Deciphering the Streptococcus mutans essentialome: multi-omic resolution of hypothetical genes and identification of a functional RocS equivalent

Genomic landscape of 340 virulent Acinetobacter bacteriophages reveals anti-CRISPR-enriched candidates for therapeutic prioritization.

PURPOSE Carbapenem-resistant Acinetobacter baumannii (CRAB) represents a critical global health threat for which existing antibiotics are increasingly inadequate. This study aimed to establish a comprehensive genomic framework for the rational prioritization of virulent Acinetobacter bacteriophages as therapeutic candidates. METHODS We performed large-scale comparative genomic analysis of 340 virulent Acinetobacter bacteriophages, integrating phylogenetic reconstruction, pangenome analysis, CRISPR spacer-based host interaction mapping, Anti-CRISPR protein identification, and systematic antimicrobial resistance (AMR) gene screening. RESULTS Genome sizes spanned a nearly 20-fold range, with a significant negative correlation between genome size and GC content (R² = 0.139, ρ = -0.630). Phylogenetic analysis revealed extensive divergence across multiple lineages with no dominant clade. Pangenome analysis identified 20,982 unique protein families, of which 76.2% were cloud genes, confirming a highly open genome architecture. CRISPR spacer matching yielded 1,480 high-confidence matches across 100 phage genomes, providing molecular evidence of broad historical infectivity. Anti-CRISPR profiling identified Acinetobacter phage XC1 as an exceptional therapeutic candidate harboring 55 predicted Anti-CRISPR proteins with canonical regulatory locus architecture. AMR screening identified 21 distinct AMR gene homologs (Loose RGI hits, 22.5 to 47.1% amino acid identity) distributed heterogeneously across the dataset, confirming abundant therapeutically clean candidates while flagging a subset warranting further scrutiny before therapeutic exclusion. CONCLUSION These findings provide a multi-criteria genomic framework for rational phage candidate prioritization against multidrug-resistant Acinetobacter infections, with direct implications for evidence-based phage therapy development.

Predicting the protein interaction landscape of a mycobacterial pathogen

Methyltransferases in Candidate Phyla Radiation: A Weapon or a Simple Shield?

A Vibrio parahaemolyticus transcriptome map captures conserved and specific regulators

How bacterial sRNAs arise, evolve, and decay is poorly understood, despite their importance as posttranscriptional regulators. Here, we provide a high-resolution transcriptome map and curated sRNA set for the pathogen Vibrio parahaemolyticus. We identify over 100 sRNAs, including broadly conserved, lineage-specific, and previously unidentified transcripts, as well as dual-function (regulatory/coding) sRNAs. We show that broadly conserved VcrX represses chitin utilization genes and may regulate Vibrio Spot 42, which we also confirm is translated. We expand on FlaX regulation of polar flagella by demonstrating that it differentially activates/represses downstream flagellins across the genus, and identifying a potential FlaX sponge in only some clades. We further show that RyhB is translated in V. parahaemolyticus but not V. cholerae and encodes a Cys-rich small protein. Together, this provides a resource for Vibrio and foundation for comparative study of posttranscriptional regulation to understand how sRNAs and their networks evolve.

Chromosome-scale CRISPR screening reveals secretory pathway genes as drivers of aneuploidy-mediated antifungal tolerance

The gain or loss of chromosomes in eukaryotes often drives aberrant phenotypes by altering the expression levels of hundreds or thousands of genes. In the case of beneficial aneuploidies, the genetic basis of fitness improvement has rarely been pinpointed, and identifying the causal genes remains a major challenge in engineering biology. The leading cause of human fungal infections, Candida albicans, frequently acquires extra copies of chromosome R (ChrR) following exposure to azole antifungal drugs, resulting in heightened antifungal tolerance. Here, we combine RNA-seq with parallel chromosome-wide CRISPR activation (CRISPRa) and CRISPR interference (CRISPRi) screens to systematically profile the ChrR genes contributing to azole tolerance. Using multiplexed CRISPR-dCas12a tools, we further characterize the combinatorial effects of candidate genes and uncover a central role for post-Golgi secretory trafficking in antifungal tolerance. Specifically, we demonstrate that the secretory pathway regulators SEC4 and YPT31 are both necessary and sufficient for ChrR-mediated azole tolerance. By leveraging a large-scale CRISPRa screen in a fungal pathogen, our work functionally dissects one of the most common aneuploidies observed in C. albicans, provides mechanistic insight into the molecular basis of antifungal tolerance, and establishes a generalizable framework for studying aneuploidy-mediated phenotypes across eukaryotic organisms.

Minimal organism JCVI-syn3.0, an engineered derivative of Mycoplasma mycoides, encodes an assortment of unrecognized membrane transporters and immunity factors

ABSTRACT In 2016, researchers at the J. Craig Venter Institute produced JCVI-Syn3.0, a derivative of Mycoplasma mycoides subsp. capri Large Colony strain GM12 with a re-engineered, minimized genome. The minimal synthetic genome of 532 kbp encodes 473 genes, selected from ∼910 parent genes on the basis of their indispensability, that is, a lethal phenotype caused by a knockout of each such gene. Among these genes, 438 genes encode proteins, of which ∼150 were not assigned a molecular function or predicted cellular role. A decade later, despite community annotation efforts, 50–60 of these putative proteins still lack any assigned function. We analyzed the intrinsic sequence characteristics, evolutionary conservation, and three-dimensional AlphaFold models of this least understood subset of the JCVI-Syn3.0 genes. We predicted conserved sequence motifs and structural features and inferred molecular functions of six proteins likely involved in membrane transport or membrane interactions, and six proteins that apparently belong to bacterial immunity systems, such as putative antitoxins, an essential toxin-like protein, and a member of a previously unnoticed clade of Cas4-like nucleases. The spatial models produced for this challenging data set by the AlphaFold pipeline help dissect proteins into discrete domains, which are often more convenient to analyze by sequence similarity than whole proteins. In several cases, AlphaFold models also enable functional inference more directly, based on spatial matching of the models to the databases of known protein structures. IMPORTANCE The smallest synthetic genome, JCVI-Syn3.0, encodes 438 proteins. JCVI-Syn3.0 was obtained by combining all genes whose deletion from a larger parental genome of M. mycoides subsp. capri Large Colony strain GM12 causes a complete stop or a significant delay in bacterial growth. Thus, nearly every protein in this minimal genome is essential; however, we do not know what ∼15% of these genes do. Using computational methods, we predicted molecular functions for about a quarter of these enigmatic proteins, including several proteins that are related to the components of recently discovered bacterial immunity systems. The smallest synthetic genome, JCVI-Syn3.0, encodes 438 proteins. JCVI-Syn3.0 was obtained by combining all genes whose deletion from a larger parental genome of M. mycoides subsp. capri Large Colony strain GM12 causes a complete stop or a significant delay in bacterial growth. Thus, nearly every protein in this minimal genome is essential; however, we do not know what ∼15% of these genes do. Using computational methods, we predicted molecular functions for about a quarter of these enigmatic proteins, including several proteins that are related to the components of recently discovered bacterial immunity systems.