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Diego Mora

Publications and source records attributed to Diego Mora.

15 recordsLinked to original sources
PubMed2006

Molecular characterization of Bifidobacterium longum biovar longum NAL8 plasmids and construction of a novel replicon screening system.

In this study, we performed molecular characterization and sequence analysis of three plasmids from the human intestinal isolate Bifidobacterium longum biovar longum NAL8 and developed a novel vector screening system. Plasmids pNAL8H (10 kb) and pNAL8M (4.9 kb) show close sequence similarity to and the same gene organization as the already characterized B. longum plasmids. The B. longum plasmid pNAC1 was identified as being most closely related to pNAL8L (3.5 kb). However, DNA sequence analysis suggested that direct repeat-rich sites could have promoted several recombination events to diversify the two plasmid molecules. We verified the likely rolling circle replication of plasmid pNAL8L and studied the phylogenetic relationship in all the Bifidobacterium plasmids fully sequenced to date based on in silico comparative sequence analysis of their replication proteins and iteron regions. Our transformation experiments confirmed that the ColE1 replication origin from high-copy-number pUC vectors could interfere with the replication apparatus of Bifidobacterium plasmids and give rise to false positive clones. As a result, we developed a system suitable for avoiding possible interference by other functional replication modules on the vector and for screening functional replicons from wild-type plasmids.

Bifidobacterium↗
PubMed2006

Usefulness of length heterogeneity-PCR for monitoring lactic acid bacteria succession during maize ensiling.

The use of length-heterogeneity PCR was explored to monitor lactic acid bacteria succession during ensiling of maize. Bacterial diversity was studied during the fermentation of 30-day-old maize in optimal and spoilage-simulating conditions. A length heterogeneity PCR profile database of lactic acid bacteria isolated from the silage and identified by 16S rRNA gene sequencing was established. Although interoperonic 16S rRNA gene length polymorphisms were detected in some isolates, strain analysis showed that most of the lactic acid bacteria species thriving in silage could be discriminated by this method. The length heterogeneity PCR profiles of bacterial communities during maize fermentation were compared with those on a database. Under optimal fermentation conditions all the ecological indices of bacterial diversity, richness and evenness, deduced from community profiles, increased until day thirteen of fermentation and then decreased to the initial values. Pediococcus and Weissella dominated, especially in the first days of fermentation. Lactococcus lactis ssp. lactis and Lactobacillus brevis were mainly found after six days of fermentation. A peak corresponding to Lactobacillus plantarum was present in all the fermentation phases, but was only a minor fraction of the population. Unsuitable fermentation conditions and withered maize leaves in the presence of oxygen and water excess caused an enrichment of Enterococcus sp. and Enterobacter sp.

Base Sequence↗
PubMed2005

Urease biogenesis in Streptococcus thermophilus.

Urease biogenesis was monitored in the lactic acid bacterium Streptococcus thermophilus during the growth cycle using in-gel detection and a phenol-hypochloride assay. Zymogram analysis, performed in a non-denaturing polyacrylamide gel, enabled visualization of a complex profile of bands whose number and intensity were dependent on the growth phase and culture conditions. The monitoring of urease biogenesis in batch fermentations revealed the onset of enzyme synthesis starting from the mid-exponential growth phase, with a maximum reached during the late exponential phase. Urease activity strongly increased at acidic pH but to a lesser extent when urea and nickel ions were added to the culture medium. When S. thermophilus cells were cultured with pH maintained at a neutral value, urease activity was detectable only in gel with extremely low signals. Evaluation of beta-glucuronidase activity in strain DSM 20617(T) harboring a transcriptional fusion between a DNA fragment containing the putative urease promoter and the gusA reporter evidenced significant expression at neutral pH that strongly increased in an acidic environment. Further experiments carried out on p(ureI)-gusA recombinant strain revealed that expression of ure genes was not affected by carbohydrates, nickel or urea availability. The presence of consistent expression of ure genes at neutral pH and the absence of induction of expression by carbohydrate availability demonstrated that the transcription of ure genes in S. thermophilus is regulated differently compared with that of the closely related S. salivarius. These differences are discussed taking into consideration the different habitats colonized by the two bacterial species.

Bacterial Proteins↗
PubMed2004

Isolation of Elaeagnus-compatible Frankia from soils collected in Tunisia.

The occurrence and diversity of Frankia nodulating Elaeagnus angustifolia in Tunisia were evaluated in 30 soils from different regions by a Frankia-capturing assay. Despite the absence of actinorhizal plants in 24 of the 30 soils, nodules were captured from all the samples. Eight pure strains were isolated from single colonies grown in agar medium. On the basis of 16S rRNA and GlnII sequences, seven strains were clustered with Frankia, colonizing Elaeagnaceae and Rhamnaceae in two different phylogenetic groups while one strain described a new lineage in the Frankia assemblage, indicating that Frankia strains genetically diverse from previously known Elaeagnus-infective strains are present in tunisian soils. Genomic fingerprinting determined by rep-PCR, and tDNA-PCR-SSCP, confirmed the wide genetic diversity of the strains.

Base Sequence↗
PubMed2004

Homologies among three Bacillus licheniformis plasmids and molecular characterization of their replication module.

To assess to what extent three Bacillus licheniformis plasmids had the same molecular organization a physical map of the 9.34, 8.40 and 7.90 kb plasmids was achieved by using seventeen restriction enzymes. Southern hybridization was performed on plasmids using restriction fragments of the smallest plasmid as probes. Data from different hybridization patterns show a close homology among the three plasmids hypothesizing a similar molecular organization. The lack of plasmid diversity observed, seem to support the hypothesis of a similar phylogeny among these plasmids. This investigation provides more information concerning phylogeny, interrelationships and level of diversity among Bacillus plasmids and a molecular characterization of three plasmids useful for the construction of cloning vectors.

Amino Acid Sequence↗
PubMed2003

Reclassification of Lactobacillus maltaromicus (Miller et al. 1974) DSM 20342(T) and DSM 20344 and Carnobacterium piscicola (Collins et al. 1987) DSM 20730(T) and DSM 20722 as Carnobacterium maltaromaticum comb. nov.

Phenotypic and genotypic characterizations of Lactobacillus maltaromicus strains DSM 20342(T) and DSM 20344 provided evidence for the reclassification of this species in the genus Carnobacterium. Moreover, phenotypic and genotypic comparisons made between L. maltaromicus and Carnobacterium piscicola highlighted that these two species should be considered synonyms. For these reasons, the species Carnobacterium maltaromaticum comb. nov. (type strain DSM 20342(T) = ATCC 27865(T) = CCUG 30142(T) = CIP 103135(T) = JCM 1154(T) = LMG 6903(T) = NRRL B-14852(T)) is proposed to accommodate L. maltaromicus and C. piscicola.

Bacterial Typing Techniques↗
PubMed2003

16S-23S rRNA intergenic spacer region sequence variation in Streptococcus thermophilus and related dairy streptococci and development of a multiplex ITS-SSCP analysis for their identification.

The 16S-23S rRNA internal transcribed spacer (ITS) region of several Streptococcus thermophilus strains and some related dairy streptococci, S. macedonicus, S. salivarius and S. bovis, was analysed by sequence analysis. All the Streptococcus species were easily discriminated on the basis of sequence variations principally located upstream and downstream of the region encompassing the double-stranded processing sites and the tRNA(Ala) gene. Comparison between tRNA(Ala) gene sequences highlighted a high level of sequence conservation among the Streptococcus species investigated despite their belonging to separated phylogenetic clusters, i.e. the S. salivarius and S. bovis rRNA groups. A low but significant degree of variability was detected among the S. thermophilus strains, allowing the identification of four different ITS sequences. Similarity analysis of the ITS sequences showed that the Streptococcus species were clustered in two main branches, one containing S. macedonicus and S. bovis strains, and one containing S. thermophilus and S. salivarius strains. With the aim of developing a rapid tool for the identification of the dairy streptococci species a multiplex ITS-SSCP analysis of two discrete regions within the ITS locus was carried out.

Bacterial Typing Techniques↗
PubMed2003

Unusual organization for lactose and galactose gene clusters in Lactobacillus helveticus.

The nucleotide sequences of the Lactobacillus helveticus lactose utilization genes were determined, and these genes were located and oriented relative to one another. The lacLM genes (encoding the beta-galactosidase protein) were in a divergent orientation compared to lacR (regulatory gene) and lacS (lactose transporter). Downstream from lacM was an open reading frame (galE) encoding a UDP-galactose 4 epimerase, and the open reading frame had the same orientation as lacM. The lacR gene was separated from the downstream lacS gene by 2.0 kb of DNA containing several open reading frames that were derived from fragmentation of another permease gene (lacS'). Northern blot analysis revealed that lacL, lacM, and galE made up an operon that was transcribed in the presence of lactose from an upstream lacL promoter. The inducible genes lacL and lacM were regulated at the transcriptional level by the LacR repressor. In the presence of glucose and galactose galE was transcribed from its promoter, suggesting that the corresponding enzyme can be expressed constitutively. Lactose transport was inducible by addition of lactose to the growth medium.

Amino Acid Sequence↗
PubMed2003

Nature of polymorphisms in 16S-23S rRNA gene intergenic transcribed spacer fingerprinting of Bacillus and related genera.

The intergenic transcribed spacers (ITS) between the 16S and 23S rRNA genetic loci are frequently used in PCR fingerprinting to discriminate bacterial strains at the species and intraspecies levels. We investigated the molecular nature of polymorphisms in ITS-PCR fingerprinting of low-G+C-content spore-forming bacteria belonging to the genera Bacillus, Brevibacillus, Geobacillus, and Paenibacillus: We found that besides the polymorphisms in the homoduplex fragments amplified by PCR, heteroduplex products formed during PCR between amplicons from different ribosomal operons, with or without tRNA genes in the ITS, contribute to the interstrain variability in ITS-PCR fingerprinting patterns obtained in polyacrylamide-based gel matrices. The heteroduplex nature of the discriminating bands was demonstrated by fragment separation in denaturing polyacrylamide gels, by capillary electrophoresis, and by cloning, sequencing, and recombination of purified short and tRNA gene-containing long ITS. We also found that heteroduplex product formation is enhanced by increasing the number of PCR cycles. Homoduplex-heteroduplex polymorphisms (HHP) in a conserved region, such as the 16S and 23S rRNA gene ITS, allowed discrimination of closely related strains and species undistinguishable by other methods, indicating that ITS-HHP analysis is an easy and reproducible additional tool for strain typing.

Bacillus↗
PubMed2002

Development of genus/species-specific PCR analysis for identification of Carnobacterium strains.

Heterofermentative lactic acid bacteria belonging to the genus Carnobacterium are currently divided into seven different species, C. piscicola, C. mobile, C. gallinarum, C. inhibens, C. divergens, C. funditum, and C. alterfunditum. 16S rDNA-targeted PCR assay was carried out for the identification of the genus Carnobacterium. In addition, type strains of all Carnobacterium species were analyzed by 16S-23S rDNA intergenic spacer analysis in comparison with type strains of phylogenetically related lactic acid bacteria. These methods enabled the identification and the discrimination among Carnobacterium species and the other phylogenetically related lactic acid bacteria. Likewise, analogous results were obtained by restriction analysis of amplified 16S rDNA performed with HaeIII and HinfI as restriction enzymes.

Base Sequence↗
PubMed2002

Multilocus hybridization typing in Pediococcus acidilactici strains.

In previous a study we demonstrated the presence of several genomic subpopulations within a collection of Pediococcus acidilactici strains isolated from different environments, through a multilocus typing analysis taking into consideration housekeeping conserved loci and protein coding genes of the primary metabolism. In this study, representative strains of five genomic subpopulations previously described (I, II, III, V, VII) were analyzed by restriction analysis of chromosomal DNA and subsequent hybridization assays using as probes amplified fragments obtained from five housekeeping genes (16S rDNA, rpoC, ldhD, ldhL, and metS). A computer similarity and clustering analysis of hybridization data showed the subdivision of P. acidilactici strains in five distinct genotypes according to the grouping previously obtained confirming that pediocin AcH/PA-1 producer strains represent one genomic lineage within the species P. acidilactici.

5-Methyltetrahydrofolate-Homocysteine S-Methyltran↗
PubMed2002

Comparison between Streptococcus macedonicus and Streptococcus waius strains and reclassification of Streptococcus waius (Flint et at. 1999) as Streptococcus macedonicus (Tsakalidou et al. 1998).

Two species of dairy streptococci, Streptococcus waius and Streptococcus macedonicus, were originally characterized by 16S-23S intergenic spacer sequence analysis, random amplified polymorphic DNA fingerprinting, PFGE analysis and DNA-DNA reassociation experiments. All genetic data suggested that S. waius strains belong to the previously described species S. macedonicus. Likewise, the phenotypic characterization showed that strains of S. macedonicus and S. waius were highly related and easily differentiated from the closest phylogenetic neighbour, Streptococcus bovis, principally by their failure to produce a blackening reaction in medium containing aesculin. The utilization of maltose and cellobiose by S. macedonicus/S. waius strains allowed their differentiation from the most studied dairy species, Streptococcus thermophilus. On the basis of genetic and phenotypic data S. macedonicus and S. waius species should be considered synonyms and S. macedonicus has the priority.

Bacterial Typing Techniques↗
PubMed2000

Genomic subpopulations within the species Pediococcus acidilactici detected by multilocus typing analysis: relationships between pediocin AcH/PA-1 producing and non-producing strains.

A high degree of genetic polymorphism among P. acidilactici strains was highlighted by a multilocus typing approach analysing several housekeeping genes and by sampling the whole genome using random amplified polymorphic DNA (RAPD) fingerprint analysis performed by using a single primer pedA gene targeted in low-stringency amplification conditions. Restriction fragment length polymorphism of the rpoC, ldhD/L and mle genes, and a modified RAPD analysis, permitted the grouping of Pediococcus acidilactici strains in seven genotypes (I-VII). Genotypic results obtained by analysing housekeeping genes involved in the transcription/translation machinery and in primary metabolism were supported by phylogenetic analysis based on the partial 16S rDNA sequencing of a reference strain of each of the seven clusters obtained. Three of the seven genotypes detected showed relationships with pediocin AcH/PA-1 production and carbohydrate fermentation patterns: all pediocin-producing and sucrose-positive strains were grouped in genotype VII, melibiose-, sucrose- and raffinose-positive strains in genotype VI, and arabinose-positive strains in genotype V.

Bacterial Typing Techniques↗
PubMedDate not supplied

Conditions affecting cell surface properties of human intestinal bifidobacteria.

The cell surface properties of human intestinal bifidobacteria have been characterized for 30 strains isolated from a fecal sample. Strain identification to the species level was obtained by restriction analysis of the amplified 16S rRNA gene and confirmed by DNA/DNA reassociation experiments. The isolates were grouped in four genetically homogeneous clusters whose members belonged to Bifidobacterium bifidum, Bifidobacterium adolescentis, Bifidobacterium longum and Bifidobacterium pseudocatenulatum species. Cell surface properties of Bifidobacterium strains were evaluated by determining the level of hydrophobicity, adhesion to hydrocarbons and contact angle measurements, and their autoaggregation ability. The results showed high and homogeneous level of hydrophobicity in all tested strains when contact angle measurements values were considered. On the contrary, autoaggregation assays and bacterial adhesion to hydrocarbons detected interesting differences in cell surface properties among the tested Bifidobacterium strains. The highest levels of autoaggregation, detected in B. bifidum and B. adolescentis strains, were strictly dependent on the pH of the medium. Moreover, protease treatment experiments suggested that proteins had a key role in the autoaggregating ability of B. bifidum and B. adolescentis strains.

Adult↗