Single-cell approaches in human microbiome research.

Microbial culturing and meta-omic profiling technologies have significantly advanced our understanding of the taxonomic and functional variation of the human microbiome and its impact on host processes. The next increase in resolution will come by understanding the role of low-abundant and less-prevalent bacteria and the study of individual cell behaviors that underlie the complexity of microbial ecosystems. To this aim, single-cell techniques are being rapidly developed to isolate, culture, and characterize the genomes and transcriptomes of individual microbes in complex communities. Here, we discuss how these single-cell technologies are providing unique insights into the biology and behavior of human microbiomes.

Distinguishing between productive and abortive promoters using a random forest classifier in Mycoplasma pneumoniae.

Distinguishing between promoter-like sequences in bacteria that belong to true or abortive promoters, or to those that do not initiate transcription at all, is one of the important challenges in transcriptomics. To address this problem, we have studied the genome-reduced bacterium Mycoplasma pneumoniae, for which the RNAs associated with transcriptional start sites have been recently experimentally identified. We determined the contribution to transcription events of different genomic features: the -10, extended -10 and -35 boxes, the UP element, the bases surrounding the -10 box and the nearest-neighbor free energy of the promoter region. Using a random forest classifier and the aforementioned features transformed into scores, we could distinguish between true, abortive promoters and non-promoters with good -10 box sequences. The methods used in this characterization of promoters can be extended to other bacteria and have important applications for promoter design in bacterial genome engineering.

Defining a minimal cell: essentiality of small ORFs and ncRNAs in a genome-reduced bacterium.

Identifying all essential genomic components is critical for the assembly of minimal artificial life. In the genome-reduced bacterium Mycoplasma pneumoniae, we found that small ORFs (smORFs; < 100 residues), accounting for 10% of all ORFs, are the most frequently essential genomic components (53%), followed by conventional ORFs (49%). Essentiality of smORFs may be explained by their function as members of protein and/or DNA/RNA complexes. In larger proteins, essentiality applied to individual domains and not entire proteins, a notion we could confirm by expression of truncated domains. The fraction of essential non-coding RNAs (ncRNAs) non-overlapping with essential genes is 5% higher than of non-transcribed regions (0.9%), pointing to the important functions of the former. We found that the minimal essential genome is comprised of 33% (269,410 bp) of the M. pneumoniae genome. Our data highlight an unexpected hidden layer of smORFs with essential functions, as well as non-coding regions, thus changing the focus when aiming to define the minimal essential genome.

Comprehensive methylome characterization of Mycoplasma genitalium and Mycoplasma pneumoniae at single-base resolution.

In the bacterial world, methylation is most commonly associated with restriction-modification systems that provide a defense mechanism against invading foreign genomes. In addition, it is known that methylation plays functionally important roles, including timing of DNA replication, chromosome partitioning, DNA repair, and regulation of gene expression. However, full DNA methylome analyses are scarce due to a lack of a simple methodology for rapid and sensitive detection of common epigenetic marks (ie N(6)-methyladenine (6 mA) and N(4)-methylcytosine (4 mC)), in these organisms. Here, we use Single-Molecule Real-Time (SMRT) sequencing to determine the methylomes of two related human pathogen species, Mycoplasma genitalium G-37 and Mycoplasma pneumoniae M129, with single-base resolution. Our analysis identified two new methylation motifs not previously described in bacteria: a widespread 6 mA methylation motif common to both bacteria (5'-CTAT-3'), as well as a more complex Type I m6A sequence motif in M. pneumoniae (5'-GAN(7)TAY-3'/3'-CTN(7)ATR-5'). We identify the methyltransferase responsible for the common motif and suggest the one involved in M. pneumoniae only. Analysis of the distribution of methylation sites across the genome of M. pneumoniae suggests a potential role for methylation in regulating the cell cycle, as well as in regulation of gene expression. To our knowledge, this is one of the first direct methylome profiling studies with single-base resolution from a bacterial organism.

Assessing the hodgepodge of non-mapped reads in bacterial transcriptomes: real or artifactual RNA chimeras?

BACKGROUND: RNA sequencing methods have already altered our view of the extent and complexity of bacterial and eukaryotic transcriptomes, revealing rare transcript isoforms (circular RNAs, RNA chimeras) that could play an important role in their biology. RESULTS: We performed an analysis of chimera formation by four different computational approaches, including a custom designed pipeline, to study the transcriptomes of M. pneumoniae and P. aeruginosa, as well as mixtures of both. We found that rare transcript isoforms detected by conventional pipelines of analysis could be artifacts of the experimental procedure used in the library preparation, and that they are protocol-dependent. CONCLUSION: By using a customized pipeline we show that optimal library preparation protocol and the pipeline to analyze the results are crucial to identify real chimeric RNAs.

Bacteria in metastatic sites: Unveiling hidden players in cancer progression.

Bacteria exhibit key features of cancer metastasis, such as motility, invasion, and modulation of the tumor microenvironment. They migrate through lymphatic and blood systems, invade metastatic tissues, and alter local microenvironments to support metastatic growth. Bacteria also shape the tumor microenvironment, affecting immune responses and inflammation, which influence tumor progression and therapy response. While they hold therapeutic potential, challenges like contamination and complex characterization persist, necessitating advanced sequencing and research for clinical application.

Microbiome confounders and quantitative profiling challenge predicted microbial targets in colorectal cancer development.

Despite substantial progress in cancer microbiome research, recognized confounders and advances in absolute microbiome quantification remain underused; this raises concerns regarding potential spurious associations. Here we study the fecal microbiota of 589 patients at different colorectal cancer (CRC) stages and compare observations with up to 15 published studies (4,439 patients and controls total). Using quantitative microbiome profiling based on 16S ribosomal RNA amplicon sequencing, combined with rigorous confounder control, we identified transit time, fecal calprotectin (intestinal inflammation) and body mass index as primary microbial covariates, superseding variance explained by CRC diagnostic groups. Well-established microbiome CRC targets, such as Fusobacterium nucleatum, did not significantly associate with CRC diagnostic groups (healthy, adenoma and carcinoma) when controlling for these covariates. In contrast, the associations of Anaerococcus vaginalis, Dialister pneumosintes, Parvimonas micra, Peptostreptococcus anaerobius, Porphyromonas asaccharolytica and Prevotella intermedia remained robust, highlighting their future target potential. Finally, control individuals (age 22-80 years, mean 57.7 years, standard deviation 11.3) meeting criteria for colonoscopy (for example, through a positive fecal immunochemical test) but without colonic lesions are enriched for the dysbiotic Bacteroides2 enterotype, emphasizing uncertainties in defining healthy controls in cancer microbiome research. Together, these results indicate the importance of quantitative microbiome profiling and covariate control for biomarker identification in CRC microbiome studies.

Benchmarking microbiome transformations favors experimental quantitative approaches to address compositionality and sampling depth biases.

While metagenomic sequencing has become the tool of preference to study host-associated microbial communities, downstream analyses and clinical interpretation of microbiome data remains challenging due to the sparsity and compositionality of sequence matrices. Here, we evaluate both computational and experimental approaches proposed to mitigate the impact of these outstanding issues. Generating fecal metagenomes drawn from simulated microbial communities, we benchmark the performance of thirteen commonly used analytical approaches in terms of diversity estimation, identification of taxon-taxon associations, and assessment of taxon-metadata correlations under the challenge of varying microbial ecosystem loads. We find quantitative approaches including experimental procedures to incorporate microbial load variation in downstream analyses to perform significantly better than computational strategies designed to mitigate data compositionality and sparsity, not only improving the identification of true positive associations, but also reducing false positive detection. When analyzing simulated scenarios of low microbial load dysbiosis as observed in inflammatory pathologies, quantitative methods correcting for sampling depth show higher precision compared to uncorrected scaling. Overall, our findings advocate for a wider adoption of experimental quantitative approaches in microbiome research, yet also suggest preferred transformations for specific cases where determination of microbial load of samples is not feasible.

Clinical practices underlie COVID-19 patient respiratory microbiome composition and its interactions with the host.

Understanding the pathology of COVID-19 is a global research priority. Early evidence suggests that the respiratory microbiome may be playing a role in disease progression, yet current studies report contradictory results. Here, we examine potential confounders in COVID-19 respiratory microbiome studies by analyzing the upper (n = 58) and lower (n = 35) respiratory tract microbiome in well-phenotyped COVID-19 patients and controls combining microbiome sequencing, viral load determination, and immunoprofiling. We find that time in the intensive care unit and type of oxygen support, as well as associated treatments such as antibiotic usage, explain the most variation within the upper respiratory tract microbiome, while SARS-CoV-2 viral load has a reduced impact. Specifically, mechanical ventilation is linked to altered community structure and significant shifts in oral taxa previously associated with COVID-19. Single-cell transcriptomics of the lower respiratory tract of COVID-19 patients identifies specific oral bacteria in physical association with proinflammatory immune cells, which show higher levels of inflammatory markers. Overall, our findings suggest confounders are driving contradictory results in current COVID-19 microbiome studies and careful attention needs to be paid to ICU stay and type of oxygen support, as bacteria favored in these conditions may contribute to the inflammatory phenotypes observed in severe COVID-19 patients.

Determination of the Gene Regulatory Network of a Genome-Reduced Bacterium Highlights Alternative Regulation Independent of Transcription Factors.

Here, we determined the relative importance of different transcriptional mechanisms in the genome-reduced bacterium Mycoplasma pneumoniae, by employing an array of experimental techniques under multiple genetic and environmental perturbations. Of the 143 genes tested (21% of the bacterium's annotated proteins), only 55% showed an altered phenotype, highlighting the robustness of biological systems. We identified nine transcription factors (TFs) and their targets, representing 43% of the genome, and 16 regulators that indirectly affect transcription. Only 20% of transcriptional regulation is mediated by canonical TFs when responding to perturbations. Using a Random Forest, we quantified the non-redundant contribution of different mechanisms such as supercoiling, metabolic control, RNA degradation, and chromosome topology to transcriptional changes. Model-predicted gene changes correlate well with experimental data in 95% of the tested perturbations, explaining up to 70% of the total variance when also considering noise. This analysis highlights the importance of considering non-TF-mediated regulation when engineering bacteria.

Integrated culturing, modeling and transcriptomics uncovers complex interactions and emergent behavior in a three-species synthetic gut community.

The composition of the human gut microbiome is well resolved, but predictive understanding of its dynamics is still lacking. Here, we followed a bottom-up strategy to explore human gut community dynamics: we established a synthetic community composed of three representative human gut isolates (Roseburia intestinalis L1-82, Faecalibacterium prausnitzii A2-165 and Blautia hydrogenotrophica S5a33) and explored their interactions under well-controlled conditions in vitro. Systematic mono- and pair-wise fermentation experiments confirmed competition for fructose and cross-feeding of formate. We quantified with a mechanistic model how well tri-culture dynamics was predicted from mono-culture data. With the model as reference, we demonstrated that strains grown in co-culture behaved differently than those in mono-culture and confirmed their altered behavior at the transcriptional level. In addition, we showed with replicate tri-cultures and simulations that dominance in tri-culture sensitively depends on the initial conditions. Our work has important implications for gut microbial community modeling as well as for ecological interaction detection from batch cultures.

Alternative transcriptional regulation in genome-reduced bacteria.

Transcription is a core process of bacterial physiology, and as such it must be tightly controlled, so that bacterial cells maintain steady levels of each RNA molecule in homeostasis and modify them in response to perturbations. The major regulators of transcription in bacteria (and in eukaryotes) are transcription factors. However, in genome-reduced bacteria, the limited number of these proteins is insufficient to explain the variety of responses shown upon changes in their environment. Thus, alternative regulators may play a central role in orchestrating RNA levels in these microorganisms. These alternative mechanisms rely on intrinsic features within DNA and RNA molecules, suggesting they are ancestral mechanisms shared among bacteria that could have an increased relevance on transcriptional regulation in minimal cells. In this review, we summarize the alternative elements that can regulate transcript abundance in genome-reduced bacteria and how they contribute to the RNA homeostasis at different levels.

The yin-yang of kinase activation and unfolding explains the peculiarity of Val600 in the activation segment of BRAF.

Many driver mutations in cancer are specific in that they occur at significantly higher rates than - presumably - functionally alternative mutations. For example, V600E in the BRAF hydrophobic activation segment (AS) pocket accounts for >95% of all kinase mutations. While many hypotheses tried to explain such significant mutation patterns, conclusive explanations are lacking. Here, we use experimental and in silico structure-energy statistical analyses, to elucidate why the V600E mutation, but no other mutation at this, or any other positions in BRAF's hydrophobic pocket, is predominant. We find that BRAF mutation frequencies depend on the equilibrium between the destabilization of the hydrophobic pocket, the overall folding energy, the activation of the kinase and the number of bases required to change the corresponding amino acid. Using a random forest classifier, we quantitatively dissected the parameters contributing to BRAF AS cancer frequencies. These findings can be applied to genome-wide association studies and prediction models.

Insights into the Mechanisms of Basal Coordination of Transcription Using a Genome-Reduced Bacterium.

Coordination of transcription in bacteria occurs at supra-operonic scales, but the extent, specificity, and mechanisms of such regulation are poorly understood. Here, we tackle this problem by profiling the transcriptome of the model organism Mycoplasma pneumoniae across 115 growth conditions. We identify three qualitatively different levels of co-expression corresponding to distinct relative orientations and intergenic properties of adjacent genes. We reveal that the degree of co-expression between co-directional adjacent operons, and more generally between genes, is tightly related to their capacity to be transcribed en bloc into the same mRNA. We further show that this genome-wide pervasive transcription of adjacent genes and operons is specifically repressed by DNA regions preferentially bound by RNA polymerases, by intrinsic terminators, and by large intergenic distances. Taken together, our findings suggest that the basal coordination of transcription is mediated by the physical entities and mechanical properties of the transcription process itself, and that operon-like behaviors may strongly vary from condition to condition.

Bacterial antisense RNAs are mainly the product of transcriptional noise.

cis-Encoded antisense RNAs (asRNAs) are widespread along bacterial transcriptomes. However, the role of most of these RNAs remains unknown, and there is an ongoing discussion as to what extent these transcripts are the result of transcriptional noise. We show, by comparative transcriptomics of 20 bacterial species and one chloroplast, that the number of asRNAs is exponentially dependent on the genomic AT content and that expression of asRNA at low levels exerts little impact in terms of energy consumption. A transcription model simulating mRNA and asRNA production indicates that the asRNA regulatory effect is only observed above certain expression thresholds, substantially higher than physiological transcript levels. These predictions were verified experimentally by overexpressing nine different asRNAs in Mycoplasma pneumoniae. Our results suggest that most of the antisense transcripts found in bacteria are the consequence of transcriptional noise, arising at spurious promoters throughout the genome.

Comparative "-omics" in Mycoplasma pneumoniae Clinical Isolates Reveals Key Virulence Factors.

The human respiratory tract pathogen M. pneumoniae is one of the best characterized minimal bacterium. Until now, two main groups of clinical isolates of this bacterium have been described (types 1 and 2), differing in the sequence of the P1 adhesin gene. Here, we have sequenced the genomes of 23 clinical isolates of M. pneumoniae. Studying SNPs, non-synonymous mutations, indels and genome rearrangements of these 23 strains and 4 previously sequenced ones, has revealed new subclasses in the two main groups, some of them being associated with the country of isolation. Integrative analysis of in vitro gene essentiality and mutation rates enabled the identification of several putative virulence factors and antigenic proteins; revealing recombination machinery, glycerol metabolism and peroxide production as possible factors in the genetics and physiology of these pathogenic strains. Additionally, the transcriptomes and proteomes of two representative strains, one from each of the two main groups, have been characterized to evaluate the impact of mutations on RNA and proteins levels. This study has revealed that type 2 strains show higher expression levels of CARDS toxin, a protein recently shown to be one of the major factors of inflammation. Thus, we propose that type 2 strains could be more toxigenic than type 1 strains of M. pneumoniae.