All posts filed under “Bacteria

comment 0

CRISPR Antimicrobials: A bacterial defense strategy turned on itself

Phage introduction of CRISPR-Cas9, DNA break and eventual cell killing.

Phage introduction of CRISPR-Cas9, DNA break and eventual cell killing.

Bacteria are innovators. Life as a bacterium means lethal and frequent chemical assaults from antibiotic exposure and physical assault from the most abundant “biological entities” living on earth, bacteriophages. The myriad of resistance mechanisms used in response to these threats illustrates the creativity of bacterial survival. The CRISPR-Cas system is one shining example of how microbes resist killing by viruses. Bacteria recognize foreign DNA injected from viruses and degrade it, along with keep a record of all viral DNA sequences from previous infections, as a kind of ‘memory’ for the bacterial immune system.

 Components of a CRISPR system.

Encoded on the bacterial chromosome of numerous species, the CRISPR immune system has 3 main components:

  • An enzyme that cuts double stranded DNA (Cas gene)
  • A short RNA guide (tracRNA)
  • CRISPR array of “clustered regularly interspaced short palindromic repeat” sequences separated with “spacer” regions that contain the target DNA

The original function of CRISPR/Cas system is to degrade incoming plasmid or viral DNA, but it has been developed as a powerful genome engineering tool. It is easy and cheap to employ this pioneering system towards cutting any desired target DNA, which explains its widespread and rapid usage in many academic research labs. The CRISPR technology permits DNA cutting, editing, introducing mutations, deletions or insertions, with unprecedented precision. There can be “off-target”mutations that arise and this is an area where the technology needs refining to limit the creation of potentially harmful DNA changes.

Game changing applications of CRISPR and the patent race to own it.

The list of potential applications is growing and underscores its transformative potential. CRISPR may give the field of gene therapy a boost, with the ability to correct numerous human genetic diseases including blindness, Duchenne muscular dystrophy, Cystic Fibrosis, sickle cell anemia and others. CRISPR gene drives are being developed to engineer sterile mosquitoes to help eliminate vector-borne diseases like malaria, dengue fever, West Nile and Zika. Plants could be edited for resistance to drought and pathogens, which can proceed without the addition of any foreign genes, but rather editing existing genes. This approach to not introduce foreign genes offers a solution to the problems associated with GM organisms. The other huge opportunity is to engineer T cells to combat HIV infection or to treat cancer. Since HIV infects T cells, it is proposed to engineer T cells to either resist HIV infection, or to redirect HIV immune responses. As part of the oversimplified “cancer moonshot”, CRISPR is also being considered for use in treating cancer.

Despite the many hurdles faced to obtain these diverse goals, it is no surprise that there is a mad rush to patent the CRISPR technology and researchers at the Broad Institute (MIT) (Feng Zhang) and the University of California Berkeley (Dr Jennifer Doudna – along with Emmanuelle Charpentier) are engaged in a combative patent dispute. The value of the technology is difficult to estimate but the impact will be comparable to the invention of PCR by Kary Mullis, and will also likely lead to a Nobel prize. Professor, open science advocate, and co-founder of the Public Library of Science (PLOS) journals, Michael Eiesen, has been refreshingly honest and critical of the tactics used in the CRISPR patent debate. He even went so far as to propose that neither group should be able to patent a technology that was government funded research, and therefore ultimately owned by the tax-paying citizens.

Novel CRISPR antimicrobial treatment that does not involve antibiotics.

Widespread antibiotic resistance is an increasing, global problem due to the excessive use of antibiotics in hospitals and more importantly, in agriculture. The arms race between microbes and antibiotics is a losing battle for antibiotic discoverers. As soon as a new antibiotic is discovered and employed, there is often the immediate discovery of clinical resistant isolates in response to treatment. It has also been shown than antibiotic resistance even precedes the clinical usage of antibiotics, as bacteria need to defend against antimicrobial attack our in the real world. Resistance is out there.

As an alternative to continuing our search for new antibiotics (which is ongoing in new and exciting ways), scientists are developing new approaches to kill microbes and treat bacterial infections. The CRISPR-based antimicrobial approach described here is one such alternative approach to killing microbes without antibiotics. A bacterial defense strategy against viruses is being turned on itself.

Early work showed that the CRISPR-cas system can be directed to cleave DNA sequences in the bacterial genome, which led to irreparable damage and bacterial death (1). Recent studies have moved this concept further by developing a system to deliver cytotoxic, CRISPR-Cas9 systems to microbes. Phagemids are plasmids derived from phage genomes, viruses that specifically target bacteria. The phagemids have been engineered to express all the components of a CRISPR system, in addition to the information required to package the phagemid DNA into a phage particle. Bacteriophages are experts at landing on the outer bacterial surface, injecting its DNA into the bacterium, taking over the bacterial machinery and producing hundreds of viruses. The factory ultimately bursts, leading to death and release of an army of viruses to maintain the attack on neighbouring cells.

CRISPR Antimicrobials.

In back-to-back papers in Nature Biotechnology, two groups have published remarkably similar reports on the use of the Streptococcus pyogenes type II Cas 9 protein as a killing enzyme (2,3). The basic concept was to develop a CRISPR-mediated approach that can cause specific bacterial death. The element of specificity is particularly interesting in light of our increasing awareness of the dangers of broad-spectrum killing of healthy and helpful commensal, microbiome members. Drugs that cause little collateral damage is a new principal of antimicrobial design, which is a large shift from conventional thinking that broad-spectrum is best. Also, these papers explored the use of CRISPR to limit the transfer of antibiotic resistance genes.

Both approaches used phage-derived vectors to encode the CRISPR components of the the Cas9 enzyme, the short RNA guide (tracRNA) and a CRISPR array containing the desired cut sites within the spacer regions. When directing cleavage of kanamycin or ampicillin resistance genes, both groups could successfully show a loss of antibiotic resistance in the target organism, after delivery of the phagemid-encoded CRISPR systems. The same principal was applied to monitoring the efficiency of transformation, by measuring the number of viable cells in strains containing the target genes compared to strains that lacked the target. There was a 1000-fold decrease viability (and in the efficiency of conjugation) in hosts that contained the targeted cleavage site, indicating that successful DNA disruption and killing. Interestingly, there was never a complete killing of the target population in these experimental conditions.

To further examine the feasibility of this approach, various infection models were used to determine the capacity of CRISPR killing when applied to pathogens during an infection. In both a skin infection and the Galleria larval infection models, the pathogen numbers could be reduced by CRISPR-Cas antimicrobials, which permitted skin healing and survival of the larvae. The final experiments were to test the system when exposed to simple, mixed communities, in order to show specific pathogen targeting and non-targeting of commensals.

Although phages are being revisited for use as an alternative to antibiotics, engineered phages with CRISPR capacity have perhaps the added value of narrow spectrum killing. The biggest obstacle to improving these transmissable, programmed antimicrobials is to improve the method of delivery. It is not unreasonable to image a scenario where due to the low costs of DNA synthesis, custom, specific CRISPR-Cas-phage particles could be synthesized within a few days, and used as needed to treat any possible infectious disease.


1 . CRISPR design for next-generation antimicrobials. Beisel CL, Gomaa AA, Barrangou R. Genome Biol. 2014 Nov 8;15(11):516. doi: 10.1186/s13059-014-0516-x.

2.  Exploiting CRISPR-Cas nucleases to produce sequence-specific antimicrobials. Bikard D, Euler CW, Jiang W, Nussenzweig PM, Goldberg GW, Duportet X, Fischetti VA, Marraffini LA.                  Nat Biotechnol. 2014 Nov;32(11):1146-50. doi: 10.1038/nbt.3043..

3. Sequence-specific antimicrobials using efficiently delivered RNA-guided nucleases.Citorik RJ, Mimee M, Lu TK. Nat Biotechnol. 2014 Nov;32(11):1141-5. doi: 10.1038/nbt.3011.

comment 0

Kill switches prevent escape of genetically modified bacteria

bacterial kill switch

There is considerable public concern about the use of genetic engineering, particularly around the production of food. The most recent example is the FDA-approved, fast growing salmon from the company AquaBounty. This fast growing fish is grown in land-locked tanks as sterile females so that they cannot reproduce if they were to escape into the wild. Interestingly, this fish grows almost twice as fast as salmon in the wild due to the increased production of a fish growth hormone, genetically engineered to be expressed from multiple organs instead of in limiting amounts from the pituitary gland. The basic research that led to this transgenic fish was described over 20 years ago from a Canadian researcher, Dr Garth Fletcher of Memorial University.

What is less appreciated by the public are the numerous examples of genetic modification that result in medicines or other useful and less controversial products. One of the first products resulting from genetic modification was the production of synthetic insulin used to treat diabetes, which is expressed and purified from bacteria.  And the exploding field of synthetic biology has the potential of providing a bounty of new products, medicines, bioremediation strategies using engineered bacteria or biofuel production by microbes. If genetically engineered bacteria are to be employed in the field, at sites of contamination, or in production factories, there is a significant worry about their escape and the possible implications of release into the wild. To address this concern, numerous research groups are designing so-called ‘kill switch’ strategies, which will prevent the growth of these microbes in the environment. A creative new strategy was described earlier this year.

Microbiologists frequently work with ‘auxotrophs’, which are strains that cannot synthesize a specific nutrient, and therefore do not grow without its supply to the microbe. As standard auxotrophs might be capable somehow of scavenging these needed nutrients, ‘synthetic auxotrophs’ were designed that rely on the supply of unusual amino acids that are not commonly found in nature. There are several uncommon amino acids that are coded for in the triplet DNA code, but that are not found very often in proteins. This suggests the possibility to change the genetic code and introduce new codons that would introduce  non-standard amino acids into proteins.

The first stage of the kill switch technology was founded on an earlier landmark paper that used whole genome editing,  where all 321 UAG stop codons were replaced with UAA codons (ref 1). UAG was convenient to alter, since it appears rarely in the genome and only at the end of genes. These strains are referred to as genetically recoded organisms, or GROs.  Note, there were 355 off target mutations created in the process, which did not affect viability but may need to be corrected in future. It should be noted that this GRO strain was now quite resistant to infection from viruses, another advantage to this type of biocontainment strategy. The new genetic code mistranslates viral proteins that hijack the bacterial protein synthesis machinery, which ultimately disables new virus assembly leading to resistance.

Several other sophisticated, genetic manipulations were performed in order to create a growth dependence. It was now possible to reassign UAG as a dedicated sense codon for “plug and play” incorporation of rare amino acids. In order to translate the UAG codon to a  meaningful response by the protein synthesis machinery,  they expressed a specific tRNA synthetase that recognizes the UAG codon and introduces  1-4,4′-biphenylalanine (bipA) into target proteins during synthesis. As there were no longer any functional UAG codons in the genome, they selected a handful of essential proteins and redesigned these genes to encode UAG at various positions. Structure predictions were used to select the candidate essential genes to recode, which were confirmed by protein structure determinations. Mass spectrometry also confirmed the introduction of bipA into target proteins.

 Unfortunately, some synthetic auxotroph strains were still viable at low frequencies when grown in the the absence of bipA, indicating that compensatory mutations occurred which restored viability. In other words, the supposed ‘kill switch’  was not perfect in the earliest attempts. Several additional mutations were needed, including the addition of conventional auxotrophs, to really limit growth and escape of this strain in the absence of the non-standard amino acids. They ultimately identified a highly engineered strain could only survive and produce functional essential proteins when grown in the presence of the non-standard amino acid, 1-4,4′-biphenylalanine (ref 2). This ‘tour de force’ approach created a synthetic auxotroph which now can now be used as the ‘chassis’ for the future development of a particular biotechnology.

In the same issue, a second paper on the same subject was also published. They generally used a similar approach to construct synthetic auxotrophs that were dependent on the exogenous supply of unnatural analogs of phenylalanine (ref 3). The end result was the same, a strain that could not escape its amino acid dependency through mutation or the acquisition of multiple genes through horizontal gene transfer. The NIH standard for an effective kill switch is an approach where survivors do not occur with a frequency of 1 per 108 cells. Other non-auxotroph strategies have been used that meet this minimum requirement.  Environmental microbes capable of degrading environmental pollutants have been designed to express a lethal toxin when in the environment, but only once the pollutants have been degraded. This permits the deliberate use of bacteria for bioremediation of contaminated soils but limits their survival after the job is done. These bacterial suicide strategies were developed to mitigate possible risks and should permit the safe use of microbes in the environment to help resolve environmental or health crises, with the benefit greatly outweighing the risk.

Ref 1. Genomically Recoded Organisms Expand Biological Functions. 10.1126/science.1241459

Ref 2. Biocontainment of genetically modified organisms by synthetic protein design. doi:10.1038/nature14121

Ref 3.  Recoded organisms engineered to depend on synthetic amino acids. doi:10.1038/nature14095


comment 0

Biosensor screening of metagenomes for lignin biodegradation genes


Coal on the edge of the Bow River, near the old coal mines in Canmore, Alberta.

Coal on the edge of the Bow River, near the old coal mines in Canmore, Alberta.

Second Generation Biofuel

The term ‘second generation biofuel’ refers to the use of lignocellulosic material as the feedstock for fermentation reactions to produce ethanol or other hydrocarbons. The advantage over ‘first generation biofuels’ is that cellulose is an extremely abundant polysaccharide from woody plants and agricultural waste can be used as a source of fermentable sugars, in the place of sugars from food crops, such as wheat, corn, beets or sugar cane.

The challenge is that lignocellulose has evolved towards resisting biodegradation because of its important structural role in plant cell walls.  The polysaccharides in cellulose and hemicellulose are heavily cross-linked to lignin, which explains the recalcitrance.  Therefore, in order to improve the process of making second generation biofuels, we need to improve the ability to remove and isolate cellulose from lignin. Fungi are the best known lignin degraders and most enzymes used for lignin transformation have been characterized from fungi. The focus of this blog is to highlight a new approach to identify bacterial enzymes that are involved in lignin transformation, the early stages of degrading lignin, to aid in producing the most abundant source of fermentable sugars for biofuel production1. The ultimate goal is sustainability, to replace the use of fossil fuels and provide a carbon neutral source of energy.

Most bacteria in nature and on the human body are unculturable. Despite this limitation, microbiologists have developed innovative methods to probe these unknown communities and exploit their genetic potential. While we are also improving our ability to culture these organisms in the lab, a common approach is to recover the total DNA from a sample containing a unique bacterial population and to screen this DNA for novel properties, when it’s expressed in a recombinant bacterial host. This ‘shotgun’ approach is also useful to capture the DNA for metagenomic sequencing projects, where bacterial numbers are low.

Microbial processes are essential for the production of oil in the oilsands and coal beds. The Hydrocarbon Metagenomic Project ( is focussed on understanding the complex microbiology in these important sites. Researchers have isolated DNA from oil sands and coalbed samples, and used 16SrRNA metagenomics to identify the bacteria in these environments2. Given the high abundance of aerobic and anaerobic bacteria in these samples, it was hypothesized that these microorganisms would be excellent sources of bacterial lignin degradation enzymes1.

Biosensor of monoaromatic degradation products of lignin

The genius of microbiology is the creativity of genetic screens to identify new stuff. As touched on in our previous post, biosensors are the use of engineered microbial cells used as a detection kit for almost any kind of small molecule, toxin, antibiotic or compound with any interesting application. This first step in identifying lignin transformation genes was the creation of a biosensor to detect the degradation products of lignin.  A library of strains expressing each intergenic region (E. coli promoter) to GFP was screened to identify genes responsive to the presence of lignin degradation products; the monoaromatic compounds vanillin, vacillate and p-coumarate. The emrRAB promoter was very responsive to these and other structurally related compounds, as well as to lignin treated with solvents and enzymes. This operon encodes a transcriptional regulator, and a multidrug resistance pump EmrAB. This pump extrudes a large variety of antibiotics, and is also responding to exposure to these monoaromatic compounds.

Metagenomic libraries

Samples were obtained from Alberta coal beds  and the total DNA was extracted from these samples. It’s safe to say that the bulk of DNA present in these samples will reflect the microbial diversity in a coal bed.  The total DNA is digested into sizeable fragments and cloned into a fosmid cloning vector. Roughly 60,000 fragments each with a size ~ 40 kb were cloned and about a third of these fragments were sequenced. The exciting part was to employ high throughput approaches screen this library of 60,000 strains for the ability to degrade lignin. Each strain is robotically picked and arrayed into 384-well plates, and grown in very small culture volumes for a few hours in the presence of commercially purified lignin. Using a co-culture system, the biosensor strain was then added to each strain in the arrayed library, and co-cultures were screened for increased fluorescence from the biosensor that detects lignin degradation products. I love putting robots to good use!

This high throughput robotic screen identified 24 fosmids that activated the PemrR-GFP biosensor and 8 were selected for further sequencing and characterization. GC-MS was used to specifically identify the degradation products from many of the recombinant strains expressing lignin degrading genes, as detected with the biosensor. The variety and difference in end products produced by the various lignin transforming strains indicates a variety of enzymatic pathways to transform lignin. A limited characterization of the some of the genes encoded on the fosmid was performed. Mutation of the emrR regulator led to an inability to detect lignin degradation products, as well as slight growth defects in their presence. This observation supports the role of this efflux pump in coping with the toxic affects of these aromatic degradation  products. Random transposon mutagenesis was performed to identify specific genes in the large fragments of DNA that were required for degradation.

Functional and bioinformatic analysis revealed the presence of six functional classes of genes that contribute to degrading lignin. These included oxidoreductases, hydrogen peroxide generating enzymes, multidrug efflux systems, protein secretion systems to secrete the degradative enzymes, as well as motility and signal transduction systems that may be required to move toward these nutrient sources in the microscale environments within coal beds. Interestingly, many of the genes identified within the sequenced fosmid clones were associated with genetic elements that facilitate horizontal gene transfer. This evidence suggested that these genes are freely exchanged between microbes in this environment. This is conceptually analogous to pathogens found in hospitals that share advantageous antibiotic resistance genes.

This exciting approach included the construction of a novel biosensor to probe a unique gene pool isolated from lignin-rich coal bed microbial communities and discovered multiple classes of lignin degrading genes. These enzymes may ultimately aid in the refinement of wood waste and lignocellulose to produce fermentable sugars for 2nd generation biofuels. This is the kind of green chemistry discovery that can support new biotechnologies, such as the startup that resulted from this innovative project, MetaMixis Inc. I wish them the best of luck in moving this technology forward.


1. Metagenomic scaffolds enable combinatorial lignin transformation.  Strachan CR, Singh R, VanInsberghe D, Ievdokymenko K, Budwill K, Mohn WW, Eltis LD, Hallam SJ. Proc Natl Acad Sci U S A. 2014 Jul 15;111(28):10143-8. doi: 10.1073/pnas.1401631111. Epub 2014 Jun 30.

2. Metagenomics of hydrocarbon resource environments indicates aerobic taxa and genes to be unexpectedly common.  An D, Caffrey SM, Soh J, Agrawal A, Brown D, Budwill K, Dong X, Dunfield PF, Foght J, Gieg LM, Hallam SJ, Hanson NW, He Z, Jack TR, Klassen J, Konwar KM, Kuatsjah E, Li C, Larter S, Leopatra V, Nesbø CL, Oldenburg T, Pagé AP, Ramos-Padron E, Rochman FF, Saidi-Mehrabad A, Sensen CW, Sipahimalani P, Song YC, Wilson S, Wolbring G, Wong ML, Voordouw G. Environ Sci Technol. 2013 Sep 17;47(18):10708-17. doi: 10.1021/es4020184. Epub 2013 Aug 26.


Project Leader of the Hydrocarbon Metagenomic Project

Gerrit Voordouw, Professor, University of Calgary

Canadian Synthetic Biology Researchers

Steven Hallam, Associate Professor, University of British Columbia

Lindsay Eltis, Professor University of British Columbia