Construction of a broad-host-range Anderson promoter series and particulate methane monooxygenase promoter variants expand the methanotroph genetic toolbox.

Methanotrophic bacteria are currently used industrially for the bioconversion of methane-rich natural gas and anaerobic digestion-derived biogas to valuable products. These bacteria may also serve to mitigate the negative effects of climate change by capturing atmospheric greenhouse gases. Several genetic tools have previously been developed for genetic and metabolic engineering of methanotrophs. However, the available tools for use in methanotrophs are significantly underdeveloped compared to many other industrially relevant bacteria, which hinders genetic and metabolic engineering of these biocatalysts. As such, expansion of the methanotroph genetic toolbox is needed to further our understanding of methanotrophy and develop biotechnologies that leverage these unique microbes for mitigation and conversion of methane to valuable products. Here, we determined the copy number of three broad-host-range plasmids in Methylococcus capsulatus Bath and Methylosinus trichosporium OB3b, representing phylogenetically diverse Gammaproteobacterial and Alphaproteobacterial methanotrophs, respectively. Further, we show that the commonly used synthetic Anderson series promoters are functional and exhibit similar relative activity in M. capsulatus and M. trichosporium OB3b, but the synthetic series had limited range. Thus, we mutagenized the native M. capsulatus particulate methane monooxygenase promoter and identified variants with activity that expand the activity range of synthetic, constitutive promoters functional not only in M. capsulatus, but also in Escherichia coli. Collectively, the tools developed here advance the methanotroph genetic engineering toolbox and represent additional synthetic genetic parts that may have broad applicability in Pseudomonadota bacteria.

Optimized Tools and Methods for Methanotroph Genome Editing.

Microbes with the capacity to use methane (CH4) as a carbon source (methanotrophs) have significant potential for the bioconversion of CH4-containing natural gas and anaerobic digestion-derived biogas to high value products. These organisms also play a vital role in the biogeochemical cycling of atmospheric CH4 by serving as the only known biological sink of this gas in terrestrial and aquatic ecosystems. Much is known regarding the enzymes and central metabolic pathways mediating CH4 utilization in these bacteria. However, large fundamental knowledge gaps exist regarding methanotroph physiology and responses to environmental stimuli, primarily due to a lack of efficient molecular tools to probe gene-function relationships. In this chapter, we describe several recently developed genetic tools and optimized genome editing methods that can be used for methanotroph metabolic engineering and to probe metabolic and physiological governing mechanisms in these unique bacteria.

Ribulose-1,5-Bisphosphate Carboxylase/Oxygenase (RubisCO) Is Essential for Growth of the Methanotroph Methylococcus capsulatus Strain Bath.

The ribulose-1,5-bisphosphate carboxylase/oxygenase (RubisCO) enzyme found in plants, algae, and an array of autotrophic bacteria is also encoded by a subset of methanotrophs, but its role in these microbes has largely remained elusive. In this study, we showed that CO2 was requisite for RubisCO-encoding Methylococcus capsulatus strain Bath growth in a bioreactor with continuous influent and effluent gas flow. RNA sequencing identified active transcription of several carboxylating enzymes, including key enzymes of the Calvin and serine cycles, that could mediate CO2 assimilation during cultivation with both CH4 and CO2 as carbon sources. Marker exchange mutagenesis of M. capsulatus Bath genes encoding key enzymes of potential CO2-assimilating metabolic pathways indicated that a complete serine cycle is not required, whereas RubisCO is essential for growth of this bacterium. 13CO2 tracer analysis showed that CH4 and CO2 enter overlapping anaplerotic pathways and implicated RubisCO as the primary enzyme mediating CO2 assimilation in M. capsulatus Bath. Notably, we quantified the relative abundance of 3-phosphoglycerate and ribulose-1,5-bisphosphate 13C isotopes, which supported that RubisCO-produced 3-phosphoglycerate is primarily converted to ribulose-1-5-bisphosphate via the oxidative pentose phosphate pathway in M. capsulatus Bath. Collectively, our data establish that RubisCO and CO2 play essential roles in M. capsulatus Bath metabolism. This study expands the known capacity of methanotrophs to fix CO2 via RubisCO, which may play a more pivotal role in the Earth's biogeochemical carbon cycling and greenhouse gas regulation than previously recognized. Further, M. capsulatus Bath and other CO2-assimilating methanotrophs represent excellent candidates for use in the bioconversion of biogas waste streams that consist of both CH4 and CO2. IMPORTANCE The importance of RubisCO and CO2 in M. capsulatus Bath metabolism is unclear. In this study, we demonstrated that both CO2 and RubisCO are essential for M. capsulatus Bath growth. 13CO2 tracing experiments supported that RubisCO mediates CO2 fixation and that a noncanonical Calvin cycle is active in this organism. Our study provides insights into the expanding knowledge of methanotroph metabolism and implicates dually CH4/CO2-utilizing bacteria as more important players in the biogeochemical carbon cycle than previously appreciated. In addition, M. capsulatus and other methanotrophs with CO2 assimilation capacity represent candidate organisms for the development of biotechnologies to mitigate the two most abundant greenhouse gases, CH4 and CO2.

Evaluation of Systematic Blood Testing at the Time of Recruitment to the Armed Forces: Retrospective Monocentric Study Among 726 French Army Soldiers.

INTRODUCTION: In the French armed forces, the biological checkup required during the recruitment process comprises a urinalysis (urinary dipstick), a complete blood count (CBC), and measurement of serum levels of aspartate aminotransferase, alanine aminotransferase, fasting blood glucose, and creatinine. This study aimed to evaluate the benefits of this biological checkup and to determine the most relevant parameters. MATERIALS AND METHODS: We conducted a monocentric retrospective study of all standardized and systematically conducted blood tests (CBC and measurement of aspartate aminotransferase, alanine aminotransferase, fasting blood glucose, and creatinine) over a 15-month period among 726 French Army recruits. RESULTS: The population included mainly young males (85.4%, mean age 21.6 years). More than half (54.1%) of the blood tests had at least one abnormal parameter, most often concerning the CBC. Anemia occurred in 5.3% of the population and was mostly normocytic. Microcytosis was mostly not associated with anemia (72.3% of cases). Lymphopenia occurred in 20.1% of the population and was mostly mild. Eosinophilia was present in 5.1% of the population and was never severe. Thrombocytopenia occurred in 0.7% of the population and was never severe. Serum levels of aminotransferases were elevated in 8.1% of the population. Fasting plasma glucose averaged 84 mg/dL (SD: 0.07) ranging from 64 to 123 mg/dL, was abnormal in 0.4% of the population, and one case of diabetes was diagnosed. Serum creatinine concentration was elevated in 0.7% of the population. CONCLUSION: CBCs gave useful information but iron deficiency was common and insufficiently detected by this single analysis. Assessing aminotransferase levels without screening for viral hepatitis and systematic measurement of fasting plasma glucose levels did not appear to be efficient. In addition, the only interest in systematic measurement of creatinine serum levels was to obtain a reference level for long-term follow-up. In addition to the urinary dipstick, the systematic biological checkup at recruitment could be limited to a CBC with measurement of plasma ferritin levels and Hepatitis B virus serology, providing that any CBC abnormalities, in particular cytopenia, eosinophilia, and microcytosis, are systematically investigated. For a public health approach, systematic screening for other sexually transmitted infections could be proposed.