PMID- 37699129
OWN - NLM
STAT- MEDLINE
DCOM- 20230929
LR  - 20240210
IS  - 1098-5336 (Electronic)
IS  - 0099-2240 (Print)
IS  - 0099-2240 (Linking)
VI  - 89
IP  - 9
DP  - 2023 Sep 28
TI  - Methyl halide transferase-based gas reporters for quantification of filamentous 
      bacteria in microdroplet emulsions.
PG  - e0076423
LID - 10.1128/aem.00764-23 [doi]
LID - e00764-23
AB  - 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.
FAU - Song, Xinhao
AU  - Song X
AUID- ORCID: 0009-0009-4776-2428
AD  - Department of BioSciences, Rice University , Houston, Texas, USA.
FAU - Kong, Sarah J
AU  - Kong SJ
AD  - Department of BioSciences, Rice University , Houston, Texas, USA.
FAU - Seo, Seokju
AU  - Seo S
AD  - Department of BioSciences, Rice University , Houston, Texas, USA.
FAU - Prabhakar, Ramya Ganiga
AU  - Prabhakar RG
AD  - Department of BioSciences, Rice University , Houston, Texas, USA.
FAU - Shamoo, Yousif
AU  - Shamoo Y
AD  - Department of BioSciences, Rice University , Houston, Texas, USA.
LA  - eng
GR  - R01 AI080714/AI/NIAID NIH HHS/United States
PT  - Journal Article
PT  - Research Support, N.I.H., Extramural
DEP - 20230908
PL  - United States
TA  - Appl Environ Microbiol
JT  - Applied and environmental microbiology
JID - 7605801
RN  - 0 (Emulsions)
RN  - 0 (Gases)
RN  - EC 2.- (Transferases)
SB  - IM
MH  - Emulsions
MH  - Fluorescence
MH  - *Gases
MH  - *Transferases
PMC - PMC10537575
OTO - NOTNLM
OT  - Streptomyces
OT  - microfluidics
OT  - soil microbiology
COIS- The authors declare no conflict of interest.
EDAT- 2023/09/12 18:41
MHDA- 2023/09/29 06:44
PMCR- 2023/09/28
CRDT- 2023/09/12 15:03
PHST- 2023/09/29 06:44 [medline]
PHST- 2023/09/12 18:41 [pubmed]
PHST- 2023/09/12 15:03 [entrez]
PHST- 2023/09/28 00:00 [pmc-release]
AID - 00764-23 [pii]
AID - aem.00764-23 [pii]
AID - 10.1128/aem.00764-23 [doi]
PST - ppublish
SO  - Appl Environ Microbiol. 2023 Sep 28;89(9):e0076423. doi: 10.1128/aem.00764-23. 
      Epub 2023 Sep 8.