Tobacco, Microbes, and Carcinogens: Correlation Between Tobacco Cure Conditions, Tobacco-Specific Nitrosamine Content, and Cured Leaf Microbial Community
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Audrey D Law1, Colin Fisher2, Anne Jack2
1Department of Plant & Soil Sciences, University of Kentucky, Lexington, KY, 40546-0312, USA.
Abstract:
Tobacco-specific nitrosamines are carcinogenic N-nitrosamine compounds present at very low levels in freshly harvested tobacco leaves that accumulate during leaf curing. Formation of N-nitrosamine compounds is associated with high nitrate levels in the leaf at harvest, and nitrate is presumed to be the source from which the N-nitrosation species originates. More specifically, nitrite is considered to be a direct precursor, and nitrite is linked with N-nitrosation in many environmental matrices where it occurs via microbial nitrate reduction. Here, we initiate work exploring the role of leaf microbial communities in formation of tobacco-specific nitrosamines. Leaves from burley tobacco line TN90H were air cured under various temperature and relative humidity levels, and 22 cured tobacco samples were analyzed for their microbial communities and leaf chemistry. Analysis of nitrate, nitrite, and total tobacco-specific nitrosamine levels revealed a strong positive correlation between the three variables, as well as a strong positive correlation with increasing relative humidity during cure conditions. 16S rRNA gene amplicon sequencing was used to assess microbial communities in each of the samples. In most samples, Proteobacteria predominated at the phylum level, accounting for >90 % of the OTUs. However, a distinct shift was noted among members of the high tobacco-specific nitrosamine group, with increases in Firmicutes and Actinobacteria. Several OTUs were identified that correlate strongly (positive and negative) with tobacco-specific nitrosamine content. Copy number of bacterial nitrate reductase genes, obtained using quantitative PCR, did not correlate strongly with tobacco-specific nitrosamine content. Incomplete denitrification is potentially implicated in tobacco-specific nitrosamine levels.
Insights
Microbial communities in tobacco leaves influence tobacco-specific nitrosamine (TSN) formation. Higher levels of nitrate, nitrite, and TSNs correlate with specific bacterial shifts, particularly increased Firmicutes and Actinobacteria during curing.
Area of Science:
- Agricultural Chemistry
- Microbiology
- Environmental Science
Background:
- Tobacco-specific nitrosamines (TSNs) are carcinogenic compounds found in cured tobacco.
- TSN formation is linked to high nitrate levels in tobacco leaves at harvest.
- Nitrite is a direct precursor to TSNs, often formed via microbial nitrate reduction.
Purpose of the Study:
- To investigate the role of leaf microbial communities in the formation of tobacco-specific nitrosamines.
- To explore correlations between microbial composition, leaf chemistry (nitrate, nitrite), and TSN levels.
- To identify specific microbial taxa associated with TSN accumulation during tobacco curing.
Main Methods:
- Air curing of burley tobacco (line TN90H) under varied temperature and humidity.
- Chemical analysis of nitrate, nitrite, and total TSN levels in 22 cured samples.
- 16S rRNA gene amplicon sequencing for microbial community profiling.
- Quantitative PCR to assess bacterial nitrate reductase gene copy number.
Main Results:
- Strong positive correlations observed between nitrate, nitrite, and TSN levels.
- Increasing relative humidity during curing also correlated positively with TSNs.
- Microbial communities shifted in high-TSN samples, with increased Firmicutes and Actinobacteria.
- Specific operational taxonomic units (OTUs) showed strong positive or negative correlations with TSN content.
- Bacterial nitrate reductase gene copy number did not strongly correlate with TSN levels.
Conclusions:
- Leaf microbial communities play a significant role in tobacco-specific nitrosamine formation.
- Environmental conditions like relative humidity influence both microbial communities and TSN levels.
- Specific bacterial phyla (Firmicutes, Actinobacteria) and OTUs are associated with TSN accumulation, suggesting their involvement in the biochemical pathways.
- Incomplete denitrification by microbes is a potential factor contributing to elevated TSN levels in cured tobacco.
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