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PubMed1998

Cloning and characterization of the lactococcal plasmid-encoded type II restriction/modification system, LlaDII.

The LlaDII restriction/modification (R/M) system was found on the naturally occurring 8.9-kb plasmid pHW393 in Lactococcus lactis subsp. cremoris W39. A 2.4-kb PstI-EcoRI fragment inserted into the Escherichia coli-L. lactis shuttle vector pCI3340 conferred to L. lactis LM2301 and L. lactis SMQ86 resistance against representatives of the three most common lactococcal phage species: 936, P335, and c2. The LlaDII endonuclease was partially purified and found to recognize and cleave the sequence 5'-GC decreases NGC-3', where the arrow indicates the cleavage site. It is thus an isoschizomer of the commercially available restriction endonuclease Fnu4HI. Sequencing of the 2.4-kb PstI-EcoRI fragment revealed two open reading frames arranged tandemly and separated by a 105-bp intergenic region. The endonuclease gene of 543 bp preceded the methylase gene of 954 bp. The deduced amino acid sequence of the LlaDII R/M system showed high homology to that of its only sequenced isoschizomer, Bsp6I from Bacillus sp. strain RFL6, with 41% identity between the endonucleases and 60% identity between the methylases. The genetic organizations of the LlaDII and Bsp6I R/M systems are identical. Both methylases have two recognition sites (5'-GCGGC-3' and 5'-GCCGC-3') forming a putative stemloop structure spanning part of the presumed -35 sequence and part of the intervening region between the -35 and -10 sequences. Alignment of the LlaDII and Bsp6I methylases with other m5C methylases showed that the protein primary structures possessed the same organization.

Amino Acid Sequence↗
PubMed1991

Molecular organization of the minimal replicon of novel, narrow-host-range, lactococcal plasmid pCI305.

Plasmid pCI305 is an 8.7-kb, narrow-host-range, cryptic plasmid originating from Lactococcus lactis subsp. lactis UC317. The nucleotide sequence of the pCI305 replication region was determined. A single open reading frame of 1158 bp was identified in the trans-active domain repB. The size of the predicted repB protein (46 kDa) is in close agreement with the size of the repB product visualized in vivo in Escherichia coli when repB was placed under control of the inducible phi T7 RNA polymerase promoter. In vivo substitution of the native repB promoter sequence with a Tn5-derived promoter sequence was demonstrated. repA, a 344-bp cis-acting region which is the probable pCI305 replication origin region, was noncoding, was AT-rich, and possessed a unique set of inverted and direct repeat sequences. No significant homology between repA or repB and other gram-positive replication regions was evident. Combined with the absence of a detectable single-stranded DNA intermediate during replication, these results indicate that the pCI305 replication region differs markedly from most gram-positive replicons examined to date. The presence on other lactococcal plasmids of replication regions related to that of pCI305 was demonstrated.

Amino Acid Sequence↗
PubMed2000

Cloning and expression of the genes involved in the production of and immunity against the bacteriocin lacticin RM.

The production of lacticin RM, a novel bacteriocin produced by Lactococcus lactis subsp. lactis EZ26, is associated with the presence of a 6-kb plasmid, pHU1. The information necessary for lacticin RM production and immunity was localized to a 2.5-kb SalI-Eco47III fragment. Sequencing analysis of this fragment revealed the presence of six open reading frames (ORFs). Deletion and mutation analyses showed that orfX and orfY are not required for lacticin RM production or immunity, whereas the other ORFs (lacA, lacF, lacG and lacI) are necessary for the bacteriocin's production. Transcription analysis indicated that lacA, lacF and lacG are organized in an operon. lacA is probably the lacticin RM structural gene. It putatively encodes a 134-amino acid peptide, and it does not share homology with known bacteriocins. The deduced LacG protein is hydrophobic and consists of six potential trans-membrane helices. lacF encodes a conserved ATP-binding domain homologous to ABC transporters known in bacteriocin immunity systems. LacF and LacG may form an active ABC transporter. Gene-disruption mutations indicated that both are required for immunity against lacticin RM. lacI encodes a small cationic protein, which is required for the production of and immunity to lacticin RM. Protection was obtained only when lacF, lacG and lacI were present together.

ATP-Binding Cassette Transporters↗
PubMed1997

Proteolytic enzyme activity in lactococci grown in different pretreated milk media.

Lactococcus lactis ssp. lactis MG1363, harbouring plasmid pNZ521, which encodes the extracellular serine proteinase (PrtP) from strain SK110, was used to study the effect of two different treatments of the growth medium milk on the activity levels of PrtP and the intracellular localized aminopeptidase PepN and X-prolyl-dipeptidyl aminopeptidase PepXP. All three proteolytic enzymes showed lower activity levels in cells grown in high heat-treated milk as compared to cells grown in non-heat-treated milk. Highest activity levels of the three studied enzymes were found in cells grown in milk heat treated for 30 min at 63 degrees C. Using cells of strain L. lactis ssp. lactis MG1363, harbouring plasmid pNZ544, which encodes reporter gene gusA under control of the prtP promoter, it was demonstrated that the regulation of PrtP takes place at the transcription initiation level. After separation of the pH 4.6 soluble fraction of high heat-treated milk with reverse phase HPLC, it was found that the hydrophilic small peptide fraction of the milk was responsible for this regulation. Amino acid analysis of this fraction confirmed that this fraction consisted of peptides only. Ultrafiltration of milk, which increases the dry matter of the milk specifically through increase of its protein content, only significantly affected the levels of PrtP and PepN in cells of strain MG1363. Highest activity was found during growth in unconcentrated milk, and the lowest level was found during growth in four times concentrated retentate. Using cells of strain MG1363(pNZ544), it was demonstrated that also in this case regulation of PrtP takes place at the level of transcription initiation. Approximately 40% of the decrease in activity of PrtP and PepN could be explained by the presence of higher amounts of purified whey proteins and higher amounts of dry mass. This suggests the presence of another factor, concentrated by ultrafiltration, which controls the production different proteolytic enzymes in concentrated retentate.

Aminopeptidases↗
PubMed2003

Purification and genetic characterization of plantaricin NC8, a novel coculture-inducible two-peptide bacteriocin from Lactobacillus plantarum NC8.

A new, coculture-inducible two-peptide bacteriocin named plantaricin NC8 (PLNC8) was isolated from Lactobacillus plantarum NC8 cultures which had been induced with Lactococcus lactis MG1363 or Pediococcus pentosaceus FBB63. This bacteriocin consists of two distinct peptides, named alpha and beta, which were separated by C(2)-C(18) reverse-phase chromatography and whose complementary action is necessary for full plantaricin NC8 activity. N-terminal sequencing of both purified peptides showed 28 and 34 amino acids residues for PLNC8 alpha and PLNC8 beta, respectively, which showed no sequence similarity to other known bacteriocins. Mass spectrometry analysis showed molecular masses of 3,587 Da (alpha) and 4,000 Da (beta). The corresponding genes, designated plNC8A and plNC8B, were sequenced, and their nucleotide sequences revealed that both peptides are produced as bacteriocin precursors of 47 and 55 amino acids, respectively, which include N-terminal leader sequences of the double-glycine type. The mature alpha and beta peptides contain 29 and 34 amino acids, respectively. An open reading frame, orfC, which encodes a putative immunity protein was found downstream of plNC8B and overlapping plNC8A. Upstream of the putative -35 region of plNC8B, two direct repeats of 9 bp were identified, which agrees with the consensus sequence and structure of promoters of class II bacteriocin operons whose expression is dependent on an autoinduction mechanism.

Amino Acid Sequence↗
PubMed2006

Nonstarter lactic acid bacteria biofilms and calcium lactate crystals in Cheddar cheese.

A sanitized cheese plant was swabbed for the presence of nonstarter lactic acid bacteria (NSLAB) biofilms. Swabs were analyzed to determine the sources and microorganisms responsible for contamination. In pilot plant experiments, cheese vats filled with standard cheese milk (lactose:protein = 1.47) and ultrafiltered cheese milk (lactose:protein = 1.23) were inoculated with Lactococcus lactis ssp. cremoris starter culture (8 log cfu/mL) with or without Lactobacillus curvatus or Pediococci acidilactici as adjunct cultures (2 log cfu/mL). Cheddar cheeses were aged at 7.2 or 10 degrees C for 168 d. The raw milk silo, ultrafiltration unit, cheddaring belt, and cheese tower had NSLAB biofilms ranging from 2 to 4 log cfu/100 cm2. The population of Lb. curvatus reached 8 log cfu/g, whereas P. acidilactici reached 7 log cfu/g of experimental Cheddar cheese in 14 d. Higher NSLAB counts were observed in the first 14 d of aging in cheese stored at 10 degrees C compared with that stored at 7.2 degrees C. However, microbial counts decreased more quickly in Cheddar cheeses aged at 10 degrees C compared with 7.2 degrees C after 28 d. In cheeses without specific adjunct cultures (Lb. curvatus or P. acidilactici), calcium lactate crystals were not observed within 168 d. However, crystals were observed after only 56 d in cheeses containing Lb. curvatus, which also had increased concentration of D(-)-lactic acid compared with control cheeses. Our research shows that low levels of contamination with certain NSLAB can result in calcium lactate crystals, regardless of lactose:protein ratio.

Animals↗
PubMed2006

Importance of bacterial surface properties to control the stability of emulsions.

In colloidal media such as emulsions or food matrixes, the stability results from physicochemical interactions. The same type of interaction is involved in the attachment processes of microorganisms, through their surface properties, to interfaces. When bacteria are present in a food matrix, it is probable that their surface interacts with the other constituents. In this paper, the involvement of bacterial surface properties of Lactococcus lactis subsp lactis biovar diacetylactis (LLD) on the stability of model emulsions has been studied. The hydrophobic and electrostatic cell-surface properties were characterized by the MATH method and by microelectrophoresis, respectively. The oil-in-water emulsions were stabilized by various surface-active compounds, CTAB, SDS or Tween 20, giving differently charged droplets. Two strains with different surface characteristics were added to the emulsion. Contrasting with emulsions made with the non-ionic surfactant, for which the stability was not modified by the addition of bacteria, the emulsions made with ionic surface-active compounds were unstable in the presence of bacteria when the bacterial surface charge was opposite to the one of the emulsion droplets. Moreover, aggregation and flocculation phenomena were observed for emulsions stabilized with the cationic surfactant, particularly for more negatively charged bacteria. The effect of bacteria on the emulsion stability depended on the strain which shows the importance of the choice of the microorganism according to of the characteristics of the colloidal media to obtain a stable system. In addition, these results suggest that the interactions between bacteria and other food components can influence the position of bacteria in food matrixes.

Bacterial Adhesion↗
PubMed2006

Single-molecule kinetics reveals signatures of half-sites reactivity in dihydroorotate dehydrogenase A catalysis.

Subunit activity and cooperativity of a homodimeric flavoenzyme, dihydroorotate dehydrogenase A (DHODA) from Lactococcus lactis, were characterized by employing single-molecule spectroscopy to follow the turnover kinetics of individual DHODA molecules, eliminating ensemble averaging. Because the enzyme-bound FMN is fluorescent in its oxidized state but not when reduced, a single DHODA molecule exhibits stepwise fluorescence changes during turnover, providing a signal to determine reaction kinetics and study cooperativity. Our results showed significant heterogeneity in the catalytic behaviors of individual dimer molecules, with only 40% interconverting between the three possible redox states: the fully fluorescent (both subunits oxidized), the half-fluorescent (one subunit oxidized and the other reduced), and the nonfluorescent (both subunits reduced). Forty percent of the single dimer traces showed turnovers between only the fully fluorescent and half-fluorescent states. The remaining 20% of the molecules interconverted only between the half-fluorescent state and the nonfluorescent state. Kinetic analysis revealed very similar reaction rates in both the reductive and oxidative half-reactions for different DHODA dimers. Our single-molecule data provide strong evidence for half-sites reactivity, in which only one subunit reacts at a time. The present study presents an effective way to explore the subunit catalytic activity and cooperativity of oligomeric enzymes by virtue of single-molecule fluorescence.

Bacterial Proteins↗
PubMed2005

Gene targeting using randomly inserted group II introns (targetrons) recovered from an Escherichia coli gene disruption library.

The Lactococcus lactis Ll.LtrB group II intron retrohomes by reverse-splicing into one strand of a double-stranded DNA target site, while the intron-encoded protein cleaves the opposite strand and uses it to prime reverse transcription of the inserted intron RNA. The protein and intron RNA function in a ribonucleoprotein particle, with much of the DNA target sequence recognized by base-pairing of the intron RNA. Consequently, group II introns can be reprogrammed to insert into specific or random DNA sites by substituting specific or random nucleotide residues in the intron RNA. Here, we show that an Escherichia coli gene disruption library obtained using such randomly inserting Ll.LtrB introns contains most viable E.coli gene disruptions. Further, each inserted intron is targeted to a specific site by its unique base-pairing regions, and in most cases, could be recovered by PCR and used unmodified to obtain the desired single disruptant. Additionally, we identified a subset of introns that insert at sites lacking T+5, a nucleotide residue critical for second-strand cleavage. All such introns tested individually gave the desired specific disruption, some by switching to an alternate retrohoming mechanism targeting single-stranded DNA and using a nascent lagging DNA strand to prime reverse transcription.

Base Pairing↗
PubMed2003

Molecular structure of galactokinase.

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

Adenosine Triphosphate↗
PubMed2006

Nisin induction without nisin secretion.

Nisin Z, a post-translationally modified antimicrobial peptide of Lactococcus lactis, is positively autoregulated by extracellular nisin via the two-component regulatory proteins NisRK. A mutation in the nisin NisT transporter rendered L. lactis incapable of nisin secretion, and nisin accumulated inside the cells. Normally nisin is activated after secretion by the serine protease NisP in the cell wall. This study showed that when secretion of nisin was blocked, intracellular proteolytic activity could cleave the N-terminal leader peptide of nisin precursor, resulting in active nisin. The isolated cytoplasm of a non-nisin producer could also cleave the leader from the nisin precursor, showing that the cytoplasm of L. lactis cells does contain proteolytic activity capable of cleaving the leader from fully modified nisin precursor. Nisin could not be detected in the growth supernatant of the NisT mutant strain with a nisin-sensing strain (sensitivity 10 pg ml(-1)), which has a green fluorescent protein gene connected to the nisin-inducible nisA promoter and a functional nisin signal transduction circuit. Northern analysis of the NisT mutant cells revealed that even though the cells could not secrete nisin, the nisin-inducible promoter P(nisZ) was active. In a nisB or nisC background, where nisin could not be fully modified due to the mutations in the nisin modification machinery, the unmodified or partly modified nisin precursor accumulated in the cytoplasm. This immature nisin could not induce the P(nisZ) promoter. The results suggest that when active nisin is accumulated in the cytoplasm, it can insert into the membrane and from there extrude parts of the molecule into the pseudoperiplasmic space to interact with the signal-recognition domain of the histidine kinase NisK. Potentially, signal presentation via the membrane represents a general pathway for amphiphilic signals to interact with their sensors for signal transduction.

Artificial Gene Fusion↗
PubMed1993

High-efficiency gene inactivation and replacement system for gram-positive bacteria.

A system for high-efficiency single- and double-crossover homologous integration in gram-positive bacteria has been developed, with Lactococcus lactis as a model system. The system is based on a thermosensitive broad-host-range rolling-circle plasmid, pG+host5, which contains a pBR322 replicon for propagation in Escherichia coli at 37 degrees C. A nested set of L. lactis chromosomal fragments cloned onto pG+host5 were used to show that the single-crossover integration frequency was logarithmically proportional to the length of homology for DNA fragments between 0.35 and 2.5 kb. Using random chromosomal 1-kb fragments, we showed that homologous integration can occur along the entire chromosome. We made use of the reported stimulatory effect of rolling-circle replication on intramolecular recombination to develop a protocol for gene replacement. Cultures were first maintained at 37 degrees C to select for a bacterial population enriched for plasmid integrants; activation of the integrated rolling-circle plasmid by a temperature shift to 28 degrees C resulted in efficient plasmid excision by homologous recombination and replacement of a chromosomal gene by the plasmid-carried modified copy. More than 50% of cells underwent replacement recombination when selection was applied for the replacing gene. Between 1 and 40% of cells underwent replacement recombination when no selection was applied. Chromosomal insertions and deletions were obtained in this way. These results show that gene replacement can be obtained at an extremely high efficiency by making use of the thermosensitive rolling-circle nature of the delivery vector. This procedure is applicable to numerous gram-positive bacteria.

DNA Replication↗
PubMed2005

Structural characterization and assembly of the distal tail structure of the temperate lactococcal bacteriophage TP901-1.

The tail structures of bacteriophages infecting gram-positive bacteria are largely unexplored, although the phage tail mediates the initial interaction with the host cell. The temperate Lactococcus lactis phage TP901-1 of the Siphoviridae family has a long noncontractile tail with a distal baseplate. In the present study, we investigated the distal tail structures and tail assembly of phage TP901-1 by introducing nonsense mutations into the late transcribed genes dit (orf46), tal(TP901-1) (orf47), bppU (orf48), bppL (orf49), and orf50. Transmission electron microscopy examination of mutant and wild-type TP901-1 phages showed that the baseplate consisted of two different disks and that a central tail fiber is protruding below the baseplate. Evaluation of the mutant tail morphologies with protein profiles and Western blots revealed that the upper and lower baseplate disks consist of the proteins BppU and BppL, respectively. Likewise, Dit and Tal(TP901-1) were shown to be structural tail proteins essential for tail formation, and Tal(TP901-1) was furthermore identified as the tail fiber protein by immunogold labeling experiments. Determination of infection efficiencies of the mutant phages showed that the baseplate is fundamental for host infection and the lower disk protein, BppL, is suggested to interact with the host receptor. In contrast, ORF50 was found to be nonessential for tail assembly and host infection. A model for TP901-1 tail assembly, in which the function of eight specific proteins is considered, is presented.

Genes, Viral↗
PubMed1999

Restrictive use of detergents in the functional reconstitution of the secondary multidrug transporter LmrP.

The histidine-tagged secondary multidrug transporter LmrP was overexpressed in Lactococcus lactis, using a novel protein expression system for cytotoxic proteins based on the tightly regulated, nisin-inducible nisA promoter. LmrP-mediated H+/drug antiport activity in inside-out membrane vesicles was inhibited by detergents, such as Triton X-100, Triton X-114, and Tween 80, at low concentrations that did not affect the magnitude or composition of the proton motive force. The inhibition of the activity of LmrP by detergents restricted the range of compounds that could be used for the solubilization and reconstitution of the protein because low concentrations of detergent are retained in proteoliposomes. Surprisingly, dodecyl maltoside did not modulate the activity of LmrP. Therefore, LmrP was solubilized with dodecyl maltoside, purified by nickel-chelate affinity chromatography, and reconstituted in dodecyl maltoside-destabilized, preformed liposomes prepared from Escherichia coli phospholipids and egg phosphatidylcholine. Reconstituted LmrP mediated the transport of multiple drugs in response to an artificially imposed pH gradient, demonstrating that the protein functions as a proton motive force-dependent multidrug transporter, independent of accessory proteins. These observations are relevant for the effective solubilization and reconstitution of multidrug transporters belonging to the major facilitator superfamily, which, in view of their broad drug specificity, may strongly interact with detergents.

Bacterial Proteins↗
PubMed2001

Resolvase-like recombination performed by the TP901-1 integrase.

The site-specific recombination system of temperate lactococcal bacteriophage TP901-1 is unusual in several respects. First, the integrase belongs to the family of extended resolvases rather than to the lambda integrase family and second, in the presence of this integrase, a 56 bp attP fragment is sufficient for efficient recombination with the chromosomal attB site in the host Lactococcus lactis subsp. cremoris MG1363. In the present work, this attB site was analysed and a 43 bp attB region was found to be the smallest fragment able to participate fully in recombination. In vitro studies showed that the TP901-1 integrase binds this 43 bp attB fragment, the 56 bp attP and a larger attP fragment with equal affinity. Mutational analysis of the 5 bp common core region (TCAAT) showed that the TC dinucleotide is essential for recombination, but not for binding of the integrase, whereas none of the last three bases are important for recombination. When a number of attL sites, obtained by recombination between an attB site containing a mutation in this TC dinucleotide and a wild-type attP site, were sequenced, a mix of sites with the wild-type or the mutated sequence was obtained. These results are consistent with the hypothesis that the TC dinucleotide constitutes the TP901-1 overlap region. A 2 bp overlap region has been observed in recombination reactions catalysed by all other members of the resolvase/invertase family tested so far. By selecting for attB sites with a decreased ability to participate in recombination, two bases located outside the core region of attB were shown to be involved in the in vitro binding of the TP901-1 integrase.

Attachment Sites, Microbiological↗
PubMed2001

Regulation of immunity to the two-component lantibiotic, lacticin 3147, by the transcriptional repressor LtnR.

Lacticin 3147 is a membrane-active, two-component lantibiotic produced by Lactococcus lactis ssp. lactis DPC3147. In this study, the promoters of the lacticin 3147 gene cluster were mapped to the intergenic region between ltnR and ltnA1 (the genes encoding the regulatory protein LtnR and the first structural gene, LtnA1), and Northern analyses revealed that the biosynthetic and immunity genes are divergently transcribed in two operons, ltnA1A2M1TM2D and ltnRIFE respectively. Although the promoter controlling biosynthesis (Pbac) appears to be constitutive, characterization of a downstream beta-galactosidase (beta-gal) fusion beyond an intragenic stem-loop structure in ltnM1 confirmed that this putative transcriptional attenuator allows limited readthrough to the downstream biosynthetic genes, thus maintaining the correct stoichiometry between structural peptides and biosynthetic machinery. The promoter of the ltnRIFE operon (Pimm) was shown to be regulated by the transcriptional repressor LtnR. A mutant with a truncated ltnR gene exhibited a hyperimmune phenotype, whereas overexpression of ltnR resulted in cells with increased sensitivity to lacticin 3147. Gel mobility shift analysis indicated that LtnR binds to the Pimm promoter region, and fusion of this promoter to the beta-gal gene of pAK80 revealed that expression from Pimm is significantly reduced in the presence of LtnR. Thus, we have demonstrated that lacticin 3147 uses a regulatory mechanism not previously identified in lantibiotic systems.

Anti-Bacterial Agents↗
PubMed2006

Fibronectin-binding proteins of Staphylococcus aureus mediate activation of human platelets via fibrinogen and fibronectin bridges to integrin GPIIb/IIIa and IgG binding to the FcgammaRIIa receptor.

Staphylococcus aureus is a leading cause of infective endocarditis (IE). Platelet activation promoted by S. aureus resulting in aggregation and thrombus formation is an important step in the pathogenesis of IE. Here, we report that the fibrinogen/fibronectin-binding proteins FnBPA and FnBPB are major platelet-activating factors on the surface of S. aureus from the exponential phase of growth. Truncated derivatives of FnBPA, presenting either the fibrinogen-binding A domain or the fibronectin-binding BCD region, each promoted platelet activation when expressed on the surface of S. aureus or Lactococcus lactis, indicating two distinct mechanisms of activation. FnBPA-promoted platelet activation is mediated by fibrinogen and fibronectin bridges between the A domain and the BCD domains, respectively, to the low affinity form of the integrin GPIIb/IIIa on resting platelets. Antibodies recognizing the FnBPA A domain or the complex between the FnBPA BCD domains and fibronectin were essential for activation promoted by bacteria expressing the A domain or the BCD domain respectively. Activation was inhibited by a monoclonal antibody (IV-3) specific for the FcgammaRIIa IgG receptor on platelets. We propose that the activation of quiescent platelets by bacteria expressing FnBPs involves the formation of a bridge between the bacterial cell and the platelet surface by (i) fibronectin and fibrinogen interacting with the low affinity form of GPIIb/IIIa and (ii) by antibodies specific to FnBPs that engage the platelet Fc receptor FcgammaRIIa. Platelet activation by S. aureus clinical IE isolates from both the exponential and stationary phases of growth was completely inhibited by monoclonal antibody IV-3 suggesting that the IgG-FcgammaRIIa interaction is of fundamental importance for platelet activation mediated by this organism. This suggests new avenues for development of therapeutics against vascular infections.

Adhesins, Bacterial↗
PubMed2005

Application of exopolysaccharide-producing cultures in reduced-fat Cheddar cheese: texture and melting properties.

Textural, melting, and sensory characteristics of reduced-fat Cheddar cheeses made with exopolysaccharide (EPS)-producing and nonproducing cultures were monitored during ripening. Hardness, gumminess, springiness, and chewiness significantly increased in the cheeses as fat content decreased. Cheese made with EPS-producing cultures was the least affected by fat reduction. No differences in hardness, springiness, and chewiness were found between young reduced fat cheese made with a ropy Lactococcus lactis ssp. cremoris [JFR1; the culture that produced reduced-fat cheese with moisture in the nonfat substance (MNFS) similar to that in its full-fat counterpart] and its full-fat counterpart. Whereas hardness of full-fat cheese and reduced-fat cheese made with JFR1 increased during ripening, a significant decrease in its value was observed in all other cheeses. After 6 mo of ripening, reduced fat cheeses made with all EPS-producing cultures maintained lower values of all texture profile analysis parameters than did those made with no EPS. Fat reduction decreased cheese meltability. However, no differences in meltability were found between the young full-fat cheese and the reduced-fat cheese made with the ropy culture JFR1. Both the aged full- and reduced-fat cheeses made with JFR1 had similar melting patterns. When heated, they both became soft and creamy without losing shape, whereas reduced-fat cheese made with no EPS ran and separated into greasy solids and liquid. No differences were detected by panelists between the textures of the full-fat cheese and reduced-fat cheese made with JFR1, both of which were less rubbery or firm, curdy, and crumbly than all other reduced-fat cheeses.

Cheese↗

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