Clostridium chromiireducens sp. nov., isolated from Cr(VI)-contaminated soil.

A Cr(VI)-resistant, Gram-positive, spore-forming, obligate anaerobe, designated GCAF-1(T), was isolated from chromium-contaminated soil by its ability to reduce Cr(VI) in low concentrations. Mixed acid fermentation during growth on glucose resulted in accumulation of acetate, butyrate, formate and lactate. Morphological studies indicated the presence of peritrichous flagella, pili and an S-layer. The major cellular fatty acids (>5 %) were C(16 : 0), C(14 : 0), summed feature 3 (comprising iso-C(15 : 0) 2-OH and/or C(16 : 1)ω7c), C(18 : 1)ω7c, C(16 : 1)ω9c, summed feature 4 (comprising iso-C(17 : 1) I and/or anteiso-C(17 : 1) B) and C(18 : 1)ω9c. The DNA G+C content of strain GCAF-1(T) was 30.7 mol%. Phylogenetic interference indicated that strain GCAF-1(T) clustered with group I of the genus Clostridium. Of strains within this cluster, strain GCAF-1(T) shared the highest 16S rRNA gene sequence similarities (98.1-98.9 %) with Clostridium beijerinckii DSM 791(T), C. saccharobutylicum NCP 262(T), C. saccharoperbutylacetonicum N1-4(T), C. puniceum DSM 2619(T) and C. roseum DSM 51(T). However, strain GCAF-1(T) could be clearly distinguished from its closest phylogenetic neighbours by low levels of DNA-DNA relatedness (<50 %) and some phenotypic features. Based on the evidence presented here, strain GCAF-1(T) ( = DSM 23318(T) = KCTC 5935(T)) represents a novel species of the genus Clostridium, for which the name Clostridium chromiireducens sp. nov. is proposed.

Detection of Pasteuria penetrans infection in Meloidogyne arenaria race 1 in planta by polymerase chain reaction.

We report on the development of a PCR-based assay to detect Pasteuria penetrans infection of Meloidogyne arenaria in planta using specific primers for recently sequenced sigE, spoIIAB and atpF genes of P. penetrans biotype P20. Amplification of these genes in crude DNA extracts of ground tomato root galls using real-time kinetic PCR distinguished infected from uninfected M. arenaria race 1 by analysis of consensus thresholds for single copy genes. Fluorescent in situ hybridization (FISH) using the sigE primer sequence as a probe shows hybridization to P. penetrans cells in various stages of vegetative (pre-endospore) development. Ratios of gene copies for sigE and 16S rDNA were obtained for P. penetrans and compared to Bacillus subtilis as a genomic paradigm of endospore-forming bacteria. Phylogenetic analysis of the sigE gene from Gram-positive, endospore-forming bacteria finds P. penetrans most closely related Paenbacillus polymyxa. The sporulation genes (spo genes), particularly sigE, have sequence diversity that recommends them for species and biotype differentiation of the numerous Pasteuria isolates that infect a large number of plant-parasitic nematodes.

Cloning, characterization, and functional expression of the Klebsiella oxytoca xylodextrin utilization operon (xynTB) in Escherichia coli.

Escherichia coli is being developed as a biocatalyst for bulk chemical production from inexpensive carbohydrates derived from lignocellulose. Potential substrates include the soluble xylodextrins (xyloside, xylooligosaccharide) and xylobiose that are produced by treatments designed to expose cellulose for subsequent enzymatic hydrolysis. Adjacent genes encoding xylobiose uptake and hydrolysis were cloned from Klebsiella oxytoca M5A1 and are functionally expressed in ethanologenic E. coli. The xylosidase encoded by xynB contains the COG3507 domain characteristic of glycosyl hydrolase family 43. The xynT gene encodes a membrane protein containing the MelB domain (COG2211) found in Na(+)/melibiose symporters and related proteins. These two genes form a bicistronic operon that appears to be regulated by xylose (XylR) and by catabolite repression in both K. oxytoca and recombinant E. coli. Homologs of this operon were found in Klebsiella pneumoniae, Lactobacillus lactis, E. coli, Clostridium acetobutylicum, and Bacillus subtilis based on sequence comparisons. Based on similarities in protein sequence, the xynTB genes in K. oxytoca appear to have originated from a gram-positive ancestor related to L. lactis. Functional expression of xynB allowed ethanologenic E. coli to metabolize xylodextrins (xylosides) containing up to six xylose residues without the addition of enzyme supplements. 4-O-methylglucuronic acid substitutions at the nonreducing termini of soluble xylodextrins blocked further degradation by the XynB xylosidase. The rate of xylodextrin utilization by recombinant E. coli was increased when a full-length xynT gene was included with xynB, consistent with xynT functioning as a symport. Hydrolysis rates were inversely related to xylodextrin chain length, with xylobiose as the preferred substrate. Xylodextrins were utilized more rapidly by recombinant E. coli than K. oxytoca M5A1 (the source of xynT and xynB). XynB exhibited weak arabinosidase activity, 3% that of xylosidase.

Temporal Formation and Immunolocalization of an Endospore Surface Epitope During Pasteuria penetrans Sporogenesis.

The synthesis and localization of an endospore surface epitope associated with the development of Pasteuria penetrans was determined using a monoclonal antibody (MAb) as a probe. Nematodes, uninfected or infected with P. penetrans, were harvested at 12, 16, 24, and 38 days after inoculation (DAI) and then examined to determine the developmental stage of the bacterium. Vegetative growth of P. penetrans was observed only in infected nematodes harvested at 12 and 16 DAI, whereas cells at different stages of sporulation and mature endospores were observed at 24 and 38 DAI. ELISA and immunoblot analysis revealed that the adhesin-associated epitope was first detected at 24 DAI, and increased in the later stages of sporogenesis. These results indicate that the synthesis of adhesin-related proteins occurred at a certain developmental stage relative to the sporulation process, and was associated with endospore maturation. Immunofluorescence microscopy indicated that the distribution of the epitope is nearly uniform on the periphery of each spore, as defined by parasporal fibers. Immunocytochemistry at the ultrastructural level indicated a distribution of the epitope over the parasporal fibers. The epitope also was detected over other structures such as sporangium and exosporium during the sporogenesis process, but it was not observed over the cortex, inner-spore coat, outer-spore coat, or protoplasm. The appearance of the adhesin epitope first at stage III of sporogenesis and its presence on the parasporal fibers are consistent with an adhesin-related role in the attachment of the mature endospore to the cuticle of the nematode host.

Sustaining olfaction at low salinities: evidence for a paracellular route of ion movement from the hemolymph to the sensillar lymph in the olfactory sensilla of the blue crab Callinectes sapidus.

Evidence reported previously suggests that in low-salinity conditions the integrity of the olfactory dendrites of the blue crab is sustained by a diffusion-generated ionic microenvironment within the aesthetascs. Diffusion of ions from the hemolymph to the sensillar lymph is proposed to maintain this microenvironment. In this study, using lanthanum as an electron-dense marker of extracellular fluid space, we find morphological evidence for paracellular continuity between the hemolymph and the sensillar lymph. Lanthanum penetrates extracellular fluid spaces within the aesthetascs when antennules are either perfused or bathed externally with solutions containing lanthanum nitrate. This was found in both freshwater- and seawater-acclimated animals. Evidence for ion diffusion from the aesthetascs was obtained using self-referencing, ion-selective microelectrodes. Both Ca2+ and K+ exhibit outwardly directed flux gradients associated with the aesthetasc tuft in low-salinity conditions. These findings are consistent with the concept that ion diffusion from the hemolymph to the sensillar lymph generates an ionic/osmotic microenvironment within the aesthetascs at low salinities.

Identification and localization of the carboxysome peptide Csos3 and its corresponding gene in Thiobacillus neapolitanus.

Four genes encoding carboxysome shell peptides (csoS1A, csoS1B, csoS1C, csoS2), the genes encoding the large and small subunits of RuBisCO (cbbL, cbbS), and three unidentified ORFs constitute an operon in Thiobacillus neapolitanus. An unidentified ORF 1.54 kb in size is predicted from sequence analysis to encode a protein with a molecular mass of approximately 57 kDa. When this ORF was expressed in Escherichia coli under the control of its endogenous ribosome-binding site, no peptide product was observed. In order to correlate this ORF with a carboxysome peptide, the ORF was overexpressed in E. coli by cloning it into pProExHTb, a prokaryotic expression vector containing an E. coli ribosome binding site. When antibodies raised against the recombinant protein were used to probe an immunoblot containing carboxysome peptides, a 60-kDa peptide was recognized. The peptide was subsequently named CsoS3. CsoS3 is a minor component of the carboxysome; a peptide of this size is commonly not observed or is very faint on Coomassie blue-stained SDS-polyacrylamide gels of purified carboxysomes. Immunogold labeling established CsoS3 to be a component of the carboxysome shell.

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 alpha subunit of the G(q) family and an inositol 1,4, 5-trisphosphate-gated channel or an inositol 1,4,5-trisphosphate (IP(3)) receptor. Both proteins localize to the site of olfactory transduction, the outer dendrite of the olfactory receptor neurons. Furthermore, the IP(3) 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 IP(3) receptor is associated with the plasma membrane and provides the first definitive evidence for plasma membrane localization of an IP(3)R in neurons. The association of the IP(3) receptor with the plasma membrane may be a novel mechanism for regulating intracellular cations in restricted cellular compartments of neurons.

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.

Enteric bacterial catalysts for fuel ethanol production.

The technology is available to produce fuel ethanol from renewable lignocellulosic biomass. The current challenge is to assemble the various process options into a commercial venture and begin the task of incremental improvement. Current process designs for lignocellulose are far more complex than grain to ethanol processes. This complexity results in part from the complexity of the substrate and the biological limitations of the catalyst. Our work at the University of Florida has focused primarily on the genetic engineering of Enteric bacteria using genes encoding Zymomonas mobilis pyruvate decarboxylase and alcohol dehydrogenase. These two genes have been assembled into a portable ethanol production cassette, the PET operon, and integrated into the chromosome of Escherichia coli B for use with hemicellulose-derived syrups. The resulting strain, KO11, produces ethanol efficiently from all hexose and pentose sugars present in the polymers of hemicellulose. By using the same approach, we integrated the PET operon into the chromosome of Klebsiella oxytoca to produce strain P2 for use in the simultaneous saccharification and fermentation (SSF) process for cellulose. Strain P2 has the native ability to ferment cellobiose and cellotriose, eliminating the need for one class of cellulase enzymes. Recently, the ability to produce and secrete high levels of endoglucanase has also been added to strain P2, further reducing the requirement for fungal cellulase. The general approach for the genetic engineering of new biocatalysts using the PET operon has been most successful with Enteric bacteria but was also extended to Gram positive bacteria, which have other useful traits for lignocellulose conversion. Many opportunities remain for further improvements in these biocatalysts as we proceed toward the development of single organisms that can be used for the efficient fermentation of both hemicellulosic and cellulosic substrates.

Halophilic 20S proteasomes of the archaeon Haloferax volcanii: purification, characterization, and gene sequence analysis.

A 20S proteasome, composed of alpha(1) and beta subunits arranged in a barrel-shaped structure of four stacked rings, was purified from a halophilic archaeon Haloferax volcanii. The predominant peptide-hydrolyzing activity of the 600-kDa alpha(1)beta-proteasome on synthetic substrates was cleavage carboxyl to hydrophobic residues (chymotrypsin-like [CL] activity) and was optimal at 2 M NaCl, pH 7.7 to 9.5, and 75 degrees C. The alpha(1)beta-proteasome also hydrolyzed insulin B-chain protein. Removal of NaCl inactivated the CL activity of the alpha(1)beta-proteasome and dissociated the complex into monomers. Rapid equilibration of the monomers into buffer containing 2 M NaCl facilitated their reassociation into fully active alpha(1)beta-proteasomes of 600 kDa. However, long-term incubation of the halophilic proteasome in the absence of salt resulted in hydrolysis and irreversible inactivation of the enzyme. Thus, the isolated proteasome has unusual salt requirements which distinguish it from any proteasome which has been described. Comparison of the beta-subunit protein sequence with the sequence deduced from the gene revealed that a 49-residue propeptide is removed to expose a highly conserved N-terminal threonine which is proposed to serve as the catalytic nucleophile and primary proton acceptor during peptide bond hydrolysis. Consistent with this mechanism, the known proteasome inhibitors carbobenzoxyl-leucinyl-leucinyl-leucinal-H (MG132) and N-acetyl-leucinyl-leucinyl-norleucinal (calpain inhibitor I) were found to inhibit the CL activity of the H. volcanii proteasome (K(i) = 0.2 and 8 microM, respectively). In addition to the genes encoding the alpha(1) and beta subunits, a gene encoding a second alpha-type proteasome protein (alpha(2)) was identified. All three genes coding for the proteasome subunits were mapped in the chromosome and found to be unlinked. Modification of the methods used to purify the alpha(1)beta-proteasome resulted in the copurification of the alpha(2) protein with the alpha(1) and beta subunits in nonstoichometric ratios as cylindrical particles of four stacked rings of 600 kDa with CL activity rates similar to the alpha(1)beta-proteasome, suggesting that at least two separate 20S proteasomes are synthesized. This study is the first description of a prokaryote which produces two separate 20S proteasomes and suggests that there may be distinct physiological roles for the two different alpha subunits in this halophilic archaeon.

The propanediol utilization (pdu) operon of Salmonella enterica serovar Typhimurium LT2 includes genes necessary for formation of polyhedral organelles involved in coenzyme B(12)-dependent 1, 2-propanediol degradation.

The propanediol utilization (pdu) operon of Salmonella enterica serovar Typhimurium LT2 contains genes needed for the coenzyme B(12)-dependent catabolism of 1,2-propanediol. Here the completed DNA sequence of the pdu operon is presented. Analyses of previously unpublished pdu DNA sequence substantiated previous studies indicating that the pdu operon was acquired by horizontal gene transfer and allowed the identification of 16 hypothetical genes. This brings the total number of genes in the pdu operon to 21 and the total number of genes at the pdu locus to 23. Of these, six encode proteins of unknown function and are not closely related to sequences of known function found in GenBank. Two encode proteins involved in transport and regulation. Six probably encode enzymes needed for the pathway of 1,2-propanediol degradation. Two encode proteins related to those used for the reactivation of adenosylcobalamin (AdoCbl)-dependent diol dehydratase. Five encode proteins related to those involved in the formation of polyhedral organelles known as carboxysomes, and two encode proteins that appear distantly related to those involved in carboxysome formation. In addition, it is shown that S. enterica forms polyhedral bodies that are involved in the degradation of 1,2-propanediol. Polyhedra are formed during either aerobic or anaerobic growth on propanediol, but not during growth on other carbon sources. Genetic tests demonstrate that genes of the pdu operon are required for polyhedral body formation, and immunoelectron microscopy shows that AdoCbl-dependent diol dehydratase is associated with these polyhedra. This is the first evidence for a B(12)-dependent enzyme associated with a polyhedral body. It is proposed that the polyhedra consist of AdoCbl-dependent diol dehydratase (and perhaps other proteins) encased within a protein shell that is related to the shell of carboxysomes. The specific function of these unusual polyhedral bodies was not determined, but some possibilities are discussed.

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.

Enhancement of expression and apparent secretion of Erwinia chrysanthemi endoglucanase (encoded by celZ) in Escherichia coli B.

Escherichia coli B has been engineered as a biocatalyst for the conversion of lignocellulose into ethanol. Previous research has demonstrated that derivatives of E. coli B can produce high levels of Erwinia chrysanthemi endoglucanase (encoded by celZ) as a periplasmic product and that this enzyme can function with commercial fungal cellulase to increase ethanol production. In this study, we have demonstrated two methods that improve celZ expression in E. coli B. Initially, with a low-copy-number vector, two E. coli glycolytic gene promoters (gap and eno) were tested and found to be less effective than the original celZ promoter. By screening 18,000 random fragments of Zymomonas mobilis DNA, a surrogate promoter was identified which increased celZ expression up to sixfold. With this promoter, large polar inclusion bodies were clearly evident in the periplasmic space. Sequencing revealed that the most active surrogate promoter is derived from five Sau3A1 fragments, one of which was previously sequenced in Z. mobilis. Visual inspection indicated that this DNA fragment contains at least five putative promoter regions, two of which were confirmed by primer extension analysis. Addition of the out genes from E. chrysanthemi EC16 caused a further increase in the production of active enzyme and facilitated secretion or release of over half of the activity into the extracellular environment. With the most active construct, of a total of 13,000 IU of active enzyme per liter of culture, 7,800 IU was in the supernatant. The total active endoglucanase was estimated to represent 4 to 6% of cellular protein.

Insertion mutation of the form I cbbL gene encoding ribulose bisphosphate carboxylase/oxygenase (RuBisCO) in Thiobacillus neapolitanus results in expression of form II RuBisCO, loss of carboxysomes, and an increased CO2 requirement for growth.

It has been previously established that Thiobacillus neapolitanus fixes CO2 by using a form I ribulose bisphosphate carboxylase/oxygenase (RuBisCO), that much of the enzyme is sequestered into carboxysomes, and that the genes for the enzyme, cbbL and cbbS, are part of a putative carboxysome operon. In the present study, cbbL and cbbS were cloned and sequenced. Analysis of RNA showed that cbbL and cbbS are cotranscribed on a message approximately 2,000 nucleotides in size. The insertion of a kanamycin resistance cartridge into cbbL resulted in a premature termination of transcription; a polar mutant was generated. The mutant is able to fix CO2, but requires a CO2 supplement for growth. Separation of cellular proteins from both the wild type and the mutant on sucrose gradients and subsequent analysis of the RuBisCO activity in the collected fractions showed that the mutant assimilates CO2 by using a form II RuBisCO. This was confirmed by immunoblot analysis using antibodies raised against form I and form II RuBisCOs. The mutant does not possess carboxysomes. Smaller, empty inclusions are present, but biochemical analysis indicates that if they are carboxysome related, they are not functional, i.e., do not contain RuBisCO. Northern analysis showed that some of the shell components of the carboxysome are produced, which may explain the presence of these inclusions in the mutant.

Biochemical characterization of the 20S proteasome from the methanoarchaeon Methanosarcina thermophila.

The 20S proteasome from the methanoarchaeon Methanosarcina thermophila was produced in Escherichia coli and characterized. The biochemical properties revealed novel features of the archaeal 20S proteasome. A fully active 20S proteasome could be assembled in vitro with purified native alpha ring structures and beta prosubunits independently produced in Escherichia coli, which demonstrated that accessory proteins are not essential for processing of the beta prosubunits or assembly of the 20S proteasome. A protein complex with a molecular mass intermediate to those of the alpha7 ring and the 20S proteasome was detected, suggesting that the 20S proteasome is assembled from precursor complexes. The heterologously produced M. thermophila 20S proteasome predominately catalyzed cleavage of peptide bonds carboxyl to the acidic residue Glu (postglutamyl activity) and the hydrophobic residues Phe and Tyr (chymotrypsinlike activity) in short chromogenic and fluorogenic peptides. Low-level hydrolyzing activities were also detected carboxyl to the acidic residue Asp and the basic residue Arg (trypsinlike activity). Sodium dodecyl sulfate and divalent or monovalent ions stimulated chymotrypsinlike activity and inhibited postglutamyl activity, whereas ATP stimulated postglutamyl activity but had little effect on the chymotrypsinlike activity. The results suggest that the 20S proteasome is a flexible protein which adjusts to binding of substrates. The 20S proteasome also hydrolyzed large proteins. Replacement of the nucleophilic Thr1 residue with an Ala in the beta subunit abolished all activities, which suggests that only one active site is responsible for the multisubstrate activity. Replacement of beta subunit active-site Lys33 with Arg reduced all activities, which further supports the existence of one catalytic site; however, this result also suggests a role for Lys33 in polarization of the Thr1 N, which serves to strip a proton from the active-site Thr1 Ogamma nucleophile. Replacement of Asp51 with Asn had no significant effect on trypsinlike activity, enhanced postglutamyl and trypsinlike activities, and only partially reduced lysozyme-hydrolyzing activity, which suggested that this residue is not essential for multisubstrate activity.

Production of a cell wall-specific monoclonal antibody to Fibrobacter succinogenes.

A monoclonal antibody (mAb) was raised against Fibrobacter succinogenes and produced after fusion as ascites in BALB/c mice. An ELISA was used to test for specificity and sensitivity of the mAb to detect F. succinogenes. The mAb BD1 was tested for sensitivity and cross-reactivity in detecting F. succinogenes with ELISA. The lower limits for F. succinogenes detection in pure and mixed culture-using mAb BD1 with ELISA was 10(5) cells ml-1. Twenty-six other species of bacteria, including 12 cellulolytic species, were tested for cross-reactivity with the ELISA but none was detected. Electron micrographs of F. succinogenes cells with immunogold labelling showed that the mAb BD1 reacted exclusively with cell wall epitopes but not intracellular material, as confirmed by ELISA.

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.

Biofouling of groundwater systems by Thiothrix spp.

Thiothrix spp., sulfide-oxidizing filamentous bacteria, were found to be a principal bacterial component of aquatic biofilms causing biofouling in selected municipal water storage tanks, private wells, and drip irrigation systems in Florida. Treatments of up to 200 ppm chlorine in the affected systems could not prevent return of the biofouling problem. The water originated from the upper Floridan aquifer and associated surficial aquifers in central and north Florida. Samples were examined where visible biofilms had a white, filamentous appearance, indicative of Thiothrix spp. The detection of Thiothrix spp. was confirmed by enzyme-liked immunosorbent assay (ELISA), indirect immunofluorescence (IIF), and microbiological procedures. It was estimated through immunocytochemical procedures that Thiothrix spp. comprised 18% of the biofilm in the municipal water storage tanks. These observations confirm that specific biological and chemical interactions may induce physical changes leading to significant biofouling.

Ultrasound stimulates ethanol production during the simultaneous saccharification and fermentation of mixed waste office paper.

The commercial production of ethanol from cellulose by simultaneous saccharification and fermentation (SSF) is prevented in part by the high cost of fungal cellulase enzymes. Intermittent exposure of SSF processes to ultrasonic energy under selected conditions (5 FPU of cellulase/g of substrate; 15 min of exposure/240 min cycle during the latter half of SSF) was found to increase ethanol production from mixed waste office paper by approximately 20%, producing 36.6 g/L ethanol after 96 h (70% of the maximum theoretical yield). Without ultrasound, 10 FPU of cellulase/g of substrate was required to achieve similar results. Continuous exposure of the organism to ultrasonic energy was bacteriostatic and decreased ethanol production but may be useful for the controlling bacterial growth in other processes.