Probiotic Plants: Newly Discovered Protein Regulates the Plant Biome

Britt Bistis | britt.bistis@yale.edu January 29, 2020

Probiotic Plants: Newly Discovered Protein Regulates the Plant Biome

Image courtesy of Jennifer Yoon

A new gene cluster discovered by Professor Konstantin Severinov’s lab at Rutgers encodes a peptide that provides legumous plants with extra power. This peptide acts as an antibiotic that endows the plants with enhanced resistance to certain harmful bacteria—leaving room for bacteria which promote health. Probiotic bacteria support their host organism’s health in two main ways: first, they produce compounds necessary to the organism’s fitness, and, secondly, they protect against harmful bacteria. Probiotic bacteria called Rhizobium are nitrogen-fixing bacteria that exist in a symbiotic relationship with many plants, helping them by converting free atmospheric nitrogen into necessary nitrogen compounds that plants can uptake and use. Severinov’s lab discovered that Rhizobium also acts as a probiotic by producing a protein that targets harmful bacteria. By characterizing a new gene cluster, the lab discovered a new protein that is thought to have antimicrobial and anticancer effects.

Severinov and his colleagues used cutting-edge genetic data processing to identify this new gene cluster. Before the age of bioinformatics, the classic forward genetic screen was, and still is, prevalent in research. This approach entails randomly mutating DNA and subsequently carefully characterizing random mutations that affect the model system, allowing researchers to screen for the desired phenotype being studied and trace the phenotype back to the random mutation made. However, in some cases, this is rather slow and inefficient, often returning previously discovered hits.

The researchers screened for new proteins belonging to the family of peptide regions called Linear Azol(in)e-containing Proteins (LAPs), which have several common sequences in the DNA that encode them and are of particular interest to researchers because of their antibiotic effects. LAP regions contain a few key genetic elements. First, these gene clusters encode enzymes which facilitate the modification of proteins, such as the addition of chemical functional groups required for the protein to function. In addition, they need an ABC transporter, a type of transporter that facilitates the secretion of the mature protein. Lastly, they include the sequence of the core peptide itself. Using bioinformatics, Severinov and his colleagues were able to circumvent the challenges of traditional genetic screens and efficiently screen for novel gene clusters.

Having discovered a novel LAP gene cluster that fit their research criteria, the team then needed to purify and characterize this new central protein. However, it is difficult to purify an unknown protein whose properties have not yet been classified. The Rutgers lab had some sense of the polarity of the protein, and so they used a process that separates proteins based on this characteristic. Then, using knowledge of the optical properties of the protein family, they were able to find and further purify the fraction of their new protein. To analyze the structure of the purified protein, they used a variation of mass spectrometry, a laboratory technique that allows researchers to determine the exact molecular weight and atomic composition of a compound.

Severinov and his team called the new protein phazolicin (PHZ). PHZ is produced by the species Rhizobium Pop5 and belongs to the LAP family, which contains a five-atom ring in their structure with one nitrogen. PHZ has more of three specific amino acids (protein building blocks)—serine, cysteine, and threonine—all strategically placed to allow the mature PHZ protein to form a cycle. These LAPs belong to an even larger family of proteins called Ribosomally-synthesized and Post-translationally modified Peptides (RiPPs). The PHZ protein, like all peptides in this family, is synthesized at the ribosome and undergoes significant post-translational modifications, which, for PHZ specifically, involves cyclization. Empowered with the knowledge of what type of motifs the LAP-encoding gene cluster would have, Severinov and his colleagues used genome-mining techniques to screen the genome for these motifs.

To determine which pathway PHZ inhibits, researchers investigated whether PHZ’s mode of bacterial targeting involves inhibiting the DNA replication or protein synthesis machinery in bacteria. To accomplish this, they used two different fluorescent proteins. One fluorescent protein is expressed when DNA replication is lowered, and the other fluorescent protein is expressed only in the presence of translational inhibitors. They then looked for processes which caused the fluorophore to fluoresce. They found that functional PHZ protein inhibits protein translation.

The next step was to determine the exact binding sites of PHZ to further elucidate the role of this antibacterial protein in blocking translation at the ribosome. To this end, the researchers first attempted to crystallize the ribosome and its associated proteins, including PHZ. They then used an advanced imaging technique called x-ray crystallography to determine the three-dimensional structure of the complex. However, the ribosome with associated proteins proved a poor candidate for crystallization.

Severinov and his colleagues also used cryo-electron microscopy to confirm the structure predicted by x-ray crystallography. In contrast to x-ray crystallography, cryo-electron microscopy allows for precise determination of structure without crystallization. “Cryo-EM is a new technology, which is very powerful and doesn’t need crystals. Essentially, you simply look at molecules lying on an electron microscopy grid, average tens of thousands of molecules at different angles lying on the surface, and then make computers make three dimensional models at high resolution,” Severinov explained. This technique revealed that PHZ binds the protein-ribosome complex in the narrowest part of the peptide exit tunnel, through which newly formed protein leave the ribosome, and blocks it, thereby blocking translation. This eventually stops protein synthesis and results in bacterial death.

Like many other proteins altered by post-transcriptionally modifications, PHZ has the strongest activity in bacteria closely related to the bacterial species that produce the PHZ protein. Specifically, PHZ has the most antibiotic activity in Rhizobium and significantly lower antibiotic activity in E. coli. A likely evolutionary explanation for why PHZ has strong antibiotic effects in similar bacteria is that the molecule is made for a competitive advantage over those in a similar niche. “It needs to take care of its competing bacteria that live in the same habitat in the soil, and E. coli is not one of them as it lives in human intestines” Severinov explained. The Rutgers team found that the Rhizobium Pop5 are immune to the PHZ they produce due to another gene in the cluster they called phzE that confers self-immunity to the bacteria. PhzE encodes an exporter pump that enables Pop5 to efficiently excrete PHZ upon entry into the cell, thereby immunizing the bacteria to its harmful effects.  

In the near future, Severinov and his colleagues project that PHZ-like proteins could be used in agriculture and employed as biocontrol agents. Understanding the antibacterial mechanism of PHZ will enhance our understanding of how antibacterial LAPs function, as many protein members of this family induce antibiotic effects via significantly different mechanisms, which makes them an excellent candidate for antibiotic research. Investigations into this class of proteins can further inform bacterial immunobiology studies and aid research and medicine in the quest for new antibiotics.

References:

Blankenfeldt, W., Kuzin, A., Skarina, T., Korniyenko, Y., Tong, L., Bayer, P., … Mavrodi, D. (2004). Structure and function of the phenazine biosynthetic protein PhzF from Pseudomonas fluorescens. doi: 10.2210/pdb1xua/pdb

Carazo, J.-M. (2019). F1000Prime recommendation of Single particle cryo-EM reconstruction of 52 kDa streptavidin at 3.2 Angstrom resolution. F1000 – Post-Publication Peer Review of the Biomedical Literature. doi: 10.3410/f.735891717.793561147

Travin, D. Y., Watson, Z. L., Metelev, M., Ward, F. R., Osterman, I. A., Khven, I. M., … Severinov, K. (2019). Structure of ribosome-bound azole-modified peptide phazolicin rationalizes its species-specific mode of bacterial translation inhibition. Nature Communications10(1). doi: 10.1038/s41467-019-12589-5

Blankenfeldt, W., Kuzin, A., Skarina, T., Korniyenko, Y., Tong, L., Bayer, P., … Mavrodi, D. (2004). Structure and function of the phenazine biosynthetic protein PhzF from Pseudomonas fluorescens. doi: 10.2210/pdb1xua/pdb

Travin, D. Y., Watson, Z. L., Metelev, M., Ward, F. R., Osterman, I. A., Khven, I. M., … Severinov, K. (2019). Structure of ribosome-bound azole-modified peptide phazolicin rationalizes its species-specific mode of bacterial translation inhibition. Nature Communications10(1). doi: 10.1038/s41467-019-12589-5

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