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Summary Bioluminescence generated by the Vibrio fischeri Lux system consumes oxygen and reducing power, and it has been proposed that cells use this to counteract either oxidative stress or the accumulation of excess reductant. These models predict that lux expression should respond to redox conditions; yet no redox- responsive regulator of lux is known. We found that the luxICDABEG operon responsible for biolumines- cence is repressed by the ArcAB system, which is activated under reducing conditions. Consistent with a role for ArcAB in connecting redox monitoring to lux regulation, adding reductant decreased lumines- cence in an arc-dependent manner. ArcA binds to and regulates transcription from the luxICDABEG pro- moter, and it represses luminescence both in the bright strain MJ1 and in ES114, an isolate from the squid Euprymna scolopes that is not visibly lumines- cent in culture. In ES114, deleting arcA increased luminescence in culture ~500-fold to visible levels comparable to that of symbiotic cells. ArcA did not repress symbiotic luminescence, but by 48 h after inoculation, ArcA did contribute to colonization competitiveness. We hypothesize that inactivation of ArcA in response to oxidative stress during initial colonization derepresses luxICDABEG, but that ArcAB actively regulates other metabolic pathways in the more reduced environment of an established infection.

Biochemically, the syntrophic bacteria constitute the missing link in our understanding of anaerobic flow of carbon in the biosphere. The completed genome sequence of Syntrophus aciditrophicus SB, a model fatty acid- and aromatic acid-degrading syntrophic bacterium, provides a glimpse of the composition and architecture of the electron transfer and energy-transducing systems needed to exist on marginal energy economies of a syntrophic lifestyle. The genome contains 3,179,300 base pairs and 3,169 genes where 1,618 genes were assigned putative functions. Metabolic reconstruction of the gene inventory revealed that most biosynthetic pathways of a typical Gram-negative microbe were present. A distinctive feature of syntrophic metabolism is the need for reverse electron transport; the presence of a unique Rnf-type ion-translocating electron transfer complex, menaquinone, and membrane-bound Fe-S proteins with associated heterodisulfide reductase domains suggests mechanisms to accomplish this task. Previously undescribed approaches to degrade fatty and aromatic acids, including multiple AMP-forming CoA ligases and acyl-CoA synthetases seem to be present as ways to form and dissipate ion gradients by using a sodium-based energy strategy. Thus, S. aciditrophicus , although nutritionally self-sufficient, seems to be a syntrophic specialist with limited fermentative and respiratory metabolism. Genomic analysis confirms the S. aciditrophicus metabolic and regulatory commitment to a nonconventional mode of life compared with our prevailing understanding of microbiology.

© 2015 Leon, Ytterberg, Boontheung, Kim, Loo, Gunsalus and Ogorzalek Loo. Proteomic tools identify constituents of complex mixtures, often delivering long lists of identified proteins. The high-throughput methods excel at matching tandem mass spectrometry data to spectra predicted from sequence databases. Unassigned mass spectra are ignored, but could, in principle, provide valuable information on unanticipated modifications and improve protein annotations while consuming limited quantities of material. Strategies to "mine" information from these discards are presented, along with discussion of features that, when present, provide strong support for modifications. In this study we mined LC-MS/MS datasets of proteolytically-digested concanavalin A pull down fractions from Methanosarcina mazei Gö1 cell lysates. Analyses identified 154 proteins. Many of the observed proteins displayed post-translationally modified forms, including O-formylated and methyl-esterified segments that appear biologically relevant (i.e., not artifacts of sample handling). Interesting cleavages and modifications (e.g., S-cyanylation and trimethylation) were observed near catalytic sites of methanogenesis enzymes. Of 31 Methanosarcina protein N-termini recovered by concanavalin A binding or from a previous study, only M. mazei S-layer protein MM1976 and its M. acetivorans C2A orthologue, MA0829, underwent signal peptide excision. Experimental results contrast with predictions from algorithms SignalP 3.0 and Exprot, which were found to over-predict the presence of signal peptides. Proteins MM0002, MM0716, MM1364, and MM1976 were found to be glycosylated, and employing chromatography tailored specifically for glycopeptides will likely reveal more. This study supplements limited, existing experimental datasets of mature archaeal N-termini, including presence or absence of signal peptides, translation initiation sites, and other processing. Methanosarcina surface and membrane proteins are richly modified.

Many archaeal cell envelopes contain a protein coat or sheath composed of one or more surface exposed proteins. These surface layer (S-layer) proteins contribute structural integrity and protect the lipid membrane from environmental challenges. To explore the species diversity of these layers in the Methanosarcinaceae, the major S-layer protein inMethanosarcina barkeristrain Fusaro was identified using proteomics. The Mbar_A1758 gene product was present in multiple forms with apparent sizes of 130, 120, and 100 kDa, consistent with post-translational modifications including signal peptide excision and protein glycosylation. A protein with features related to the surface layer proteins found inMethanosarcina acetivoransC2A andMethanosarcina mazeiGoel was identified in theM. barkerigenome. These data reveal a distinct conserved protein signature with features and implied cell surface architecture in the Methanosarcinaceae that is absent in other archaea. Paralogous gene expression patterns in twoMethanosarcinaspecies revealed abundant expression of a single S-layer paralog in each strain. Respective promoter elements were identified and shown to be conserved in mRNA coding and upstream untranslated regions. PriorM. acetivoransgenome annotations assigned S-layer or surface layer associated roles of eighty genes: however, of 68 examined none was significantly expressed relative to the experimentally determined S-layer gene.

The outermost cell envelope structure of many archaea and bacteria contains a proteinaceous lattice termed the surface layer or S-layer. It is typically composed of only one or two abundant, often post-translationally modified proteins that self-assemble to form the highly organized arrays. Surprisingly, over a hundred proteins were annotated to be S-layer components in the archaeal species Methanosarcina acetivorans C2A and Methanosarcina mazei Gö1, reflecting limitations of current predictions. An in vivo biotinylation methodology was devised to affinity tag surface-exposed proteins while overcoming unique challenges in working with these fragile organisms. Cells were adapted to growth under N2 fixing conditions, thus minimizing free amines reactive to the NHS-label, and high pH media compatible with the acylation chemistry was used. A 3-phase separation procedure was employed to isolate intact, labeled cells from lysed-cell derived proteins. Streptavidin affinity enrichment followed by stringent wash conditions removed non-specifically bound proteins. This methodology revealed S-layer proteins in M. acetivorans C2A and M. mazei Gö1 to be MA0829 and MM1976, respectively. Each was demonstrated to exist as multiple glycosylated forms using SDS-PAGE coupled with glycoprotein-specific staining, and by interaction with the lectin, Concanavalin A. A number of additional surface-exposed proteins and glycoproteins were identified and included all three subunits of the thermosome: the latter suggests that the chaperonin complex is both surface- and cytoplasmically-localized. This approach provides an alternative strategy to study surface proteins in the archaea.

The heat shock response of the hyperthermophilic archaeon Archaeoglobus fulgidus strain VC-16 was studied using whole-genome microarrays. On the basis of the resulting expression profiles, approximately 350 of the 2,410 open reading frames (ORFs) (ca. 14%) exhibited increased or decreased transcript abundance. These span a range of cell functions, including energy production, amino acid metabolism, and signal transduction, where the majority are uncharacterized. One ORF called AF1298 was identified that contains a putative helix-turn-helix DNA binding motif. The gene product, HSR1, was expressed and purified from Escherichia coli and was used to characterize specific DNA recognition regions upstream of two A. fulgidus genes, AF1298 and AF1971. The results indicate that AF1298 is autoregulated and is part of an operon with two downstream genes that encode a small heat shock protein, Hsp20, and cdc48, an AAA + ATPase. The DNase I footprints using HSR1 suggest the presence of a cis -binding motif upstream of AF1298 consisting of CTAAC-N5-GTTAG. Since AF1298 is negatively regulated in response to heat shock and encodes a protein only distantly related to the N-terminal DNA binding domain of Phr of Pyrococcus furiosus , these results suggest that HSR1 and Phr may belong to an evolutionarily diverse protein family involved in heat shock regulation in hyperthermophilic and mesophilic Archaea organisms.