New drugs from mutant bugs

Scientists from the Universities of Birmingham and Bristol have discovered how marine bacteria join together two antibiotics they make independently to produce a potent chemical that can kill drug-resistant strains of the MRSA superbug.

Working with Japanese pharmaceutical company Daiichi-Sankyo, and funded by the UK Biotechnology and Biological Sciences Research Council (BBSRC), the researchers’ work paves the way for the creation of new hybrid antibiotics that may help to solve the growing problem of bacterial infections that are resistant to essentially all antibiotics.

The research is published online in the journal PLoS ONE.

The team, comprising microbial geneticists from Birmingham and chemists from Bristol, determined the sequence of the complete DNA content of the marine bacterium that produces the new antibiotic, thiomarinol, owned by Daiichi-Sankyo. They then identified the genes responsible for making the antibiotic on the basis of their similarity to genes that make the related but less potent antibiotic, mupirocin, which is currently used to combat MRSA (methicillin resistant Staphylococcus aureus).

They found the genes are on a relatively small, separate DNA molecule called a plasmid, which is just big enough to carry the genes for making the antibiotic plus genes to allow the plasmid to replicate autonomously in the bacterium. The plasmid thus carries genes that make both the mupirocin-like antibiotic as well a second antibiotic, holomycin, and a gene responsible for joining both antibiotics together, forming a more potent molecule.

Tests showed that by joining the antibiotics together the resulting chemical is able to inhibit the growth of MRSA strains that have become resistant to mupirocin. ‘This shows how mupirocin can be modified to make it more potent and suggests that related molecules could be used against the increasingly problematic Enterobacteriacae like Escherichia coli and Klebsiella pneumoniae,’ says University of Birmingham research lead Professor Chris Thomas.

By using mutant strains that were unable to make either the mupirocin part or the holomycin part the team was able to feed alternative compounds to the bacteria – so-called mutasynthesis - so that a family of novel molecules was created, and tests showed some of these had biological activity. ‘This provides hope that the system will allow the production of new antibiotics that may help to combat the growing problem of antibiotic resistance in pathogenic bacteria,’ adds University of Bristol research lead Professor Tom Simpson.

More information: A Natural Plasmid Uniquely Encodes Two Biosynthetic Pathways Creating a Potent Anti-MRSA Antibiotic is published in PLoS ONE. It is available online at http://dx.plos.org … pone.0018031
The detailed chemical analysis was recently published in Angewandte Chemie International Edition at http://dx.doi.org/ … ie.201007029

Provided by University of Birmingham

Mutant proteins weigh in: Researchers 'see' binding with DNA through quartz crystal microbalance

 Rice University scientists have demonstrated a new way to see and quickly measure DNA/protein binding, a discovery that prompted one journal reviewer to write, "This study has made my day."

"I've never had that happen," said co-author Kathleen Matthews, Rice's Stewart Memorial Professor of Biochemistry and Cell Biology and former dean of the Wiess School of Natural Sciences.

Sibani Lisa Biswal, an assistant professor in chemical and biomolecular engineering, led the study with Matthews that made novel use of a quartz crystal microbalance (QCM) to dynamically measure the binding activity of wild-type and mutant proteins. The process let them monitor what happened in real time when DNA was first introduced to and then removed from protein molecules attached to the microbalance.

The study published this week in the American Chemical Society journal Langmuir is of interest to researchers who work in biological and chemical sensing. Co-authors include Jia Xu, a Welch postdoctoral fellow in biochemistry and cell biology, and Kai-Wei Liu, a graduate student in chemical and biomolecular engineering.

There's nothing new about QCM, a device Biswal said probably exists at most research universities. "It's mainly used for chemical and biomolecular sensing," she said. "QCM works by measuring the resonance frequency of a quartz crystal. When you apply current, it resonates, and we translate that through electronic measurements into a frequency. When you add something to the surface of the crystal, the frequency changes. That's what we're looking for."

The sensitivity is so fine that she can see the difference in a protein when a DNA molecule attaches itself to the protein. "When you apply sufficient mass to the quartz crystal, it's going to change its resonance frequency," Biswal said. "The change in mass is proportional to the change in frequency."

The researchers chose to look at an Escherichia coli protein, a lactose repressor called LacI, primarily because Matthews has the wild-type protein and multiple variants. "She has studied LacI for a long time," Biswal said. "It's a well-characterized system, and we wanted to use something we know about."

The team compared two types of LacI via QCM. The first, wild-type LacI, was used as a control because it bonds strongly to the gold-coated QCM surface using its DNA-binding domain, which spreads out like a mold and is then unable to bind DNA.

The second was a mutant form of LacI, engineered with a sulfur-based amino acid introduced far from the protein's DNA binding site. Unlike the wild-type, this mutant LacI puts down tight roots at the site of the mutation and stands like a tree, waving in the liquid breeze. "The mutation will actually orient the protein, so we can immobilize it onto a solid support," Biswal said.

Experimenting on both types, the researchers flowed liquid containing DNA and then IPTG, an inducer that releases the DNA, over the proteins bound to the QCM. In the process that took between 90 minutes and two hours, the wild-type Lacl, adsorbed by the gold surface, largely ignored the offerings.

But the mutant LacI responded with a clear change in the signal as it first grabbed passing DNA and then released it when IPTG was introduced to the stream.

"It's dynamic," Matthew said of the process. "We're flowing very small volumes of liquid over the sensor. We start with a buffer, then add the protein. When the protein binds, we can see how much goes on the surface of the crystal. Then we add the DNA and we can see that binding process with the mutant (for the wild-type, nothing happens) and then we add the IPTG and see the release."

As a bonus, the QCM technique also provides information about the viscoelastic properties of bound proteins. "It's nice that we can measure dissipation," Biswal said. "When we turn off the current, we can watch how the resonance decays -- and that tells us things like the elasticity of the material on top of the quartz."

Biswal, thinking about that reviewer, noted he had studied proteins on surfaces for the past 25 years and was delighted that one could study how mutations to a protein would change its binding properties and functionalities.

"People who develop biosensors need to be able to attach proteins onto surfaces," she said. "Our work indicates that you can't just immobilize proteins nonspecifically onto a surface. You actually do need to make some type of mutation to make sure the binding site is accessible. This platform allows us to easily screen and study what type of mutations are needed."

More information: Read the abstract at http://pubs.acs.or … 21/la200056h

Provided by Rice University (news : web)