James F. Holden

Alterations of the human gut microbiome in multiple sclerosis.

The gut microbiome plays an important role in immune function and has been implicated in several autoimmune disorders. Here we use 16S rRNA sequencing to investigate the gut microbiome in subjects with multiple sclerosis (MS, n=60) and healthy controls (n=43). Microbiome alterations in MS include increases in Methanobrevibacter and Akkermansia and decreases in Butyricimonas, and correlate with variations in the expression of genes involved in dendritic cell maturation, interferon signalling and NF-kB signalling pathways in circulating T cells and monocytes. Patients on disease-modifying treatment show increased abundances of Prevotella and Sutterella, and decreased Sarcina, compared with untreated patients. MS patients of a second cohort show elevated breath methane compared with controls, consistent with our observation of increased gut Methanobrevibacter in MS in the first cohort. Further study is required to assess whether the observed alterations in the gut microbiome play a role in, or are a consequence of, MS pathogenesis.

Key Role for Sulfur in Peptide Metabolism and in Regulation of Three Hydrogenases in the Hyperthermophilic Archaeon Pyrococcus furiosus

The hyperthermophilic archaeon Pyrococcus furiosus grows optimally at 100 degrees C by the fermentation of peptides and carbohydrates. Growth of the organism was examined in media containing either maltose, peptides (hydrolyzed casein), or both as the carbon source(s), each with and without elemental sulfur (S(0)). Growth rates were highest on media containing peptides and S(0), with or without maltose. Growth did not occur on the peptide medium without S(0). S(0) had no effect on growth rates in the maltose medium in the absence of peptides. Phenylacetate production rates (from phenylalanine fermentation) from cells grown in the peptide medium containing S(0) with or without maltose were the same, suggesting that S(0) is required for peptide utilization. The activities of 14 of 21 enzymes involved in or related to the fermentation pathways of P. furiosus were shown to be regulated under the five different growth conditions studied. The presence of S(0) in the growth media resulted in decreases in specific activities of two cytoplasmic hydrogenases (I and II) and of a membrane-bound hydrogenase, each by an order of magnitude. The primary S(0)-reducing enzyme in this organism and the mechanism of the S(0) dependence of peptide metabolism are not known. This study provides the first evidence for a highly regulated fermentation-based metabolism in P. furiosus and a significant regulatory role for elemental sulfur or its metabolites.

Biological colonization of new hydrothermal vents following an eruption on Juan de Fuca Ridge

Abstract A recent eruption on CoAxial Segment of Juan de Fuca Ridge initiated hydrothermal conditions with rapid changes in water chemistry and growth of microbial communities. Vent animals recruited from distal sources within a year. One site with newly erupted lava attracted no animals to high-iron and low-sulphide conditions. However, sustained release of flocculent material at a second site suggests extensive subterranean microbial production; here, the dissolved sulphide/heat ratio peaked during the first year. The first larval recruits included vestimentiferans, alvinellid polychaetes and nemerteans; despite the small areal extent of venting, one-third of the regional vent species pool had arrived by 2 years. Near-optimal growth conditions and recruitment by many species continued in the centre of the system but several habitats went extinct within 2 years. Rapid response and exploitation by vent animals must be an important adaptation to such ephemeral conditions.

Identification of 2D-gel proteins: A comparison of MALDI/TOF peptide mass mapping to μ LC-ESI tandem mass spectrometry

A comparative analysis of protein identification for a total of 162 protein spots separated by two-dimensional gel electrophoresis from two fully sequenced archaea, Methanococcus jannaschii and Pyrococcus furiosus, using MALDI-TOF peptide mass mapping (PMM) and μLC-MS/MS is presented. 100% of the gel spots analyzed were successfully matched to the predicted proteins in the two corresponding open reading frame databases by μLC-MS/MS while 97% of them were identified by MALDI-TOF PMM. The high success rate from the PMM resulted from sample desalting/concentrating with ZipTipC18 and optimization of several PMM search parameters including a 25 ppm average mass tolerance and the application of two different protein molecular weight search windows. By using this strategy, low-molecular weight (<23 kDa) proteins could be identified unambiguously with less than 5 peptide matches. Nine percent of spots were identified as containing multiple proteins. By using μLC-MS/MS, 50% of the spots analyzed were identified as containing multiple proteins. μLC-MS/MS demonstrated better protein sequence coverage than MALDI-TOF PMM over the entire mass range of proteins identified. MALDI-TOF and PMM produced unique peptide molecular weight matches that were not identified by μLC-MS/MS. By incorporating amino acid sequence modifications into database searches, combined sequence coverage obtained from these two complimentary ionization methods exceeded 50% for ∼70% of the 162 spots analyzed. This improved sequence coverage in combination with enzymatic digestions of different specificity is proposed as a method for analysis of post-translational modification from 2D-gel separated proteins.

Microbe-metal interactions in marine hydrothermal environments

Marine hydrothermal microorganisms respond rapidly to changes in the concentrations and availability of metals within their environment. Hyperthermophilic archaea appear to possess novel mechanisms for metal detoxification, dissimilatory metal reduction and metal assimilation that may be absent in their mesophilic and bacterial counterparts. For example, tungsten was found in high concentrations in a hydrothermal sulfide deposit where hyperthermophiles were also most abundant, consistent with the unique requirement of these organisms for this element. Furthermore, newly isolated genera of iron-reducing hyperthermophiles expand the scope of carbon cycling in hydrothermal environments. The advent of genome sequences and new molecular techniques will facilitate our further understanding of microbe-mineral interactions in these environments.

Hydrogen-limited growth of hyperthermophilic methanogens at deep-sea hydrothermal vents

Microbial productivity at hydrothermal vents is among the highest found anywhere in the deep ocean, but constraints on microbial growth and metabolism at vents are lacking. We used a combination of cultivation, molecular, and geochemical tools to verify pure culture H2 threshold measurements for hyperthermophilic methanogenesis in low-temperature hydrothermal fluids from Axial Volcano and Endeavour Segment in the northeastern Pacific Ocean. Two Methanocaldococcus strains from Axial and Methanocaldococcus jannaschii showed similar Monod growth kinetics when grown in a bioreactor at varying H2 concentrations. Their H2 half-saturation value was 66 μM, and growth ceased below 17–23 μM H2, 10-fold lower than previously predicted. By comparison, measured H2 and CH4 concentrations in fluids suggest that there was generally sufficient H2 for Methanocaldococcus growth at Axial but not at Endeavour. Fluids from one vent at Axial (Marker 113) had anomalously high CH4 concentrations and contained various thermal classes of methanogens based on cultivation and mcrA/mrtA analyses. At Endeavour, methanogens were largely undetectable in fluid samples based on cultivation and molecular screens, although abundances of hyperthermophilic heterotrophs were relatively high. Where present, Methanocaldococcus genes were the predominant mcrA/mrtA sequences recovered and comprised ∼0.2–6% of the total archaeal community. Field and coculture data suggest that H2 limitation may be partly ameliorated by H2 syntrophy with hyperthermophilic heterotrophs. These data support our estimated H2 threshold for hyperthermophilic methanogenesis at vents and highlight the need for coupled laboratory and field measurements to constrain microbial distribution and biogeochemical impacts in the deep sea.

Phosphoenolpyruvate Synthetase from the Hyperthermophilic Archaeon Pyrococcus furiosus

Phosphoenolpyruvate synthetase (PpsA) was purified from the hyperthermophilic archaeon Pyrococcus furiosus. This enzyme catalyzes the conversion of pyruvate and ATP to phosphoenolpyruvate (PEP), AMP, and phosphate and is thought to function in gluconeogenesis. PpsA has a subunit molecular mass of 92 kDa and contains one calcium and one phosphorus atom per subunit. The active form has a molecular mass of 690+/-20 kDa and is assumed to be octomeric, while approximately 30% of the protein is purified as a large ( approximately 1.6 MDa) complex that is not active. The apparent K(m) values and catalytic efficiencies for the substrates pyruvate and ATP (at 80 degrees C, pH 8.4) were 0.11 mM and 1.43 x 10(4) mM(-1). s(-1) and 0.39 mM and 3.40 x 10(3) mM(-1) x s(-1), respectively. Maximal activity was measured at pH 9.0 (at 80 degrees C) and at 90 degrees C (at pH 8.4). The enzyme also catalyzed the reverse reaction, but the catalytic efficiency with PEP was very low [k(cat)/K(m) = 32 (mM. s(-1)]. In contrast to several other nucleotide-dependent enzymes from P. furiosus, PpsA has an absolute specificity for ATP as the phosphate-donating substrate. This is the first PpsA from a nonmethanogenic archaeon to be biochemically characterized. Its kinetic properties are consistent with a role in gluconeogenesis, although its relatively high cellular concentration ( approximately 5% of the cytoplasmic protein) suggests an additional function possibly related to energy spilling. It is not known whether interconversion between the smaller, active and larger, inactive forms of the enzyme has any functional role.

Constraints on Anaerobic Respiration in the Hyperthermophilic Archaea Pyrobaculum islandicum and Pyrobaculum aerophilum

ABSTRACT Pyrobaculum islandicum uses iron, thiosulfate, and elemental sulfur for anaerobic respiration, while Pyrobaculum aerophilum uses iron and nitrate; however, the constraints on these processes and their physiological mechanisms for iron and sulfur reduction are not well understood. Growth rates on sulfur compounds are highest at pH 5 to 6 and highly reduced (<−420-mV) conditions, while growth rates on nitrate and iron are highest at pH 7 to 9 and more-oxidized (>−210-mV) conditions. Growth on iron expands the known pH range of growth for both organisms. P. islandicum differs from P. aerophilum in that it requires direct contact with insoluble iron oxide for growth, it did not produce any extracellular compounds when grown on insoluble iron, and it lacked 2,6-anthrahydroquinone disulfonate oxidase activity. Furthermore, iron reduction in P. islandicum appears to be completely independent of c-type cytochromes. Like that in P. aerophilum, NADH-dependent ferric reductase activity in P. islandicum increased significantly in iron-grown cultures relative to that in non-iron-grown cultures. Proteomic analyses showed that there were significant increases in the amounts of a putative membrane-bound thiosulfate reductase in P. islandicum cultures grown on thiosulfate relative to those in cultures grown on iron and elemental sulfur. This is the first evidence of this enzyme being used in either a hyperthermophile or an archaeon. Pyrobaculum arsenaticum and Pyrobaculum calidifontis also grew on Fe(III) citrate and insoluble iron oxide, but only P. arsenaticum could grow on insoluble iron without direct contact.

Characterization of Dissimilatory Fe(III) versus NO3− Reduction in the Hyperthermophilic Archaeon Pyrobaculum aerophilum

The hyperthermophilic archaeon Pyrobaculum aerophilum used 20 mM Fe(III) citrate, 100 mM poorly crystalline Fe(III) oxide, and 10 mM KNO3 as terminal electron acceptors. The two forms of iron were reduced at different rates but with equal growth yields. The insoluble iron was reduced when segregated spatially by dialysis tubing, indicating that direct contact with the iron was not necessary for growth. When partitioned, there was no detectable Fe(III) or Fe(II) outside of the tubing after growth, suggesting that an electron shuttle, not a chelator, may be used as an extracellular mediator of iron reduction. The addition of 25 and 50% (vol vol(-1)) cell-free spent insoluble iron media to fresh media led to growth without a lag phase. Liquid chromatography analysis of spent media showed that cultures grown in iron, especially insoluble iron, produced soluble extracellular compounds that were absent or less abundant in spent nitrate medium. NADH-dependent ferric reductase activity increased approximately 100-fold, while nitrate reductase activity decreased 10-fold in whole-cell extracts from iron-grown cells relative to those from nitrate-grown cells, suggesting that dissimilatory iron reduction was regulated. A novel 2,6-anthrahydroquinone disulfonate oxidase activity was more than 580-fold higher in iron-grown cells than in nitrate-grown cells. The activity was primarily (>95%) associated with the membrane cellular fraction, but its physiological function is unknown. Nitrate-grown cultures produced two membrane-bound, c-type cytochromes that are predicted to be monoheme and part of nitrite reductase and a bc1 complex using genome analyses. Only one cytochrome was present in cells grown on Fe(III) citrate whose relative abundance was unchanged.

Overview of Hyperthermophiles and Their Heat-Shock Proteins

Publisher Summary This chapter focuses on the diversity, phylogeny, and heat-shock proteins of hyperthermophilic micro-organisms, organisms that grow at 90°C and above. Hyperthermophiles are defined as the organisms that grow optimally at 80°C or higher with a maximal growth temperature of 90°C or higher. The term extreme thermophile is also used to describe the same group of organisms. Hyperthermophiles are detected in environments having temperatures of less than 50°C to greater than 110°C and at pH ranges from 0.5 to 11.0 provided suitable carbon and nitrogen sources and needed electron acceptors and donors exist. Such environments are found in both the shallow and deep sea. Physiological characteristics, phylogeny and evolution of thermophiles are discussed. At present, there are too few data on heat-shock proteins in hyperthermophilic species to make sweeping conclusions about the role of hyperthermophiles in the origins of the heat-shock and other stress-response proteins.