Silvia Argimón

Expanding the biotechnology potential of lactobacilli through comparative genomics of 213 strains and associated genera

Lactobacilli are a diverse group of species that occupy diverse nutrient-rich niches associated with humans, animals, plants and food. They are used widely in biotechnology and food preservation, and are being explored as therapeutics. Exploiting lactobacilli has been complicated by metabolic diversity, unclear species identity and uncertain relationships between them and other commercially important lactic acid bacteria. The capacity for biotransformations catalysed by lactobacilli is an untapped biotechnology resource. Here we report the genome sequences of 213 Lactobacillus strains and associated genera, and their encoded genetic catalogue for modifying carbohydrates and proteins. In addition, we describe broad and diverse presence of novel CRISPR-Cas immune systems in lactobacilli that may be exploited for genome editing. We rationalize the phylogenomic distribution of host interaction factors and bacteriocins that affect their natural and industrial environments, and mechanisms to withstand stress during technological processes. We present a robust phylogenomic framework of existing species and for classifying new species.

Developmental regulation of an adhesin gene during cellular morphogenesis in the fungal pathogen Candida albicans

ABSTRACT Candida albicans expresses specific virulence traits that promote disease establishment and progression. These traits include morphological transitions between yeast and hyphal growth forms that are thought to contribute to dissemination and invasion and cell surface adhesins that promote attachment to the host. Here, we describe the regulation of the adhesin gene ALS3, which is expressed specifically during hyphal development in C. albicans. Using a combination of reporter constructs and regulatory mutants, we show that this regulation is mediated by multiple factors at the transcriptional level. The analysis of ALS3 promoter deletions revealed that this promoter contains two activation regions: one is essential for activation during hyphal development, while the second increases the amplitude of this activation. Further deletion analyses using the Renilla reniformis luciferase reporter delineate the essential activation region between positions −471 and −321 of the promoter. Further 5′ or 3′ deletions block activation. ALS3 transcription is repressed mainly by Nrg1 and Tup1, but Rfg1 contributes to this repression. Efg1, Tec1, and Bcr1 are essential for the transcriptional activation of ALS3, with Tec1 mediating its effects indirectly through Bcr1 rather than through the putative Tec1 sites in the ALS3 promoter. ALS3 transcription is not affected by Cph2, but Cph1 contributes to full ALS3 activation. The data suggest that multiple morphogenetic signaling pathways operate through the promoter of this adhesin gene to mediate its developmental regulation in this major fungal pathogen.

Oral Lactobacilli and Dental Caries A Model for Niche Adaptation in Humans

Lactobacilli have been associated with dental caries for over a century. Here, we review the pertinent literature along with findings from our own study to formulate a working hypothesis about the natural history and role of lactobacilli. Unlike most indigenous microbes that stably colonize a host, lactobacilli appear to be planktonic, opportunistic settlers that can gather and multiply only in certain restrictive niches of the host, at least within the oral cavity. We postulate that the following essential requirements are necessary for sustained colonization of lactobacilli in humans: 1) a stagnant, retentive niche that is mostly anaerobic; 2) a low pH milieu; and 3) ready access to carbohydrates. Three sites on the human body meet these specifications: caries lesions, the stomach, and the vagina. Only a handful of Lactobacillus species is found in caries lesions, but they are largely absent in caries-free children. Lactobacilli present in caries lesions represent both a major contributor to caries progression and a major reservoir to the gastrointestinal (GI) tract. We extend the assertion from other investigators that lactobacilli found in the GI tract originate in the oral cavity by proposing that lactobacilli in the oral cavity arise from caries lesions. This, in turn, leads us to reflect on the health implications of the lactobacilli in the mouth and downstream GI and to ponder whether these or any of the Lactobacillus species are truly indigenous to the human GI tract or the oral cavity.

Distribution of Putative Virulence Genes in Streptococcus mutans Strains Does Not Correlate with Caries Experience

ABSTRACT Streptococcus mutans, a member of the human oral flora, is a widely recognized etiological agent of dental caries. The cariogenic potential of S. mutans is related to its ability to metabolize a wide variety of sugars, form a robust biofilm, produce copious amounts of lactic acid, and thrive in the acid environment that it generates. The remarkable genetic variability present within the species is reflected at the phenotypic level, notably in the differences in the cariogenic potential between strains. However, the genetic basis of these differences is yet to be elucidated. In this study, we surveyed by PCR and DNA hybridization the distribution of putative virulence genes, genomic islands, and insertion sequences across a collection of 33 strains isolated from either children with severe early childhood caries (S-ECC) or those who were caries free (CF). We found this genetically diverse group of isolates to be remarkably homogeneous with regard to the distribution of the putative virulence genes and genetic elements analyzed. Our findings point to the role of other factors in the pathogenesis of S-ECC, such as uncharacterized virulence genes, differences in gene expression and/or enzymatic activity, cooperation between S. mutans strains or with other members of the oral biota, and host factors.

Determining the genetic diversity of lactobacilli from the oral cavity

Several methods for determining the diversity of Lactobacillus spp were evaluated with the purpose of developing a realistic approach for further studies. The patient population was comprised of young children with an oral disease called severe early childhood caries. The ultimate goal of these studies was to ascertain the role of lactobacilli in the caries process. To accomplish that goal, we evaluated several methods and approaches for determining diversity including AP-PCR, chromosomal DNA fingerprinting, denaturing gradient gel electrophoresis, and 16S rRNA gene sequencing. Central to these methods was the gathering and screening of isolates from cultivation medium. Using various estimates of diversity, we addressed the question as to how many isolates represent the overall diversity and how cultivation compares to non-cultivation techniques. Finally, we proposed a working approach for achieving the goals outlined framed by both practical constraints in terms of time, effort and efficacy while yielding a reliable outcome.

Phylogenetic Analysis of Glucosyltransferases and Implications for the Coevolution of Mutans Streptococci with Their Mammalian Hosts

Glucosyltransferases (Gtfs) catalyze the synthesis of glucans from sucrose and are produced by several species of lactic-acid bacteria. The oral bacterium Streptococcus mutans produces large amounts of glucans through the action of three Gtfs. GtfD produces water-soluble glucan (WSG), GtfB synthesizes water-insoluble glucans (WIG) and GtfC produces mainly WIG but also WSG. These enzymes, especially those synthesizing WIG, are of particular interest because of their role in the formation of dental plaque, an environment where S. mutans can thrive and produce lactic acid, promoting the formation of dental caries. We sequenced the gtfB, gtfC and gtfD genes from several mutans streptococcal strains isolated from the oral cavity of humans and searched for their homologues in strains isolated from chimpanzees and macaque monkeys. The sequence data were analyzed in conjunction with the available Gtf sequences from other bacteria in the genera Streptococcus, Lactobacillus and Leuconostoc to gain insights into the evolutionary history of this family of enzymes, with a particular emphasis on S. mutans Gtfs. Our analyses indicate that streptococcal Gtfs arose from a common ancestral progenitor gene, and that they expanded to form two clades according to the type of glucan they synthesize. We also show that the clade of streptococcal Gtfs synthesizing WIG appeared shortly after the divergence of viviparous, dentate mammals, which potentially contributed to the formation of dental plaque and the establishment of several streptococci in the oral cavity. The two S. mutans Gtfs capable of WIG synthesis, GtfB and GtfC, are likely the product of a gene duplication event. We dated this event to coincide with the divergence of the genomes of ancestral early primates. Thus, the acquisition and diversification of S. mutans Gtfs predates modern humans and is unrelated to the increase in dietary sucrose consumption.

Protein A‐tagging for purification of native macromolecular complexes from Candida albicans

Protein A‐tagging has become an important tool in characterization of protein–protein interactions in many systems, allowing purification of multicomponent complexes under native conditions. Here we provide a set of vectors that allow protein A‐tagging in Candida albicans, through addition of the tag to open reading frames. These vectors were successfully used to generate stably tagged proteins that were functional, shown to be localized appropriately or assembled into complexes. These new vectors comprise a useful addition to the C. albicans molecular toolbox. Copyright

Comparative genomics of oral isolates of Streptococcus mutans by in silico genome subtraction does not reveal accessory DNA associated with severe early childhood caries

Comparative genomics is a popular method for the identification of microbial virulence determinants, especially since the sequencing of a large number of whole bacterial genomes from pathogenic and non-pathogenic strains has become relatively inexpensive. The bioinformatics pipelines for comparative genomics usually include gene prediction and annotation and can require significant computer power. To circumvent this, we developed a rapid method for genome-scale in silico subtractive hybridization, based on blastn and independent of feature identification and annotation. Whole genome comparisons by in silico genome subtraction were performed to identify genetic loci specific to Streptococcus mutans strains associated with severe early childhood caries (S-ECC), compared to strains isolated from caries-free (CF) children. The genome similarity of the 20 S. mutans strains included in this study, calculated by Simrank k-mer sharing, ranged from 79.5% to 90.9%, confirming this is a genetically heterogeneous group of strains. We identified strain-specific genetic elements in 19 strains, with sizes ranging from 200 to 39 kb. These elements contained protein-coding regions with functions mostly associated with mobile DNA. We did not, however, identify any genetic loci consistently associated with dental caries, i.e., shared by all the S-ECC strains and absent in the CF strains. Conversely, we did not identify any genetic loci specific with the healthy group. Comparison of previously published genomes from pathogenic and carriage strains of Neisseria meningitidis with our in silico genome subtraction yielded the same set of genes specific to the pathogenic strains, thus validating our method. Our results suggest that S. mutans strains derived from caries active or caries free dentitions cannot be differentiated based on the presence or absence of specific genetic elements. Our in silico genome subtraction method is available as the Microbial Genome Comparison (MGC) tool, with a user-friendly JAVA graphical interface.

Mucor rouxii Rho1 protein; characterization and possible role in polarized growth

We have previously shown that protein kinase A of the medically important zygomycete Mucor rouxii participates in fungal morphology through cytoskeletal organization. As a first step towards finding the link between protein kinase A and cytoskeletal organization we here demonstrate the cloning of the Rho1 gene and the characterization of its protein product. The RHO1 protein primary sequence shows 70–85% identity with fungal RHO1 or mammalian RhoA. Two protein kinase A phosphorylation sequences in adequate context are predicted, Ser73 and Ser135. The peptide IRRNSQKFV, containing Ser135 proved to be a good substrate for M. rouxii protein kinase A catalytic subunit. The over-expressed Rho1 fully complements a Saccharomyces cerevisiae null mutant. The endogenous protein was identified by western blot against a developed antibody and by ADP-ribosylation. Localization in germlings was visualized by immunofluorescence; the protein was localized in patches in the mother cell surface and excluded from the germ tube. Measurement of Rho1 expression during germination indicates that Rho1, at both the mRNA and protein levels, correlates with differentiation and not with growth. Rho1 has been shown to be the regulatory protein of the β-1,3-glucan synthase complex in fungi in which β-1,3-glucans are major components of the cell wall. Even though glucans have not been detected in zygomycetes, caspofungin, an echinochandin known to be an inhibitor of β-1,3-glucan synthase complex, is shown here to have a negative effect on growth and to produce an alteration on morphology when added to M. rouxii growth culture medium. This result has an important impact on the possible participation of β-1,3-glucans on the regulation of morphology of zygomycetes.