Amazonian Superstars

Darwin’s theory of evolution and origin of species teach us that nature is the greatest engineer. In this aspect, we will show the identification and a few steps of characterization of specimen isolated from water samples of rivers nearby Manaus city, which is full of small streams of water that cross the city and end at bigger rivers.

In this scenario, we have the release of residual effluents from industrial processes, full of numerous substances, including chemical compounds highly damaging to environment and heavy metals. Among them, mercury is one of the most abundant and it is deployed through our whole hydrological basin. With this evolutionary pressure microorganism need to adapt themselves to survive.

At first water samples were collected in certain places at Manaus metropolitan zone in march 2014. Water samples were diluted and plated in Petri dishes containing LM medium (50% less NaCl than LB medium) and HgCl2 under 50µg/mL and 100µg/mL concentrations.

Then we named these colonies after the spot where the sample came from and isolated them by grooving in Petri dishes. We proceeded to antibiogram of bacteria to get to know their antibiotic resistance profile. We found out that:

A1, A2, P1 and P2 are resistant to: tetracycline (12ug/mL), ampicilina (100ug/mL) and streptomycin (20ug/mL).

P1: tetracycline (12ug/mL) and streptomycin (20ug/mL)

We reactivated the bactéria cryopreserved and extracted their chromosomal and plasmidial DNA. Unfortunately we couldn’t extract chromosomal DNA from A2.

In parallel to identification, we began to characterize them. We started with setting growth curves, because we noted that some bacteria grew better than others in 37ºC. In the tests, temperature ranged from 30 to 37ºC in LB medium. They were cultivated in LM, since we observed that they didn’t grow well with half the NaCl concentration. We believe that this result is due to these bacteria originally inhabit freshwater.

Take a look at these crazy results! Every bacteria reached the 1.0 O.D. way faster in every temperature when salt concentration is lower in the medium. We also had this graphic below, where it is possible to see that the growth curve went better at 30ºC, excepting to sample A2, which was hard to cultivate in laboratory.

We also did Gram coloring and chemical Gram on them and confirmed that they all are Gram-positive bacteria, which is unexpected since most bacteria that are metal resistant are Gram-negative.

After many failed attempts, adjusting PCR protocols, new dilutions, we amplified the 16S region with the primers 530F/1490R and 400F/400R for the samples A1, P1, P2 and S1!!! Take a look at the BLAST of the outstanding bacterium.

Acinetobacter radioresistens was not isolated and identified in Amazon before, and we were the first ones to do it!!! This organism is utilized in many processes and is described by other autors as an excellent natural bioremediator. So, we did plasmid extraction and transformed competent cells of E. coli DH5ɑ and the transformed cells obtained mercury resistance. See the growth curve demonstrating that the transforming cell with plasmids from A. radioresistens grows better than our own genetic construction!!

And we didn’t stop! We extracted total DNA and sequenced the genome, so we could study in details what were the differences between genome of amazonian A. radioresistens and the others from all around the world. So, we’re proud to emphasise that we’re waiting the result for the FIRST AMAZONIAN BACTERIAL GENOME TO BE SEQUENCED FOR THE COMPETITION!!!