Staphylococcus aureus HemX Modulates Glutamyl-tRNA Reductase Abundance To Regulate Heme Biosynthesis.

Staphylococcus aureus is responsible for a significant amount of devastating disease. Its ability to colonize the host and cause infection is supported by a variety of proteins that are dependent on the cofactor heme. Heme is a porphyrin used broadly across kingdoms and is synthesized de novo from common cellular precursors and iron. While heme is critical to bacterial physiology, it is also toxic in high concentrations, requiring that organisms encode regulatory processes to control heme homeostasis. In this work, we describe a posttranscriptional regulatory strategy in S. aureus heme biosynthesis. The first committed enzyme in the S. aureus heme biosynthetic pathway, glutamyl-tRNA reductase (GtrR), is regulated by heme abundance and the integral membrane protein HemX. GtrR abundance increases dramatically in response to heme deficiency, suggesting a mechanism by which S. aureus responds to the need to increase heme synthesis. Additionally, HemX is required to maintain low levels of GtrR in heme-proficient cells, and inactivation of hemX leads to increased heme synthesis. Excess heme synthesis in a ΔhemX mutant activates the staphylococcal heme stress response, suggesting that regulation of heme synthesis is critical to reduce self-imposed heme toxicity. Analysis of diverse organisms indicates that HemX is widely conserved among heme-synthesizing bacteria, suggesting that HemX is a common factor involved in the regulation of GtrR abundance. Together, this work demonstrates that S. aureus regulates heme synthesis by modulating GtrR abundance in response to heme deficiency and through the activity of the broadly conserved HemX.IMPORTANCEStaphylococcus aureus is a leading cause of skin and soft tissue infections, endocarditis, bacteremia, and osteomyelitis, making it a critical health care concern. Development of new antimicrobials against S. aureus requires knowledge of the physiology that supports this organism's pathogenesis. One component of staphylococcal physiology that contributes to growth and virulence is heme. Heme is a widely utilized cofactor that enables diverse chemical reactions across many enzyme families. S. aureus relies on many critical heme-dependent proteins and is sensitive to excess heme toxicity, suggesting S. aureus must maintain proper intracellular heme homeostasis. Because S. aureus provides heme for heme-dependent enzymes via synthesis from common precursors, we hypothesized that regulation of heme synthesis is one mechanism to maintain heme homeostasis. In this study, we identify that S. aureus posttranscriptionally regulates heme synthesis by restraining abundance of the first heme biosynthetic enzyme, GtrR, via heme and the broadly conserved membrane protein HemX.

A strictly monofunctional bacterial hydroxymethylpyrimidine phosphate kinase precludes damaging errors in thiamin biosynthesis.

The canonical kinase (ThiD) that converts the thiamin biosynthesis intermediate hydroxymethylpyrimidine (HMP) monophosphate to the diphosphate can also very efficiently convert free HMP to the monophosphate in prokaryotes, plants, and fungi. This HMP kinase activity enables salvage of HMP, but it is not substrate-specific and so allows toxic HMP analogs and damage products to infiltrate the thiamin biosynthesis pathway. Comparative analysis of bacterial genomes uncovered a gene, thiD2 , that is often fused to the thiamin synthesis gene thiE and could potentially encode a replacement for ThiD. Standalone ThiD2 proteins and ThiD2 fusion domains are small (~130-residues) and do not belong to any previously known protein family. Genetic and biochemical analyses showed that representative standalone and fused ThiD2 proteins catalyze phosphorylation of HMP monophosphate, but not of HMP or its toxic analogs and damage products such as bacimethrin and 5-(hydroxymethyl)-2-methylpyrimidin-4-ol. As strictly monofunctional HMP monophosphate kinases, ThiD2 proteins eliminate a potentially fatal vulnerability of canonical ThiD, at the cost of the ability to reclaim HMP formed by thiamin turnover.

CtaM Is Required for Menaquinol Oxidase aa3 Function in Staphylococcus aureus.

UNLABELLED: Staphylococcus aureus is the leading cause of skin and soft tissue infections, bacteremia, osteomyelitis, and endocarditis in the developed world. The ability of S. aureus to cause substantial disease in distinct host environments is supported by a flexible metabolism that allows this pathogen to overcome challenges unique to each host organ. One feature of staphylococcal metabolic flexibility is a branched aerobic respiratory chain composed of multiple terminal oxidases. Whereas previous biochemical and spectroscopic studies reported the presence of three different respiratory oxygen reductases (o type, bd type, and aa3 type), the genome contains genes encoding only two respiratory oxygen reductases, cydAB and qoxABCD Previous investigation showed that cydAB and qoxABCD are required to colonize specific host organs, the murine heart and liver, respectively. This work seeks to clarify the relationship between the genetic studies showing the unique roles of the cydAB and qoxABCD in virulence and the respiratory reductases reported in the literature. We establish that QoxABCD is an aa3-type menaquinol oxidase but that this enzyme is promiscuous in that it can assemble as a bo3-type menaquinol oxidase. However, the bo3 form of QoxABCD restricts the carbon sources that can support the growth of S. aureus In addition, QoxABCD function is supported by a previously uncharacterized protein, which we have named CtaM, that is conserved in aerobically respiring Firmicutes In total, these studies establish the heme A biosynthesis pathway in S. aureus, determine that QoxABCD is a type aa3 menaquinol oxidase, and reveal CtaM as a new protein required for type aa3 menaquinol oxidase function in multiple bacterial genera. IMPORTANCE: Staphylococcus aureus relies upon the function of two terminal oxidases, CydAB and QoxABCD, to aerobically respire and colonize distinct host tissues. Previous biochemical studies support the conclusion that a third terminal oxidase is also present. We establish the components of the S. aureus electron transport chain by determining the heme cofactors that interact with QoxABCD. This insight explains previous observations by revealing that QoxABCD can utilize different heme cofactors and confirms that the electron transport chain of S. aureus is comprised of two terminal menaquinol oxidases. In addition, a newly identified protein, CtaM, is found to be required for the function of QoxABCD. These results provide a more complete assessment of the molecular mechanisms that support staphylococcal respiration.

Systematic identification and analysis of frequent gene fusion events in metabolic pathways.

BACKGROUND: Gene fusions are the most powerful type of in silico-derived functional associations. However, many fusion compilations were made when <100 genomes were available, and algorithms for identifying fusions need updating to handle the current avalanche of sequenced genomes. The availability of a large fusion dataset would help probe functional associations and enable systematic analysis of where and why fusion events occur. RESULTS: Here we present a systematic analysis of fusions in prokaryotes. We manually generated two training sets: (i) 121 fusions in the model organism Escherichia coli; (ii) 131 fusions found in B vitamin metabolism. These sets were used to develop a fusion prediction algorithm that captured the training set fusions with only 7 % false negatives and 50 % false positives, a substantial improvement over existing approaches. This algorithm was then applied to identify 3.8 million potential fusions across 11,473 genomes. The results of the analysis are available in a searchable database at http://modelseed.org/projects/fusions/ . A functional analysis identified 3,000 reactions associated with frequent fusion events and revealed areas of metabolism where fusions are particularly prevalent. CONCLUSIONS: Customary definitions of fusions were shown to be ambiguous, and a stricter one was proposed. Exploring the genes participating in fusion events showed that they most commonly encode transporters, regulators, and metabolic enzymes. The major rationales for fusions between metabolic genes appear to be overcoming pathway bottlenecks, avoiding toxicity, controlling competing pathways, and facilitating expression and assembly of protein complexes. Finally, our fusion dataset provides powerful clues to decipher the biological activities of domains of unknown function.

Evolution of proline biosynthesis: enzymology, bioinformatics, genetics, and transcriptional regulation.

Proline is not only an essential component of proteins but it also has important roles in adaptation to osmotic and dehydration stresses, redox control, and apoptosis. Here, we review pathways of proline biosynthesis in the three domains of life. Pathway reconstruction from genome data for hundreds of eubacterial and dozens of archaeal and eukaryotic organisms revealed evolutionary conservation and variations of this pathway across different taxa. In the most prevalent pathway of proline synthesis, glutamate is phosphorylated to γ-glutamyl phosphate by γ-glutamyl kinase, reduced to γ-glutamyl semialdehyde by γ-glutamyl phosphate reductase, cyclized spontaneously to Δ(1)-pyrroline-5-carboxylate and reduced to proline by Δ(1)-pyrroline-5-carboxylate reductase. In higher plants and animals the first two steps are catalysed by a bi-functional Δ(1) -pyrroline-5-carboxylate synthase. Alternative pathways of proline formation use the initial steps of the arginine biosynthetic pathway to ornithine, which can be converted to Δ(1)-pyrroline-5-carboxylate by ornithine aminotransferase and then reduced to proline or converted directly to proline by ornithine cyclodeaminase. In some organisms, the latter pathways contribute to or could be fully responsible for the synthesis of proline. The conservation of proline biosynthetic enzymes and significance of specific residues for catalytic activity and allosteric regulation are analysed on the basis of protein structural data, multiple sequence alignments, and mutant studies, providing novel insights into proline biosynthesis in organisms. We also discuss the transcriptional control of the proline biosynthetic genes in bacteria and plants.

Whole-genome detection of conditionally essential and dispensable genes in Escherichia coli via genetic footprinting.

We present a whole-genome approach to genetic footprinting in Escherichia coli using Tn5-based transposons to determine gene essentiality. A population of cells is mutagenized and subjected to outgrowth under selective conditions. Transposon insertions in the surviving mutants are detected using nested polymerase chain reaction (PCR), agarose gel electrophoresis, and software-assisted PCR product size determination. Genomic addresses of these inserts are then mapped onto the E. coli genome sequence based on the PCR product lengths and the addresses of the corresponding genome-specific primers. Gene essentiality conclusions were drawn based on a semiautomatic analysis of the number and relative positions of inserts retained within each gene after selective outgrowth.

Modeling competitive outgrowth of mutant populations: why do essentiality screens yield divergent results?

Mutant propagation (outgrowth) is an important step in all large-scale gene essentiality experiments, profoundly influencing essentiality assignment produced. Using a simplified mathematical model of competitive outgrowth in a diverse mutant population, we have identified several technological factors (duration of outgrowth, sensitivity of the scoring technique, initial cell titer of each mutant in the population) that have the largest impact on the outcome of the essentiality screen. The model can be used for planning a large-scale gene essentiality screen as well as for analyzing its results, including meaningful comparisons of "essential" gene lists generated by different techniques.

Comparative genomics of NAD biosynthesis in cyanobacteria.

Biosynthesis of NAD(P) cofactors is of special importance for cyanobacteria due to their role in photosynthesis and respiration. Despite significant progress in understanding NAD(P) biosynthetic machinery in some model organisms, relatively little is known about its implementation in cyanobacteria. We addressed this problem by a combination of comparative genome analysis with verification experiments in the model system of Synechocystis sp. strain PCC 6803. A detailed reconstruction of the NAD(P) metabolic subsystem using the SEED genomic platform (http://theseed.uchicago.edu/FIG/index.cgi) helped us accurately annotate respective genes in the entire set of 13 cyanobacterial species with completely sequenced genomes available at the time. Comparative analysis of operational variants implemented in this divergent group allowed us to elucidate both conserved (de novo and universal pathways) and variable (recycling and salvage pathways) aspects of this subsystem. Focused genetic and biochemical experiments confirmed several conjectures about the key aspects of this subsystem. (i) The product of the slr1691 gene, a homolog of Escherichia coli gene nadE containing an additional nitrilase-like N-terminal domain, is a NAD synthetase capable of utilizing glutamine as an amide donor in vitro. (ii) The product of the sll1916 gene, a homolog of E. coli gene nadD, is a nicotinic acid mononucleotide-preferring adenylyltransferase. This gene is essential for survival and cannot be compensated for by an alternative nicotinamide mononucleotide (NMN)-preferring adenylyltransferase (slr0787 gene). (iii) The product of the slr0788 gene is a nicotinamide-preferring phosphoribosyltransferase involved in the first step of the two-step non-deamidating utilization of nicotinamide (NMN shunt). (iv) The physiological role of this pathway encoded by a conserved gene cluster, slr0787-slr0788, is likely in the recycling of endogenously generated nicotinamide, as supported by the inability of this organism to utilize exogenously provided niacin. Positional clustering and the co-occurrence profile of the respective genes across a diverse collection of cellular organisms provide evidence of horizontal transfer events in the evolutionary history of this pathway.

Genome-scale identification of conditionally essential genes in E. coli by DNA microarrays.

Identifying the genes required for the growth or viability of an organism under a given condition is an important step toward understanding the roles these genes play in the physiology of the organism. Currently, the combination of global transposon mutagenesis with PCR-based mapping of transposon insertion sites is the most common method for determining conditional gene essentiality. In order to accelerate the detection of essential gene products, here we test the utility and reliability of a DNA microarray technology-based method for the identification of conditionally essential genes of the bacterium, Escherichia coli, grown in rich medium under aerobic or anaerobic growth conditions using two different DNA microarray platforms. Identification and experimental verification of five hypothetical E. coli genes essential for anaerobic growth directly demonstrated the utility of the method. However, the two different DNA microarray platforms yielded largely non-overlapping results after a two standard deviations cutoff and were subjected to high false positive background levels. Thus, further methodological improvements are needed prior to the use of DNA microarrays to reliably identify conditionally essential genes on genome-scale.

A genetic screen for the identification of thiamin metabolic genes.

A genetic screen was developed for the identification of genes related to thiamin biosynthesis and degradation. Genes conferring resistance to bacimethrin or 4-amino-2-trifluoromethyl-5-hydroxymethylpyrimidine were selected from Escherichia coli and Bacillus subtilis genomic libraries. Hits from the selection included the known thiamin biosynthetic genes thiC, thiE, and dxs as well as five genes of previously unknown function (E. coli yjjX, yajO, ymfB, and cof and B. subtilis yveN). The gene products YmfB and Cof catalyze the hydrolysis of 4-amino-2-methyl-5-hydroxymethylpyrimidine pyrophosphate to 4-amino-2-methyl-5-hydroxymethylpyrimidine phosphate. YmfB also converts thiamin pyrophosphate into thiamin phosphate.

Ribosylnicotinamide kinase domain of NadR protein: identification and implications in NAD biosynthesis.

NAD is an indispensable redox cofactor in all organisms. Most of the genes required for NAD biosynthesis in various species are known. Ribosylnicotinamide kinase (RNK) was among the few unknown (missing) genes involved with NAD salvage and recycling pathways. Using a comparative genome analysis involving reconstruction of NAD metabolism from genomic data, we predicted and experimentally verified that bacterial RNK is encoded within the 3' region of the nadR gene. Based on these results and previous data, the full-size multifunctional NadR protein (as in Escherichia coli) is composed of (i) an N-terminal DNA-binding domain involved in the transcriptional regulation of NAD biosynthesis, (ii) a central nicotinamide mononucleotide adenylyltransferase (NMNAT) domain, and (iii) a C-terminal RNK domain. The RNK and NMNAT enzymatic activities of recombinant NadR proteins from Salmonella enterica serovar Typhimurium and Haemophilus influenzae were quantitatively characterized. We propose a model for the complete salvage pathway from exogenous N-ribosylnicotinamide to NAD which involves the concerted action of the PnuC transporter and NRK, followed by the NMNAT activity of the NadR protein. Both the pnuC and nadR genes were proven to be essential for the growth and survival of H. influenzae, thus implicating them as potential narrow-spectrum drug targets.

From genetic footprinting to antimicrobial drug targets: examples in cofactor biosynthetic pathways.

Novel drug targets are required in order to design new defenses against antibiotic-resistant pathogens. Comparative genomics provides new opportunities for finding optimal targets among previously unexplored cellular functions, based on an understanding of related biological processes in bacterial pathogens and their hosts. We describe an integrated approach to identification and prioritization of broad-spectrum drug targets. Our strategy is based on genetic footprinting in Escherichia coli followed by metabolic context analysis of essential gene orthologs in various species. Genes required for viability of E. coli in rich medium were identified on a whole-genome scale using the genetic footprinting technique. Potential target pathways were deduced from these data and compared with a panel of representative bacterial pathogens by using metabolic reconstructions from genomic data. Conserved and indispensable functions revealed by this analysis potentially represent broad-spectrum antibacterial targets. Further target prioritization involves comparison of the corresponding pathways and individual functions between pathogens and the human host. The most promising targets are validated by direct knockouts in model pathogens. The efficacy of this approach is illustrated using examples from metabolism of adenylate cofactors NAD(P), coenzyme A, and flavin adenine dinucleotide. Several drug targets within these pathways, including three distantly related adenylyltransferases (orthologs of the E. coli genes nadD, coaD, and ribF), are discussed in detail.