Effects of oxygen exposure on relative nucleic acid content and membrane integrity in the human gut microbiota

While the diversity of the human gut microbiota is becoming increasingly well characterized, bacterial physiology is still a critical missing link in understanding how the gut microbiota may be implicated in disease. The current best practice for studying bacterial physiology involves the immediate storage of fecal samples in an anaerobic chamber. This reliance on immediate access to anaerobic chambers greatly limits the scope of sample populations that can be studied. Here, we assess the effects of short-term oxygen exposure on gut bacterial physiology and diversity. We use relative nucleic acid content and membrane integrity as markers of bacterial physiology, and 16S rRNA gene amplicon sequencing to measure bacterial diversity. Samples were stored for up to 6 h in either ambient conditions or in anoxic environments created with gas packs or in an anaerobic chamber. Our data indicate that AnaeroGen sachets preserve bacterial membrane integrity and nucleic acid content over the course of 6 h similar to storage in an anaerobic chamber. Short-term oxygen exposure increases bacterial membrane permeability, without exceeding inter-individual differences. As oxygen exposure remains an important experimental consideration for bacterial metabolism, our data suggest that AnaeroGen sachets are a valid alternative limiting loss of membrane integrity for short-term storage of samples from harder-to-access populations.

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Translational activity is uncoupled from nucleic acid content in bacterial cells of the human gut microbiota

Gut Microbes ◽

10.1080/19490976.2021.1903289 ◽

2021 ◽

Vol 13 (1) ◽

pp. 1-15

Author(s):

Mariia Taguer ◽

B. Jesse Shapiro ◽

Corinne F. Maurice

Keyword(s):

Nucleic Acid ◽

Gut Microbiota ◽

Acid Content ◽

Nucleic Acid Content ◽

Bacterial Cells ◽

Human Gut ◽

Human Gut Microbiota ◽

Translational Activity

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Translational activity is uncoupled from nucleic acid content in bacterial cells of the human gut microbiota

10.1101/2020.10.05.327163 ◽

2020 ◽

Author(s):

Mariia Taguer ◽

B. Jesse Shapiro ◽

Corinne F. Maurice

Keyword(s):

Nucleic Acid ◽

Gut Microbiota ◽

Gut Microbiome ◽

Acid Content ◽

Membrane Damage ◽

Bacterial Activity ◽

Nucleic Acid Content ◽

Bacterial Cells ◽

Human Gut ◽

Human Gut Microbiota

AbstractBackgroundChanges in bacterial diversity in the human gut microbiome, characterized primarily though DNA sequencing methods, have been associated with many different adverse health conditions. However, these changes do not always reflect changes in bacterial activity, and thus how the gut microbiome is implicated in disease is still not often understood. New methods that link together bacterial function to bacterial identity are needed to further explore the role of the gut microbiome in health and disease. We optimized bioorthogonal non-canonical amino acid tagging (BONCAT) for the gut microbiota and combined it with fluorescently activated cell sorting and sequencing (FACS-Seq) to identify the translationally active members of the community. We then used this novel technique to compare and contrast to other methods of bulk community measurements of activity and viability: physiological staining of relative nucleic acid content and membrane damage. Relative nucleic acid content has previously been linked to metabolic activity, yet remains currently undefined for the human gut microbiota.ResultsTen healthy, unrelated individuals were sampled to determine the proportion and diversity of distinct physiological fractions of their gut microbiota. The translationally active bacteria represent about half of the gut microbiota, and are not distinct from the whole community. The high nucleic acid content (HNA) bacteria also represent about half of the gut microbiota, but are distinct from the whole community and correlate with the damaged subset. Perturbing the community with xenobiotics previously shown to alter bacterial activity but not diversity resulted in stronger changes in the distinct physiological fractions than in the whole community.ConclusionsBONCAT is a suitable method to probe the translationally active members of the human gut microbiota, and combined with FACS-Seq, allows for their identification. The high nucleic acid content bacteria are not necessarily the protein-producing bacteria in the community, and so further work is needed to understand the relationship between nucleic acid content and bacterial metabolism in the human gut. Taking into account physiologically distinct subsets of the gut microbiota may be more informative than relying on whole community profiling.

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Weekly variations of viruses and heterotrophic nanoflagellates and their potential impact on bacterioplankton in shallow waters of the central Red Sea

FEMS Microbiology Ecology ◽

10.1093/femsec/fiaa033 ◽

2020 ◽

Vol 96 (4) ◽

Cited By ~ 2

Author(s):

Eman I Sabbagh ◽

Tamara M Huete-Stauffer ◽

Maria L l Calleja ◽

Luis Silva ◽

Miguel Viegas ◽

...

Keyword(s):

Nucleic Acid ◽

Red Sea ◽

Acid Content ◽

Temporal Dynamics ◽

Heterotrophic Bacteria ◽

Membrane Integrity ◽

Heterotrophic Nanoflagellates ◽

Nucleic Acid Content ◽

Top Down ◽

The Impact

ABSTRACT Bacterioplankton play a pivotal role in marine ecosystems. However, their temporal dynamics and underlying control mechanisms are poorly understood in tropical regions such as the Red Sea. Here, we assessed the impact of bottom-up (resource availability) and top-down (viruses and heterotrophic nanoflagellates) controls on bacterioplankton abundances by weekly sampling a coastal central Red Sea site in 2017. We monitored microbial abundances by flow cytometry together with a set of environmental variables including temperature, salinity, dissolved organic and inorganic nutrients and chlorophyll a. We distinguished five groups of heterotrophic bacteria depending on their physiological properties relative nucleic acid content, membrane integrity and cell-specific respiratory activity, two groups of Synechococcus cyanobacteria and three groups of viruses. Viruses controlled heterotrophic bacteria for most of the year, as supported by a negative correlation between their respective abundances and a positive one between bacterial mortality rates and mean viral abundances. On the contrary, heterotrophic nanoflagellates abundance covaried with that of heterotrophic bacteria. Heterotrophic nanoflagellates showed preference for larger bacteria from both the high and low nucleic acid content groups. Our results demonstrate that top-down control is fundamental in keeping heterotrophic bacterioplankton abundances low (< 5 × 10 5 cells mL−1) in Red Sea coastal waters.

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Application of Ogur & Rosen's Method for the Determination of Nucleic Acid Content of Bacteria

Nippon Saikingaku Zasshi ◽

10.3412/jsb.12.125 ◽

1957 ◽

Vol 12 (2) ◽

pp. 125-129 ◽

Cited By ~ 3

Author(s):

Nobuyasu KAWASAKI ◽

Ichiro TAKI ◽

Chiaki WATANABE ◽

Kiyoshi MATOBA ◽

Mokichiro NISHIO ◽

...

Keyword(s):

Nucleic Acid ◽

Acid Content ◽

Nucleic Acid Content

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Using flow cytometry and light-induced fluorescence technique to characterizethe variability and characteristics of bioaerosols in springtime at Metro Atlanta, Georgia

10.5194/acp-2018-1073 ◽

2018 ◽

Author(s):

Arnaldo Negron ◽

Natasha DeLeon-Rodriguez ◽

Samantha M. Waters ◽

Luke D. Ziemba ◽

Bruce Anderson ◽

...

Keyword(s):

Flow Cytometry ◽

Nucleic Acid ◽

Acid Content ◽

Fungal Spores ◽

Epifluorescence Microscopy ◽

Nucleic Acid Content ◽

Rain Event ◽

Bacterial Cells ◽

Sampling System ◽

Spores And Pollen

Abstract. The abundance and speciation of primary biological aerosol particles (PBAP) is important for understanding their impacts on human health, cloud formation and ecosystems. Towards this, we have developed a protocol for quantifying PBAP collected from large volumes of air with a portable wet-walled cyclone bioaerosol sampler. A flow cytometry (FCM) protocol was then developed to quantify and characterize the PBAP populations from the sampler, which were confirmed against epifluorescence microscopy. The sampling system and FCM analysis were used to study PBAP in Atlanta, GA over a two-month period and showed clearly defined populations of DNA-containing particles: Low Nucleic Acid-content particles (bioLNA), High Nucleic Acid-content particles (HNA) being fungal spores and pollen. We find that daily-average springtime PBAP concentration (1 to 5 μm diameter) ranged between 1.4 × 104 and 1.1 × 105 m−3. The BioLNA population dominated PBAP during dry days (72 ± 18 %); HNA dominated the PBAP during humid days and following rain events, where HNA (e.g., wet-ejected fungal spores) comprised up to 92 % of the PBAP number. Concurrent measurements with a Wideband Integrated Bioaerosol Sensor (WIBS-4A) showed that FBAP and total FCM counts are similar; HNA (from FCM) significantly correlated with ABC type FBAP concentrations throughout the sampling period (and for the same particle size range, 1–5 μm diameter). However, the FCM bioLNA population, possibly containing bacterial cells, did not correlate to any FBAP type. The lack of correlation of any WIBS FBAP type with the bioLNA suggest bacterial cells may be more difficult to detect with autofluorescence than previously thought. Ιdentification of bacterial cells even in the FCM (bioLNA population) is challenging, given that the fluorescence level of stained cells at times may be comparable to that seen from abiotic particles. HNA and ABC displayed highest concentration on a humid and warm day after a rain event (4/14), suggesting that both populations correspond to wet-ejected fungal spores. Overall, information from both instruments combined reveals a highly dynamic airborne bioaerosol community over Atlanta, with a considerable presence of fungal spores during humid days, and a bioLNA population dominating bioaerosol community during dry days.

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