Deeply branching Bacillota species exhibit atypical Gram-negative staining.

The Gram staining method differentiates bacteria based on their cell envelope structure, with the monoderm and diderm cell envelope types traditionally being synonymous with Gram-positive and Gram-negative stain results, respectively. Monoderms have a single phospholipid membrane surrounded by a thick layer of peptidoglycan, while diderms have a lipopolysaccharide outer membrane exterior to a thin peptidoglycan layer. The Bacillota (formerly Firmicutes) phylum has members with both cell wall types, and recent phylogenetic analyses have shown that monoderm Bacillota evolved from diderm ancestors on multiple occasions. Here, we compiled Gram staining and ultrastructural data for Bacillota species with complete genomes to further investigate the evolution of Gram-positive and Gram-negative cell wall types. The results indicate that many deeply branching lineages at the root of Bacillota phylum stain Gram-negative but do not harbor genes for outer membrane protein or lipopolysaccharide biosynthesis. Phylogenetic reconstructions suggest that several deeply branching Bacillota species have retained a thin peptidoglycan layer in their cell walls, which was inherited from a diderm ancestor. Taxa with this atypical Gram-negative-staining cell wall structure include the thermophilic anaerobe Symbiobacterium thermophilum and members of the Desulfotomaculia, Syntrophamonadia, Desulfitobacteriia, Thermosediminibacteria, and Thermoanaerobacteria. Using Gram-staining results as a proxy for cell wall thickness, our analysis indicates that several independent peptidoglycan thickening events may have occurred in the evolution of the Gram-positive cell envelope. IMPORTANCE: In this study, we examined the evolution of bacterial cell envelopes, specifically focusing on Gram-positive and Gram-negative cell wall types in the Bacillota phylum. Our results indicate that certain bacteria can stain Gram-negative despite having a monoderm cell wall structure, thus challenging the conventional interpretation of Gram-staining results. Our observations also question the assumption that Gram-negative staining is always indicative of a diderm structure. These findings have broader implications for understanding how and when cell walls thicken during the evolution of bacterial cell envelopes.

Genome sequence of Methylobacterium fujisawaense strain C14, isolated from concrete.

Methylobacterium fujisawaense strain C14 was isolated from a weathered concrete cylinder. Using PacBio sequencing, we generated a complete genome for strain C14, which includes one circular chromosome (6,656,731 bp) and six putative plasmids (35,452 to 85,428 bp).

Genome sequences of four bacterial strains isolated from organofluorine enrichment cultures.

Because fluorinated organic compounds are broadly used and highly persistent, microbes isolated from wastewater may be able to degrade these contaminants. Here, we report the genome sequences of Flavobacterium sp. str. WV_118_3, Nocardioides sp. str. WV_118_6, Ochrobactrum anthropi str. WV_118_8, and Sphingomonas sp. str. VL_57B, isolated from wastewater.

Mixed waste contamination selects for a mobile genetic element population enriched in multiple heavy metal resistance genes.

Mobile genetic elements (MGEs) like plasmids, viruses, and transposable elements can provide fitness benefits to their hosts for survival in the presence of environmental stressors. Heavy metal resistance genes (HMRGs) are frequently observed on MGEs, suggesting that MGEs may be an important driver of adaptive evolution in environments contaminated with heavy metals. Here, we report the meta-mobilome of the heavy metal-contaminated regions of the Oak Ridge Reservation subsurface. This meta-mobilome was compared with one derived from samples collected from unimpacted regions of the Oak Ridge Reservation subsurface. We assembled 1615 unique circularized DNA elements that we propose to be MGEs. The circular elements from the highly contaminated subsurface were enriched in HMRG clusters relative to those from the nearby unimpacted regions. Additionally, we found that these HMRGs were associated with Gamma and Betaproteobacteria hosts in the contaminated subsurface and potentially facilitate the persistence and dominance of these taxa in this region. Finally, the HMRGs were associated with conjugative elements, suggesting their potential for future lateral transfer. We demonstrate how our understanding of MGE ecology, evolution, and function can be enhanced through the genomic context provided by completed MGE assemblies.

Genomic and environmental controls on Castellaniella biogeography in an anthropogenically disturbed subsurface.

Castellaniella species have been isolated from a variety of mixed-waste environments including the nitrate and multiple metal-contaminated subsurface at the Oak Ridge Reservation (ORR). Previous studies examining microbial community composition and nitrate removal at ORR during biostimulation efforts reported increased abundances of members of the Castellaniella genus concurrent with increased denitrification rates. Thus, we asked how genomic and abiotic factors control the Castellaniella biogeography at the site to understand how these factors may influence nitrate transformation in an anthropogenically impacted setting. We report the isolation and characterization of several Castellaniella strains from the ORR subsurface. Five of these isolates match at 100% identity (at the 16S rRNA gene V4 region) to two Castellaniella amplicon sequence variants (ASVs), ASV1 and ASV2, that have persisted in the ORR subsurface for at least 2 decades. However, ASV2 has consistently higher relative abundance in samples taken from the site and was also the dominant blooming denitrifier population during a prior biostimulation effort. We found that the ASV2 representative strain has greater resistance to mixed metal stress than the ASV1 representative strains. We attribute this resistance, in part, to the large number of unique heavy metal resistance genes identified on a genomic island in the ASV2 representative genome. Additionally, we suggest that the relatively lower fitness of ASV1 may be connected to the loss of the nitrous oxide reductase (nos) operon (and associated nitrous oxide reductase activity) due to the insertion at this genomic locus of a mobile genetic element carrying copper resistance genes. This study demonstrates the value of integrating genomic, environmental, and phenotypic data to characterize the biogeography of key microorganisms in contaminated sites.