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 . Fur