Diversity in CO2-Concentrating Mechanisms among Chemolithoautotrophs from the Genera Hydrogenovibrio, Thiomicrorhabdus, and Thiomicrospira, Ubiquitous in Sulfidic Habitats Worldwide.

Members of the genera Hydrogenovibrio, Thiomicrospira, and Thiomicrorhabdus fix carbon at hydrothermal vents, coastal sediments, hypersaline lakes, and other sulfidic habitats. The genome sequences of these ubiquitous and prolific chemolithoautotrophs suggest a surprising diversity of mechanisms for the uptake and fixation of dissolved inorganic carbon (DIC); these mechanisms are verified here. Carboxysomes are apparent in the transmission electron micrographs of most of these organisms but are lacking in Thiomicrorhabdus sp. strain Milos-T2 and Thiomicrorhabdus arctica, and the inability of Thiomicrorhabdus sp. strain Milos-T2 to grow under low-DIC conditions is consistent with the absence of carboxysome loci in its genome. For the remaining organisms, genes encoding potential DIC transporters from four evolutionarily distinct families (Tcr_0853 and Tcr_0854, Chr, SbtA, and SulP) are located downstream of carboxysome loci. Transporter genes collocated with carboxysome loci, as well as some homologs located elsewhere on the chromosomes, had elevated transcript levels under low-DIC conditions, as assayed by reverse transcription-quantitative PCR (qRT-PCR). DIC uptake was measureable via silicone oil centrifugation when a representative of each of the four types of transporter was expressed in Escherichia coli The expression of these genes in the carbonic anhydrase-deficient E. coli strain EDCM636 enabled it to grow under low-DIC conditions, a result consistent with DIC transport by these proteins. The results from this study expand the range of DIC transporters within the SbtA and SulP transporter families, verify DIC uptake by transporters encoded by Tcr_0853 and Tcr_0854 and their homologs, and introduce DIC as a potential substrate for transporters from the Chr family.IMPORTANCE Autotrophic organisms take up and fix DIC, introducing carbon into the biological portion of the global carbon cycle. The mechanisms for DIC uptake and fixation by autotrophic Bacteria and Archaea are likely to be diverse but have been well characterized only for "Cyanobacteria" Based on genome sequences, members of the genera Hydrogenovibrio, Thiomicrospira, and Thiomicrorhabdus have a variety of mechanisms for DIC uptake and fixation. We verified that most of these organisms are capable of growing under low-DIC conditions, when they upregulate carboxysome loci and transporter genes collocated with these loci on their chromosomes. When these genes, which fall into four evolutionarily independent families of transporters, are expressed in E. coli, DIC transport is detected. This expansion in known DIC transporters across four families, from organisms from a variety of environments, provides insight into the ecophysiology of autotrophs, as well as a toolkit for engineering microorganisms for carbon-neutral biochemistries of industrial importance.

The OSUMMER lines: A series of ultraviolet-accelerated NRAS-mutant mouse melanoma cell lines syngeneic to C57BL/6.

An increasing number of cancer subtypes are treated with front-line immunotherapy. However, approaches to overcome primary and acquired resistance remain limited. Preclinical mouse models are often used to investigate resistance mechanisms, novel drug combinations, and delivery methods; yet most of these models lack the genetic diversity and mutational patterns observed in human tumors. Here we describe a series of 13 C57BL/6J melanoma cell lines to address this gap in the field. The Ohio State University-Moffitt Melanoma Exposed to Radiation (OSUMMER) cell lines are derived from mice expressing endogenous, melanocyte-specific, and clinically relevant Nras driver mutations (Q61R, Q61K, or Q61L). Exposure of these animals to a single, non-burning dose of ultraviolet B accelerates the onset of spontaneous melanomas with mutational patterns akin to human disease. Furthermore, in vivo irradiation selects against potent tumor antigens, which could prevent the outgrowth of syngeneic cell transfers. Each OSUMMER cell line possesses distinct in vitro growth properties, trametinib sensitivity, mutational signatures, and predicted antigenicity. Analysis of OSUMMER allografts shows a correlation between strong, predicted antigenicity and poor tumor outgrowth. These data suggest that the OSUMMER lines will be a valuable tool for modeling the heterogeneous responses of human melanomas to targeted and immune-based therapies.

Cell-intrinsic melanin fails to protect melanocytes from ultraviolet-mutagenesis in the absence of epidermal melanin.

Melanin is a free-radical scavenger, antioxidant, and broadband absorber of ultraviolet (UV) radiation which protects the skin from environmental carcinogenesis. However, melanin synthesis and UV-induced reactive melanin species are also implicated in melanocyte genotoxicity. Here, we attempted to reconcile these disparate functions of melanin using a UVB-sensitive, NRAS-mutant mouse model, TpN. We crossed TpN mice heterozygous for an inactivating mutation in Tyrosinase to produce albino and black littermates on a C57BL/6J background. These animals were then exposed to a single UVB dose on postnatal day three when keratinocytes in the skin have yet to be melanized. Approximately one-third (35%) of black mice were protected from UVB-accelerated tumor formation. However, melanoma growth rates, tumor mutational burdens, and gene expression profiles were similar in melanomas from black and albino mice. Skin from albino mice contained more cyclobutane pyrimidine dimer (CPD) positive cells than black mice 1-h post-irradiation. However, this trend gradually reversed over time with CPDs becoming more prominent in black than albino melanocytes at 48 h. These results show that in the absence of epidermal pigmentation, melanocytic melanin limits the tumorigenic effects of acute UV exposure but fails to protect melanocytes from UVB-induced mutagenesis.