First, there was biology which began in earnest in the 19th century. Then came molecular biology in the 1920s and the foundation of mutagenesis set forth by Herman Muller in 1927. Then, genetic engineering was first applied in 1972 the lab of Paul Berg. Finally, humans had the ability to manipulate living organisms in a specific, directed way. Fast forward 38 years to the announcement by J. Craig Venter that the first synthetic organism was created with a completely synthetic genome. However, Mother Nature is very particular about what exactly humans can do with respect to organismal manipulation. The naive thought that simple addition of genes from one organism into a more suitable organism would lead to theoretical, effective production of desired chemicals was soon the way of the albatros.
This is when scientists had to take a step back and rethink their strategy. They had to consider gene regulation (positive and negative feedback), build-up of secondary metabolites, toxicity of produced end products, etc. It wasn’t enough to add genes coding for enzymes necessary for desired chemical production. Through the advancements of bioinformatics, computation biology, and a nascent field called systems biology, scientists are just now starting to see the fruits of their labor.
Humor me; type in “engineering bacteria” into Google News. Take a look at the headlines that pop up in your browser. Look at the amazing advancements that are happening currently and imagine what is to come…
There is one thing that can be said about scientists: they’re never satisfied…thankfully. Observation and curiosity leave them on a never-ending quest to understand Mother Nature and improve humanity. One great example of this is the field of alternative energy science. Through the efforts of the Bioenergy Research Centers (BRCs) and Joint Genome Institute within the U.S. Department of Energy‘s Office of Science, there is a perpetual search for Nature’s best metabolic machinery. This search requires thinking outside the box and sometimes outside your comfort zone. For example, last year researchers from the Joint BioEnergy Institute published findings that originated in the El Yunque National Forest in Puerto Rico, a rain forest and home to Enterobacter lignolyticus, a bacterium that is tolerant to ionic liquids (liquids with salts that are not crystaline, but are liquid). This discovery began with the observation that soil microbes at El Yunque have a high rate of organic decomposition and tolerance to osmotic pressure.
Another example are bacteria from the genus Caldicellulosiruptor that are able to degrade biomass, however, they live in extremely thermophilic environments like hot springs from New Zealand to Russia to Yellowstone. Researchers at the BioEnergy Science Center were able to isolate these microbes and start characterizing the enzymes responsible for degrading woody biomass into simple sugars.
Or what about researchers at the Great Lakes Bioenergy Research Center essentially dissecting a leaf-cutter ant colony in Panama to examine its ecology; from the fungus the ants use as food, to the bacteria that help degrade the leaves. Or what about isolating microbes from termite guts or wasp guts?
Then there is the champion for raising scientific curiosities, Clostridium thermocellumwhich holsters woody biomass degradation factories attached to the outside of its cell membrane. These factories are known as cellulosomes.
Welcome to part 4 of how bacteria make decisions. Parts 1 and 2 dealt with chemotaxis. Part 3 was a look at two component signaling systems. This part will deal with my favorite aspect of bacterial decisions for several reasons. Second messengers are common from bacteria to humans. The major second messenger we all learn about in biochemistry class is cyclic AMP (cAMP). However, bacteria use several nucleotides as second messengers. Many are used as determinants in the decision making process, but one of the most recently discovered (and personal favorite) is cyclic-di-GMP, or c-di-GMP.
Bacteria are constantly processing signals both inside and outside their cell membranes. Hard to believe that one of the most abundant response molecules was only discovered in the late 1980s while researching how a certain species, Acetobacter xylinumnow known as Gluconacetobacter xylinus, produced cellulose. Almost by accident, the Benziman lab discovered the enzyme responsible for cellulose production (cellulose synthase) was regulated by a nucleotide, later found to be c-di-GMP. Since that discovery, c-di-GMP has become a hot topic among microbiologists and immunologists due to the decisions bacteria make as the level of c-di-GMP increases within the cell. As I learned it, the concentration of c-di-GMP had predictable outcomes on the decisions of bacteria: high levels leads to loss of motility, increase in biofilm formation, changes in cell morphology, and increase in cell-cell communication. When low levels of c-di-GMP are present, the cell decides to move around (motility), become resistant to heavy metals, and, most importantly, becomes virulent. For example, Vibrio cholerae, the bad guy responsible for cholera, only decides to move around and produce cholera toxin when c-di-GMP levels are low in the cell. If levels increase, V. cholerae will produce biofilms via extracellular polysaccharide (EPS) production.
You might be asking yourself what controls the c-di-GMP levels of a bacterial cell. The initial discovery in the Benziman lab also found the enzymes/proteins that were responsible for making and breaking the second messenger. The long (and short) names are; for making c-di-GMP from 2 GTP molecules, diguanylate cyclases (DGCs aka GGDEF proteins) and degradation by phosphodiesterases (PDEs aka EAL proteins). GGDEF and EAL proteins are so called due to important amino acids necessary for their functions, GGDEF is glycine, glycine, aspartate, glutamate, phenylalanine, and EAL is glutamate, alanine, leucine. These enzymatic activities are usually controlled by regulatory protein domains common in bacteria (and humans). Signals from the environment (internal or external) can trigger changes in enzyme activity of GGDEFs and EALs thus changing the cellular concentration of c-di-GMP. This mechanism is well understood after 30 years of research. However, what happens next is still essentially unknown.
Cyclic-di-GMP levels rise within a bacterial cell. Now what? It was known early on that c-di-GMP itself could then interact with GGDEFs to inhibit activity. But what other proteins interact with c-di-GMP and help these bacteria decide to make major lifestyle changes? It wasn’t until 2006 that bioinformaticians predicted c-di-GMP binding to a protein, or protein domain. PilZ, an obscure protein of unknown function but necessary for Type IV pili motility, was hypothesized to bind c-di-GMP. By the end of 2007, this prediction was verified and PilZ domain proteins were the first shown linking c-di-GMP to downstream proteins in pathways, or circus rings.
Transitioning from a free swimming/moving cell to life in a biofilm community is a major lifestyle change for bacteria. This decision takes commitment which is initiated by a small molecule. In the next installment, we will get to the heart of current research.
Continuing on the theme that bacteria are Nature’s smallest circus, I want to highlight the most glaring problem with our knowledge of these 2000 ring circuses. We have discussed how proteins encoded by genes within a microbe’s genome often work together to carry out their function, i.e. pathways (or rings). To date, according to the NCBI genome site 4019 bacterial genomes have been sequenced to the point that we know the number of genes and proteins each organism contains. Moreover, this equates to 7,309,205 genes total or roughly 1818 genes per genome. These are astonishing numbers. To show our futility as experts of all things natural, over 30% of these genes are considered hypothetical or uncharacterized. In some genomes, these genes make up 60% of the total genes. These terms are a technical way of saying “hell if we know what they do”. Computers have recognized them as genes or open reading frames, however, the gene itself isn’t similar enough to known or characterized genes for scientists or computers to call it “the same”. If these gene products (proteins) functions are unknown, they cannot be assigned to a ring in the circus therefore making the largest ring by far in any bacterial circus the “unknown” ring.
A simple GIF to give a look at the complexity of a 1000-ring circus going on in cells all the time. Each ‘ring’ is a different pathway necessary for this generic microbe to survive. Wish I had 40 years to make an accurate depiction of an actual bacterial circus with its 2000 ‘rings’. Enjoy!
Time again for My Tiny Highlight (MyTH) of a particular bacterium. This week is the infamous Pseudomonas aeruginosa. This trooper can be found almost anywhere on earth due to its ability to use all kinds of material as food including diesel and jet fuel. Most of us know it as a common infection you get while in the hospital because it is so darn hard to kill. If you know someone that suffers from cystic fibrosis, this bacterium can be fatal as it is commonly found in the lungs of these patients causing pneumonia. I personally learned about this bacterium in school when talking about hot tubs due to its resistance to disinfectants.
P. aeruginosa is one of the most famous microbes for forming biofilms. A biofilm is a group of cells that attach to each other and to some surface usually by secretion of sticky sugars from the cells. A common sticky sugar, or exopolysaccharide (EPS), excreted by P. aeruginosa is alginate, an uncommon sugar for bacteria since it is most commonly found associated with brown algae. Its existence in Pseudomonas has led to significant knowledge about the synthesis of alginate (alginate biosynthesis; another ring in the bacterial circus). This knowledge is leading to novel medical applications for alginate.
P. aeruginosa is also known for its role in a multicellular behavior which seems strange for a single-celled organism but is becoming more and more common. Quorum sensing was first described over 40 years ago. I beg you to check out Bonnie Bassler‘s TED talk about this. QS is how single cell bacteria are able to send signals into the environment to communicate with members of their own species and with other types of bacteria as a way to assess their population.
Another feature of P. aeruginosa is their modes of transportation, i.e. motility. P. aeruginosa has two major modes of getting around their environment, swimming motility with flagella or “twitching motility” on a surface using Type IV pili. We have discussed flagellar motility before (Chemotaxis). Type IV pili (TFP) is different. Pili are somewhat similar to flagella because they protrude out of the cell and are used to move around. Unlike flagella, pili don’t rotate but rather extend out before retracting back to the cell pulling the cell in the direction of the pili tip. The coordination of pili extension and retraction is guided by a chemotaxis-like pathway. Many bacteria have evolved to use chemotaxis proteins that have adapted to new roles within the cell.
I will end this MyTH with a special shout out to one of my favorite researchers, Carrie Harwood. Caroline Harwood is a professor at the University of Washington in Seattle. Carrie’s daughter suffers from cystic fibrosis and this led to Carrie’s curiosity in P. aeruginosa. She is a pioneer and a role model for many female scientists including Becky Parales. For her tireless career, Carrie was inducted into the National Academy of Science a few years back. A recognition well deserved.