Highlights d Quantified the proteomes of 375 cell lines from diverse lineages in the CCLE d Correlated expression of proteins across many pathways d Downregulation of multiple protein complexes in microsatellite instability d Protein complexes associated with sensitivity to gene knockdown and mutation

Overexpression of the HipA protein of the HipBA toxin/antitoxin module leads to multidrug tolerance in Escherichia coli. HipA is a "toxin" that causes reversible dormancy, whereas HipB is an antitoxin that binds HipA and acts as a transcriptional repressor of the hipBA operon. Comparative sequence analysis shows that HipA is a member of the phosphatidylinositol 3/4-kinase superfamily. The kinase activity of HipA was examined. HipA was autophosphorylated in the presence of ATP in vitro, and the purified protein appeared to carry a single phosphate group on serine 150. Thus, HipA is a serine kinase that is at least partially phosphorylated in vivo. Overexpression of HipA caused inhibition of cell growth and increase in persister formation. Replacing conserved aspartate 309 in the conserved kinase active site or aspartate 332 in the Mg 2؉ -binding site with glutamine produced mutant proteins that lost the ability to stop cellular growth upon overexpression. Replacing serine 150 with alanine yielded a similarly inactive protein. The mutant proteins were then examined for their ability to increase antibiotic tolerance. Cells overexpressing wild-type HipA were highly tolerant to cefotaxime, a cell wall synthesis inhibitor, to ofloxacin, a fluoroquinolone inhibitor of DNA gyrase, and to topoisomerase IV and were almost completely resistant to killing by mitomycin C, which forms DNA adducts. The mutant proteins did not protect cells from cefotaxime or ofloxacin and had an impaired ability to protect from mitomycin C. Taken together, these results suggest that the protein kinase activity of HipA is essential for persister formation.

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The results indicate that oxidation of CO to CO 2 supplies electrons for reduction of CO 2 to a methyl group by steps and enzymes of the pathway for CO 2 reduction determined for other methane-producing species. However, proteomic and quantitative RT-PCR results suggest that reduction of the methyl group to methane involves novel methyltransferases and a coenzyme F 420H2:heterodisulfide oxidoreductase system that generates a proton gradient for ATP synthesis not previously described for pathways reducing CO 2 to methane. Biochemical assays support a role for the oxidoreductase, and transcriptional mapping identified an unusual operon structure encoding the oxidoreductase. The proteomic results further indicate that acetate is synthesized from the methyl group and CO by a reversal of initial steps in the pathway for conversion of acetate to methane that yields ATP by substrate level phosphorylation. The results indicate that M. acetivorans utilizes a pathway distinct from all known CO 2 reduction pathways for methane formation that reflects an adaptation to the marine environment. Finally, the pathway supports the basis for a recently proposed primitive CO-dependent energy-conservation cycle that drove and directed the early evolution of life on Earth.anaerobic ͉ Archaea ͉ carbon monoxide C arbon monoxide (CO), an atmospheric pollutant that binds tightly to hemoglobin, is held below toxic levels in part by both aerobic and anaerobic microbes (1). The microbial metabolism of CO is an important component of the global carbon cycle (1, 2), and CO is believed to have been present in the atmosphere of early Earth that fueled the evolution of primitive metabolisms (3-7). Investigations of aerobic species from the Bacteria domain have contributed important insights into microbial CO oxidation (8, 9), as have investigations of anaerobes from the Bacteria domain that conserve energy by coupling CO oxidation to H 2 evolution (10-12). Further understanding has been derived from studies of CO-using anaerobes from the Bacteria domain that conserve energy by oxidizing CO and reducing CO 2 to acetate (13,14) or reducing sulfate to sulfide (15). Far less is known for pathways of the few CO-using species in the Archaea domain that have been described. Methanothermobacter thermautotrophicus, Methanosarcina barkeri, and Methanosarcina acetivorans obtain energy for growth by converting CO to methane (16)(17)(18)(19)(20). Although methane formation from CO first was reported in 1947 (21), a comprehensive understanding of the overall pathway for any species has not been reported. It is postulated that M. barkeri oxidizes CO to H 2 , and the H 2 is reoxidized to provide electrons for reducing CO 2 to methane (16). It is postulated further that H 2 production is essential for ATP synthesis during growth on CO (16,22,23). M. acetivorans was isolated from marine sediments where giant kelp is decomposed to methane (24). The flotation bladders of kelp contain CO that is a presumed substrate for M. acetivorans in nature. M. acetivorans produ...

Methanosarcina acetivorans strain C2A is an acetate- and methanol-utilizing methane-producing organism for which the genome, the largest yet sequenced among the Archaea, reveals extensive physiological diversity. LC linear ion trap-FTICR mass spectrometry was employed to analyze acetate- vs methanol-grown cells metabolically labeled with 14N vs 15N, respectively, to obtain quantitative protein abundance ratios. DNA microarray analyses of acetate- vs methanol-grown cells was also performed to determine gene expression ratios. The combined approaches were highly complementary, extending the physiological understanding of growth and methanogenesis. Of the 1081 proteins detected, 255 were > or =3-fold differentially abundant. DNA microarray analysis revealed 410 genes that were > or =2.5-fold differentially expressed of 1972 genes with detected expression. The ratios of differentially abundant proteins were in good agreement with expression ratios of the encoding genes. Taken together, the results suggest several novel roles for electron transport components specific to acetate-grown cells, including two flavodoxins each specific for growth on acetate or methanol. Protein abundance ratios indicated that duplicate CO dehydrogenase/acetyl-CoA complexes function in the conversion of acetate to methane. Surprisingly, the protein abundance and gene expression ratios indicated a general stress response in acetate- vs methanol-grown cells that included enzymes specific for polyphosphate accumulation and oxidative stress. The microarray analysis identified transcripts of several genes encoding regulatory proteins with identity to the PhoU, MarR, GlnK, and TetR families commonly found in the Bacteria domain. An analysis of neighboring genes suggested roles in controlling phosphate metabolism (PhoU), ammonia assimilation (GlnK), and molybdopterin cofactor biosynthesis (TetR). Finally, the proteomic and microarray results suggested roles for two-component regulatory systems specific for each growth substrate.