Biofilm colonization and succession in a full-scale partial nitritation-anammox moving bed biofilm reactor.

BACKGROUND: Partial nitritation-anammox (PNA) is a biological nitrogen removal process commonly used in wastewater treatment plants for the treatment of warm and nitrogen-rich sludge liquor from anaerobic digestion, often referred to as sidestream wastewater. In these systems, biofilms are frequently used to retain biomass with aerobic ammonia-oxidizing bacteria (AOB) and anammox bacteria, which together convert ammonium to nitrogen gas. Little is known about how these biofilm communities develop, and whether knowledge about the assembly of biofilms in natural communities can be applied to PNA biofilms. RESULTS: We followed the start-up of a full-scale PNA moving bed biofilm reactor for 175 days using shotgun metagenomics. Environmental filtering likely restricted initial biofilm colonization, resulting in low phylogenetic diversity, with the initial microbial community comprised mainly of Proteobacteria. Facilitative priority effects allowed further biofilm colonization, with the growth of initial aerobic colonizers promoting the arrival and growth of anaerobic taxa like methanogens and anammox bacteria. Among the early colonizers were known 'oligotrophic' ammonia oxidizers including comammox Nitrospira and Nitrosomonas cluster 6a AOB. Increasing the nitrogen load in the bioreactor allowed colonization by 'copiotrophic' Nitrosomonas cluster 7 AOB and resulted in the exclusion of the initial ammonia- and nitrite oxidizers. CONCLUSIONS: We show that complex dynamic processes occur in PNA microbial communities before a stable bioreactor process is achieved. The results of this study not only contribute to our knowledge about biofilm assembly and PNA bioreactor start-up but could also help guide strategies for the successful implementation of PNA bioreactors. Video Abstract.

Inoculation with adapted bacterial communities promotes development of full scale slow sand filters for drinking water production.

Gravity-driven filtration through slow sand filters (SSFs) is one of the oldest methods for producing drinking water. As water passes through a sand bed, undesired microorganisms and chemicals are removed by interactions with SSF biofilm and its resident microbes. Despite their importance, the processes through which these microbial communities form are largely unknown, as are the factors affecting these processes. In this study, two SSFs constructed using different sand sources were compared to an established filter and observed throughout their maturation process. One SSF was inoculated through addition of sand scraped from established filters, while the other was not inoculated. The operational and developing microbial communities of SSFs, as well as their influents and effluents, were studied by sequencing of 16S ribosomal rRNA genes. A functional microbial community resembling that of the established SSF was achieved in the inoculated SSF, but not in the non-inoculated SSF. Notably, the non-inoculated SSF had significantly (p < 0.01) higher abundances of classes Armatimonadia, Elusimicrobia, Fimbriimonadia, OM190 (phylum Planctomycetota), Parcubacteria, Vampirivibrionia and Verrucomicrobiae. Conversely, it had lower abundances of classes Anaerolineae, Bacilli, bacteriap25 (phylum Myxococcota), Blastocatellia, Entotheonellia, Gemmatimonadetes, lineage 11b (phylum Elusimicrobiota), Nitrospiria, Phycisphaerae, subgroup 22 (phylum Acidobacteriota) and subgroup 11 (phylum Acidobacteriota). Poor performance of neutral models showed that the assembly and dispersal of SSF microbial communities was mainly driven by selection. The temporal turnover of microbial species, as estimated through the scaling exponent of the species-time relationship, was twice as high in the non-inoculated filter (0.946 ± 0.164) compared to the inoculated filter (0.422 ± 0.0431). This study shows that the addition of an inoculum changed the assembly processes within SSFs. Specifically, the rate at which new microorganisms were observed in the biofilm was reduced. The reduced temporal turnover may be driven by inoculating taxa inhibiting growth, potentially via secondary metabolite production. This in turn would allow the inoculation community to persist and contribute to SSF function.

Characterizing natural degradation of tetrachloroethene (PCE) using a multidisciplinary approach.

A site in mid-western Sweden contaminated with chlorinated solvents originating from a previous dry cleaning facility, was investigated using conventional groundwater analysis combined with compound-specific isotope data of carbon, microbial DNA analysis, and geoelectrical tomography techniques. We show the value of this multidisciplinary approach, as the different results supported each interpretation, and show where natural degradation occurs at the site. The zone where natural degradation occurred was identified in the transition between two geological units, where the change in hydraulic conductivity may have facilitated biofilm formation and microbial activity. This observation was confirmed by all methods and the examination of the impact of geological conditions on the biotransformation process was facilitated by the unique combination of the applied methods. There is thus significant benefit from deploying an extended array of methods for these investigations, with the potential to reduce costs involved in remediation of contaminated sediment and groundwater.

Vanillin Production in Pseudomonas: Whole-Genome Sequencing of Pseudomonas sp. Strain 9.1 and Reannotation of Pseudomonas putida CalA as a Vanillin Reductase.

Microbial degradation of lignin and its related aromatic compounds has great potential for the sustainable production of chemicals and bioremediation of contaminated soils. We previously isolated Pseudomonas sp. strain 9.1 from historical waste deposits (forming so-called fiber banks) released from pulp and paper mills along the Baltic Sea coast. The strain accumulated vanillyl alcohol during growth on vanillin, and while reported in other microbes, this phenotype is less common in wild-type pseudomonads. As the reduction of vanillin to vanillyl alcohol is an undesired trait in Pseudomonas strains engineered to accumulate vanillin, connecting the strain 9.1 phenotype with a genotype would increase the fundamental understanding and genetic engineering potential of microbial vanillin metabolism. The genome of Pseudomonas sp. 9.1 was sequenced and assembled. Annotation identified oxidoreductases with homology to Saccharomyces cerevisiae alcohol dehydrogenase ScADH6p, known to reduce vanillin to vanillyl alcohol, in both the 9.1 genome and the model strain Pseudomonas putida KT2440. Recombinant expression of the Pseudomonas sp. 9.1 FEZ21_09870 and P. putida KT2440 PP_2426 (calA) genes in Escherichia coli revealed that these open reading frames encode aldehyde reductases that convert vanillin to vanillyl alcohol, and that P. putida KT2440 PP_3839 encodes a coniferyl alcohol dehydrogenase that oxidizes coniferyl alcohol to coniferyl aldehyde (i.e., the function previously assigned to calA). The deletion of PP_2426 in P. putida GN442 engineered to accumulate vanillin resulted in a decrease in by-product (vanillyl alcohol) yield from 17% to ∼1%. Based on these results, we propose the reannotation of PP_2426 and FEZ21_09870 as areA and PP_3839 as calA-IIIMPORTANCE Valorization of lignocellulose (nonedible plant matter) is of key interest for the sustainable production of chemicals from renewable resources. Lignin, one of the main constituents of lignocellulose, is a heterogeneous aromatic biopolymer that can be chemically depolymerized into a heterogeneous mixture of aromatic building blocks; those can be further converted by certain microbes into value-added aromatic chemicals, e.g., the flavoring agent vanillin. We previously isolated a Pseudomonas sp. strain with the (for the genus) unusual trait of vanillyl alcohol production during growth on vanillin. Whole-genome sequencing of the isolate led to the identification of a vanillin reductase candidate gene whose deletion in a recombinant vanillin-accumulating P. putida strain almost completely alleviated the undesired vanillyl alcohol by-product yield. These results represent an important step toward biotechnological production of vanillin from lignin using bacterial cell factories.

Bacterial release from pipe biofilm in a full-scale drinking water distribution system.

Safe drinking water is delivered to the consumer through kilometres of pipes. These pipes are lined with biofilm, which is thought to affect water quality by releasing bacteria into the drinking water. This study describes the number of cells released from this biofilm, their cellular characteristics, and their identity as they shaped a drinking water microbiome. Installation of ultrafiltration (UF) at full scale in Varberg, Sweden reduced the total cell count to 1.5 × 103 ± 0.5 × 103 cells mL-1 in water leaving the treatment plant. This removed a limitation of both flow cytometry and 16S rRNA amplicon sequencing, which have difficulties in resolving small changes against a high background cell count. Following installation, 58% of the bacteria in the distributed water originated from the pipe biofilm, in contrast to before, when 99.5% of the cells originated from the treatment plant, showing that UF shifts the origin of the drinking water microbiome. The number of bacteria released from the biofilm into the distributed water was 2.1 × 103 ± 1.3 × 103 cells mL-1 and the percentage of HNA (high nucleic acid) content bacteria and intact cells increased as it moved through the distribution system. DESeq2 analysis of 16S rRNA amplicon reads showed increases in 29 operational taxonomic units (OTUs), including genera identified as Sphingomonas, Nitrospira, Mycobacterium, and Hyphomicrobium. This study demonstrated that, due to the installation of UF, the bacteria entering a drinking water microbiome from a pipe biofilm could be both quantitated and described.

Monitoring biofilm function in new and matured full-scale slow sand filters using flow cytometric histogram image comparison (CHIC).

While slow sand filters (SSFs) have produced drinking water for more than a hundred years, understanding of their associated microbial communities is limited. In this study, bacteria in influent and effluent water from full-scale SSFs were explored using flow cytometry (FCM) with cytometric histogram image comparison (CHIC) analysis; and routine microbial counts for heterotrophs, total coliforms and Escherichia coli. To assess if FCM can monitor biofilm function, SSFs differing in age and sand composition were compared. FCM profiles from two established filters were indistinguishable. To examine biofilm in the deep sand bed, SSFs were monitored during a scraping event, when the top layer of sand and the schmutzdecke are removed to restore flow through the filter. The performance of an established SSF was stable: total organic carbon (TOC), pH, numbers of heterotrophs, coliforms, E. coli, and FCM bacterial profile were unaffected by scraping. However, the performance of two newly-built SSFs containing new and mixed sand was compromised: breakthrough of both microbial indicators and TOC occurred following scraping. The compromised performance of the new SSFs was reflected in distinct effluent bacterial communities; and, the presence of microbial indicators correlated to influent bacterial communities. This demonstrated that FCM can monitor SSF performance. Removal of the top layer of sand did not alter the effluent water from the established SSF, but did affect that of the SSFs containing new sand. This suggests that the impact of the surface biofilm on effluent water is greater when the deep sand bed biofilm is not established.

Luminometric Label Array for Counting and Differentiation of Bacteria.

Methods for simple and fast detection and differentiation of bacterial species are required, for instance, in medicine, water quality monitoring, and the food industry. Here, we have developed a novel label array method for the counting and differentiation of bacterial species. This method is based on the nonspecific interactions of multiple unstable lanthanide chelates and selected chemicals within the sample leading to a luminescence signal profile that is unique to the bacterial species. It is simple, cost-effective, and/or user-friendly compared to many existing methods, such as plate counts on selective media, automatic (hemocytometer-based) cell counters, flow cytometry, and polymerase chain reaction (PCR)-based methods for identification. The performance of the method was demonstrated with nine single strains of bacteria in pure culture. The limit of detection for counting was below 1000 bacteria per mL, with an average coefficient of variation of 10% achieved with the developed label array. A predictive model was trained with the measured luminescence signals and its ability to differentiate all tested bacterial species from each other, including members of the same genus Bacillus licheniformis and Bacillus subtilis, was confirmed via leave-one-out cross-validation. The suitability of the method for analysis of mixtures of bacterial species was shown with ternary mixtures of Bacillus licheniformis, Escherichia coli JM109, and Lactobacillus reuteri ATCC PTA 4659. The potential future application of the method could be monitoring for contamination in pure cultures; analysis of mixed bacterial cultures, where examining one species in the presence of another could inform industrial microbial processes; and the analysis of bacterial biofilms, where nonspecific methods based on physical and chemical characteristics are required instead of methods specific to individual bacterial species.

Bacterial community analysis of drinking water biofilms in southern Sweden.

Next-generation sequencing of the V1-V2 and V3 variable regions of the 16S rRNA gene generated a total of 674,116 reads that described six distinct bacterial biofilm communities from both water meters and pipes. A high degree of reproducibility was demonstrated for the experimental and analytical work-flow by analyzing the communities present in parallel water meters, the rare occurrence of biological replicates within a working drinking water distribution system. The communities observed in water meters from households that did not complain about their drinking water were defined by sequences representing Proteobacteria (82-87%), with 22-40% of all sequences being classified as Sphingomonadaceae. However, a water meter biofilm community from a household with consumer reports of red water and flowing water containing elevated levels of iron and manganese had fewer sequences representing Proteobacteria (44%); only 0.6% of all sequences were classified as Sphingomonadaceae; and, in contrast to the other water meter communities, markedly more sequences represented Nitrospira and Pedomicrobium. The biofilm communities in pipes were distinct from those in water meters, and contained sequences that were identified as Mycobacterium, Nocardia, Desulfovibrio, and Sulfuricurvum. The approach employed in the present study resolved the bacterial diversity present in these biofilm communities as well as the differences that occurred in biofilms within a single distribution system, and suggests that next-generation sequencing of 16S rRNA amplicons can show changes in bacterial biofilm communities associated with different water qualities.

A GH57 4-α-glucanotransferase of hyperthermophilic origin with potential for alkyl glycoside production.

4-α-Glucanotransferase (GTase) enzymes (EC 2.4.1.25) modulate the size of α-glucans by cleaving and reforming α-1,4 glycosidic bonds in α-glucans, an essential process in starch and glycogen metabolism in plants and microorganisms. The glycoside hydrolase family 57 enzyme (GTase57) studied in the current work catalyzes both disproportionation and cyclization reactions. Amylose was converted into cyclic amylose (with a minimum size of 17 glucose monomers) as well as to a spectrum of maltodextrins, but in contrast to glycoside hydrolase family 13 cyclodextrin glucanotransferases (CGTases), no production of cyclodextrins (C6-C8) was observed. GTase57 also effectively produced alkyl-glycosides with long α-glucan chains from dodecyl-ß-D-maltoside and starch, demonstrating the potential of the enzyme to produce novel variants of surfactants. Importantly, the GTase57 has excellent thermostability with a maximal activity at 95 °C and an activity half-life of 150 min at 90 °C which is highly advantageous in this manufacturing process suggesting that enzymes from this relatively uncharacterized family, GH57, can be powerful biocatalysts for the production of large head group glucosides from soluble starch.

Characterization of the properties of Pediococcus parvulus for probiotic or protective culture use.

Pediococcus parvulus 2.6 (previously Pediococcus damnosus 2.6, here confirmed as P. parvulus by 16S DNA sequencing) displayed antibacterial activity toward several bacterial species, including isolates found as contaminants in oats, herein genetically identified as Bacillus cereus. No inhibition of Listeria monocytogenes was found under the conditions used. Antibacterial activity was retrieved after ammonium sulfate or acetone precipitation showed it to be peptide mediated. P. parvulus 2.6 has previously shown good technological properties in oat-based products. This, together with the currently found inhibition of food spoilage microorganisms like B. cereus, makes it suitable as a food protective culture. Survival trials of P. parvulus 2.6 at conditions mimicking the gastrointestinal tract were prompted by previously found cholesterol-lowering effects in humans after consumption of oat products cofermented by using P. parvulus 2.6 and Bifidobacterium spp. Viability was measured with in vitro, gutlike simulations at 37 degrees C. High survival was shown under two of three conditions (gastric juice, bile, and small intestine juice), defined as main obstacles of the gastrointestinal tract. The critical step was bile exposure. At a concentration of 20%, viability was low, but 0.3% bile (mean concentration in the intestine) did not have a major influence on growth. Viability of P. parvulus 2.6 was significantly decreased in gastric juice at pH 1.5 (with pepsin), but it was not significantly affected at pH 2.5, and was also improved at a lower pH in 20% oat milk. Viability was judged sufficient for colonization at gutlike conditions, qualifying the strain for further probiotic studies.

Independent evolution of neurotoxin and flagellar genetic loci in proteolytic Clostridium botulinum.

BACKGROUND: Proteolytic Clostridium botulinum is the causative agent of botulism, a severe neuroparalytic illness. Given the severity of botulism, surprisingly little is known of the population structure, biology, phylogeny or evolution of C. botulinum. The recent determination of the genome sequence of C. botulinum has allowed comparative genomic indexing using a DNA microarray. RESULTS: Whole genome microarray analysis revealed that 63% of the coding sequences (CDSs) present in reference strain ATCC 3502 were common to all 61 widely-representative strains of proteolytic C. botulinum and the closely related C. sporogenes tested. This indicates a relatively stable genome. There was, however, evidence for recombination and genetic exchange, in particular within the neurotoxin gene and cluster (including transfer of neurotoxin genes to C. sporogenes), and the flagellar glycosylation island (FGI). These two loci appear to have evolved independently from each other, and from the remainder of the genetic complement. A number of strains were atypical; for example, while 10 out of 14 strains that formed type A1 toxin gave almost identical profiles in whole genome, neurotoxin cluster and FGI analyses, the other four strains showed divergent properties. Furthermore, a new neurotoxin sub-type (A5) has been discovered in strains from heroin-associated wound botulism cases. For the first time, differences in glycosylation profiles of the flagella could be linked to differences in the gene content of the FGI. CONCLUSION: Proteolytic C. botulinum has a stable genome backbone containing specific regions of genetic heterogeneity. These include the neurotoxin gene cluster and the FGI, each having evolved independently of each other and the remainder of the genetic complement. Analysis of these genetic components provides a high degree of discrimination of strains of proteolytic C. botulinum, and is suitable for clinical and forensic investigations of botulism outbreaks.

Characterization of the cell surface glycolipid from Spirochaeta aurantia.

Spirochaeta aurantia is a free-living saprophytic spirochete that grows easily in simple laboratory media, and thus can be used as a model for the investigation of surface carbohydrate structures in spirochetae, which are normally not available in sufficient amounts. Freeze-substitution electron microscopy indicated the presence of a capsule-like material projecting from the surface of S. aurantia. Extraction of cells gave two major glycolipids, the one with a higher molecular mass glycolipid was designated large glycolipid A (LGLA). LGLA contained small amount of branched and unsaturated O-linked fatty acids, L: -rhamnose, L: -fucose, D: -xylose, D: -mannose, D: -glucosamine, D: -glycero-D: -gluco-heptose (DDglcHep), D: -glycero-D: -manno-heptose (DDHep), and a novel branched tetradeoxydecose monosaccharide, which we proposed to call aurantose (Aur). The carbohydrate structure of LGLA was extremely complex and consisted of the repeating units built of 11 monosaccharides, arrangement of nine of them was determined as: - [- 3 - beta - DDglcHep - 3 - beta - D - GlcNAc - 2 - beta - D - Man - ] - which wasdeduced from the NMR and chemical data on the LGLA and its fragments, obtained by various degradations. Tentative position of two remaining sugars is proposed. LGLA was negative for gelation of Limulus amebocyte lysate, did not contain lipid A, and was unable to activate any known Toll-like receptors.

Flagellar glycosylation in Clostridium botulinum.

Flagellins from Clostridium botulinum were shown to be post-translationally modified with novel glycan moieties by top-down MS analysis of purified flagellin protein from strains of various toxin serotypes. Detailed analyses of flagellin from two strains of C. botulinum demonstrated that the protein is modified by a novel glycan moiety of mass 417 Da in O-linkage. Bioinformatic analysis of available C. botulinum genomes identified a flagellar glycosylation island containing homologs of genes recently identified in Campylobacter coli that have been shown to be responsible for the biosynthesis of legionaminic acid derivatives. Structural characterization of the carbohydrate moiety was completed utilizing both MS and NMR spectroscopy, and it was shown to be a novel legionaminic acid derivative, 7-acetamido-5-(N-methyl-glutam-4-yl)-amino-3,5,7,9-tetradeoxy-D-glycero-alpha-D-galacto-nonulosonic acid, (alphaLeg5GluNMe7Ac). Electron transfer dissociation MS with and without collision-activated dissociation was utilized to map seven sites of O-linked glycosylation, eliminating the need for chemical derivatization of tryptic peptides prior to analysis. Marker ions for novel glycans, as well as a unique C-terminal flagellin peptide marker ion, were identified in a top-down analysis of the intact protein. These ions have the potential for use in for rapid detection and discrimination of C. botulinum cells, indicating botulinum neurotoxin contamination. This is the first report of glycosylation of Gram-positive flagellar proteins by the 'sialic acid-like' nonulosonate sugar, legionaminic acid.

A unique restriction site in the flaA gene allows rapid differentiation of group I and group II Clostridium botulinum strains by PCR-restriction fragment length polymorphism analysis.

Clostridium botulinum produces the potent botulinum neurotoxin, the causative agent of botulism. Based on distinctive physiological traits, strains of C. botulinum can be divided into four groups: however, only groups I and II are associated with human illness. Alignment of the flaA gene sequences from 40 group I and 40 group II strains identified a single BsrG1 restriction cut site that was present at base pair 283 in all group II flaA sequences and was not found in any group I sequence. The flaA gene was amplified by rapid colony PCR from 22 group I strains and 18 group II strains and digested with BsrGI restriction enzyme. Standard agarose gel electrophoresis with ethidium bromide staining showed two fragments, following restriction digestion of group II flaA gene amplicons with BsrGI, but only a single band of uncut flaA from group I strains. Combining rapid colony PCR with BsrGI restriction digest of the flaA gene at 60 degrees C is a significant improvement over current methods, such as meat digestion or amplified fragment length polymorphism, as a strain can be identified as either group I or group II in under 5 h when starting with a visible plated C. botulinum colony.

Genome sequence of a proteolytic (Group I) Clostridium botulinum strain Hall A and comparative analysis of the clostridial genomes.

Clostridium botulinum is a heterogeneous Gram-positive species that comprises four genetically and physiologically distinct groups of bacteria that share the ability to produce botulinum neurotoxin, the most poisonous toxin known to man, and the causative agent of botulism, a severe disease of humans and animals. We report here the complete genome sequence of a representative of Group I (proteolytic) C. botulinum (strain Hall A, ATCC 3502). The genome consists of a chromosome (3,886,916 bp) and a plasmid (16,344 bp), which carry 3650 and 19 predicted genes, respectively. Consistent with the proteolytic phenotype of this strain, the genome harbors a large number of genes encoding secreted proteases and enzymes involved in uptake and metabolism of amino acids. The genome also reveals a hitherto unknown ability of C. botulinum to degrade chitin. There is a significant lack of recently acquired DNA, indicating a stable genomic content, in strong contrast to the fluid genome of Clostridium difficile, which can form longer-term relationships with its host. Overall, the genome indicates that C. botulinum is adapted to a saprophytic lifestyle both in soil and aquatic environments. This pathogen relies on its toxin to rapidly kill a wide range of prey species, and to gain access to nutrient sources, it releases a large number of extracellular enzymes to soften and destroy rotting or decayed tissues.

Flagellin diversity in Clostridium botulinum groups I and II: a new strategy for strain identification.

Strains of Clostridium botulinum are traditionally identified by botulinum neurotoxin type; however, identification of an additional target for typing would improve differentiation. Isolation of flagellar filaments and analysis by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) showed that C. botulinum produced multiple flagellin proteins. Nano-liquid chromatography-tandem mass spectrometry (nLC-MS/MS) analysis of in-gel tryptic digests identified peptides in all flagellin bands that matched two homologous tandem flagellin genes identified in the C. botulinum Hall A genome. Designated flaA1 and flaA2, these open reading frames encode the major structural flagellins of C. botulinum. Colony PCR and sequencing of flaA1/A2 variable regions classified 80 environmental and clinical strains into group I or group II and clustered isolates into 12 flagellar types. Flagellar type was distinct from neurotoxin type, and epidemiologically related isolates clustered together. Sequencing a larger PCR product, obtained during amplification of flaA1/A2 from type E strain Bennett identified a second flagellin gene, flaB. LC-MS analysis confirmed that flaB encoded a large type E-specific flagellin protein, and the predicted molecular mass for FlaB matched that observed by SDS-PAGE. In contrast, the molecular mass of FlaA was 2 to 12 kDa larger than the mass predicted by the flaA1/A2 sequence of a given strain, suggesting that FlaA is posttranslationally modified. While identification of FlaB, and the observation by SDS-PAGE of different masses of the FlaA proteins, showed the flagellin proteins of C. botulinum to be diverse, the presence of the flaA1/A2 gene in all strains examined facilitates single locus sequence typing of C. botulinum using the flagellin variable region.

The structure and biological characteristics of the Spirochaeta aurantia outer membrane glycolipid LGLB.

In an attempt to isolate lipopolysaccharide from Spirochaeta aurantia, Darveau-Hancock extraction of the cell mass was performed. While no lipopolysaccharide was found, two carbohydrate-containing compounds were detected. They were resolved by size-exclusion chromatography into high molecular mass (LGLA) and low molecular mass (LGLB) fractions. Here we present the results of the analysis of the glycolipid LGLB. Deacylation of LGLB with hydrazine and separation of the products by using anion-exchange chromatography gave two major products. Their structure was determined by using chemical methods, NMR and mass spectrometry. All monosaccharides had the D-configuration, and aspartic acid had the L-configuration. Intact LGLB contained two fatty groups at O-2 and O-3 of the glycerol residue. Nonhydroxylated C14 to C18 fatty acids were identified, which were predominantly unsaturated or branched. LGLB was able to gel Limulus amebocyte lysate, albeit at a lower level than that observed for Escherichia coli O113 lipopolysaccharide. However, even large amounts of LGLB were unable to stimulate any Toll-like receptor (TLR) examined, including TLR4 and TLR2, previously shown to be sensitive to lipopolysaccharide and glycolipids from diverse bacterial origins, including other spirochetes.

Nature of the genome of the saprophytic spirochete Spirochaeta aurantia and its ribosomal RNA operons.

Using restriction endonucleases DraI, AseI, and I-CeuI in conjunction with pulsed-field gel electrophoresis, we have shown that Spirochaeta aurantia M1 possesses a circular 3.98-Mb genome. This is the second largest spirochete chromosome yet analyzed. The observation that the latter enzyme cuts in 3 places suggests the presence of 3 copies of the large subunit (23S) rRNA gene (rrl), which was confirmed by Southern hybridizations. The complete sequence of 2 of the ribosomal RNA operons was determined, revealing that their structure resembled that of the typical member of the bacterial superkingdom: rrs (16S; 1561 bp), tRNA, rrl (23S; 2972 bp), and rrf (5S; 110 bp). The S. aurantia rrs-rrl intergenic regions, as with Treponema denticola, contain genes specifying a 73-nt tRNA(Ala) (anticodon TGC) and a 77-nt tRNA(Ile) (anticodon GAT).