MicrobiologyBytes

The advent of genetic engineering – the ability to edit and insert DNA into living organisms – in the latter half of the 20th century created visions of a new era of synthetic biology, where novel biological functions could be designed and implemented for useful purposes. We are witnessing an exciting revolution of scale, wherein technical progresses allow for the manipulation of genetic material at the whole genome level. This will enable the manufacture of increasingly complex genetic designs to solve pressing challenges in health, energy and the environment-if and when such designs can be specified.

This paper argues that the organized development of key common application organisms, engineered for engineerability, and attendant libraries of parts, pathways and standardized manufacturing are necessary for this genome-scale technology to realize its promise.

Toward rational design of bacterial genomes. Curr Opin Microbiol. Aug 22 2011

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The mechanism by which dsDNA is packaged into tailed bacteriophage virions has fascinated molecular biologists since it was realized, over four decades ago, that these structures contain long dsDNA molecules that are hundreds of times more compact than dsDNA in solution. Experiments in the 1970s showed that, rather than condensing the DNA first and then assembling a shell around this DNA core, tailed-phage DNA is inserted into a preformed protein container (called a prohead or procapsid). Mmolecular genetics studies indicate that the basic machinery of dsDNA packaging is similar in all tailed phages.

The recent burst of structural progress concerning phage DNA-packaging proteins, as well as the introduction of optical tweezer technology into this field, has allowed the formulation of much more detailed ideas for the mechanism by which the DNA-packaging motor pumps DNA into phage procapsids. Potential applications of packaging nanomotors in nanotechnology and biology are beginning to be described, for example these nanomotors could be used for efficient delivery of nucleic acids or related molecules across barriers such as cell membranes or for applications in single-molecule DNA sequencing. There is little doubt that this will be a fertile research area for some time into the future.

The DNA-packaging nanomotor of tailed bacteriophages. 2011 Nat Rev Microbiol. 9(9):647-57 doi: 10.1038/nrmicro2632
Tailed bacteriophages use nanomotors, or molecular machines that convert chemical energy into physical movement of molecules, to insert their double-stranded DNA genomes into virus particles. These viral nanomotors are powered by ATP hydrolysis and pump the DNA into a preformed protein container called a procapsid. As a result, the virions contain very highly compacted chromosomes. Here, I review recent progress in obtaining structural information for virions, procapsids and the individual motor protein components, and discuss single-molecule in vitro packaging reactions, which have yielded important new information about the mechanism by which these powerful molecular machines translocate DNA.

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The emergence of resistance against most current drugs emphasizes the need to develop new approaches to control bacterial pathogens, particularly Staphylococcus aureus. Bacterial fatty acid synthesis is one such target that is being actively pursued by several research groups to develop anti-Staphylococcal agents. Recently, the wisdom of this approach has been challenged based on the ability of a Gram-positive bacterium to incorporate extracellular fatty acids and thus circumvent the inhibition of de novo fatty acid synthesis. The generality of this conclusion has been challenged, and there is enough diversity in the enzymes and regulation of fatty acid synthesis in bacteria to conclude that there is not a single organism that can be considered typical and representative of bacteria as a whole. We are left without a clear resolution to this ongoing debate and await new basic research to define the pathways for fatty acid uptake and that determine the biochemical and genetic mechanisms for the regulation of fatty acid synthesis in Gram-positive bacteria. These crucial experiments will determine whether diversity in the control of this important pathway accounts for the apparently different responses of Gram-positive bacteria to the inhibition of de novo fatty acid synthesis in presence of extracellular fatty acid supplements.

Is bacterial fatty acid synthesis a valid target for antibacterial drug discovery? Curr Opin Microbiol. Aug 20 2011

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Pseudomonas aeruginosa is a common bacterium that can infect and cause disease in a wide variety of hosts, ranging from humans to plants. In healthy individuals, the innate immune system can counteract this microorganism effectively; however immunocompromised patients and cystic fibrosis patients suffer from severe infections with this bacterium. P. aeruginosa can propel itself through tissue by rotation of its long tail, called the flagellum, which is essential to establish colonization and infection of the host. The building blocks of the bacterial flagellum are over a thousand copies of the highly conserved protein flagellin. Mammals and plants have developed recognition systems to detect many different bacteria by sensing flagellin via Toll-like receptor 5 and Flagellin. Bacteria actively try to interfere with this recognition (immune evasion).

This new study describes a novel mechanism of P. aeruginosa to escape flagellin recognition. The secreted protein alkaline protease of P. aeruginosa, degrades immunity activating free flagellin. Bacterial motility is maintained, because flagellin present as building block of flagella is not degraded. In this way, the bacterium impairs recognition and hides itself from destruction by the immune system. Understanding these immune evasion strategies is of extreme importance for the development of new therapeutic approaches.

Pseudomonas Evades Immune Recognition of Flagellin in Both Mammals and Plants. 2011 PLoS Pathog 7(8): e1002206. doi:10.1371/journal.ppat.1002206
The building blocks of bacterial flagella, flagellin monomers, are potent stimulators of host innate immune systems. Recognition of flagellin monomers occurs by flagellin-specific pattern-recognition receptors, such as Toll-like receptor 5 (TLR5) in mammals and flagellin-sensitive 2 (FLS2) in plants. Activation of these immune systems via flagellin leads eventually to elimination of the bacterium from the host. In order to prevent immune activation and thus favor survival in the host, bacteria secrete many proteins that hamper such recognition. In our search for Toll like receptor (TLR) antagonists, we screened bacterial supernatants and identified alkaline protease (AprA) of Pseudomonas aeruginosa as a TLR5 signaling inhibitor as evidenced by a marked reduction in IL-8 production and NF-κB activation. AprA effectively degrades the TLR5 ligand monomeric flagellin, while polymeric flagellin (involved in bacterial motility) and TLR5 itself resist degradation. The natural occurring alkaline protease inhibitor AprI of P. aeruginosa blocked flagellin degradation by AprA. P. aeruginosa aprA mutants induced an over 100-fold enhanced activation of TLR5 signaling, because they fail to degrade excess monomeric flagellin in their environment. Interestingly, AprA also prevents flagellin-mediated immune responses (such as growth inhibition and callose deposition) in Arabidopsis thaliana plants. This was due to decreased activation of the receptor FLS2 and clearly demonstrated by delayed stomatal closure with live bacteria in plants. Thus, by degrading the ligand for TLR5 and FLS2, P. aeruginosa escapes recognition by the innate immune systems of both mammals and plants.

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