,6-bisphosphate were increased and the concentration of P(i) was decreased compared with the concentrations in growing cells . Addition of an ionophore (monensin or valinomycin) to resting cultures of L . lactis 65.1 induced mixed acid product formation concomitant with decreases in the ADP, ATP, and fructose 1,6-bisphosphate concentrations . ADP and ATP were shown to inhibit glyceraldehyde-3-phosphate dehydrogenase, lactate dehydrogenase, and alcohol dehydrogenase in vitro . Alcohol dehydrogenase was the most sensitive enzyme and was totally inhibited at an adenine nucleotide concentration of 16 mM, which is close to the sum of the intracellular concentrations of ADP and ATP of resting cells . This inhibition of alcohol dehydrogenase might be partially responsible for the homolactic behavior of resting cells . A hypothesis regarding the level of the ATP-ADP pool as a regulating mechanism for the glycolytic flux and product formation in L . lactis is discussed.

Appl Environ Microbiol, 2004 Sep, 70(9), 5398 - 406
New expression system tightly controlled by zinc availability in Lactococcus lactis; Llull D et al.; Here we developed the new expression system P(Zn) zitR, based on the regulatory signals (P(Zn) promoter and zitR repressor) of the Lactococcus lactis zit operon, involved in Zn(2+) high-affinity uptake and regulation . A P(Zn) zitR-controlled expression vector was constructed, and expression regulation was studied with two reporter genes, uspnuc and lacLM; these genes encode, respectively, a protein derived from Staphylococcus aureus secreted nuclease and Leuconostoc mesenteroides cytoplasmic beta-galactosidase . Nuclease and beta-galactosidase activities of L . lactis MG1363 cells expressing either uspnuc or lacLM under the control of P(Zn) zitR were evaluated on plates and quantified from liquid cultures as a function of divalent metal ion, particularly Zn(2+), availability in the environment . Our results demonstrate that P(Zn) zitR is highly inducible upon divalent cation starvation, obtained either through EDTA addition or during growth in chemically defined medium, and is strongly repressed in the presence of excess Zn(2+) . The efficiency of the P(Zn) zitR expression system was compared to that of the well-known nisin-controlled expression (NICE) system with the same reporter genes cloned under either P(Zn) zitR or P(nisA) nisRK control . lacLM induction levels reached with both systems were on the same order of magnitude, even though the NICE system is fivefold more efficient than the P(Zn) zitR system . An even smaller difference or no difference was observed after 3 h of induction when nuclease was used as a reporter for Western blotting detection . P(Zn) zitR proved to be a powerful expression system for L . lactis, as it is tightly controlled by the zinc concentration in the medium.

Appl Environ Microbiol, 2004 Sep, 70(9), 5132 - 7
Clonality and diversity of the fish pathogen Lactococcus garvieae in Mediterranean countries; Eyngor M et al.; Infection with Lactococcus garvieae is considered the most important risk factor for the European trout industry, and the losses are approximately 50% of the total production . To improve our understanding of the genetic links among strains originating from different countries, we examined the population structure of L . garvieae by comparing 81 strains isolated from different sources and ecosystems (41 farms in six countries) in which the bacterium is commonly found . Genetic similarities (as assessed with molecular tools, including restriction fragment length polymorphism ribotyping with two endonucleases) were compared with serological data . The combined results reveal that in endemic sites the bacterial population displays a clonal structure, whereas bacterial diversity characterizes sites where the infection is sporadic.

Arch Microbiol, 2004 Oct, 182(2-3), 175 - 81 Epub 2004 Aug 31.
Ferrihydrite reduction by Geobacter species is stimulated by secondary bacteria; Straub KL et al.; Geobacter species such as G . bremensis, G . pelophilus, and G . sulfurreducens are obligately anaerobic and grow in anoxic, non-reduced medium by fast reduction of soluble ferric citrate . In contrast, insoluble ferrihydrite was either only slowly or not reduced when supplied as electron acceptor in similar growth experiments . Ferrihydrite reduction was stimulated by addition of a reducing agent or by concomitant growth of secondary bacteria that were physiologically and phylogenetically as diverse as Escherichia coli, Lactococcus lactis, or Pseudomonas stutzeri . In control experiments with heat-inactivated Geobacter cells and viable secondary bacteria, no ( E . coli, P . stutzeri) or only little ( L . lactis) ferrihydrite was reduced . Redox indicator dyes showed that growing E . coli, P . stutzeri, or L . lactis cells lowered the redox potential of the medium in a similar way as a reducing agent did . The lowered redox potential was presumably the key factor that stimulated ferrihydrite reduction by all three Geobacter species . The observed differences in anoxic non-reduced medium with ferric citrate versus ferrihydrite as electron acceptor indicated that reduction of these electron acceptors involved different cellular components or different biochemical strategies . Furthermore, it appears that redox-sensitive components are involved, and/or that gene expression of components needed for ferrihydrite reduction is controlled by the redox state.

FEMS Microbiol Lett, 2004 Aug 15, 237(2), 279 - 88
Expression of the Staphylococcus aureus surface proteins HtrA1 and HtrA2 in Lactococcus lactis; Rigoulay C et al.; Staphylococcus aureus encodes two HtrA-like serine surface proteases, called HtrA1 and HtrA2 . The roles of these HtrA homologs were distinguished by expression studies in a heterologous host, Lactococcus lactis, whose single extracellular protease, HtrA(Ll), was absent . HtrA(Ll) is involved in stress resistance, and processing and/or degradation of extracellular proteins . Controlled expression of staphylococcal htrA1 and htrA2 was achieved in L . lactis strain NZ9000 DeltahtrA, as confirmed with anti-HtrA1 and anti-HtrA2 specific antibodies . HtrA1 fully restored thermo-resistance to the htrA-defective L . lactis strain, despite a poor capacity to degrade abnormal or truncated proteins . We therefore propose that activities of HtrA1 other than proteolysis may be sufficient for high-temperature growth complementation . HtrA2 is 36% identical to HtrA(Ll), and was highly expressed in L . lactis; nevertheless, it displayed nearly no detectable activities . The poor proteolytic activities of staphylococcal HtrA proteins in L . lactis may reflect a requirement for S . aureus-specific co-factors.

J Bacteriol, 2004 Sep, 186(17), 5649 - 60
Acid-inducible transcription of the operon encoding the citrate lyase complex of Lactococcus lactis Biovar diacetylactis CRL264; Martin MG et al.; Although Lactococcus is one of the most extensively studied lactic acid bacteria and is the paradigm for biochemical studies of citrate metabolism, little information is available on the regulation of the citrate lyase complex . In order to fill this gap, we characterized the genes encoding the subunits of the citrate lyase of Lactococcus lactis CRL264, which are located on an 11.4-kb chromosomal DNA region . Nucleotide sequence analysis revealed a cluster of eight genes in a new type of genetic organization . The citM-citCDEFXG operon (cit operon) is transcribed as a single polycistronic mRNA of 8.6 kb . This operon carries a gene encoding a malic enzyme (CitM, a putative oxaloacetate decarboxylase), the structural genes coding for the citrate lyase subunits (citD, citE, and citF), and the accessory genes required for the synthesis of an active citrate lyase complex (citC, citX, and citG) . We have found that the cit operon is induced by natural acidification of the medium during cell growth or by a shift to media buffered at acidic pHs . Between the citM and citC genes is a divergent open reading frame whose expression was also increased at acidic pH, which was designated citI . This inducible response to acid stress takes place at the transcriptional level and correlates with increased activity of citrate lyase . It is suggested that coordinated induction of the citrate transporter, CitP, and citrate lyase by acid stress provides a mechanism to make the cells (more) resistant to the inhibitory effects of the fermentation product (lactate) that accumulates under these conditions.

Scand J Infect Dis, 2004, 36(6-7), 490 - 1
Liver abscess caused by Lactococcus lactis cremoris: a new pathogen; Antolin J et al.; We describe the first case reported in the literature of liver abscess due to Lactococcus lactis cremoris in an immunocompetent adult patient . The patient was treated with catheter drainage and antibiotics, which resulted in improvement and resolution.

Langmuir, 2004 Aug 17, 20(17), 6985 - 7
Lipid-mediated light activation of a mechanosensitive channel of large conductance; Folgering JH et al.; This paper describes the reversible activation of a mechanosensitive channel via a light-sensitive lipid mimic . For these experiments, the mechanosensitive channel of large conductance (MscL) protein from Lactococcus lactis and Escherichia coli was reconstituted in lipid bilayers composed of 80 mol % 1,2-dioleoyl-sn-glycero-3-phosphocholine and 20 mol % di-(5-{{4-(4-butylphenyl)azo}phenoxy}pentyl)phosphate (4-Azo-5P) . Light-induced isomerization of the azobenzene moiety of 4-Azo-5P from trans to cis was used to activate MscL.

Gastroenterology, 2004 Aug, 127(2), 502 - 13
Active delivery of trefoil factors by genetically modified Lactococcus lactis prevents and heals acute colitis in mice; Vandenbroucke K et al.; BACKGROUND & AIMS: Effective therapeutics for treating acute colitis, caused by disruption of the intestinal epithelial barrier, are scarce . Trefoil factors (TFF) are cytoprotective and promote epithelial wound healing and reconstitution of the gastrointestinal tract, which makes them good candidate therapeutics for acute colitis . However, orally administered TFF stick to the mucus of the small intestine and are absorbed at the cecum . METHODS: We have engineered the food-grade bacterium Lactococcus lactis to secrete bioactive murine TFF . The protective and therapeutic potentials of these TFF-secreting L . lactis were evaluated in parallel with purified TFF in the dextran sodium sulfate (DSS)-induced murine model for acute colitis and in established chronic colitis in interleukin (IL)-10(-/-) mice . Disease was evaluated by blinded macroscopic and microscopic inflammatory scores and by myeloperoxidase activity . RESULTS: Intragastric administration of TFF-secreting L . lactis led to active delivery of TFF at the mucosa of the colon and, in contrast to administration of purified TFF, proved to be very effective in prevention and healing of acute DSS-induced colitis . The in situ secreted murine TFF significantly decreased morbidity and mortality and stimulated prostaglandin-endoperoxide synthase 2 expression, which represents a major therapeutic pathway . In addition, this approach was successful in improving established chronic colitis in IL-10(-/-) mice . CONCLUSIONS: We have positively evaluated a new therapeutic approach for acute and chronic colitis that involves in situ secretion of murine TFF by orally administered L . lactis . This novel approach may lead to effective management of acute and chronic colitis and epithelial damage in humans.

Acta Crystallogr D Biol Crystallogr, 1997 Nov, 53(Pt 6), 802 - 4
Crystallization and preliminary X-ray diffraction analysis of the heterotetrameric dihydroorotate dehydrogenase B of Lactococcus lactis, a flavoprotein enzyme system consisting of two PyrDB subunits and two iron-sulfur cluster containing PyrK subunits; Rowland P; Dihydroorotate dehydrogenases are flavin-containing enzymes which catalyze the conversion of (S)-dihydroorotate to orotate . Dihydroorotate dehydrogenase B (DHODB) from Lactococcus lactis is a heterotetramer containing two subunits of the protein encoded by the pyrDb gene (PyrDB) and two subunits of the protein encoded by the pyrK gene (PyrK) . In addition, DHODB contains two molecules of flavin mononucleotide, two molecules of flavin adenine dinucleotide and two {2Fe-2S} iron-sulfur clusters as tightly bound cofactors . Yellow crystals of this enzyme have been grown using the hanging-drop vapour-diffusion technique from solutions of 2.5 M ammonium sulfate and 0.1 M sodium acetate, pH 4.6 . The crystals have been shown to contain both the PyrDB and the PyrK subunits and fluorescence measurements indicate that the two different subunits interact very closely with each other in the active-site region . Native data sets have been collected to 2.6 A with a conventional X-ray source and to 2.2 A using synchrotron radiation . The crystals are rhombohedral, space group R32, with correspondin8 hexagonal unit-cell dimensions a = b = 202.3 and c = 81.0 A . The asymmetric unit in the crystal contains one PyrDB subunit and one PyrK subunit, which suggests that the two halves of the heterotetramer are related by a crystallographic twofold axis.

Acta Crystallogr D Biol Crystallogr, 1996 Nov, 52(Pt 6), 1199 - 201
Crystallization and preliminary structural studies of lactose-specific enzyme IIA from Lactococcus lactis; Sliz P; Lactose-specific enzyme IIA of the phosphoenol:pyruvate-dependent sugar phosphotransferase system from Lactococcus lactis has been crystallized in phosphate buffer . The crystals belong to space group P4(1)2(1)2 or its enantiomorph P4(3)2(1)2 with unit-cell axes a = b = 90.9 and c = 82.4 A . The packing parameter (Matthews parameter) V(m) of 2.48 A(3) Da(-1) is consistent with one trimer per asymmetric unit and non-crystallographic threefold symmetry has been confirmed by calculating a selfrotation function . The crystals diffract X-rays to at least 2.3 A resolution, are stable in an X-ray beam and are therefore appropriate for structure determination . Native data to 2.3 A resolution have been collected using a MAR image-plate system at a synchrotron source . One isomorphous heavy-atom derivative has been identified and the presence of an isomorphous signal in the data has been confirmed by Patterson methods.

J Clin Microbiol, 2004 Aug, 42(8), 3686 - 95
Use of tuf sequences for genus-specific PCR detection and phylogenetic analysis of 28 streptococcal species; Picard FJ et al.; A 761-bp portion of the tuf gene (encoding the elongation factor Tu) from 28 clinically relevant streptococcal species was obtained by sequencing amplicons generated using broad-range PCR primers . These tuf sequences were used to select Streptococcus-specific PCR primers and to perform phylogenetic analysis . The specificity of the PCR assay was verified using 102 different bacterial species, including the 28 streptococcal species . Genomic DNA purified from all streptococcal species was efficiently detected, whereas there was no amplification with DNA from 72 of the 74 nonstreptococcal bacterial species tested . There was cross-amplification with DNAs from Enterococcus durans and Lactococcus lactis . However, the 15 to 31% nucleotide sequence divergence in the 761-bp tuf portion of these two species compared to any streptococcal tuf sequence provides ample sequence divergence to allow the development of internal probes specific to streptococci . The Streptococcus-specific assay was highly sensitive for all 28 streptococcal species tested (i.e., detection limit of 1 to 10 genome copies per PCR) . The tuf sequence data was also used to perform extensive phylogenetic analysis, which was generally in agreement with phylogeny determined on the basis of 16S rRNA gene data . However, the tuf gene provided a better discrimination at the streptococcal species level that should be particularly useful for the identification of very closely related species . In conclusion, tuf appears more suitable than the 16S ribosomal RNA gene for the development of diagnostic assays for the detection and identification of streptococcal species because of its higher level of species-specific genetic divergence.

Appl Environ Microbiol, 2004 Aug, 70(8), 5051 - 3
Nisin-producing Lactococcus lactis strains isolated from human milk; Beasley SS et al.; Characterization by partial 16S rRNA gene sequencing, ribotyping, and green fluorescent protein-based nisin bioassay revealed that 6 of 20 human milk samples contained nisin-producing Lactococcus lactis bacteria . This suggests that the history of humans consuming nisin is older than the tradition of consuming fermented milk products.

Appl Environ Microbiol, 2004 Aug, 70(8), 5030 - 2
Nisin-controlled production of pediocin PA-1 and colicin V in nisin- and non-nisin-producing Lactococcus lactis strains; Horn N et al.; The introduction of chimeric genes encoding the fusion leader of lactococcin A-propediocin PA-1 or procolicin V under the control of the inducible nisA promoter and the lactococcin A-dedicated secretion genes (lcnCD) into Lactococcus lactis strains, including a nisin producer, expressing the two component regulator NisRK led to the production or pediocin PA-1 or colicin V, respectively.

J Dairy Sci, 2004 May, 87(5), 1151 - 7
Predominant microflora of downgraded Danish bulk tank milk; Holm C et al.; The microflora of downgraded Danish bulk tank milk was examined to identify the main causes of increased microbial counts . Seventy-five representative samples with a microbial count exceeding 3.0 x 10(4) cfu/mL were selected for a more detailed microbial examination . A total of 1237 isolates from these samples were identified . Gram-negative, oxidase-positive bacteria were found in 72% of the samples . Coliforms were found in 20% of the samples, and non-coliforms were found in 49% of the samples . Coryneforms, other gram-positive rods, Lactococcus spp., Micrococcus spp., and coagulase-negative Staphylococcus spp . were found in 28 to 53% of the samples . Bacillus spp., Enterococcus spp., Staphylococcus aureus, Streptococcus dysgalactiae, Streptococcus uberis, and yeasts were found in <25% of the samples . Additionally, the isolates were divided into 3 groups, based on the main cause of an elevated microbial count . Microorganisms primarily associated with poor hygiene dominated the microflora in 64% of the samples; bacteria also related to poor hygiene, but in addition associated with growth at low temperatures (psychrotrophic bacteria) dominated the microflora in 28% of the samples; and bacteria mainly associated with mastitis dominated the microflora in 8% of the samples . A bulk tank milk storage period of 48 h instead of 24 h did not affect the proportion of downgraded milk samples and could not be associated with a specific group of microorganisms . Further, no relationship was found between somatic cell counts and the presence of mastitis bacteria.

Microbiology, 2004 Aug, 150(Pt 8), 2663 - 8
Expression of mptC of Listeria monocytogenes induces sensitivity to class IIa bacteriocins in Lactococcus lactis; Ramnath M et al.; Sensitivity to class IIa bacteriocins from lactic acid bacteria was recently associated with the mannose phosphotransferase system (PTS) permease, in Listeria monocytogenes . To assess the involvement of this protein complex in class IIa bacteriocin activity, the mptACD operon, encoding, was heterologously expressed in an insensitive species, namely Lactococcus lactis, using the NICE double plasmid system . Upon induction of the cloned operon, the recombinant Lc . lactis became sensitive to leucocin A . Pediocin PA-1 and enterocin A also showed inhibitory activity against Lc . lactis cultures expressing mptACD . Furthermore, the role of the three genes of the mptACD operon was investigated . Derivative plasmids containing various combinations of these three genes were made from the parental mptACD plasmid by divergent PCR . The results showed that expression of mptC alone is sufficient to confer sensitivity to class IIa bacteriocins in Lc . lactis.

Microbiology, 2004 Aug, 150(Pt 8), 2503 - 12
Controlled expression of CluA in Lactococcus lactis and its role in conjugation; Stentz R et al.; CluA is a 136 kDa surface-bound protein encoded by the chromosomally located sex factor of Lactococcus lactis MG1363 and is associated with cell aggregation linked to high-frequency transfer of the sex factor . To further investigate the involvement of CluA in these phenomena, the cluA gene was cloned on a plasmid, downstream from the lactococcal nisA promoter . In a sex-factor-negative MG1363 derivative, nisin-controlled CluA expression resulted in aggregation, despite the absence of the other genes of the sex factor . Therefore, CluA is the only sex factor component responsible for aggregation . The direct involvement of CluA in the establishment of cell-to-cell contact for aggregate formation was observed by electron microscopy using immunogold-labelled CluA antibodies . Inactivation of cluA in an MG1363 background led to a dramatic decrease in sex factor conjugation frequency compared to the parental strain . Increasing levels of CluA expressed in trans in the cluA-inactivated donor strain facilitated a gradual restoration of conjugation frequency, reaching that of the parental strain . In conclusion, CluA is essential for efficient sex factor transfer in conjugation of L . lactis.

RNA, 2004 Sep, 10(9), 1433 - 43 Epub 2004 Jul 23.
High-affinity binding site for a group II intron-encoded reverse transcriptase/maturase within a stem-loop structure in the intron RNA; Watanabe K et al.; Mobile group II introns encode proteins that have reverse transcriptase and maturase activities and bind specifically to the intron RNA to promote both RNA splicing and intron mobility . Previous studies with the Lactococcus lactis Ll.LtrB intron showed that the intron-encoded protein (LtrA) has a high-affinity binding site in intron subdomain DIVa, an idiosyncratic structure containing the translation initiation region of the LtrA open reading frame, and that this binding site consists of a small stem-loop emanating from a purine-rich internal loop . The binding of LtrA to DIVa is important for translational regulation, RNA splicing, and intron mobility . Here, we show by in vitro selection that part of the purine-rich internal loop can be closed by base pairing, enabling the LtrA binding site to be represented as an extended stem-loop structure with a bulged A (A556) required for tight binding of LtrA . The deletion or pairing of A556 has relatively little effect on maturase-promoted RNA splicing, but significantly inhibits intron mobility . The wild-type DIVa structure has a second bulged A (A553), which is selected against in tightly binding variants . As expected from the selection, the deletion or pairing of A553 results in tighter binding of LtrA, but surprisingly, also inhibits intron mobility . These findings suggest that the binding of LtrA to DIVa is delicately balanced, so that either too weak or too tight binding can be deleterious . The nature of the maturase/DIVa interaction and its role in translational regulation are reminiscent of the coat protein/RNA hairpin interactions of single-stranded RNA phages.

Protein Sci, 2004 Aug, 13(8), 2009 - 21
Insights into the DNA repair process by the formamidopyrimidine-DNA glycosylase investigated by molecular dynamics; Amara P et al.; Formamidopyrimidine-DNA glycosylase (Fpg) identifies and removes 8-oxoguanine from DNA . All of the X-ray structures of Fpg complexed to an abasic site containing DNA exhibit a common disordered region present in the C-terminal domain of the enzyme . However, this region is believed to be involved in the damaged base binding site when the initial protein/DNA complex is formed . The dynamic behavior of the disordered polypeptide (named Loop) in relation to the supposed scenario for the DNA repair mechanism was investigated by molecular dynamics on different models, derived from the X-ray structure of Lactococcus lactis Fpg bound to an abasic site analog-containing DNA and of Bacillus stearothermophilus Fpg bound to 8-oxoG . This study shows that the presence of the damaged base influences the dynamics of the whole enzyme and that the Loop location is dependent on the presence and on the conformation of the 8-oxoG in its binding site . In addition, from our results, the conformation of the 8-oxoG seems to be favored in syn in the L . lactis models, in agreement with the available X-ray structure from B . stearothermophilus Fpg and with a possible catalytic role of the flexibility of the Loop region.

FEMS Microbiol Lett, 2004 Aug 1, 237(1), 147 - 56
Occurrence of tetracycline resistance genes tet(M) and tet(S) in bacteria from marine aquaculture sites; Kim SR et al.; Occurrence of tetracycline resistance genes encoding ribosomal protection proteins was examined in 151 tetracycline-resistant bacterial isolates from fish and seawater at coastal aquaculture sites in Japan and Korea . The tet(M) gene was detected in 34 Japanese and Korean isolates, which included Vibrio sp., Lactococcus garvieae, Photobacterium damsela subsp . piscicida, and unidentified Gram-positive bacteria . The majority of these bacterial isolates displayed high-level resistance with a minimum inhibitory concentrations (MICs) equal to or greater than 250 microg/ml of oxytetracycline and only four isolates had MICs less than 31.3 microg/ml . 16S rDNA RFLP typing of tet(M)-positive Vibrio isolates suggests that these are clonal populations of the same phylotype specific to a particular location . One Vibrio clone (phylotype III), however, is widely disseminated, being detected during different sampling years, at different locations, and in different fish species in both Japan and Korea . The tet(S) gene was detected in L . garvieae from yellowtail in Japan and in Vibrio sp . from seawater in Korea . This is the first report of tet(S) occurrence in Gram-negative facultative anaerobes . These results suggest that tet(M) and tet(S) genes are present in fish intestinal and seawater bacteria at aquaculture sites and could be an important reservoir of tetracycline resistance genes in the marine environment.

Genet Mol Res, 2004 Jun 30, 3(2), 273 - 81
Genetic organization and expression of citrate permease in lactic acid bacteria; Drider D et al.; Citrate is present in many natural substrates, such as milk, vegetables and fruits, and its metabolism by lactic acid bacteria (LAB) plays an important role in food fermentation . The industrial importance of LAB stems mainly from their ability to convert carbohydrates into lactic acid and, in some species, like Lactococcus lactis and Leuconostoc mesenteroides, to produce C4 flavor compounds (diacetyl, acetoin) through citrate metabolism . Three types of genetic organization and gene locations, involving citrate metabolism, have been found in LAB . Citrate uptake is mediated by a citrate permease, which leads to a membrane potential upon electrogenic exchange of divalent citrate and monovalent lactate . The internal citrate is cleaved into acetate and oxaloacetate by a citrate lyase, and oxaloacetate is decarboxylated into pyruvate by an oxaloacetate decarboxylase, yielding a pH gradient through the consumption of scalar protons.

FEBS Lett, 2004 Jul 16, 570(1-3), 217 - 22
Characterization of an oxaloacetate decarboxylase that belongs to the malic enzyme family; Sender PD et al.; The citM gene from Lactococcus lactis CRL264 was demonstrated to encode for an oxaloacetate decarboxylase . The enzyme exhibits high levels of similarity to malic enzymes (MEs) from other organisms . CitM was expressed in Escherichia coli, purified and its oxaloacetate decarboxylase activity was demonstrated by biochemical and genetic studies . The highest oxaloacetate decarboxylation activity was found at low pH in the presence of manganese, and the Km value for oxaloacetate was 0.52+/-0.03 mM . However, no malic activity was found for this enzyme . Our studies clearly show a new group of oxaloacetate decarboxylases associated with the citrate fermentation pathway in gram-positive bacteria . Furthermore, the essential catalytic residues were found to be conserved in all members of the ME family, suggesting a common mechanism for oxaloacetate decarboxylation.

J Biol Chem, 2004 Oct 15, 279(42), 44074 - 83 Epub 2004 Jul 10.
Structural basis for the recognition of the FapydG lesion (2,6-diamino-4-hydroxy-5-formamidopyrimidine) by formamidopyrimidine-DNA glycosylase; Coste F et al.; Formamidopyrimidine-DNA glycosylase (Fpg) is a DNA repair enzyme that excises oxidized purines such as 7,8-dihydro-8-oxoguanine (8-oxoG) and 2,6-diamino-4-hydroxy-5-formamidopyrimidine (FapyG) from damaged DNA . Here, we report the crystal structure of the Fpg protein from Lactococcus lactis (LlFpg) bound to a carbocyclic FapydG (cFapydG)-containing DNA . The structure reveals that Fpg stabilizes the cFapydG nucleoside into an extrahelical conformation inside its substrate binding pocket . In contrast to the recognition of the 8-oxodG lesion, which is bound with the glycosidic bond in a syn conformation, the cFapydG lesion displays in the complex an anti conformation . Furthermore, Fpg establishes interactions with all the functional groups of the FapyG base lesion, which can be classified in two categories: (i) those specifying a purine-derived lesion (here a guanine) involved in the Watson-Crick face recognition of the lesion and probably contributing to an optimal orientation of the pyrimidine ring moiety in the binding pocket and (ii) those specifying the imidazole ring-opened moiety of FapyG and probably participating also in the rotameric selection of the FapydG nucleobase . These interactions involve strictly conserved Fpg residues and structural water molecules mediated interactions . The significant differences between the Fpg recognition modes of 8-oxodG and FapydG provide new insights into the Fpg substrate specificity.

J Appl Microbiol, 1998 Jan, 84(1), 90 - 6
Cell membrane integrity and lysis in Lactococcus lactis: the detection of a population of permeable cells in post-logarithmic phase cultures; Niven GW et al.; A method was developed that enabled an analysis of the proportion of permeable cells in a culture of Lactococcus lactis . This used the fluorescence of propidium iodide (PI) when in contact with DNA and the impermeability of the intact cell membrane to this compound . A permeability index was suggested that expresses the PI-induced fluorescence of a cell suspension as a percentage of the value obtained from wholly permeabilized cells after treatment with cetyltrimethylammonium bromide . This method was applied to the determination of cell permeability in death phase cultures . A large proportion of unlysed cells was freely permeable to PI, a finding that may have some significance for the investigation of the role of cell lysis in cheese maturation . This method is suggested as a useful addition to the techniques available for the study of cell damage in a variety of fields, and for the screening of cheese starter bacteria.

Appl Environ Microbiol, 2004 Jul, 70(7), 4318 - 25
Restoration of a defective Lactococcus lactis xylose isomerase; Park JH et al.; The genes (xylA) encoding xylose isomerase (XI) from two Lactococcus lactis subsp . lactis strains, 210 (Xyl(-)) and IO-1 (Xyl(+)), were cloned, and the activities of their expressed proteins in recombinant strains of Escherichia coli were investigated . The nucleotide and amino acid sequence homologies between the xylA genes were 98.4 and 98.6%, respectively, and only six amino acid residues differed between the two XIs . The purified IO-1 XI was soluble with K(m) and k(cat) being 2.25 mM and 184/s, respectively, while the 210 XI was insoluble and inactive . Site-directed mutagenesis on 210 xylA showed that a triple mutant possessing R202M/Y218D/V275A mutations regained XI activity and was soluble . The K(m) and k(cat) of this mutant were 4.15 mM and 141/s, respectively . One of the IO-1 XI mutants, S388T, was insoluble and showed negligible activity similar to that of 210 XI . The introduction of a K407E mutation to the IO-1 S388T XI mutant restored its activity and solubility . The dissolution of XI activity in L . lactis subsp . lactis involves a series of mutations that collectively eliminate enzyme activity by reducing the solubility of the enzyme.

J Appl Microbiol, 2004, 97(2), 306 - 13
Development of a high throughput screening method to test flavour-forming capabilities of anaerobic micro-organisms; Smit BA et al.; AIM: Development of a fast, automated and reliable screening method for screening of large collections of bacterial strains with minimal handling time . METHODS AND RESULTS: The method is based on the injection of a small headspace sample (100 microl) from culture vials (2 ml) in 96-well format directly into the mass spectrometry (MS) . A special sample tray has been developed for liquid media, and anaerobically grown cultures . In principle, all volatile components can be measured, but a representative mass fragment has to be obtained in the MS . Representative masses for 3-methylbutanal, 2-methylpropanal and benzaldehyde are 58, 72 and 105, respectively . In 1 day over 1500 samples could be analysed and the coefficient of variation for the response was <5% . CONCLUSION: Screening of 72 strains belonging to the genus Lactococcus in quadruple on the production of the key-flavour compound 3-methylbutanal illustrated the effectiveness of the method . Furthermore, knowledge of the biochemistry and physiology of 3-methylbutanal formation was used to optimize the composition of the growth medium to enhance 3-methylbutanal production, and thereby improve the screening . SIGNIFICANCE AND IMPACT OF THE STUDY: A commonly used method to control flavour formation in fermented food products is the selection of bacterial strains, which are able to produce the desired flavour compounds . As large collections of strains are available for such screenings, studying biodiversity of micro-organisms on the level of metabolic routes is strongly facilitated by highly automated high throughput screening methods for measuring enzyme activities or production of metabolites . Therefore, this method will be a useful tool for selecting flavour-producing strains and for enhancing starter culture development.

Virology, 2004 Jul 20, 325(1), 82 - 95
Diversity in the lysis-integration region of oenophage genomes and evidence for multiple tRNA loci, as targets for prophage integration in Oenococcus oeni; Sao-Jose C et al.; The central genomic regions of Oenococcus oeni phages fOg30 and fOgPSU1 have been compared with the equivalent regions of oenophages fOg44 and phi 10MC . In all cases, an almost identical endolysin gene was followed by one of two orfs, encoding putative holins (orf117 and orf163) . The fOg44 endolysin was established as a secretory protein when expressed in Lactococcus lactis . Orf117 (from fOg44) promoted lysis of Escherichia coli cultures upon induction of a defective lambda Sam7 prophage, but Orf163 (from fOg30) failed to elicit a lysis response in this system . fOg44 and fOgPSU1 were shown to integrate at the 3' end of a tRNA(Glu) and a tRNA(Lys), respectively . Searching the available sequence of the O . oeni MCW genome for attP-like elements, two other tRNA targets could be proposed for prophage establishment . Between the lysis and integration elements, a diverse cluster of genes (absent in phi 10MC) was observed . One common gene in this "lysogenic conversion cluster" was experimentally confirmed as a transcriptional repressor, affecting the expression of a putative permease gene.

Mol Microbiol, 2004 Jul, 53(2), 613 - 21
Intracellular effectors regulating the activity of the Lactococcus lactis CodY pleiotropic transcription regulator; Petranovic D et al.; CodY is a pleiotropic transcriptional regulator conserved in low-G+C Gram-positive bacteria . Two distinct signals have been shown independently to influence the activity of this regulator: the level of intracellular GTP in Bacillus subtilis and the level of intracellular branched-chain amino acids (BCAA) isoleucine, leucine and valine in Lactococcus lactis . Measurement of BCAA and GTP levels in several environmental conditions showed that L . lactis CodY responded to the intracellular BCAA concentrations but not to physiological fluctuations in intracellular GTP . In addition, we demonstrated that CodY from L . lactis did not respond to intracellular GTP even when complementing CodY activity in B . subtilis . However, L . lactis CodY activity could still be modulated in B . subtilis by adding a rich nitrogen source to the growth media . This finding suggests that only BCAA are sensed by L . lactis CodY, whereas both GTP and BCAA signals may be integrated by B . subtilis CodY . The difference in the function of CodY from B . subtilis and L . lactis seems to reflect the difference in the physiology of these two bacteria.

Gene, 2004 Jul 7, 336(1), 73 - 80
Engineering large fragment insertions into the chromosome of Escherichia coli; Rong R et al.; An effective DNA replacement system has been established for engineering large fragment insertions into the chromosome of Escherichia coli . The DNA replacement plasmid, pHybrid I, was first constructed based on the bacterial artificial chromosome (BAC) vector . Two fragments of the E . coli genome, 5.5 and 6.5 kb in length, were introduced into the vector for homologous recombination . In addition to the chloramphenicol gene, a second gene neo was introduced for double marker screening for recombinant clones . By shot-gun cloning and homologous recombination techniques, using our new recombinant vector (pHybrid I), a 20-kb fragment from Lactococcus lactis genomic DNA has been successfully integrated into the chromosome of the E . coli strain J93-140 . Plating tests and PCR amplification indicated that the integration remained stable after many generations in cell culture . This system will be especially useful for the chromosome engineering of large heterologous fragment insertions, which is necessary for pathway engineering.

FEMS Microbiol Lett, 2004 Jul 1, 236(1), 85 - 90
Methylthioacetaldehyde, a possible intermediate metabolite for the production of volatile sulphur compounds from L-methionine by Lactococcus lactis; Bonnarme P et al.; Volatile sulphur compounds (VSCs) production from L-methionine was studied in Lactococcus lactis . In vitro studies with radiolabelled L-methionine and resting cells of L . lactis revealed that L-methionine was initially converted to alpha-keto-gamma-methylthiobutyrate (KMBA) by a transamination reaction . A part of KMBA was subsequently chemically converted to methylthioacetaldehyde, methanethiol and dimethylsulphides . Chemical conversion of KMBA to methylthioacetaldehyde was dependent on pH, Mn(II) and oxygen . Since methanethiol and dimethylsulphide production was highly related to that of methylthioacetaldehyde, the latter compound was proposed as being an intermediate in VSCs production by L . lactis.

FEMS Microbiol Lett, 2004 Jul 1, 236(1), 53 - 60
A new vector, pGID052, for genetic transfer in Oenococcus oeni; Beltramo C et al.; Despite the large number of techniques available for the transformation of bacteria, several species are still resistant to the introduction of foreign DNA . Oenococcus oeni are among the organisms that are particularly refractory to transformation . However, conjugal experiments from Lactococcus lactis to O . oeni with a new plasmid, pGID052, were performed via mobilization with success . This plasmid, a derivative of pORI19, encompasses: (i) the oriT of pIP501, which permitted the transfer to O . oeni, (ii) the replication genes of a native Leuconostoc citreum plasmid . Frequencies of 10(-7) conjugants per recipient were found . The transfer did not affect the structure of this low-copy-number plasmid . Moreover, pGID052 seems segregationally stable and could be used in the future as an expression vector.

J Drug Target, 2003, 11(8-10), 489 - 93
Induction of partial protection in mice after oral administration of Lactococcus lactis producing Brucella abortus L7/L12 antigen; Pontes DS et al.; The Brucella abortus ribosomal protein L7/L12 is an immunodominant antigen and an interesting candidate for the development of oral live vaccines against brucellosis . Here, a recombinant Lactococcus lactis strain producing L7/L12 under the control of nisin inducible promoter was orally administered to BALB/c mice . Significant levels of anti-L7/L12 specific IgA detected in feces revealed an induced local humoral immune response . However, serum analysis did not reveal any anti-L7/L12 antibodies suggesting the absence of a systemic response . Nevertheless, the vaccinated mice showed a partial protective immunity against B . abortus virulent strain (S2308) challenged by intraperitoneal inoculation.

J Dairy Sci, 2004 Mar, 87(3), 574 - 82
Factors affecting calcium lactate and liquid expulsion defects in Cheddar cheese; Swearingen PA et al.; This paper summarizes the results of 2 studies designed to investigate the influence of several manufacturing and curing treatments on the appearance of Cheddar cheese defects . Specifically, 2 defects, calcium lactate crystal formation and the expulsion of free liquid (weeping) were monitored in Cheddar cheese . Both studies were conducted at a commercial cheese manufacturing facility that produces Cheddar in 18.14-kg (40-lb) blocks . In the first study we monitored cheese calcium, both total and soluble during manufacture and early curing . In the second study we measured cheese pH from 3 d through 8 mo, as well as some factors that are influenced by cheese pH . Early cheese pH (3 d to 7 d) patterns were used to select vats of cheese for retail packaging . Mild Cheddar packaged at 30 d postmanufacture and sharp Cheddar packaged at 8 mo postmanufacture from the same vats were monitored for the incidence and severity of the defects . Our results indicated that factors measured in early stages of manufacture and curing (less than 7 d) such as cheese pH at mill, lactic acid concentration, nonprotein nitrogen, and calcium (total and soluble) in cheese did not correlate with the appearance of either calcium lactate or expulsion of free liquid in packaged cheeses . Factors including pH, lactic acid concentrations, and soluble calcium measured during curing (greater than 7 d) of cheese were found to be statistically significant in the development of defects and appeared to be associated with use of specific starter culture groups . In the study, 5 different starter culture groups, each consisting of a 4-strain blend of Lactococcus lactis ssp . cremoris and Lactococcus lactis ssp . lactis, were used to manufacture the cheeses . Cheese manufactured with one particular culture group showed no incidence of calcium lactate crystal formation or weeping during curing and shelf-life of cheeses in this study . This starter group also generated the least amount of pH change in cheese during the first month of curing . From these results we conclude that starter culture group, more than any other factor measured, played an important role in the development of calcium lactate and liquid expulsion defects in Cheddar cheese . Starter culture group appeared to strongly influence cheese pH, lactic acid, and soluble calcium concentrations during curing and storage.

J Mol Biol, 2004 Jul 2, 340(2), 211 - 31
A group II intron-encoded maturase functions preferentially in cis and requires both the reverse transcriptase and X domains to promote RNA splicing; Cui X et al.; Mobile group II introns encode proteins with both reverse transcriptase activity, which functions in intron mobility, and maturase activity, which promotes RNA splicing by stabilizing the catalytically active structure of the intron RNA . Previous studies with the Lactococcus lactis Ll.LtrB intron suggested a model in which the intron-encoded protein binds first to a high-affinity binding site in intron subdomain DIVa, an idiosyncratic structure at the beginning of its own coding region, and then makes additional contacts with conserved catalytic core regions to stabilize the active RNA structure . Here, we developed an Escherichia coli genetic assay that links the splicing of the Ll.LtrB intron to the expression of green fluorescent protein and used it to study the in vivo splicing of wild-type and mutant introns and to delineate regions of the maturase required for splicing . Our results show that the maturase functions most efficiently when expressed in cis from the same transcript as the intron RNA . In agreement with previous in vitro assays, we find that the high-affinity binding site in DIVa is required for efficient splicing of the Ll.LtrB intron in vivo, but in the absence of DIVa, 6-10% residual splicing occurs by the direct binding of the maturase to the catalytic core . Critical regions of the maturase were identified by statistically analyzing ratios of missense to silent mutations in functional LtrA variants isolated from a library generated by mutagenic PCR ("unigenic evolution") . This analysis shows that both the reverse transcriptase domain and domain X, which likely corresponds to the reverse transcriptase thumb, are required for RNA splicing, while the C-terminal DNA-binding and DNA endonuclease domains are not required . Within the reverse transcriptase domain, the most critical regions for maturase activity include parts of the fingers and palm that function in template and primer binding in HIV-1 reverse transcriptase, but the integrity of the reverse transcriptase active site is not required . Biochemical analysis of LtrA mutants indicates that the N terminus of the reverse transcriptase domain is required for high-affinity binding of the intron RNA, possibly via direct interaction with DIVa, while parts of domain X interact with conserved regions of the catalytic core . Our results support the hypothesis that the intron-encoded protein adapted to function in splicing by using, at least in part, interactions used initially to recognize the intron RNA as a template for reverse transcription.

FEBS Lett, 2004 Jun 18, 568(1-3), 117 - 21
Amino acid type selective isotope labelling of the multidrug ABC transporter LmrA for solid-state NMR studies; Mason AJ et al.; The ABC transporter LmrA in Lactococcus lactis confers resistance to a wide range of antibiotics and cytotoxic drugs and is a functional homologue of P-glycoprotein . Recently, solid-state NMR methods have shown potential for structural- and non-perturbing, site directed functional studies . These experiments require isotopic labelling of selected sites . We have developed a strategy to produce large quantities of selectively labelled LmrA reconstituted at a high density in lipid membranes . This makes the 64 kDa integral membrane protein LmrA and therefore the ABC transporter superfamily accessible to NMR analysis.

J Biol Chem, 2004 Aug 13, 279(33), 34449 - 55 Epub 2004 Jun 10.
ydaG and ydbA of Lactococcus lactis encode a heterodimeric ATP-binding cassette-type multidrug transporter; Lubelski J et al.; Multidrug resistance (MDR)-type transporters mediate the active extrusion of structurally and functionally dissimilar compounds from the cells, thereby rendering cells resistant to a range of drugs . The ydaG and ydbA genes of Lactococcus lactis encode two ATP-binding cassette half-transporters, which both share homology with MDR proteins such as LmrA from L . lactis or the mammalian P-glycoprotein . The ydaG/ydbA genes were cloned and expressed separately and jointly in L . lactis using the nisin-inducible system . When both proteins are co-expressed, several structurally dissimilar drugs such as ethidium, daunomycin, and BCECF-AM are extruded from the cell . YdaG and YdbA could be co-purified as a stable heterodimer . ATPase activity was found to be associated with the YdaG/YdbA heterodimer only and not with the individual subunits . Both the ydaG and ydbA genes are up-regulated in multidrug-resistant L . lactis strains selected for growth in the presence of a variety of toxic compounds . This is the first demonstration of a functional heterodimeric ATP-binding cassette-type MDR transporter.

J Dairy Res, 2004 May, 71(2), 216 - 21
High-level coproduction of the bacteriocins nisin A and lactococcin A by Lactococcus lactis; Fernandez A et al.; In this study, a two-plasmid system for enhanced and consistent biosynthesis of the model lactococcal bacteriocin lactococcin A in non-producing Lactococcus lactis hosts was developed . The system comprised a plasmid carrying the genes lcnA and lciA under the control of the nisin-inducible nisA promoter, and a second plasmid harbouring the lcnC and lcnD genes . The introduction of both plasmids into two strains containing the nisRK genes required for nisin-controlled expression, Lc . lactis FI5876 (a nisin A-producer strain) and FI7847, resulted in production of extracellular lactococcin A at a higher level than that for the parental strain, Lc . lactis WM4 . In addition, transformation of the nisin-producing host with both plasmids led to a high-level production of both lactococcal bacteriocins, which may provide a means to exploit their complementary properties in cheese ripening.

Proteomics, 2004 May, 4(5), 1293 - 304
Setting up standards and a reference map for the alkaline proteome of the Gram-positive bacterium Lactococcus lactis; Drews O et al.; Despite the fact that almost 39% of the theoretical expressed proteins of Lactococcus lactis have a predicted isoelectric point above 7, these proteins have not been studied in previous proteome analyses . In the present study, we set up a reference map of alkaline lactococcal proteins by using immobilized pH gradients (IPG) spanning pH 6 to 12 and 9 to 12, and protein identification by matrix-assisted laser desorption/ionization-time of flight mass spectrometry (MALDI-TOF MS) . Different electrophoresis systems for isoelectric focusing were evaluated to optimize the first dimension . Best results were obtained by sample application using cup-loading at the anodic side and increasing the final voltage up to 8000 V for IPGs, using N,N-dimethylacrylamide as monomer . After two-dimensional gel electrophoresis of extracts obtained from exponentially growing cells, about 200 protein spots were selected for identification by peptide mass fingerprinting . With MALDI-TOF MS, 153 proteins were identified that were the products of 85 different genes . Their predicted isoelectric points range from as high as 11.31 to as low as 6.34 . Ribosomal proteins, hypothetical proteins and proteins with unknown function represent the largest groups of identified proteins . For further classification, the codon adaptation index (CAI) and grand average of hydropathicity (GRAVY) for each lactococcal protein were calculated . The protein with the lowest CAI identified in this study is the manganese ABC transporter ATP-binding protein . Less than 10% of the alkaline lactococcal proteins have a smaller CAI . The highest GRAVY for an identified protein is 0.26 . The complete in silico data of Lactococcus lactis as well as clickable reference maps are available at www.wzw.tum.de/proteomik/lactis.

Appl Microbiol Biotechnol, 2004 Nov, 66(1), 48 - 52 Epub 2004 Nov.
Enhanced production of lactococcin 972 in chemostat cultures; de Rojas AH et al.; Lactococcus lactis subsp . lactis IPLA972 is a wild lactococcal strain suitable as a single starter in the manufacture of dairy products . This strain synthesizes lactococcin 972 (Lcn972), a unique bacteriocin that blocks septum formation . In this work, we report on the conditions to optimize biomass and Lcn972 production . In batch cultures, pH 6.8 was found to be optimum for bacteriocin synthesis and both glucose and lactose supported Lcn972 production . The inhibitory activity improved up to eight-fold with increasing carbohydrate concentration . In chemostat cultures, steady states were achieved even at dilution rates higher than mu(max), due to the strong wall growth . Lcn972 behaved as a true primary metabolite, as it was maximally produced when the cells were actively growing . Bacteriocin yields were improved up to ten-fold in chemostat cultures compared with those achieved in batch.

Appl Environ Microbiol, 2004 Jun, 70(6), 3695 - 9
Transformation of Leuconostoc carnosum 4010 and evidence for natural competence of the organism; Helmark S et al.; Plasmid transformation in Leuconostoc carnosum 4010 was analyzed . A successful transformation protocol for L . carnosum was established by modifying an existing protocol for Lactococcus lactis . Several parameters, including the number of generations that the cells had grown at the time of harvest, glycine concentration, the time of incubation for phenotypic expression, and the electrical field strength, were investigated and proved to have influence on the transformation frequency . Electrocompetence was found to be transient and to peak in the early exponential growth phase . Optimized conditions resulted in transformation frequencies of up to 6.7 x 10(5) transformants per microgram of plasmid DNA . A total of five plasmids in L . carnosum were successfully introduced and maintained . Interestingly, we discovered that DNA uptake was of a frequency of 3 x 10(-6) to 19 x 10(-6) transformants per CFU in the absence of an applied electrical field . We concluded that L . carnosum is naturally competent.

Appl Environ Microbiol, 2004 Jun, 70(6), 3493 - 9
Analysis of the peptidoglycan hydrolase complement of Lactococcus lactis: identification of a third N-acetylglucosaminidase, AcmC; Huard C et al.; The peptidoglycan hydrolase (PGH) complement of Lactococcus lactis was identified by amino acid sequence similarity searching of the L . lactis IL-1403 complete genome sequence . Five PGHs that are not encoded by prophages were detected, including the previously characterized AcmA and AcmB proteins . Four of these PGHs, AcmA to AcmD, contain a catalytic domain homologous to that of enterococcal muramidase, but they have different domain structures . The fifth one (YjgB) has sequence similarity with the active-site domain of peptidoglycan-specific endopeptidases . The three new PGH-encoding genes identified in this study are all actively transcribed in L . lactis subsp . cremoris MG1363 . The relative abundance of their transcripts varied during growth and was maximal during the early exponential growth phase . The three encoded proteins have peptidoglycan-hydrolyzing activities which are detected only at acidic pHs by zymography . Like AcmA and AcmB, AcmC has N-acetylglucosaminidase activity rather than the N-acetylmuramidase activity predicted by sequence similarity.

Eur J Biochem, 2004 Jun, 271(12), 2438 - 45
Expression of the pyrG gene determines the pool sizes of CTP and dCTP in Lactococcus lactis; Jorgensen CM et al.; The pyrG gene from Lactococcus lactis encodes CTP synthase (EC 6.4.3.2), an enzyme converting UTP to CTP . A series of strains were constructed with different levels of pyrG expression by insertion of synthetic constitutive promoters with different strengths in front of pyrG . These strains expressed pyrG levels in a range from 3 to 665% relative to the wild-type expression level . Decreasing the level of CTP synthase to 43% had no effect on the growth rate, showing that the capacity of CTP synthase in the cell is in excess in a wild-type strain . We then studied how pyrG expression affected the intracellular pool sizes of nucleotides and the correlation between pyrG expression and nucleotide pool sizes was quantified using metabolic control analysis in terms of inherent control coefficients . At the wild-type expression level, CTP synthase had full control of the CTP concentration with a concentration control coefficient close to one and a negative concentration control coefficient of -0.28 for the UTP concentration . Additionally, a concentration control coefficient of 0.49 was calculated for the dCTP concentration . Implications for the homeostasis of nucleotide pools are discussed.

Biochemistry, 2004 Jun 15, 43(23), 7491 - 502
Characterization of YvcC (BmrA), a multidrug ABC transporter constitutively expressed in Bacillus subtilis; Steinfels E et al.; The involvement of transporters in multidrug resistance of bacteria is an increasingly challenging problem, and most of the pumps identified so far use the protonmotive gradient as the energy source . A new member of the ATP-binding cassette (ABC) family, known in Bacillus subtilis as YvcC and homologous to each half of mammalian P-glycoprotein and to LmrA of Lactococcus lactis, has been studied here . The yvcC gene was constitutively expressed in B . subtilis throughout its growth, and a knockout mutant showed a lower rate of ethidium efflux than the wild-type strain . Overexpression of yvcC in Escherichia coli allowed the preparation of highly enriched inverted-membrane vesicles that exhibited high transport activities of three fluorescent drugs, namely, Hoechst 33342, doxorubicin, and 7-aminoactinomycin D . After solubilization with n-dodecyl beta-D-maltoside, the hexahistidine-tagged YvcC was purified by a one-step affinity chromatography, and its ability to bind many P-glycoprotein effectors was evidenced by fluorescence spectroscopy experiments . Collectively, these results showed that YvcC is a multidrug ABC transporter functionally active in wild-type B . subtilis, and YvcC was therefore renamed BmrA for Bacillus multidrug resistance ATP . Besides, reconstitution of YvcC into liposomes led to the highest, vanadate-sensitive, ATPase activity reported so far for an ABC transporter . Interestingly, such a high ATP hydrolysis proceeds with a positive cooperativity mechanism, a property only found so far with ABC importers.

Biosci Biotechnol Biochem, 2004 May, 68(5), 1149 - 52
Catalytic activity of tripeptidase from Lactococcus lactis to which amino acid substitution was introduced according to natural mutation; Mori S et al.; Four mutations observed between tripeptidases from Lactococcus lactis subsp . lactis and subsp . cremoris were introduced one by one to the corresponding points in wild-type tripeptidase from L . lactis subsp . lactis . The k(cat) values of four resultant mutants were analyzed and discussed in stereographical terms . Change in catalytic activity appeared to be related to the sequential and steric location of mutation point within the enzyme protein, even though no drastic change was observed with one point mutation.

Int J Food Microbiol, 2004 Jun 15, 93(3), 335 - 47
Enhancement of 2-methylbutanal formation in cheese by using a fluorescently tagged Lacticin 3147 producing Lactococcus lactis strain; Fernandez de Palencia P et al.; The amino acid conversion to volatile compounds by lactic acid bacteria is important for aroma formation in cheese . In this work, we analyzed the effect of the lytic bacteriocin Lacticin 3147 on transamination of isoleucine and further formation of the volatile compound 2-methylbutanal in cheese . The Lacticin 3147 producing strain Lactococcus lactis IFPL3593 was fluorescently tagged (IFPL3593-GFP) by conjugative transfer of the plasmid pMV158GFP from Streptococcus pneumoniae, and used as starter in cheese manufacture . Starter adjuncts were the bacteriocin-sensitive strains L . lactis T1 and L . lactis IFPL730, showing branched chain amino acid aminotransferase and alpha-keto acid decarboxylase activity, respectively . Adjunct strains were selected to complete the isoleucine conversion pathway and, hence, increase formation of 2-methylbutanal conferring aroma to the cheese . The non-bacteriocin-producing strain L . lactis IFPL359-GFP was included as starter in the control batch . Fluorescent tagging of the starter strains allowed their tracing in cheese during ripening by fluorescence microscopy and confocal scanning laser microscopy . The bacteriocin produced by L . lactis IFPL3593-GFP enhanced lysis of the adjuncts with a concomitant increase in isoleucine transamination and about a two-fold increase of the derived volatile compound 2-methylbutanal . This led to an enhancement of the cheese aroma detected by a sensory panel . The improvement of cheese flavour and aroma may be of significant importance for the dairy industry.

Biochemistry, 2004 Jun 1, 43(21), 6498 - 510
Thermodynamic basis of electron transfer in dihydroorotate dehydrogenase B from Lactococcus lactis: analysis by potentiometry, EPR spectroscopy, and ENDOR spectroscopy; Mohsen AW et al.; Dihydroorotate dehydrogenase B (DHODB) is a complex iron-sulfur flavoprotein that catalyzes the conversion of dihydroorotate to orotate and the reduction of NAD(+) . The enzyme is a dimer of heterodimers containing an FMN, an FAD, and a 2Fe-2S center . UV-visible, EPR, and ENDOR spectroscopies have been used to determine the reduction potentials of the flavins and the 2Fe-2S center and to characterize radicals and their interactions . Reductive titration using dithionite indicates a five-electron capacity for DHODB . The midpoint reduction potential of the 2Fe-2S center (-212 +/- 3 mV) was determined from analysis of absorption data at 540 nm, where absorption contributions from the two flavins are small . The midpoint reduction potentials of the oxidized/semiquinone (E(1)) and semiquinone/hydroquinone (E(2)) couples for the FMN (E(1) = -301 +/- 6 mV; E(2) = -252 +/- 8 mV) and FAD (E(1) = -312 +/- 6 mV; E(2) = -297 +/- 5 mV) were determined from analysis of spectral changes at 630 nm . Corresponding values for the midpoint reduction potentials for FMN (E(1) = -298 +/- 4 mV; E(2) = -259 +/- 5 mV) in the isolated catalytic subunit (subunit D, which lacks the 2Fe-2S center and FAD) are consistent with the values determined for the FMN couples in DHODB . During reductive titration of DHODB, small amounts of the neutral blue semiquinone are observed at approximately 630 nm, consistent with the measured midpoint reduction potentials of the flavins . An ENDOR spectrum of substrate-reduced DHODB identifies hyperfine couplings to proton nuclei similar to those recorded for the blue semiquinone of free flavins in aqueous solution, thus confirming the presence of this species in DHODB . Spectral features observed during EPR spectroscopy of dithionite-reduced DHODB are consistent with the midpoint reduction potentials determined using UV-visible spectroscopy and further identify an unusual EPR signal with very small rhombic anisotropy and g values of 2.02, 1.99, and 1.96 . This unusual signal is assigned to the formation of a spin interacting state between the FMN semiquinone species and the reduced 2Fe-2S center . Reduction of DHODB using an excess of NADH or dihydroorotate produces EPR spectra that are distinct from those produced by dithionite . From potentiometric studies, the reduction of the 2Fe-2S center and the reduction of the FMN occur concomitantly . The study provides a detailed thermodynamic framework for electron transfer in this complex iron-sulfur flavoprotein.

Biochemistry, 2004 Jun 1, 43(21), 6486 - 97
Assembly of an active group II intron-maturase complex by protein dimerization; Rambo RP et al.; Group II intron-encoded proteins promote both splicing and mobility of the intron RNA through formation of a specific RNA-protein complex . The Lactococcus lactis L1.LtrB intron encodes a maturase, LtrA, with reverse transcriptase homology and specific binding affinity for two domains of the intron RNA . The catalytically active ribonucleoprotein (RNP) has splicing, endonuclease, and reverse transcriptase activity, enabling efficient insertion of the intron sequence by a retro-homing mechanism . To determine the composition and assembly mechanism of the RNP complex, purified LtrA protein was analyzed for its ability to recognize a series of intron-derived RNAs . Equilibrium dissociation measurements show that LtrA recognizes two intronic domains, DI and DIV . However, distinct electrostatic requirements for binding imply different modes of molecular recognition in each case . Stoichiometric binding experiments show that the functional RNP complex consists of a dimeric protein species bound to a single intron RNA . Significant differences between the measured equilibrium dissociation constants and kinetically derived values suggest that conformational changes accompany assembly of the intron-maturase complex, and results of limited proteolysis and fluorescence spectroscopy experiments suggest that significant RNA-dependent structural changes within the maturase occur upon association with the intron . These results support a mutually induced fit model in which RNA-dependent conformational changes within LtrA enable stable association of the protein dimer with two independent intron domains to form a functional RNP.

Nucleic Acids Res, 2004 May 20, 32(9), 2880 - 8 Print 2004.
The RmInt1 group II intron has two different retrohoming pathways for mobility using predominantly the nascent lagging strand at DNA replication forks for priming; Martinez-Abarca F et al.; Sinorhizobium meliloti RmInt1 is an efficient mobile group II intron that uses an unknown reverse transcriptase priming mechanism as the intron ribonucleoprotein complex can reverse splice into DNA target substrates but cannot carry out site-specific second strand cleavage due to the lack of a C-terminal DNA endonuclease domain . We show here that, like other mobile group II introns, RmInt1 moves around by an efficient RNA-based retrohoming mechanism . We found evidence of two distinct RmInt1 retrohoming pathways for mobility depending on the orientation of the target site relative to the direction of DNA replication . The preferred retrohoming pathway is consistent with reverse splicing of the intron RNA into single-stranded DNA at a replication fork, using a nascent lagging DNA strand as the primer for reverse transcription . This strand bias is the opposite of that reported for mobility of the lactococcal Ll.ltrB intron in the absence of second strand cleavage . The mobility mechanism found here for RmInt1 may be used for dissemination by many bacterial group II introns encoding proteins lacking the DNA endonuclease domain.

Infect Immun, 2004 Jun, 72(6), 3444 - 50
Mucosal vaccine made from live, recombinant Lactococcus lactis protects mice against pharyngeal infection with Streptococcus pyogenes; Mannam P et al.; A novel vaccine (LL-CRR) made from live, nonpathogenic Lactococcus lactis that expresses the conserved C-repeat region (CRR) of M protein from Streptococcus pyogenes serotype 6 was tested in mice . Nasally vaccinated mice produced CRR-specific salivary immunoglobulin A (IgA) and serum IgG . Subcutaneously vaccinated mice produced CRR-specific serum IgG but not salivary IgA . A combined regimen produced responses similar to the salivary IgA of nasally vaccinated mice and serum IgG of subcutaneously vaccinated mice . Mice vaccinated nasally or with the combined regimen were significantly protected against pharyngeal infection following a nasal challenge with S . pyogenes M serotype 14 . Mice vaccinated subcutaneously were not protected against pharyngeal infection . Mice in all three LL-CRR vaccination groups were significantly protected against the lethal effects of S . pyogenes . Only 1 of 77 challenged mice that were vaccinated with LL-CRR died, whereas 60 of 118 challenged mice that were vaccinated with a control strain or phosphate-buffered saline died . In conclusion, mucosal vaccination with LL-CRR produced CRR-specific salivary IgA and serum IgG, prevented pharyngeal infection with S . pyogenes, and promoted survival.

J Food Prot, 2004 May, 67(5), 947 - 51
Isolation, identification, and selection of lactic acid bacteria from alfalfa sprouts for competitive inhibition of foodborne pathogens; Wilderdyke MR et al.; Several studies have investigated the control of pathogens on alfalfa sprouts, and some treatments have been shown to be effective in reducing pathogen populations . However, control methods investigated thus far only provide pathogen control at a given point in the sprouting process and can affect germination . Competitive inhibition of pathogens with lactic acid bacteria might provide pathogen control throughout the sprouting process and up to consumption . The purpose of this study was to isolate and identify lactic acid bacteria from alfalfa sprouts to inhibit the growth of foodborne pathogens . Fifty-eight lactic acid bacteria isolates were obtained from alfalfa seeds and sprouts . These isolates were evaluated for inhibitory action against Salmonella enterica, Escherichia coli O157:H7, and Listeria monocytogenes by agar spot tests . All pathogens were inhibited by 32 (55%) of the isolates, S . enterica by 56 (97%), E . coli O157:H7 by 49 (84%), and L . monocytogenes by 41 (71%) . The isolates were identified by the Analytical Profile Index evaluation of carbohydrate utilization . Isolates obtained from a sample of alfalfa seeds and identified as Lactococcus lactis subsp . lactis showed zones of inhibition of 4.0 mm or greater for all pathogens . One of these isolates, Lactococcus lactis subsp . lactis (L7), and an isolate previously obtained, Pediococcus acidilactici (D3), were evaluated for competitive inhibition of S . enterica, E . coli O157:H7, and L . monocytogenes in deMan Rogosa Sharpe agar and broth . Pathogen populations were significantly reduced by day 5 . The selected isolates will be further evaluated in future studies for inhibitory action toward S . enterica, E . coli O157:H7, and L . monocytogenes during sprouting.

J Food Prot, 2004 May, 67(5), 928 - 33
Inhibition of a methicillin-resistant Staphylococcus aureus strain in Afuega'l Pitu cheese by the nisin Z-producing strain Lactococcus lactis subsp . lactis IPLA 729; Rilla N et al.; Methicillin-resistant Staphylococcus aureus strains are a potential threat for food safety because foodborne illness caused by methicillin-resistant Staphylococcus aureus has been reported even though these strains were only associated with nosocomial infections until recently . This article focuses on the inhibitory effect of the nisin Z-producing strain Lactococcus lactis subsp . lactis IPLA 729 on the growth of Staphylococcus aureus CECT 4013, a methicillin-resistant strain . S . aureus was inhibited by the presence of the nisin producer IPLA 729 in buffered Trypticase soy broth, milk, and Afuega'l Pitu cheese, an acid-coagulated cheese manufactured in Asturias, Northern Spain . A reduction of 3.66 log units was observed in Trypticase soy broth at the end of the incubation period . In milk, viable counts of S . aureus were undetectable or were reduced by 2.16 log units in 24 h depending on the initial inoculum (1.8 x 10(4) and 7.2 x 10(6) CFU/ml) . The staphylococcal strain was also undetected in test cheeses in which the nisin Z producer was present whereas 2 log units were detected in control cheeses at the end of ripening.

J Bacteriol, 2004 Jun, 186(11), 3649 - 52
Lactococcal phage genes involved in sensitivity to AbiK and their relation to single-strand annealing proteins; Bouchard JD et al.; Lactococcal phage mutants insensitive to the antiviral abortive infection mechanism AbiK are divided into two classes . One comprises virulent phages that result from DNA exchanges between a virulent phage and the host chromosome . Here, we report the analysis of the second class of phage mutants, which are insensitive to AbiK as a result of a single nucleotide change causing an amino acid substitution . The mutated genes occupy the same position in the various lactococcal phage genomes, but the deduced proteins do not share amino acid sequence similarity . Four nonsimilar proteins involved in the sensitivity to AbiK (Sak) were identified . Two of these Sak proteins are related to Erf and RAD52, single-strand annealing proteins involved in homologous recombination.

J Bacteriol, 2004 Jun, 186(11), 3480 - 91
Bacteriophage Tuc2009 encodes a tail-associated cell wall-degrading activity; Kenny JG et al.; Tuc2009 is a P335-type member of the tailed-phage supergroup Siphoviridae and was originally identified as a resident prophage of the gram-positive bacterium Lactococcus lactis UC509 . A Tuc2009 gene designated tal2009 which is located within the morphogenic module was shown to specify a lytic activity within the 3' portion of its coding region . Comparative sequence analysis indicated that the cell wall-degrading part of Tal2009 is a member of the M37 protein family and that Tal2009 lacks a cell-binding domain, a finding supported by binding studies . Tal2009 appears to undergo self-mediated posttranslational processing in both L . lactis and Escherichia coli . Antibodies directed against a purified C-terminal portion of Tal2009 were used for immunoelectron microscopy, which showed that Tal2009 is located at the tail tip of Tuc2009 . Antibody neutralization studies demonstrated that Tal2009-directed antibodies inhibited the ability of phage to mediate host lysis by more than 100-fold . These data indicate that tal2009 encodes a tail-associated lysin involved in localized cell wall degradation, thus allowing the Tuc2009 DNA injection machinery access to the membrane of its bacterial host.

Nahrung, 2004 Apr, 48(2), 145 - 8
Mode of action of lactococcin R produced by Lactococcus lactis R; Yildirim Z et al.; We investigated the mode of action and factors affecting adsorption of lactoccocin R produced by Lactococcus lactis R . It was found that lactococcin R adsorbed to all Gram-positive but not to the Gram-negative bacteria tested and its adsorption was dependent on pH . It was observed that the binding of lactococcin R was prevented by anions of several salts (Cl-, PO4(-3)) and lipoteichoic acid . Pretreatments of sensitive cells and cell walls with detergents, organic solvents or enzymes did not reduce subsequent binding of lactococcin R . However, treatment of cell wall preparations with methanol:chloroform and hot 20% trichloroacetic acid (TCA) caused such walls to lose their ability to adsorb lactococcin R . Sensitive cells treated with lactococcin R lost high amounts of intracellular K+ ions, UV-absorbing materials and became more permeable to o-nitrophenol-beta-D-glactopyranoside (ONPG) . In addition, different lactococcin R concentrations (0-2560 AU/mL) decreased the colony counts of Listeria monocytogenes by 99% and also a reduction in the absorbance values . These results show that the mode of action of lactococcin R is bactericidal rather than bacteriostatic.

J Appl Microbiol, 2004, 96(6), 1347 - 53
Protective immunity of SpaA-antigen producing Lactococcus lactis against Erysipelothrix rhusiopathiae infection; Cheun HI et al.; AIMS: To develop an economical, safe and simple vaccination system against swine erysipelas using SpaA-antigen producing Lactococcus lactis . METHODS AND RESULTS: The spaA gene of Erysipelothrix rhusiopathiae was inserted into a shuttle plasmid pSECE1 to construct pSECE1.3 . The SpaA produced in L . lactis maintained a stable antigenicity without degrading in growth . After mice were inoculated intranasally and orally with pSECE1.3-carrying L . lactis cells, IgG and IgA specific to SpaA were detected, and all the mice survived a challenge with 100 LD(50) of E . rhusiopathiae Tama-96 in the inner thigh . CONCLUSIONS: SpaA-producing L . lactis appears useful as an effective subunit vaccine against swine erysipelas . SIGNIFICANCE AND IMPACT OF THE STUDY: In this vaccination system, purification of the antigen and injection are unnecessary, leading to a reduced production cost, reduced labour and less stress to the animals . This vaccination system of the lactic acid bacteria should be a safe and suitable vehicle for a polyvalent vaccine.

Appl Environ Microbiol, 2004 May, 70(5), 3183 - 7
Multiplex PCR assay for detection of bacterial pathogens associated with warm-water Streptococcosis in fish; Mata AI et al.; A multiplex PCR-based method was designed for the simultaneous detection of the main pathogens involved in warm-water streptococcosis in fish (Streptococcus iniae, Streptococcus difficilis, Streptococcus parauberis, and Lactococcus garvieae) . Each of the four pairs of oligonucleotide primers exclusively amplified the targeted gene of the specific microorganism . The sensitivity of the multiplex PCR using purified DNA was 25 pg for S . iniae, 12.5 pg for S . difficilis, 50 pg for S . parauberis, and 30 pg for L . garvieae . The multiplex PCR assay was useful for the specific detection of the four species of bacteria not only in pure culture but also in inoculated fish tissue homogenates and naturally infected fish . Therefore, this method could be a useful alternative to the culture-based method for the routine diagnosis of warm-water streptococcal infections in fish.

J Bacteriol, 2004 May, 186(10), 3278 - 81
A phage protein confers resistance to the lactococcal abortive infection mechanism AbiP; Domingues S et al.; Phage bIL66M1 is sensitive to the lactococcal abortive infection mechanism AbiP . No spontaneous AbiP-resistant variant could be obtained at a frequency of <10(-10) . However, AbiP-resistant variants were readily obtained during infection with both bIL66M1 and the highly homologous AbiP-resistant phage bIL170 . Gain of AbiP resistance was due to the acquisition of the e6 gene from bIL170.

Fish Shellfish Immunol, 2004 Feb, 16(2), 207 - 14
Role of capsule in serotypic differences and complement fixation by Lactococcus garvieae; Barnes AC et al.; Seventeen geographically distinct isolates of Lactococcus garvieae, isolated from diseased fish, were compared serologically using antiserum raised against the various isolates in rainbow trout . Sera raised against a capsule deficient isolate did not agglutinate capsulated isolates, regardless of origin . In contrast, all antisera raised against capsulated isolates cross reacted strongly with non-capsulated isolates . Antisera raised against capsulated Japanese isolates cross reacted with other capsulated Japanese isolates including isolates from geographically distinct prefectures within Japan (Ehime and Oita) . However, antisera against these virulent capsulated isolates did not cross react with European capsulated isolates . Antisera raised against European capsulated isolates cross reacted with other European isolates, regardless of origin within Europe (UK, Italy, Spain), but did not cross-react with Japanese capsulated isolates . Agglutination assays performed with a range of fifteen lectins revealed differences in surface carbohydrate structure: capsule deficient isolates agglutinated with concanavalin A, Ricinis communis agglutinin, Pisum sativum agglutinin, Lens culinaris agglutinin, wheat germ agglutinin and succinylated wheat germ agglutinin . European capsulated isolates agglutinated with concanavalin A only . The Japanese capsulated isolates were not agglutinated by any of the lectins used in this study . Representative isolates from each group (Japanese capsulated and non-capsulated, European capsulated and non-capsulated) were investigated for their ability to fix complement . Non-capsulated isolates fixed complement regardless of origin, and antibody did not markedly enhance complement fixation . In contrast, the capsulated isolates were less efficient at fixing complement, but complement fixation was markedly increased by homologous antibody.

Metab Eng, 2004 Apr, 6(2), 109 - 15
Multivitamin production in Lactococcus lactis using metabolic engineering; Sybesma W et al.; The dairy starter bacterium Lactococcus lactis has the potential to synthesize both folate (vitamin B11) and riboflavin (vitamin B2) . By directed mutagenesis followed by selection and metabolic engineering we have modified two complicated biosynthetic pathways in L . lactis resulting in simultaneous overproduction of both folate and riboflavin: Following exposure to the riboflavin analogue roseoflavin we have isolated a spontaneous mutant of L . lactis strain NZ9000 that was changed from a riboflavin consumer into a riboflavin producer . This mutant contained a single base change in the regulatory region upstream of the riboflavin biosynthetic genes . By the constitutive overproduction of GTP cyclohydrolase I in this riboflavin-producing strain, the production of folate was increased as well . Novel foods, enriched through fermentation using these multivitamin-producing starters, could compensate the B-vitamin-deficiencies that are common even in highly developed countries and could specifically be used in dietary foods for the large fraction of the Caucasian people (10-15%) with mutations in the methylene tetrahydrofolate reductase (MTHFR).

Plasmid, 2004 May, 51(3), 265 - 71
General and specialized vectors derived from pBM02, a new rolling circle replicating plasmid of Lactococcus lactis; Sanchez C et al.; This paper reports the construction of several general cloning vectors and a specialized depurative vector based on a new lactococcal plasmid that replicates by the rolling circle mechanism {pBM02; Plasmid 49 (2003) 118} . Most vectors are shuttle vectors for Escherichia coli-Lactococcus lactis and carry replicons of both ColE1 and pBM02 plasmids (ColE1 is used even though the pBM02 replicon is fully active in both Gram-positive and Gram-negative organisms) . Segregational and structural studies indicated that the new vectors were stable enough for the majority of applications . Further, since the basic replicon is compatible with plasmid derivatives of pWV01 and pSH71, they can be maintained in the same cell with members of the two largest vector series for L . lactis and other lactic acid bacteria, the pGK, and the pNZ series .

Plasmid, 2004 May, 51(3), 256 - 64
Development of an inducible system to control and easily monitor gene expression in Lactococcus lactis; Viegas SC et al.; This report describes the implementation and use of a maltose-inducible system for regulated gene expression in Lactococcus lactis . The system was established using Green Fluorescent Protein as reporter . The transcription of a gene of interest from the inducible promoter of pLS1RGFP plasmid vector can be easily monitored by fluorescence spectroscopy and microscopy . As an example, the lactococcal ribonuclease III was overproduced in an active form .

Infect Immun, 2004 May, 72(5), 2753 - 61
Mucosal and cellular immune responses elicited by recombinant Lactococcus lactis strains expressing tetanus toxin fragment C; Robinson K et al.; The mucosal and cellular responses of mice were studied, following mucosal-route administration of recombinant Lactococcus lactis expressing tetanus toxin fragment C (TTFC), which is a known immunogen protective against tetanus . A TTFC-specific T-cell response with a mixed profile of T-helper (Th) subset-associated cytokines was elicited in the intestine, with a Th2 bias characteristic of a mucosal response . These results correlated with the humoral response, where equivalent titers of anti-TTFC immunoglobulin G1 (IgG1) and IgG2a in serum were accompanied by an elevated IgA-specific response at more than one mucosal site . The route of vaccination had an important role in determining the immune response phenotype, as evidenced by the fact that an IgG1-biased subclass profile was obtained when lactococci were administered parenterally . Stimulation of splenic or mesenteric lymph node cells with lactococci resulted in their proliferation and the secretion of gamma interferon via antigen-specific and innate immune mechanisms . The data therefore provide further evidence of the potential of recombinant lactococcal vaccines for inducing systemic and mucosal immune responses.

J Med Microbiol, 2004 May, 53(Pt 5), 427 - 33
An inducible surface presentation system improves cellular immunity against human papillomavirus type 16 E7 antigen in mice after nasal administration with recombinant lactococci; Bermudez-Humaran LG et al.; Human papillomavirus type 16 (HPV-16) is the major causative agent of cervical cancer . To date, vaccine strategies against HPV-16 are based on the ability of the E7 oncoprotein to elicit an immune response against this virus . In this study, the use of an inducible or a constitutive system to produce the HPV-16 E7 protein in Lactococcus lactis, a non-pathogenic and non-invasive Gram-positive bacterium, was compared . The highest E7 production was obtained with the inducible system . When mice were immunized intranasally with recombinant lactococci expressing either inducible or constitutive E7, an antigen-specific cellular response (i.e . secretion of IL2 and IFN-gamma cytokines) was evoked and was substantially higher in mice receiving L . lactis expressing E7 with the inducible system . As bacterial antigen location may influence the immune response, recombinant L . lactis strains that produced E7 in three cellular locations, intracellular, secreted or cell-wall-anchored were evaluated . The highest immune response was elicited by administration of L . lactis producing an inducible cell-wall-anchored form of E7 protein . These promising results represent a step towards the development of a new, safe mucosal vector to treat HPV-related cervical cancer.

Protein Sci, 2004 May, 13(5), 1295 - 303 Epub 2004 Apr 09.
Deciphering the function of an ORF: Salmonella enterica DeoM protein is a new mutarotase specific for deoxyribose; Assairi L et al.; We identified in Salmonella enterica serovar Typhi a cluster of four genes encoding a deoxyribokinase (DeoK), a putative permease (DeoP), a repressor (DeoQ), and an open reading frame encoding a 337 amino acid residues protein of unknown function . We show that the latter protein, called DeoM, is a hexamer whose synthesis is increased by a factor over 5 after induction with deoxyribose . The CD spectrum of the purified recombinant protein indicated a dominant contribution of betatype secondary structure and a small content of alpha-helix . Temperature and guanidinium hydrochloride induced denaturation of DeoM indicated that the hexamer dissociation and monomer unfolding are coupled processes . DeoM exhibits 12.5% and 15% sequence identity with galactose mutarotase from Lactococcus lactis and respectively Escherichia coli, which suggested that these three proteins share similar functions . Polarimetric experiments demonstrated that DeoM is a mutarotase with high specificity for deoxyribose . Site-directed mutagenesis of His183 in DeoM, corresponding to a catalytically active residue in GalM, yielded an almost inactive deoxyribose mutarotase . DeoM was crystallized and diffraction data collected for two crystal systems, confirmed its hexameric state . The possible role of the protein and of the entire gene cluster is discussed in connection with the energy metabolism of S . enterica under particular growth conditions.

Microbiology, 2004 Apr, 150(Pt 4), 1103 - 11
Effect of pyruvate kinase overproduction on glucose metabolism of Lactococcus lactis; Ramos A et al.; Lactococcus lactis strain NZ9000(pNZpyk), which overproduces pyruvate kinase (PK), was constructed . The pNZpyk plasmid carries the P(nisA)-pyk transcriptional fusion, and the overexpression of its pyk gene was accomplished by using the nisin-inducible expression system of the NZ9000 strain . In vivo (13)C- and (31)P-NMR spectroscopy was used to evaluate the effect of this modification on the metabolism of glucose in non-growing cells . A detailed description of the kinetics of glucose, end products, glycolytic intermediates, NAD(+) and NADH was obtained . A 15-fold increase in the level of PK did not increase the overall glycolytic flux, which, on the contrary, was slightly reduced . Significant differences were observed in (i) the level of 3-phosphoglycerate (3-PGA) and phosphoenolpyruvate (PEP), metabolites associated with starvation; (ii) the rate of fructose 1,6-bisphosphate (FBP) depletion upon glucose exhaustion; and (iii) the NAD(+)/NADH ratio during glucose catabolism . In the mutant, the rate of FBP consumption after glucose depletion was notably accelerated under anaerobic conditions, whereas 3-PGA and PEP decreased to undetectable levels . Furthermore, the level of NAD(+) decreased steadily during the utilization of glucose, probably due to the unanticipated reduction in the lactate dehydrogenase activity in comparison with the control strain, NZ9000(pNZ8020) . The results show that PK is an important bottleneck to carbon flux only when glucose becomes limiting; in the overproducer this constriction was no longer present, as evidenced by the faster FBP consumption and lack of accumulation of 3-PGA and PEP in anaerobic as well as aerobic conditions . Despite these clear changes, the PK-overproducing strain showed typical homolactic metabolism under anaerobic conditions, as did the strain harbouring the vector plasmid without the pyk insert . However, under an oxygen atmosphere, there was increased channelling of carbon to the production of acetate and acetoin, to the detriment of lactate production.

Appl Environ Microbiol, 2004 Apr, 70(4), 2061 - 71
Receptor binding domain of Escherichia coli F18 fimbrial adhesin FedF can be both efficiently secreted and surface displayed in a functional form in Lactococcus lactis; Lindholm A et al.; Adherence of F18 fimbrial Escherichia coli to porcine intestinal epithelial cells is mediated by the adhesin (FedF) of F18 fimbriae . In a previous study, we demonstrated the specificity of the amino acid residues between 60 and 109 as the receptor binding domain of FedF . In this study, different expression, secretion, and anchoring systems for the receptor binding domain of the FedF adhesin in Lactococcus lactis were evaluated . Two partially overlapping receptor binding domains (42 and 62 amino acid residues) were expressed as fusions with L . lactis subsp . cremoris protein PrtP for evaluation of secretion efficiency . To evaluate the cell surface display of these FedF-PrtP fusions, they were further combined with different lengths of PrtP spacers fused with either the L . lactis AcmA anchor or the PrtP cell wall binding domain . An HtrA-defective L . lactis NZ9000 mutant was constructed to determine its effect on the level of secreted or anchored fusion proteins . Recombinant L . lactis clones secreting the receptor binding domain of F18 fimbriae as a fusion with the H domains of L . lactis protein PrtP were first constructed by using two different signal peptides . FedF-PrtP fusions, directed by the signal sequence of L . brevis SlpA, were throughout found to be secreted at significantly higher quantities than corresponding fusions with the signal peptide of L . lactis Usp45 . In the surface display systems tested, the L . lactis AcmA anchor performed significantly better, particularly in the L . lactis NZ9000DeltahtrA strain, compared to the L . lactis PrtP anchor region . Of the cell surface display constructs with the AcmA anchor, only those with the longest PrtP spacer regions resulted in efficient binding of recombinant L . lactis cells to porcine intestinal epithelial cells . These results confirmed that it is possible to efficiently produce the receptor binding domain of the F18 adhesin in a functionally active form in L . lactis.

Appl Environ Microbiol, 2004 Apr, 70(4), 2013 - 20
Protective effect of sucrose and sodium chloride for Lactococcus lactis during sublethal and lethal high-pressure treatments; Molina-Hoppner A et al.; The bactericidal effect of hydrostatic pressure is reduced when bacteria are suspended in media with high osmolarity . To elucidate mechanisms responsible for the baroprotective effect of ionic and nonionic solutes, Lactococcus lactis was treated with pressures ranging from 200 to 600 MPa in a low-osmolarity buffer or with buffer containing 0.5 M sucrose or 4 M NaCl . Pressure-treated cells were characterized in order to determine viability, the transmembrane difference in pH (DeltapH), and multiple-drug-resistance (MDR) transport activity . Furthermore, pressure effects on the intracellular pH and the fluidity of the membrane were determined during pressure treatment . In the presence of external sucrose and NaCl, high intracellular levels of sucrose and lactose, respectively, were accumulated by L . lactis; 4 M NaCl and, to a lesser extent, 0.5 M sucrose provided protection against pressure-induced cell death . The transmembrane DeltapH was reversibly dissipated during pressure treatment in any buffer system . Sucrose but not NaCl prevented the irreversible inactivation of enzymes involved in pH homeostasis and MDR transport activity . In the presence 0.5 M sucrose or 4 M NaCl, the fluidity of the cytoplasmic membrane was maintained even at low temperatures and high pressure . These results indicate that disaccharides protect microorganisms against pressure-induced inactivation of vital cellular components . The protective effect of ionic solutes relies on the intracellular accumulation of compatible solutes as a response to the osmotic stress . Thus, ionic solutes provide only asymmetric protection, and baroprotection with ionic solutes requires higher concentrations of the osmolytes than of disaccharides.

J Bacteriol, 2004 Apr, 186(8), 2393 - 401
Conserved target for group II intron insertion in relaxase genes of conjugative elements of gram-positive bacteria; Staddon JH et al.; The lactococcal group II intron Ll.ltrB interrupts the ltrB relaxase gene within a region that encodes a conserved functional domain . Nucleotides essential for the homing of Ll.ltrB into an intronless version of ltrB are found exclusively at positions required to encode amino acids broadly conserved in a family of relaxase proteins of gram-positive bacteria . Two of these relaxase genes, pcfG from the enterococcal plasmid pCF10 and the ORF4 gene in the streptococcal conjugative transposon Tn5252, were shown to support Ll.ltrB insertion into the conserved motif at precisely the site predicted by sequence homology with ltrB . Insertion occurred through a mechanism indistinguishable from retrohoming . Splicing and retention of conjugative function was demonstrated for pCF10 derivatives containing intron insertions . Ll.ltrB targeting of a conserved motif of a conjugative element suggests a mechanism for group II intron dispersal among bacteria . Additional support for this mechanism comes from sequence analysis of the insertion sites of the E.c.I4 family of bacterial group II introns.

Appl Biochem Biotechnol, 2004 Spring, 113-116, 627 - 38
Simultaneous production of nisin and lactic acid from cheese whey: optimization of fermentation conditions through statistically based experimental designs; Liu C et al.; A biorefinery process that utilizes cheese whey as substrate to simultaneously produce nisin, a natural food preservative, and lactic acid, a raw material for biopolymer production, was studied . The conditions for nisin biosynthesis and lactic acid coproduction by Lactococcus lactis subsp . lactis (ATCC 11454) in a whey-based medium were optimized using statistically based experimental designs . A Plackett-Burman design was applied to screen seven parameters for significant factors for the production of nisin and lactic acid . Nutrient supplements, including yeast extract, MgSO4, and KH2PO4, were found to be the significant factors affecting nisin and lactic acid formation . As a follow-up, a central-composite design was applied to optimize these factors . Second-order polynomial models were developed to quantify the relationship between nisin and lactic acid production and the variables . The optimal values of these variables were also determined . Finally, a verification experiment was performed to confirm the optimal values that were predicted by the models . The experimented results agreed well with the model prediction, giving a similar production of 19.3 g/L of lactic acid and 92.9 mg/L of nisin.

J Biol Chem, 2004 Jun 11, 279(24), 24923 - 8 Epub 2004 Mar 29.
Quantitative 2H NMR at natural abundance can distinguish the pathway used for glucose fermentation by lactic acid bacteria; Roger O et al.; For any given metabolic pathway, isotope redistribution coefficients (a(ij)) that characterize the specific derivation of each hydrogen atom can be defined . By using quantitative deuterium NMR, the redistribution of deuterium at natural abundance in lactic acid produced by the bacterial fermentation of glucose has been determined for each non-labile hydrogen atom of glucose or water and the hydrogen atoms of lactic acid . Distinct differences are observed in the lactic acid isolated from Lactococcus lactis and Leuconostoc mesenteroides that can be interpreted in terms of the different fermentative pathways used . Specifically, the affiliations observed between the H1, H3, and H4 positions of glucose with methyl and hydroxymethylene of lactic acid can give quantitative information on whether the glycolytic or the reductive pentose-phosphate pathway was involved in glucose catabolism.

Biotechnol Lett, 2004 Feb, 26(3), 235 - 8
Nisin production by Lactococcus lactis subsp . lactis under nutritional limitation in fed-batch culture; Lv W et al.; Nisin production was improved under nutritional limitation in fed-batch culture of Lactococcus lactis . Nisin titre reached 3887 IU ml(-1) by a slow feeding of sucrose and 4131 IU ml(-1) by slow feeding of nitrogen source, which were, respectively, 64% and 74% above the values in batch culture.

Biochem Biophys Res Commun, 2004 Apr 23, 317(1), 269 - 75
Interaction of the breast cancer resistance protein with plant polyphenols; Cooray HC et al.; Multidrug transporters influence drug distribution in vivo and are often associated with tumour drug resistance . Here we show that plant-derived polyphenols that interact with P-glycoprotein can also modulate the activity of the recently discovered ABC transporter, breast cancer resistance protein (BCRP/ABCG2) . In two separate BCRP-overexpressing cell lines, accumulation of the established BCRP substrates mitoxantrone and bodipy-FL-prazosin was significantly increased by the flavonoids silymarin, hesperetin, quercetin, and daidzein, and the stilbene resveratrol (each at 30 microM) as measured by flow cytometry, though there was no corresponding increase in the respective wild-type cell lines . These compounds also stimulated the vanadate-inhibitable ATPase activity in membranes prepared from bacteria (Lactococcus lactis) expressing BCRP . Given the high dietary intake of polyphenols, such interactions with BCRP, particularly in the intestines, may have important consequences in vivo for the distribution of these compounds as well as other BCRP substrates.

J Biol Chem, 2004 May 21, 279(21), 22176 - 82 Epub 2004 Mar 24.
NisT, the transporter of the lantibiotic nisin, can transport fully modified, dehydrated, and unmodified prenisin and fusions of the leader peptide with non-lantibiotic peptides; Kuipers A et al.; Lantibiotics are lanthionine-containing peptide antibiotics . Nisin, encoded by nisA, is a pentacyclic lantibiotic produced by some Lactococcus lactis strains . Its thioether rings are posttranslationally introduced by a membrane-bound enzyme complex . This complex is composed of three enzymes: NisB, which dehydrates serines and threonines; NisC, which couples these dehydrated residues to cysteines, thus forming thioether rings; and the transporter NisT . We followed the activity of various combinations of the nisin enzymes by measuring export of secreted peptides using antibodies against the leader peptide and mass spectroscopy for detection . L . lactis expressing the nisABTC genes efficiently produced fully posttranslationally modified prenisin . Strikingly, L . lactis expressing the nisBT genes could produce dehydrated prenisin without thioether rings and a dehydrated form of a non-lantibiotic peptide . In the absence of the biosynthetic NisBC enzymes, the NisT transporter was capable of excreting unmodified prenisin and fusions of the leader peptide with non-lantibiotic peptides . Our data show that NisT specifies a broad spectrum (poly)peptide transporter that can function either in conjunction with or independently from the biosynthetic genes . NisT secretes both unmodified and partially or fully posttranslationally modified forms of prenisin and non-lantibiotic peptides . These results open the way for efficient production of a wide range of peptides with increased stability or novel bioactivities.

FEMS Microbiol Lett, 2004 Apr 1, 233(1), 37 - 43
Distribution and composition of the lysis cassette of Lactococcus lactis phages and functional analysis of bacteriophage ul36 holin; Labrie S et al.; The bacteriophage lysis cassette, which comprises a lysin and a holin gene, was analyzed in 18 Lactococcus lactis phages . A muramidase motif was found in the lysins of c2-like phages, while an amidase motif was observed in the lysins of 936-like phages . Both amidase and muramidase types were detected among the P335 phages . The P335 lysins were separated into three groups based on amino acid sequence identity . A class I holin was recognized in 936-like and c2-like phages, whereas P335-like phages possess class II holins . The P335 holins were further divided into four groups based on sequence identity . Only the holins of 936-like phages contained putative dual-start motifs . The unusual lysis cassette of the highly virulent P335-like phage ul36 contains a unique holin (orf74B) upstream of a lysin which is present in several other P335-like phages . Using the lambdadelta Sthf system, we demonstrated that gpORF74B induces cell lysis at the same time as lambdadelta Sthf::S105, the effector of lambda lysis . Transcriptional analysis of ul36 lysis cassette showed that first transcripts are detected 35 min after infection of L . lactis cells . The lysis clock of phage ul36 appears to be controlled by the late expression of the holin and lysin genes.

Int J Food Microbiol, 2004 Apr 1, 92(1), 1 - 14
A bacterial gene homologous to ABC transporters protect Oenococcus oeni from ethanol and other stress factors in wine; Bourdineaud JP et al.; The wine lactic acid bacteria Oenococcus oeni has to cope with harsh environmental conditions including an acidic pH, a high alcoholic content, non-optimal growth temperatures, and growth inhibitory compounds such as fatty acids, phenolic acids and tannins . We here describe characterisation and cloning of the O . oeni omrA gene encoding a protein belonging to the ATP-binding cassette superfamily of transporters . The OmrA protein displays the highest sequence similarity with the subfamily of ATP-dependent multidrug resistance (MDR) proteins, most notably the bacterial Lactococcus lactis LmrA homologue of the human MDR1 P-glycoprotein . The omrA gene proved to be a stress-responsive gene since its expression was increased at high temperature or under osmotic shock . The OmrA protein function was tested in Escherichia coli, and consistent with the omrA gene expression pattern, OmrA conferred protection to bacteria grown on a high salt medium . OmrA also triggered bacterial resistance to sodium laurate, wine and ethanol toxicity . The homologous LmrA protein featured the same stress-protective pattern than OmrA when expressed in E . coli, and the contribution to resistance of both OmrA and LmrA transporters was decreased by verapamil, a well-known inhibitor of the human MDR1 protein . Genes homologous to omrA were detected in other wine lactic acid bacteria, suggesting that this type of genes might constitute a well-conserved stress-protective molecular device.

J Bacteriol, 2004 Apr, 186(7), 1991 - 8
A conjugation-based system for genetic analysis of group II intron splicing in Lactococcus lactis; Klein JR et al.; The conjugative element pRS01 from Lactococcus lactis encodes the putative relaxase protein LtrB . The ltrB gene is interrupted by the functional group II intron Ll.ltrB . Accurate splicing of the two ltrB exons is required for synthesis of the mRNA encoding the LtrB conjugative relaxase and subsequent plasmid transfer . A conjugation-based genetic assay was developed to identify Ll.ltrB mutations that affect splicing . In this assay a nonsplicing, transfer-defective pRS01 derivative (pM1014) and a shuttle vector carrying the ltrB region, including the Ll.ltrB intron (pCOM9), are used . pCOM9 provides splicing-dependent complementation of the transfer defect of pM1014 . Site-directed mutations within Ll.ltrB, either in the catalytic RNA or in the intron-encoded protein gene ltrA, were generated in the context of pCOM9 . When these mutants were tested in the conjugation-based assay, significantly reduced mating was observed . Quantitative molecular analysis of in vivo splicing activity confirmed that the observed mating defects resulted from reduced splicing . Once the system was validated for the engineered mutants, random mutagenesis of the intron followed by genetic and molecular screening for splicing defects resulted in identification of point mutations that affect splicing.

J Biol Chem, 2004 May 28, 279(22), 23431 - 7 Epub 2004 Mar 16.
Molecular structure of human galactose mutarotase; Thoden JB et al.; Galactose mutarotase catalyzes the conversion of beta-d-galactose to alpha-d-galactose during normal galactose metabolism . The enzyme has been isolated from bacteria, plants, and animals and is present in the cytoplasm of most cells . Here we report the x-ray crystallographic analysis of human galactose mutarotase both in the apoform and complexed with its substrate, beta-d-galactose . The polypeptide chain folds into an intricate array of 29 beta-strands, 25 classical reverse turns, and 2 small alpha-helices . There are two cis-peptide bonds at Arg-78 and Pro-103 . The sugar ligand sits in a shallow cleft and is surrounded by Asn-81, Arg-82, His-107, His-176, Asp-243, Gln-279, and Glu-307 . Both the side chains of Glu-307 and His-176 are in the proper location to act as a catalytic base and a catalytic acid, respectively . These residues are absolutely conserved among galactose mutarotases . To date, x-ray models for three mutarotases have now been reported, namely that described here and those from Lactococcus lactis and Caenorhabditis elegans . The molecular architectures of these enzymes differ primarily in the loop regions connecting the first two beta-strands . In the human protein, there are six extra residues in the loop compared with the bacterial protein for an approximate longer length of 9 A . In the C . elegans protein, the first 17 residues are missing, thereby reducing the total number of beta-strands by one.

Biochemistry, 2004 Mar 23, 43(11), 3049 - 56
Structural characterization of lacticin 3147, a two-peptide lantibiotic with synergistic activity; Martin NI et al.; Lantibiotics are antibacterial peptides isolated from bacterial sources that exhibit activity toward Gram-positive organisms and are usually several orders of magnitude more potent than traditional antibiotics such as penicillin . They contain a number of unique structural features including dehydro amino acid and lanthionine (thioether) residues . Introduced following ribosomal translation of the parent peptide, these moieties render conventional methods of peptide analysis ineffective . We report herein a new method using nickel boride (Ni(2)B), in the presence of deuterium gas, to reduce dehydro side chains and reductively desulfurize lanthionine bridges found in lantibiotics . Using this approach, it is possible to identify and distinguish the original locations of dehydro side chains and lanthionine bridges by traditional peptide sequencing (Edman degradation) followed by mass spectrometry . The strategy was initially verified using nisin A, a structurally well characterized lantibiotic, and subsequently extended to the novel two-component lantibiotic, lacticin 3147, produced by Lactococcus lactis subspecies lactis DPC3147 . The primary structures of both lacticin 3147 peptides were then fully assigned by use of multidimensional NMR spectroscopy, showing that lacticin 3147 A1 has a specific lanthionine bridging pattern which resembles the globular type-B lantibiotic mersacidin, whereas the A2 peptide is a member of the elongated type-A lantibiotic class . Also obtained by NMR were solution conformations of both lacticin 3147 peptides, indicating that A1 may adopt a conformation similar to that of mersacidin and that the A2 peptide adopts alpha-helical structure . These results are the first of their kind for a synergistic lantibiotic pair (only four such pairs have been reported to date).

Arch Biochem Biophys, 2004 Apr 1, 424(1), 105 - 11
Competition between ammonia derived from internal glutamine hydrolysis and hydroxylamine present in the solution for incorporation into UTP as catalysed by Lactococcus lactis CTP synthase; Willemoes M; CTP synthase catalyses the reaction: glutamine+UTP+ATP --> glutamate+CTP+ADP+P(i) . The reaction is greatly stimulated by the allosteric binding of GTP . In addition to glutamine that is hydrolysed by the enzyme to ammonia and glutamate, CTP synthase will also utilise external sources of amino donors such as NH(4)Cl . This reaction is no longer dependent on allosteric activation by GTP . Hydroxylamine is also a substrate for Lactococcus lactis CTP synthase and results in the formation of N4-OH CTP . This product has the feature that it absorbs at 300nm where CTP absorption was shown to be greatly reduced and enabled the determination of N4-OH CTP formation in the presence of CTP synthesis derived from glutamine hydrolysis . Differences in initial rates determined for the hydroxylamine dependent reaction at 291nm in the presence and absence of glutamine and GTP were ascribed to simultaneous CTP and N4-OH CTP synthesis in the presence of these compounds . A characterisation of the apparent inhibition by GTP and glutamine of N4-OH CTP synthesis determined at 300nm showed that glutamine dependent CTP synthesis occurs at a rate of about 60% of that in the absence of hydroxylamine . GTP dependent inhibition of the ammonium chloride dependent reaction of L . lactis CTP synthase by the glutamine analog glutamate gamma-semialdehyde showed a partial inhibition with a maximum inhibition of about 60% . These results are interpreted in terms of a "half of the sites" mechanism for glutamine hydrolysis on CTP synthase.

Genet Mol Res, 2003 Dec 30, 2(4), 348 - 59
Oxidative stress in Lactococcus lactis; Miyoshi A et al.; Lactococcus lactis, the most extensively characterized lactic acid bacterium, is a mesophilic- and microaerophilic-fermenting microorganism widely used for the production of fermented food products . During industrial processes, L . lactis is often exposed to multiple environmental stresses (low and high temperature, low pH, high osmotic pressure, nutrient starvation and oxidation) that can cause loss or reduction of bacterial viability, reproducibility, as well as organoleptic and/or fermentative qualities . Among these stress factors, oxidation can be considered one of the most deleterious to the cell, causing cellular damage at both molecular and metabolic levels . During the last two decades, considerable efforts have been made to improve our knowledge of oxidative stress in L . lactis . Many genes involved with both oxidative stress resistance and control mechanisms have been identified; functionally they seem to overlap . The finding of new genes, and a better understanding of the molecular mechanisms of stress resistance in L . lactis and other lactic acid bacterium, will lead to the construction and isolation of stress-resistant strains . Such strains could be exploited for both traditional and probiotic uses.

Appl Environ Microbiol, 2004 Mar, 70(3), 1843 - 6
Reappraisal of the regulation of lactococcal L-lactate dehydrogenase; van Niel EW et al.; Lactococcal lactate dehydrogenases (LDHs) are coregulated at the substrate level by at least two mechanisms: the fructose-1,6-biphosphate/phosphate ratio and the NADH/NAD ratio . Among the Lactococcus lactis species, there are strains that are predominantly regulated by the first mechanism (e.g., strain 65.1) or by the second mechanism (e.g., strain NCDO 2118) . A more complete model of the kinetics of the regulation of lactococcal LDH is discussed.

Appl Environ Microbiol, 2004 Mar, 70(3), 1600 - 7
Influence of lipoteichoic acid D-alanylation on protein secretion in Lactococcus lactis as revealed by random mutagenesis; Nouaille S et al.; Lactococcus lactis, a food-grade nonpathogenic lactic acid bacterium, is a good candidate for the production of heterologous proteins of therapeutic interest . We examined host factors that affect secretion of heterologous proteins in L . lactis . Random insertional mutagenesis was performed with L . lactis strain MG1363 carrying a staphylococcal nuclease (Nuc) reporter cassette in its chromosome . This cassette encodes a fusion protein between the signal peptide of the Usp45 lactococcal protein and the mature moiety of a truncated form of Nuc (NucT) . The Nuc secretion efficiency (secreted NucT versus total NucT) from this construct is low in L . lactis (approximately 40%) . Twenty mutants affected in NucT production and/or in secretion capacity were selected and identified . In these mutants, several independent insertions mapped in the dltA gene (involved in D-alanine transfer in lipoteichoic acids) and resulted in a NucT secretion defect . Characterization of the dltA mutant phenotype with respect to NucT secretion revealed that it is involved in a late secretion stage by causing mature NucT entrapment at the cell surface.

Appl Environ Microbiol, 2004 Mar, 70(3), 1466 - 74
Engineering Lactococcus lactis for production of mannitol: high yields from food-grade strains deficient in lactate dehydrogenase and the mannitol transport system; Gaspar P et al.; Mannitol is a sugar polyol claimed to have health-promoting properties . A mannitol-producing strain of Lactococcus lactis was obtained by disruption of two genes of the phosphoenolpyruvate (PEP)-mannitol phosphotransferase system (PTS(Mtl)) . Genes mtlA and mtlF were independently deleted by double-crossover recombination in strain L . lactis FI9630 (a food-grade lactate dehydrogenase-deficient strain derived from MG1363), yielding two mutant (Delta ldh Delta mtlA and Delta ldh Delta mtlF) strains . The new strains, FI10091 and FI10089, respectively, do not possess any selection marker and are suitable for use in the food industry . The metabolism of glucose in nongrowing cell suspensions of the mutant strains was characterized by in vivo (13)C-nuclear magnetic resonance . The intermediate metabolite, mannitol-1-phosphate, accumulated intracellularly to high levels (up to 76 mM) . Mannitol was a major end product, one-third of glucose being converted to this hexitol . The double mutants, in contrast to the parent strain, were unable to utilize mannitol even after glucose depletion, showing that mannitol was taken up exclusively by PEP-PTS(Mtl) . Disruption of this system completely blocked mannitol transport in L . lactis, as intended . In addition to mannitol, approximately equimolar amounts of ethanol, 2,3-butanediol, and lactate were produced . A mixed-acid fermentation (formate, ethanol, and acetate) was also observed during growth under controlled conditions of pH and temperature, but mannitol production was low . The reasons for the alteration in the pattern of end products under nongrowing and growing conditions are discussed, and strategies to improve mannitol production during growth are proposed.

Vaccine, 2004 Mar 12, 22(9-10), 1188 - 98
A Plasmodium falciparum GLURP-MSP3 chimeric protein; expression in Lactococcus lactis, immunogenicity and induction of biologically active antibodies; Theisen M et al.; Plasmodium falciparum malaria is a major cause of morbidity and mortality worldwide . To evaluate the efficacy of a possible vaccine antigen against P . falciparum infection, a fusion protein, derived from P . falciparum Glutamate-rich protein (GLURP) genetically coupled to P . falciparum Merozoite surface protein 3 (MSP3) was produced in Lactococcus lactis as a secreted recombinant GLURP-MSP3 fusion protein . The hybrid protein was purified to homogeneity by ion exchange and hydrophobic-interaction chromatography and its composition was verified by MALDI MS, SDS/PAGE and Western blotting with antibodies against antigenic components of GLURP and MSP3 . Mice immunized with the hybrid protein produced higher levels of both GLURP- and MSP3-specific antibodies than mice immunized with either GLURP, MSP3 or a mix of both . The protective potential of the hybrid protein was also demonstrated by in vitro parasite-growth inhibition of mouse anti-GLURP-MSP3 IgG antibodies in a monocyte-dependent manner . These results indicate that the GLURP-MSP3 hybrid could be a valuable strategy for future P . falciparum vaccine development.

J Bacteriol, 2004 Mar, 186(6), 1648 - 57
Proteome analyses of heme-dependent respiration in Lactococcus lactis: involvement of the proteolytic system; Vido K et al.; Sugar fermentation was long considered the sole means of energy metabolism available to lactic acid bacteria . We recently showed that metabolism of Lactococcus lactis shifts progressively from fermentation to respiration during growth when oxygen and heme are available . To provide insights into this phenomenon, we compared the proteomic profiles of L . lactis under fermentative and respiratory growth conditions in rich medium . We identified 21 proteins whose levels differed significantly between these conditions . Two major groups of proteins were distinguished, one involved in carbon metabolism and the second in nitrogen metabolism . Unexpectedly, enzymes of the proteolytic system (PepO1 and PepC) which are repressed in rich medium in fermentation growth were induced under respiratory conditions despite the availability of free amino acids . A triple mutant (dtpT dtpP oppA) deficient in oligopeptide transport displayed normal respiration, showing that increased proteolytic activity is not an absolute requirement for respiratory metabolism . Transcriptional analysis confirmed that pepO1 is induced under respiration-permissive conditions . This induction was independent of CodY, the major regulator of proteolytic functions in L . lactis . We also observed that pepO1 induction is redox sensitive . In a codY mutant, pepO1 expression was increased twofold in aeration and eightfold in respiration-permissive conditions compared to static conditions . These observations suggest that new regulators activate proteolysis in L . lactis, which help to maintain the energetic needs of L . lactis during respiration.

FEMS Microbiol Lett, 2004 Feb 16, 231(2), 291 - 8
Regulation of lantibiotic lacticin 481 production at the transcriptional level by acid pH; Hindre T et al.; The lantibiotic lacticin 481 operon (lctAMTFEG) is mainly transcribed from P1 and P3, two promoters lying upstream of lctA . A weak additional promoter allows independent expression of the immunity genes (lctFEG) . Lacticin 481 production by Lactococcus lactis is stimulated by the acidification due to lactic acid production, and by artificially lowering the pH of the medium . This regulation occurs at the transcriptional level, since P1 and P3 are both acid-induced . P1 is weaker but more tightly regulated than P3 . As no specific regulator is encoded by the lacticin 481 operon, P1 and P3 are likely controlled by a general regulator.

Microbiol Immunol, 2004, 48(2), 75 - 82
New Lactococcus strain with immunomodulatory activity: enhancement of Th1-type immune response; Kimoto H et al.; Few studies exist dealing with the probiotic activity of lactococci, which are commonly used as starter bacteria in the manufacture of many kinds of fermented dairy products . Fifteen strains of the genus Lactococcus were examined for their probiotic activities, such as immunomodulatory effects . Six strains induced the production of cytokines (IL-12, IL-6, and TNF-alpha) in macrophage-like cell line J774.1, and the highest induction was observed with Lactococcus lactis subsp . lactis G50 . The cytokine induction in the J774.1 cell line was almost entirely sustained after heat-killing of the strain . Spleen cells from BALB/c mice fed G50 culture produced more IL-12 and IFN-gamma and slightly less IL-4 and IL-6 than the control (i.e., without strain G50), indicating that strain G50 can enhance Th1-type immune response in vivo . The effect of the oral administration of strain G50 on antibody response in mice was also investigated . Mice were immunized with ovomucoid (OVM), a potent egg allergen, and the antibody level in the serum was then determined . The total IgE antibody level in the group treated with strain G50 was significantly lower than that of the control . The response of OVM-specific IgG1 and IgE antibodies tended to be low in the group that was administered strain G50, compared with the response of the control group . These results suggest that strain G50 has an ability to suppress the Th2 response . Thus, Lactococcus lactis subsp . lactis G50 is a potential probiotic strain for the suppression of hypersensitive reactions caused by the Th2 response.

J Bacteriol, 2004 Mar, 186(5), 1239 - 48
Identification and functional characterization of the Lactococcus lactis rfb operon, required for dTDP-rhamnose Biosynthesis; Boels IC et al.; dTDP-rhamnose is an important precursor of cell wall polysaccharides and rhamnose-containing exopolysaccharides (EPS) in Lactococcus lactis . We cloned the rfbACBD operon from L . lactis MG1363, which comprises four genes involved in dTDP-rhamnose biosynthesis . When expressed in Escherichia coli, the lactococcal rfbACBD genes could sustain heterologous production of the Shigella flexneri O antigen, providing evidence of their functionality . Overproduction of the RfbAC proteins in L . lactis resulted in doubled dTDP-rhamnose levels, indicating that the endogenous RfbAC activities control the intracellular dTDP-rhamnose biosynthesis rate . However, RfbAC overproduction did not affect rhamnose-containing B40-EPS production levels . A nisin-controlled conditional RfbBD mutant was unable to grow in media lacking the inducer nisin, indicating that the rfb genes have an essential role in L . lactis . Limitation of RfbBD activities resulted in the production of altered EPS . The monomeric sugar of the altered EPS consisted of glucose, galactose, and rhamnose at a molar ratio of 1:0.3:0.2, which is clearly different from the ratio in the native sugar . Biophysical analysis revealed a fourfold-greater molecular mass and a twofold-smaller radius of gyration for the altered EPS, indicating that these EPS are more flexible polymers with changed viscosifying properties . This is the first indication that enzyme activity at the level of central carbohydrate metabolism affects EPS composition.

Virology, 2004 Jan 5, 318(1), 231 - 44
Complete genome sequence of the Lactococcus lactis temperate phage phiLC3: comparative analysis of phiLC3 and its relatives in lactococci and streptococci; Blatny JM et al.; Complete genome sequencing of the P335 temperate Lactococcus lactis bacteriophage phiLC3 (32, 172 bp) revealed fifty-one open reading frames (ORFs) . Four ORFs did not show any homology to other proteins in the database and twenty-one ORFs were assigned a putative biological function . phiLC3 contained a unique replication module and orf201 was identified as the putative replication initiator protein-encoding gene . phiLC3 was closely related to the L . lactis r1t phage (73% DNA identity) . Similarity was also shared with other lactococcal P335 phages and the Streptococcus pyogenes prophages 370.3, 8232.4 and 315.5 over the non-structural genes and the genes involved in DNA packaging/phage morphogenesis, respectively . phiLC3 contained small homologous regions distributed among lactococcal phages suggesting that these regions might be involved in mediating genetic exchange . Two regions of 30 and 32 bp were conserved among the streptococcal and lactococcal r1t-like phages . These two regions, as well as other homologous regions, were located at mosaic borders and close to putative transcriptional terminators indicating that such regions together might attract recombination . The conserved regions found among lactococcal and streptococcal phages might be used for identification of phages/prophages/prophage remnants in their hosts.

FEMS Microbiol Lett, 2004 Feb 9, 231(1), 85 - 90
Distribution of the NisI immunity protein and enhancement of nisin activity by the lipid-free NisI; Koponen O et al.; Lactococcus lactis cells producing the antibacterial peptide nisin protect their own cytoplasmic membrane by specific immunity proteins, NisI and NisF/E/G . We show here that approximately half of the produced NisI escaped the lipid modification (LF-NisI=lipid-free NisI) and was secreted to the medium, and that LF-NisI had no affinity to cells of L . lactis . The molar ratio of NisI and nisin was determined to be approximately 1:10 on the cell surface and 1:50 in the culture supernatant . Purified LF-NisI was shown to enhance the activity of nisin against several tested indicator strains . The enhancement of nisin activity by LF-NisI was not observed with cells containing the NisFEG transport system.

J Dairy Sci, 2004 Feb, 87(2), 300 - 7
The effect of Lactococcus lactis starter cultures on the oxidative stability of liquid whey; Tomaino RM et al.; The oxidative stability of liquid Cheddar cheese whey was evaluated using 2 Lactococcus lactis starter cultures in combination and alone along with a control, utilizing glucono-delta-lactone for acid development . Fresh and stored whey were evaluated for volatile composition, free fatty acids, and flavor by descriptive sensory analysis . A significant increase in volatile lipid oxidation products, most notably, hexanal, occurred during storage, and a corresponding decline in the free fatty acid linoleic acid was found . The flavor and aroma characteristic, cardboardy, was correlated to the increase in volatile lipid oxidation products and the decline in linoleic acid . Evidence strongly suggested that lipid oxidation was initiated during whey production and escalated during storage and that the starter cultures significantly influenced the level of volatile lipid oxidation products . Further understanding of the impact of starter cultures on whey may allow for the production of higher quality whey ingredients with wider food application.

J Bacteriol, 2004 Feb, 186(4), 1147 - 57
ArgR and AhrC are both required for regulation of arginine metabolism in Lactococcus lactis; Larsen R et al.; The DNA binding proteins ArgR and AhrC are essential for regulation of arginine metabolism in Escherichia coli and Bacillus subtilis, respectively . A unique property of these regulators is that they form hexameric protein complexes, mediating repression of arginine biosynthetic pathways as well as activation of arginine catabolic pathways . The gltS-argE operon of Lactococcus lactis encodes a putative glutamate or arginine transport protein and acetylornithine deacetylase, which catalyzes an important step in the arginine biosynthesis pathway . By random integration knockout screening we found that derepression mutants had ISS1 integrations in, among others, argR and ahrC . Single as well as double regulator deletion mutants were constructed from Lactococcus lactis subsp . cremoris MG1363 . The three arginine biosynthetic operons argCJDBF, argGH, and gltS-argE were shown to be repressed by the products of argR and ahrC . Furthermore, the arginine catabolic arcABD1C1C2TD2 operon was activated by the product of ahrC but not by that of argR . Expression from the promoter of the argCJDBF operon reached similar levels in the single mutants and in the double mutant, suggesting that the regulators are interdependent and not able to complement each other . At the same time they also appear to have different functions, as only AhrC is involved in activation of arginine catabolism . This is the first study where two homologous arginine regulators are shown to be involved in arginine regulation in a prokaryote, representing an unusual mechanism of regulation.

Appl Microbiol Biotechnol, 2004 Jul, 65(1), 61 - 7 Epub 2004 Feb 03.
A food-grade delivery system for Lactococcus lactis and evaluation of inducible gene expression; Simoes-Barbosa A et al.; The genetic improvement of Lactococcus lactis is a matter of biotechnological interest in the food industry and in the pharmaceutical and medical fields . However, to construct a food-grade delivery system, both the presence of antibiotic markers or plasmid sequences should be avoided and the maintenance and expression of the cloned gene should be guaranteed . The objective of this work was to produce crossover mutants of L . lactis with a reporter gene under the control of an inducible promoter in order to evaluate the level of gene expression . We utilized a nuclease gene of Staphylococcus aureus as a reporter gene, P(nisA) as the nisin-inducible promoter, a non-essential gene involved in histidine biosynthesis of L . lactis as the site for homologous recombination, and pRV300 as a suicide vector for the genomic integration in L . lactis NZ9000 . Single- and double-crossover mutants were identified by genotype and phenotype . Relative to episomal transformants of L . lactis, the level of expression of the heterologous protein after nisin induction was similar in the crossover mutants, suggesting that a single copy of the heterologous gene can be used to produce the protein of interest.

J Mol Biol, 2004 Feb 13, 336(2), 421 - 39
Use of computer-designed group II introns to disrupt Escherichia coli DExH/D-box protein and DNA helicase genes; Perutka J et al.; Mobile group II introns are site-specific retroelements that use a novel mobility mechanism in which the excised intron RNA inserts directly into a DNA target site and is then reverse transcribed by the associated intron-encoded protein . Because the DNA target site is recognized primarily by base-pairing of the intron RNA with only a small number of positions recognized by the protein, it has been possible to develop group II introns into a new type of gene targeting vector ("targetron"), which can be reprogrammed to insert into desired DNA targets simply by modifying the intron RNA . Here, we used databases of retargeted Lactococcus lactis Ll.LtrB group II introns and a compilation of nucleotide frequencies at active target sites to develop an algorithm that predicts optimal Ll.LtrB intron-insertion sites and designs primers for modifying the intron to insert into those sites . In a test of the algorithm, we designed one or two targetrons to disrupt each of 28 Escherichia coli genes encoding DExH/D-box and DNA helicase-related proteins and tested for the desired disruptants by PCR screening of 100 colonies . In 21 cases, we obtained disruptions at frequencies of 1-80% without selection, and in six other cases, where disruptants were not identified in the initial PCR screen, we readily obtained specific disruptions by using the same targetrons with a retrotransposition-activated selectable marker . Only one DExH/D-box protein gene, secA, which was known to be essential, did not give viable disruptants . The apparent dispensability of DExH/D-box proteins in E.coli contrasts with the situation in yeast, where the majority of such proteins are essential . The methods developed here should permit the rapid and efficient disruption of any bacterial gene, the computational analysis provides new insight into group II intron target site recognition, and the set of E.coli DExH/D-box protein and DNA helicase disruptants should be useful for analyzing the function of these proteins.

Trends Biotechnol, 2004 Feb, 22(2), 72 - 9
Engineering metabolic highways in Lactococci and other lactic acid bacteria; de Vos WM et al.; Lactic acid bacteria (LAB) are widely used in industrial food fermentations and are receiving increased attention for use as cell factories for the production of food and pharmaceutical products . Glycolytic conversion of sugars into lactic acid is the main metabolic highway in these Gram-positive bacteria and Lactococcus lactis has become the model organism because of its small genome, genetic accessibility and simple metabolism . Here we discuss the metabolic engineering of L . lactis and the value of metabolic models compared with other LAB, with a particular focus on the food-grade production of metabolites involved in flavour, texture and health.

Mol Microbiol, 2004 Jan, 51(2), 511 - 22
Chromosomal constraints in Gram-positive bacteria revealed by artificial inversions; Campo N et al.; We used artificial chromosome inversions to investigate the chromosomal constraints that preserve genome organization in the Gram-positive bacterium Lactococcus lactis . Large inversions, 80-1260 kb in length, disturbing the symmetry of the origin and terminus of the replication axis to various extents, were constructed using the site-specific Cre-loxP recombination system . These inversions were all mechanistically feasible and fell into various classes according to stability and effect on cell fitness . The L . lactis chromosome supports only to some extent unbalance in length of its replication arms . The location of detrimental inversions allowed identification of two constrained chromosomal regions: a large domain covering one fifth of the genome that encompasses the origin of replication (Ori domain), and a smaller domain located at the opposite of the chromosome (Ter domain).

Dis Aquat Organ, 2003 Dec 3, 57(1-2), 151 - 5
Effect of potassium permanganate stress on immune resistance and susceptibility to Lactococcus garvieae in the giant freshwater prawn Macrobrachium rosenbergii; Cheng W et al.; This work is part of a continuing series of investigations on the effect of commonly used aquaculture chemicals on the immune resistance and susceptibility of the giant freshwater prawn Macrobrachium rosenbergii to Lactococcus garvieae . The methodology has been described in earlier publications of the series . Potassium permanganate at 1.0 mg l(-1) in tryptic soy broth (TSB) had no effect on the growth rate of L . garvieae . The mortality of M . rosenbergii challenged with 4 x 10(6) colony-forming units (cfu) prawn(-1) of TSB-grown L . garvieae was significantly greater than that of challenged controls . Addition of potassium permanganate at 1.0 mg l(-1) in TSB significantly increased the virulence of L . garvieae to M . rosenbergii . Exposure of M . rosenbergii to potassium permanganate prior to challenge with TSB-grown L . garvieae at 4 x 10(6) and 3 x 10(6) cfu prawn(-1) revealed that 96 h mortality was significantly lower for prawns held in water containing 0.3 mg l(-1) of the chemical than for prawns in water containing 1.0 mg l(-1) or no chemical . Potassium permanganate caused no significant changes in total hemocyte counts and differential hemocyte counts, compared to the control treatments . However, a concentration of 1.0 mg l(-1) or more for 96 h resulted in decreased phenoloxidase activity, phagocytic activity and clearance efficiency . Respiratory burst increased with exposure to 0.3 mg l(-1) . In conclusion, treatment with potassium permanganate at 0.3 mg 1(-1) was effective in reducing M . rosenbergii mortality from L . garvieae infection, but higher concentrations had a negative effect, probably due to reduced prawn defenses.

Can J Microbiol, 2003 Nov, 49(11), 707 - 11
Survival of lactococci during passage through mouse digestive tract; Kimoto H et al.; One of the important properties of probiotics is the ability to survive in the intestine . There have been few studies on the probiotic property of lactococci, since they are formally not considered to be natural inhabitants of the intestine . To evaluate lactococci as probiotic bacteria, we investigated their ability to survive during gastric transit by in vitro and in vivo tests . When exposed to an in vitro simulated gastrointestinal environment, such as low pH and bile, only Lactococcus lactis subsp . lactis bv . diacetylactis N7 showed a moderate survival rate among the four strains tested . The tested strains were orally administered to mice, and intestinal passage of the ingested strains was monitored by two methods: antibiotics and PCR . Viable cells of strain N7 were recovered from feces within 24-48 h after administration but not at 72 h . Lactococcus lactis subsp . cremoris ATCC 19257, which had a poor survival rate in vitro test, was also detected at 12 h but not at 24 h . These results indicate that lactococci can reach the mouse intestine alive, but not colonize it . If administered daily, viable strain N7 may exist continuously in the intestine . The effect of strain N7 on intestinal microbial balance and on animal health will be the subject of a further study.

J Bacteriol, 2004 Feb, 186(3), 713 - 21
The Lactococcal abortive phage infection system AbiP prevents both phage DNA replication and temporal transcription switch; Domingues S et al.; We describe here a new lactococcal abortive phage infection system, designated AbiP . AbiP is effective against some lactococcal phages of one prevalent group, 936, but not against phages from the other two groups (c6A and P335) . It was identified in the Lactococcus lactis subsp . cremoris strain IL420, on the native plasmid pIL2614 . AbiP is encoded by a single gene, expressed in an operon with a second gene . In this work, abiP is shown to affect both the replication and transcription of phage DNA . In AbiP(+) cells, phage DNA replication is arrested approximately 10 min after infection . Levels of middle and late phage transcripts are lower in AbiP(+) than in AbiP(-) cells, probably due to the smaller amount of phage DNA . By contrast, early phage transcripts are more abundant in AbiP(+) than in AbiP(-) cells, suggesting that the switch-off, which occurs 15 min after infection in AbiP(-) cells, is prevented in AbiP(+) cells.

Appl Environ Microbiol, 2004 Jan, 70(1), 638 - 41
Role of aminotransferase IlvE in production of branched-chain fatty acids by Lactococcus lactis subsp . lactis; Ganesan B et al.; The objective of this study was to determine the role of a lactococcal branched-chain amino acid aminotransferase gene, ilvE, in the production of branched-chain fatty acids . Lactococcus lactis subsp . lactis LM0230 and an ilvE deletion mutant, JLS450, produced branched-chain fatty acids from amino and alpha-keto acids at levels above alpha-keto acid spontaneous degradation and the fatty acids' flavor thresholds . The deletion mutant produced the same amounts of branched-chain fatty acids from precursor amino acids as did the parent . This was not the case, however, for the production of branched-chain fatty acids from the corresponding precursor alpha-keto acids . The deletion mutant produced a set of fatty acids different from that produced by the parent . We concluded from these observations that ilvE plays a role in the specific type of fatty acids produced but has little influence on the total amount of fatty acids produced by lactococci.

Appl Environ Microbiol, 2004 Jan, 70(1), 34 - 42
Variable bacteriocin production in the commercial starter Lactococcus lactis DPC4275 is linked to the formation of the cointegrate plasmid pMRC02; Trotter M et al.; Lactococcus lactis DPC4275 is a bacteriocin-producing transconjugant of the industrial starter strain DPC4268 . Strain DPC4275 was generated through conjugal transfer by mating DPC4268 with L . lactis MG1363 containing the 60-kb plasmid pMRC01, which encodes the genetic determinants for the lantibiotic lacticin 3147 and for a phage resistance mechanism of the abortive infection type . The many significant applications of this strain prompted a genetic analysis of its apparently unstable bacteriocin-producing phenotype . Increased levels of lacticin 3147 produced by DPC4275 were associated with the appearance of an 80-kb plasmid, designated pMRC02, which was derived from DNA originating from pMRC01 (60 kb) and a resident DPC4268 proteinase plasmid, pMT60 (60 kb) . Indeed, pMRC02 was shown to be derived from the insertion of a 17-kb fragment of pMRC01, encompassing the lacticin 3147 operon, into pMT60 . The presence of pMRC02 at a high copy number was found to correlate with increased levels of lacticin 3147 in DPC4275 compared to the wild-type containing pMRC01 . Subsequent transfer of pMRC02 into the plasmid-free strain MG1363 by electroporation allowed a direct phenotypic comparison with pMRC01, also studied in the MG1363 background . Plasmid pMRC02 displayed phage resistance similar to that by pMRC01, although it was less potent, as demonstrated by a larger plaque size for phage c2 infection of MG1363(pMRC02) . While this locus is flanked by IS946 elements, the sequencing of pMT60-pMRC01 junction sites established that this event was unlikely to be insertion sequence mediated and most probably occurred by homologous recombination followed by deletion of most of pMRC01 . This was not a random occurrence, as nine other transconjugants investigated were found to have the same junction sites . Such derivatives of commercial strains producing increased levels of bacteriocin could be exploited as protection cultures for food applications.

J Microbiol Methods, 2004 Jan, 56(1), 133 - 6
Photometric assay for measuring the intracellular concentration of branched-chain amino acids in bacteria; Petranovic D et al.; The changes in intracellular pool of branched-chain amino acids (BCAA) regulate different physiological processes in bacteria . Up to date, the only available photometric test for measuring BCAA concentration was adapted for blood and plasma samples in diagnostic purposes . We have modified this method for use on bacterial cells, and tested its applicability on several model organisms: Lactococcus lactis, Bacillus subtilis and Escherichia coli.

J Bacteriol, 2004 Jan, 186(2), 287 - 95
The methyltransferase from the LlaDII restriction-modification system influences the level of expression of its own gene; Christensen LL et al.; The type II restriction-modification (R-M) system LlaDII isolated from Lactococcus lactis contains two tandemly arranged genes, llaDIIR and llaDIIM, encoding a restriction endonuclease (REase) and a methyltransferase (MTase), respectively . Interestingly, two LlaDII recognition sites are present in the llaDIIM promoter region, suggesting that they may influence the activity of the promoter through methylation status . In this study, separate promoters for llaDIIR and llaDIIM were identified, and the regulation of the two genes at the transcriptional level was investigated . DNA fragments containing the putative promoters were cloned in a promoter probe vector and tested for activity in the presence and absence of the active MTase . The level of expression of the MTase was 5- to 10-fold higher than the level of expression of the REase . The results also showed that the presence of M.LlaDII reduced the in vivo expression of the llaDIIM promoter (P(llaDIIM)) up to 1,000-fold, whereas the activity of the llaDIIR promoter (P(llaDIIR)) was not affected . Based on site-specific mutations it was shown that both of the LlaDII recognition sites within P(llaDIIM) are required to obtain complete repression of transcriptional activity . No regulation was found for llaDIIR, which appears to be constitutively expressed.

J Biomol Struct Dyn, 2004 Feb, 21(4), 527 - 36
Synonymous codon usage in Lactococcus lactis: mutational bias versus translational selection; Gupta SK et al.; In this study codon usage bias of all experimentally known genes of Lactococcus lactis has been analyzed . Since Lactococcus lactis is an AT rich organism, it is expected to occur A and/or T at the third position of codons and detailed analysis of overall codon usage data indicates that A and/or T ending codons are predominant in this organism . However, multivariate statistical analyses based both on codon count and on relative synonymous codon usage (RSCU) detect a large number of genes, which are supposed to be highly expressed are clustered at one end of the first major axis, while majority of the putatively lowly expressed genes are clustered at the other end of the first major axis . It was observed that in the highly expressed genes C and T ending codons are significantly higher than the lowly expressed genes and also it was observed that C ending codons are predominant in the duets of highly expressed genes, whereas the T endings codons are abundant in the quartets . Abundance of C and T ending codons in the highly expressed genes suggest that, besides, compositional biases, translational selection are also operating in shaping the codon usage variation among the genes in this organism as observed in other compositionally skewed organisms . The second major axis generated by correspondence analysis on simple codon counts differentiates the genes into two distinct groups according to their hydrophobicity values, but the same analysis computed with relative synonymous codon usage values could not discriminate the genes according to the hydropathy values . This suggests that amino acid composition exerts constraints on codon usage in this organism . On the other hand the second major axis produced by correspondence analysis on RSCU values differentiates the genes into two groups according to the synonymous codon usage for cysteine residues (rarest amino acids in this organism), which is nothing but a artifactual effect induced by the RSCU values . Other factors such as length of the genes and the positions of the genes in the leading and lagging strand of replication have practically no influence in the codon usage variation among the genes in this organism.

Nutr Cancer, 2003, 46(2), 197 - 201
Cell cycle dysregulation induced by cytoplasm of Lactococcus lactis ssp lactis in SNUC2A, a colon cancer cell line; Kim JY et al.; The anticancer effect of cytoplasmic fraction from Lactococcus lactis ssp . lactis, which had showed strong antiproliferative activity against SNUC2A human colon cancer cell line in the previous study, was investigated . The proliferation of SNUC2A was inhibited by the treatment with cytoplasmic fraction of Lactococcus lactis ssp . lactis in a dose-dependent and partially reversible manner . After exposure to the cytoplasmic fraction of Lc . lactis for 72 h, strong antiproliferative activity was efficiently induced through S-phase accumulation in SNUC2A cells . Analysis of cell cycle regulatory proteins demonstrated that the cytoplasmic fraction enhanced the levels of p21CIP1 and cyclin A, decreased cyclin E protein, and slightly reduced the activity of cyclin-dependent kinase 2 (CDK2).

Nature, 2003 Dec 18, 426(6968), 866 - 70
An ABC transporter with a secondary-active multidrug translocator domain; Venter H et al.; Multidrug resistance, by which cells become resistant to multiple unrelated pharmaceuticals, is due to the extrusion of drugs from the cell's interior by active transporters such as the human multidrug resistance P-glycoprotein . Two major classes of transporters mediate this extrusion . Primary-active transporters are dependent on ATP hydrolysis, whereas secondary-active transporters are driven by electrochemical ion gradients that exist across the plasma membrane . The ATP-binding cassette (ABC) transporter LmrA is a primary drug transporter in Lactococcus lactis that can functionally substitute for P-glycoprotein in lung fibroblast cells . Here we have engineered a truncated LmrA protein that lacks the ATP-binding domain . Surprisingly, this truncated protein mediates a proton-ethidium symport reaction without the requirement for ATP . In other words, it functions as a secondary-active multidrug uptake system . These findings suggest that the evolutionary precursor of LmrA was a secondary-active substrate translocator that acquired an ATP-binding domain to enable primary-active multidrug efflux in L . lactis.

J Appl Microbiol, 2004, 96(1), 144 - 8
Characterization of closely related lactococcal starter strains which show differing patterns of bacteriophage sensitivity; Ward LJ et al.; AIMS: To characterize a group of closely related Lactococcus lactis subsp . lactis casein starter strains used commercially, which differ in their sensitivity to bacteriophages isolated from the same industrial environment . METHODS AND RESULTS: Nine strains of L . lactis, six of which had been used as starter cultures for lactic casein manufacture, were shown to be closely related by pulsed-field gel electrophoresis and total DNA profiles . Nineteen phages which propagated on one or more of these starter strains were isolated from industrial casein whey samples . The phages were all small isometric-headed and could be divided into five groups on the basis of host range on the nine strains . Most of the phages did not give a PCR product with primers designed to detect the two most common lactococcal small isometric phage species (936 and P335) . The hosts could be divided into six groups depending on their phage sensitivity . Plasmids encoding genes for the cell envelope associated PI-type proteinase, lactose metabolism and specificity subunits of a type I restriction/modification system were identified . CONCLUSIONS: This work demonstrates how isolates of the same starter strain may come to be regarded as separate cultures because of their different origins, and how these closely related strains may differ in some of their industrially relevant characteristics . SIGNIFICANCE AND IMPACT OF THE STUDY: This situation may be very common among lactococci used as dairy starter cultures, and implies that the dairy industry worldwide depends on a small number of different strains.

Proc Natl Acad Sci U S A, 2003 Dec 23, 100(26), 15742 - 7 Epub 2003 Dec 12.
A bacterial group II intron favors retrotransposition into plasmid targets; Ichiyanagi K et al.; Group II introns, widely believed to be the ancestors of nuclear pre-mRNA introns, are catalytic RNAs found in bacteria, archaea, and eukaryotes . They are mobile genetic elements that move via an RNA intermediate . They retrohome to intronless alleles and retrotranspose to ectopic sites, aided by an intron-encoded protein with reverse transcriptase, maturase, and endonuclease activities . Many group II introns identified in bacteria reside on plasmid genomes rather than bacterial chromosomes, implying that plasmids are havens for these retroelements . This study demonstrates that almost one-fourth of retrotransposition events of the Ll.LtrB intron in Lactococcus lactis are into the plasmid donor . This level is more than twice that predicted based on target size and plasmid copy number relative to the chromosome . In particular, the fraction of such plasmid targeting events was elevated to more than one-third of retrotransposition events by mutation of the intron-encoded endonuclease, a situation that may resemble most bacterial group II introns, which lack the endonuclease . Target-site sequences on the plasmid are more relaxed than those on the chromosome, likely accounting for preferred integration into plasmid replicons . Furthermore, the direction of integration relative to promoters and origins of replication is consistent with group II intron retrotransposition into single-stranded DNA at replication forks . This work provides mechanistic rationales for the prevalence of group II introns in natural plasmid populations and underscores that targeting to plasmids, which are themselves mobile elements, could promote intron spread.

Dis Aquat Organ, 2003 Oct 24, 56(3), 275 - 8
Lactococcus garvieae in wild Red Sea wrasse Coris aygula (Labridae); Colorni A et al.; Lactococcus garvieae infection in wild wrasse Coris aygula is reported, and the serological and molecular characteristics of the isolate are described . This is the first evidence of the presence of this pathogen in the Red Sea, and it follows the recent diagnosis of Mycobacterium marinum and Streptococcus iniae in wild fish from the same region . Whether all 3 pathogens are strains endemic to the Red Sea, or recent introductions into the region, remains to be determined, but their appearance over a period of a few years in wild fish populations in the northern Red Sea is consistent with an emerging trend affecting marine organisms on a global level in areas subjected to intense anthropogenic impacts.

J Biol Chem, 2004 Mar 19, 279(12), 11273 - 80 Epub 2003 Dec 05.
Reversible transport by the ATP-binding cassette multidrug export pump LmrA: ATP synthesis at the expense of downhill ethidium uptake; Balakrishnan L et al.; The ATP dependence of ATP-binding cassette (ABC) transporters has led to the widespread acceptance that these systems are unidirectional . Interestingly, in the presence of an inwardly directed ethidium concentration gradient in ATP-depleted cells of Lactococcus lactis, the ABC multidrug transporter LmrA mediated the reverse transport (or uptake) of ethidium with an apparent K(t) of 2.0 microm . This uptake reaction was competitively inhibited by the LmrA substrate vinblastine and was significantly reduced by an E314A substitution in the membrane domain of the transporter . Similar to efflux, LmrA-mediated ethidium uptake was inhibited by the E512Q replacement in the Walker B region of the nucleotide-binding domain of the protein, which strongly reduced its drug-stimulated ATPase activity, consistent with published observations for other ABC transporters . The notion that ethidium uptake is coupled to the catalytic cycle in LmrA was further corroborated by studies in LmrA-containing cells and proteoliposomes in which reverse transport of ethidium was associated with the net synthesis of ATP . Taken together, these data demonstrate that the conformational changes required for drug transport by LmrA are (i) not too far from equilibrium under ATP-depleted conditions to be reversed by appropriate changes in ligand concentrations and (ii) not necessarily coupled to ATP hydrolysis, but associated with a reversible catalytic cycle . These findings and their thermodynamic implications shed new light on the mechanism of energy coupling in ABC transporters and have implications for the development of new modulators that could enable reverse transport-associated drug delivery in cells through their ability to uncouple ATP binding/hydrolysis from multidrug efflux.

Appl Environ Microbiol, 2003 Dec, 69(12), 7281 - 8
Ability of Lactococcus lactis to export viral capsid antigens: a crucial step for development of live vaccines; Dieye Y et al.; The food grade bacterium Lactococcus lactis is a potential vehicle for protein delivery in the gastrointestinal tract . As a model, we constructed lactococcal strains producing antigens of infectious bursal disease virus (IBDV) . IBDV infects chickens and causes depletion of B-lymphoid cells in the bursa of Fabricius and subsequent immunosuppression, morbidity, or acute mortality . The two major IBDV antigens, i.e., VP2 and VP3, that form the viral capsid were expressed and targeted to the cytoplasm, the cell wall, or the extracellular compartment of L . lactis . Whereas VP3 was successfully targeted to the three compartments by the use of relevant expression and export vectors, VP2 was recalcitrant to export, thus confirming the difficulty of translocating naturally nonsecreted proteins across the bacterial membrane . This defect could be partly overcome by fusing VP2 to a naturally secreted protein (the staphylococcal nuclease Nuc) that carried VP2 through the membrane . Lactococcal strains producing Nuc-VP2 and VP3 in various bacterial compartments were administered orally to chickens . The chickens did not develop any detectable immune response against VP2 and VP3 but did exhibit an immune response against Nuc when Nuc-VP2 was anchored to the cell wall of lactococci.

Appl Environ Microbiol, 2003 Dec, 69(12), 7101 - 7
Controlled modulation of folate polyglutamyl tail length by metabolic engineering of Lactococcus lactis; Sybesma W et al.; The dairy starter bacterium Lactococcus lactis is able to synthesize folate and accumulates >90% of the produced folate intracellularly, predominantly in the polyglutamyl form . Approximately 10% of the produced folate is released into the environment . Overexpression of folC in L . lactis led to an increase in the length of the polyglutamyl tail from the predominant 4, 5, and 6 glutamate residues in wild-type cells to a maximum of 12 glutamate residues in the folate synthetase overproducer and resulted in a complete retention of folate in the cells . Overexpression of folKE, encoding the bifunctional protein 2-amino-4-hydroxy-6-hydroxymethyldihydropteridine pyrophosphokinase and GTP-cyclohydrolase I, resulted in reduction of the average polyglutamyl tail length, leading to enhanced excretion of folate . By simultaneous overexpression of folKE and folC, encoding the enzyme folate synthetase or polyglutamyl folate synthetase, the average polyglutamyl tail length was increased, again resulting in normal wild-type distribution of folate . The production of bioavailable monoglutamyl folate and almost complete release of folate from the bacterium was achieved by expressing the gene for gamma-glutamyl hydrolase from human or rat origin . These engineering studies clearly establish the role of the polyglutamyl tail length in intracellular retention of the folate produced . Also, the potential application of engineered food microbes producing folates with different tail lengths is discussed.

FEMS Microbiol Lett, 2003 Dec 5, 229(1), 37 - 42
Mice immunization with live lactococci displaying a surface anchored HPV-16 E7 oncoprotein; Cortes-Perez NG et al.; E7 oncoprotein of human papillomavirus-16 (HPV-16) is constitutively produced in cervical cancer (CxCa) and is a good candidate for the design of therapeutic vaccines . In this work, the nisin-controlled expression system was used to display the E7 protein at the cell surface of the food-grade Gram-positive bacterium Lactococcus lactis . An efficient cell wall anchoring of E7 was obtained . Intranasal administration of these recombinant lactococci in mice induced an HPV-16 E7-specific immune response . This is the first report of E7 cell wall anchoring in L . lactis and represents one more step towards the use of live food-grade bacteria to fight against CxCa.

Biochimie, 2003 Oct, 85(10), 993 - 7
Purification and characterization of the Streptococcus salivarius methionine aminopeptidase (MetAP); Boufous el H et al.; Streptococcus salivarius methionine aminopeptidase (MetAP) was purified from a recombinant Escherichia coli strain containing the S . salivarius map gene, which codes for MetAP . S . salivarius map coded for a protein of 286 amino acids with a calculated molecular mass of 31,723 Da and a pI of 4.6 . The native enzyme eluted from a Superdex column as a protein with a molecular mass of 30.6 kDa and cleaved N-terminal Met of peptide only when the penultimate amino acid was Gly, Ala, Ser, Val, Pro, or Thr . The enzyme was more active against tetrapeptides than tripeptides and did not recognize dipeptides . It required the presence of a metal cation for activity, with a preference for Co(2+) over Mn(2+) . S . salivarius MetAP has a pH optimum of 8.0 and an optimal temperature at 50 degrees C . The S . salivarius protein had an extra sequence of 24 amino acids between two conserved aspartate residues involved in the coordination of the metal ion . A similar extra sequence is present in MetAP from other streptococci and from Lactococcus lactis, but not from other bacteria or eukaryotes.

J Appl Microbiol, 2003, 95(6), 1235 - 41
A lacticin 481-producing adjunct culture increases starter lysis while inhibiting nonstarter lactic acid bacteria proliferation during Cheddar cheese ripening; O'Sullivan L et al.; AIMS: The main aim of this study was to exploit a lacticin 481 producing strain, Lactococcus lactis CNRZ481, as an adjunct for Cheddar cheese manufacture, to increase starter cell lysis and control nonstarter lactic acid bacteria (NSLAB) proliferation in cheese . METHODS AND RESULTS: Lactococcus lactis CNRZ481 was exploited as an adjunct to L . lactis HP for the manufacture of Cheddar cheese at pilot scale (450 l) . In these trials, inclusion of the adjunct strain did not compromise acid production by L . lactis HP and cheese was successfully manufactured within 5 h . Experimental cheese exhibited levels of lactate dehydrogenase (LDH) up to five-fold higher than control cheese and a significant reduction in NSLAB growth was also observed throughout the ripening period . CONCLUSIONS: The aims of the study were accomplished as (i) greater enzyme release was achieved through lacticin 481-induced lysis which was associated with an improved flavoured cheese as assessed by a commercial grader and (ii) NSLAB growth was controlled, thus reducing the risk of off-flavour development . SIGNIFICANCE AND IMPACT OF THE STUDY: The use of lacticin 481-producing adjuncts for cheese manufacture may prove beneficial for manufacturers who aim to achieve faster ripening through premature and elevated intracellular enzyme release while minimizing inconsistencies in cheese quality because of NSLAB activity.

Biochem Biophys Res Commun, 2003 Nov 21, 311(3), 696 - 701
Photoaffinity labeling under non-energized conditions of a specific drug-binding site of the ABC multidrug transporter LmrA from Lactococcus lactis; Alqawi O et al.; The Lactococcus lactis multidrug resistance ABC transporter protein LmrA has been shown to confer resistance to structurally and functionally diverse antibiotics and anti-cancer drugs . Using a previously characterized photoreactive drug analogue of Rhodamine 123 (iodo-aryl azido-Rhodamine 123 or IAARh123), direct and specific photoaffinity labeling of LmrA in enriched membrane vesicles could be achieved under non-energized conditions . This photoaffinity labeling of LmrA occurs at a physiologically relevant site as it was inhibited by molar excess of ethidium bromide>Rhodamine 6G>vinblastine>doxorubicin>MK571 (a quinoline-based drug) while colchicine had no effect . The MDR-reversing agents PSC 833 and cyclosporin A were similarly effective in inhibiting IAARh123 photolabeling of LmrA and P-glycoprotein . In-gel digestion with Staphyloccocus aureus V8 protease of IAARh123-photolabeled LmrA revealed several IAARh123 labeled polypeptides, in addition to a 6.8kDa polypeptide that comprises the last two transmembrane domains of LmrA.

J Bacteriol, 2003 Dec, 185(23), 6968 - 75
Genomic organization and molecular characterization of SM1, a temperate bacteriophage of Streptococcus mitis; Siboo IR et al.; The direct binding of Streptococcus mitis to human platelets is mediated in part by two proteins (PblA and PblB) encoded by a lysogenic bacteriophage (SM1) . Since SM1 is the first prophage of S . mitis that has been identified and because of the possible role of these phage-encoded proteins in virulence, we sought to characterize SM1 in greater detail . Sequencing of the SM1 genome revealed that it consisted of 34,692 bp, with an overall G+C content of 39 mol% . Fifty-six genes encoding proteins of 40 or more amino acids were identified . The genes of SM1 appear to be arranged in a modular, life cycle-specific organization . BLAST analysis also revealed that the proteins of SM1 have homologies to proteins from a wide variety of lambdoid phages . Bioinformatic analyses, in addition to N-terminal sequencing of the proteins, led to the assignment of possible functions to a number of proteins, including the integrase, the terminase, and two major structural proteins . Examination of the phage structural components indicates that the phage head may assemble using stable multimers of the major capsid protein, in a process similar to that of phage r1t . These findings indicate that SM1 may be part of a discrete subfamily of the Siphoviridae that includes at least phages r1t of Lactococcus lactis and SF370.3 of Streptococcus pyogenes.

Proc Natl Acad Sci U S A, 2003 Nov 11, 100(23), 13184 - 9
Creation of the first anomeric D/L-sugar kinase by means of directed evolution; Hoffmeister D et al.; Chemoenzymatic routes toward complex glycoconjugates often depend on the availability of sugar-1-phosphates . Yet the chemical synthesis of these vital components is often tedious, whereas natural enzymes capable of anomeric phosphorylation are known to be specific for one or only a few monosaccharides . Herein we describe the application of directed evolution and a high-throughput multisugar colorimetric screen to enhance the catalytic capabilities of the Escherichia coli galactokinase GalK . From this approach, one particular GalK mutant carrying a single amino acid exchange (Y371H) displayed a surprisingly substantial degree of kinase activity toward sugars as diverse as d-galacturonic acid, d-talose, l-altrose, and l-glucose, all of which failed as wild-type GalK substrates . Furthermore, this mutant provides enhanced turnover of the small pool of sugars converted by the wild-type enzyme . Comparison of this mutation to the recently solved structure of Lactococcus lactis GalK begins to provide a blueprint for further engineering of this vital class of enzyme . In addition, the rapid access to such promiscuous sugar C-1 kinases will significantly enhance accessibility to natural and unnatural sugar-1-phosphates and thereby impact both in vitro and in vivo glycosylation methodologies, such as natural product glycorandomization.

EMBO J, 2003 Nov 17, 22(22), 5983 - 93
On the role of the two extracytoplasmic substrate-binding domains in the ABC transporter OpuA; Biemans-Oldehinkel E et al.; Members of two transporter families of the ATP-binding cassette (ABC) superfamily use two or even four extracytoplasmic substrate-binding domains (SBDs) for transport . We report on the role of the two SBDs in the translocation cycle of the ABC transporter OpuA from Lactococcus lactis . Heterooligomeric OpuA complexes with only one SBD or one functional and one non-functional SBD (inactivated by covalent linkage of a substrate mimic) have been constructed, and the substrate binding and transport kinetics of the purified transporters, reconstituted in liposomes, have been determined . The data indicate that the two SBDs of OpuA interact in a cooperative manner in the translocation process by stimulating either the docking of the SBDs onto the translocator or the delivery of glycine betaine to the translocator . It appears that one of these initial steps, but not the later steps in translocation or resetting of the system to the initial state, is rate determining for transport . These new insights on the functional role of the extracytoplasmic SBDs are discussed in the light of the current knowledge of substrate-binding-protein-dependent ABC transporters.

World J Gastroenterol, 2003 Nov, 9(11), 2469 - 73
Lack of inhibitory effects of lactic acid bacteria on 1,2-dimethylhydrazine-induced colon tumors in rats; Li W et al.; AIM: A myriad of healthful effects has been attributed to the probiotic lactic acid bacteria, perhaps the most controversial issue remains that of anticancer activity . This study was aimed at investigating the putative anti-cancer effects of lactic acid bacteria strains on the progression of colon tumor in 1,2-dimethylhydrazine (DMH)-treated animals . METHODS: The strain of lactic acid bacteria used in this study was lactic acid bacteria NZ9000 that conformed to the characteristics of plasmid free . Sixty male Wistar rats were given subcutaneous injections of DMH at a dose of 40 mg/kg body wt or saline once a week for 10 weeks . The rats were divided into 6 experimental groups . After the last DMH injection, animals in groups 1 and 4 were gavaged with 1 ml of lactic acid bacteria at a dose of 5 X 10(9) per day or vehicle until sacrifice at the end of week 22 or week 52 . Animals in groups 1-3 were killed at the end of week 22 for histopathological examination . The whole period of experimental observation was 52 weeks . RESULTS: By the end of 22nd week, final average body weights of the rats treated with DMH alone and all animals receiving lactic acid bacteria were significantly decreased compared with the vehicle control (P<0.05) . No differences in tumor incidence, multiplicity, dimensions and stage in the colonic mucosa were observed among the groups . At week 52, the survival rate of the rats administered lactic acid bacteria was lower than that of the rats treated with DMH that were fed on control fluids of non-lactococcus lactis . The mean survival time of lactic acid bacteria-treated animals was 39 weeks . CONCLUSION: These results indicate that lactic acid bacteria lacks inhibitory effects on the progression of colon tumor in DMH-treated animals, and does not support the hypothesis that alteration of colonic flora may exert an influence on the progression of colon tumor.

Nucleic Acids Res . 2003 Nov 15;31(22):e144.
Projector: automatic contig mapping for gap closure purposes; van Hijum SA et al.; Projector was designed for automatic positioning of contigs from an unfinished prokaryotic genome onto a template genome of a closely related strain or species . Projector mapped 84 contigs of Lactococcus lactis MG1363 (corresponding to 81% of the assembly nucleotides) against the genome of L.lactis IL1403 . Ninety three percent of subsequent gap closure PCRs were successful . Moreover, a significant improvement in the N50 and N80 values (describing the assembly quality) was observed after the use of Projector . Because increasing numbers of bacterial genomes are being sequenced, Projector provides an efficient method to close a significant number of remaining gaps in the late stages of a genome sequencing project.

Appl Environ Microbiol, 2003 Nov, 69(11), 6620 - 7
Characterization of a Lactococcus lactis strain that secretes a major epitope of bovine beta-lactoglobulin and evaluation of its immunogenicity in mice; Chatel JM et al.; Bovine beta-lactoglobulin (Blg) is one of the major cow's milk allergens . Peptide 41-60 of Blg (Blg41-60) was described as a murine T-cell determinant and a murine, rat, and human immunoglobulin E (IgE) epitope . The aim of this study was the expression of Blg41-60 as a fusion protein in the food-grade bacterium Lactococcus lactis and the characterization of its immunogenicity in mice . We constructed a recombinant strain of L . lactis capable of inducible production and secretion of Blg41-60::Nuc, a fusion protein between Blg41-60 and the mature part of the staphylococcal nuclease (Nuc) . The highest production yield of Blg41-60::Nuc (32.5 mg/liter) was reached 4 h after induction . At this time, up to 75% of Blg41-60::Nuc was secreted . When monoclonal antibodies specific for Blg41-60 were used, purified Blg41-60::Nuc and synthetic Blg41-60 exhibited very similar immunoreactivities . Subcutaneous coadministration of purified Blg41-60::Nuc and killed nonrecombinant L . lactis resulted in the induction of specific anti-Blg41-60 IgG2a and IgG1 . The IgG1/IgG2a ratio and the lack of specific IgE suggest a Th1-type immune response, i.e., a nonallergic response . Similar administrations of the killed Blg41-60::Nuc-producing L . lactis strain did not elicit a specific immune response, whereas a transitory mucosal IgA-specific immune response was induced in mice after oral administration of the live Blg41-60::Nuc-producing L . lactis strain.

Microbiology, 2003 Nov, 149(Pt 11), 3331 - 41
Characterization of the LlaCI methyltransferase from Lactococcus lactis subsp . cremoris W15 provides new insights into the biology of type II restriction-modification systems; Mruk I et al.; The gene encoding the LlaCI methyltransferase (M.LlaCI) from Lactococcus lactis subsp . cremoris W15 was overexpressed in Escherichia coli . The enzyme was purified to apparent homogeneity using three consecutive steps of chromatography on phosphocellulose, blue-agarose and Superose 12HR, yielding a protein of M(r) 31 300+/-1000 under denaturing conditions . The exact position of the start codon AUG was determined by protein microsequencing . This enzyme recognizes the specific palindromic sequence 5'-AAGCTT-3' . Purified M.LlaCI was characterized . Unlike many other methyltransferases, M.LlaCI exists in solution predominantly as a dimer . It modifies the first adenine residue at the 5' end of the specific sequence to N(6)-methyladenine and thus is functionally identical to the corresponding methyltransferases of the HindIII (Haemophilus influenzae Rd) and EcoVIII (Escherichia coli E1585-68) restriction-modification systems . This is reflected in the identity of M.LlaCI with M.HindIII and M.EcoVIII noted at the amino acid sequence level (50 % and 62 %, respectively) and in the presence of nine sequence motifs conserved among N(6)-adenine beta-class methyltransferases . However, polyclonal antibodies raised against M.EcoVIII cross-reacted with M.LlaCI but not with M.HindIII . Restriction endonucleases require Mg(2+) for phosphodiester bond cleavage . Mg(2+) was shown to be a strong inhibitor of the M.LlaCI enzyme and its isospecific homologues . This observation suggests that sensitivity of the M.LlaCI to Mg(2+) may strengthen the restriction activity of the cognate endonuclease in the bacterial cell . Other biological implications of this finding are also discussed.

Proc Natl Acad Sci U S A . 2003 Oct 31; {Epub ahead of print}
Creation of the first anomeric D/L-sugar kinase by means of directed evolution; Hoffmeister D et al.; Chemoenzymatic routes toward complex glycoconjugates often depend on the availability of sugar-1-phosphates . Yet the chemical synthesis of these vital components is often tedious, whereas natural enzymes capable of anomeric phosphorylation are known to be specific for one or only a few monosaccharides . Herein we describe the application of directed evolution and a high-throughput multisugar colorimetric screen to enhance the catalytic capabilities of the Escherichia coli galactokinase GalK . From this approach, one particular GalK mutant carrying a single amino acid exchange (Y371H) displayed a surprisingly substantial degree of kinase activity toward sugars as diverse as D-galacturonic acid, D-talose, L-altrose, and L-glucose, all of which failed as wild-type GalK substrates . Furthermore, this mutant provides enhanced turnover of the small pool of sugars converted by the wild-type enzyme . Comparison of this mutation to the recently solved structure of Lactococcus lactis GalK begins to provide a blueprint for further engineering of this vital class of enzyme . In addition, the rapid access to such promiscuous sugar C-1 kinases will significantly enhance accessibility to natural and unnatural sugar-1-phosphates and thereby impact both in vitro and in vivo glycosylation methodologies, such as natural product glycorandomization.

J Bacteriol, 2003 Nov, 185(22), 6562 - 74
CTP limitation increases expression of CTP synthase in Lactococcus lactis; Jorgensen CM et al.; CTP synthase is encoded by the pyrG gene and catalyzes the conversion of UTP to CTP . A Lactococcus lactis pyrG mutant with a cytidine requirement was constructed, in which beta-galactosidase activity in a pyrG-lacLM transcriptional fusion was used to monitor gene expression of pyrG . A 10-fold decrease in the CTP pool induced by cytidine limitation was found to immediately increase expression of the L . lactis pyrG gene . The final level of expression of pyrG is 37-fold higher than the uninduced level . CTP limitation has pronounced effects on central cellular metabolism, and both RNA and protein syntheses are inhibited . Expression of pyrG responds only to the cellular level of CTP, since expression of pyrG has no correlation to alterations in UTP, GTP, and ATP pool sizes . In the untranslated pyrG leader sequence a potential terminator structure can be identified, and this structure is required for regulation of the pyrG gene . It is possible to fold the pyrG leader in an alternative structure that would prevent the formation of the terminator . We suggest a model for pyrG regulation in L . lactis, and probably in other gram-positive bacteria as well, in which pyrG expression is directly dependent on the CTP concentration through an attenuator mechanism . At normal CTP concentrations a terminator is preferentially formed in the pyrG leader, thereby reducing expression of CTP synthase . At low CTP concentrations the RNA polymerase pauses at a stretch of C residues in the pyrG leader, thereby allowing an antiterminator to form and transcription to proceed . This model therefore does not include any trans-acting protein for sensing the CTP concentration as previously proposed for Bacillus subtilis.

FEMS Microbiol Lett, 2003 Oct 24, 227(2), 229 - 35
Heterologous gene expression in Lactococcus lactis; expression of the Azotobacter vinelandii algE6 gene product displaying mannuronan C-5 epimerase activity; Blatny JM et al.; The Azotobacter vinelandii mannuronan C-5 epimerases AlgE1-7 can be used to improve the properties of the commercially important polysaccharide alginate that is widely used in a variety of products, such as food and pharmaceuticals . Since lactic acid bacteria are generally regarded as safe, they are attractive candidates for production of the epimerases . A . vinelandii genes are GC-rich, in contrast to those of lactic acid bacteria, but we show here that significant expression levels of the epimerase AlgE6 can be obtained in Lactococcus lactis using the nisin-controlled expression system . A 1200-fold induction ratio was obtained resulting in an epimerase activity of 23900 dpm mg(-1) h(-1), using a tritiated alginate substrate . The epimerase was detected by Western blotting and nuclear magnetic resonance spectroscopy analysis of its reaction product showed that the enzyme displayed catalytic properties similar to those produced in Escherichia coli.

Luminescence, 2003 Sep-Oct, 18(5), 254 - 8
Use of ATP bioluminescence to determine the bacterial sensitivity threshold to a bacteriocin; Valat C et al.; A new ATP bioluminescence-based method was developed to determine the effectiveness of nisin on a sensitive strain of Lactococcus cremoris . The principle of the method is to quantify the release of adenylic-nucleotides (AN) by a sensitive strain under the action of the bacteriocin, with the complex luciferin-luciferase . Nisin-induced leakage of AN included ATP from a sensitive L . cremoris to the external medium immediately after the contact with the bacteria . The growth of L . cremoris was correlated with the extracellular AN content . The extracellular ATP and AN concentration exhibited a linear correlation to the logarithm of the nisin concentration . For the determination of the effectiveness threshold, the concentration of AN was more sensitive and more reliable than the direct quantification of ATP . The effectiveness threshold, corresponding to a 100% inhibition of L . cremoris growth, was obtained for a null concentration of intracellular nucleotides, i.e . for a AN(tot):AN(ext) ratio = 1 . For an initial concentration of 1.4 x 10(7) bacteria/mL, the nisin effectiveness threshold is 3.4 +/- 0.01 mg nisin/L . It is possible to detect effectiveness threshold concentration by taking into account the physiological state of the cells .

Biochemistry, 2003 Nov 4, 42(43), 12466 - 80
Effects of maturase binding and Mg2+ concentration on group II intron RNA folding investigated by UV cross-linking; Noah JW et al.; The Lactococcus lactis Ll.LtrB group II intron encodes a reverse transcriptase/maturase (LtrA protein) that promotes RNA splicing by stabilizing the catalytically active RNA structure . Here, we mapped 17 UV cross-links induced in both wild-type Ll.LtrB RNA and Ll.LtrB-Delta2486 RNA, which has a branch-point deletion that prevents splicing, and we used these cross-links to follow tertiary structure formation under different conditions in the presence or absence of the LtrA protein . Twelve of the cross-links are long-range, with six near known tertiary interaction sites in the active RNA structure . In a reaction medium containing 0.5 M NH(4)Cl, eight of the 17 cross-links were detected in the absence of Mg(2+) or the presence of EDTA, and all were detected at 5 mM Mg(2+), where efficient splicing requires the LtrA protein . The frequencies of all but four cross-links increased with increasing Mg(2+) concentrations, becoming maximal between 4 and 50 mM Mg(2+), where the intron is self-splicing . These findings suggest that a high Mg(2+) concentration induces self-splicing by globally stabilizing tertiary structure, including key tertiary interactions that are required for catalytic activity . Significantly, the binding of the maturase under protein-dependent splicing conditions (0.5 M NH(4)Cl and 5 mM Mg(2+)) increased the frequency of only nine cross-links, seven of which are long-range, suggesting that, in contrast to a high Mg(2+) concentration, LtrA promotes splicing by stabilizing critical tertiary structure interactions, while leaving other regions of the intron relatively flexible . This difference may contribute to the high rate of protein-dependent splicing, relative to the rate of self-splicing . The propensity of the intron RNA to form tertiary structure even at relatively low Mg(2+) concentrations raises the possibility that the maturase functions at least in part by tertiary structure capture . Finally, an abundant central wheel cross-link, present in >50% of the molecules at 5 mM Mg(2+), suggests models in which group II intron domains I and II are either coaxially stacked or aligned in parallel, bringing the 5'-splice site together with the 3'-splice site and catalytic core elements at JII/III . This and other cross-links provide new constraints for three-dimensional structural modeling of the group II intron catalytic core.

FEMS Microbiol Lett, 2003 Oct 10, 227(1), 33 - 8
Specificity of the second binding protein of the peptide ABC-transporter (Dpp) of Lactococcus lactis IL1403; Sanz Y et al.; The genome sequence of Lactococcus lactis IL1403 revealed the presence of a putative peptide-binding protein-dependent ABC-transporter (Dpp) . The genes for two peptide-binding proteins (dppA and dppP) precede the membrane components, which include two transmembrane protein genes (dppB and dppC) and two ATP-binding protein genes (dppD and dppF) . In this work, the gene specifying the second peptide-binding protein (DppP) was cloned under the control of the nisin promoter . The protein fused to a carboxyl-terminal histidine tag (DppP-His(6)) was purified and its binding properties were determined by monitoring the intrinsic fluorescence changes observed upon ligand binding . The major features of peptide binding to DppP-His(6) include: (i) a requirement for a free N-terminal alpha-amino group in the ligand; (ii) a high affinity for di-, tri- and tetra-peptides; (iii) affinity constants for peptide binding independent of pH; and (iv) a high affinity for D-isomer-containing peptides . Remarkably, the features (ii), (iii) and (iv) differ from those previously reported for DppA-His(6), suggesting that DppP-His(6) is a more versatile peptide-binding protein that could have additional functions.

Nucleosides Nucleotides Nucleic Acids, 2003 May-Aug, 22(5-8), 1563 - 5
Recognition of cyclonucleoside lesions by the Lactococcus lactis FPG protein; Muller E et al.; Several purine and pyrimidine cyclonucleosides were found to be not recognized by several Escherichia coli and yeast DNA N-glycosylases . Interestingly, a non covalent complex was observed between the Lactoccocus lactis formamidopyrimidine-DNA glycosylases (Fpg-Ll) and the cyclonucleosides . This may provide new information on the mechanism involved in the activity of the latter enzyme.

J Biol Chem, 2004 Jan 2, 279(1), 103 - 8 Epub 2003 Oct 14.
Facilitated drug influx by an energy-uncoupled secondary multidrug transporter; Mazurkiewicz P et al.; The majority of bacterial multidrug resistance transporters belong to the class of secondary transporters . LmrP is a proton/drug antiporter of Lactococcus lactis that extrudes positively charged lipophilic substrates from the inner leaflet of the membrane to the external medium . This study shows that LmrP is a true secondary transporter . In the absence of a proton motive force, LmrP facilitates downhill fluxes of ethidium in both directions . These fluxes are inhibited by other substrates of LmrP . The cysteine-reactive agent p-chloromercuri-benzene sulfonate inhibits these fluxes in wild type LmrP but not in the cysteine-less LmrP C270A mutant . Cysteine mutagenesis of LmrP resulted in three mutants, D68C/C270A, D128C/C270A, and E327C/C270A, with an energy-uncoupled phenotype . Asp68 is located in the conserved motif GXXX(D/E)(R/K)XGRK for the major facilitator superfamily of secondary transporters and was found to play an important role in energy coupling, whereas the negatively charged residues Asp128 and Glu327 have indirect effects on the transport process . L . lactis strains expressing these uncoupled mutants of LmrP show an increased rate of ethidium influx and an increased drug susceptibility compared with cells harboring an empty vector . The rate of influx in these mutants is enhanced by a transmembrane electrical potential, inside negative . These observations suggest a new strategy for eliminating drug-resistant microbial pathogens, i.e . the design and use of modulators of secondary multidrug resistance transporters that uncouple drug efflux from proton influx, thereby allowing transmembrane electrical potential-driven influx of cationic drugs.

Fish Shellfish Immunol, 2003 Nov, 15(5), 425 - 31
Streptococcus iniae expresses a cell surface non-immune trout immunoglobulin-binding factor when grown in normal trout serum; Barnes AC et al.; Three capsulated isolates of S . iniae representing serotype I and II and being arginine dihydrolase positive, negative or variable (AD+ve, AD-ve, AD+-ve) were investigated for their ability to bind rainbow trout serum immunoglobulin by the Fc region . Using a coagglutination assay with bacteria grown in Todd-Hewitt broth (THB), no evidence of non-specific Fc-binding of trout immunoglobulin (Ig) was obtained . However, when grown in normal trout serum, all isolates produced similar protein patterns in SDS-PAGE, but they were markedly different from the patterns of the bacteria grown in THB . Some bands with MW 70 kDa and over 100 kDa were very intense in the profiles of the serum-grown isolates . In Western blots, these bands of all isolates were immunostained with the conjugated goat antiserum to trout Ig, after blocking with normal goat serum, demonstrating that the bacteria had bound the trout Ig during growth in the serum . When the isolates were grown overnight in trout antiserum against Lactococcus garvieae they coagglutinated with L . garvieae cells but S . iniae isolates grown in normal trout serum did not . These data indicate that S . iniae grown in serum express surface factors which can bind trout Ig by the Fc-region.

Appl Environ Microbiol, 2003 Oct, 69(10), 5739 - 45
Glutathione protects Lactococcus lactis against oxidative stress; Li Y et al.; Glutathione was found in several dairy Lactococcus lactis strains grown in M17 medium . None of these strains was able to synthesize glutathione . In chemically defined medium, L . lactis subsp . cremoris strain SK11 was able to accumulate up to approximately 60 mM glutathione when this compound was added to the medium . Stationary-phase cells of strain SK11 grown in chemically defined medium supplemented with glutathione showed significantly increased resistance (up to fivefold increased resistance) to treatment with H2O2 compared to the resistance of cells without intracellular glutathione . The resistance to H2O2 treatment was found to be dependent on the accumulation of glutathione in 16 strains of L . lactis tested . We propose that by taking up glutathione, L . lactis might activate a glutathione-glutathione peroxidase-glutathione reductase system in stationary-phase cells, which catalyzes the reduction of H2O2 . Glutathione reductase, which reduces oxidized glutathione, was detectable in most strains of L . lactis, but the activities of different strains were very variable . In general, the glutathione reductase activities of L . lactis subsp . lactis are higher than those of L . lactis subsp . cremoris, and the activities were much higher when strains were grown aerobically . In addition, glutathione peroxidase is detectable in strain SK11, and the level was fivefold greater when the organism was grown aerobically than when the organism was grown anaerobically . Therefore, the presence of glutathione in L . lactis could result in greater stability under storage conditions and quicker growth upon inoculation, two important attributes of successful starter cultures.

Int J Food Microbiol, 2003 Nov 1, 87(3), 293 - 300
Identification of proteins induced at low pH in Lactococcus lactis; Frees D et al.; The Gram-positive bacterium Lactococcus lactis is of major importance to the dairy industry due to its conversion of lactose to lactic acid leading to the acidification of milk . To investigate which proteins are induced when L . lactis is exposed to conditions of low pH, we used two-dimensional gel electrophoresis to follow how protein expression changes with the degree of acidification . We found that reducing the pH of the growth medium with hydrochloric acid induced the synthesis of a small subset of proteins . The majority of these proteins were induced both after a minor (pH 5.5) and a major (pH 4.5) reduction in pH . Among the most strongly induced proteins, we identified the oxidative stress proteins superoxide dismutase and alkylhydroperoxidase as well as the autoinducer synthesis protein, LuxS . We also observed a differential induction of heat shock proteins by low pH as members of the CtsR regulon, ClpE and ClpP were induced at both pH 5.5 and 4.5, while HrcA-regulated chaperones, GroEL, GroES, DnaK and GrpE were induced only at pH 4.5 . In addition, we identified two proteins repressed by low pH that proved to be the L . lactis HPr protein of the phosphoenolpyruvate sugar phosphotransferase system and the trigger factor known to participate in the folding of newly synthesized polypeptides.

Microbiology, 2003 Oct, 149(Pt 10), 2759 - 67
The Staphylococcus aureus surface protein SasG and its homologues promote bacterial adherence to human desquamated nasal epithelial cells; Roche FM et al.; Staphylococcus aureus binds to human desquamated nasal epithelial cells, a phenomenon likely to be important in nasal colonization . ClfB was identified previously as one staphylococcal adhesin that promoted binding to nasal epithelia . In this study, it is shown that the S . aureus surface protein SasG, identified previously by in silico analysis of genome sequences, and two homologous proteins, Pls of S . aureus and AAP of Staphylococcus epidermidis, also promote bacterial adherence to nasal epithelial cells . Conditions for in vitro expression of SasG by S . aureus were not found . Adherence assays were therefore performed with S . aureus and Lactococcus lactis expressing SasG from an expression plasmid . These studies showed that SasG did not bind several ligands typically bound by S . aureus . Significantly, SasG and Pls did promote bacterial adherence to nasal epithelial cells . Furthermore, pre-incubation of epithelial cells with purified recombinant proteins revealed that the N-terminal A regions of SasG, Pls and AAP, but not the B repeats of SasG, inhibited adherence of L . lactis expressing SasG in a dose-dependent fashion . These results suggest that SasG, Pls and AAP bind to the same as-yet-unidentified receptor on the surface of nasal epithelial cells . In addition, expression of SasG, like Pls, reduced adherence of S . aureus to fibronectin and fibrinogen.

J Biol Chem, 2003 Dec 19, 278(51), 51494 - 503 Epub 2003 Sep 30.
Beta-ketoacyl-acyl carrier protein synthase III (FabH) is essential for bacterial fatty acid synthesis; Lai CY et al.; beta-Ketoacyl-acyl carrier protein (ACP) synthase III (KAS III, also called acetoacetyl-ACP synthase) encoded by the fabH gene is thought to catalyze the first elongation reaction (Claisen condensation) of type II fatty acid synthesis in bacteria and plant plastids . However, direct in vivo evidence that KAS III catalyzes an essential reaction is lacking, because no mutant organism deficient in this activity has been isolated . We report the first bacterial strain lacking KAS III, a fabH mutant constructed in the Gram-positive bacterium Lactococcus lactis subspecies lactis IL1403 . The mutant strain carries an in-frame deletion of the KAS III active site region and was isolated by gene replacement using a medium supplemented with a source of saturated and unsaturated long-chain fatty acids . The mutant strain is devoid of KAS III activity and fails to grow in the absence of supplementation with exogenous long-chain fatty acids demonstrating that KAS III plays an essential role in cellular metabolism . However, the L . lactis fabH deletion mutant requires only long-chain unsaturated fatty acids for growth, a source of long-chain saturated fatty acids is not required . Because both saturated and unsaturated fatty acids are required for growth when fatty acid synthesis is blocked by biotin starvation (which prevents the synthesis of malonyl-CoA), another pathway for saturated fatty acid synthesis must remain in the fabH deletion strain . Indeed, incorporation of {1-14C}acetate into fatty acids in vivo showed that the fabH mutant retained about 10% of the fatty acid synthetic ability of the wild-type strain and that this residual synthetic capacity was preferentially diverted to the saturated branch of the pathway . Moreover, mass spectrometry showed that the fabH mutant retained low levels of palmitic acid upon fatty acid starvation . Derivatives of the fabH deletion mutant strain were isolated that were octanoic acid auxotrophs consistent with biochemical studies indicating that the major role of FabH is production of short-chain fatty acid primers . We also confirmed the essentiality of FabH in Escherichia coli by use of a plasmid-based gene insertion/deletion system . Together these results provide the first genetic evidence demonstrating that FabH conducts the major condensation reaction in the initiation of type II fatty acid biosynthesis in both Gram-positive and Gram-negative bacteria.

Mol Microbiol, 2003 Oct, 50(1), 183 - 92
CcpA regulation of aerobic and respiration growth in Lactococcus lactis; Gaudu P et al.; The catabolic control protein CcpA is the highly conserved regulator of carbon metabolism in Gram-positive bacteria . We recently showed that Lactococcus lactis, a fermenting bacterium in the family of Streptococcaceae, is capable of respiration late in growth when haem is added to aerated cultures . As the start of respiration coincides with glucose depletion from the medium, we hypothesized that CcpA is involved in this metabolic switch and investigated its role in lactococcal growth under aeration and respiration conditions . Compared with modest changes observed in fermentation growth, inactivation of ccpA shifts metabolism to mixed acid fermentation under aeration conditions . This shift is due to a modification of the redox balance via derepression of NADH oxidase, which eliminates oxygen and decreases the NADH pool . CcpA also plays a decisive role in respiration metabolism . Haem addition to lag phase ccpA cells results in growth arrest and cell mortality . Toxicity is due to oxidative stress provoked by precocious haem uptake . We identify the repressor of the haem transport system and show that it is a target of CcpA activation . We propose that CcpA-mediated repression of haem uptake is a means of preventing oxidative damage at the start of exponential growth . CcpA thus appears to govern a regulatory network that coordinates oxygen, iron and carbon metabolism.

Arch Microbiol, 2003 Nov, 180(5), 367 - 73 Epub 2003 Sep 20.
Conjugative plasmid pIP501 undergoes specific deletions after transfer from Lactococcus lactis to Oenococcus oeni; Zuniga M et al.; Conjugal transfer of plasmids pIP501 and its derivative pVA797 from Lactococcus lactis to Oenococcus oeni was assayed by filter mating . Plasmid pIP501 was transferred to a number of O . oeni strains whereas a single transconjugant of O . oeni M42 was recovered when pVA797 was used . Physical analysis of the transconjugant plasmids revealed that pIP501 and pVA797 underwent extensive deletions in O . oeni that affected the tra region (conjugal transfer) and SegB region (stability) . All derivatives showed segregational instability in O . oeni, but were stably maintained in L . lactis . These differences correlated with the different plasmid copy numbers and the extent of deletions within the SegB region.

Infect Immun, 2003 Oct, 71(10), 5823 - 30
M1 protein triggers a phosphoinositide cascade for group A Streptococcus invasion of epithelial cells; Purushothaman SS et al.; Invasion of nonphagocytic cells by bacteria provides a favorable niche for persistence and evasion of host defenses and antibiotics . M protein is a major virulence factor because it promotes high-frequency invasion of epithelial cells by group A Streptococcus (GAS) and also renders the bacterium resistant to phagocytosis . In this study, we investigated the role of M1 protein from serotype M1 strain 90-226 in regulating mammalian signal transduction and cytoskeletal rearrangement for bacterial entry . LY294002 and wortmannin, which are inhibitors of phosphatidylinositol 3-kinase (PI 3-K) blocked invasion of epithelial cells by GAS by 75 and 80%, respectively, but failed to inhibit invasion by Salmonella enterica serovar Typhimurium . Also, epithelial cells transiently transfected with dominant negative p85 and p110 genes, the regulatory and catalytic subunits of PI 3-K, respectively, were less able to be invaded by GAS . To separate the influence of other streptococcal virulence factors from M protein, Lactococcus lactis was engineered to express M1 protein on its surface . L . lactis(pLM1) invaded epithelial cells efficiently in vitro, and PI 3-K inhibitors blocked 90% of this invasion . Purified soluble M1 protein stimulated the formation of stress fibers and actin tuffs on epithelial cells . LY294002 and wortmannin inhibited these cellular changes . A phosphoinositide analogue also inhibited the invasion of epithelial cells by GAS . Therefore, M1 protein, either directly or via bound fibronectin, initiates signals that depend on the lipid kinase PI 3-K pathway, which paves the way for cytoskeletal rearrangement that internalize the bacterium.

J Microbiol Methods, 2003 Oct, 55(1), 187 - 200
Modification of A-stat for the characterization of microorganisms; Kasemets K et al.; Two novel modifications of continuous culture with gradual change of dilution rate (A-stat): D-stat and auxo-accelerostat were evaluated in the studies of the effect of changing individual environmental parameters (T, pH, pO(2), substrate concentration, etc.) on growth characteristics of different microorganisms . Common for those cultivation methods is that one environmental parameter is programmed to change with constant change rate (change-stat) while the others are kept constant or in the range not affecting the growth characteristics . The environment response growth curves were obtained starting with chemostat (in A-stat and D-stat) or auxostat (in auxo-accelerostat) steady-state cultures followed by change of set-point value of the desired cultivation parameter . Physiological studies of Saccharomyces sp . and Lactococcus lactis were combined with validation of the different modifications of the A-stat method based on well-known cultivation techniques: chemostat, pH-auxostat, pO(2)-auxostat CO(2)-auxostat and fed-batch . The auxo-accelerostat was shown to be very efficient for cell characterization and dynamic studies in growth environments with excess of essential substrates . Choosing the rate of change of environmental parameters was shown to be critical in comparative physiological studies of microorganisms.

Appl Environ Microbiol, 2003 Sep, 69(9), 5290 - 6
Effect of acidic pH on expression of surface-associated proteins of Streptococcus oralis; Wilkins JC et al.; Streptococcus oralis, a member of the mitis group of oral streptococci, is implicated in the pathogenesis of infective endocarditis and is the predominant aciduric non-mutans-group streptococcus in dental plaque . We undertook to identify the most abundant surface-associated proteins of S . oralis and to investigate changes in protein expression when the organism was grown under acidic culture conditions . Surface-associated proteins were extracted from cells grown in batch culture, separated by two-dimensional gel electrophoresis, excised, digested with trypsin, and analyzed by matrix-assisted laser desorption ionization-time of flight mass spectrometry and liquid chromatography-tandem mass spectrometry . Putative functions were assigned by homology to a translated genomic database of Streptococcus pneumoniae . A total of 27 proteins were identified; these included a lipoprotein, a ribosome recycling factor, and the glycolytic enzymes phosphoglycerate kinase, fructose bisphosphate aldolase, glyceraldehyde-3-phosphate dehydrogenase, and enolase . The most abundant protein, phosphocarrier protein HPr, was present as three isoforms . Neither lactate dehydrogenase nor pyruvate oxidase, dominant intracellular proteins, were present among the proteins on the gels, demonstrating that proteins in the surface-associated pool did not arise as a result of cell lysis . Eleven of the proteins identified were differentially expressed when cells were grown at pH 5.2 versus pH 7.0, and these included superoxide dismutase, a homologue of dipeptidase V from Lactococcus lactis, and the protein translation elongation factors G, Tu, and Ts . This study has extended the range of streptococcal proteins known to be expressed at the cell surface . Further investigations are required to ascertain their functions at this extracellular location and determine how their expression is influenced by other environmental conditions.

Appl Environ Microbiol, 2003 Sep, 69(9), 5104 - 14
Sequence diversity and functional conservation of the origin of replication in lactococcal prolate phages; Rakonjac J et al.; Prolate or c2-like phages are a large homologous group of viruses that infect the bacterium Lactococcus lactis . In a collection of 122 prolate phages, three distinct, non-cross-hybridizing groups of origins of DNA replication were found . The nonconserved sequence was confined to the template for an untranslated transcript, P(E)1-T, 300 to 400 nucleotides in length, while the flanking sequences were conserved . All three origin types, despite the low sequence homology, have the same functional characteristics: they express abundant P(E)1-T transcripts and can function as origins of plasmid replication in the absence of phage proteins . Using chimeric constructs, we showed that hybrids of two nonhomologous origin sequences failed to function as replication origins, suggesting that preservation of a particular secondary structure of the P(E)1-T transcript is required for replication . This is the first systematic survey of the sequence and function of origins of replication in a group of lactococcal phages.

EMBO J, 2003 Sep 1, 22(17), 4555 - 65
Group II intron mobility using nascent strands at DNA replication forks to prime reverse transcription; Zhong J et al.; The Lactococcus lactis Ll.LtrB group II intron uses a major retrohoming mechanism in which the excised intron RNA reverse splices into one strand of a DNA target site, while the intron-encoded protein uses a C-terminal DNA endonuclease domain to cleave the opposite strand and then uses the cleaved 3' end as a primer for reverse transcription of the inserted intron RNA . Here, experiments with mutant introns and target sites indicate that Ll.LtrB can retrohome without second-strand cleavage by using a nascent strand at a DNA replication fork as the primer for reverse transcription . This mechanism connecting intron mobility to target DNA replication may be used by group II intron species that encode proteins lacking the C-terminal DNA endonuclease domain and for group II intron retrotransposition to ectopic sites.

J Bacteriol, 2003 Sep, 185(17), 5117 - 24
ClpE from Lactococcus lactis promotes repression of CtsR-dependent gene expression; Varmanen P et al.; The heat shock response in bacterial cells is characterized by rapid induction of heat shock protein expression, followed by an adaptation period during which heat shock protein synthesis decreases to a new steady-state level . In this study we found that after a shift to a high temperature the Clp ATPase (ClpE) in Lactococcus lactis is required for such a decrease in expression of a gene negatively regulated by the heat shock regulator (CtsR) . Northern blot analysis showed that while a shift to a high temperature in wild-type cells resulted in a temporal increase followed by a decrease in expression of clpP encoding the proteolytic component of the Clp protease complex, this decrease was delayed in the absence of ClpE . Site-directed mutagenesis of the zinc-binding motif conserved in ClpE ATPases interfered with the ability to repress CtsR-dependent expression . Quantification of ClpE by Western blot analysis revealed that at a high temperature ClpE is subjected to ClpP-dependent processing and that disruption of the zinc finger domain renders ClpE more susceptible . Interestingly, this domain resembles the N-terminal region of McsA, which was recently reported to interact with the CtsR homologue in Bacillus subtilis . Thus, our data point to a regulatory role of ClpE in turning off clpP gene expression following temporal heat shock induction, and we propose that this effect is mediated through CtsR.

Genet Mol Res, 2003 Mar 31, 2(1), 112 - 6
Challenges when transferring technology from Lactococcus laboratory strains to industrial strains; Johansen E; Many genetically modified Lactococcus strains have been constructed in research laboratories around the world . Most of these have originated from laboratory strains and therefore there are several barriers to using them in an industrial setting . Laboratory strains are often plasmid-free and consequently Lac- and Prt-, rendering them unable to grow in milk . Many of the commonly used techniques have been optimised for laboratory strains and their application to industrial strains may require a great deal of effort . Often genetically modified organisms produced in the laboratory do not fit the published definition of 'food-grade' (Johansen, 1999, Encyclopedia of Food Microbiology, Academic Press, London, pp . 917-921) and a great deal of effort is required to eliminate undesirable DNA sequences . As a consequence, it is often necessary to recreate the strains in industrial backgrounds before the innovations described in the scientific literature can be applied to the real-world dairy industry.

Genet Mol Res, 2003 Mar 31, 2(1), 102 - 11
Heterologous protein production and delivery systems for Lactococcus lactis; Nouaille S et al.; Lactic acid bacteria (LAB), widely used in the food industry, are present in the intestine of most animals, including humans . The potential use of these bacteria as live vehicles for the production and delivery of heterologous proteins of vaccinal, medical or technological interest has therefore been extensively investigated . Lactococcus lactis, a LAB species, is a potential candidate for the production of biologically useful proteins . Several delivery systems have been developed to target heterologous proteins to a specific cell location (i.e., cytoplasm, cell wall or extracellular medium) . A promising application of L . lactis is its use as an antigen delivery vehicle, for the development of live mucosal vaccines . The expression of heterologous proteins and antigens as well as the various delivery systems developed in L . lactis, and its use as an oral vaccine carrier are discussed.

Int J Food Microbiol, 2003 Sep 15, 86(3), 213 - 22
Characterisation of technologically proficient wild Lactococcus lactis strains resistant to phage infection; Madera C et al.; The aim of this work was to establish whether Lactococcus lactis strains isolated from spontaneous dairy fermentations exhibited useful milk-processing capabilities and resistance to bacteriophage infection in order to be used as components in starter formulations . The 33 out of 100 isolates of L . lactis, originated from farmhouse cheeses, were found to be resistant to a collection of 34 phages belonging to the c2 and 936 groups . Six of the isolates were discarded as potential starters because they were lysogenic and other five because they produced tyramine . Plasmid and chromosomal profiles of the 22 remaining isolates allowed their classification into 16 different strains . All of these were good lactic acid producers from lactose, moderately proteolytic and, in eight cases, diacetyl production from citrate was observed . The mechanism(s) leading to the phenotype of phage resistance was identified for all the strains used in this study . Inhibition of adsorption was the most frequent one, although genetic determinants for some abortive infection systems were also detected (abiB, abiG and abiI) . Frequently, more than one mechanism was present in the same strain . One of the strains, L . lactis IPLA542, was selected as a model starter for pilot fermentations . It clotted milk normally both in the absence and in the presence of phage at concentrations that completely abolished the process when promoted by a phage-susceptible strain.

Biosci Biotechnol Biochem, 2003 Jul, 67(7), 1616 - 9
Identification of the lantibiotic nisin Q, a new natural nisin variant produced by Lactococcus lactis 61-14 isolated from a river in Japan; Zendo T et al.; Lactococcus lactis 61-14 isolated from river water produced a bacteriocin active against a wide range of Gram-positive bacteria . N-terminal amino acid sequencing, mass spectral analysis of the purified bacteriocin, and genetic analysis using nisin-specific primers showed that the bacteriocin was a new natural nisin variant, termed nisin Q . Nisin Q and nisin A differ in four amino acids in the mature peptide and two in the leader sequence.

Appl Microbiol Biotechnol, 2004 Feb, 63(6), 659 - 65 Epub 2003 Aug 09.
Production of nisin with continuous adsorption to Amberlite XAD-4 resin using Lactococcus lactis N8 and L . lactis LAC48; Tolonen M et al.; The production of nisin, biomass and lactic acid in pH-controlled and uncontrolled batch fermentation and batch fermentation (pH 5.5) with continuous removal of nisin was examined in the parent strain Lactococcus lactis N8 and LAC48 . Strain LAC48 in batch fermentor (pH not controlled) gave a maximum nisin concentration of 2.5 x 10(6) IU g dcw(-1) . The nisin concentration remained high (2.0 x 10(6) IU g dcw(-1)) after the logarithmic growth phase (10-22 h), whereas nisin production of strain N8 decreased after the logarithmic growth phase . The maximum nisin production of strain LAC48 was not directly related to the biomass formation and not associated with growth . In order to study end product inhibition in nisin production, a system was built for adsorption of nisin during fermentation . The adsorbent Amberlite XAD-4 was found to have an effective binding capacity for nisin . Cells of LAC48 and N8 compensated for the removal of nisin, indicating that nisin production also occurs in the stationary phase.

Microbiology, 2003 Aug, 149(Pt 8), 2193 - 201
Optimization of signal peptide SP310 for heterologous protein production in Lactococcus lactis; Ravn P et al.; The authors have previously reported the identification of novel signal peptides (SPs) from Lactococcus lactis using transposon insertion . Of these, SP310 caused the highest level of secretion . However, the levels were lower than those obtained using the signal peptide from Usp45 (SPUSP), the major secreted lactococcal protein . In this study, site-directed mutagenesis of signal peptide SP310 was used to investigate the effect of amino acid alterations on lactococcal secretion and to improve secretion efficiency . Several mutated SPs caused higher secretion . This increase in secretion was due to modifications in the cleavage region . In fermenter experiments, the signal peptide SP310mut2 resulted in an extracellular Staphylococcus aureus nuclease (Nuc) yield which was 45 % higher than that with the natural SP310 . Surprisingly, increasing the hydrophobicity of the hydrophobic core or increasing the number of positively charged amino acids in the N-terminal region of SP310 decreased secretion . High extracellular yields of Nuc resulted from more efficient secretion, as strains with less efficient SPs accumulated more intracellular SP-Nuc precursor.

Appl Environ Microbiol, 2003 Aug, 69(8), 5029 - 31
Increased exopolysaccharide production in Lactococcus lactis due to increased levels of expression of the NIZO B40 eps gene cluster; Boels IC et al.; Exopolysaccharides (EPS) play an important role in the rheology and texture of fermented food products . This is the first report demonstrating that homologous overexpression of a complete eps gene cluster in Lactococcus lactis leads to increased EPS production levels . A ninefold-elevated EPS plasmid copy number led to an almost threefold increase in the eps expression level, resulting in an almost fourfold increase in the NIZO B40 EPS production level . It was previously reported that increased EPS precursor levels did not influence NIZO B40 EPS production levels . However, the present results indicate that the maximal NIZO B40 EPS production level is limited by the activity level of the expression products of the eps gene cluster rather than by the level of EPS precursors.

Appl Environ Microbiol, 2003 Aug, 69(8), 4367 - 74
Microbial and physiological characterization of weakly amylolytic but fast-growing lactic acid bacteria: a functional role in supporting microbial diversity in pozol, a Mexican fermented maize beverage; Diaz-Ruiz G et al.; Pozol is an acid beverage obtained from the natural fermentation of nixtamal (heat- and alkali-treated maize) dough . The concentration of mono- and disaccharides from maize is reduced during nixtamalization, so that starch is the main carbohydrate available for lactic acid fermentation . In order to provide some basis to understand the role of amylolytic lactic acid bacteria (ALAB) in this fermented food, their diversity and physiological characteristics were determined . Forty amylolytic strains were characterized by phenotypic and molecular taxonomic methods . Four different biotypes were distinguished via ribotyping; Streptococcus bovis strains were found to be predominant . Streptococcus macedonicus, Lactococcus lactis, and Enterococcus sulfureus strains were also identified . S . bovis strain 25124 showed extremely low amylase yield relative to biomass (139 U g {cell dry weight}(-1)) and specific rate of amylase production (130.7 U g {cell dry weight}(-1) h(-1)) . In contrast, it showed a high specific growth rate (0.94 h(-1)) and an efficient energy conversion yield to bacterial cell biomass (0.31 g of biomass g of substrate(-1)) . These would confer on the strain a competitive advantage and are the possible reasons for its dominance . Transient accumulation of maltooligosaccharides during fermentation could presumably serve as energy sources for nonamylolytic species in pozol fermentation . This would explain the observed diversity and the dominance of nonamylolytic lactic acid bacteria at the end of fermentation . These results are the first step to understanding the importance of ALAB during pozol fermentation.

FEMS Microbiol Lett, 2003 Jul 29, 224(2), 307 - 13
Controlled intra- or extracellular production of staphylococcal nuclease and ovine omega interferon in Lactococcus lactis; Bermudez-Humaran LG et al.; A system for controlled targeting of heterologous protein was developed in the food-grade bacterium Lactococcus lactis . It is composed of the L . lactis strain NZ9000 and of two broad host range expression vectors pCYT:Nuc and pSEC:Nuc for, respectively, cytoplasmic and secreted staphylococcal nuclease (Nuc) nisin-inducible production . The level of intracellular production of Nuc measured with pCYT:Nuc (3 mg x l(-1)) is significantly lower than the one obtained with pSEC:Nuc ( approximately 20 mg x l(-1)) . The secretion efficiency (SE) of Nuc is estimated to be approximately 70%, corresponding to approximately 15 mg of secreted Nuc x l(-1) . Furthermore, we established that Nuc production continued in L . lactis 10 h after a 1-h nisin-pulse induction . This system was then used for intra- and extracellular production of a protein of therapeutical interest in L . lactis, the ovine interferon-omega (IFN-omega) . The SE and the quantity of secreted active IFN-omega were evaluated respectively to be approximately 70% and approximately 1 mg x l(-1) ( approximately two-fold higher than the cytoplasmic form).

Biotechnol Lett, 2003 Jul, 25(13), 1093 - 8
L-Lactic acid production from raw cassava starch in a circulating loop bioreactor with cells immobilized in loofa (Luffa cylindrica); Roble ND et al.; L-Lactic acid was produced from raw cassava starch, by simultaneous enzyme production, starch saccharification and fermentation in a circulating loop bioreactor with Aspergillus awamori and Lactococcus lactis spp . lactis immobilized in loofa sponge . A . awamori was immobilized directly in cylindrical loofa sponge while the L . lactis was immobilized in a loofa sponge alginate gel cube . In the loofa sponge alginate gel cube, the sponge serves as skeletal support for the gel with the cells . The alginate gel formed a hard outer layer covering the soft porous gel inside . By controlling the rate and frequency of broth circulation between the riser and downcomer columns, the riser could be maintained under aerobic condition while the downcomer was under anaerobic condition . Repeated fed-batch L-lactic acid production was performed for more than 400 h and the average lactic acid yield and productivity from raw cassava starch were 0.76 g lactic acid g(-1) starch and 1.6 g lactic acid l(-1) h(-1), respectively.

Biotechnol Lett, 2003 Jun, 25(11), 877 - 82
Application of statistically-based experimental designs for the optimization of nisin production from whey; Liu C et al.; Statistically-based experimental designs were applied for the optimization of nisin production by Lactococcus lactis in a whey-based medium . Yeast extract, KH2PO4, and MgSO4 were identified to have significant effects on nisin biosynthesis by a Plackett-Burman design . These three significant factors were subsequently optimized using central composite design, and the optimal conditions were determined to be 12.067 g l(-1) for yeast extract, 0.569 g l(-1) for KH2PO4, and 0.572 g l(-1) for MgSO4 . The validity of the optimal conditions was verified by a separate experiment.

Biotechnol Lett, 2003 Apr, 25(8), 599 - 602
Lacticin 3147 favours isoleucine transamination by Lactococcus lactis IFPL359 in a cheese-model system; Martinez-Cuesta C et al.; The bacteriocin, lacticin 3147, increased isoleucine transamination by Lactococcus lactis IFPL359 in a cheese model system . The formation of alpha-keto-beta-methyl-n-valeric acid and 2-hydroxy-3-methyl-valeric acid increased by three times in cheese slurries at 12 degrees C and cheese aroma intensity increased as well, which corresponded with a higher 2-methylbutanal formation.

Biotechnol Lett, 2003 Apr, 25(7), 573 - 77
Lactic acid production using two food processing wastes, canned pineapple syrup and grape invertase, as substrate and enzyme; Ueno T et al.; Canned pineapple syrup, a food processing waste, was utilized as a substrate for lactic acid production by Lactococcus lactis . To improve the utilization of sucrose from the syrup, grape invertase from grape juice derived from wine production was used for sucrose hydrolysis . The highest lactic acid concentrations achieved were 20 and 92 g l-1 from 20 and 100 g total sugars l-1, respectively, without a lag period for sucrose consumption.

Antimicrob Agents Chemother, 2003 Aug, 47(8), 2413 - 7
Molecular cloning and characterization of an ABC multidrug efflux pump, VcaM, in Non-O1 Vibrio cholerae; Huda N et al.; A gene responsible for multidrug resistance was cloned from the chromosomal DNA of non-O1 Vibrio cholerae NCTC 4716 by using as a host drug-hypersensitive Escherichia coli strain KAM32, which lacks major multidrug efflux pumps . E . coli cells transformed with the gene showed elevated levels of resistance to a number of structurally dissimilar drugs, such as tetracycline, norfloxacin, ciprofloxacin, doxorubicin, daunomycin, 4',6-diamidino-2-phenylindole, and Hoechst 33342 . We determined the nucleotide sequence and found one open reading frame . We designated the gene vcaM . The deduced product, VcaM, seems to be a polypeptide with 619 amino acid residues (69 kDa) that has a putative topology of six transmembrane segments in the N-terminal hydrophobic domain, followed by an ATP binding domain in the C-terminal hydrophilic region . The sequence of VcaM was shown to be similar to those of human multidrug resistance proteins P-glycoprotein MDR1 and lactococcal LmrA, which are driven by ATP . The efflux of Hoechst 33342 and doxorubicin from cells possessing VcaM was detected . The efflux activity was inhibited by reserpine and sodium o-vanadate, which are potent inhibitors of MDR1 and LmrA . Thus, we conclude that VcaM is a member of the family of multidrug efflux pumps of the ATP binding cassette type and the first experimentally proven example of a multidrug efflux pump of this family in gram-negative bacteria.

J Bacteriol, 2003 Aug, 185(15), 4499 - 507
IS981-mediated adaptive evolution recovers lactate production by ldhB transcription activation in a lactate dehydrogenase-deficient strain of Lactococcus lactis; Bongers RS et al.; Lactococcus lactis NZ9010 in which the las operon-encoded ldh gene was replaced with an erythromycin resistance gene cassette displayed a stable phenotype when grown under aerobic conditions, and its main end products of fermentation under these conditions were acetate and acetoin . However, under anaerobic conditions, the growth of these cells was strongly retarded while the main end products of fermentation were acetate and ethanol . Upon prolonged subculturing of this strain under anaerobic conditions, both the growth rate and the ability to produce lactate were recovered after a variable number of generations . This recovery was shown to be due to the transcriptional activation of a silent ldhB gene coding for an Ldh protein (LdhB) with kinetic parameters different from those of the native las operon-encoded Ldh protein . Nevertheless, cells producing LdhB produced mainly lactate as the end product of fermentation . The mechanism underlying the ldhB gene activation was primarily studied in a single-colony isolate of the recovered culture, designated L . lactis NZ9015 . Integration of IS981 in the upstream region of ldhB was responsible for transcription activation of the ldhB gene by generating an IS981-derived -35 promoter region at the correct spacing with a natively present -10 region . Subsequently, analysis of 10 independently isolated lactate-producing derivatives of L . lactis NZ9010 confirmed that the ldhB gene is transcribed in all of them . Moreover, characterization of the upstream region of the ldhB gene in these derivatives indicated that site-specific and directional IS981 insertion represents the predominant mechanism of the observed recovery of the ability to produce lactate.

Microbiology, 2003 Jul, 149(Pt 7), 1935 - 44
Transcriptional, translational and metabolic regulation of glycolysis in Lactococcus lactis subsp . cremoris MG 1363 grown in continuous acidic cultures; Even S et al.; The physiological behaviour of Lactococcus lactis subsp . cremoris MG 1363 was characterized in continuous culture under various acidic conditions (pH 4.7-6.6) . Biomass yield was diminished in cultures with low pH and the energy dedicated to maintenance increased due to organic acid inhibition and cytoplasmic acidification . Under such acidic conditions, the specific rate of glucose consumption by the bacterium increased, thereby enhancing energy supply . This acceleration of glycolysis was regulated by both an increase in the concentrations of glycolytic enzymes (hierarchical regulation) and the specific modulation of enzyme activities (metabolic regulation) . However, when the inhibitory effect of intracellular pH on enzyme activity was taken into account in the model of regulation, metabolite regulation was shown to be the dominant factor controlling pathway flux . The changes in glycolytic enzyme concentrations were not correlated directly to modifications in transcript concentrations . Analyses of the relative contribution of the phenomena controlling enzyme synthesis indicated that translational regulation had a major influence compared to transcriptional regulation . An increase in the translation efficiency was accompanied by an important decrease of total cellular RNA concentrations, confirming that the translation apparatus of L . lactis was optimized under acid stress conditions.

FEMS Microbiol Lett, 2003 Jul 15, 224(1), 53 - 9
HtrA is a key factor in the response to specific stress conditions in Lactococcus lactis; Foucaud-Scheunemann C et al.; We investigated the physiological role of Lactococcus lactis housekeeping surface protease HtrA . It is involved in surface properties under regular growth conditions, as the htrA mutant strain forms longer chains in liquid medium . It participates in cellular defence against environmental stress conditions: compared to the wild-type strain, the htrA mutant strain exhibited increased sensitivity to heat, ethanol, puromycin, and NaCl, but not to pH, H2O2, bile salts or to carbon or nitrogen starvation . htrA transcription in the wild-type strain showed a transient increase under stress conditions determined as requiring htrA, but not under overexpression of a secreted heterologous protein . Our results demonstrate that in L . lactis, htrA is a key factor in the response to specific stress conditions.

J Exp Med, 2003 Jul 7, 198(1), 5 - 17
Cellular prion protein promotes Brucella infection into macrophages; Watarai M et al.; The products of the Brucella abortus virB gene locus, which are highly similar to conjugative DNA transfer system, enable the bacterium to replicate within macrophage vacuoles . The replicative phagosome is thought to be established by the interaction of a substrate of the VirB complex with macrophages, although the substrate and its host cellular target have not yet been identified . We report here that Hsp60, a member of the GroEL family of chaperonins, of B . abortus is capable of interacting directly or indirectly with cellular prion protein (PrPC) on host cells . Aggregation of PrPC tail-like formation was observed during bacterial swimming internalization into macrophages and PrPC was selectively incorporated into macropinosomes containing B . abortus . Hsp60 reacted strongly with serum from human brucellosis patients and was exposed on the bacterial surface via a VirB complex-associated process . Under in vitro and in vivo conditions, Hsp60 of B . abortus bound to PrPC . Hsp60 of B . abortus, expressed on the surface of Lactococcus lactis, promoted the aggregation of PrPC but not PrPC tail formation on macrophages . The PrPC deficiency prevented swimming internalization and intracellular replication of B . abortus, with the result that phagosomes bearing the bacteria were targeted into the endocytic network . These results indicate that signal transduction induced by the interaction between bacterial Hsp60 and PrPC on macrophages contributes to the establishment of B . abortus infection.

J Biol Chem, 2003 Sep 12, 278(37), 35193 - 8 Epub 2003 Jul 02.
The ATP binding cassette multidrug transporter LmrA and lipid transporter MsbA have overlapping substrate specificities; Reuter G et al.; LmrA is an ATP binding cassette (ABC) multidrug transporter in Lactococcus lactis that is a structural and functional homologue of the human multidrug resistance P-glycoprotein MDR1 (ABCB1) . LmrA is also homologous to MsbA, an essential ABC transporter in Escherichia coli involved in the trafficking of lipids, including Lipid A . We have compared the substrate specificities of LmrA and MsbA in detail . Surprisingly, LmrA was able to functionally substitute for a temperature-sensitive mutant MsbA in E . coli WD2 at non-permissive temperatures, suggesting that LmrA could transport Lipid A . LmrA also exhibited a Lipid A-stimulated, vanadate-sensitive ATPase activity . Reciprocally, the expression of MsbA conferred multidrug resistance on E . coli . Similar to LmrA, MsbA interacted with photoactivatable substrate {3H}azidopine, displayed a daunomycin, vinblastine, and Hoechst 33342-stimulated vanadate-sensitive ATPase activity, and mediated the transport of ethidium from cells and Hoechst 33342 in proteoliposomes containing purified and functionally reconstituted protein . Taken together, these data demonstrate that MsbA and LmrA have overlapping substrate specificities . Our observations imply the presence of structural elements in the recently published crystal structures of MsbA in E . coli and Vibrio cholera (Chang, G., and Roth, C . B . (2001) Science 293, 1793-1800; Chang, G . (2003) J . Mol . Biol . 330, 419-430) that support drug-protein interactions and suggest a possible role for LmrA in lipid trafficking in L . lactis.

Appl Environ Microbiol, 2003 Jul, 69(7), 4214 - 8
Microplate bioassay for nisin in foods, based on nisin-induced green fluorescent protein fluorescence; Reunanen J et al.; A plasmid coding for the nisin two-component regulatory proteins, NisK and NisR, was constructed; in this plasmid a gfp gene (encoding the green fluorescent protein) was placed under control of the nisin-inducible nisF promoter . The plasmid was transformed into non-nisin-producing Lactococcus lactis strain MG1614 . The new strain could sense extracellular nisin and transduce it to green fluorescent protein fluorescence . The amount of fluorescence was dependent on the nisin concentration, and it could be measured easily . By using this strain, an assay for quantification of nisin was developed . With this method it was possible to measure as little as 2.5 ng of pure nisin per ml in culture supernatant, 45 ng of nisin per ml in milk, 0.9 microg of nisin in cheese, and 1 microg of nisin per ml in salad dressings.

Appl Environ Microbiol, 2003 Jul, 69(7), 4049 - 56
Characterization of genes involved in the metabolism of alpha-galactosides by Lactococcus raffinolactis; Boucher I et al.; Lactococcus raffinolactis, unlike most lactococci, is able to ferment alpha-galactosides, such as melibiose and raffinose . More than 12 kb of chromosomal DNA from L . raffinolactis ATCC 43920 was sequenced, including the alpha-galactosidase gene and genes involved in the Leloir pathway of galactose metabolism . These genes are organized into an operon containing aga (alpha-galactosidase), galK (galactokinase), and galT (galactose 1-phosphate uridylyltransferase) . Northern blotting experiments revealed that this operon was induced by galactosides, such as lactose, melibiose, raffinose, and, to a lesser extent, galactose . Similarly, alpha-galactosidase activity was higher in lactose-, melibiose-, and raffinose-grown cells than in galactose-grown cells . No alpha-galactosidase activity was detected in glucose-grown cells . The expression of the aga-galKT operon was modulated by a regulator encoded by the upstream gene galR . The product of galR belongs to the LacI/GalR family of transcriptional regulators . In L . lactis, L . raffinolactis GalR acted as a repressor of aga and lowered the enzyme activity by more than 20-fold . We suggest that the expression of the aga operon in lactococci is negatively controlled by GalR and induced by a metabolite derived from the metabolism of galactosides.

Acta Biochim Pol, 2003, 50(2), 455 - 9
Overproduction and purification of the CcpA protein from Lactococcus lactis; Kowalczyk M et al.; In this work we present cloning and overexpression of lactococcal CcpA protein in Escherichia coli Xl1blue strain as a fusion with 6 x His tag . A high yield of the CcpA protein was obtained when the cells were cultured in liquid medium LB with 100 microg/ml ampicillin at 37 degrees C and subsequently for 4 h after induction by IPTG . The procedure let us obtain 5 mg of homogenous CcpA protein . Glutaraldehyde crosslinking analysis indicated the formation of dimer or tetramer forms of the CcpA protein.

Virology, 2003 Jun 20, 311(1), 144 - 56
Identification of operator sites of the CI repressor of phage TP901-1: evolutionary link to other phages; Johansen AH et al.; The repressor encoded by the cI gene of the temperate Lactococcus lactis subsp . cremoris bacteriophage TP901-1 has been purified . Gel-retardation and footprinting analyses identified three palindromic operator sites (O(R), O(L), and O(D)) . The operator site O(R) is located between the two divergent early promoters P(R) and P(L), O(L) overlaps the transcriptional start of the lytic P(L) promoter, and O(D) is located downstream of the mor gene, the first gene in the lytic gene cluster . The function of O(L) was verified by mutational analysis . Binding was found to be specific and cooperative . Multimeric forms of the repressor were observed, thus indicating that the repressor may bind simultaneously to all three operator sites . Inverted repeats with homology to the operator sites of TP901-1 were identified in phage genomes encoding repressors homologous to CI of TP901-1 . Interestingly, the locations of these repeats on the phage genomes correspond to those found in TP901-1, indicating that the same system of cooperative repression of early phage promoters has been inherited by modular evolution.

Nat Biotechnol, 2003 Jul, 21(7), 785 - 9 Epub 2003 Jun 15.
Biological containment of genetically modified Lactococcus lactis for intestinal delivery of human interleukin 10; Steidler L et al.; Genetically modified Lactococcus lactis secreting interleukin 10 provides a therapeutic approach for inflammatory bowel disease . However, the release of such genetically modified organisms through clinical use raises safety concerns . In an effort to address this problem, we replaced the thymidylate synthase gene thyA of L . lactis with a synthetic human IL10 gene . This thyA- hIL10+ L . lactis strain produced human IL-10 (hIL-10), and when deprived of thymidine or thymine, its viability dropped by several orders of magnitude, essentially preventing its accumulation in the environment . The biological containment system and the bacterium's capacity to secrete hIL-10 were validated in vivo in pigs . Our approach is a promising one for transgene containment because, in the unlikely event that the engineered L . lactis strain acquired an intact thyA gene from a donor such as L . lactis subsp . cremoris, the transgene would be eliminated from the genome.

ScientificWorldJournal, 2001 May 11, 1, 216 - 7
Lactococcus lactis, a tool for the delivery of therapeutic proteins treatment of IBD; Steidler L; Inflammatory bowel disease (IBD) is a group of chronic intestinal inflammatory diseases that consists of ulcerative colitis (UC), an inflammation of the large intestine, and Crohn's disease (CD), which can affect any part of the gastrointestinal tract . IBD affects approximately 1 in every 1000 individuals in western countries . There is a marked tendency in the age of onset toward gradually younger people . IBD represents a genuine problem in public health because of the absence of etiologic treatment . The clinical image is characterized by recurrent segmental or total inflammatory involvement of the large and/or small intestine, often resulting in a chronic, unpredictable course . The symptoms of both are extremely unpleasant and impact all aspects of quality of life . They include diarrhea, abdominal pain, rectal bleeding, fever, nausea, weight loss, lethargy, and loss of appetite . If left untreated, malnutrition, dehydration, and anemia follow, which, in extreme cases, can even lead to death . Although many patients are managed successfully with conventional medical therapy, such as anti-inflammatory corticosteroid treatment, some stay refractory to treatment, most will have recurrent activity of disease, and two thirds will require surgery . Administered orally or by injection, only a fraction of the active components of most conventional drugs reaches the intended target site, the inflamed intestinal lining . This is not only an inefficient way to deliver drugs, but, more important, means that patients are often subject to a spectrum of unpleasant side effects that result from the high levels of the drugs in other, otherwise healthy tissues and organs of the body.

Lett Appl Microbiol, 2003, 37(1), 51 - 5
Influence of pH drop on both nisin and pediocin production by Lactococcus lactis and Pediococcus acidilactici; Guerra NP et al.; AIMS: To develop a kinetic model for describing the specific effect of pH drop on nisin and pediocin production in whey . METHODS AND RESULTS: The effect of pH drop on both bacteriocin productions was tested in non-buffered whey and whey buffered at initial pH 6.3 with 0.03, 0.10 and 0.25 mol l-1 of potassium hydrogen phthalate-NaOH . An accurate description of the experimental data of nisin and pediocin obtained at different pH drops is obtained with the proposed model . CONCLUSIONS: The proposed model was able to typify both bacteriocins as pH-dependent primary metabolites . SIGNIFICANCE AND IMPACT OF THE STUDY: The decisive role of pH drop for bacteriocin production on whey was demonstrated and modelled . This study contributes to a better understanding of underlying metabolic regulatory mechanisms, which could facilitate the optimization of bacteriocin production for upscaling.

Lett Appl Microbiol, 2003, 37(1), 26 - 30
Modelling the individual cell lag phase . Isolating single cells: protocol development; Francois K et al.; AIMS: To develop a protocol to isolate single cells in wells of a microtitre plate, having a high certainty of individual cells, combined with a sufficient yield . METHODS AND RESULTS: Single cells were obtained using 1/2 dilution series in microtitre plates . Seventy-two Lactococcus lactis dilution series were checked by plate counting . When the last five columns of the plates were observed, the chance of having one single cell was 80%, while the yield was 75 wells containing cells . A simulation model confirmed these results . This method was compared with the commonly applied method . CONCLUSIONS: This method makes it possible to combine a higher chance of having one cell in a microtitre well with a slightly higher yield . SIGNIFICANCE AND IMPACT OF THE STUDY: A tool is developed to isolate single cells to provide a suitable base for investigating and modelling the individual cell lag phase.

J Biol Chem, 2003 Sep 5, 278(36), 34291 - 8 Epub 2003 Jun 11.
Novel mechanism of bacteriocin secretion and immunity carried out by lactococcal multidrug resistance proteins; Gajic O et al.; A natural isolate of Lactococcus lactis was shown to produce two narrow spectrum class II bacteriocins, designated LsbA and LsbB . The cognate genes are located on a 5.6-kb plasmid within a gene cluster specifying LmrB, an ATP-binding cassette-type multidrug resistance transporter protein . LsbA is a hydrophobic peptide that is initially synthesized with an N-terminal extension . The housekeeping surface proteinase HtrA was shown to be responsible for the cleavage of precursor peptide to yield the active bacteriocin . LsbB is a relatively hydrophilic protein synthesized without an N-terminal leader sequence or signal peptide . The secretion of both polypeptides was shown to be mediated by LmrB . An L . lactis strain lacking plasmid-encoded LmrB and the chromosomally encoded LmrA is unable to secrete either of the two bacteriocins . Complementation of the strain with an active LmrB protein resulted in restored export of the two polypeptides across the cytoplasmic membrane . When expressed in an L . lactis strain that is sensitive to LsbA and LsbB, LmrB was shown to confer resistance toward both bacteriocins . It does so, most likely, by removing the two polypeptides from the cytoplasmic membrane . This is the first report in which a multidrug transporter protein is shown to be involved in both secretion and immunity of antimicrobial peptides.

J Biol Chem, 2003 Aug 29, 278(35), 33305 - 11 Epub 2003 Jun 09.
Molecular structure of galactokinase; Thoden JB et al.; Galactokinase plays a key role in normal galactose metabolism by catalyzing the ATP-dependent phosphorylation of alpha-D-galactose to galactose 1-phosphate . In humans, mutations in the galactokinase gene can lead to the diseased state referred to as Type II galactosemia . Here we describe the three-dimensional structure of galactokinase from Lactococcus lactis determined to 2.1-A resolution . As expected from amino acid sequence alignments, galactokinase adopts a similar topology to that observed for members of the GHMP superfamily . The N-terminal domain is characterized by a five-stranded mixed beta-sheet while the C-terminal motif is dominated by two distinct four-stranded anti-parallel beta-sheets . The structure was solved in the presence of alpha-D-galactose and inorganic phosphate . These ligands are wedged between the N- and C-terminal domains . Amino acid side chains responsible for anchoring the sugar ligand to the protein include Arg36, Glu42, Asp45, Asp183, and Tyr233 . Both Arg36 and Asp183 are strictly conserved in the amino acid sequences available in the literature thus far for galactokinases . Interestingly, the carboxylate side chain of Asp183 is positioned within 3.5 A of the C-1 hydroxyl group of galactose, whereas the guanidinium group of Arg36 is situated between both the C-1 hydroxyl group and the inorganic phosphate . Most likely these residues play key roles in catalysis . The structure of galactokinase described here serves as a model for understanding the functional consequences of point mutations known to result in Type II galactosemia in humans.

Biotechnol Appl Biochem, 2003 Oct, 38(Pt 2), 157 - 67
Enhancement of nisin production by Lactococcus lactis in periodically re-alkalized cultures; Guerra NP et al.; Synthesis of nisin as well as biomass production by Lactococcus lactis subsp . lactis CECT (Coleccion Espanola de Cultivos Tipo) 539 on both hydrolysed mussel-processing waste and whey medium were followed in three fixed volume fed-batch fermentations, with re-alkalization cycles . The two cultures on mussel-processing waste (MPW) were fed with a 240 g/l concentrated glucose and with a concentrated MPW (about 100 g of glucose/l) . The culture on whey was fed with a mixture of concentrated whey (48 g of total sugars/l) and a 400 g/l concentrated lactose . The three cultures were mainly characterized with higher nisin titres {49.7, 109.6 and 124.7 bacteriocin activity units (AU)/ml respectively} compared with the batch process on de Man, Rogosa and Sharpe {(1960) J . Appl . Bacteriol . 23, 130-135} medium (49.6 AU/ml), MPW (9.5 AU/ml) and whey (22.5 AU/ml) {1 AU/ml is the amount of antibacterial compound needed to obtain 50% growth inhibition (LD50) compared with control tubes} . In the three fed-batch cultures a shift from homolactic to mixed-acid fermentation was observed, and other products (acetic acid, butane-2,3-diol or ethanol) in addition to lactic acid were detectable in the medium . However, their contributions to the total antibacterial activity of the post-incubates (the cell-free culture supernatant obtained at the end of the fermentation process) of L . lactis CECT 539 against Carnobacterium piscicola CECT 4020 were very low.

Biotechnol Prog, 2003 May-Jun, 19(3), 1101 - 4
Fusion to a carrier protein and a synthetic propeptide enhances E7 HPV-16 production and secretion in Lactococcus lactis; Bermudez-Humaran LG et al.; An inducible system to improve and stabilize the production of an extremely labile protein (E7 antigen of human papillomavirus type 16) was developed in the food-grade bacterium Lactococcus lactis . A protein carrier, the staphylococcal nuclease Nuc, was fused either to N- or C-termini of E7 protein, and the resulting hybrid proteins were rescued from intracellular proteolysis but poorly secreted by L . lactis . A synthetic propeptide (LEISSTCDA) was then fused and significantly improved the secretion efficiency of the hybrid protein Nuc-E7 by L . lactis.

Appl Environ Microbiol, 2003 Jun, 69(6), 3668 - 71
Behavior of psychrotrophic lactic acid bacteria isolated from spoiling cooked meat products; Hamasaki Y et al.; Three kinds of lactic acid bacteria were isolated from spoiling cooked meat products stored below 10 degrees C . They were identified as Leuconostoc mesenteroides subsp . mesenteroides, Lactococcus lactis subsp . lactis, and Leuconostoc citreum . All three strains grew well in MRS broth at 10 degrees C . In particular, L . mesenteroides subsp . mesenteroides and L . citreum grew even at 4 degrees C, and their doubling times were 23.6 and 51.5 h, respectively . On the other hand, although the bacteria were initially below the detection limit (<10 CFU/g) in model cooked meat products, the bacterial counts increased to 10(8) CFU/g at 10 degrees C after 7 to 12 days.

Appl Environ Microbiol, 2003 Jun, 69(6), 3462 - 8
Glucose metabolism in Lactococcus lactis MG1363 under different aeration conditions: requirement of acetate to sustain growth under microaerobic conditions; Nordkvist M et al.; Lactococcus lactis subsp . lactis MG1363 was grown in batch cultures on a defined medium with glucose as the energy source under different aeration conditions, namely, anaerobic conditions, aerobic conditions, and microaerobic conditions with a dissolved oxygen tension of 5% (when saturation with air was used as the reference) . The maximum specific growth rate was high (0.78 to 0.91 h(-1)) under all aeration conditions but decreased with increasing aeration, and more than 90% of the glucose was converted to lactate . However, a shift in by-product formation was observed . Increasing aeration resulted in acetate, CO(2), and acetoin replacing formate and ethanol as end products . Under microaerobic conditions, growth came to a gradual halt, although more than 60% of the glucose was still left . A decline in growth was not observed during microaerobic cultivation when acetate was added to the medium . We hypothesize that the decline in growth was due to a lack of acetyl coenzyme A (acetyl-CoA) needed for fatty acid synthesis since acetyl-CoA can be synthesized from acetate by means of acetate kinase and phosphotransacetylase activities.

Appl Environ Microbiol, 2003 Jun, 69(6), 3069 - 76
Increased production of folate by metabolic engineering of Lactococcus lactis; Sybesma W et al.; The dairy starter bacterium Lactococcus lactis is able to synthesize folate and accumulates large amounts of folate, predominantly in the polyglutamyl form . Only small amounts of the produced folate are released in the extracellular medium . Five genes involved in folate biosynthesis were identified in a folate gene cluster in L . lactis MG1363: folA, folB, folKE, folP, and folC . The gene folKE encodes the biprotein 2-amino-4-hydroxy-6-hydroxymethyldihydropteridine pyrophosphokinase and GTP cyclohydrolase I . The overexpression of folKE in L . lactis was found to increase the extracellular folate production almost 10-fold, while the total folate production increased almost 3-fold . The controlled combined overexpression of folKE and folC, encoding polyglutamyl folate synthetase, increased the retention of folate in the cell . The cloning and overexpression of folA, encoding dihydrofolate reductase, decreased the folate production twofold, suggesting a feedback inhibition of reduced folates on folate biosynthesis.

Appl Environ Microbiol, 2003 Jun, 69(6), 3061 - 8
CodY-regulated aminotransferases AraT and BcaT play a major role in the growth of Lactococcus lactis in milk by regulating the intracellular pool of amino acids; Chambellon E et al.; Aminotransferases, which catalyze the last step of biosynthesis of most amino acids and the first step of their catabolism, may be involved in the growth of Lactococcus lactis in milk . Previously, we isolated two aminotransferases from L . lactis, AraT and BcaT, which are responsible for the transamination of aromatic amino acids, branched-chain amino acids, and methionine . In this study, we demonstrated that double inactivation of AraT and BcaT strongly reduced the growth of L . lactis in milk . Supplementation of milk with amino acids and keto acids that are substrates of both aminotransferases did not improve the growth of the double mutant . On the contrary, supplementation of milk with isoleucine or a dipeptide containing isoleucine almost totally inhibited the growth of the double mutant, while it did not affect or only slightly affected the growth of the wild-type strain . These results suggest that AraT and BcaT play a major role in the growth of L . lactis in milk by degrading the intracellular excess isoleucine, which is responsible for the growth inhibition . The growth inhibition by isoleucine is likely to be due to CodY repression of the proteolytic system, which is necessary for maximal growth of L . lactis in milk, since the growth of the CodY mutant was not affected by addition of isoleucine to milk . Moreover, we demonstrated that AraT and BcaT are part of the CodY regulon and therefore are regulated by nutritional factors, such as the carbohydrate and nitrogen sources.

Curr Microbiol, 2003 Jul, 47(1), 17 - 21
Growth kinetics of Lactococcus lactis ssp diacetylactis harboring different plasmid content; Lee K et al.; The effect of plasmid content on growth of Lactococcus lactis ssp . diacetylactis harboring different plasmids and on plasmid stability was studied . Strain DRC-2C is a plasmid Lac(+)- and Prt(+)-free strain . Strain DRC-2 utilizes lactose as carbohydrate and has proteinase activity . The plasmid-free strain DRC-2C exhibited none of these features . Plasmid-encoded properties were clearly identified . Results showed that plasmid content decreased bacterial growth in terms of the specific growth rate determined . Slightly lower specific growth rate and lactic acid production were observed in the strain of higher plasmid content owing to the plasmid presence, causing metabolic burden to the host cell . The plasmid profile results showed that the number of bands in the two strains before and after fermentation were the same . This indicated that the plasmids were stably maintained and unchanged during the fermentation.

J Biol Chem, 2003 Aug 8, 278(32), 29546 - 51 Epub 2003 May 23.
The ATP/substrate stoichiometry of the ATP-binding cassette (ABC) transporter OpuA; Patzlaff JS et al.; ATP-binding cassette (ABC) transport proteins catalyze the translocation of substrates at the expense of hydrolysis of ATP, but the actual ATP/substrate stoichiometry is still controversial . In the osmoregulated ABC transporter (OpuA) from Lactococcus lactis, ATP hydrolysis and substrate translocation are tightly coupled, and the activity of right-side-in and inside-out reconstituted OpuA can be determined accurately . Although the ATP/substrate stoichiometry determined from the uptake of glycine betaine and intravesicular ATP hydrolysis tends to increase with decreasing average size of the liposomes, the data from inside-out reconstituted OpuA indicate that the mechanistic stoichiometry is 2 . Moreover, the two orientations of OpuA in proteoliposomes allowed possible contributions from substrate (glycine betaine) inhibition on the trans-side of the membrane and inhibition by ADP to be determined . Here we show that OpuA is not inhibited by up to 400 mm glycine betaine on the trans-side of the membrane . ADP is an inhibitor, but accumulation of ADP was negligible in the assays with inside-out-oriented OpuA, and potential effects of the ATP/ADP ratio on the ATP/substrate stoichiometry determinations could be eliminated.

Mol Genet Genomics, 2003 Jul, 269(4), 487 - 98 Epub 2003 May 21.
Transcriptional analysis of the genetic elements involved in the lysogeny/lysis switch in the temperate lactococcal bacteriophage phiLC3, and identification of the Cro-like protein ORF76; Blatny JM et al.; A transcriptional analysis of the lysogeny-related genes of the temperate bacteriophage Lactococcus lactis phiLC3 was performed using Northern blot hybridization during lysogeny and lytic infection by the phage . The lysogeny-related gene cluster was found to contain four promoters (P(1), P(2), Pint and P(173)), while the P(87) promoter directed transcription of orf80 and the putative gene orf87, which are located between the integrase gene and the cell lysis genes . The start sites of the transcripts were determined by primer extension . The divergently oriented lysogenic P(1) and lytic P(2) promoters located in the genetic switch region are responsible for transcription of orf286 which encodes the phage repressor, and the genes orf63 - orf76 - orf236 - orf110 - orf82 - orf57, respectively, while orf173 is transcribed from P(173) . orf76 was identified as the gene encoding the Cro-like protein of phiLC3, and it was shown that ORF76 is able to bind specifically to the genetic switch region, albeit with lower affinity than does the phage repressor ORF286 . ORF76 also competed with ORF286 for binding to this region . The functionality of P(1) and P(2), and their regulation by ORF286 and ORF76, was investigated using a reporter gene . In general, P(2) was a stronger promoter than P(1), but expression from both promoters, especially P(2), was regulated and modulated by flanking sequences and the presence of orf286 and orf76 . ORF286 and ORF76 were both able to repress transcription from P(1) and P(2), while ORF286 was able to stimulate its own synthesis by tenfold . This work reveals the complex interplay between the regulatory elements that control the genetic switch between lysis and lysogeny in phiLC3 and other temperate phages of Lactococcus.

Appl Microbiol Biotechnol, 2003 Oct, 62(5-6), 489 - 97 Epub 2003 May 15.
Co-fermentation of glucose and citrate by Lactococcus lactis diacetylactis: quantification of the relative metabolic rates by isotopic analysis at natural abundance; Goupry S et al.; The simultaneous catabolism of citrate and glucose by growing Lactococcus lactis subsp . lactis biovar . diacetylactis to obtain energy was followed quantitatively, using a non-enrichment isotopic technique . Both citrate and glucose are precursors of pyruvate, which may either be reduced to lactate, the principle product that accumulates, or be converted to diacetyl and acetoin . Under suitable conditions, both routes regenerate NAD+ . Until recently, however, the quantitative relationships between the two substrates and these three products were poorly defined . It was recently shown, by exploiting differences in natural abundance 13C/12C ratios in the two substrates, that there is no metabolic separation of the catabolism of these two carbon sources . In this study, it is shown that the relative consumption rates change throughout the growth phase, citrate being preferentially metabolised at the onset of a culture of energy-depleted cells, with a subsequent evolution towards a metabolism dominated by glucose consumption . Additionally, it is shown that the relative consumption rates are influenced by environmental factors, notably initial pH and temperature.

Proteomics, 2003 May, 3(5), 786 - 97
Proteome analysis of the purine stimulon from Lactococcus lactis; Beyer NH et al.; A comparative expression proteome analysis was carried out by analyzing differential expression patterns of pulse-labelled proteins on two-dimensional gels under standard conditions and during purine nucleotide starvation, followed by mass spectrometric identification of regulated proteins . Based upon the expression patterns, three stimulons could be identified in Lactococcus lactis subsp . cremoris . The Psu proteins (purine starvation up-regulated) had increased synthesis during purine depletion in a purine auxotroph . Among these proteins were enzymes of the purine biosynthesis pathways (PurE, PurS, PurM, PurL), and enzymes involved in the generation of C1 units (GlyA, Fhs) . C1 units are primarily required for purine biosynthesis . Upon analysis of the nucleotide sequence preceding the structural genes for these proteins in the L . lactis IL1403 genome sequence showed that all contained PurBox-Pribnov box structures resembling the PurR activated promoters for the purDEK and purCSQLF operons . Most, and possibly all members of the Psu stimulon are thus members of the PurR regulon . Five Psu proteins could not be identified . The second stimulon, the Psd stimulon (purine starvation decreased), whose members are down-regulated during purine depletion, contained proteins related to protein synthesis (PpsB, EF-TS, trigger factor), or to GTPases (FtsZ, EF-TS); or are involved in energy metabolism (GapB, CcpA) . No common regulatory elements could be found for members of this stimulon . Two Psd proteins escaped identification . The last, Dcu (decoynine up-regulated), stimulon contained proteins whose synthesis escaped the severe general depression during inhibition of the GMP synthetase by decoynine . This regulon was comprised of mostly glycolytic enzymes (fructose bisphosphate aldolase, enolase, pyruvate kinase) and translation elongation factors (GTPases: EF-TU, EF-G) . Two Dcu proteins could not be identified . Out of 28 proteins subjected to mass spectrometry, 19 could be readily identified despite the fact that only the genome sequence of a strain of L . lactis subsp . lactis was available . The two subspecies share about 85% sequence identity, comparable to the genetic distance between Escherichia coli and Salmonella typhimurium . A success rate of 68% indicates that it may be feasible to perform proteomics based upon genomic sequences of relatives outside the genus.

Dis Aquat Organ, 2003 Mar 31, 54(2), 127 - 34
Development of sensitive, high-throughput one-tube RT-PCR-enzyme hybridisation assay to detect selected bacterial fish pathogens; Wilson T et al.; Bacterial monitoring and surveillance is critical for the early detection of pathogens to avoid the spread of disease . To facilitate this, an efficient, high-performance and high-throughput method to detect the presence of femotgram amounts of ribosomal RNA from 4 bacterial fish pathogens: Aeromonas salmonicida; Tenacibaculum maritimum (formerly Flexibacter maritimus); Lactococcus garvieae; and Yersinia ruckeri was developed . The system uses NucleoLink strips for liquid- and solid-phase PCR in 1 tube, to perform RT-PCR-enzyme hybridisation assays (RT-PCR-EHA) detecting 4 fg or less of rRNA from pure cultures and between 1 and 9 CFU per 200 microl sample volume from selective-enrichment culture media . The liquid-phase amplicons were visualised by gel electrophoresis and the solid-phase amplicons detected using internal probes and visualised using colorimetric detection and p-nitrophenylphosphate.

J Dairy Sci, 2003 Apr, 86(4), 1472 - 5
Technical note: Use of RFLP to characterize Lactococcus lactis strains producing exopolysaccharides; Deveau H et al.; Restriction fragment length polymorphism (RFLP) is used to differentiate microorganisms by analysis of their DNA restriction patterns . A modified RFLP procedure is proposed for the rapid characterization of Lactococcus lactis strains producing exopolysaccharides (EPS) . The availability of such effective cataloging system is likely to benefit research aimed at identifying lactococcal strains that produce novel EPS.

J Dairy Sci, 2003 Apr, 86(4), 1139 - 46
High pressure effects on proteolytic and glycolytic enzymes involved in cheese manufacturing; Malone AS et al.; The activity of chymosin, plasmin, and Lactococcus lactis enzymes (cell envelope proteinase, intracellular peptidases, and glycolytic enzymes) were determined after 5-min exposures to pressures up to 800 MPa . Plasmin was unaffected by any pressure treatment . Chymosin activity was unaffected up to 400 MPa and decreased at 500 to 800 MPa . Fifty percent of control chymosin activity remained after the 800 MPa treatment . The lactococcal cell envelope proteinase (CEP) and intracellular peptidase activities were monitored in cell extracts of pressure-treated cells . A pressure of 100 MPa increased the CEP activity, whereas 200 MPa had no effect . At 300 MPa, CEP activity was reduced, and 400 to 800 MPa inactivated the enzyme . X-Prolyl-dipeptidyl aminopeptidase was insensitive to 5-min pressure treatments of 100 to 300 MPa, but was inactivated at 400 to 800 MPa . Aminopeptidase N was unaffected by 100 and 200 MPa . However, 300 MPa significantly reduced its activity, and 400 to 800 MPa inactivated it . Aminopeptidase C activity increased with increasing pressures up to 700 MPa . High pressure did not affect aminopeptidase A activity at any level . Hydrolysis of Lys-Ala-p-NA doubled after 300-MPa exposure, and was eliminated at 400 to 800 MPa . Glycolytic enzyme activities of pressure-treated cells were evaluated collectively by determining the titratable acidity as lactic acid produced by cell extracts in the presence of glucose . The titratable acidities produced by the 100 and 200 MPa samples were slightly increased compared to the control . At 300 to 800 MPa, no significant acid production was observed . These data demonstrate that high pressure causes no effect, activation, or inactivation of proteolytic and glycolytic enzymes depending on the pressure level and enzyme . Pressure treatment of cheese may alter enzymes involved in ripening, and pressure-treating L . lactis may provide a means to generate attenuated starters with altered enzyme profiles.

J Biol Chem, 2003 Aug 1, 278(31), 28812 - 22 Epub 2003 May 05.
Lactococcus lactis dihydroorotate dehydrogenase A mutants reveal important facets of the enzymatic function; Norager S et al.; Dihydroorotate dehydrogenases (DHODs) are flavoenzymes catalyzing the oxidation of (S)-dihydroorotate to orotate in the biosynthesis of UMP, the precursor of all other pyrimidine nucleotides . On the basis of sequence, DHODs can be divided into two classes, class 1, further divided in subclasses 1A and 1B, and class 2 . This division corresponds to differences in cellular location and the nature of the electron acceptor . Herein we report a study of Lactococcus lactis DHODA, a representative of the class 1A enzymes . Based on the DHODA structure we selected seven residues that are highly conserved between both main classes of DHODs as well as three residues representing surface charges close to the active site for site-directed mutagenesis . The availability of both kinetic and structural data on the mutant enzymes allowed us to define the roles individual structural segments play in catalysis . We have also structurally proven the presence of an open active site loop in DHODA and obtained information about the interactions that control movements of loops around the active site . Furthermore, in one mutant structure we observed differences between the two monomers of the dimer, confirming an apparent asymmetry between the two substrate binding sites that was indicated by the kinetic results.

Appl Environ Microbiol, 2003 May, 69(5), 2638 - 50
Genetic organization and molecular analysis of the EcoVIII restriction-modification system of Escherichia coli E1585-68 and its comparison with isospecific homologs; Mruk I et al.; The EcoVIII restriction-modification (R-M) system is carried by the Escherichia coli E1585-68 natural plasmid pEC156 (4,312 bp) . The two genes were cloned and characterized . The G+C content of the EcoVIII R-M system is 36.1%, which is significantly lower than the average G+C content of either plasmid pEC156 (43.6%) or E . coli genomic DNA (50.8%) . The difference suggests that there is a possibility that the EcoVIII R-M system was recently acquired by the genome . The 921-bp EcoVIII endonuclease (R . EcoVIII) gene (ecoVIIIR) encodes a 307-amino-acid protein with an M(r) of 35,554 . The convergently oriented EcoVIII methyltransferase (M . EcoVIII) gene (ecoVIIIM) consists of 912 bp that code for a 304-amino-acid protein with an M(r) of 33,930 . The exact positions of the start codon AUG were determined by protein microsequencing . Both enzymes recognize the specific palindromic sequence 5'-AAGCTT-3' . Preparations of EcoVIII R-M enzymes purified to homogeneity were characterized . R . EcoVIII acts as a dimer and cleaves a specific sequence between two adenine residues, leaving 4-nucleotide 5' protruding ends . M . EcoVIII functions as a monomer and modifies the first adenine residue at the 5' end of the specific sequence to N(6)-methyladenine . These enzymes are thus functionally identical to the corresponding enzymes of the HindIII (Haemophilus influenzae Rd) and LlaCI (Lactococcus lactis subsp . cremoris W15) R-M systems . This finding is reflected by the levels of homology of M . EcoVIII with M . HindIII and M . LlaCI at the amino acid sequence level (50 and 62%, respectively) and by the presence of nine sequence motifs conserved among m(6) N-adenine beta-class methyltransferases . The deduced amino acid sequence of R . EcoVIII shows weak homology with its two isoschizomers, R . HindIII (26%) and R . LlaCI (17%) . A catalytic sequence motif characteristic of restriction endonucleases was found in the primary structure of R . EcoVIII (D(108)X(12)DXK(123)), as well as in the primary structures of R . LlaCI and R . HindIII . Polyclonal antibodies raised against R . EcoVIII did not react with R . HindIII, while anti-M . EcoVIII antibodies cross-reacted with M . LlaCI but not with M . HindIII . R . EcoVIII requires Mg(II) ions for phosphodiester bond cleavage . We found that the same ions are strong inhibitors of the M . EcoVIII enzyme . The biological implications of this finding are discussed.

Appl Environ Microbiol, 2003 May, 69(5), 2512 - 20
The ftsH gene of the wine bacterium Oenococcus oeni is involved in protection against environmental stress; Bourdineaud JP et al.; The wine bacterium Oenococcus oeni has to cope with harsh environmental conditions, including an acidic pH, a high alcoholic content, nonoptimal growth temperatures, and growth-inhibitory compounds such as fatty acids, phenolic acids, and tannins . We describe the characterization and cloning of the O . oeni ftsH gene, encoding a protease belonging to the ATP binding cassette protein superfamily . The O . oeni FtsH protein is closest in sequence similarity to the FtsH homologue of Lactococcus lactis . The O . oeni ftsH gene proved to be stress-responsive, since its expression increased at high temperatures or under osmotic shock . O . oeni FtsH protein function was tested in an Escherichia coli ftsH mutant strain, and consistent with the O . oeni ftsH gene expression pattern, the O . oeni FtsH protein provided protection for the E . coli ftsH mutant against heat shock . O . oeni and Bradyrhizobium japonicum FtsH proteins also triggered E . coli resistance to wine toxicity . Genes homologous to O . oeni ftsH were detected in many other lactic acid bacteria found in wine, suggesting that this type of gene constitutes a well-conserved stress-protective molecular device.

Curr Opin Biotechnol, 2003 Apr, 14(2), 232 - 7
Metabolic pathway engineering in lactic acid bacteria; Kleerebezem M et al.; Lactic acid bacteria (LAB) display a relatively simple carbon and energy metabolism where the sugar source is converted mainly to lactic acid . In Lactococcus lactis metabolic engineering has been very successful in the re-routing of lactococcal pyruvate metabolism to products other than lactic acid . Current metabolic engineering approaches tend to focus on more complex, biosynthetic pathways leading to end-products that generate a health benefit for the consumer (nutraceuticals) . Several examples of research on these minor pathways in L . lactis have illustrated the potential of LAB as producers of these metabolites . Whole genome sequencing efforts and corresponding global technologies will have an impact on metabolic engineering in the future.

Plasmid, 2003 Mar, 49(2), 130 - 42
Molecular organization of exopolysaccharide (EPS) encoding genes on the lactococcal bacteriophage adsorption blocking plasmid, pCI658; Forde A et al.; The lactococcal plasmid pCI658 (58 kb) isolated from Lactococcus lactis ssp . cremoris HO2 encodes the production of a hydrophilic exopolysaccharide (EPS) which consists primarily of galactose and glucuronic acid and which interferes with adsorption of phages o712 and oc2 to cell surface receptors . Examination of the nucleotide sequence of a 21.8-kb region of the plasmid revealed a large genetic cluster consisting of at least 23 putative EPS biosynthetic determinants in addition to the presence of insertion sequences at the 5(') and 3(') ends . According to homology searches, the genes were organized in specific regions involved in regulation, synthesis and export of the EPS . The predicted products of individual genes exhibited significant homology to exopolysaccharide, capsular polysaccharide (CPS), and lipopolysaccharide (LPS) gene products from a variety of Gram positive and Gram negative bacteria . Evidence of a gene encoding UDP-glucose dehydrogenase is also presented and this is the first description of such a gene in Lactococcus.

Virology, 2003 Apr 25, 309(1), 10 - 7
Identification of the host determinant of two prolate-headed phages infecting Lactococcus lactis; Stuer-Lauridsen B et al.; A gene responsible for host determination was identified in two prolate-headed bacteriophages of the c2 species infecting strains of Lactococcus lactis . The identification of the host determinant gene was based on low DNA sequence homology in a specific open reading frame (ORF) between prolate-headed phages with different host ranges . When a host carrying this ORF from one phage on a plasmid was infected with another phage, we obtained phages with an altered host range at a frequency of 10(-6) to 10(-7) . Sequencing of phage DNA originating from 10 independent single plaques confirmed that a genetic recombination had taken place at different positions between the ORF on the plasmid and the infecting phage . The adsorption of the recombinant phages to their bacterial hosts had also changed to match the phage origin of the ORF . Consequently, it is concluded that this ORF codes for the host range determinant.

Protein Sci, 2003 May, 12(5), 1051 - 9
The catalytic mechanism of galactose mutarotase; Thoden JB et al.; Galactose mutarotase catalyzes the first step in normal galactose metabolism by catalyzing the conversion of beta-D-galactose to alpha-D-galactose . The structure of the enzyme from Lactococcus lactis was recently solved in this laboratory and shown to be topologically similar to domain 5 of beta-galactosidase . From this initial X-ray analysis, four amino acid residues were demonstrated to be intimately involved in sugar binding to the protein: His 96, His 170, Asp 243, and Glu 304 . Here we present a combined X-ray crystallographic and kinetic analysis designed to examine the role of these residues in the reaction mechanism of the enzyme . For this investigation, the following site-directed mutant proteins were prepared: H96N, H170N, D243N, D243A, E304Q, and E304A . All of the structures of these proteins, complexed with either glucose or galactose, were solved to a nominal resolution of 1.95 A or better, and their kinetic parameters were measured against D-galactose, D-glucose, L-arabinose, or D-xylose . From these studies, it can be concluded that Glu 304 and His 170 are critical for catalysis and that His 96 and Asp 243 are important for proper substrate positioning within the active site . Specifically, Glu 304 serves as the active site base to initiate the reaction by removing the proton from the C-1 hydroxyl group of the sugar substrate and His 170 functions as the active site acid to protonate the C-5 ring oxygen.

Poult Sci, 2003 Apr, 82(4), 640 - 7
Alternatives to antibiotics: bacteriocins, antimicrobial peptides and bacteriophages; Joerger RD; Bacteriocins, antimicrobial peptides, and bacteriophage have attracted attention as potential substitutes for, or as additions to, currently used antimicrobial compounds . This publication will review research on the potential application of these alternative antimicrobial agents to poultry production and processing . Bacteriocins are proteinaceous compounds of bacterial origin that are lethal to bacteria other than the producing strain . It is assumed that some of the bacteria in the intestinal tract produce bacteriocins as a means to achieve a competitive advantage, and bacteriocin-producing bacteria might be a desirable part of competitive exclusion preparations . Purified or partially purified bacteriocins could be used as preservatives or for the reduction or elimination of certain pathogens . Currently only nisin, produced by certain strains of Lactococcus lactis subsp . lactis, has regulatory approval for use in certain foods, and its use for poultry products has been studied extensively . Exploration of the application of antimicrobial peptides from sources other than bacteria to poultry has not yet commenced to a significant extent . Evidence for the ability of chickens to produce such antimicrobial peptides has been provided, and it is likely that these peptides play an important role in the defense against various pathogens . Bacteriophages have received renewed attention as possible agents against infecting bacteria . Evidence from several trials indicates that phage therapy can be effective under certain circumstances . Numerous obstacles for the use of phage as antimicrobials for poultry or poultry products remain . Chiefly among them are the narrow host range of many phages, the issue of phage resistance, and the possibility of phage-mediated transfer of genetic material to bacterial hosts . Regulatory issues and the high cost of producing such alternative antimicrobial agents are also factors that might prevent application of these agents in the near future.

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