Juan E. Suárez

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

Lactobacillus gasseri 2459, isolated from the human vagina, exhibits a strongautoaggregating phenotype. Filter‐sterilized spent supernatants of this strain promote aggregationof Lact. plantarum LL441 and Enterococcus faecalis EF. Aggregation wasabolished upon exposure of the cells to proteases and, in the case of Ent. faecalis, tometaperiodate, which suggests the involvement of cell‐surface proteins and glycoproteins,respectively, in the aggregation phenotype. In accordance with this, a 75 kDa surface protein, andpossibly another of approximately 94 kDa, appears in Lact. plantarum LL441 culturesincubated with Lact. gasseri culture supernatants. The diffusible aggregation promotingfactor was purified from stationary phase culture supernatants and determined to be a 2 kDahydrophilic peptide active at pH 3–4 and stable at neutral and acid pH. The activity wasresistant to heat, chymotrypsin, chelating agents, triton X‐100 and reducing agents, but sensitiveto other proteases and SDS.

The Dilemma of Phage Taxonomy Illustrated by Comparative Genomics of Sfi21-Like Siphoviridae in Lactic Acid Bacteria

The complete genome sequences of two dairy phages, Streptococcus thermophilus phage 7201 and Lactobacillus casei phage A2, are reported. Comparative genomics reveals that both phages are members of the recently proposed Sfi21-like genus of Siphoviridae, a widely distributed phage type in low-GC-content gram-positive bacteria. Graded relatedness, the hallmark of evolving biological systems, was observed when different Sfi21-like phages were compared. Across the structural module, the graded relatedness was represented by a high level of DNA sequence similarity or protein sequence similarity, or a shared gene map in the absence of sequence relatedness. This varying range of relatedness was found within Sfi21-like phages from a single species as demonstrated by the different prophages harbored by Lactococcus lactis strain IL1403. A systematic dot plot analysis with 11 complete L. lactis phage genome sequences revealed a clear separation of all temperate phages from two classes of virulent phages. The temperate lactococcal phages share DNA sequence homology in a patchwise fashion over the nonstructural gene cluster. With respect to structural genes, four DNA homology groups could be defined within temperate L. lactis phages. Closely related structural modules for all four DNA homology groups were detected in phages from Streptococcus or Listeria, suggesting that they represent distinct evolutionary lineages that have not uniquely evolved in L. lactis. It seems reasonable to base phage taxonomy on data from comparative genomics. However, the peculiar modular nature of phage evolution creates ambiguities in the definition of phage taxa by comparative genomics. For example, depending on the module on which the classification is based, temperate lactococcal phages can be classified as a single phage species, as four distinct phage species, or as two if not three different phage genera. We propose to base phage taxonomy on comparative genomics of a single structural gene module (head or tail genes). This partially phylogeny-based taxonomical system still mirrors some aspects of the current International Committee on Taxonomy in Virology classification system. In this system the currently sequenced lactococcal phages would be grouped into five genera: c2-, sk1, Sfi11-, r1t-, and Sfi21-like phages.

Milk Contamination and Resistance to Processing Conditions Determine the Fate of Lactococcus lactis Bacteriophages in Dairies

ABSTRACT 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°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.

Purification of a glutathione S-transferase that mediates fosfomycin resistance in bacteria.

The enzyme that modifies fosfomycin by formation of an adduct with glutathione was purified 12-fold with a 56% activity yield by passage through DEAE Sephacel and high-performance liquid chromatography molecular exclusion columns. Its functional form was a homodimer of two 16,000-dalton polypeptides, which possibly showed an antiparallel alpha tertiary structure and which lacked marked hydrophobic regions. Visualization of the reaction was achieved by precolumn derivatization of glutathione and the adduct, separation by high-performance liquid chromatography, and fluorescence detection of both compounds. Temperature and pH optima were 20 to 30 degrees C and 8.25, respectively; Mn2+, Fe2+, and Co2+ enhanced the rate of modification; and Km values were 9.4 and 11 mM for fosfomycin and glutathione, respectively. Phosphoenolpyruvate did not interfere with fosfomycin modification. The enzyme was stable at 4 degrees C for at least 6 months but progressively lost its activity upon being heated for 60 min at temperatures over 30 degrees C. Images

Characterization of indigenous vaginal lactobacilli from healthy women as probiotic candidates

The probiotic relevant characteristics of 45 strains of vaginal Lactobacillus isolated from healthy women were analyzed. Of these, 21 strains were classified as L. crispatus, 17 as L. jensenii, six as L. gasseri, and one as L. plantarum. The rate of acidification varied significantly between the strains as did their ability to form biofilms. None used glycogen as a fermentable carbohydrate. H2O2 generation was common, especially among L. jensenii isolates (88%). No bacteriocinogenic strains were detected. Most strains harbored plasmids (from 1 to 7) of various sizes, those in excess of 50 kb being frequent. One of these plasmids was found to be promiscuous since it hybridized with extrachromosomal bands of 15 isolates. All strains were resistant to metronidazole, ciprofloxacin, gentamicin, clindamycin, trimethoprim, and sulfametoxazole and susceptible to a series of beta-lactams, erythromycin, tetracycline, and benzalkonium chloride. Almost half of the strains were highly resistant to nonoxinol 9, which is commonly used as a spermicide. Based on these analyses, strains of all three common species are proposed as new probiotic candidates.

Synthesis of lactococcin 972, a bacteriocin produced by Lactococcus lactis IPLA 972, depends on the expression of a plasmid-encoded bicistronic operon

Synthesis of lactococcin 972 is plasmid-encoded. An operon composed of two genes that encode pre-bacteriocin and a putative immunity protein has been identified. The first gene encodes a 91-residue polypeptide that is exported via a sec-dependent system to give the mature 66-aa bacteriocin. The immunity protein is a 563-residue polypeptide with seven potential transmembrane domains. Two transcripts were observed from this region: one comprises the whole operon and is synthesized during the exponential phase of growth while the other, which corresponds just to the bacteriocin structural gene, presents a maximum in exponential cultures but is still present in late-stationary-phase cells.

Generation of Food-Grade Recombinant Lactic Acid Bacterium Strains by Site-Specific Recombination

ABSTRACT 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 (β-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 orientedsix 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 β-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 β-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.

Biosynthesis and Degradation of H2O2 by Vaginal Lactobacilli

ABSTRACT Hydrogen peroxide production by vaginal lactobacilli represents one of the most important defense mechanisms against vaginal colonization by undesirable microorganisms. To quantify the ability of a collection of 45 vaginal Lactobacillus strains to generate H2O2, we first compared three published colorimetric methods. It was found that the use of DA-64 as a substrate rendered the highest sensitivity, while tetramethyl-benzidine (TMB) maintained its linearity from nanomolar to millimolar H2O2 concentrations. Generation of H2O2 was found to be especially common and strong for L. jensenii strains, while it was variable among L. crispatus and L. gasseri strains. Biosynthesis of H2O2 only occurred upon agitation of the cultures, but the H2O2-producing machinery was already present in them before aeration started. Calcium, magnesium, manganese, and zinc ions did not affect H2O2 production, while Cu2+ inhibited the growth of Lactobacillus jensenii CECT 4306, which was chosen as a model strain. Cultures with Fe3+, hemin, and hemoglobin did not accumulate H2O2. Fe3+ activated an extracellular peroxidase that destroyed the H2O2 being produced by the cultures. This protected the lactobacilli against its antimicrobial effect. The production of the enzyme appears to be constitutive, the Fe3+ ions being a necessary cofactor of the reaction.

Lactococcin 972, a bacteriocin that inhibits septum formation in lactococci.

Addition of lactococcin 972 to exponentially growing sensitive cultures of Lactococcus lactis resulted in cell elongation and widening. Thin sections revealed that septum invagination was blocked. Cell growth progressed until most cells showed equatorial constriction and even initial deposition of material at the septum ring, although cell division did not proceed any further. The increase in the incorporation of labelled precursors into the cell wall shifted from an exponential to a linear mode in treated cultures, subsequently being arrested. Gross degeneration of the cells was observed prior to cell death, followed by slow lysis of the culture. In contrast, stationary-phase cultures remained unaffected.

Lactococcin 972 : a homodimeric lactococcal bacteriocin whose primary target is not the plasma membrane

Lactococcus lactis subsp. lactis IPLA 972 was shown to produce a bacteriocin which had a bactericidal effect on sensitive lactococci. Production of lactococcin 972 reached a maximum during the late-exponential phase of growth. The bacteriocinogenic activity was heat-sensitive, active in the pH range 40-9.0 and showed low susceptibility to proteases. Purification of the bacteriocin rendered a single polypeptide of 7.5 kDa (monomer) as shown by SDS-PACE. Gels overlaid with a lawn of sensitive bacteria showed inhibitory activity at a point corresponding t o 15 kDa. Changes in the electrophoretic conditions allowed the detection of a band at a position corresponding to that expected for a hypothetical dimer. Sequencing of the NH,-terminal end of lactococcin 972 revealed the sequence NH,-EGTWQHGYGV, which is not related to any other bacteriocin sequence present in the databases. Finally, lactococcin 972 did not induce the efflux of compounds previously incorporated into the cytoplasm of sensitive cultures nor did it inhibit macromolecular synthesis,suggesting that, in contrast to other bacteriocins, its primary target is not the plasma membrane.