Utilizing novel Escherichia coli-specific conserved signature proteins for enhanced monitoring of recreational water quality.

Escherichia coli serves as a proxy indicator of fecal contamination in aquatic ecosystems. However, its identification using traditional culturing methods can take up to 24 h. The application of DNA markers, such as conserved signature proteins (CSPs) genes (unique to all species/strains of a specific taxon), can form the foundation for novel polymerase chain reaction (PCR) tests that unambiguously identify and detect targeted bacterial taxa of interest. This paper reports the identification of three new highly-conserved CSPs (genes), namely YahL, YdjO, and YjfZ, which are exclusive to E. coli/Shigella. Using PCR primers based on highly conserved regions within these CSPs, we have developed quantitative PCR (qPCR) assays for the evaluation of E. coli/Shigella species in water ecosystems. Both in-silico and experimental PCR testing confirmed the absence of sequence match when tested against other bacteria, thereby confirming 100% specificity of the tested CSPs for E. coli/Shigella. The qPCR assays for each of the three CSPs provided reliable quantification for all tested enterohaemorrhagic and environmental E. coli strains, a requirement for water testing. For recreational water samples, CSP-based quantification showed a high correlation (r > 7, p < 0.01) with conventional viable E. coli enumeration. This indicates that novel CSP-based qPCR assays for E. coli can serve as robust tools for monitoring water ecosystems and other critical areas, including food monitoring.

Identification of Molecular Markers That Are Specific to the Class Thermoleophilia.

The class Thermoleophilia is one of the deep-rooting lineages within the Actinobacteria phylum and metagenomic investigation of microbial diversity suggested that species associated with the class Thermoleophilia are abundant in hot spring and soil samples. However, very few species of this class have been cultivated and characterized. Our understanding of the phylogeny and taxonomy of Thermoleophilia is solely based on 16S rRNA sequence analysis of limited cultivable representatives, but no other phenotypic or genotypic characteristics are known that can clearly discriminate members of this class from the other taxonomic units within the kingdom bacteria. This study reports phylogenomic analysis for 12 sequenced members of this class and clearly resolves the interrelationship of not yet cultivated species with reconstructed genomes and known type species. Comparative genome analysis discovered 12 CSIs in different proteins and 32 CSPs that are specific to all species of this class. In addition, a large number of CSIs or CSPs were identified to be unique to certain lineages within this class. This study represents the first and most comprehensive phylogenetic analysis of the class Thermoleophilia, and the identified CSIs and CSPs provide valuable molecular markers for the identification and delineation of species belonging to this class or its subordinate taxa.

A Phylogenomic and Molecular Markers Based Analysis of the Class Acidimicrobiia.

Recent metagenomic surveys of microbial community suggested that species associated with the class Acidimicrobiia are abundant in diverse aquatic environments such as acidic mine water, waste water sludge, freshwater, or marine habitats, but very few species have been cultivated and characterized. The current taxonomic framework of Acidimicrobiia is solely based on 16S rRNA sequence analysis of few cultivable representatives, and no molecular, biochemical, or physiological characteristics are known that can distinguish species of this class from the other bacteria. This study reports the phylogenomic analysis for 20 sequenced members of this class and reveals another three major lineages in addition to the two recognized families. Comparative analysis of the sequenced Acidimicrobiia species identified 15 conserved signature indels (CSIs) in widely distributed proteins and 26 conserved signature proteins (CSPs) that are either specific to this class as a whole or to its major lineages. This study represents the most comprehensive phylogenetic analysis of the class Acidimicrobiia and the identified CSIs and CSPs provide useful molecular markers for the identification and delineation of species belonging to this class or its subgroups.

Phylogenomic Analyses and Comparative Studies on Genomes of the Bifidobacteriales: Identification of Molecular Signatures Specific for the Order Bifidobacteriales and Its Different Subclades.

The order Bifidobacteriales comprises a diverse variety of species found in the gastrointestinal tract of humans and other animals, some of which are opportunistic pathogens, whereas a number of others exhibit health-promoting effects. However, currently very few biochemical or molecular characteristics are known which are specific for the order Bifidobacteriales, or specific clades within this order, which distinguish them from other bacteria. This study reports the results of detailed comparative genomic and phylogenetic studies on 62 genome-sequenced species/strains from the order Bifidobacteriales. In a robust phylogenetic tree for the Bifidobacteriales constructed based on 614 core proteins, a number of well-resolved clades were observed including a clade separating the Scarodvia-related genera (Scardovia clade) from the genera Bifidobacterium and Gardnerella, as well as a number of previously reported clusters of Bifidobacterium spp. In parallel, our comparative analyses of protein sequences from the Bifidobacteriales genomes have identified numerous molecular markers that are specific for this group of bacteria. Of these markers, 32 conserved signature indels (CSIs) in widely distributed proteins and 10 signature proteins are distinctive characteristics of all sequenced Bifidobacteriales species and provide novel and highly specific means for distinguishing these bacteria. In addition, multiple other molecular signatures are specific for the following clades of Bifidobacteriales: (i) 5 CSIs specific for a clade comprising of the Scardovia-related genera; (ii) 3 CSIs and 2 CSPs specific for a clade consisting of the Bifidobacterium and Gardnerella spp.; (iii) multiple other signatures demarcating a number of clusters of the B. asteroides-and B. longum- related species. The described molecular markers provide novel and reliable means for distinguishing the Bifidobacteriales and a number of their clades in molecular terms and for the classification of these bacteria. The Bifidobacteriales-specific CSIs, found in important proteins, are predicted to play important roles in modifying the cellular functions of the affected proteins. Hence, biochemical studies on the cellular functions of these CSIs could lead to discovery of novel characteristics of either all Bifidobacteriales, or specific groups of bacteria within this order. Some of the functions affected/modified by these genetic changes could also be important for the probiotic/pathogenic activities of the bifidobacteria.

Identification of distinctive molecular traits that are characteristic of the phylum "Deinococcus-Thermus" and distinguish its main constituent groups.

The phylum "Deinococcus-Thermus" contains two heavily researched groups of extremophilic bacteria: the highly radioresistant order Deinococcales and the thermophilic order Thermales. Very few characteristics are known that are uniquely shared by members of the phylum "Deinococcus-Thermus". Comprehensive phylogenetic and comparative analyses of >65 "Deinococcus-Thermus" genomes reported here have identified numerous molecular signatures in the forms of conserved signature insertions/deletions (CSIs) and conserved signature proteins (CSPs), which provide distinguishing characteristics of the phylum "Deinococcus-Thermus" and its main groups. We have identified 58 unique CSIs and 155 unique CSPs that delineate different phylogenetic groups within the phylum. Of these identified traits, 24 CSIs and 29 CSPs are characteristic of the phylum "Deinococcus-Thermus" and they provide novel and reliable means to circumscribe/describe this phylum. An additional 3 CSIs and 3 CSPs are characteristic of the order Deinococcales, and 6 CSIs and 51 CSPs are characteristic of the order Thermales. The remaining 25 CSIs and 72 CSPs identified in this study are distinctive traits of genus level groups within the phylum "Deinococcus-Thermus". The molecular characteristics identified in this work provide novel and independent support for the common ancestry of the members of the phylum "Deinococcus-Thermus" and provide a new means to distinguish the main constituent clades of the phylum. Additionally, the CSIs and CSPs identified in this work may play a role in the unique extremophilic adaptations of the members of this phylum and further functional analyses of these characteristics could provide novel biochemical insights into the unique adaptations found within the phylum "Deinococcus-Thermus".