User:Erivere/sandbox

By Erivere and ZachWig

Eleftheria terrae (abbreviated E. terrae) is a Gram-negative bacterium that has been recently discovered by Ling and her team. E. terrae is only a temporary name for the organism, as it has only been recently discovered and is still undergoing major scientific study. Teixobactin, a seemingly powerful new antibiotic, was found in this bacterium making it a very important discovery. The discovery of E. terrae could represent a new age of antibiotics, as teixobactin is the first new antibiotic discovered since the synthetic era of the 1980s. Prior research has indicated that other uncultivable bacteria like E. terrae have great potential in the development of new antimicrobial agents.

Discovery
Currently, an estimated 99% of bacterial species are uncultured and require advanced means, such as the iChip, to be isolated. E. terrae is one such bacterium affectionately named by scientists "microbial dark matter" cultivated by emerging scientific methods. Eleftheria terrae was discovered in the fall of 2014 in a field in Maine by a team from Novobiotic Pharmaceuticals led by L. Ling. It was discovered using a technique developed at Northeastern University called the iChip or isolation chip technique. The iChip is a small plastic block that contains 192 holes going through it. The holes are filled with a culture medium that are then inoculated with soil diluted to deposit only one bacterium in each hole. After the deposit of the bacterium in the holes the iChip is covered on both sides by a semipermeable membrane and put into a box of the original soil. The permeable membranes allow nutrients and growth factors from the soil to diffuse in and allow growth of only one species. Ling et al. screened approximately 10,000 iChip growth isolates for prospective antimicrobial activity, and E. terrae seemed to be hopeful. This technology has exciting potential for discovering even more antibiotics by allowing labs to grow previously “unculturable” microorganisms.

Phylogeny
E. terrae belongs to the class beta-proteobacteria. After sequencing the organism’s genome it was concluded that E. terrae is a member of a previously unknown genus close in genetic makeup to Aquabacteria based upon its 16S rRNA gene sequencing and DNA-DNA hybridization performed by computer analysis. Organisms of the Aquabacteria genus are Gram-negative and are not known to produce antibiotics; however, E. terrae is also Gram-negative but does produce the antibiotic teixobactin.

General Characteristics
E. terrae is a Gram-negative bacterium which produces a powerful antibiotic teixobactin. It was found that E. terrae produced antibacterial activity in many different growth conditions; however, it was found to grow and produce antibacterial activity optimally in R4 fermentation broth. R4 fermentation broth consists of 10g glucose, 1g yeast extract, 0.1g casamino acids, 3g proline, 10g MgC12-6H2O, 4g CaC12-2H2O, 0.2g K2SO4, 5.6g TES free acid per liter of deionized H2O at pH 7. Due to the emerging nature of E. terrae, its metabolism and ecology have not yet been extensively documented.

Genomics
The genome of E. terrae was sequenced by Ling and her team and is estimated to be 6.6 Mbp in length, which was determined by using an in house pipeline provided by TUCF Genomics. After the draft genome was assembled it was screened for sequences closely related to adenylation domains. Contigs that were found to code for teixobactin biosynthetic pathways were manually edited and placed in order. This allowed the combination of other contigs that were separately assembled. Any gaps that remained in the genome were filled using bridging fragments developed by PCR and Sanger sequencing. The gaps were closed using the same primers used in amplification.

Antibiotic Production
The production of teixobactin by E. terrae is very prominent because recent tests have revealed that teixobactin binds differently than most normally used antibiotics which makes it harder for the bacteria being attacked to develop resistance. Experiments performed by Ling et al. have shown teixobactin is capable of binding to lipid precursors of peptidoglycan, which makes up part of bacterial cell walls. The results did not show any resistance to teixobactin in the organisms that were studied, including Staphylococcus aureus and Mycobacterium tuberculosis. These findings indicate that teixobactin’s target is not a protein, leading to the belief that the development of bacterial resistance to teixobactin is much less likely. These experiments also showed that teixobactin followed a similar mechanism of action as the antibiotic vancomycin that binds to the lipid II molecule in peptidoglycan precursors but, unlike vancomycin, teixobactin is capable of binding to modified lipid II molecules found in vancomycin resistant bacteria. Teixobactin’s inhibition of peptidoglycan synthesis is further explained by Ling’s finding of a build up of undecaprenyl-N-acetylmuramic acid-pentapeptide, a crucial step in the biosynthesis of peptidoglycan. According to Ling’s tests, teixobactin is capable of inhibiting peptidoglycan synthesis by binding to either lipid I, lipid II, and undecaprenyl-pyrophosphate. Teixobactin also seemed to be specifically involved with peptidoglycan precursors rather than blocking enzyme activity.