Agar Artist Caitlin Cahak

At ASM Microbe 2017 in New Orleans, I spoke with Medical Laboratory Scientist Caitlin Cahak about her agar art.

Prions in bacteria

prion conversionBacteria do not develop transmissible spongiform encephalopathies, but they have been found to produce prions – proteins that can adopt alternative conformations with different functions.

Prion diseases, a frequent topic on this blog, are caused by misfolding of a normal cellular prion protein (illustrated; image copyright ASM Press). Prion proteins are found in other organisms, where the alternative conformation confers a new, non-pathogenic function to the protein. At least 12 different prion proteins have been found in yeast, and they confer the ability to grow more efficiently under certain conditions. Now prions have been discovered in bacteria (link to article).

A search of 60,000 bacterial genomes for proteins with prion-forming domains revealed one in the transcription termination protein Rho from Clostridium botulinum (Cb-Rho). When produced in E. coli, the protein forms amyloid – protein aggregates in the form of fibrils – that are characteristic of prions. A 68 amino acid stretch of Cb-Rho can functionally substitute for the prion-forming domain of a yeast prion-forming protein. This protein, called Sup35, can read stop codons in the prion state, and this phenotype was recapitulated in yeast by the Clostridium prion.

The Cb-Rho prion can convert between prion and non-prion conformations in E. coli. This property was demonstrated by placing a Rho-dependent terminator between a promoter and the lacZ gene, the product of which produces a blue color. In the prion state, Rho has decreased activity, leading to blue cells. In the non-prion state, normal termination leads to pale blue colonies. A mixture of blue and pale blue colonies was observed, showing that Rho exists in the prion and non-prion states.

The prion conformation was also shown to be heritable. Blue colonies always gave rise to blue colonies, while pale blue colonies formed pale blue colonies. The blue colony color lasted for over 120 generations.

The finding of a prion in bacteria indicates that this form of protein-based heredity arose before eukaryotes emerged on Earth. Similar prion-like protein domains have also been found in other phyla of bacteria, suggesting the existence of an important source of epigenetic diversity that can allow bacterial growth under diverse conditions. Exactly how bacterial prions confer new functions will be exciting to discover.

Last time we learned that eukaryotes probably didn’t invent the nucleus. Now we find that prions likely emerged first in bacteria. Did eukaryotes invent anything?

Viruses help form biofilms

Inoviridae virionBacteria frequently grow in communities called biofilms, which are aggregates of cells and polymers. An example of a biofilm is the dental plaque on your teeth. Biofilms are medically important as they can allow bacteria to persist in host tissues and on catheters, and confer increased resistance to antibiotics and dessication. Therefore understanding how biofilms form is crucial for controlling microbial infections. An advance in our understanding of biofilms formation is the observation that filamentous phages help them assemble, and contribute to their fundamental properties.

Pseudomonas aeruginosa is an important human pathogen which is a particular problem in patients with cystic fibrosis. The ability of this bacterium to form biofilms in the lung is linked to its ability to cause chronic infections. Pseudomonas aeruginosa biofilms contain large numbers of filamentous Pf bacteriophages (pictured; image credit). These viruses lyse cells and release DNA, which becomes one component of the biofilm matrix.

Mixing supernatants of P. aeruginosa cultures with hyaluronan, which is present in airways of cystic fibrosis patients, resulted in the formation of a biofilm – in the absence of bacteria. A major component of P. aeruginosa biofilms was found to be Pf bacteriophages. When purifed Pf bacteriophages were mixed with hyaluronan, biofilms formed. Similar biofilms also formed when the filamentous bacteriophage fd of E. coli was mixed with hyaluronan. Mixtures of Pf bacteriophages and various polymers (alginate, DNA, hyaluronan, polyethylene glycol) formed liquid crystals (matter in a state between a liquid and a solid crystal).

Pf phages were detected in sputum from patients with cystic fibrosis, but not in uninfected patients. Addition of Pf phage to sputum from patients infected with P. aeruginosa made the samples more birefringent, a property of liquid crystals. Compared with a strain of P. aeruginosa that does not produce Pf phage, colonies of virus-producing strains formed liquid crystals. These observations indicate that Pf phage help organize the bacteria into a biofilm matrix.

Some features of biofilms include their ability to adhere to surfaces, to protect bacteria from dessication, and to increase resistance to antibiotics. Addition of phage Pf increased biofilm adhesion and tolerance against dessication. Such addition also made the biofilm more resistant to aminoglycoside antibiotics, because these were sequestered in the biofilm. No phage-mediated increased resistance to ciprofloxacin was observed, probably because this antimicrobial does not interact with polyanions of the biofilm as do aminoglycosides.

These results show that presence of bacteriophage in a biofilm of P. aeruginosa helps organize the matrix while contributing to some of its fundamental properties. It seems likely that filamentous phages of other bacteria will play roles in biofilm formation, suggesting that targeting the phages in these matrices could be effectie strategies for treating biofilm infections.

TWiV 323: A skid loader full of viromes

On episode #323 of the science show This Week in Virology, the family TWiVidae discuss changes in the human fecal virome associated with Crohn’s disease and ulcerative colitis.

You can find TWiV #323 at www.microbe.tv/twiv.

TWiM 90: Think globally, act locally

I usually don’t post TWiM episodes here, but #90 has a lot of virology. In this episode, recorded in La Jolla, CA at the annual meeting of the Southern California Branch of the American Society for Microbiology, I first speak with Laurene Mascola, Chief of Acute Communicable Diseases at the Los Angeles County Department of Public Health. Dr. Mascola talks about how Los Angeles county has prepared for an outbreak of Ebola virus. Next up is David Persing, Executive Vice President and Chief Medical and Technology Officer at Cepheid. His company has developed an amazing, modular PCR machine that is brining rapid diagnosis everywhere, including the United States Post Office. And it might even be available on your refrigerator one day.

Watch TWiM #90 below, or listen at microbeworld.org/twim or iTunes.

 

Michael Schmidt on copper at TEDx

On TWiM #55 we discussed the remarkable ability of copper to reduce hospital acquired infections. Now you can watch Michael Schmidt, TWiM co-host and a co-author on this work, discuss the findings at a recent TEDx talk in Charleston, South Carolina.

An RNA virus that infects Archaea?

Nymph Lake, Yellowstone National ParkEvery different life form on earth can probably be infected with at least one type of virus, if not many more. Most of these viruses have not yet been discovered: just over 2,000 viral species are recognized. While the majority of the known viruses infect bacteria and eukaryotes, there are only about 50 known viruses of the Archaea, and these all have DNA genomes. The first archaeal RNA viruses might have been recently discovered in a hot, acidic spring in Yellowstone National Park.

Archaea are single-cell organisms that are similar in size and shape to bacteria, but are evolutionarily and biochemically quite distinct. They inhabit a broad range of environments including those with extreme conditions such as high temperature, acidity, and salinity. Identification of archaeal RNA viruses is important because their study could provide information about the ancestors of RNA viruses that infect eukaryotes. Direct sequencing of viral communities from the environment, known as viral metagenomics, is one approach being taken to discover archaeal viruses.

The acidic (pH <4) and hot (>80°C) springs in Yellowstone National Park were examined for the presence of archaeal RNA viruses because these bodies of water contain mainly Archaea. Samples were obtained from 28 different sites and extracted nucleic acids were treated with DNAase (to remove DNA genomes) and then reverse transcriptase (to copy RNA to DNA). If reverse transcription was reduced by treatment with RNAse, it was concluded that the sample contained mostly RNA. The results narrowed the sample size to three, all from Nymph Lake. New samples obtained twelve months later also showed a predominance of RNA and were used for metagenomic analysis by deep sequencing.

Analysis of the RNA viral sequences revealed coding regions for a predicted RNA dependent RNA polymerase (RdRp), a hallmark of RNA viruses. One assembled sequence of 5,662 nucleotides, believed to be a complete viral genome, encodes a single open reading frame containing a RdRp and a putative capsid protein similar to that of the positive-strand RNA containing nodaviruses, tetraviruses, and birnaviruses. Another viral sequence encoded a protein with 70% amino acid homology to the predicted RdRp. The sequences are from a novel virus which does not belong to any known virus family.

These results clearly show that at least two related but distinct RNA viruses are present in Nymph Lake. However whether or not the hosts of these viruses are Archaea or Bacteria cannot be determined by these metagenomic analyses. What is needed to resolve this question is old-fashioned virology:  isolating RNA virus particles that can infect an archaeal host and produce new infectious viruses.

B Bolduc, DP Shaughnessy, YI Wolf, EV Koonin, FF Roberto and M Young J. Virol. 2012, 86(10):5562. DOI: 10.1128/JVI.07196-11.

TWiM 34: Doing the DISCO with Emiliania

On episode #34 of the science show This Week in Microbiology, Vincent, Michael, and Elio discuss changing populations of Emiliania huxleyi and their viruses in the North and Black Seas.

You can find TWiM #34 at microbeworld.org/twim.

TWiM 33: Tuning the immune organ

On episode #33 of the science show This Week in Microbiology, Vincent, Michael, and Ivo review the requirement for segmented, filamentous bacteria for the induction of a specific type of helper T cell in the gut.

You can find TWiM #33 at microbeworld.org/twim.

A spike for piercing the cell membrane

bacteriophage t4Some viruses that infect bacteria (bacteriophages) deliver their DNA into the host cell with an amazing injection machine. The tailed bacteriophages (such as T4, illustrated) store their DNA in a capsid attached to a long tail tube that is surrounded by a sheath. At the bottom of the tube is a baseplate with a spike in the center. When the baseplate contacts the host cell, the sheath contracts, driving the spike into the cell membrane. The viral DNA travels down the tube and enters the cell through the opening produced by the spike. The structure of the spike has now been determined, providing insight into how it makes a hole in the cell membrane.spike

Structures of the spike from P2, a well studied virus of E. coli, and the choleraphage phi92 were determined.  The spikes are built from three copies of a single protein (trimers). The trimers are indeed shaped like spikes: they are wider at one end and taper to a rather sharp tip (figure at right). The bulk of the spike is made up of alternating beta-strands which form a corkscrew-like beta-helix. The sharp tip is composed of three beta-hairpins which the authors say “come together like petals in a flower bud”.

iron in spikeAn interesting feature on the interior of the spike tip are three pairs of histidine residues that hold a single iron atom (figure at left). The authors believe that the iron helps the trimers form by keeping the protein chains in register, and also provides increased strength to the tip. The latter would be important as it pierces the cell membrane. This idea could be tested by changing one or more histidines to another amino acid so that iron cannot be held in the tip.

To verify that these structures are those of the spike that is attached to the baseplate, the authors solved the structure of the phage particle by cryo-electron microscopy and image reconstruction. The image clearly shows the spike protruding from the base plate (figure below). The structures of the spike proteins solved by X-ray crystallography could then be computationally fitted in the correct location in the cryo-EM image of the baseplate.baseplate with spike

These structures support the idea that the spike is a rigid needle that pierces the bacterial membrane and forms a channel through which the DNA can pass. An interesting question is how the DNA gets past the spike, which plugs the end of the tail tube. The authors believe that the spike is loosely attached to the tube and might be easily dissociated once it passes through the cell membrane. The spike of phage T4 can be dissociated at low pH, a condition that is found in the periplasm, the space between the inner and outer bacterial membranes.

There are distinct signatures of the spike structure that can be identified in proteins of other contractile injection systems, including diverse bacteriophages. They can also be found in bacterial type VI secretion systems, which are membrane complexes used to transport proteins outside of the cell. Once evolution builds a useful machine, it is often put to many diverse uses.

 
Browning, C., Shneider, M., Bowman, V., Schwarzer, D., & Leiman, P. (2012). Phage Pierces the Host Cell Membrane with the Iron-Loaded Spike Structure, 20 (2), 326-339 DOI: 10.1016/j.str.2011.12.009