Henry C. Aldrich

A Novel Lineage of Proteobacteria Involved in Formation of Marine Fe-Oxidizing Microbial Mat Communities

Background For decades it has been recognized that neutrophilic Fe-oxidizing bacteria (FeOB) are associated with hydrothermal venting of Fe(II)-rich fluids associated with seamounts in the worlds oceans. The evidence was based almost entirely on the mineralogical remains of the microbes, which themselves had neither been brought into culture or been assigned to a specific phylogenetic clade. We have used both cultivation and cultivation-independent techniques to study Fe-rich microbial mats associated with hydrothermal venting at Loihi Seamount, a submarine volcano. Methodology/Principle Findings Using gradient enrichment techniques, two iron-oxidizing bacteria, strains PV-1 and JV-1, were isolated. Chemolithotrophic growth was observed under microaerobic conditions; Fe(II) and Fe0 were the only energy sources that supported growth. Both strains produced filamentous stalk-like structures composed of multiple nanometer sized fibrils of Fe-oxyhydroxide. These were consistent with mineralogical structures found in the iron mats. Phylogenetic analysis of the small subunit (SSU) rRNA gene demonstrated that strains PV-1 and JV-1 were identical and formed a monophyletic group deeply rooted within the Proteobacteria. The most similar sequence (85.3% similarity) from a cultivated isolate came from Methylophaga marina. Phylogenetic analysis of the RecA and GyrB protein sequences confirmed that these strains are distantly related to other members of the Proteobacteria. A cultivation-independent analysis of the SSU rRNA gene by terminal-restriction fragment (T-RF) profiling showed that this phylotype was most common in a variety of microbial mats collected at different times and locations at Loihi. Conclusions On the basis of phylogenetic and physiological data, it is proposed that isolate PV-1T ( = ATCC BAA-1019: JCM 14766) represents the type strain of a novel species in a new genus, Mariprofundus ferrooxydans gen. nov., sp. nov. Furthermore, the strain is the first cultured representative of a new candidatus class of the Proteobacteria that is widely distributed in deep-sea environments, Candidatus ζ (zeta)-Proteobacteria cl. nov.

Bacillus infernus sp. nov., an Fe(III)- and Mn(IV)-reducing anaerobe from the deep terrestrial subsurface.

Bacillus infernus sp. nov. was isolated from ca. 2,700 m below the land surface in the Taylorsville Triassic Basin in Virginia. B. infernus was a strict anaerobe that grew on formate or lactate with Fe(III), MnO2, trimethylamine oxide, or nitrate (reduced to nitrite) as an electron acceptor, and it also grew fermentatively on glucose. Type strain TH-23 and five reference strains were gram-positive rods that were thermophilic (growth occurred at 61 degrees C), halotolerant (good growth occurred in the presence of Na+ concentrations up to 0.6 M), and very slightly alkaliphilic (good growth occurred at pH 7.3 to 7.8). A phylogenetic analysis of its 16S rRNA indicated that B. infernus should be classified as a new species of the genus Bacillus. B. infernus is the only strictly anaerobic species in the genus Bacillus.

Characterization of the anaerobic propionate-degrading syntrophs Smithella propionica gen. nov., sp. nov. and Syntrophobacter wolinii.

A strain of anaerobic, syntrophic, propionate-oxidizing bacteria, strain LYPT (= OCM 661T; T = type strain), was isolated and proposed as representative of a new genus and new species, Smithella propionica gen. nov., sp. nov. The strain was enriched from an anaerobic digestor and isolated. Initial isolation was as a monoxenic propionate-degrading co-culture containing Methanospirillum hungateii JF-1T as an H2- and formate-using partner. Later, an axenic culture was obtained by using crotonate as the catabolic substrate. The previously described propionate-degrading syntrophs of the genus Syntrophobacter also grow in co-culture with methanogens such as Methanospirillum hungateii, forming acetate, CO2 and methane from propionate. However, Smithella propionica differs by producing less methane and more acetate; in addition, it forms small amounts of butyrate. Smithella propionica and Syntrophobacter wolinii grew within similar ranges of pH, temperature and salinity, but they differed significantly in substrate ranges and catabolic products. Unlike Syntrophobacter wolinii, Smithella propionica grew axenically on crotonate, although very slowly. Co-cultures of Smithella propionica grew on propionate, and grew slowly on crotonate or butyrate. Syntrophobacter wolinii and Syntrophobacter pfennigii grow on propionate plus sulfate, whereas Smithella propionica did not. Comparisons of 16S rDNA genes indicated that Smithella propionica is most closely related to Syntrophus, and is more distantly related to Syntrophobacter.

Microcompartments in prokaryotes: carboxysomes and related polyhedra.

All cyanobacteria and many chemoautotrophs contain polyhedral inclusion bodies that are bound by a unilamellar protein shell (15, 63). Isolation and enzymatic analysis of the bodies from Halothiobacillus neapolitanus (previously Thiobacillus neapolitanus) revealed that they are filled with ribulose 1,5bisphosphate carboxylase/oxygenase (RuBisCO); therefore they were given the name “carboxysomes” (59). Subsequent studies of both cyanobacteria and chemoautotrophic bacteria have led to the well-accepted conclusion that the “organelles” or microcompartments function to enhance the catalytic properties of the RuBisCO they contain, although the mechanism of this catalytic enhancement is unclear (51, 65). Localization and characterization of the genes encoding carboxysome components has underscored the apparent common function of these bodies in carboxysome-containing autotrophic bacteria. More surprising is the finding that a number of heterotrophic prokaryotes harbor genes homologous to those for carboxysome shell proteins (9, 32, 62). Under proper growth conditions, these bacteria produce polyhedral inclusion bodies that are morphologically similar to carboxysomes, although the cells expressing these bodies contain no RuBisCO and do not fix CO 2 via the Calvin-Benson-Bassham cycle as a major part of their carbon metabolism. This review evaluates the evidence that relates carboxysome structure to function in the carbon metabolism of autotrophic prokaryotes and examines similarities to newly discovered particles found in heterotrophs. The possibility is explored that microcompartmentalization of key metabolic enzymes by carboxysomes and their relatives is a more widely utilized regulatory mechanism in prokaryotes than was previously envisioned.

Isolation and Characterization of Acid-Tolerant, Thermophilic Bacteria for Effective Fermentation of Biomass-Derived Sugars to Lactic Acid

ABSTRACT Biomass-derived sugars, such as glucose, xylose, and other minor sugars, can be readily fermented to fuel ethanol and commodity chemicals by the appropriate microbes. Due to the differences in the optimum conditions for the activity of the fungal cellulases that are required for depolymerization of cellulose to fermentable sugars and the growth and fermentation characteristics of the current industrial microbes, simultaneous saccharification and fermentation (SSF) of cellulose is envisioned at conditions that are not optimal for the fungal cellulase activity, leading to a higher-than-required cost of cellulase in SSF. We have isolated bacterial strains that grew and fermented both glucose and xylose, major components of cellulose and hemicellulose, respectively, to l(+)-lactic acid at 50°C and pH 5.0, conditions that are also optimal for fungal cellulase activity. Xylose was metabolized by these new isolates through the pentose-phosphate pathway. As expected for the metabolism of xylose by the pentose-phosphate pathway, [13C]lactate accounted for more than 90% of the total 13C-labeled products from [13C]xylose. Based on fatty acid profile and 16S rRNA sequence, these isolates cluster with Bacillus coagulans, although the B. coagulans type strain, ATCC 7050, failed to utilize xylose as a carbon source. These new B. coagulans isolates have the potential to reduce the cost of SSF by minimizing the amount of fungal cellulases, a significant cost component in the use of biomass as a renewable resource, for the production of fuels and chemicals.

Methanogenium frigidum sp. nov., a psychrophilic, H2-Using methanogen from Ace Lake, Antarctica

Methanogenium frigidum sp. nov. was isolated from the perennially cold, anoxic hypolimnion of Ace Lake in the Vesfold Hills of Antarctica. The cells were psychrophilic, exhibiting most rapid growth at 15 degrees C and no growth at temperatures above 18 to 20 degrees C. The cells were irregular, nonmotile coccoids (diameter, 1.2 to 2.5 microns) that occurred singly and grew by CO2 reduction by using H2 as a reductant. Formate could replace H2, but growth was slower. Acetate, methanol, and trimethylamine were not catabolized. Cells grew with acetate as the only organic compounds in the culture medium, but growth was much faster in medium also supplemented with peptones and yeast extract. The cells were slightly halophilic; good growth occurred in medium supplemented with 350 to 600 mM Na+, but no growth occurred with 100 or 850 mM Na+. The pH range for growth was 6.5 to 7.9; no growth occurred at pH 6.0 or 8.5. Growth was slow (maximum specific growth rate, 0.24 day-1; doubling time, 2.9 days). This is the first report of a psychrophilic methanogen growing by CO2 reduction.

Electron microscopy of liquid crystalline DNA: direct evidence for cholesteric-like organization of DNA in dinoflagellate chromosomes

Freeze-fracture-etch replicas of concentrated DNA solutions which appeared, by polarized light microscopy, to be in a cholesteric-like liquid crystalline state were examined by high resolution transmission electron microscopy (TEM). Individual DNA molecules were resolvable, and the microscopic morphologies observed for such replicas confirmed the cholesteric organization of DNA molecules in this liquid crystalline state. Furthermore, replica morphologies were strikingly similar to TEM images of dinoflagellate chromosomes in both thin section and freeze-etch replicas, providing strong support for the cholesteric DNA packing model proposed for the organization of DNA in these chromosomes by Bouligand and Livolant.

Biochemical and Physical Properties of the Methanococcus jannaschii 20S Proteasome and PAN, a Homolog of the ATPase (Rpt) Subunits of the Eucaryal 26S Proteasome

The 20S proteasome is a self-compartmentalized protease which degrades unfolded polypeptides and has been purified from eucaryotes, gram-positive actinomycetes, and archaea. Energy-dependent complexes, such as the 19S cap of the eucaryal 26S proteasome, are assumed to be responsible for the recognition and/or unfolding of substrate proteins which are then translocated into the central chamber of the 20S proteasome and hydrolyzed to polypeptide products of 3 to 30 residues. All archaeal genomes which have been sequenced are predicted to encode proteins with up to approximately 50% identity to the six ATPase subunits of the 19S cap. In this study, one of these archaeal homologs which has been named PAN for proteasome-activating nucleotidase was characterized from the hyperthermophile Methanococcus jannaschii. In addition, the M. jannaschii 20S proteasome was purified as a 700-kDa complex by in vitro assembly of the alpha and beta subunits and has an unusually high rate of peptide and unfolded-polypeptide hydrolysis at 100 degrees C. The 550-kDa PAN complex was required for CTP- or ATP-dependent degradation of beta-casein by archaeal 20S proteasomes. A 500-kDa complex of PAN(Delta1-73), which has a deletion of residues 1 to 73 of the deduced protein and disrupts the predicted N-terminal coiled-coil, also facilitated this energy-dependent proteolysis. However, this deletion increased the types of nucleotides hydrolyzed to include not only ATP and CTP but also ITP, GTP, TTP, and UTP. The temperature optimum for nucleotide (ATP) hydrolysis was reduced from 80 degrees C for the full-length protein to 65 degrees C for PAN(Delta1-73). Both PAN protein complexes were stable in the absence of ATP and were inhibited by N-ethylmaleimide and p-chloromercuriphenyl-sulfonic acid. Kinetic analysis reveals that the PAN protein has a relatively high V(max) for ATP and CTP hydrolysis of 3.5 and 5.8 micromol of P(i) per min per mg of protein as well as a relatively low affinity for CTP and ATP with K(m) values of 307 and 497 microM compared to other proteins of the AAA family. Based on electron micrographs, PAN and PAN(Delta1-73) apparently associate with the ends of the 20S proteasome cylinder. These results suggest that the M. jannaschii as well as related archaeal 20S proteasomes require a nucleotidase complex such as PAN to mediate the energy-dependent hydrolysis of folded-substrate proteins and that the N-terminal 73 amino acid residues of PAN are not absolutely required for this reaction.

Structure of the aesthetasc (olfactory) sensilla of the blue crab, Callinectes sapidus: transformations as a function of salinity

Abstract.The aesthetasc sensilla of the euryhaline blue crab, Callinectes sapidus, are innervated by the dendrites of from 40 to 160 bipolar chemosensory neurons. Each dendrite forms two cilia within the basal portion of the sensillum, and these subsequently branch yielding approximately 10 outer dendritic segments per neuron. Auxiliary cells surround the inner dendritic segments and also ensheathe the outer dendritic segments up to the terminus of the ”constricted region” (a zone in which there is a slight narrowing of the aesthetasc). Crystal violet staining suggesting access of odor stimuli is limited to that portion of the sensillum distal to the constricted region. In freshwater-acclimated blue crabs the length and level of branching in the dendrites extending beyond the constricted region is significantly reduced relative to that of seawater-acclimated animals (mean lengths: 150 µm versus 517 µm, respectively). After transfer of freshwater-acclimated crabs to seawater there is a rapid increase in length of the outer dendritic segments, reaching 60% of that for seawater-acclimated crabs by 48 h. A similar time course for regrowth is seen for seawater-acclimated crabs in which the outer dendritic segments have been osmotically ablated. Conversely, with rapid transfer of seawater-acclimated animals to lower salinities, there is a correspondingly rapid reduction in length of the outer dendritic segments. The reduced length of the outer dendritic segments in freshwater-acclimated animals may reflect the effective distance over which an appropriate osmotic/ionic microenvironment for neural function can be maintained within the aesthetasc.

Characterization of a Phosphoinositide-mediated Odor Transduction Pathway Reveals Plasma Membrane Localization of an Inositol 1,4,5-Trisphosphate Receptor in Lobster Olfactory Receptor Neurons

The role of phosphoinositide signaling in olfactory transduction is still being resolved. Compelling functional evidence for the transduction of odor signals via phosphoinositide pathways in olfactory transduction comes from invertebrate olfactory systems, in particular lobster olfactory receptor neurons. We now provide molecular evidence for two components of the phosphoinositide signaling pathway in lobster olfactory receptor neurons, a G protein α subunit of the Gq family and an inositol 1,4,5-trisphosphate-gated channel or an inositol 1,4,5-trisphosphate (IP3) receptor. Both proteins localize to the site of olfactory transduction, the outer dendrite of the olfactory receptor neurons. Furthermore, the IP3 receptor localizes to membranes in the ciliary transduction compartment of these cells at both the light microscopic and electron microscopic levels. Given the absence of intracellular organelles in the sub-micron diameter olfactory cilia, this finding indicates that the IP3receptor is associated with the plasma membrane and provides the first definitive evidence for plasma membrane localization of an IP3R in neurons. The association of the IP3receptor with the plasma membrane may be a novel mechanism for regulating intracellular cations in restricted cellular compartments of neurons.