BackgroundEndolithic microbes in coral skeletons are known to be a nutrient source for the coral host. In addition to aerobic endolithic algae and Cyanobacteria, which are usually described in the various corals and form a green layer beneath coral tissues, the anaerobic photoautotrophic green sulfur bacteria (GSB) Prosthecochloris is dominant in the skeleton of Isopora palifera. However, due to inherent challenges in studying anaerobic microbes in coral skeleton, the reason for its niche preference and function are largely unknown.ResultsThis study characterized a diverse and dynamic community of endolithic microbes shaped by the availability of light and oxygen. In addition, anaerobic bacteria isolated from the coral skeleton were cultured for the first time to experimentally clarify the role of these GSB. This characterization includes GSB’s abundance, genetic and genomic profiles, organelle structure, and specific metabolic functions and activity. Our results explain the advantages endolithic GSB receive from living in coral skeletons, the potential metabolic role of a clade of coral-associated Prosthecochloris (CAP) in the skeleton, and the nitrogen fixation ability of CAP.ConclusionWe suggest that the endolithic microbial community in coral skeletons is diverse and dynamic and that light and oxygen are two crucial factors for shaping it. This study is the first to demonstrate the ability of nitrogen uptake by specific coral-associated endolithic bacteria and shed light on the role of endolithic bacteria in coral skeletons.Electronic supplementary materialThe online version of this article (10.1186/s40168-018-0616-z) contains supplementary material, which is available to authorized users.

The environmental release and fate of estrogens are becoming an increasing public concern. Bacterial degradation has been considered the main process for eliminating estrogens from wastewater treatment plants. Various bacterial isolates are reportedly capable of aerobic estrogen degradation, and several estrogen degradation pathways have been proposed in proteobacteria and actinobacteria. However, the ecophysiological relevance of estrogen-degrading bacteria in the environment is unclear. In this study, we investigated the estrogen degradation pathway and corresponding degraders in activated sludge collected from the Dihua Sewage Treatment Plant, Taipei, Taiwan. Cultivation-dependent and cultivation-independent methods were used to assess estrogen biodegradation in the collected activated sludge. Estrogen metabolite profile analysis revealed the production of pyridinestrone acid and two A/B-ring cleavage products in activated sludge incubated with estrone (1 mM), which are characteristic of the 4,5- pathway. PCR-based functional assays detected sequences closely related to alphaproteobacterial , a key gene of the 4,5- pathway. Metagenomic analysis suggested that spp. are major estrogen degraders in estrone-amended activated sludge. sp. strain SLCC, an estrone-degrading alphaproteobacterium, was isolated from the examined activated sludge. The general physiology and metabolism of this strain were characterized. Pyridinestrone acid and the A/B-ring cleavage products were detected in estrone-grown strain SLCC cultures. The production of pyridinestrone acid was also observed during the aerobic incubation of strain SLCC with 3.7 nM (1 μg/liter) estrone. This concentration is close to that detected in many natural and engineered aquatic ecosystems. The presented data suggest the ecophysiological relevance of spp. in activated sludge. Estrogens, which persistently contaminate surface water worldwide, have been classified as endocrine disruptors and human carcinogens. We contribute new knowledge on the major estrogen biodegradation pathway and estrogen degraders in wastewater treatment plants. This study considerably advances the understanding of environmental estrogen biodegradation, which is instrumental for the efficient elimination of these hazardous pollutants. Moreover, this study substantially improves the understanding of microbial estrogen degradation in the environment.

Lactobacillus casei, Lactobacillus paracasei, and Lactobacillus rhamnosus are phenotypically and genotypically closely related, and together comprise the L. casei group. Although the strains of this group are commercially valuable as probiotics, the taxonomic status and nomenclature of the L. casei group have long been contentious because of the difficulties in identifying these three species by using the most frequently used genotypic methodology of 16S rRNA gene sequencing. Long used as the gold standard for species classification, DNA–DNA hybridization is laborious, requires expert skills, and is difficult to use routinely in laboratories. Currently, genome-based comparisons, including average nucleotide identity (ANI) and digital DNA–DNA hybridization (dDDH), are commonly applied to bacterial taxonomy as alternatives to the gold standard method for the demarcating phylogenetic relationships. To establish quick and accurate methods for identifying strains in the L. casei group at the species and subspecies levels, we developed species- and subspecies-specific identification methods based on housekeeping gene sequences and whole-cell matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) spectral pattern analysis. By phylogenetic analysis based on concatenated housekeeping gene sequences (dnaJ, dnaK, mutL, pheS, and yycH), 53 strains were separated into four clusters corresponding to the four species: L. casei, L. paracasei and L. rhamnosus, and Lactobacillus chiayiensis sp. nov. A multiplex minisequencing assay using single nucleotide polymorphism (SNP)-specific primers based on the dnaK gene sequences and species-specific primers based on the mutL gene sequences provided high resolution that enabled the strains at the species level to be identified as L. casei, L. paracasei, and L. rhamnosus. By MALDI-TOF MS analysis coupled with an internal database and ClinProTools software, species- and subspecies-level L. casei group strains were identified based on reliable scores and species- and subspecies-specific MS peaks. The L. paracasei strains were distinguished clearly at the subspecies level based on subspecies-specific MS peaks. This article describes the rapid and accurate methods used for identification and classification of strains in the L. casei group based on housekeeping gene sequences and MALDI-TOF MS analysis as well as the novel speciation of this group including L. chiayiensis sp. nov. and ‘Lactobacillus zeae’ by genome-based methods.

Using common taxonomic methods such as 16S rDNA sequencing and physiological and biochemical analysis to identify members of the Lactobacillus casei group (LCG) is time-consuming, expensive and inaccurate. In this study, we applied matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) to rapid discriminate LCG strains by creating an analytical in-house database (IHDB) and to develop a classification model for subspecies-level differentiation based on MS biomarkers using ClinProTools bioinformatics software (Bruker Daltonics, Billerica, MA). Genotypic methods (housekeeping gene sequencing and species-specific PCR) were also established to validate the MALDI-TOF MS platform. A total of 48 LCG reference strains were correctly identified at the species level (mean score: 2.45 ± 0.1) by using MALDI-TOF MS with an IHDB and had high score values, which was in accordance with results from mutL gene sequencing and specific PCR-based methods. However, one strain that was identified as L. casei had a relatively low score value (2.02 ± 0.02), lower sequence similarities (mutL: 90.4%), and failed to amplify a species-specific amplicon; it may therefore represent an undescribed novel species. In addition, after implementation of the classification model (based on 2 biomarker peaks: m/z 4,930 and 5,303), L. paracasei strains could be clearly and easily differentiated to the subspecies level. Afterward, 7 LCG-related isolates from different probiotic samples were analyzed and accurately identified. Our data demonstrate the high-resolution performance of MALDI-TOF MS for fast and accurate demarcation of LCG strains when used with an IHDB coupled to ClinProTools; this methodology can serve as an alternative for quality control of probiotic products.

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