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PubMed2004

A nudix enzyme removes pyrophosphate from dihydroneopterin triphosphate in the folate synthesis pathway of bacteria and plants.

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. Because 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 (approximately 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 reported previously to have NADH pyrophosphatase activity in the presence of 5 mM Mn(2+) (Dobrzanska, M., Szurmak, B., Wyslouch-Cieszynska, A., and Kraszewska, E. (2002) J. Biol. Chem. 277, 50482-50486). 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.

Amino Acid Sequence↗
PubMed2006

Ion specificity and ionic strength dependence of the osmoregulatory ABC transporter OpuA.

The ATPase subunit of the osmoregulatory ATP-binding cassette transporter OpuA from Lactococcus lactis has a C-terminal extension, the tandem cystathionine beta-synthase (CBS) domain, which constitutes the sensor that allows the transporter to sense and respond to osmotic stress (Biemans-Oldehinkel, E., Mahmood, N. A. B. N., and Poolman, B. (2006) Proc. Natl. Acad. Sci. U. S. A. 103, 10624-10629). C-terminal of the tandem CBS domain is an 18-residue anionic tail (DIPDEDEVEEIEKEEENK). To investigate the ion specificity of the full transporter, we probed the activity of inside-out reconstituted wild-type OpuA and the anionic tail deletion mutant OpuADelta12; these molecules have the tandem CBS domains facing the external medium. At a mole fraction of 40% of anionic lipids in the membrane, the threshold ionic strength for activation of OpuA was approximately 0.15, irrespective of the electrolyte composition of the medium. At equivalent concentrations, bivalent cations (Mg(2+) and Ba(2+)) were more effective in activating OpuA than NH(4)(+), K(+), Na(+), or Li(+), consistent with an ionic strength-based sensing mechanism. Surprisingly, Rb(+) and Cs(+) were potent inhibitors of wild-type OpuA, and 0.1 mM RbCl was sufficient to completely inhibit the transporter even in the presence of 0.2 M KCl. Rb(+) and Cs(+) were no longer inhibitory in OpuADelta12, indicating that the anionic C-terminal tail participates in the formation of a binding site for large alkali metal ions. Compared with OpuADelta12, wild-type OpuA required substantially less potassium ions (the dominant ion under physiological conditions) for activation. Our data lend new support for the contention that the CBS module in OpuA constitutes the ionic strength sensor whose activity is modulated by the C-terminal anionic tail.

ATP-Binding Cassette Transporters↗
PubMed2001

Subtraction hybridization in microplates: an improved method to generate strain-specific PCR primers.

An improved subtraction hybridization technique was developed and evaluated. The hybridization is performed in a microplate with the subtractor-DNA immobilized in the plate while the probe-DNA is in solution. After hybridization the probe-specific DNA can easily be removed from the microwell and submitted to further analysis. This new technique has been successfully applied to generate several strain-specific PCR-primers for Lactococcus lactis subsp. lactis, Pediococcus spec., Saccharomyces spec. and Listeria monocytogenes.

DNA Primers↗
PubMed1997

The cross-linking by o-phthalaldehyde of two amino acid residues at the active site of 6-phosphogluconate dehydrogenase.

o-phthalaldehyde inactivates homodimeric, NADP+ dependent, 6-phosphogluconate dehydrogenase from sheep liver, upon formation of a single isoindole derivative per enzyme subunit. This indicates that the thiol group of a cysteine residue or the epsilon-amino group of a lysine residue located within 3 A and crosslinked by the reagent is essential for catalysis. Fluorescence analyses of the modified enzyme suggest that the isoindole derivative forms at the binding site of the nicotinamide moiety of NADP+. The enzymes from Trypanosoma brucei and Lactococcus lactis are also inactivated suggesting a similar three-dimensional structure in this domain. The isoindole derivative does not form with two mutants of the T. brucei enzyme (Lys185His and Lys185Leu), this allowing to identify not only the lysine but also the cysteine involved in the cross-linking. The formation of the isoindole derivative inactivates not only the oxidative decarboxylation, but also two partial reactions catalysed by the enzyme.

Animals↗
PubMed2005

Fibrinogen and fibronectin binding cooperate for valve infection and invasion in Staphylococcus aureus experimental endocarditis.

The expression of Staphylococcus aureus adhesins in Lactococcus lactis identified clumping factor A (ClfA) and fibronectin-binding protein A (FnBPA) as critical for valve colonization in rats with experimental endocarditis. This study further analyzed their role in disease evolution. Infected animals were followed for 3 d. ClfA-positive lactococci successfully colonized damaged valves, but were spontaneously eradicated over 48 h. In contrast, FnBPA-positive lactococci progressively increased bacterial titers in vegetations and spleens. At imaging, ClfA-positive lactococci were restricted to the vegetations, whereas FnBPA-positive lactococci also invaded the adjacent endothelium. This reflected the capacity of FnBPA to trigger cell internalization in vitro. Because FnBPA carries both fibrinogen- and fibronectin-binding domains, we tested the role of these functionalities by deleting the fibrinogen-binding domain of FnBPA and supplementing it with the fibrinogen-binding domain of ClfA in cis or in trans. Deletion of the fibrinogen-binding domain of FnBPA did not alter fibronectin binding and cell internalization in vitro. However, it totally abrogated valve infectivity in vivo. This ability was restored in cis by inserting the fibrinogen-binding domain of ClfA into truncated FnBPA, and in trans by coexpressing full-length ClfA and truncated FnBPA on two separate plasmids. Thus, fibrinogen and fibronectin binding could cooperate for S. aureus valve colonization and endothelial invasion in vivo.

Adhesins, Bacterial↗
PubMed2006

Human lysozyme expressed in the mammary gland of transgenic dairy goats can inhibit the growth of bacteria that cause mastitis and the cold-spoilage of milk.

The addition of human milk components with intrinsic antimicrobial activity to livestock milk by genetic engineering has the potential to benefit milk safety and production as well as the health of the lactating animal. As a model for the dairy cow, we generated transgenic goats that expressed human lysozyme in their milk at 68% of the levels found in human milk. Milk from these transgenic animals had a bacteriostatic effect on both in vitro and in vivo growth of several microorganisms important to the dairy industry. In vitro, milk from transgenic animals was capable of slowing the growth of mastitis-causing strains of Escherichia coli (P < 0.02) and Staphylococcus aureus (P < 0.05) as well as the cold-spoilage organism Pseudomonas fragi (P < 0.02). The growth of an organism involved in cheese-making, Lactococcus lactis, was not affected by the presence of lysozyme in milk. The supplementation of control milk with purified lysozyme did not achieve the same inhibitory effect as milk from transgenic animals. In vivo, milk from transgenic animals supported less bacterial growth than control milk. This transgenic model demonstrates the possibilities offered by genetic engineering to enhance the antimicrobial nature of milk and the udder.

Animals↗
PubMed2001

On the osmotic signal and osmosensing mechanism of an ABC transport system for glycine betaine.

The osmosensing mechanism of the ATP-binding cassette (ABC) transporter OpuA of Lactococcus lactis has been elucidated for the protein reconstituted in liposomes. Activation of OpuA by osmotic upshift was instantaneous and reversible and followed changes in volume and membrane structure of the proteoliposomes. Osmotic activation of OpuA was dependent on the fraction of anionic lipids present in the lipid bilayer. Also, cationic and anionic lipophilic amphiphiles shifted the activation profile in a manner indicative of an osmosensing mechanism, in which electrostatic interactions between lipid headgroups and the OpuA protein play a major role. Further support for this notion came from experiments in which ATP-driven uptake and substrate-dependent ATP hydrolysis were measured with varying concentrations of osmolytes at the cytoplasmic face of the protein. Under iso-osmotic conditions, the transporter could be activated by high concentrations of ionic osmolytes, whereas neutral ones had no effect, demonstrating that intracellular ionic strength, rather than a specific signaling molecule or water activity, signals osmotic stress to the transporter. The data indicate that OpuA is under the control of a mechanism in which the membrane and ionic strength act in concert to signal osmotic changes.

ATP-Binding Cassette Transporters↗
PubMed2003

Group II intron mobility using nascent strands at DNA replication forks to prime reverse transcription.

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

Bacterial Proteins↗
PubMed2003

On the role of the two extracytoplasmic substrate-binding domains in the ABC transporter OpuA.

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

ATP-Binding Cassette Transporters↗
PubMed1996

Augmented production of extracellular superoxide by blood isolates of Enterococcus faecalis.

To assess the frequency of extracellular superoxide (O-2) production by enterococci, multiple species were surveyed in a whole organism assay for their ability to reduce ferricytochrome c in a superoxide dismutase-inhibitable fashion. For stool and clinical enterococcal isolates and 12 type strains, only Enterococcus faecalis (87/91 isolates and ATCC 19433), Enterococcus faecium (5/13 isolates), Enterococcus casseliflavus (ATCC 25778), and Enterococcus gallinarum (ATCC 35038) produced extracellular O-2. Among 106 strains comprising 13 species of enteric gram-negative bacilli and gram-positive cocci, only Lactococcus lactis subspecies lactis produced extracellular O-2. Mean (+/-SE) rates of extracellular O-2 production in vitro by E . faecalis for isolates associated with bacteremia and endocarditis and for isolates from stool were 2.4+/-0.2, 1.9+/-0.2, 1.5+/-0.3 nmol of O-2 min(-1) 10(9) cfu(-1), respectively (P=.025, analysis of variance), suggesting an association between invasiveness and extracellular O-2 production.

Bacteremia↗
PubMed2006

Two families of rep-like genes that probably originated by interspecies recombination are represented in viral, plasmid, bacterial, and parasitic protozoan genomes.

Two families of genes related to, and including, rolling circle replication initiator protein (Rep) genes were defined by sequence similarity and by evidence of intergene family recombination. The Rep genes of circoviruses were the best characterized members of the "RecRep1 family." Other members of the RecRep1 family were Rep-like genes found in the genomes of the Canarypox virus, Entamoeba histolytica, and Giardia duodenalis and in a plasmid, p4M, from the Gram-positive bacterium, Bifidobacterium pseudocatenulatum. The "RecRep2 family" comprised some previously identified Rep-like genes from plasmids of phytoplasmas and similar Rep-like genes from the genomes of Lactobacillus acidophilus, Lactococcus lactis, and Phytoplasma asteris. Both RecRep1 and RecRep2 proteins have a nucleotide-binding domain significantly similar to the helicases (2C proteins) of picorna-like viruses. On the N-terminal side of the nucleotide binding domain, RecRep1 proteins have a domain significantly similar to one found in nanovirus Reps, whereas RecRep2 proteins have a domain significantly similar to one in the Reps of pLS1 plasmids. We speculate that RecRep genes have been transferred from viruses or plasmids to parasitic protozoan and bacterial genomes and that Rep proteins were themselves involved in the original recombination events that generated the ancestral RecRep genes.

Animals↗
PubMed1999

AP site structural determinants for Fpg specific recognition.

The binding of Escherichia coli and Lactococcus lactis Fapy-DNA glyosylase (Fpg) proteins to DNA containing either cyclic or non-cyclic abasic (AP) site analogs was investigated by electrophoretic mobility shift assay (EMSA) and by footprinting experiments. We showed that the reduced AP site is the best substrate analog for the E.coli and L.lactis enzymes ( K Dapp = 0.26 and 0.5 nM, respectively) as compared with the other analogs tested in this study ( K Dapp >2.8 nM). The 1,3-propanediol (Pr) residue-containing DNA seems to be the minimal AP site structure allowing a Fpg specific DNA binding, since the ethyleneglycol residue is not specifically bound by these enzymes. The newly described cyclopentanol residue is better recognized than tetrahydrofuran (for the E.coli Fpg, K Dapp = 2.9 and 25 nM, respectively). These results suggest that the hemiacetal form of the AP site is negatively discriminated by the Fpg protein suggesting a hydrogen bond between the C4'-hydroxyl group of the sugar and a Fpg residue. High-resolution hydroxyl radical footprinting using a duplex containing Pr shows that Fpg binds to six nucleotides on the strand containing the AP site and only the base opposite the lesion on the undamaged complementary strand. This comparative study provides new information about the molecular mechanism involved in the Fpg AP lyase activity.

Cyclopentanes↗
PubMed2000

Rapid genome walking: a simplified oligo-cassette mediated polymerase chain reaction using a single genome-specific primer.

In the present report we show that unknown DNA fragments are easily amplified in a single PCR reaction from an oligo-cassette library with a single genome-specific primer in combination with a cassette-specific primer. The novelty of the system, in comparison to the vectorette PCR method, lies in the use of unphosphorylated in contrast with phosphorylated oligo-cassettes in the ligation to the chromosomal DNA fragments. After denaturation of the DNA library, all chromosomal fragments carry a single-stranded linker attached to the 5'-end only. Therefore, the presence of the vectorette mismatched region is not required when unphosphorylated cassettes are used. As an example we report the amplification of the era gene from Lactococcus lactis.

Bacterial Proteins↗
PubMed2002

Frequent oligonucleotide motifs in genomes of three streptococci.

Complete genomes of three closely related Gram-positive bacteria Streptococcus pyogenes, Streptococcus pneumoniae and Lactococcus lactis are analyzed for abundances of short DNA sequence motifs (frequent words). The character and extent of frequent words are strikingly different among these genomes. The frequent words of S.pneumoniae split into three categories: parts of the previously characterized RUP and BOX repetitive elements and a 24 bp tandem repeat in the gene SP1772. The most abundant frequent words of L.lactis are all related to the 13 bp motif, WWNTTACTGACRR or its inverted complement YYGTCAGTAANWW. Distributional analysis of this motif, which we called highly repetitive motif (HRM), indicates its possible dual role. Frequent occurrences immediately downstream of genes suggest a possible role in transcription termination whereas spacings of consecutive HRMs consistent with the DNA helical period are indicative of a protein-binding site. Two regions of the L.lactis genome feature an intriguing pattern of several periodically occurring HRMs separated by precisely 59 bp. In a striking contrast to S.pneumoniae and L.lactis, S.pyogenes contains hardly any frequent words.

Base Sequence↗
PubMed2005

DynaProt 2D: an advanced proteomic database for dynamic online access to proteomes and two-dimensional electrophoresis gels.

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.

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↗
PubMed2005

Localization, mobility and fidelity of retrotransposed Group II introns in rRNA genes.

We previously showed that the group II Lactococcus lactis Ll.LtrB intron could retrotranspose into ectopic locations on the genome of its native host. Two integration events, which had been mapped to unique sequences, were localized in the present study to separate copies of the six L.lactis 23S rRNA genes, within operon B or D. Although further movement within the bacterial chromosome was undetectable, the retrotransposed introns were able to re-integrate into their original homing site provided on a plasmid. This finding indicates not only that retrotransposed group II introns retain mobility properties, but also that movement occurs back into sequence that is heterologous to the sequence of the chromosomal location. Sequence analysis of the retrotransposed introns and the secondary mobility events back to the homing site showed that the introns retain sequence integrity. These results are illuminating, since the reverse transcriptase (RT) of the intron-encoded protein, LtrA, has no known proofreading function, yet the mobility events have a low error rate. Enzymatic digests were used to monitor sequence changes from the wild-type intron. The results indicate that retromobility events have approximately 10(-5) misincorporations per nucleotide inserted. In contrast to the high RT error rates for retroviruses that must escape host defenses, the infrequent mutations of group II introns would ensure intron spread through retention of sequences essential for mobility.

Base Sequence↗
PubMed2005

Structural insights into abasic site for Fpg specific binding and catalysis: comparative high-resolution crystallographic studies of Fpg bound to various models of abasic site analogues-containing DNA.

Fpg is a DNA glycosylase that recognizes and excises the mutagenic 8-oxoguanine (8-oxoG) and the potentially lethal formamidopyrimidic residues (Fapy). Fpg is also associated with an AP lyase activity which successively cleaves the abasic (AP) site at the 3' and 5' sides by betadelta-elimination. Here, we present the high-resolution crystal structures of the wild-type and the P1G defective mutant of Fpg from Lactococcus lactis bound to 14mer DNA duplexes containing either a tetrahydrofuran (THF) or 1,3-propanediol (Pr) AP site analogues. Structures show that THF is less extrahelical than Pr and its backbone C5'-C4'-C3' diverges significantly from those of Pr, rAP, 8-oxodG and FapydG. Clearly, the heterocyclic oxygen of THF is pushed back by the carboxylate of the strictly conserved E2 residue. We can propose that the ring-opened form of the damaged deoxyribose is the structure active form of the sugar for Fpg catalysis process. Both structural and functional data suggest that the first step of catalysis mediated by Fpg involves the expulsion of the O4' leaving group facilitated by general acid catalysis (involving E2), rather than the immediate cleavage of the N-glycosic bond of the damaged nucleoside.

Bacterial Proteins↗

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