Biology subjects

Carini, P.

Publications and source records attributed to Carini, P..

7 recordsLinked to original sources

bioRxiv2025

Transcriptional and metabolic stasis define desiccation-induced dormancy in a soil bacterium until water vapor initiates resuscitation.

Microbes inhabiting soils experience periodic water deprivation. The effects of desiccation on DNA, protein, and membrane integrity are well-described. However, the effects of drying and rehydration on the composition of cellular RNA and metabolites are still poorly understood. Here, we describe how slow drying and rehydration with water vapor influence the composition of RNAs and metabolites in a soil Arthrobacter. While drying reduced cultivability relative to hydrated controls, water vapor rehydration fully restored it. Ribosomal RNA proportions remained constant throughout all treatments, and mRNA profiles showed stable composition during desiccation--changing only during transitions into and out of desiccation-induced dormancy. Six transcriptional modules displayed distinct expression patterns in desiccated-rehydrated samples relative to hydrated controls, including desiccation-rehydration responsive and rehydration-specific profiles. Targeted intracellular metabolomics revealed similarly static profiles during desiccation, with a cluster of ribonucleosides and nucleobases increasing in response to desiccation and returning to baseline levels upon rehydration with water vapor. These findings demonstrate that both mRNA and metabolite profiles remain essentially frozen in desiccated Arthrobacter, with dynamic changes occurring only during state transitions. These results have important implications in environments with frequent drying cycles where stable mRNA in dormant cells combined with intracellular RNA recycling may obscure interpretations of RNA-based environmental analyses that use RNA as a marker of microbial activity. Our results suggest that RNA-based activity assessments in periodically dry environments require careful consideration of dormancy-associated molecular preservation. SIGNIFICANCE STATEMENTMetabolic activity quickly ceases in drying bacteria as they enter desiccation-induced dormancy. We show mRNA and metabolite profiles were variable during drying and rewetting but did not change while desiccated. Additionally, water vapor stimulated the shift from the static to active state when exiting desiccation-induced dormancy. These shifts coincided with increased cultivability, indicating water vapor resuscitated dry cells. Because RNAs are transient, labile molecules that are turned over rapidly in growing bacteria, the presence of RNA in the environment is used as a marker for microbial activity. Our research shows this assumption may not hold for desiccated cells, indicating reliance on RNA as a marker of activity in environments that experience drying may obscure estimates of in situ microbial activity.

microbiology↗

bioRxiv2024

Predicting microbial growth conditions from amino acid composition

The ability to grow a microbe in the laboratory enables reproducible study and engineering of its genetics. Unfortunately, the majority of microbes in the tree of life remain uncultivated because of the effort required to identify culturing conditions. Predictions of viable growth conditions to guide experimental testing would be highly desirable. While carbon and energy sources can be computationally predicted with annotated genes, it is harder to predict other requirements for growth such as oxygen, temperature, salinity, and pH. Here, we developed genome-based computational models capable of predicting oxygen tolerance (92% balanced accuracy), optimum temperature (R2=0.73), salinity (R2=0.81) and pH (R2=0.48) for novel taxonomic microbial families without requiring functional gene annotations. Using growth conditions and genome sequences of 15,596 bacteria and archaea, we found that amino acid frequencies are predictive of growth requirements. As little as two amino acids can predict oxygen tolerance with 88% balanced accuracy. Using cellular localization of proteins to compute amino acid frequencies improved prediction of pH (R2 increase of 0.36). Because these models do not rely on the presence or absence of specific genes, they can be applied to incomplete genomes, requiring as little as 10% completeness. We applied our models to predict growth requirements for all 85,205 species of sequenced bacteria and archaea and found that uncultivated species are enriched in thermophiles, anaerobes, and acidophiles. Finally, we applied our models to 3,349 environmental samples with metagenome-assembled genomes and showed that individual microbes within a community have differing growth requirements. This work guides identification of growth constraints for laboratory cultivation of diverse microbes.

microbiology↗

bioRxiv2023

Cultivating efficiency: High-throughput growth analysis of anaerobic bacteria in compact microplate readers.

Anaerobic microbes play crucial roles in environmental processes, industry, and human health. Traditional methods for monitoring the growth of anaerobes, including plate counts or subsampling broth cultures for optical density measurements, are time and resource intensive. The advent of microplate readers revolutionized bacterial growth studies by enabling high-throughput and real-time monitoring of microbial growth kinetics but their use in anaerobic microbiology has remained limited. Here, we present a workflow for using small-footprint microplate readers and the Growthcurver R package to analyze the kinetic growth metrics of anaerobic bacteria. We benchmarked the small-footprint Cerillo Stratus microplate reader against a BioTek Synergy HTX microplate reader in aerobic conditions using Escherichia coli DSM 28618 cultures. The growth rates and carrying capacities obtained from the two readers were statistically indistinguishable. However, the area under the logistic curve was significantly higher in cultures monitored by the Stratus reader. We used the Stratus to quantify the growth responses of anaerobically grown E. coli and Clostridium bolteae DSM 29485 to different doses of the toxin sodium arsenite. The growth of E. coli and C. bolteae was sensitive to arsenite doses of 1.3 M and 0.4 M, respectively. Complete inhibition of growth was achieved at 38 M arsenite for C. bolteae, and 338 M in E. coli. These results show that the Stratus performs similarly to a leading brand of microplate reader and can be reliably used in anaerobic conditions. We discuss the advantages of the small format microplate readers and our experiences with the Stratus. Importance statementWe present a workflow that facilitates the production and analysis of growth curves for anaerobic microbes using small-footprint microplate readers and an R script. This workflow is a cost and space-effective solution to most high-throughput solutions for collecting growth data from anaerobic microbes. This technology can be used for applications in which high-throughput would advance discovery, including microbial isolation, bioprospecting, co-culturing, host-microbe interactions, and drug/toxin-microbial interactions.

microbiology↗

bioRxiv2020

Genome evolution in bacteria isolated from million-year-old subseafloor sediments

Beneath the seafloor, microbial life subsists in isolation from the surface world under persistent energy limitation. The nature and extent of genomic evolution in subseafloor microbes has been unknown. Here we show that the genomes of Thalassospira bacterial populations cultured from million-year-old subseafloor sediments evolve by point mutation, with a relatively low rate of homologous recombination and a high frequency of pseudogenes. Ratios of synonymous to non-synonymous mutation rates correlate with the accumulation of pseudogenes, consistent with a dominant role for genetic drift in the subseafloor strains, but not in type strains of Thalassospira isolated from the surface world. Our findings demonstrate that the long term physical isolation of these bacteria, in the absence of recombination, has resulted in clonal populations that evolve consistent with Mullers Ratchet, whereby reduced access to novel genetic material from neighbors has resulted in fixation of new mutations that accumulate in genomes over millions of years. Significance statementThe nature and extent of genomic evolution in subseafloor microbial populations subsisting for millions of years below the seafloor is unknown. Subseafloor populations have ultra-slow metabolic rates that are hypothesized to restrict reproduction and, consequently, the spread of new traits. Our findings demonstrate that genomes of cultivated bacterial strains from the genus Thalassospira isolated from million-year-old abyssal sediment exhibit greatly reduced levels of homologous recombination, elevated numbers of pseudogenes, and genome-wide evidence of relaxed purifying selection. These substitutions and pseudogenes are fixed into the population, suggesting the genome evolution of these bacteria has been dominated by genetic drift, whereby under long-term physical isolation in small population sizes, and in the absence of homologous recombination, newly acquired mutations accumulate in the genomes of clonal populations over millions of years.

microbiology↗

bioRxiv2019

The concentration of heterotrophic growth substrates in culture medium is a crucial determinant of the culturability of subsurface soil microbes

The vast majority of microbes inhabiting oligotrophic shallow subsurface soil environments have not been isolated or studied under controlled laboratory conditions. In part, the challenges associated with isolating shallow subsurface microbes may persist because microbes in deeper soils are adapted to low nutrient availability or quality. Here we use high-throughput dilution-to-extinction culturing to isolate shallow subsurface microbes from a conifer forest in Arizona, USA. We hypothesized that the concentration of heterotrophic substrates in microbiological growth medium would affect which microbial taxa were culturable from these soils. To test this, we diluted extracted cells into one of two custom-designed defined growth media that differed only by a 100-fold difference in the concentration of amino acids and organic carbon. Across both media, we isolated a total of 133 pure cultures, all of which were classified as Actinobacteria or Alphaproteobacteria. The substrate availability dictated which actinobacterial phylotypes were culturable but had no significant effect on the culturability of Alphaproteobacteria. We isolated cultures that were representative of the most abundant phylotype in the soil microbial community (Bradyrhizobium spp.) and representatives of five of the top 10 most abundant Actinobacteria phylotypes, including Nocardioides spp., Mycobacterium spp., and several other phylogenetically-divergent lineages. Flow cytometry of nucleic acid-stained cells showed that cultures isolated on low-substrate medium had significantly lower nucleic-acid fluorescence than those isolated on high-substrate medium. These results show that dilution-to-extinction is an effective method to isolate abundant soil microbes and the concentration of substrates in culture medium influences the culturability of specific microbial lineages. ImportanceIsolating environmental microbes and studying their physiology under controlled conditions is an essential aspect of understanding their ecology. Subsurface ecosystems are typically nutrient-poor environments that harbor diverse microbial communities--the majority of which are thus far uncultured. In this study, we use modified high-throughput cultivation methods to isolate subsurface soil microbes. We show that a component of whether a microbe is culturable from subsurface soils is the concentration of growth substrates in the culture medium. Our results offer new insight into technical approaches and growth medium design that can be used to access the uncultured diversity of soil microbes.

microbiology↗

bioRxiv2018

Unraveling the effects of spatial variability and relic DNA on the temporal dynamics of soil microbial communities

Few studies have comprehensively investigated the temporal variability in soil microbial communities despite widespread recognition that the belowground environment is dynamic. In part, this stems from the challenges associated with the high degree of spatial heterogeneity in soil microbial communities and because the presence of relic DNA (DNA from non-living cells) may dampen temporal signals. Here we disentangle the relationships among spatial, temporal, and relic DNA effects on bacterial, archaeal, and fungal communities in soils collected from contrasting hillslopes in Colorado, USA. We intensively sampled plots on each hillslope over six months to discriminate between temporal variability, intra-plot spatial heterogeneity, and relic DNA effects on the soil prokaryotic and fungal communities. We show that the intra-plot spatial variability in microbial community composition was strong and independent of relic DNA effects with these spatial patterns persisting throughout the study. When controlling for intra-plot spatial variability, we identified significant temporal variability in both plots over the six-month study. These microbial communities were more dissimilar over time after relic DNA was removed, suggesting that relic DNA hinders the detection of important temporal dynamics in belowground microbial communities. We identified microbial taxa that exhibited shared temporal responses and show these responses were often predictable from temporal changes in soil conditions. Our findings highlight approaches that can be used to better characterize temporal shifts in soil microbial communities, information that is critical for predicting the environmental preferences of individual soil microbial taxa and identifying linkages between soil microbial community composition and belowground processes.\n\nImportanceNearly all microbial communities are dynamic in time. Understanding how temporal dynamics in microbial community structure affect soil biogeochemistry and fertility are key to being able to predict the responses of the soil microbiome to environmental perturbations. Here we explain the effects of soil spatial structure and relic DNA on the determination of microbial community fluctuations over time. We found that intensive spatial sampling is required to identify temporal effects in microbial communities because of the high degree of spatial heterogeneity in soil and that DNA from non-living microbial cells masks important temporal patterns. We identified groups of microbes that display correlated behavior over time and show that these patterns are predictable from soil characteristics. These results provide insight into the environmental preferences and temporal relationships between individual microbial taxa and highlight the importance of considering relic DNA when trying to detect temporal dynamics in belowground communities.

microbiology↗

bioRxiv2017

Correlated expression of archaeal ammonia oxidation machinery across disparate environmental and culture conditions

Thaumarchaea are ubiquitous in marine habitats where they participate in carbon and nitrogen cycling. Although metatranscriptomes suggest thaumarchaea are active microbes in marine waters, we understand little about how thaumarchaeal gene expression patterns relate to substrate utilization and activity. Here, we report the global transcriptional response of the marine ammonia-oxidizing thaumarchaeon Candidatus Nitrosopelagicus brevis str. CN25 to ammonia limitation using RNA-Seq. We further describe the genome and transcriptome of Ca. N. brevis str. U25, a new strain capable of urea utilization. Ammonia limitation in CN25 resulted in reduced expression of transcripts coding for ammonia oxidation proteins, and increased expression of a gene coding an Hsp20-like chaperone. Despite significantly different transcript abundances across treatments, two ammonia monooxygenase subunits (amoAB), a nitrite reductase (nirK), and both ammonium transporter genes were always among the most abundant transcripts, regardless of growth state. Ca. N. brevis str. U25 cells expressed a urea transporter 139-fold more than the urease catalytic subunit ureC. Gene co-expression networks derived from culture transcriptomes and ten thaumarchaea-enriched metatranscriptomes revealed a high degree of correlated gene expression across disparate environmental conditions and identified a module of genes, including amoABC and nirK, that we hypothesize to represent the core ammonia oxidation machinery.\n\nOriginality-Significance StatementDiscovering gene function in fastidious or uncultivated lineages remains one of the biggest challenges in environmental microbiology. Here, we use an approach that combines controlled laboratory experiments with in situ transcript abundance data from the environment to identify genes that share similar transcription patterns in marine ammonia-oxidizing thaumarchaea. These findings demonstrate how transcriptomes from microbial cultures can be used together with complex environmental samples to identify suites of co-expressed genes that are otherwise enigmatic and provide new insights into the mechanism of ammonia oxidation. Our results add to the growing body of literature showing that relatively small changes in transcript abundance are linked to large changes in growth in organisms with reduced genomes, suggesting they have limited capacity for metabolic regulation or that they rely on mechanisms other than transcriptional regulation to deal with a fluctuating environment.

microbiology↗