Bacterial Approaches for Assembling Iron-Sulfur Proteins.

Building iron-sulfur (Fe-S) clusters and assembling Fe-S proteins are essential actions for life on Earth. The three processes that sustain life, photosynthesis, nitrogen fixation, and respiration, require Fe-S proteins. Genes coding for Fe-S proteins can be found in nearly every sequenced genome. Fe-S proteins have a wide variety of functions, and therefore, defective assembly of Fe-S proteins results in cell death or global metabolic defects. Compared to alternative essential cellular processes, there is less known about Fe-S cluster synthesis and Fe-S protein maturation. Moreover, new factors involved in Fe-S protein assembly continue to be discovered. These facts highlight the growing need to develop a deeper biological understanding of Fe-S cluster synthesis, holo-protein maturation, and Fe-S cluster repair. Here, we outline bacterial strategies used to assemble Fe-S proteins and the genetic regulation of these processes. We focus on recent and relevant findings and discuss future directions, including the proposal of using Fe-S protein assembly as an antipathogen target.

Genetic Approaches to Uncover Gene Products Involved in Iron-Sulfur Protein Maturation: High-Throughput Genomic Screening Using Transposon Sequencing.

Iron-sulfur (Fe-S) clusters are one of the most ubiquitous and versatile prosthetic groups exploited by nature. Fe-S clusters aid in conducting redox reactions, carbon activation, and environmental sensing. This chapter presents an overview of the genetic approaches that have been useful for identifying and characterizing bacterial factors involved in Fe-S protein assembly. Traditional genetic screens that assess viability or conditional auxotrophies and bioinformatic approaches have identified the majority of the described genes utilized for Fe-S protein assembly. Herein, we expand upon this list of genetic methods by detailing the use of transposon sequencing (TnSeq) to identify gene products that are necessary for the proper function of metabolic pathways that require Fe-S enzymes. TnSeq utilizes the power of genomics and massively parallel DNA sequencing to allow researchers to quantify the necessity of individual gene products for a specific growth condition. This allows for the identification of gene products or gene networks that have a role in a given metabolic process but are not essential for the process. An advantage of this approach is that it allows researchers to identify mutants that have partial phenotypes that are often missed using traditional plate-based selections. Applying TnSeq to address questions of Fe-S protein maturation will result in a more comprehensive understanding of genetic interactions and factors utilized in Fe-S biogenesis and Fe-S protein assembly.

An Orphan MbtH-Like Protein Interacts with Multiple Nonribosomal Peptide Synthetases in Myxococcus xanthus DK1622.

One mechanism by which bacteria and fungi produce bioactive natural products is the use of nonribosomal peptide synthetases (NRPSs). Many NRPSs in bacteria require members of the MbtH-like protein (MLP) superfamily for their solubility or function. Although MLPs are known to interact with the adenylation domains of NRPSs, the role MLPs play in NRPS enzymology has yet to be elucidated. MLPs are nearly always encoded within the biosynthetic gene clusters (BGCs) that also code for the NRPSs that interact with the MLP. Here, we identify 50 orphan MLPs from diverse bacteria. An orphan MLP is one that is encoded by a gene that is not directly adjacent to genes predicted to be involved in nonribosomal peptide biosynthesis. We targeted the orphan MLP MXAN_3118 from Myxococcus xanthus DK1622 for characterization. The M. xanthus DK1622 genome contains 15 NRPS-encoding BGCs but only one MLP-encoding gene (MXAN_3118). We tested the hypothesis that MXAN_3118 interacts with one or more NRPS using a combination of in vivo and in vitro assays. We determined that MXAN_3118 interacts with at least seven NRPSs from distinct BGCs. We show that one of these BGCs codes for NRPS enzymology that likely produces a valine-rich natural product that inhibits the clumping of M. xanthus DK1622 in liquid culture. MXAN_3118 is the first MLP to be identified that naturally interacts with multiple NRPS systems in a single organism. The finding of an MLP that naturally interacts with multiple NRPS systems suggests it may be harnessed as a "universal" MLP for generating functional hybrid NRPSs.IMPORTANCE MbtH-like proteins (MLPs) are essential accessory proteins for the function of many nonribosomal peptide synthetases (NRPSs). We identified 50 MLPs from diverse bacteria that are coded by genes that are not located near any NRPS-encoding biosynthetic gene clusters (BGCs). We define these as orphan MLPs because their NRPS partner(s) is unknown. Investigations into the orphan MLP from Myxococcus xanthus DK1622 determined that it interacts with NRPSs from at least seven distinct BGCs. Support for these MLP-NRPS interactions came from the use of a bacterial two-hybrid assay and copurification of the MLP with various NRPSs. The flexibility of this MLP to naturally interact with multiple NRPSs led us to hypothesize that this MLP may be used as a "universal" MLP during the construction of functional hybrid NRPSs.