Methyl halide transferase-based gas reporters for quantification of filamentous bacteria in microdroplet emulsions

Abstract

ABSTRACT The application of microfluidic techniques in experimental and environmental studies is a rapidly emerging field. Water-in-oil microdroplets can serve readily as controllable micro-vessels for studies that require spatial structure. In many applications, it is useful to monitor cell growth without breaking or disrupting the microdroplets. To this end, optical reporters based on color, fluorescence, or luminescence have been developed. However, optical reporters suffer from limitations when used in microdroplets such as inaccurate readings due to strong background interference or limited sensitivity during early growth stages. In addition, optical detection is typically not amenable to filamentous or biofilm-producing organisms that have significant nonlinear changes in opacity and light scattering during growth. To overcome such limitations, we show that volatile methyl halide gases produced by reporter cells expressing a methyl halide transferase (MHT) can serve as an alternative nonoptical detection approach suitable for microdroplets. In this study, an MHT-labeled Streptomyces venezuelae reporter strain was constructed and characterized. Protocols were established for the encapsulation and incubation of S. venezuelae in microdroplets. We observed the complete life cycle for S. venezuelae including the vegetative expansion of mycelia, mycelial fragmentation, and late-stage sporulation. Methyl bromide (MeBr) production was detected by gas chromatography-mass spectrometry (GC-MS) from S. venezuelae gas reporters incubated in either liquid suspension or microdroplets and used to quantitatively estimate bacterial density. Overall, using MeBr production as a means of quantifying bacterial growth provided a 100- to 1,000-fold increase in sensitivity over optical or fluorescence measurements of a comparable reporter strain expressing fluorescent proteins. IMPORTANCE Quantitative measurement of bacterial growth in microdroplets in situ is desirable but challenging. Current optical reporter systems suffer from limitations when applied to filamentous or biofilm-producing organisms. In this study, we demonstrate that volatile methyl halide gas production can serve as a quantitative nonoptical growth assay for filamentous bacteria encapsulated in microdroplets. We constructed an S. venezuelae gas reporter strain and observed a complete life cycle for encapsulated S. venezuelae in microdroplets, establishing microdroplets as an alternative growth environment for Streptomyces spp. that can provide spatial structure. We detected MeBr production from both liquid suspension and microdroplets with a 100- to 1,000-fold increase in signal-to-noise ratio compared to optical assays. Importantly, we could reliably detect bacteria with densities down to 10 6 CFU/mL. The combination of quantitative gas reporting and microdroplet systems provides a valuable approach to studying fastidious organisms that require spatial structure such as those found typically in soils.