bluuebunny/crossref_metadata_2025 huggingface dataset:
Identification of putative insect brush border membrane-binding molecules specific to Bacillus thuringiensis delta-endotoxin by protein blot analysis
Authors: Garczynski S F , Crim J W , Adang M J
Published: January 2020

Binding sites for insecticidal toxins of Bacillus thuringiensis are located in the brush border membranes of insect midguts. Two approaches were used to investigate the interactions of B. thuringiensis subsp. kurstaki HD-73 CryIA(c) toxin with brush border membrane vesicles from sensitive and naturally resistant insects: 125I-toxin-vesicle binding assays and protein blots probed with 125I-CryIA(c) toxin. In bioassays, Manduca sexta and Heliothis virescens larvae were highly sensitive, Helicoverpa zea larvae were moderately sensitive, and Spodoptera frugiperda larvae were resistant to CryIA(c) toxin. Studies of binding of 125I-CryIA(c) toxin to brush border membrane vesicles from the larval midguts revealed that all insects tested had high-affinity, saturable binding sites. Significantly, S. frugiperda larvae bind but are not killed by CryIA(c) toxin. Labeled CryIA(c) toxin incubated with protein blots identifies a major binding molecule of 120 kDa for M. sexta and 148 kDa for S. frugiperda. H. virescens and H. zea are more complex, containing 155-, 120-, 103-, 90-, and 63-kDa proteins as putative toxin-binding molecules. H. virescens also contains a minor toxin-binding protein of 81 kDa. These experiments provide information that can be applied toward a more detailed characterization of B. thuringiensis toxin-binding proteins.
Osmotically regulated transport of proline by Lactobacillus acidophilus IFO 3532
Authors: Jewell J B , Kashket E R
Published: January 2020

We reported previously that, when exposed to high osmotic pressure, Lactobacillus acidophilus IFO 3532 cells accumulated N,N,N-trimethylglycine (glycine betaine), which serves as a compatible intracellular solute. When grown in medium with high osmotic pressure, these cells also accumulated one amino acid, proline. The uptake of [3H]proline by resting, glucose-energized cells was stimulated by increasing the osmotic pressure of the assay medium with 0.5 to 1.0 M KCl, 1.0 M NaCl, or 0.5 M sucrose. The accumulated [3H]proline was not metabolized further. In contrast, there was no osmotic stimulation of [3H]leucine uptake. The uptake of proline was activated rather than induced by exposure of the cells to high osmotic pressure. Only one proline transport system could be discerned from kinetics plots. The affinity of the carrier for proline remained constant over a range of osmotic pressures from 650 to 1,910 mosM (Kt, 7.8 to 15.5 mM). The Vmax, however, increased from 15 nmol/min/mg of dry weight in 0.5 M sucrose to 27 and 40 nmol/min/mg of dry weight in 0.5 M KCl and in 1.0 M KCl or NaCl, respectively. The efflux of proline from preloaded cells occurred rapidly when the osmotic pressure of the suspending buffer was lowered.
7-Mercaptoheptanoylthreonine phosphate substitutes for heat-stable factor (mobile factor) for growth of Methanomicrobium mobile
Authors: Kuhner C H , Smith S S , Noll K M , Tanner R S , Wolfe R S
Published: January 2020

Methanomicrobium mobile requires a heat-stable factor present in ruminal fluid and in boiled cell extract from Methanobacterium thermoautotrophicum for growth. By comparing the growth of M. mobile with boiled cell extract with that observed with various methanogenic cofactors, we found that 7-mercaptoheptanoylthreonine phosphate (HS-HTP) supported sustained growth of M. mobile, at an optimal concentration of 100 microM. No derivatives or possible biosynthetic precursors of HS-HTP could replace HS-HTP as the sole source of growth factor. Results suggest that the growth requirement might be satisfied by 7-mercaptoheptanoic acid plus a second, unidentified heat-stable factor.
Self-Cycling Fermentation Applied to
Acinetobacter calcoaceticus
RAG-1
Authors: Brown Wayne A. , Cooper David G.
Published: January 2020

The self-cycling fermentation (SCF) technique was applied to a culture of
Acinetobacter calcoaceticus
RAG-1. This method was shown to result in synchronization of the cells, achieving a 77% improvement in cell synchrony over that of the batch case. Cellular occurrences, averaged out by asynchronous batch cultures, were magnified by the temporal alignment of metabolic events brought about by the synchronization associated with SCFs. The cell population doubled only once per cycle, thus establishing an equality between cycle time and doubling time. Parameters of interest were biomass concentration, total bioemulsifier (emulsan) production, cycle time, and residual carbon concentration. Cycle-to-cycle variation of these parameters was, in most cases, insignificant. Repeatability of doubling time estimates (based on 95% confidence intervals) was roughly 7 to 10 times better between cycles in an SCF than between batch replicates. The carbon substrate was completely utilized in all cases in which it was measured, giving this technique an advantage over chemostat-type fermentations. The dissolved-oxygen profiles monitored throughout a cycle were found to be repeatable. A characteristic shape, which can be related to the growth of the organism, was associated with each carbon source. The specific emulsan productivity of SCFs was found to be approximately 50 times greater than that of the batch process and 2 to 9 times greater than that of the chemostat, depending on the dilution rate considered. With respect to specific emulsan production, a 25-fold improvement over that in an immobilized cell system recently introduced was obtained. Thus, SCFs are a viable alternative to established fermentation techniques.
Possible mechanisms underlying the slow lactose fermentation phenotype in Shigella spp
Authors: Ito H , Kido N , Arakawa Y , Ohta M , Sugiyama T , Kato N
Published: January 2020

A Southern hybridization analysis revealed that the region homologous to Escherichia coli lacZ was present on the chromosomal DNAs of beta-galactosidase-positive Shigella strains, such as Shigella dysenteriae serovar 1 and Shigella sonnei strains, whereas this region was absent from chromosomal DNAs of beta-galactosidase-negative strains of Shigella flexneri and Shigella boydii. We found that the lacY-A region was deficient in S. dysenteriae serovar 1 and believe that this is the reason for the slow fermentation of lactose by this strain. S. sonnei strains possessed the region which hybridized with E. coli lacY-A despite their slow hydrolysis of lactose. The whole lactose-fermenting region was cloned from S. sonnei and compared with the cloned lac operon of E. coli K-12. Both clones directed the synthesis of beta-galactosidase in an E. coli K-12 strain lacking indigenous beta-galactosidase activity (strain JM109-1), and we observed no difference in the expression of beta-galactosidase activity in S. sonnei and E. coli. However, E. coli JM109-1 harboring the lactose-fermenting genes of S. sonnei exhibited the slow lactose fermentation phenotype like the parental strain. S. sonnei strains had no detectable lactose permease activities. E. coli JM109-1 harboring the lactose-fermenting genes of S. sonnei had a detectable permease activity, possibly because of the multicopy nature of the cloned genes, but this permease activity was much lower than that of strain JM109-1 harboring the lac operon of E. coli K-12. From these results we concluded that slow lactose fermentation by S. sonnei is due to weak lactose permease activity.
Genetic analysis of the antifungal activity of a soilborne Pseudomonas aureofaciens strain
Authors: Vincent M N , Harrison L A , Brackin J M , Kovacevich P A , Mukerji P , Weller D M , Pierson E A
Published: January 2020

Pseudomonas aureofaciens Q2-87 produces the antibiotic 2,4-diacetophloroglucinol (Phl), which inhibits Gaeumannomyces graminis var. tritici and other fungi in vitro. Strain Q2-87 also provides biological control of take-all, a root disease of wheat caused by this fungus. To assess the role of Phl in the antifungal activity of strain Q2-87, a genetic analysis of antibiotic production was conducted. Two mutants of Q2-87 with altered antifungal activity were isolated by site-directed mutagenesis with Tn5. One mutant, Q2-87::Tn5-1, did not inhibit G. graminis var. tritici in vitro and did not produce Phl. Two cosmids were isolated from a genomic library of the wild-type strain by probing with the mutant genomic fragment. Antifungal activity and Phl production were coordinately restored in Q2-87::Tn5-1 by complementation with either cosmid. Mobilization of one of these cosmids into two heterologous Pseudomonas strains conferred the ability to synthesize Phl and increased their activity against G. graminis var. tritici, Pythium ultimum, and Rhizoctonia solani in vitro. Subcloning and deletion analysis of these cosmids identified a 4.8-kb region which was necessary for Phl synthesis and antifungal activity.
Genetic Transformation System for the Fungal Soybean Pathogen
Cercospora kikuchii
Authors: Upchurch R. G. , Ehrenshaft M. , Walker D. C. , Sanders L. A.
Published: January 2020

An altered β-tubulin gene that confers resistance to the fungicide benomyl was isolated from a genomic library of a UV-induced mutant of
Cercospora kikuchii
and used as a selectable marker for transformation. The level of benomyl resistance conferred to the transformants was at least 150-fold greater than the intrinsic resistance of the
C. kikuchii
recipient protoplasts. In the majority of cases, the tubulin fragment was integrated at the native β-tubulin locus, apparently by gene replacement or gene conversion. The frequency of transformation ranged from 0.2 to 6 transformants per μg of DNA, depending on the recipient strain. Transformation with linearized plasmid resulted in a higher frequency, without changing the type of integration event. Transformants were phenotypically stable after eight consecutive transfers on medium without benomyl. This is the first report of a genetic transformation system for a
Cercospora
species.
Deletion of pgi alters tryptophan biosynthesis in a genetically engineered strain of Escherichia coli
Authors: Mascarenhas D , Ashworth D J , Chen C S
Published: January 2020

Deletion of the structural gene for phosphoglucose isomerase (pgi) of Escherichia coli dramatically alters the path of glucose catabolism by diverting carbon into the hexose monophosphate shunt. The effect of this genetic alteration on the conversion of glucose to tryptophan by strains optimized for the biosynthesis of this amino acid was determined by using 13C-nuclear magnetic resonance spectroscopy in vivo. Pgi- strains converted glucose to tryptophan almost twice as efficiently as did their Pgi+ counterparts.
NH
4
+
-Excreting
Azospirillum brasilense
Mutants Enhance the Nitrogen Supply of a Wheat Host
Authors: Christiansen-Weniger C. , Van Veen J. A.
Published: January 2020

Spontaneous ethylenediamine-resistant mutants of
Azospirillum brasilense
were selected on the basis of their excretion of NH
4
+
. Two mutants exhibited no repression of their nitrogenase enzyme systems in the presence of high (20 mM) concentrations of NH
4
+
. The nitrogenase activities of these mutants on nitrogen-free minimal medium were two to three times higher than the nitrogenase activity of the wild type. The mutants excreted substantial amounts of ammonia when they were grown either under oxygen-limiting conditions (1 kPa of O
2
) or aerobically on nitrate or glutamate. The mutants grew well on glutamate as a sole nitrogen source but only poorly on NH
4
Cl. Both mutants failed to incorporate [
14
C]methylamine. We demonstrated that nitrite ammonification occurs in the mutants. Wild-type
A. brasilense
, as well as the mutants, became established in the rhizospheres of axenically grown wheat plants at levels of > 10
7
cells per g of root. The rhizosphere acetylene reduction activity was highest in the preparations containing the mutants. When plants were grown on a nitrogen-free nutritional medium, both mutants were responsible for significant increases in root and shoot dry matter compared with wild-type-treated plants or with noninoculated controls. Total plant nitrogen accumulation increased as well. When they were exposed to a
15
N
2
-enriched atmosphere, both
A. brasilense
mutants incorporated significantly higher amounts of
15
N inside root and shoot material than the wild type did. The results of our nitrogen balance and
15
N enrichment studies indicated that NH
4
+
-excreting
A. brasilense
strains potentially support the nitrogen supply of the host plants.
Influence of CO
2
-HCO
3
−
Levels and pH on Growth, Succinate Production, and Enzyme Activities of
Anaerobiospirillum succiniciproducens
Authors: Samuelov N. S. , Lamed R. , Lowe S. , Zeikus J. G.
Published: January 2020

Growth and succinate versus lactate production from glucose by
Anaerobiospirillum succiniciproducens
was regulated by the level of available carbon dioxide and culture pH. At pH 7.2, the generation time was almost doubled and extensive amounts of lactate were formed in comparison with growth at pH 6.2. The succinate yield and the yield of ATP per mole of glucose were significantly enhanced under excess-CO
2
-HCO
3
−
growth conditions and suggest that there exists a threshold level of CO
2
for enhanced succinate production in
A. succiniciproducens
. Glucose was metabolized via the Embden-Meyerhof-Parnas route, and phosphoenolpyruvate carboxykinase levels increased while lactate dehydrogenase and alcohol dehydrogenase levels decreased under excess-CO
2
-HCO
3
−
growth conditions. Kinetic analysis of succinate and lactate formation in continuous culture indicated that the growth rate-linked production rate coefficient (K) cells was much higher for succinate (7.2 versus 1.0 g/g of cells per h) while the non-growth-rate-related formation rate coefficient (K′) was higher for lactate (1.1 versus 0.3 g/g of cells per h). The data indicate that
A. succiniciproducens
, unlike other succinate-producing anaerobes which also form propionate, can grow rapidly and form high final yields of succinate at pH 6.2 and with excess CO
2
-HCO
3
−
as a consequence of regulating electron sink metabolism.
Contemporaneous N
2
Fixation and Oxygenic Photosynthesis in the Nonheterocystous Mat-Forming Cyanobacterium
Lyngbya aestuarii
Authors: Paerl Hans W. , Prufert Leslie E. , Ambrose Wallace W.
Published: January 2020

The nonheterocystous filamentous cyanobacterial genus
Lyngbya
is a widespread and frequently dominant component of marine microbial mats. It is suspected of contributing to relatively high rates of N
2
fixation associated with mats. The ability to contemporaneously conduct O
2
-sensitive N
2
fixation and oxygenic photosynthesis was investigated in
Lyngbya aestuarii
isolates from a North Carolina intertidal mat. Short-term (<4-h) additions of the photosystem II (O
2
evolution) inhibitor 3(3,4-dichlorophenyl)-1,1-dimethylurea stimulated light-mediated N
2
fixation (nitrogenase activity), indicating potential inhibition of N
2
fixation by O
2
production. However, some degree of light-mediated N
2
fixation in the absence of 3(3,4-dichlorophenyl)-1,1-dimethylurea was observed. Electron microscopic immunocytochemical localization of nitrogenase, coupled to microautoradiographic studies of
14
CO
2
fixation and cellular deposition of the tetrazolium salt 2,4,5-triphenyltetrazolium chloride, revealed that (i) nitrogenase was widely distributed throughout individual filaments during illuminated and dark periods, (ii)
14
CO
2
fixation was most active in intercalary regions, and (iii) daylight 2,4,5-triphenyltetrazolium chloride reduction (formazan deposition) was most intense in terminal regions. Results suggest lateral partitioning of photosynthesis and N
2
fixation during illumination, with N
2
fixation being confined to terminal regions. During darkness, a larger share of the filament appears capable of N
2
fixation.
Comparative inactivation of poliovirus type 3 and MS2 coliphage in demand-free phosphate buffer by using ozone
Authors: Finch G R , Fairbairn N
Published: January 2020

MS2 coliphage (ATCC 15597-B1) has been proposed by the U.S. Environmental Protection Agency as a surrogate for enteric viruses to determine the engineering requirements of chemical disinfection systems on the basis of previous experience with chlorine. The objective of this study was to determine whether MS2 coliphage was a suitable indicator for the inactivation of enteric viruses when ozone disinfection systems were used. Bench-scale experiments were conducted in 2-liter-batch shrinking reactors containing ozone demand-free 0.05 M phosphate buffer (pH 6.9) at 22 degrees C. Ozone was added as a side stream from a concentrated stock solution. It was found that an ozone residual of less than 40 micrograms/liter at the end of 20 s inactivated greater than 99.99% of MS2 coliphage in the demand-free buffer. When MS2 was compared directly with poliovirus type 3 in paired experiments, 1.6 log units more inactivation was observed with MS2 coliphage than with poliovirus type 3. It was concluded that the use of MS2 coliphage as a surrogate organism for studies of enteric virus with ozone disinfection systems overestimated the inactivation of enteric viruses. It is recommended that the regulatory agencies evaluate their recommendations for using MS2 coliphage as an indicator of enteric viruses.
Isolation of a bacterium resembling Pirellula species from primary tissue culture of the giant tiger prawn (Penaeus monodon)
Authors: Fuerst J A , Sambhi S K , Paynter J L , Hawkins J A , Atherton J G
Published: January 2020

During attempts to establish tissue cultures from hepatopancreas, heart, and hemolymph of the giant tiger prawn (Penaeus monodon), using a medium including penicillin, streptomycin, and amphotericin B, bacterial contamination in the form of a sheet of growth attached to the tissue culture vessel was a persistent problem. Contaminant bacteria were teardrop-shaped cells arranged in rosettes, and electron microscopy revealed buds, crateriform structures, and the absence of a peptidoglycan layer in the cell wall, features characteristic of bacteria in the Planctomyces-Pirellula group, a phylogenetically distinct group of eubacteria. Two strains of contaminant bacteria were isolated in pure culture. Both exhibited morphology and antibiotic resistance consistent with their membership in the Planctomyces-Pirellula group (order Planctomycetales) of eubacteria. Tissue culture media for marine invertebrates may select for such bacteria if high concentrations of cell wall synthesis-inhibiting antibiotics are included.
Comparison of animal infectivity, excystation, and fluorogenic dye as measures of Giardia muris cyst inactivation by ozone
Authors: Labatiuk C W , Schaefer F W , Finch G R , Belosevic M
Published: January 2020

Giardia muris cyst viability after ozonation was compared by using fluorescein diacetate-ethidium bromide staining, the C3H/HeN mouse-G. muris model, and in vitro excystation. Bench-scale batch experiments were conducted under laboratory conditions (pH 6.7, 22 degrees C) in ozone-demand-free phosphate buffer. There was a significant difference between fluorogenic staining and infectivity (P less than or equal to 0.05), with fluorogenic staining overestimating viability compared with infectivity estimates of viability. This suggests that viable cysts as indicated by fluorogenic dyes may not be able to complete the life cycle and produce an infection. No significant differences between infectivity and excystation and between fluorogenic staining and excystation (P less than or equal to 0.05) were detected for inactivations up to 99.9%. Only animal infectivity had the sensitivity to detect inactivations greater than 99.9%. Therefore, the animal model is the best method currently available for detecting high levels of G. muris cyst inactivation.
Characterization of phiLC3, a Lactococcus lactis subsp. cremoris temperature bacteriophage with cohesive single-stranded DNA ends
Authors: Lillehaug D , Lindqvist B , Birkeland N K
Published: January 2020

The temperate bacteriophage phiLC3, isolated from Lactococcus lactis subsp. cremoris, has an isometric head and a flexible tail containing 1 major protein and 8 minor proteins. Infection of a permissive L. lactis host strain yields a burst of about 50 phages per infected cell with a latent period of 60 min. A detailed restriction map of the phage chromosome was constructed by using 12 different restriction enzymes. The phage chromosome is a 33-kb linear double-stranded DNA molecule with unique cohesive ends and with a G + C content of 36.5%. Chemical sequencing of the DNA ends revealed 13-base 3' extended complementary single strands with a relatively high percentage of G + C. Pulsed-field gel electrophoretic analysis of DNA from a strain lysogenized with phiLC3 was used to localize the prophage to a 320-kb BamHI restriction endonuclease fragment from the host chromosomal DNA. This result indicates that lysogeny involves integration of the phage into the host chromosome. A spontaneous phiLC3 clear plaque mutant that was unable to give rise to lysogens was isolated.
Determination of killer yeast activity in fermenting grape juice by using a marked Saccharomyces wine yeast strain
Authors: Petering J E , Symons M R , Langridge P , Henschke P A
Published: January 2020

The Escherichia coli beta-glucuronidase gene has been used as a marker gene to monitor a killer Saccharomyces cerevisiae strain in mixed-culture ferments. The marked killer strain was cured of its M-dsRNA genome to enable direct assessment of the efficiency of killer toxin under fermentation conditions. Killer activity was clearly evident in fermenting Rhine Riesling grape juice of pH 3.1 at 18 degrees C, but the extent of killing depended on the proportion of killer to sensitive cells at the time of inoculation. Killer activity was detected only when the ratio of killer to sensitive cells exceeded 1:2. At the highest ratio of killer to sensitive cells tested (2:1), complete elimination of sensitive cells was not achieved.
Production and Consumption of Dimethylsulfoniopropionate in Marine Microbial Mats
Authors: Visscher Pieter T. , van Gemerden Hans
Published: January 2020

The fate of dimethylsulfoniopropionate (DMSP), a major sulfonium compound in marine ecosystems, was examined in
Microcoleus chthonoplastes
-dominated microbial mats. Chemical decomposition of DMSP was observed under laboratory conditions at pH values higher than 10.0. pH profiles measured in situ showed that these highly alkaline conditions occurred in microbial mats. Axenic cultures of
M. chthonoplastes
contained 37.3 μmol of DMSP g of protein
−1
, which was partially liberated when the cells were subjected to an osmotic shock. DMSP-amended mat slurries showed a rapid turnover of this compound. The addition of glutaraldehyde blocked DMSP decrease, indicating biological consumption. Populations of potential dimethyl sulfide consumers were found in the top 10 mm of the mat.
Transfer of the Pea Symbiotic Plasmid pJB5JI in Nonsterile Soil
Authors: Kinkle Brian K. , Schmidt Edwin L.
Published: January 2020

Transfer of the pea (
Pisum sativum
L.) symbiotic plasmid pJB5JI between strains of rhizobia was examined in sterile and nonsterile silt loam soil.
Sinorhizobium fredii
USDA 201 and HH003 were used as plasmid donors, and symbiotic plasmid-cured
Rhizobium leguminosarum
6015 was used as the recipient. The plasmid was carried but not expressed in
S. fredii
strains, whereas transfer of the plasmid to
R. leguminosarum
6015 rendered the recipient capable of nodulating pea plants. Confirmation of plasmid transfer was obtained by acquisition of plasmid-encoded antibiotic resistance genes, nodulation of pea plants, and plasmid profiles. Plasmid transfer in nonsterile soil occurred at frequencies of up to 10
−4
per recipient and appeared to be highest at soil temperatures and soil moisture levels optimal for rhizobial growth. Conjugation frequencies were usually higher in sterile soil than in nonsterile soil. In nonsterile soil, transconjugants were recovered only with strain USDA 201 as the plasmid donor. Increasing the inoculum levels of donor and recipient strains up to 10
9
cells g of soil
−1
increased the number of transconjugants; peak plasmid transfer frequencies, however, were found at the lower inoculum level of 10
7
cells g of soil
−1
. Plasmid transfer frequencies were raised in the presence of the pea rhizosphere or by additions of plant material. Transconjugants formed by the USDA 201(pJB5JI) × 6015 mating in soil formed effective nodules on peas.
Relative importance of fluorescent siderophores and other factors in biological control of Gaeumannomyces graminis var. tritici by Pseudomonas fluorescens 2-79 and M4-80R
Authors: Hamdan H , Weller D M , Thomashow L S
Published: January 2020

Pseudomonas fluorescens 2-79 suppresses take-all, a major root disease of wheat caused by Gaeumannomyces graminis var. tritici. The bacteria produce an antibiotic, phenazine-1-carboxylic acid (PCA), and a fluorescent pyoverdin siderophore. Previous studies have established that PCA has an important role in the biological control of take-all but that antibiotic production does not account fully for the suppressiveness of the strain. To define the role of the pyoverdin siderophore more precisely, mutants deficient in production of the antibiotic, the siderophore, or both factors were constructed and compared with the parental strain for control of take-all on wheat roots. In all cases, strains that produced PCA were more suppressive than those that did not, and pyoverdin-deficient mutant derivatives controlled take-all as effectively as their respective fluorescent parental strains. Thus, the phenazine antibiotic was the dominant factor in disease suppression and the fluorescent siderophore had little or no role. The siderophore also was of minor importance in a second strain, P. fluorescens M4-80R, that does not produce PCA. Strains 2-79 and M4-80R both produced substances distinct from the pyoverdin siderophore that were responsible for fungal inhibition in vitro under iron limitation, but these substances also had, at most, a minor role in disease suppression in situ.
Shuttle plasmid vectors for Lactobacillus casei and Escherichia coli with a minus origin
Authors: Shimizu-Kadota M , Shibahara-Sone H , Ishiwa H
Published: January 2020

Recombinant plasmids which can be used as shuttle vectors between Escherichia coli and the industrially used strains of Lactobacillus casei were constructed. They have replication regions closely related to those of pUB110 and are likely to replicate by a rolling-circle mechanism via a plus-strand-specific DNA intermediate in L. casei. Both orientations of palA from the staphylococcal plasmid pC194 and those of the intergenic region from coliphage M13 are identified as active minus origins in L. casei, in contrast to the pAM alpha 1 delta 1-derived BA3 minus origin which does not function in L. casei. Stability of the plasmids increased in L. casei when one of these two active minus origins was inserted. All the DNA sequences of the constructed vectors were known.
Double-antibody sandwich enzyme-linked immunosorbent assay for quantitation of endoglucanase I of Trichoderma reesei
Authors: Bühler R
Published: January 2020

A sensitive and specific enzyme-liked immunosorbent assay for endoglucanase I (EG-I) has been developed. The monoclonal antibody a-EG-I 2, directed against an epitope on the core part of the enzyme, was used to capture the antigen in microtiter plate wells. A second, polyclonal antibody against the enzyme was then used to detect and quantitate the bound antigen. The test was specific for EG-I; neither endoglucanase II nor cellobiohydrolase I or II interfered. As little as 20 pg of EG-I protein could be detected. The coefficients of variation were 3.8% within plates and 6% between plates for a diluted Trichoderma reesei culture supernatant that contained 31 ng of EG-I per ml. Binding of the antigen to the monoclonal antibody was pH dependent and restricted to values between pH 6.5 and 10.5 with a maximum around pH 9. Standard solutions of EG-I were very stable at concentrations as low as 5 ng/ml when prepared in buffer that contained 1% bovine serum albumin and that was stored at -20 degrees C. After 37 weeks the antigenicity was still 97%. With this test it was possible to monitor the production of EG-I in a cellulase-producing strain of T. reesei and to demonstrate the apparent absence of the enzyme in a strain with the eglI gene deleted.
Streptomyces marker plasmids for monitoring survival and spread of streptomycetes in soil
Authors: Wipat A , Wellington E M , Saunders V A
Published: January 2020

Plasmid constructs pNW1 through pNW6 containing a controllable xylE gene (for catechol 2,3-dioxygenase) were introduced into Streptomyces lividans strains to provide a selectable marker system. xylE functions in S. lividans under the control of bacteriophage lambda promoters lambda pL and lambda pR. Thermoregulated expression of xylE is provided through the lambda repressor cI857. Catechol 2,3-dioxygenase activity was increased 2.8-fold from plasmid construct pNW2 (lambda pL, xylE, cI857) and 9.5- and 7.4-fold from constructs pNW3 (lambda pR, xylE, cI857) and pNW5 (lambda pR, xylE, cI857), respectively, when the temperature was shifted from 28 degrees C to 37 degrees C. The stability of the constructs varied from 4.7% for pNW2 to 99.4% for pNW4 (lambda pL, xylE) over two rounds of sporulation. Marked S. lividans strains released into soil systems retained the XylE phenotype for more than 80 days, depending on the marker plasmid, when examined by a selective plating method. Furthermore, S. lividans harboring plasmid pNW5 was detectable by nucleic acid hybridization at less than 10 CFU g-1 (dry weight) of soil as mycelium and 10(3) CFU g-1 (dry weight) of soil as spores with the xylE marker DNA extracted from soil and amplified by using the polymerase chain reaction.
Fractionation of cellulases from the ruminal fungus Neocallimastix frontalis EB188
Authors: Li X L , Calza R E
Published: January 2020

Cellulases from the ruminal fungus Neocallimastix frontalis EB188 were separated by using hydroxylapatite column chromatography. Seven carboxymethylcellulases, six avicelases, and four beta-glucosidases accounted for the majority of the activities. The separation of enzymes was confirmed by using polyacrylamide gel electrophoresis. Electrophoretic migration, analysis of hydrolysis products, and substrate specificity measurements suggested that several different cellulases were secreted in N. frontalis EB188. The possible relationship of cellulase diversity to protein glycosylation is discussed.
Study of the Citrate Metabolism of
Lactococcus lactis
subsp.
lactis
Biovar Diacetylactis by Means of
13
C Nuclear Magnetic Resonance
Authors: Verhue Walter M. , Tjan Frans S. B.
Published: January 2020

The metabolic fate of citrate and pyruvate in four strains of
Lactococcus lactis
subsp.
lactis
biovar diacetylactis has been studied by means of
13
C nuclear magnetic resonance, using as a substrate either [3-
13
C]pyruvic acid or custom-synthesized citric acid that is
13
C labeled either at carbons 2 and 4 or at carbon 3. The fermentations were carried out batchwise in modified M17 broth. For the actual conversions of the
13
C-labeled substrates, cells at the end of their logarithmic growth phase were used to minimize the conversion to lactic acid. A mass balance of the main citric acid metabolites was obtained; the four strains produced from 50 to 70% (on a molar basis) lactic acid from either citrate or pyruvate. The remaining 50 to 30% was converted mainly to either α-acetolactic acid (for one strain) or acetoin (for the other three strains). One of the strains produced an exceptionally high concentration of the diacetyl precursor α-acetolactic acid. Another strain (SDC6) also produced α-acetolactic acid, but this was decarboxylated to acetoin at a high rate. The
13
C nuclear magnetic resonance method confirmed that the biosynthesis of α-acetolactic acid occurs via condensation of pyruvate and “active” acetaldehyde. Diacetyl was not found as a direct metabolite of citrate or pyruvate metabolism.
Gibberellins and Carotenoids in the Wild Type and Mutants of
Gibberella fujikuroi
Authors: Candau Reyes , Ávalos Javier , Cerdá-Olmedo Enrique
Published: January 2020

A new screening procedure was used to isolate 14
gib
mutants of
Gibberella fujikuroi
with modifications in the production of gibberellins. The production of carotenoids and gibberellins was investigated in the
gib
mutants and in representative
car
mutants with various modifications of carotenoid biosynthesis. The determinations of gibberellins were carried out with a simplified fluorescence method. One of the mutants lacked both gibberellins and carotenoids. In many mutants the two pathways compensated each other: an increase in the production of one group of compounds was accompanied by a decrease in the production of the other. Under certain conditions the compensation was quantitative when the output of the two pathways was measured in moles of the common precursor, geranylgeranyl pyrophosphate. α-Picoline, an inhibitor of lycopene cyclase in
G. fujikuroi
, inhibits gibberellin biosynthesis. Other agents that affect the accumulation of carotenoids have no noticeable effect on the accumulation of gibberellins; such is the case with diphenylamine and β-ionone, two inhibitors of phytoene dehydrogenation, and visible light, which stimulates carotenogenesis.
Production and Extracellular Secretion of Aqualysin I (a Thermophilic Subtilisin-Type Protease) in a Host-Vector System for
Thermus thermophilus
Authors: Touhara Nami , Taguchi Hayao , Koyama Yoshinori , Ohta Takahisa , Matsuzawa Hiroshi
Published: January 2020

Aqualysin I is synthesized as a large precursor, processed, and secreted into the culture medium by
Thermus aquaticus
YT-1. An expression plasmid for the aqualysin I gene in
T. thermophilus
HB27 was constructed.
T. thermophilus
cells harboring the recombinant plasmid produced correctly processed aqualysin I, and the mature enzyme was secreted into the culture medium.
Bacterial nitrification in chloraminated water supplies
Authors: Cunliffe D A
Published: January 2020

Nitrifying bacteria were detected in 64% of samples collected from five chloraminated water supplies in South Australia and in 20.7% of samples that contained more than 5.0 mg of monochloramine per liter. Laboratory experiments confirmed that nitrifying bacteria are relatively resistant to the disinfectant. Increased numbers of the bacteria were associated with accelerated decays of monochloramine within distribution systems. The combination of increased concentrations of oxidized nitrogen with decreased total chlorine residuals can be used as a rapid indicator of bacterial nitrification.
Construction of a Bacteriophage-Resistant Derivative of
Lactococcus lactis
subsp.
lactis
425A by Using the Conjugal Plasmid pNP40
Authors: Harrington Aidan , Hill Colin
Published: January 2020

Lactococcus lactis
subsp.
lactis
425A is an atypical strain which excretes a high concentration of α-acetolactate when grown in milk. The conjugative lactococcal plasmid pNP40, which encodes phage and nisin resistance, was introduced to strain 425A by conjugation, using resistance to phage and nisin as a selection. No phage-nisin resistance mutants were encountered. Transconjugants display complete resistance at both 21 and 39°C to those phage previously identified as lytic for 425A. Transconjugants lose their resistance characteristics when spontaneously cured of pNP40. The commercially important property of 425A—production of high levels of α-acetolactic acid—is unaffected by the presence of pNP40.
Detection of Giardia cysts by using the polymerase chain reaction and distinguishing live from dead cysts
Authors: Mahbubani M H , Bej A K , Perlin M , Schaefer F W , Jakubowski W , Atlas R M
Published: January 2020

A method was developed for the detection of Giardia cysts by using the polymerase chain reaction (PCR) and the giardin gene as the target. DNA amplification by PCR, using giardin DNA as the target, resulted in detection of both live and dead cysts. When giardin mRNA was used as the target, the ability to amplify cDNA by PCR depended on the mode of killing. Cysts killed by freezing were not detected by PCR when giardin mRNA was the target. Cysts killed by heating or exposure to monochloramine, however, gave positive detection signals for both DNA and giardin mRNA targets. The amount of giardin mRNA and total RNA was significantly increased in live cysts following the induction of excystation. Cysts killed by freezing, heating, or exposure to monochloramine did not show a change in RNA content. The detection of the giardin gene by PCR permits a sensitive and specific diagnosis for Giardia spp. Discrimination between live and dead cysts can be made by measuring the amounts of RNA or PCR-amplified product from the giardin mRNA target before and after the induction of excystation.
Indigenous and enhanced mineralization of pyrene, benzo[a]pyrene, and carbazole in soils
Authors: Grosser R J , Warshawsky D , Vestal J R
Published: January 2020

We studied the mineralization of pyrene, carbazole, and benzo[a]pyrene in soils obtained from three abandoned coal gasification plants in southern Illinois. The soils had different histories of past exposure to hydrocarbon contamination and different amounts of total organic carbon, microbial biomass, and microbial activity. Mineralization was measured by using serum bottle radiorespirometry. The levels of indigenous mineralization of 14C-labeled compounds ranged from 10 to 48% for pyrene, from undetectable to 46% for carbazole, and from undetectable to 25% for benzo[a]pyrene following long-term (greater than 180-day) incubations. Pyrene and carbazole were degraded with short or no lag periods in all soils, but benzo[a]pyrene mineralization occurred after a 28-day lag period. Mineralization was not dependent on high levels of microbial biomass and activity in the soils. Bacterial cultures that were capable of degrading pyrene and carbazole were isolated by enrichment, grown in pure culture, and reintroduced into soils. Reintroduction of a pyrene-degrading bacterium enhanced mineralization to a level of 55% within 2 days, compared with a level of 1% for the indigenous population. The carbazole degrader enhanced mineralization to a level of 45% after 7 days in a soil that showed little indigenous carbazole mineralization. The pyrene and carbazole degraders which we isolated were identified as a Mycobacterium sp. and a Xanthamonas sp., respectively. Our results indicated that mineralization of aromatic hydrocarbons can be significantly enhanced by reintroducing isolated polycyclic aromatic hydrocarbon-degrading bacteria.
Competitive inhibition of an energy-dependent nickel transport system by divalent cations in Bradyrhizobium japonicum JH
Authors: Fu C L , Maier R J
Published: January 2020

Both nickel-specific transport and nickel transport by a magnesium transporter have been described previously for a variety of nickel-utilizing bacteria. The derepression of hydrogenase activity in Bradyzhizobium japonicum JH and in a gene-directed mutant of strain JH (in an intracellular Ni metabolism locus), strain JHK7, was inhibited by MgSO4. For both strains, Ni2+ uptake was also markedly inhibited by Mg2+, and the Mg(2+)-mediated inhibition could be overcome by high levels of Ni2+ provided in the assay buffer. The results indicate that both B. japonicum strains transport Ni2+ via a high-affinity magnesium transport system. Dixon plots (1/V versus inhibitor) showed that the divalent cations Co2+, Mn2+, and Zn2+, like Mg2+, were competitive inhibitors of Ni2+ uptake. The KiS for nickel uptake inhibition by Mg2+, Co2+, Mn2+, and Zn2+ were 48, 22, 12, and 8 microM, respectively. Cu2+ strongly inhibited Ni2+ uptake, and molybdate inhibited it slightly. Respiratory inhibitors cyanide and azide, the uncoupler carbonyl cyanide m-chlorophenylhydrazone, the ATPase inhibitor N,N'-dicyclohexylcarbodiimide, and ionophores nigericin and valinomycin significantly inhibited short-term (5 min) Ni2+ uptake, showing that Ni2+ uptake in strain JH is energy dependent. Most of these conclusions are quite different from those reported previously for a different B. japonicum strain belonging to a different serogroup.
New Routes for Aerobic Biodegradation of Dimethylsulfoniopropionate
Authors: Taylor Barrie F. , Gilchrist Darrin C.
Published: January 2020

Dimethylsulfoniopropionate (DMSP), an osmolyte in marine plants, is biodegraded by cleavage of dimethyl sulfide (DMS) or by demethylation to 3-methiolpropionate (MMPA) and 3-mercaptopropionate (MPA). Sequential demethylation has been observed only with anoxic slurries of coastal sediments. Bacteria that grew aerobically on MMPA and DMSP were isolated from marine environments and phytoplankton cultures. Enrichments with DMSP selected for bacteria that generated DMS, whereas MMPA enrichments selected organisms that produced methanethiol (CH
3
SH) from either DMSP or MMPA. A bacterium isolated on MMPA grew on MMPA and DMSP, but rapid production of CH
3
SH from DMSP occurred only with DMSP-grown cells. Low levels of MPA accumulated during growth on MMPA, indicating demethylation as well as demethiolation of MMPA. The alternative routes for DMSP biodegradation via MMPA probably impact on net DMS fluxes to the marine atmosphere.
Separation of Methanotrophic Bacteria by Using Percoll and Its Application to Isolation of Mixed and Pure Cultures
Authors: Putzer K. P. , Buchholz L. A. , Lidstrom M. E. , Remsen C. C.
Published: January 2020

Type I and II methanotrophic bacteria can be cleanly separated by using Percoll buoyant density gradients. This simple and inexpensive method can be used to screen new isolates rapidly for preliminary classification. In addition, Percoll gradients can also be used to enhance standard enrichment techniques for methanotrophs from natural water samples.
Development of a 16S rRNA-based oligomer probe specific for Listeria monocytogenes
Authors: Wang R F , Cao W W , Johnson M G
Published: January 2020

Crude rRNA was isolated from Listeria monocytogenes, L. innocua, and L. ivanovii and sequenced by a reverse transcriptase method. Only two sequence regions were found to differ for L. monocytogenes versus L. innocua or L. ivanovii. Two oligonucleotide probes (RL-1 and RL-2) complementary to these two regions of rRNA of L. monocytogenes were synthesized. The RL-1 probe had one base while the RL-2 probe had two bases which differed for L. monocytogenes versus L. innocua and L. ivanovii. Use of a dried gel hybridization in place of Northern (RNA) hybridization or dot blot hybridization indicated that the RL-2 probe hybridized with all 36 strains of L. monocytogenes tested but not with 6 other Listeria species and 11 other bacteria tested. The RL-2 probe is specific for L. monocytogenes, while the RL-1 probe showed some cross-reactions with other Listeria species. An alkaline phosphatase-labeled RL-2 probe could be used in a dot blot hybridization test and gave good results, but a 32P-labeled RL-2 probe was more sensitive than the nonradioactive probe and the 32P-labeled probe was useable for up to 2 months, even though the 32P was highly degenerated.
Secretion of TEM beta-lactamase with signal sequences isolated from the chromosome of Lactococcus lactis subsp. lactis
Authors: Sibakov M , Koivula T , von Wright A , Palva I
Published: January 2020

With TEM beta-lactamase as a reporter gene, a set of expression-secretion-promoting fragments were isolated from the chromosome of Lactococcus lactis subsp. lactis. The fact that only translocated beta-lactamase renders cells resistant to ampicillin allowed direct ampicillin selection with an Escherichia coli vector (pKTH33). The clones showing the greatest ampicillin resistance were subcloned onto a replicon capable of replication in lactic acid bacteria (pVS2), and the nucleotide sequences of the relevant fragments were determined. The structure of the secretion-promoting fragments in general resembled that of gram-positive true signal sequences, with a strongly positively charged N terminus, a long hydrophobic core, and a putative signal peptidase recognition site. The promoterlike sequences preceding the signal sequences matched well with those of previously published lactococcal promoters. In addition to E. coli, the functioning of these expression-secretion cassettes was studied in three gram-positive hosts: Bacillus subtilis, L. lactis, and Lactobacillus plantarum. Efficient expression and secretion of TEM beta-lactamase into the culture medium of each gram-positive host was obtained. Furthermore, when a strain of L. lactis subsp. lactis showing increased sensitivity to lysozyme was compared with a standard laboratory strain, threefold-higher secreted enzyme activities were detected.
Molecular cloning, expression, and characterization of endoglucanase genes from Fibrobacter succinogenes AR1
Authors: Cavicchioli R , Watson K
Published: January 2020

A cosmid gene library was constructed in Escherichia coli from genomic DNA isolated from the ruminal anaerobe Fibrobacter succinogenes AR1. Clones were screened on carboxymethyl cellulose, and 8 colonies that produced large clearing zones and 25 colonies that produced small clearing zones were identified. Southern blot hybridization revealed the existence of at least three separate genes encoding cellulase activity. pRC093, which is representative of cosmid clones that produce large clearing zones, was subcloned in pGem-1, and the resulting hybrid pRCEH directed synthesis of endoglucanase activity localized on a 2.1-kb EcoRI-HindIII insert. Activity was expressed from this fragment when it was cloned in both orientations in pGem-1 and pGem-2, indicating that F. succinogenes promoters functioned successfully in E. coli. A high level of endoglucanase activity was detected on acid-swollen cellulose, ball-milled cellulose, and carboxymethyl cellulose; and a moderate level was detected on filter paper, Avicel, lichenan, and xylan. Most activity (80%) was localized in the periplasm of E. coli, with low but significant levels (16%) being detected in the extracellular medium. The periplasmic endoglucanase had an estimated molecular weight of 46,500, had an optimum temperature of 39 degrees C, and exhibited activity over a broad pH range, with a maximum at pH 5.0.
Multilaboratory evaluation of methods for detecting enteric viruses in soils
Authors: Hurst C J , Schaub S A , Sobsey M D , Farrah S R , Gerba C P , Rose J B , Goyal S M , Larkin E P , Sullivan R , Tierney J T
Published: January 2020

Two candidate methods for the recovery and detection of viruses in soil were subjected to round robin comparative testing by members of the American Society for Testing and Materials D19:24:04:04 Subcommittee Task Group. Selection of the methods, designated "Berg" and "Goyal," was based on results of an initial screening which indicated that both met basic criteria considered essential by the task group. Both methods utilized beef extract solutions to achieve desorption and recovery of viruses from representative soils: a fine sand soil, an organic muck soil, a sandy loam soil, and a clay loam soil. One of the two methods, Goyal, also used a secondary concentration of resulting soil eluants via low-pH organic flocculation to achieve a smaller final assay volume. Evaluation of the two methods was simultaneously performed in replicate by nine different laboratories. Each of the produced samples was divided into portions, and these were respectively subjected to quantitative viral plaque assay by both the individual, termed independent, laboratory which had done the soil processing and a single common reference laboratory, using a single cell line and passage level. The Berg method seemed to produce slightly higher virus recovery values; however, the differences in virus assay titers for samples produced by the two methods were not statistically significant (P less than or equal to 0.05) for any one of the four soils. Despite this lack of a method effect, there was a statistically significant laboratory effect exhibited by assay titers from the independent versus reference laboratories for two of the soils, sandy loam and clay loam.