Stable-isotope probing and metagenomics reveal predation by protozoa drives E. coli removal in slow sand filters

Raman stable isotope probing (Raman-SIP) is an excellent technique that can be used to access the overall metabolism of microorganisms. Recent studies have mainly used an excitation wavelength in the visible range to characterize isotopically labeled bacteria. In this work, we used UV resonance Raman spectroscopy (UVRR) to evaluate the spectral red-shifts caused by the uptake of isotopes (13C, 15N, 2H(D) and 18O) in E. coli cells. Moreover, we present a new approach based on the extraction of labeled DNA in combination with UVRR to identify metabolically active cells. The proof-of-principle study on E. coli revealed heterogeneities in the Raman features of both the bacterial cells and the extracted DNA after labeling with 13C, 15N, and D. The wavelength of choice for studying 18O- and deuterium-labeled cells is 532 nm is, while 13C-labeled cells can be investigated with visible and deep UV wavelengths. However, 15N-labeled cells are best studied at the excitation wavelength of 244 nm since nucleic acids are in resonance at this wavelength. These results highlight the potential of the presented approach to identify active bacterial cells. This work can serve as a basis for the development of new techniques for the rapid and efficient detection of active bacteria cells without the need for a cultivation step.

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Bacterial predation on T4 phages

10.1101/2021.06.01.446436 ◽

2021 ◽

Author(s):

jean-jacques godon ◽

ariane bize ◽

hoang ngo ◽

laurent cauquil ◽

mathieu almeida ◽

...

Keyword(s):

Stable Isotope ◽

Bacterial Species ◽

Sole Source ◽

Stable Isotope Probing ◽

Wastewater Sludge ◽

Nitrogen And Phosphorus ◽

E Coli ◽

Starting Point ◽

T4 Phage

Bacterial consumption of viruses has never yet been reported, even though bacteria feed on almost anything. Viruses are omnipresent predators for all organisms, but have no acknowledged active biocontrol. The viral biomass undoubtedly reintegrates the trophic cycles, however the mechanisms of this phase still remain unknown. Here, we show that an E. coli infecting virus - the coliphage T4- was actively consumed by bacteria. Using stable isotope probing with 13C labelled T4 phages, T4 phage disappearance in wastewater sludge was found to occur mainly through predation by Aeromonadacea. Phage consumption also favours significant in situ bacterial growth. Furthermore, an isolated strain of Aeromonas was observed to grow on T4 phages as sole source of carbon, nitrogen and phosphorus. Results demonstrate how bacterial species are capable of consuming bacteriophages in situ, which is likely a widespread and underestimated type of biocontrol. This assay is anticipated as a starting point for harnessing the bacterial potential in limiting the diffusion of harmful viruses within environments such as gut or water.

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Using Stable Isotope Probing and Raman Microspectroscopy to measure growth rates of heterotrophic bacteria

Applied and Environmental Microbiology ◽

10.1128/aem.01460-21 ◽

2021 ◽

Author(s):

Felix Weber ◽

Tatiana Zaliznyak ◽

Virginia P. Edgcomb ◽

Gordon T. Taylor

Keyword(s):

Stable Isotope ◽

Single Cell ◽

Bacterial Growth ◽

Growth Rates ◽

Heterotrophic Bacteria ◽

Raman Microspectroscopy ◽

Stable Isotope Probing ◽

Single Cell Level ◽

Cell Level ◽

E Coli

The suitability of stable isotope probing (SIP) and Raman microspectroscopy to measure growth rates of heterotrophic bacteria at the single-cell level was evaluated. Label assimilation into E. coli biomass during growth on a complex 13 C-labeled carbon source was monitored in time course experiments. 13 C-incorporation into various biomolecules was measured by spectral “red shifts” of Raman-scattered emissions. The 13 C- and 12 C-isotopologues of the amino acid phenylalanine (Phe) proved to be a quantitatively accurate reporter molecules of cellular isotopic fractional abundances ( f cell ). Values of f cell determined by Raman microspectroscopy and independently by isotope-ratio mass spectrometry (IRMS) over a range of isotopic enrichments were statistically indistinguishable. Progressive labeling of Phe in E. coli cells among a range of 13 C/ 12 C organic substrate admixtures occurred predictably through time. Relative isotopologue abundances of Phe determined by Raman spectral analysis enabled accurate calculation of bacterial growth rates as confirmed independently by optical density (OD) measurements. Results demonstrate that combining stable isotope probing (SIP) and Raman microspectroscopy can be a powerful tool for studying bacterial growth at the single-cell level when grown on defined or complex organic 13 C-carbon sources even in mixed microbial assemblages. Importance: Population growth dynamics and individual cell growth rates are the ultimate expressions of a microorganism’s fitness to its environmental conditions, whether natural or engineered. Natural habitats and many industrial settings harbor complex microbial assemblages. Their heterogeneity in growth responses to existing and changing conditions is often difficult to grasp by standard methodologies. In this proof of concept study, we tested whether Raman microspectroscopy can reliably quantify assimilation of isotopically-labeled nutrients into E. coli cells and enable determination of individual growth rates among heterotrophic bacteria. Raman-derived growth rate estimates were statistically indistinguishable from those derived by standard optical density measurements of the same cultures. Raman microspectroscopy also can be combined with methods for phylogenetic identification. We report development of Raman-based techniques that enable researchers to directly link genetic identity to functional traits and rate measurements of single cells within mixed microbial assemblages, currently a major technical challenge in microbiological research.

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