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At least 37 records · Page 2
All sourcesNASA NTRSDOE OSTI
DOE OSTI2024

Targeting Mycobacterium tuberculosis Persistence through Inhibition of the Trehalose Catalytic Shift

Tuberculosis (TB), caused by Mycobacterium tuberculosis (Mtb), is the leading cause of death worldwide by infectious disease. Treatment of Mtb infection requires a six-month course of multiple antibiotics, an extremely challenging regimen necessitated by Mtb’s ability to form drug-tolerant persister cells. Mtb persister formation is dependent on the trehalose catalytic shift, a stress-responsive metabolic remodeling mechanism in which the disaccharide trehalose is liberated from cell surface glycolipids and repurposed as an internal carbon source to meet energy and redox demands. Here, using a biofilm-persister model, metabolomics, and cryo-electron microscopy (EM), we found that azidodeoxy- and aminodeoxy-d-trehalose analogues block the Mtb trehalose catalytic shift through inhibition of trehalose synthase TreS (Rv0126), which catalyzes the isomerization of trehalose to maltose. Out of a focused eight-member compound panel constructed by chemoenzymatic synthesis, the natural product 2-trehalosamine exhibited the highest potency and significantly potentiated first- and second-line TB drugs in broth culture and macrophage infection assays. We also report the first structure of TreS bound to a substrate analogue inhibitor, obtained via cryo-EM, which revealed conformational changes likely essential for catalysis and inhibitor binding that can potentially be exploited for future therapeutic development. Our results demonstrate that inhibition of the trehalose catalytic shift is a viable strategy to target Mtb persisters and advance trehalose analogues as tools and potential adjunctive therapeutics for investigating and targeting mycobacterial persistence.

60 APPLIED LIFE SCIENCES↗
DOE OSTI2020

SPLIT: Stable Protein Coacervation Using a Light Induced Transition

Protein coacervates serve as hubs to concentrate and sequester proteins and nucleotides and thus function as membraneless organelles to manipulate cell physiology. We have engineered a coacervating protein to create tunable, synthetic membraneless organelles that assemble in response to a single pulse of light. Coacervation is driven by the intrinsically disordered RGG domain from the protein LAF-1, and opto-responsiveness is coded by the protein PhoCl, which cleaves in response to 405 nm light. We developed a fusion protein containing a solubilizing maltose-binding protein domain, PhoCl, and two copies of the RGG domain. Several seconds of illumination at 405 nm is sufficient to cleave PhoCl, removing the solubilization domain and enabling RGG-driven coacervation within minutes in cellularsized water-in-oil emulsions. An optimized version of this system displayed light-induced coacervation in Saccharomyces cerevisiae. Finally, the methods described here provide novel strategies for inducing protein phase separation using light.

32 ENERGY CONSERVATION, CONSUMPTION, AND UTILIZATI↗
DOE OSTI2025

Active Humidity Control Chamber for Desorption Electrospray Ionization-Mass Spectrometry Imaging Applications

Ambient ionization techniques enable mass spectrometry (MS) to expand into broader experimental contexts, although it is increasingly clear that results are influenced by the environmental conditions at the site of sampling. Desorption electrospray ionization (DESI), in particular, is affected by variations in relative humidity (RH) levels. Here we describe the design, development, and construction of an environmental control chassis that can actively modulate RH within ± 3% of user-defined set points across a broad humidity range (15%–70% RH). Preliminary characterization demonstrated differential analyte responses across a range of set points, with observed enhancement of leucine-enkephalin, sulfadimethoxine (negative mode), and maltose (positive mode) in response to increased humidity. The measurable differences in analyte signals across discrete humidity set points underscore the importance of environmental control in ambient ionization strategies. The humidity control system outlined here can be translated to other DESI platforms, with construction information provided herein.

DESI↗
DOE OSTI2023

Rational design of a genetically encoded NMR zinc sensor

Elucidating the biochemical roles of the essential metal ion, Zn 2+ , motivates detection strategies that are sensitive, selective, quantitative, and minimally invasive in living systems. Fluorescent probes have identified Zn 2+ in cells but complementary approaches employing nuclear magnetic resonance (NMR) are lacking. Recent studies of maltose binding protein (MBP) using ultrasensitive 129 Xe NMR spectroscopy identified a switchable salt bridge which causes slow xenon exchange and elicits strong hyperpolarized 129 Xe chemical exchange saturation transfer (hyper-CEST) NMR contrast. To engineer the first genetically encoded, NMR-active sensor for Zn 2+ , we converted the MBP salt bridge into a Zn 2+ binding site, while preserving the specific xenon binding cavity. The zinc sensor (ZS) at only 1 μM achieved ‘turn-on’ detection of Zn 2+ with pronounced hyper-CEST contrast. This made it possible to determine different Zn 2+ levels in a biological fluid via hyper-CEST. ZS was responsive to low-micromolar Zn 2+ , only modestly responsive to Cu 2+ , and nonresponsive to other biologically important metal ions, according to hyper-CEST NMR spectroscopy and isothermal titration calorimetry (ITC). Protein X-ray crystallography confirmed the identity of the bound Zn 2+ ion using anomalous scattering: Zn 2+ was coordinated with two histidine side chains and three water molecules. Penta-coordinate Zn 2+ forms a hydrogen-bond-mediated gate that controls the Xe exchange rate. Metal ion binding affinity, 129 Xe NMR chemical shift, and exchange rate are tunable parameters via protein engineering, which highlights the potential to develop proteins as selective metal ion sensors for NMR spectroscopy and imaging.

37 INORGANIC, ORGANIC, PHYSICAL, AND ANALYTICAL CH↗
DOE OSTI2021

Cryo-EM structure determination of small proteins by nanobody-binding scaffolds (Legobodies)

We describe a general method that allows structure determination of small proteins by single-particle cryo-electron microscopy (cryo-EM). The method is based on the availability of a target-binding nanobody, which is then rigidly attached to two scaffolds: 1) a Fab fragment of an antibody directed against the nanobody and 2) a nanobody-binding protein A fragment fused to maltose binding protein and Fab-binding domains. The overall ensemble of ~120 kDa, called Legobody, does not perturb the nanobody–target interaction, is easily recognizable in EM images due to its unique shape, and facilitates particle alignment in cryo-EM image processing. The utility of the method is demonstrated for the KDEL receptor, a 23-kDa membrane protein, resulting in a map at 3.2-Å overall resolution with density sufficient for de novo model building, and for the 22-kDa receptor-binding domain (RBD) of SARS-CoV-2 spike protein, resulting in a map at 3.6-Å resolution that allows analysis of the binding interface to the nanobody. The Legobody approach thus overcomes the current size limitations of cryo-EM analysis.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2021

Phosphorolytic degradation of leaf starch via plastidic α-glucan phosphorylase leads to optimized plant growth and water use efficiency over the diel phases of Crassulacean acid metabolism

In plants with Crassulacean acid metabolism (CAM), it has been proposed that the requirement for nocturnal provision of phosphoenolpyruvate as a substrate for CO 2 uptake has resulted in a re-routing of chloroplastic starch degradation from the amylolytic route to the phosphorolytic route. To test this hypothesis, we generated and characterized four independent RNAi lines of the obligate CAM species Kalanchoë fedtschenkoi with a >10-fold reduction in transcript abundance of plastidic α-glucan phosphorylase (PHS1). The rPHS1 lines showed diminished nocturnal starch degradation, reduced dark CO 2 uptake, a reduction in diel water use efficiency (WUE), and an overall reduction in growth. A re-routing of starch degradation via the hydrolytic/amylolytic pathway was indicated by hyperaccumulation of maltose in all rPHS1 lines. Further examination indicated that whilst operation of the core circadian clock was not compromised, plasticity in modulating net dark CO 2 uptake in response to changing photoperiods was curtailed. The data show that phosphorolytic starch degradation is critical for efficient operation of the CAM cycle and for optimizing WUE. This finding has clear relevance for ongoing efforts to engineer CAM into non-CAM species as a means of boosting crop WUE for a warmer, drier future.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2023

The catalytic domains of Streptococcus mutans glucosyltransferases: a structural analysis

Streptococcus mutans, found in the human oral cavity, is a significant contributor to the pathogenesis of dental caries. This bacterium expresses three genetically distinct types of glucosyltransferases named GtfB (GTF-I), GtfC (GTF-SI) and GtfD (GTF-S) that play critical roles in the development of dental plaque. The catalytic domains of GtfB, GtfC and GtfD contain conserved active-site residues for the overall enzymatic activity that relate to hydrolytic glycosidic cleavage of sucrose to glucose and fructose, release of fructose and generation of a glycosyl-enzyme intermediate in the reducing end. In a subsequent transglycosylation step, the glucosyl moiety is transferred to the nonreducing end of an acceptor to form a growing glucan polymer chain made up of glucose molecules. It has been proposed that both sucrose breakdown and glucan synthesis occur in the same active site of the catalytic domain, although the active site does not appear to be large enough to accommodate both functions. These three enzymes belong to glycoside hydrolase family 70 (GH70), which shows homology to glycoside hydrolase family 13 (GH13). GtfC synthesizes both soluble and insoluble glucans (α-1,3 and α-1,6 glycosidic linkages), while GtfB and GtfD synthesize only insoluble or soluble glucans, respectively. Here, crystal structures of the catalytic domains of GtfB and GtfD are reported. These structures are compared with previously determined structures of the catalytic domain of GtfC. With this work, apo structures and inhibitor-complex structures with acarbose are now available for the catalytic domains of GtfC and GtfB. The structure of GtfC with maltose allows further identification and comparison of active-site residues. A model of sucrose binding to GtfB is also included. The new structure of the catalytic domain of GtfD affords a structural comparison of the three S. mutans glycosyltransferases. Unfortunately, the catalytic domain of GtfD is not complete since crystallization resulted in the structure of a truncated protein lacking approximately 200 N-terminal residues of domain IV.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2024

Genetic and enzymatic characterization of Amy13E from Cellvibrio japonicus reclassifies it as a cyclodextrinase also capable of α-diglucoside degradation

ABSTRACT Cyclodextrinases are carbohydrate-active enzymes involved in the linearization of circular amylose oligosaccharides. Primarily thought to function as part of starch metabolism, there have been previous reports of bacterial cyclodextrinases also having additional enzymatic activities on linear malto-oligosaccharides. This substrate class also includes environmentally rare α-diglucosides such as kojibiose (α−1,2), nigerose (α−1,3), and isomaltose (α−1,6), all of which have valuable properties as prebiotics or low-glycemic index sweeteners. Previous genome sequencing of threeCellvibrio japonicusstrains adapted to utilize these α-diglucosides identified multiple, but uncharacterized, mutations in each strain. One of the mutations identified was in theamy13Egene, which was annotated to encode a neopullulanase. In this report, we functionally characterized this gene and determined that it in fact encodes a cyclodextrinase with additional activities on α-diglucosides. Deletion analysis ofamy13Efound that this gene was essential for kojibiose and isomaltose metabolism inC. japonicus. Interestingly, a Δamy13Emutant was not deficient for cyclodextrin or pullulan utilization inC. japonicus; however, heterologous expression of the gene inE. coliwas sufficient for cyclodextrin-dependent growth. Biochemical analyses found thatCjAmy13E cleaved multiple substrates but preferred cyclodextrins and maltose, but had no activity on pullulan. Our characterization of theCjAmy13E cyclodextrinase is useful for refining functional enzyme predictions in related bacteria and for engineering enzymes for biotechnology or biomedical applications. IMPORTANCE Understanding the bacterial metabolism of cyclodextrins and rare α-diglucosides is increasingly important, as these sugars are becoming prevalent in the foods, supplements, and medicines humans consume that subsequently feed the human gut microbiome. Our analysis of a cyclomaltodextrinase with an expanded substrate range is significant because it broadens the potential applications of the GH13 family of carbohydrate active enzymes (CAZymes) in biotechnology and biomedicine. Specifically, this study provides a workflow for the discovery and characterization of novel activities in bacteria that possess a high number of CAZymes that otherwise would be missed due to complications with functional redundancy. Furthermore, this study provides a model from which predictions can be made why certain bacteria in crowded niches are able to robustly utilize rare carbon sources, possibly to gain a competitive growth advantage.

Biotechnology & Applied Microbiology↗
DOE OSTI2020

Amylases in the Human Vagina

ABSTRACT Dominance of Lactobacillus species in vaginal communities is a hallmark of healthy conditions in the female genital tract. Key nutrients for lactobacilli include sugars produced when glycogen is degraded by α-amylase in the vagina. While α-amylase activity has been demonstrated in vaginal fluids, it is unclear whether α-amylases are produced solely by the host, bacteria in the vagina, or both. We screened cervicovaginal mucus from 23 reproductive-age women, characterized the species composition of vaginal communities, measured vaginal pH, and determined levels of amylase activity, glycogen, and lactic acid. Based on differences in these measured variables, one sample from each of four individual donors was selected for metagenomic and proteomic analyses. Of eight putative bacterial amylases identified in the assembled bacterial metagenomes, we detected four in vaginal fluids. These amylases were produced by various bacteria in different vaginal communities. Moreover, no two communities were the same in terms of which bacteria were producing amylases. Although we detected bacterial amylases in vaginal fluids, there was no clear association between the bacterial species that was dominant in a community and the level of amylase activity. This association was likely masked by the presence of human α-amylase, which was also detected in vaginal fluids. Finally, the levels of amylase activity and glycogen were only weakly associated. Our findings show, for the first time, that multiple amylases from both bacterial and human origins can be present simultaneously in the vagina. This work also suggests that the link between glycogen, amylase, and Lactobacillus in the vagina is complex. IMPORTANCE In this study, we show that multiple bacteria in the vaginal community produce amylases that hydrolyze glycogen into simpler sugars (i.e., maltose and maltotriose). These sugars serve as “common goods” that sustain bacterial populations in vaginal communities. Given the temporal changes that are observed in the human vaginal microbiome, we expect the kinds of bacterial amylases produced will also vary over time. These differences influence the pool of resources that are broadly shared and shape the species composition of the vaginal bacterial community.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2023

Data for Evaluation of Strategies to Narrow the Product Chain-Length Distribution of Microbially Synthesized Free Fatty Acids

The dominant strategy for tailoring the chain-length distribution of free fatty acids (FFA) synthesized by heterologous hosts is expression of a selective acyl-acyl carrier protein (ACP) thioesterase. However, few of these enzymes can generate a precise (greater than 90% of a desired chain-length) product distribution when expressed in a microbial or plant host. The presence of alternative chain-lengths can complicate purification in situations where blends of fatty acids are not desired. We report the assessment of several strategies for improving the dodecanoyl-ACP thioesterase from the California bay laurel to exhibit more selective production of medium-chain free fatty acids to near exclusivity. We demonstrated that matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-ToF MS) was an effective library screening technique for identification of thioesterase variants with favorable shifts in chain-length specificity. This strategy proved to be a more effective screening technique than several rational approaches discussed herein. With this data, we isolated four thioesterase variants which exhibited a more selective FFA distribution over wildtype when expressed in the fatty acid accumulating E. coli strain, RL08. We then combined mutations from the MALDI isolates to generate BTE-MMD19, a thioesterase variant capable of producing free fatty acids consisting of 90% of C12 products. Of the four mutations which conferred a specificity shift, we noted that three affected the shape of the binding pocket, while one occurred on the positively charged acyl carrier protein landing pad. Finally, we fused the maltose binding protein (MBP) from E. coli to the N – terminus of BTE-MMD19 to improve enzyme solubility and achieve a titer of 1.9 g per L of twelve-carbon fatty acids in a shake flask.

Conversion↗
DOE OSTI2022

Genome assembly PYCC 8100

The yeast Torulaspora delbrueckii is gaining importance for biotechnology due to its ability to increase wine sensorial complexity and for enhancing pre-frozen bread dough leavening. However, little is known about its population structure, and variation in gene content, or possible domestication routes have not been investigated. Here, we address these issues and update the circumscription of T. delbrueckii, which is composed of five major clades. Among the three European clades, a lineage associated with the wild arboreal niche is sister to the two other lineages that are linked with anthropic environments, one to wine fermentations and the other one to diverse sources including dairy products and bread dough (Mix- Anthropic clade). Using 62 genomes we identified 5629 genes in the pangenome of T. delbrueckii and 270 genes in the cloud genome. A pangenome tree analysis showed that wine strains have a genome composition were more similar to European wild arboreal strains than to those of the Mix Anthropic clade, in contradiction with the phylogenetic analysis. An association of gene content and ecology gave further support to the hypothesis that the Mix - Anthropic clade has the most specialized genome content and indicated that some of the exclusive genes were implicated in galactose and maltose utilization. More detailed analyses traced the acquisition of a cluster of GAL genes in strains associated with dairy products and the expansion and functional diversification of MAL genes in strains isolated from bread dough. Contrary to S. cerevisiae, domestication in T. delbrueckii is not primed by alcoholic fermentation and appears to be a recent event.

Sampaio, Jose P.↗
NASA NTRS1974

Reduction of blood serum cholesterol

By feeding a human subject as the sole source of sustenance a defined diet wherein the carbohydrate consists substantially entirely of glucose, maltose or a polysaccharide of glucose, the blood serum cholesterol level of the human subject is substantially reduced. If 25 percent of the carbohydrate is subsequently supplied in the form of sucrose, an immediate increase from the reduced level is observed. The remainder of the defined diet normally includes a source of amino acids, such as protein or a protein hydrolysate, vitamins, minerals and a source of essential fatty acid.

Winitz, M.↗
NASA NTRS2002

Spirochaeta americana sp. nov.: A New Haloalkaliphilic, Obligately Anaerobic Spirochete Isolated from Soda Mono Lake, California

A novel obligately anaerobic, mesophilic, haloalkaliphilic spirochete, strain ASpG1, was isolated from sediments of the alkaline, hypersaline Mono Lake in California, U.S.A. The gram-negative cells are motile and spirochete-shaped with sizes of 0.22 x 10-15 micron. Growth was observed over the temperature range of 10 C to 44 C (optimum 37 C), NaCl concentration range of greater than 1 - 12 % (wt/vol) (optimum 3%), and pH range 7.5 - 10.5 (optimum pH 9.5). The novel isolate is strictly alkaliphilic, requires high concentrations of carbonate in the medium, and is capable of utilizing D-glucose, fructose, maltose, sucrose, starch, and D-mannitol. Main end products of glucose fermentation are: H2, acetate, ethanol, and formate. Strain AspG1 is resistant to kanamycin, but sensitive to chloramphenicol, gentamycin and tetracycline. The G+C content of its DNA is 58.5 mol%. On the basis of its physiological and molecular properties, the isolate appears to be a novel species among the genus Spirochaeta; and the name Spirochaeta americana sp. nov., is proposed for the taxon (type strain ASpG1(sup T) = ATCC BAA_392(sup T) = DSMZ 14872(sup T)).

Hoover, Richard B.↗
NASA NTRS2002

Spirochaeta Americana Sp. Nov., A new Haloalkaliphilic, Obligately Anaerobic Spirochete Isolated from Soda Mono Lake in California

A novel obligately anaerobic, mesophilic, haloalkaliphilic spirochete, strain ASpG1(sup T), was isolated from sediments of the alkaline, hypersaline Mono Lake in California, U.S.A. The Gram-negative cells are motile and spirochete-shaped with sizes of 0.2 - 0.22 X 8-15 microns. Growth was observed over the following ranges: temperature 10 C to 44 C; optimum +37 C; NaCl concentration 2 - 12 % (w/v); optimum NaCl3 % and pH 8 - 10.5; optimum pH 9.5. The novel isolate is strictly alkaliphilic, requires high concentrations of carbonate in the medium, and is capable of utilizing D-glucose, fructose, maltose, sucrose, starch, and D-mannitol. The main end products of glucose fermentation are: H2, acetate, ethanol, and formate. Strain ASpG(sup T) is resistant to kanamycin, and rifampin, but sensitive to chloramphenicol, gentamycin and tetracycline. The G+C content of its DNA is 58.5 mol%, genome size is 2.98 x l0(exp 9) Daltons, Tm of the genomic DNA is 68 +/- 2 C, and DNA-DNA hybridization with the most closely related species, Spirocheta alkalica Strain Z-7491(sup T), exhibited 48.7% homology. On the basis of its physiological and molecular properties, the isolate appears to be a novel species of the genus Spirochaeta; and the name Spirochaeta americana sp. nov., is proposed for the taxon (type strain ASpG1(sup T) = ATCC BAA-392(sup T) = DSMZ 14872(sup T)).

Hoover, Richard B.↗
NASA NTRS2004

Enzymatic Properties of an Alkaline and Chelator Resistant alpha-amylase from the Alkaliphilic Bacillus sp. Isolate L1711

An alkaliphilic amylase producing bacterium, Bacillus sp. strain L 711, was selected among 13 soda lakes isolates. When grown at pH 10.5 and 37 C, strain L711 produced multiple forms of amylases in the culture broth. One of these, BAA, was purified from the culture supernatant by QAE column chromatography and preparative native gel electrophoresis. The molecular weight of BAA was determined to be 51 kDa by denaturing gel electrophoresis. The pH optima for activity below and above 40 C were 9.5 - 10.0 and 7.0 - 7.5 respectively. BAA was stable in the pH range 6-11 and was completely inactivated at 55 C. The thermostability was not increased in the presence of Ca(2+). The enzyme was strongly inhibited by Ca(2+), Zn(2+), Mg(2+), Mn(2+), Ba(2+) and Cu(2+), whereas the presence of Na(+), Co(2+) and EDTA (10 mM) enhanced enzymatic activity. The K(sub m), and specific activity of BAA on soluble starch were 1.9 mg/ml and 18.5 U/mg respectively. The main end products of hydrolysis were maltotetraose, maltose and glucose.

Bernhardsdotter, Eva C. M. J.↗
NASA NTRS2004

Enzymatic Properties of an Alkaline and Chelator Resistant Proportional to alpha-Amylase from the Alkaliphilic Bacillus sp. Isolate L1711

An alkaliphilic amylase producing bacterium, Bacillus sp. strain L1711, was selected among 13 soda lakes isolates. When grown at pH 10.5 and 370 C, strain L1711 produced multiple forms of amylases in the culture broth. One of these, BAA, was purified from the culture supernatant by QAE column chromatography and preparative native gel electrophoresis. The molecular weight of BAA was determined to be 51 kDa by denaturing gel electrophoresis. The pH optima for activity below and above 40 C were 9.5-10.0 and 7.0-7.5 respectively. BAA was stable in the pH range 6-11 and was completely inactivated at 55?C. The thermostability was not increased in the presence of Ca(2+). The enzyme was strongly inhibited by Ca(2+), Zn(2+), Mg(2+), Mn(2+), Ba(2+) and Cu(2+), whereas the presence of Na(+), Co2+ and EDTA (10 mM) enhanced enzymatic activity. The K(sub m) and specific activity of BAA on soluble starch were 1.9 mg/ml and 18.5 U/mg respectively. The main end products of hydrolysis were maltotetraose, maltose and glucose .

Bernhardsdotter, Eva C. M. J.↗
NASA NTRS2003

Spirochaeta Americana sp. Nov., A New Haloalkaliphilic, Obligately Anaerobic Spirochaete Isolated from Soda Mona Lake in California

A novel, obligately anaerobic, mesophilic, haloalkaliphilic spirochaete, strain ASpG1(sup T), was isolated from sediments of the alkaline, hypersaline Mono Lake in California, USA. Cells of the Gram-negative strain were motile and spirochaete-shaped with sizes of 0.2-0.22 x 8-18 microns. Growth of the strain was observed between 10 and 44 C (optimum 37 C), in 2-12% (w/v) NaCl (optimum 3 % NaCl) and between pH 8 and 10.5 (optimum pH 9.5). The novel strain was strictly alkaliphilic, required high concentrations of carbonates in the medium and was capable of utilizing D-glucose, fructose, maltose, sucrose, starch and D-mannitol. End products of glucose fermentation were H2, acetate, ethanol and formate. Strain ASpG1(sup T) was resistant to kanamycin and rifampicin, but sensitive to gentamicin, tetracycline and chloramphenicol. The G + C content of its DNA was 58.5 mol%. DNA-DNA hybridization analysis of strain ASpG1(sup T) with its most closely related species, Spirochaeta alkalica Z-7491(sup T) revealed a hybridization value of only 48.7 %. On the basis of its physiological and molecular properties, strain ASpG1(sup T) appears to represent a novel species of the genus Spirochaeta, for which the name Spirochaeta americana is proposed (type strain ASpG1(sup T) = ATCC 13AA-392(sup T) = DSM 14872(sup T)).

Hoover, Richard B.↗
NASA NTRS2003

Spirochaeta americana sp. nov., a new haloalkaliphilic, obligately anaerobic spirochaete isolated from soda Mono Lake in California

A novel, obligately anaerobic, mesophilic, haloalkaliphilic spirochaete, strain ASpG1(T), was isolated from sediments of the alkaline, hypersaline Mono Lake in California, USA. Cells of the Gram-negative strain were motile and spirochaete-shaped with sizes of 0.2-0.22 x 8-18 microm. Growth of the strain was observed between 10 and 44 degrees C (optimum 37 degrees C), in 2-12% (w/v) NaCl (optimum 3% NaCl) and between pH 8 and 10.5 (optimum pH 9.5). The novel strain was strictly alkaliphilic, required high concentrations of carbonates in the medium and was capable of utilizing D-glucose, fructose, maltose, sucrose, starch and D-mannitol. End products of glucose fermentation were H2, acetate, ethanol and formate. Strain ASpG(T) was resistant to kanamycin and rifampicin, but sensitive to gentamicin, tetracycline and chloramphenicol. The G + C content of its DNA was 58.5 mol%. DNA-DNA hybridization analysis of strain ASpG1(T) with its most closely related species, Spirochaeta alkalica Z-7491(T), revealed a hybridization value of only 48.7%. On the basis of its physiological and molecular properties, strain ASpG1(T) appears to represent a novel species of the genus Spirochaeta, for which the name Spirochaeta americana is proposed (type strain ASpG1(T) =ATCC BAA-392(T) = DSM 14872(T)).

Sodium Chloride/metabolism↗