An l-2-hydroxyglutarate biosensor based on specific transcriptional regulator LhgR

Abstractl-2-Hydroxyglutarate (l-2-HG) plays important roles in diverse physiological processes, such as carbon starvation response, tumorigenesis, and hypoxic adaptation. Despite its importance and intensively studied metabolism, regulation of l-2-HG metabolism remains poorly understood and none of regulator specifically responded to l-2-HG has been identified. Based on bacterial genomic neighborhood analysis of the gene encoding l-2-HG oxidase (LhgO), LhgR, which represses the transcription of lhgO in Pseudomonas putida W619, is identified in this study. LhgR is demonstrated to recognize l-2-HG as its specific effector molecule, and this allosteric transcription factor is then used as a biorecognition element to construct an l-2-HG-sensing FRET sensor. The l-2-HG sensor is able to conveniently monitor the concentrations of l-2-HG in various biological samples. In addition to bacterial l-2-HG generation during carbon starvation, biological function of the l-2-HG dehydrogenase and hypoxia induced l-2-HG accumulation are also revealed by using the l-2-HG sensor in human cells.

Identification and Classification of Reverse Transcriptases in Bacterial Genomes and Metagenomes

ABSTRACTReverse Transcriptases (RTs) are found in different systems including group II introns, Diversity Generating Retroelements (DGRs), retrons, CRISPR-Cas systems, and Abortive Infection (Abi) systems in prokaryotes. Different classes of RTs can play different roles, such as template switching and mobility in group II introns, spacer acquisition in CRISPR-Cas systems, mutagenic retrohoming in DGRs, programmed cell suicide in Abi systems, and recently discovered phage defense in retrons. While some classes of RTs have been studied extensively, others remain to be characterized. There is a lack of computational tools for identifying and characterizing various classes of RTs. In this study, we built a tool (called myRT) for identification and classification of prokaryotic RTs. In addition, our tool provides information about the genomic neighborhood of each RT, providing potential functional clues. We applied our tool to predict and classify RTs in all complete and draft bacterial genomes, and created a collection that can be used for exploration of putative RTs. Application of myRT to gut metagenomes showed that gut metagenomes encode proportionally more RTs related to DGRs, outnumbering retron-related RTs, as compared to the collection of reference genomes. MyRT is both available as a standalone software and also through a website.

IurV, Encoded by ORF VCA0231, Is Involved in the Regulation of Iron Uptake Genes in Vibrio cholerae

The pathogen Vibrio cholerae has multiple iron acquisition systems which allow bacteria to exploit a variety of iron sources across the different environments on which it thrives. The expression of such iron uptake systems is highly regulated, mainly by the master iron homeostasis regulator Fur but also by other mechanisms. Recently, we documented that the expression of many of the iron-responsive genes is also modulated by riboflavin. Among them, the open reading frame VCA0231, repressed both by riboflavin and iron, encodes a putative transcriptional regulator of the AraC/XylS family. Nonetheless, the genes or functions affected by this factor are unknown. In the present study, a series of in silico analyses was performed in order to identify the putative functions associated with the product of VCA0231. The STRING database predicted many iron uptake genes as functional partners for the product of VCA0231. In addition, a genomic neighborhood analysis with the Enzyme Function Initiative tools detected many Pfam families involved in iron homeostasis genetically associated with VCA0231. Moreover, a phylogenetic tree showed that other AraC/XylS members known to regulate siderophore utilization in bacteria clustered together and the product of VCA0231 localized in this cluster. This suggested that the product of VCA0231, here named IurV, is involved in the regulation of iron uptake processes. RNAseq was performed to determine the transcriptional effects of a deletion in VCA0231. A total of 52 genes were overexpressed and 21 genes were downregulated in response to the iurV deletion. Among these, several iron uptake genes and other iron homeostasis-related genes were found. Six gene ontology (GO) functional terms were enriched in the upregulated genes, of which five were related to iron metabolism. The regulatory pattern observed in the transcriptomics of a subset of genes was independently confirmed by quantitative real time PCR analysis. The results indicate that IurV is a novel regulator of the AraC/XylS family involved in the repression of iron uptake genes. Whether this effect is direct or indirect remains to be determined.

A fluorescent l-2-hydroxyglutarate biosensor

Abstractl-2-Hydroxyglutarate (l-2-HG) plays important roles in diverse physiological processes, such as carbon starvation response, tumorigenesis, and hypoxic adaptation. Despite its importance and intensively studied metabolism, regulation of l-2-HG metabolism remains poorly understood and a regulator specifically responded to l-2-HG has never been identified. Based on the genomic neighborhood analysis of the gene encoding l-2-HG oxidase (LhgO), LhgR, which represses the transcription of lhgO, was identified in Pseudomonas putida W619 in this study. LhgR was demonstrated to recognize l-2-HG as its specific effector molecule, and this allosteric transcription factor was then used as a biorecognition element for construction of l-2-HG-sensing FRET sensor. The newly developed l-2-HG sensor can conveniently monitor the concentrations of l-2-HG in various biological samples. In addition to bacterial l-2-HG generation during carbon starvation, biological functions of the l-2-HG dehydrogenase and hypoxia induced l-2-HG accumulation were also revealed by using the l-2-HG sensor in human cells.

Conserved genomic neighborhood is a strong but no perfect indicator for a direct interaction of microbial gene products

Background The order of genes in bacterial genomes is not random; for example, the products of genes belonging to an operon work together in the same pathway. The cotranslational assembly of protein complexes is deemed to conserve genomic neighborhoods even stronger than a common function. This is why a conserved genomic neighborhood can be utilized to predict, whether gene products form protein complexes. Results We were interested to assess the performance of a neighborhood-based classifier that analyzes a large number of genomes. Thus, we determined for the genes encoding the subunits of 494 experimentally verified hetero-dimers their local genomic context. In order to generate phylogenetically comprehensive genomic neighborhoods, we utilized the tools offered by the Enzyme Function Initiative. For each subunit, a sequence similarity network was generated and the corresponding genome neighborhood network was analyzed to deduce the most frequent gene product. This was predicted as interaction partner, if its abundance exceeded a threshold, which was the frequency giving rise to the maximal Matthews correlation coefficient. For the threshold of 16%, the true positive rate was 45%, the false positive rate 0.06%, and the precision 55%. For approximately 20% of the subunits, the interaction partner was not found in a neighborhood of ± 10 genes. Conclusions Our phylogenetically comprehensive analysis confirmed that complex formation is a strong evolutionary factor that conserves genome neighborhoods. On the other hand, for 55% of the cases analyzed here, classification failed. Either, the interaction partner was not present in a ± 10 gene window or was not the most frequent gene product.

Large-scale analyses of human microbiomes reveal thousands of small, novel genes and their predicted functions

Small proteins likely abound in prokaryotes, and may mediate much of the communication that occurs between organisms within a microbiome and their host. Unfortunately, small proteins are traditionally overlooked in biology, in part due to the computational and experimental difficulties in detecting them. To systematically identify novel small proteins, we carried out a large comparative genomics study on 1,773 HMP human-associated metagenomes from four different body sites (mouth, gut, skin and vagina). We describe more than four thousand conserved protein families, the majority of which are novel; ~30% of these protein families are predicted to be secreted or transmembrane. Over 90% of the small protein families have no known domain, and almost half are not represented in reference genomes, emphasizing the incompleteness of knowledge in this space. Our analysis exposes putative novel ‘housekeeping’ small protein families, including a potential novel ribosomally associated protein, as well as ‘mammalian-specific’ or ‘human-specific’ protein families. By analyzing the genomic neighborhood of small genes, we pinpoint a subset of families that are potentially associated with defense against bacteriophage. Finally, we identify families that may be subject to horizontal transfer and are thus potentially involved in adaptation of bacteria to the changing human environment. Our study suggest that small proteins are highly abundant and that those of the human microbiome, in particular, may perform diverse functions that have not been previously reported.

Tandem Duplicate Genes in Maize are Abundant and Date to Two Distinct Periods of Time

ABSTRACTTandem duplicate genes are proximally duplicated and as such occur in the same genomic neighborhood. Using the maize B73 and PH207 de novo genome assemblies, we identified thousands of tandem gene duplicates that account for ~10% of the genes. These tandem duplicates have a bimodal distribution of estimated ages corresponding to known periods of genomic instability. Tandem duplicates had a number of associated features that suggest origins in nonhomologous recombination based on smaller size distribution and higher rate of containing LTRs than non-tandem duplicates. Within relatively recent tandem duplicate genes, ~26% appear to be undergoing degeneration or divergence in function from the ancestral copy. Our results show that tandem duplicates are abundant in maize, arose in bursts throughout maize evolutionary history under multiple potential mechanisms, and may provide a substrate for novel phenotypic variation.

Expanding the catalog of cas genes with metagenomes

The CRISPR (clusters of regularly interspaced short palindromic repeats)–Cas adaptive immune system is an important defense system in bacteria, providing targeted defense against invasions of foreign nucleic acids. CRISPR–Cas systems consist of CRISPR loci and cas (CRISPR-associated) genes: sequence segments of invaders are incorporated into host genomes at CRISPR loci to generate specificity, while adjacent cas genes encode proteins that mediate the defense process. We pursued an integrated approach to identifying putative cas genes from genomes and metagenomes, combining similarity searches with genomic neighborhood analysis. Application of our approach to bacterial genomes and human microbiome datasets allowed us to significantly expand the collection of cas genes: the sequence space of the Cas9 family, the key player in the recently engineered RNA-guided platforms for genome editing in eukaryotes, is expanded by at least two-fold with metagenomic datasets. We found genes in cas loci encoding other functions, for example, toxins and antitoxins, confirming the recently discovered potential of coupling between adaptive immunity and the dormancy/suicide systems. We further identified 24 novel Cas families; one novel family contains 20 proteins, all identified from the human microbiome datasets, illustrating the importance of metagenomics projects in expanding the diversity of cas genes.