Bacillus: The Dark Side vs. The Good Side

Contributed by Darwin Bandoy, DVM

This particular talk by Bill Widner of Novozymes tantalized my interest as I only associate Bacillus with things a veterinarian must know to avoid, which is Bacillus anthracis, a deadly disease causing gram-positive bacteria in animals and humans particularly in ruminants. Anthrax gained notoriety recently in the US when letters post 9/11 were mailed to media and congressional offices. So with all this negative association with Bacillus, what makes it a good expression host? Bacillus are strong secretors and the speaker had prior experience with this working with Bacillus thurigiensis that secrete crystal proteins with insecticidal properties for his PhD in microbiology. Bacillus is also cheap to grow, does not have the endotoxin of E. coli, and without much intellectual property barrier. Putting this together makesBacillus an attractive expression host. But if you have a good expression host, how do you make things better? Technologies to improve production strains include developing stronger promoters, knocking protease activity, overexpressing chaperones and increasing copy numbers.

What is the path to products using Bacillus as expression host? A new product is first identified, the cloning of the gene product is performed, followed by construction of expression cassettes and introduction intro various Bacillus host. But it is not enough for Bacillus to express it, it must do so at a larger scale. This is done sequentially from lab scale (2 L) fermenters for yield optimization to pilot plant level (500 to 2000 L) and full-scale production. Novozymes core technologies in genomics are used for quality control. Experimentation is unavoidable as some genes are expressed better in oneBacillusthan the other. Even within a given species, some isolates are better expressors than others. This lecture on using Bacillus reminds us of the Star Wars quote by Luke Skywalker, “there is good in him”, as though the Bacillus genera contains pathogens that secrete toxins, that secreting capability can be harnessed for good purposes.

 

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Assessing Contamination of Whole Genome Sequences for Microbial Isolates

Contributed by Shawn Higdon

You are working tirelessly in the lab attempting to generate cultures of microbes that are pure, meaning a single organism comprising a single well-isolated colony. You finally have what appears to be an isolate of pure culture and wish to sequence the microorganism’s genome. After running through the isolate through the sequencing pipeline, you come to find that what was surely a pure isolate may, in fact, be multiple microbes living closely together after assembling the genome…or it could be contamination.

As a graduate student working with whole genome sequencing data from many banked microbial isolates, this is a situation that I am now faced with along the road to achieving my Ph.D. A common finding is that the draft genome assembly for a given isolate will be greater than 10 Mbp, which I interpret as a strong indication that the isolate may be a co-culture or a pure isolate that became contaminated. My challenge is in determining which is actually the case.

My initial approach, which was recently referred to as “round-about” by a colleague, has been to assemble PE150 Illumina reads for each isolate, map the reads back to the assembly and subsequently form contiguous sequence bins that theoretically indicate the presence of whole genomes from distinguished organisms. These contig bins can then be classified independently of one another using software programs such as Sourmash. An alternative approach was recently proposed to me, which is to use the CheckM software suite from Donnovan Parks et al. This is something I plan on looking into moving forward.

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Largest E. Coli O157:H7 Outbreak in Romaine Lettuce in Over a Decade with No Source Identified

Contributed by Nguyet Kong

FDA and CDC have reported that 98 people in 22 different states have become ill with E. coli O157: H7 after ingesting whole head romaine lettuce. No deaths have been reported, but many have been hospitalized. The FDA has investigated and found the source of the lettuce growing area to the Yuma, Arizona region. They are looking at all possibilities including contamination at different phases such as growing, harvesting, packaging and distribution.

The agency has figured out that the lettuce was harvested between March 5 and March 16, which when ingested by people it is past the 21-day shelf life. The growing season is ending and they are no longer growing romaine lettuce at this time. Also, another illness was not linked to Yuma region, but people were reporting illness after eating bagged romaine salads. Since the bacteria is causing many people to be sick, everyone should avoid eating romaine lettuce unless it’s clearly not from the Yuma region. If the packaging does not clearly state the growing location, the CDC has advised the public to toss the lettuce.

Symptoms of the Shiga toxin producing (STEC) E. coli O157: H7 infection is different with every person. It can include nausea, vomiting, bloody diarrhea, stomach cramps, loss of appetite, fever and mild infections. Most people will get better within a week. However, for less than 10% of people, it can have life-threatening complications due to their kidneys not functioning correctly.  This is known as hemolytic uremic syndrome (HUS).

The FDA & CDC are continuing their investigation to over 2 dozen other romaine lettuce growers in the country.

References:

https://www.cdc.gov/ecoli/2018/o157h7-04-18/index.html

https://www.cdc.gov/ecoli/ecoli-symptoms.html

https://www.fda.gov/Food/RecallsOutbreaksEmergencies/Outbreaks/ucm604254.htm

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Bacillus as Expression Hosts

Contributed by Darwin Bandoy, DVM

This particular talk by (Bill Widner, Ph.D., Novozymes Staff Scientist) tantalized my interest as I only associate Bacillus with things a veterinarian must know to avoid, which is Bacillus anthracis, a deadly disease causing gram-positive bacteria in animals and humans particularly in ruminants. Anthrax gained notoriety recently in the PhDUS when letters post 9/11 were mailed to media and congressional offices. So with all this negative association with Bacillus, what makes it a good expression host? Bacillusare strong secretors and the speaker had prior experience with this working with Bacillus thurigiensis that secrete crystal proteins with insecticidal properties for hPh.D.in microbiology. Bacillus is also cheap to grow, does not have the endotoxin of E. coli, and without much intellectual property barrier. Putting this together makesBacillus an attractive expression host. But if you have a good expression host, how do you make things better? Technologies to improve production strains include developing stronger promoters, knocking protease activity, overexpressing chaperones and increasing copy numbers.

What is the path to the product using Bacillus as expression host? A new products is first identified, the cloning of the gene product is performed, followed by construction of expression cassettes and introduction intro various bacillus host. But it is not enough for Bacillusto to express it, it must do so on a larger scale. This is done sequentially from lab scale (2 L) fermenters for yield optimization to pilot plant level (500 to 2000 L) and full scale production. Novozymes core technologies in genomics are used for quality control. Experimentation is unavoidable as some genes are expressed better in one Bacillusthan the other. Even within a given species, some isolates are better expressors than others. This lecture on using Bacillus reminds of the Star Wars quote by Luke Skywalker, “there is good in him”, as though the Bacillusgenera contains pathogens that secrete toxins, that secreting capability can be harnessed for good purposes.

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Mucilage Microbial Isolates 2.0: future directions for the corn project

Contributed by Shawn Higdon

In the summer of 2013, I joined the lab of Alan Bennett at U.C. Davis as a Junior Specialist with the Department of Plant Sciences. All I knew at that time is that I would be working on a research project with an isolated variety of corn from Mexico that had a very unusual phenotype. I soon discovered that the project was highly collaborative with multiple principle investigators on board, the project had already been going on for several years, and that the Weimer lab was working on the project to generate a collection of microbes that were isolated from this intriguing variety of corn. While my role in the project began as a technician processing dried tissue samples for elemental analysis by isotopic ratio mass spectrometry in a robot-like fashion, I had no idea of how much potential this project had to offer both me and the scientific community.

Fast-forward nearly five years to the present and I am now a graduate student in the Plant Biology Graduate Group at U.C. Davis working on a different facet of the same project. The tentative title of my Ph.D. dissertation project is, “Characterization of plant growth promoting functions from the maize aerial root microbiota.” The foundation of my project stems directly from the amazing work performed by the Weimer lab to generate a robust collection of microbial isolates from the aerial root mucilage of maize that grows naturally in the Sierra Juarez region of Mexico. Specifically, the variety of maize that we are working with exhibits the extended development of aerial roots that exude copious volumes of a polysaccharide-rich mucilage. Metagenomic analysis indicated that the mucilage environment supports a distinguished microbiota, and compositional analysis of the mucilage polysaccharide has also revealed that the host secrets a highly diverse complex-carbohydrate that may be the driving force behind associations with microbes that confer plant growth promotion or PGP.

The work that I have achieved so far during my pursuit of a Ph.D. has largely been centered around generating and analyzing whole genome sequence data from subsets of the microbes within the Weimer lab’s collection of maize microbial isolates. This work, while initially unanticipated, has changed my way of thinking and opened the door for me to an area of science that lies at the interface of Microbiology, Plant Biology, and Bioinformatics. I favor the classification of the research that I do within the area of sustainable agriculture, which is something that I am very passionate about being involved in both currently as a student and hopefully in the future. Given the context of the system I am working with, my time spent conducting research on this project, and the experiences that I have acquired thus far, I have developed the following hypotheses that I would like to test in some form during my Ph.D.:

1) mucilage polysaccharide derivatives are utilized by maize aerial root microbes for their own growth

2) mucilage polysaccharide derivatives are capable of sustaining modes of PGP conferred by maize aerial root microbes

3) members of the aerial root mucilage microbiota cooperate through metabolic specialization to confer PGP

4) microbial isolates are capable of utilizing their specialized functionalities to confer PGP upon other plant species (essentially challenging host specificity)

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Insights from UC Davis Biotechnology Seminar Class MIC 292 From Discovery to Product: An Introduction to Biotechnology at the Industrial Level

Contributed by Darwin Bandoy, DVM

I am completely amazed to find somebody working for the same company for more than twenty years, considering my own experience of changing employment several times within the last ten years. So Novozymes must be doing something exciting to motivate employees to stay for decades. The most exciting part for me is when the speaker showed diverse environments where they hunt for microbes with unusual and interesting enzymatic properties. Since Nature has different environments like oceans and deep forests, microbes evolved different modes of adoption thru enzyme production. So I would describe this as the Indiana Jones mode of Novozymes. But when Indiana Jones finds a treasure, the division gets tricky. Fortunately, Novozymes honors the Rio Convention on Biological Diversity, which stipulates fair sharing of the benefits derived from the commercialization of genetic diversity. While Nature has a diverse library of microbes and enzymes, they can still be improved to meet specific uses and purposes of customers. This next phase still employs an Indiana Jones mode of treasure hunting, but instead of exotic places, the genome is the map and the work is done bioinformatically using algorithms. Hence, a person who wants to be a genome treasure hunter needs to acquire data science skills. The speaker emphasized this as a core skill that is always in demand and is always in need for biotech companies.

So what’s a typical day for an Indiana Jones enzyme hunter in Novozymes? The case provided is the improvement of staleness in bread using maltogenic alpha-amylase that inhibits retrogradation of amylopectin. They can choose to do initial screening with their 60,000 microbes preserved in nitrogen tanks to screen for amylase activity, or do this using genome mining if the microbes are already sequenced to search for genes encoding for amylase (this is where the bioinformatics skill enter). Once enzyme activity is confirmed, several modes of improvement are done to enhance thermal stability and optimize pH (which is the case for Novamyl thermostable-amylase). A protein can be modified using a long-term engineered design or random mutagenesis. Both modes are used extensively by Novozymes which just shows that even how hard you design something, random and chance still yields totally unpredictable results (which is also crucial in patenting strategy as surprising and unexpected are the most used words in patent applications). Novozymes seems to have not only discovered the key to designing wonderful enzymes but long-term relationships, intentionally crafted with ample legroom for random surprising events.

 

 

 

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A Study Found the Common Bug Beat the Last Resort Antibiotic

Contributed by Nguyet Kong

A recent study in the publication mBio, a group from Emory University found that some common bugs, Klebsiella pneumoniae has developed a resistance to the last resort antibiotic. This is dangerous since bacteria found a way to get around one of the few last resort treatments available. Klebsiella is a bacterium that causes many different infections such as pneumonia, urinary tract infections, and bloodstream infections. In healthcare facilities, Klebsiella is responsible for 10% of the infections and kill up to 50% of the people who are infected. They can be found in the human gut, but normally doesn’t cause any disease.

An old antibiotic, Colistin that doctors give to patients as a last resort antibiotic is only used when the bacteria have developed a resistance to the other antibiotics that are given. World Health Organization (WHO) mention that it’s a lack of new drugs development and overprescribing the current antibiotics that have made common bugs to become resistance to the current antibiotics.

In the mBio paper, the bacterial isolated was not detectable with the current diagnostic tests. With an additional 24 hours, the resistance strain of Klebsiella that is resistance to Colistin was detected. The additional time needed for the resistance strain of Klebsiella to grow is a downside since diagnostics is needed for life or death situations. In a mouse study, the infection was either lethal or was not treatable by colistin.

References:

https://www.cdc.gov/hai/organisms/klebsiella/klebsiella.html

https://www.sciencedaily.com/releases/2018/03/180306085401.htm

http://mbio.asm.org/content/9/2/e02448-17

http://www.who.int/mediacentre/factsheets/fs194/en/

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Thoughts about Genome Distance Analysis for Pathogen Outbreak Isolates

Contributed by Carol Huang

The recent work on bioinformatics analysis of pathogen strains from outbreaks made me consider the aspects that can be used for this type of analysis. When we are facing a pathogen outbreak, the first thing we need to do is to identify the source and genetic variation. Understanding evolutionary dynamics and the route of transmission of the pathogens is the key to developing effective preventive strategies.

Whole genome sequencing has the potential of answering most of the key questions of related evolutionary questions. To answer these key questions, different strains of the same species were collected from different resources then sequenced with the Illumina NGS platform.   Draft de novo assemblies then were further analyzed for genome-to-genome distance calculation, which blast each individual genome against all genomes in the group. This analysis will help to determine the phylogeny of these genomes and geological association. On this aspect, it should include as many relevant strains to be representative and accurate ie: having a big enough inclusive population. Further genetic phenotypic characterization like SNP antibiotic resistance ability will enable us to develop more effective strategies.

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Congratulations Poyin Chen, PhD

Poyin Chen (aka Po) has completed her degree in my group. She graduated and is moving on to a great post doc position to study Shigella at Harvard University. Congratulations to Po!!

Her energy, lab skills, and crafting skills will be missed. However, as with all students, she is moving to a better place. I’m really proud to have Po move to another lab and expand her experience and knowledge. While in my group Po studied Listeria monocytogenes. She made some great contributions to the field. First, she showed methylation differences along with genotype was not predictive of virulence – while no link was found she did find the largest genetic variability to date in Listeria. Using PacBio sequencing she also found that large sections of the genome lift and invert at tRNA genes (Chen et al. 2017. App. Environ. Microbiol. 83:e02091-16. (PMID: 27836852)).

Secondly, Po used prebiotics with pathogens to discover that the complex oligosaccharide modulates the host intrinsic immune response to handle Listeria monocytogenes invasion differently. HMO leads to clearing via a different mechanism that does mannosyl-oligosaccharides. This observation also excluded that option that prebiotics block bacterial association using a physical method, but rather induce host changes that subsequently change the bacterial infection outcome (Chen et al., 2017. Pathogens 6:68 (invited contribution in – Special issue on Listeria monocytogenes and host interactions; Feature Article)).

Additionally, she was involved with identifying unique host/microbe receptor/ligands using a novel method to cross-link host cells together. That work is currently in review and it demonstrates that there are many touch points that bacteria use to bind the cell that were not previously known. It has implications for microbiome interactions and host response networks as well as bacterial metabolism changes during host association.

Lastly, Po was part of the 100K Pathogen Genome Project to provide methods and protocols for best practice in whole genome sequencing source tracking (Chen et al. 2017. Genome Announcements 5:e00967-16). I’m sure Po will have a productive career and post doc. All the best Po. We all wish you the very best for your new home and lab!!

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Visualizing Genomic Data

Contributed by Dr. DJ Bandoy, DVM

Next-generation sequencing opened the floodgates of biological information. However, the torrential amount of data that is now becoming the challenge itself from data storage to analytical tools. This gap is now more pronounced with visualizing millions of data points as to what is meaningful and intuitive in data analysis. Phylogenetic trees are the classical way to visualize genomic differences but fail to capture key interactions in microbial species such as gene exchange and hybrids in higher order species. The tree of life, might not even be a tree, but a tangled web of gains and losses of modular genomic components. One way to approach this technical problem is to explore the genome visually, using Hilbert curves. Hilbert curves are continuous fractal space-filling curves and were put in the spotlight with the science paper titled Comprehensive Mapping of Long-Range Interactions Reveals Folding Principles of the Human Genome. Anders published a paper with an accompanying software HilbertVis for genome data visualization using Hilbert curve. A more recent iteration of genome visualization using Hilbert curve is Meander, developed by Pavlopoulos. Visually presenting genomic data is more intuitive and pattern recognition is easier to accomplished.

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