In situ reverse transcription to detect the cbbL gene and visualize RuBisCO in chemoautotrophic nitrifying bacteria

In situ methodologies targeting the cbbL gene were used to visualize cells of nitrifying bacteria. Both procaryotic in situ PCR (IS-PCR) and in situ reverse transcription (ISRT) protocols were employed to determine gene presence and expression, respectively.

Aged-oligotropic seawater samples were inoculated with microbial assemblages containing a mixture of actively growing nitrifying bacteria, starved nitrifying bacteria, and heterotrophic bacteria without cbbL. After the molecular manipulations, we found that while all the nitrifiers (healthy or starved) with the cbbL gene were detected by IS-PCR, only the actively growing autotrophic nitrifiers with detectable levels of carbon fixation and nitrification activity were detected by ISRT analysis.

These results show how IS-PCR and ISRT supplement each other, and their potential for the analysis of heterogeneous populations where an assortment of healthy and starved/dormant cells are expected.