Paulsson:Journal 2007/09-11

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Cell

• A Common Mechanism of Cellular Death Induced by Bactericidal Antibiotics

Michael A. Kohanski1, 2, 5, 6, Daniel J. Dwyer1, 3, 6, Boris Hayete1, 4, Carolyn A. Lawrence1, 2 and James J. Collins

Antibiotic mode-of-action classification is based upon drug-target interaction and whether the resultant inhibition of cellular function is lethal to bacteria. Here we show that the three major classes of bactericidal antibiotics, regardless of drug-target interaction, stimulate the production of highly deleterious hydroxyl radicals in Gram-negative and Gram-positive bacteria, which ultimately contribute to cell death. We also show, in contrast, that bacteriostatic drugs do not produce hydroxyl radicals. We demonstrate that the mechanism of hydroxyl radical formation induced by bactericidal antibiotics is the end product of an oxidative damage cellular death pathway involving the tricarboxylic acid cycle, a transient depletion of NADH, destabilization of iron-sulfur clusters, and stimulation of the Fenton reaction. Our results suggest that all three major classes of bactericidal drugs can be potentiated by targeting bacterial systems that remediate hydroxyl radical damage, including proteins involved in triggering the DNA damage response, e.g., RecA. [1]

EMBO

• Structural analysis of the ParR/parC plasmid partition complex

Jakob Møller-Jensen1, 4, Simon Ringgaard2, Christopher P Mercogliano1, Kenn Gerdes3 and Jan Löwe1
The EMBO Journal (2007) 26, 4413–4422, doi:10.1038/sj.emboj.7601864

Accurate DNA partition at cell division is vital to all living organisms. In bacteria, this process can involve partition loci, which are found on both chromosomes and plasmids. The initial step in Escherichia coli plasmid R1 partition involves the formation of a partition complex between the DNA-binding protein ParR and its cognate centromere site parC on the DNA. The partition complex is recognized by a second partition protein, the actin-like ATPase ParM, which forms filaments required for the active bidirectional movement of DNA replicates. Here, we present the 2.8 Å crystal structure of ParR from E. coli plasmid pB171. ParR forms a tight dimer resembling a large family of dimeric ribbon–helix–helix (RHH)2 site-specific DNA-binding proteins. Crystallographic and electron microscopic data further indicate that ParR dimers assemble into a helix structure with DNA-binding sites facing outward. Genetic and biochemical experiments support a structural arrangement in which the centromere-like parC DNA is wrapped around a ParR protein scaffold. This structure holds implications for how ParM polymerization drives active DNA transport during plasmid partition. [2]

Journal of Bacteriology

• Lon Protease Degrades Transfer-Messenger RNA-Tagged Proteins

Jennifer S. Choy,1,2 Latt Latt Aung,1 and A. Wali Karzai

Bacterial trans translation is activated when translating ribosomes are unable to elongate or terminate properly. Small protein B (SmpB) and transfer-messenger RNA (tmRNA) are the two known factors required for and dedicated to trans translation. tmRNA, encoded by the ssrA gene, is a bifunctional molecule that acts both as a tRNA and as an mRNA during trans translation. The functions of tmRNA ensure that stalled ribosomes are rescued, the causative defective mRNAs are degraded, and the incomplete polypeptides are marked for targeted proteolysis. We present in vivo and in vitro evidence that demonstrates a direct role for the Lon ATP-dependent protease in the degradation of tmRNA-tagged proteins. In an endogenous protein tagging assay, lon mutants accumulated excessive levels of tmRNA-tagged proteins. In a reporter protein tagging assay with {lambda}-CI-N, the protein product of a nonstop mRNA construct designed to activate trans translation, lon mutant cells efficiently tagged the reporter protein, but the tagged protein exhibited increased stability. Similarly, a green fluorescent protein (GFP) construct containing a hard-coded C-terminal tmRNA tag (GFP-SsrA) exhibited increased stability in lon mutant cells. Most significantly, highly purified Lon preferentially degraded the tmRNA-tagged forms of proteins compared to the untagged forms. Based on these results, we conclude that Lon protease participates directly in the degradation of tmRNA-tagged proteins. [3]

Molecular Microbiology

• Plasmid partition and incompatibility – the focus shifts (REVIEW)

Jean-Yves Bouet, Kurt Nordström, David Lane

The mitotic apparatus that a plasmid uses to ensure its stable inheritance responds to the appearance of an additional copy of the plasmid's centromere by segregating it from the pre-existing copies: if the new copy arises by replication of the plasmid the result is partition, if it arrives on a different plasmid the result is incompatibility. Incompatibility thus serves as a probe of the partition mechanism. Coupling of distinct plasmids via their shared centromeres to form mixed pairs has been the favoured explanation for centromere-based incompatibility, because it supports a long-standing assumption that pairing of plasmid replicas is a prerequisite for their partition into daughter cells. Recent results from molecular genetic and fluorescence microscopy studies challenge this mixed pairing model. Partition incompatibility is seen to result from various processes, including titration, randomized positioning and a form of mixed pairing that is based on co-activation of the same partition event rather than direct contact between partition complexes. The perspectives thus opened onto the partition mechanism confirm the continuing utility of incompatibility as an approach to understanding bacterial mitosis. The results considered are compatible with the view that direct pairing of plasmids is not essential to plasmid partition. [4]

Nature Methods

• PCR's next frontier

Tech Feature Vol. 4 No. 10 October 2007 p869

• Keeping tabs on fluorescent tags

Tech Feature Vol. 4 No. 9 September 2007 p755

• High-throughput cloning and expression in recalcitrant bacteria

Eric R Geertsma & Bert Poolman

We developed a generic method for high-throughput cloning in bacteria that are less amenable to conventional DNA manipulations. The method involves ligation-independent cloning in an intermediary Escherichia coli vector, which is rapidly converted via vector-backbone exchange (VBEx) into an organism-specific plasmid ready for high-efficiency transformation. We demonstrated VBEx proof of principle for Lactococcus lactis, but the method can be adapted to all organisms for which plasmids are available.

Vol. 4 No. 9 September 2007

PLOS

• Recombination Speeds Adaptation by Reducing Competition between Beneficial Mutations in Populations of Escherichia coli.

Cooper TF

PLoS Biol 5(9): e225 [5]

• Quantitative Characteristics of Gene Regulation by Small RNA.

Levine E, Zhang Z, Kuhlman T, Hwa T

PLoS Biol 5(9): e229 [6]