Multimodal Spectroscopic Analysis of the Fe-S Clusters of the as-Isolated Escherichia coli SufBC2D Complex.

Iron-sulfur (Fe-S) clusters are essential inorganic cofactors dedicated to a wide range of biological functions, including electron transfer and catalysis. Specialized multiprotein machineries present in all types of organisms support their biosynthesis. These machineries encompass a scaffold protein, on which Fe-S clusters are assembled before being transferred to cellular targets. Here, we describe the first characterization of the native Fe-S cluster of the anaerobically purified SufBC2D scaffold from Escherichia coli by XAS and Mössbauer, UV-visible absorption, and EPR spectroscopies. Interestingly, we propose that SufBC2D harbors two iron-sulfur-containing species, a [2Fe-2S] cluster and an as-yet unidentified species. Mutagenesis and biochemistry were used to propose amino acid ligands for the [2Fe-2S] cluster, supporting the hypothesis that both SufB and SufD are involved in the Fe-S cluster ligation. The [2Fe-2S] cluster can be transferred to ferredoxin in agreement with the SufBC2D scaffold function. These results are discussed in the context of Fe-S cluster biogenesis.

Oxidative stress antagonizes fluoroquinolone drug sensitivity via the SoxR-SUF Fe-S cluster homeostatic axis.

The level of antibiotic resistance exhibited by bacteria can vary as a function of environmental conditions. Here, we report that phenazine-methosulfate (PMS), a redox-cycling compound (RCC) enhances resistance to fluoroquinolone (FQ) norfloxacin. Genetic analysis showed that E. coli adapts to PMS stress by making Fe-S clusters with the SUF machinery instead of the ISC one. Based upon phenotypic analysis of soxR, acrA, and micF mutants, we showed that PMS antagonizes fluoroquinolone toxicity by SoxR-mediated up-regulation of the AcrAB drug efflux pump. Subsequently, we showed that despite the fact that SoxR could receive its cluster from either ISC or SUF, only SUF is able to sustain efficient SoxR maturation under exposure to prolonged PMS period or high PMS concentrations. This study furthers the idea that Fe-S cluster homeostasis acts as a sensor of environmental conditions, and because its broad influence on cell metabolism, modifies the antibiotic resistance profile of E. coli.

Silver potentiates aminoglycoside toxicity by enhancing their uptake.

The predicted shortage in new antibiotics has prompted research for chemicals that could act as adjuvant and enhance efficacy of available antibiotics. In this study, we tested the effects of combining metals with aminoglycosides on Escherichia coli survival. The best synergizing combination resulted from mixing aminoglycosides with silver. Using genetic and aminoglycoside uptake assays, we showed that silver potentiates aminoglycoside action in by-passing the PMF-dependent step, but depended upon protein translation. We showed that oxidative stress or Fe-S cluster destabilization were not mandatory factors for silver potentiating action. Last, we showed that silver allows aminoglycosides to kill an E. coli gentamicin resistant mutant as well as the highly recalcitrant anaerobic pathogen Clostridium difficile. Overall this study delineates the molecular basis of silver's potentiating action on aminoglycoside toxicity and shows that use of metals might offer solutions for battling against increased bacterial resistance to antibiotics.

Tight regulation of the intS gene of the KplE1 prophage: a new paradigm for integrase gene regulation.

Temperate phages have the ability to maintain their genome in their host, a process called lysogeny. For most, passive replication of the phage genome relies on integration into the host's chromosome and becoming a prophage. Prophages remain silent in the absence of stress and replicate passively within their host genome. However, when stressful conditions occur, a prophage excises itself and resumes the viral cycle. Integration and excision of phage genomes are mediated by regulated site-specific recombination catalyzed by tyrosine and serine recombinases. In the KplE1 prophage, site-specific recombination is mediated by the IntS integrase and the TorI recombination directionality factor (RDF). We previously described a sub-family of temperate phages that is characterized by an unusual organization of the recombination module. Consequently, the attL recombination region overlaps with the integrase promoter, and the integrase and RDF genes do not share a common activated promoter upon lytic induction as in the lambda prophage. In this study, we show that the intS gene is tightly regulated by its own product as well as by the TorI RDF protein. In silico analysis revealed that overlap of the attL region with the integrase promoter is widely encountered in prophages present in prokaryotic genomes, suggesting a general occurrence of negatively autoregulated integrase genes. The prediction that these integrase genes are negatively autoregulated was biologically assessed by studying the regulation of several integrase genes from two different Escherichia coli strains. Our results suggest that the majority of tRNA-associated integrase genes in prokaryotic genomes could be autoregulated and that this might be correlated with the recombination efficiency as in KplE1. The consequences of this unprecedented regulation for excessive recombination are discussed.

Molecular Biology and Genetic Tools to Investigate Functional Redundancy Among Fe-S Cluster Carriers in E. coli.

Iron-sulfur (Fe-S) clusters are among the oldest protein cofactors, and Fe-S cluster-based chemistry has shaped the cellular metabolism of all living organisms. Over the last 30 years, thanks to molecular biology and genetic approaches, numerous actors for Fe-S cluster assembly and delivery to apotargets have been uncovered. In prokaryotes, Escherichia coli is the best-studied for its convenience of growth and its genetic amenability. During evolution, redundant ways to secure the supply of Fe-S clusters to the client proteins have emerged in E. coli. Disrupting gene expression is essential for gene function exploration, but redundancy can blur the interpretations as it can mask the role of important biogenesis components. This chapter describes molecular biology and genetic strategies that have permitted to reveal the E. coli Fe-S cluster conveying component network, composition, organization, and plasticity. In this chapter, we will describe the following genetic methods to investigate the importance of E. coli Fe-S cluster carriers: one-step inactivation of chromosomal genes in E. coli using polymerase chain reaction (PCR) products, P1 transduction, arabinose-inducible expression system, mevalonate (MVA) genetic by-pass, sensitivity tests to oxidative stress and iron starvation, ß-galactosidase assay, gentamicin survival test, and Hot Fusion cloning method.

A semi-automated high-throughput approach to the generation of transposon insertion mutants in the nematode Caenorhabditis elegans.

The generation of a large collection of defined transposon insertion mutants is of general interest to the Caenorhabditis elegans research community and has been supported by the European Union. We describe here a semi-automated high-throughput method for mutant production and screening, using the heterologous transposon Mos1. The procedure allows routine culture of several thousand independent nematode strains in parallel for multiple generations before stereotyped molecular analyses. Using this method, we have already generated >17 500 individual strains carrying Mos1 insertions. It could be easily adapted to forward and reverse genetic screens and may influence researchers faced with making a choice of model organism.

The sRNA RyhB regulates the synthesis of the Escherichia coli methionine sulfoxide reductase MsrB but not MsrA.

Controlling iron homeostasis is crucial for all aerobically grown living cells that are exposed to oxidative damage by reactive oxygen species (ROS), as free iron increases the production of ROS. Methionine sulfoxide reductases (Msr) are key enzymes in repairing ROS-mediated damage to proteins, as they reduce oxidized methionine (MetSO) residues to methionine. E. coli synthesizes two Msr, A and B, which exhibit substrate diastereospecificity. The bacterial iron-responsive small RNA (sRNA) RyhB controls iron metabolism by modulating intracellular iron usage. We show in this paper that RyhB is a direct regulator of the msrB gene that encodes the MsrB enzyme. RyhB down-regulates msrB transcripts along with Hfq and RNaseE proteins since mutations in the ryhB, fur, hfq, or RNaseE-encoded genes resulted in iron-insensitive expression of msrB. Our results show that RyhB binds to two sequences within the short 5'UTR of msrB mRNA as identified by reverse transcriptase and RNase and lead (II) protection assays. Toeprinting analysis shows that RyhB pairing to msrB mRNA prevents efficient ribosome binding and thereby inhibits translation initiation. In vivo site directed-mutagenesis experiments in the msrB 5'UTR region indicate that both RyhB-pairing sites are required to decrease msrB expression. Thus, this study suggests a novel mechanism of translational regulation where a same sRNA can basepair to two different locations within the same mRNA species. In contrast, expression of msrA is not influenced by changes in iron levels.

Fe-S cluster biosynthesis controls uptake of aminoglycosides in a ROS-less death pathway.

All bactericidal antibiotics were recently proposed to kill by inducing reactive oxygen species (ROS) production, causing destabilization of iron-sulfur (Fe-S) clusters and generating Fenton chemistry. We find that the ROS response is dispensable upon treatment with bactericidal antibiotics. Furthermore, we demonstrate that Fe-S clusters are required for killing only by aminoglycosides. In contrast to cells, using the major Fe-S cluster biosynthesis machinery, ISC, cells using the alternative machinery, SUF, cannot efficiently mature respiratory complexes I and II, resulting in impendence of the proton motive force (PMF), which is required for bactericidal aminoglycoside uptake. Similarly, during iron limitation, cells become intrinsically resistant to aminoglycosides by switching from ISC to SUF and down-regulating both respiratory complexes. We conclude that Fe-S proteins promote aminoglycoside killing by enabling their uptake.

A genome-wide collection of Mos1 transposon insertion mutants for the C. elegans research community.

Methods that use homologous recombination to engineer the genome of C. elegans commonly use strains carrying specific insertions of the heterologous transposon Mos1. A large collection of known Mos1 insertion alleles would therefore be of general interest to the C. elegans research community. We describe here the optimization of a semi-automated methodology for the construction of a substantial collection of Mos1 insertion mutant strains. At peak production, more than 5,000 strains were generated per month. These strains were then subject to molecular analysis, and more than 13,300 Mos1 insertions characterized. In addition to targeting directly more than 4,700 genes, these alleles represent the potential starting point for the engineered deletion of essentially all C. elegans genes and the modification of more than 40% of them. This collection of mutants, generated under the auspices of the European NEMAGENETAG consortium, is publicly available and represents an important research resource.

Protein binding sites involved in the assembly of the KplE1 prophage intasome.

The organization of the recombination regions of the KplE1 prophage in Escherichia coli K12 differs from that observed in the lambda prophage. Indeed, the binding sites characterized for the IntS integrase, the TorI recombination directionality factor (RDF) and the integration host factor (IHF) vary in number, spacing and orientation on the attL and attR regions. In this paper, we performed site-directed mutagenesis of the recombination sites to decipher if all sites are essential for the site-specific recombination reaction and how the KplE1 intasome is assembled. We also show that TorI and IntS form oligomers that are stabilized in the presence of their target DNA. Moreover, we found that IHF is the only nucleoid associated protein (NAP) involved in KplE1 recombination, although it is dispensable. This is consistent with the presence of only one functional IHF site on attR and none on attL.

The Vif protein of human immunodeficiency virus type 1 is posttranslationally modified by ubiquitin.

The viral infectivity factor (Vif), one of the six HIV-1 auxiliary genes, is absolutely necessary for productive infection in primary CD4-positive T lymphocytes and macrophages. Vif overcomes the antiviral function of the host factor APOBEC3G. To better understand this mechanism, it is of interest to characterize cellular proteins that interact with Vif and may regulate its function. Here, we show that Vif binds to hNedd4 and AIP4, two HECT E3 ubiquitin ligases. WW domains present in those HECT enzymes contribute to the binding of Vif. Moreover, the region of Vif, which includes amino acids 20-128 and interacts with the hNedd4 WW domains, does not contain proline-rich stretches. Lastly, we show that Vif undergoes post-translational modifications by addition of ubiquitin both in cells overexpressing Vif and in cells expressing HIV-1 provirus. Vif is mainly mono-ubiquitinated, a modification known to address the Gag precursor to the virus budding site.