Biomedical subjects

J E Suárez

Publications and source records attributed to J E Suárez.

At least 19 recordsLinked to original sources

PubMed2003

Analysis of the morphogenetic cluster and genome of the temperate Lactobacillus casei bacteriophage A2.

The genes that encode the morphogenetic proteins of bacteriophage A2 are clustered and expressed as a single operon which originates a late transcript of more than 20 kb. This DNA stretch is analyzed in the context of the whole phage genome, which presents the following peculiarities: a) the head presents two major proteins that share their NH(2) termini, i.e.: both are translated from a single gene (orf5), b) these two proteins suffer a proteolytic maturation process before being incorporated into the capsid, rendering a 123 NH(2) terminal putative polypeptide that is postulated to be the scaffolding protein of the phage, c) similar maturation processes occur at the portal and tail length determination proteins, having all in common a Pho-Pho-Arg downward arrow sequence (where Pho stands for any hydrophobic amino acid) at the processing point, d) the genes encoding the subunits of the terminase (orf61 and orf2) are separated by the cohesive ends, e) two genes that might mediate lysogenic conversion ( orf19 and orf22) have been identified and f) the genome presents a dispensable region (which covers at least 10 orfs, as judged from analysis of deletion mutants) that might be involved in maintaining its size between the packaging limits of the capsid.

Amino Acid Sequence↗

PubMed2000

Characterization of the DNA replication module of bacteriophage A2 and use of its origin of replication as a defense against infection during milk fermentation by Lactobacillus casei.

Adjacent to the lysis/lysogeny cassette of the A2 phage genome lies a stretch of over 8 kb, which contains a series of genes probably involved in DNA replication. Fifteen open reading frames (orfs) were identified, 13 of which are encoded on the main coding strand and only two on the complementary strand. Database searches and comparative analyses allowed the identification of an open reading frame (orf455) that shows similarity with DNA helicases and contains a variant zinc-finger motif known from the phage T7 helicase/primase. Orf770 showed similarity to putative plasmid and phage DNA primases. Downstream of orf770 is a noncoding 258-bp region rich in direct and inverted repeats, which specifically binds to proteins whose synthesis is induced during phage infection. When present in a plasmid, this region can direct a partial bacteriophage resistance phenotype due to interference with phage DNA replication, both under laboratory conditions and during milk fermentation. It is deduced that this stretch contains the origin of replication of phage A2.

Amino Acid Sequence↗

PubMed2000

Nucleotide sequence and analysis of pBL1, a bacteriocin-producing plasmid from Lactococcus lactis IPLA 972.

The complete sequence of the 10.9-kbp bacteriocinogenic plasmid pBL1 from Lactococcus lactis subsp. lactis IPLA 972 has been determined. Thirteen ORFs were encountered, of which 5 were incomplete. pBL1 proved to be a narrow-host-range plasmid which replicates neither in Bacilus subtilis nor in Lactobacillus spp. The structural organization of the pBL1 replication region was highly similar to other well-known theta-replicating plasmids of lactococci, at both the untranslated (the replication origin) and the translated (repB and orfX) sequences. As in other plasmids, the product of orfX was not necessary for plasmid replication. However, it was shown to be involved in plasmid stability. Three genes organized in an operon-like structure encompassed, most likely, the bacteriocin-encoding region. Upstream of the origin of replication a nicking site (oriT) was found. This oriT sequence proved to be functional by mobilization of plasmids wearing it. One complete and several partial IS elements were identified on pBL1.

Amino Acid Sequence↗

PubMed2000

Generation of food-grade recombinant lactic acid bacterium strains by site-specific recombination.

The construction of a delivery and clearing system for the generation of food-grade recombinant lactic acid bacterium strains, based on the use of an integrase (Int) and a resolvo-invertase (beta-recombinase) and their respective target sites (attP-attB and six, respectively) is reported. The delivery system contains a heterologous replication origin and antibiotic resistance markers surrounded by two directly oriented six sites, a multiple cloning site where passenger DNA could be inserted (e.g., the cI gene of bacteriophage A2), the int gene, and the attP site of phage A2. The clearing system provides a plasmid-borne gene encoding beta-recombinase. The nonreplicative vector-borne delivery system was transformed into Lactobacillus casei ATCC 393 and, by site-specific recombination, integrated as a single copy in an orientation- and Int-dependent manner into the attB site present in the genome of the host strain. The transfer of the clearing system into this strain, with the subsequent expression of the beta-recombinase, led to site-specific DNA resolution of the non-food-grade DNA. These methods were validated by the construction of a stable food-grade L. casei ATCC 393-derived strain completely immune to phage A2 infection during milk fermentation.

Animals↗

PubMed1999

A2 cro, the lysogenic cycle repressor, specifically binds to the genetic switch region of Lactobacillus casei bacteriophage A2.

Lysogenic induction of temperate bacteriophage A2 of Lactobacillus casei is controlled by the action of its cI and cro products at the phage operator region. Three 20-bp inverted repeated DNA segments (subsites O1, O2, and O3) and the two divergent (PL and PR) promoters were mapped within the 153-bp operator region. The A2-encoded Cro product is shown to be the functional homolog of lambda Cro. The binding of Cro to the three operator subsites is noncooperative and yields two discrete protein-DNA complexes of retarded migration in mobility shift assays. The Kapp value for the Cro-PL-PR DNA complex was estimated to be 6 nM. Cro shows a slightly higher affinity for O3 than for O1 and O2 subsites. The O3 subsite overlaps the -35 hexamer of the PL promoter, which directs cI expression. A Cro mutant protein, devoid of the last 12 residues (Cro*), allowed the assignment of the DNA-binding domain to the NH2 end of Cro. The C end enhances its affinity for the DNA and probably stabilizes bending induced by Cro.

Base Sequence↗

PubMed1999

Duplication of the beta-galactosidase gene in some Lactobacillus plantarum strains.

Curing of a plasmid that encoded a beta-galactosidase gene (beta-gal) from the Lactobacillus plantarum strain of dairy origin LL441 was not accompanied by complete loss of the lactose utilization phenotype. DNA-DNA hybridization, using an internal fragment of the beta-gal gene as a probe, revealed a second determinant located on the chromosome of the cured derivatives. The chromosomal copy was present in all of a series of beta-Gal+ L. plantarum and Lactobacillus pentosus strains from different origins. In addition, four other L. plantarum strains harboured plasmid encoded beta-gal genes as well. Since both sequences cross-hybridized and present a similar genetic organization, it is postulated that the plasmid copy was generated through gene duplication and, probably, selected by growth of the strains in lactose rich environments.

DNA, Bacterial↗

PubMed1999

Stable expression of the Lactobacillus casei bacteriophage A2 repressor blocks phage propagation during milk fermentation.

A general strategy was applied to implement resistance against temperate bacteriophages that infect food fermentation starters through cloning and expression of the phage repressor. Lactobacillus casei ATCC 393 and phage A2 were used to demonstrate its feasibility as milk fermentation is drastically inhibited when the strain is infected by this phage. The engineered strain Lact. casei EM40::cI, which has the A2 repressor gene (cI) integrated into the genome, was completely resistant and able to ferment milk whether phage was present or not. In addition, viable phages were eliminated from the milk, probably through adsorption to the cell wall. Finally, the integration of cI in the genome resulted in a stable resistance phenotype, being unnecessary selective pressure during milk fermentation.

Animals↗

PubMed1999

Solution structure of plantaricin C, a novel lantibiotic.

Plantaricin C, a bacteriocin produced by a Lactobacillus plantarum strain of dairy origin, is a lantibiotic. One dehydroalanine, one lanthionine and three beta-methyl-lanthionine residues were found in its 27 amino acid sequence. The plantaricin C structure has two parts: the first comprises the six NH2-terminal residues, four of which are lysines, which confer a strong positive charge to this stretch. The amino acids in positions 7 and 27 form the lanthionine bridge, giving a globular conformation to the rest of the molecule. The beta-methyl-lanthionine bridges are established between residues 12-15, 13-18 and 23-26. This central region has a charge distribution compatible with an amphipathic alpha-helix, through which plantaricin C would become inserted into the membrane matrix of sensitive organisms, provoking the opening of pores and leakage of the cytoplasmic content.

Amino Acid Sequence↗

PubMed1999

Cooperative interaction of CI protein regulates lysogeny of Lactobacillus casei by bacteriophage A2.

The temperate bacteriophage A2 forms stable lysogens in Lactobacillus casei. The A2-encoded cI product (CI), which is responsible for maintaining the A2 prophage in the lysogenic state, has been purified. The CI protein, which is a monomer of 25.3 kDa in solution, specifically binds to a 153-bp DNA fragment that contains two divergent promoters, PL and PR. These promoters mediate transcription from cI and a putative cro, respectively. Three similar, although not identical, 20-bp inverted repeated DNA segments (operator sites O1, O2, and O3) were found in this segment. CI selectively interacts with O1, which is placed downstream from the transcription start point of the cro gene, and with O2 and O3, which overlap with the -35 region of the two promoters. Using a heterologous RNA polymerase, we have determined the transcription start points of PL and PR. CI exerts a negative effect on the in vitro transcription of PR by repositioning the RNA polymerase in a concentration-dependent manner. CI, when bound to O1 and O2, enhances the positioning of the RNA polymerase with the PL promoter. Our data indicate that the CI protein regulates the lytic and lysogenic pathways of the A2 phage.

Bacteriophages↗

PubMed1998

The site-specific recombination system of the Lactobacillus species bacteriophage A2 integrates in gram-positive and gram-negative bacteria.

The region of the bacteriophage A2 genome involved in site-specific recombination with the DNA of Lactobacillus spp. has been identified. Two orfs, transcribed from the same strand, have been found immediately upstream of the phage attachment site (attP). The orf adjacent to attP predicts a 385-amino-acid protein that presents significant similarity with site-specific recombinases of the integrase family. The other orf encodes a basic polypeptide of 76 amino acid residues. The junctions of the prophage with the genomes of its hosts have been determined, allowing the identification of the host attachment site (attB), which has a common 19-nucleotide core region with attP. The attB site is located at the 3' end of the transfer RNALeu gene (anticodon CAA). Nonreplicative plasmids containing the A2-specific recombination cassette integrate into different lactobacilli but also into unrelated Gram-positive bacteria such as Lactococcus lactis and even into Escherichia coli. In Lc. lactis, integration occurs in a previously unknown intergenic region, whereas in E. coli, it maps within the rrnD operon, 5' of rrsD gene. Comparison of the integration sites in the different hosts indicates that some flexibility is permitted in the attB sequence, since Lc. lactis and E. coli only share 13 and 11 nucleotides, respectively, with the 19-nucleotide core sequence of the lactobacilli.

Amino Acid Sequence↗

PubMed1998

Chemostat production of plantaricin C by Lactobacillus plantarum LL441.

Plantaricin C, a bacteriocin synthesized by Lactobacillus plantarum LL441, was optimally produced in chemostats kept at pH 5.0, 30 degreesC, 150 rpm, and a dilution rate of 0.05 h-1 when glucose was used as carbon source and a dilution rate of 0.10 to 0.12 h-1 when sucrose or fructose was used instead. Production was abolished at high dilution rates, i.e., when the cells grew rapidly in all carbon sources.

Bacteriocins↗

PubMed1998

Adherence of human vaginal lactobacilli to vaginal epithelial cells and interaction with uropathogens.

Three strains of Lactobacillus, identified as Lactobacillus acidophilus, Lactobacillus gasseri, and Lactobacillus jensenii, were selected from among 70 isolates from the vaginas of healthy premenopausal women for properties relevant to mucosal colonization or antagonism. All three self-aggregated and adhered to epithelial vaginal cells, displacing well-known vaginal pathogens, such as G. vaginalis, and inhibiting the growth in vitro of Escherichia coli and Streptococcus agalactiae. The surface components involved in self-aggregation appeared to be proteins for L. gasseri and lipoproteins for L. acidophilus and L. jensenii, as judged by susceptibility to treatment with appropriate degrading enzymes. The factors responsible for adherence to epithelial vaginal cells seemed to be glycoproteins (L. acidophilus and L. gasseri) and carbohydrate (L. jensenii). The receptors of the vaginal cells were glycolipids, which presumably were the targets of the competition observed between the lactobacilli and the pathogenic microbes.

Bacterial Adhesion↗

PubMed1998

Identification of the repressor-encoding gene of the Lactobacillus bacteriophage A2.

The repressor gene of the Lactobacillus phage A2 has the following properties: it (i) encodes a 224-residue polypeptide with DNA binding and RecA cleavage motifs, (ii) is expressed in lysogenic cultures, and (iii) confers superinfection immunity on the host. Adjacent, but divergently transcribed, lies another open reading frame whose product resembles the lambda Cro protein. In the 161-bp intergenic segment, putative promoters and operators have been detected.

Amino Acid Sequence↗

PubMed1997

Characterization of the aggregation promoting factor from Lactobacillus gasseri, a vaginal isolate.

Lactobacillus gasseri 2459, isolated from the human vagina, exhibits a strong autoaggregating phenotype. Filter-sterilized spent supernatants of this strain promote aggregation of Lact. plantarum LL441 and Enterococcus faecalis EF. Aggregation was abolished upon exposure of the cells to proteases and, in the case of Ent. faecalis, to metaperiodate, which suggests the involvement of cell-surface proteins and glycoproteins, respectively, in the aggregation phenotype. In accordance with this, a 75 kDa surface protein, and possibly another of approximately 94 kDa, appears in Lact. plantarum LL441 cultures incubated with Lact. gasseri culture supernatants. The diffusible aggregation promoting factor was purified from stationary phase culture supernatants and determined to be a 2 kDa hydrophilic peptide active at pH 3-4 and stable at neutral and acid pH. The activity was resistant to heat, chymotrypsin, chelating agents, triton X-100 and reducing agents, but sensitive to other proteases and SDS.

Bacterial Adhesion↗

PubMed1997

Molecular analysis of the cos region of the Lactobacillus casei bacteriophage A2. Gene product 3, gp3, specifically binds to its downstream cos region.

The terminal nucleotide sequence of the Lactobacillus casei bacteriophage A2 DNA revealed a single-stranded extension 13 bases in length (5'-AACGGTCGGCCTC-3') at its 3' termini that defines the packaging initiation nicking site (cosN). The cosN sequence is bisected by an axis of hyphenated twofold rotational symmetry. Directly and inverted repeated sequences located to the left (cosL) and the right (cosR) of the cosN site were observed. Analysis of the 3.4 kb EcoRI DNA sequence surrounding the cos region revealed four complete and one incomplete open reading frames (orfs). Northern blots indicated that all were cotranscribed in a single mRNA molecule in excess of 10 kb that appeared late during infection. Minicell studies indicated that the four orfs were translated into protein. From the ORF3 amino acid sequence DNA-binding and NTP-binding domains can be predicted. The purified ORF3 (predicted molecular mass 16.8 kDa) shows specific binding to the A2 cos region, so it was renamed gp3. Gp3 forms a specific complex with a 369 bp cos DNA segment in the presence of ATP. Gp3 interaction with the intrinsically bent cos DNA segment induces intramolecular ligation in the presence of T4 DNA ligase. The data presented here suggest that gp3 is the small subunit of the terminase enzyme.

Amino Acid Sequence↗

PubMed1997

Cloning and characterization of cspL and cspP, two cold-inducible genes from Lactobacillus plantarum.

Two cold shock genes, cspL and cspP, have been cloned from two Lactobacillus plantarum strains. These genes, which are nonallelic, were present in all strains tested. The genes encode 66-amino-acid polypeptides related to each other and to the cold shock Csp family. Transcription of cspP rendered a single mRNA, while two cspL mRNAs were found with common 5' ends. The amounts of these transcripts increased moderately upon exposure of the cultures to cold.

Acclimatization↗

PubMed1997

Isolation and characterization of promoters from the Lactobacillus casei temperate bacteriophage A2.

Random Sau3A1 DNA fragments from the temperate Lactobacillus bacteriophage A2 were cloned into the promoter-probe plasmid pGKV210. Seven DNA fragments with promoter activity were selected, after transformation of Escherichia coli and Lactococcus lactis, subsp, lactis, through the chloramphenicol resistance they conferred to the corresponding clones. The seven promoters were functional in Lactobacillus casei. Their strength was analysed by measuring the levels of chloramphenicol resistance and chloramphenicol acetyltransferase activity induced in each host. The nucleotide sequences of these fragments were determined and primer extension analysis was used to locate the initiation site of transcription from each promoter in E coli. The promoters contained -10 and -35 regions similar to the consensus sequences of E. coli and Lactobacillus promoters.

Bacteriophages↗

PubMed1994

Cloning and expression of the plasmid encoded beta-D-galactosidase gene from a Lactobacillus plantarum strain of dairy origin.

The beta-galactosidase (beta-Gal) gene from Lactobacillus plantarum C3.8 was cloned and expressed in Lactococcus lactis and Escherichia coli. Hybridization experiments indicated that the gene is located on a plasmid and is present in other strains of Lactobacillus plantarum. Its sequence is very similar to a Leuconostoc lactis beta-Gal gene. Expression of the gene, both in Lactobacillus plantarum and in Lactococcus lactis, was four-fold higher in cells growth in lactose compared to those grown in glucose. The presence of the beta-Gal gene in Lactococcus lactis allowed this bacterium to be efficient in clotting milk.

Amino Acid Sequence↗