bluuebunny/crossref_metadata_2025 huggingface dataset:
Factors affecting the production of eremofortin C and PR toxin in Penicillium roqueforti
Authors: Chang S C , Wei Y H , Wei D L , Chen Y Y , Jong S C
Published: January 2020

Eremofortin C (EC) and PR toxin are secondary metabolites of Penicillium roqueforti. Of 17 strains from the American Type Culture Collection that were studied for their ability to produce EC and PR toxin, 13 produced these metabolites. Toxin production by strains grown in solid media (10 cereals and 8 other agricultural products) was also investigated. Production of EC and PR toxin by fungi grown on cereals was greater than production of EC and PR toxin by fungi grown on legumes; fungi grown on corn produced the greatest amount of PR toxin. Addition of corn extracts to the culture medium greatly increased the production of EC and PR toxin in a coordinated manner, with no significant change in mycelial dry weight. The fungi produced the highest levels of EC and PR toxin at 20 to 24 degrees C depending on the strain. Toxin production was higher in stationary cultures than in cultures that were gently shaken at 120 rpm. The optimum pH for production of both EC and PR toxin was around pH 4.0. With regard to spore age, toxin levels did not change significantly when we used spores obtained from fungi that were grown at 24 degrees C for 3 up to 48 days.
Effect of host diet on production of organic acids and methane by cockroach gut bacteria
Authors: Kane M D , Breznak J A
Published: January 2020

The effect of high-fiber diets on microbial populations and processes in cockroach guts was investigated by feeding American cockroaches (Periplaneta americana) milled cereal leaves, milled corn cob, or commercial bran-type breakfast cereal in place of the commonly used laboratory diet of dog chow. The activities and numbers of specific gut bacteria varied significantly with the insect's diet and developmental stage. Acetate and lactate were the principal organic acids present in the gut fluid of adult cockroaches and occurred at concentrations of up to 17 and 8 mM, respectively. These acids were most abundant in the gut fluid of dog chow-fed insects, and the greatest amounts were generally found in the foregut and midgut regions. Foreguts of dog chow-fed cockroaches contained an abundant population of lactic acid bacteria that formed acetate and lactate from endogenous hexoses present in the foregut. When adult cockroaches were fed dog chow amended with antibacterial drugs, (i) the concentrations of acetate, lactate, and total hexoses in gut fluid decreased significantly, (ii) the numbers of lactic acid bacteria in the foregut also decreased significantly, and (iii) the production of acetate and lactate by foregut homogenates was suppressed. It was estimated that acetate and lactate produced by bacteria in the foregut of dog chow-fed adult P. americana could support up to 14% of the insect's respiratory requirement if taken up and used by the animal. When insects were fed high-fiber diets of bran cereal, cereal leaves, or corn cob, bacterial production of acetate and lactate in the foregut diminished.(ABSTRACT TRUNCATED AT 250 WORDS)
Amplification, cloning, and sequencing of a nifH segment from aquatic microorganisms and natural communities
Authors: Kirshtein J D , Paerl H W , Zehr J
Published: January 2020

By use of the polymerase chain reaction and degenerate oligonucleotide primers for highly conserved regions of nifH, a segment of nifH DNA was amplified from several aquatic microorganisms, including an N2-fixing bacterium closely associated with the marine filamentous cyanobacterium Trichodesmium sp., a heterotrophic isolate from the root/rhizome of the seagrass Ruppia maritima, and the heterocystous freshwater cyanobacterium Anabaena oscillarioides. nifH segments were amplified directly from DNA extracted from the rhizosphere of roots of the seagrass Halodule wrightii. The nifH fragments were then cloned and sequenced. The DNA and deduced amino acid sequences were compared with known sequences, revealing distinct differences between taxonomic groups. This technique was shown to be useful for (i) the detection of N2-fixing microorganisms and (ii) rapidly obtaining the DNA sequence of the nifH gene, which provides information about general taxonomic groups of N2-fixing microorganisms.
Cloning of the Clostridium acetobutylicum ATCC 824 acetyl coenzyme A acetyltransferase (thiolase; EC 2.3.1.9) gene
Authors: Petersen D J , Bennett G N
Published: January 2020

Thiolase (acetyl coenzyme A acetyltransferase; EC 2.3.1.9) from Clostridium acetobutylicum is a key enzyme in the production of acids and solvents in this organism. The purification and properties of the enzyme have already been described (D. P. Wiesenborn, F. B. Rudolph, and E.T. Papoutsakis, Appl. Environ. Microbiol. 54:2717-2722, 1988). The thl gene encoding the thiolase has been cloned by using primary antibodies raised to the purified enzyme. A bacteriophage lambda EMBL3 library of C. acetobutylicum DNA was prepared and screened by immunoblots with the antithiolase antibodies. Phage DNA was purified from positive plaques, and restriction enzyme digests identified an approximately 4.8-kb AccI fragment common to all positive plaques. A corresponding fragment was also found in AccI digests of C. acetobutylicum chromosomal DNA. The fragment was purified and EcoRI linkers were attached before being subcloned into pUC19. Maxicell analysis showed the production of an approximately 42-kDa protein, whose size corresponded to the molecular size of the purified thiolase, from the clostridial insert. Enzyme activity assays and Western blot (immunoblot) analysis of sodium dodecyl sulfate-polyacrylamide gel electrophoresis-separated whole-cell extracts of Escherichia coli harboring the cloned thl confirmed the presence of the thiolase encoded within the cloned DNA.
Survival of Legionella pneumophila in the cold-water ciliate Tetrahymena vorax
Authors: Smith-Somerville H E , Huryn V B , Walker C , Winters A L
Published: January 2020

The processing of phagosomes containing Legionella pneumophila and Escherichia coli were compared in Tetrahymena vorax, a hymenostome ciliated protozoan that prefers lower temperatures. L. pneumophila did not multiply in the ciliate when incubated at 20 to 22 degrees C, but vacuoles containing L. pneumophila were retained in the cells for a substantially longer time than vacuoles with E. coli. Electron micrographs showed no evidence of degradation of L. pneumophila cells through 12 h, while E. coli cells in the process of being digested were observed in vacuoles 75 min after the addition of the bacterium. T. vorax ingested L. pneumophila normally, but by 10 to 15 min, the vacuolar membrane appeared denser than that surrounding nascent or newly formed phagosomes. In older vacuoles, electron-dense particles lined portions of the membrane. Acidification of the phagosomes indicated by the accumulation of neutral red was similar in T. vorax containing L. pneumophila or E. coli. This ciliate could provide a model for the analysis of virulence-associated intracellular events independent of the replication of L. pneumophila.
Induction of Cellulase by Gentiobiose and Its Sulfur-Containing Analog in
Penicillium purpurogenum
Authors: Kurasawa Takashi , Yachi Makoto , Suto Manabu , Kamagata Yoichi , Takao Shoichi , Tomita Fusao
Published: January 2020

Cellulase induction by β-glucodisaccharides was investigated by using non-cellulase-induced mycelia of
Penicillium purpurogenum
P-26, a highly-cellulase-producing fungus. Gentiobiose induced significant amounts of cellulase compared with cellobiose when nojirimycin was added to the induction medium to inhibit extracellular β-glucosidase activity. Thiogentiobiose (6-
S
-β-
d
-glucopyranosyl-6-thio-
d
-glucose), a sulfur-containing analog of gentiobiose, was more effective for cellulase induction than gentiobiose even in the absence of nojirimycin. Thiogentiobiose appeared to be a gratuitous inducer since it was not metabolized during cellulase induction. Gentiobiose was formed from cellobiose by the intracellular β-glucosidase of
P. purpurogenum.
These findings indicate that gentiobiose is an active inducer of cellulase for this fungus and may possibly be formed by intracellular β-glucosidase from cellobiose.
Changes in bacterial composition and enzymatic activity in ileostomy and ileal reservoir during intermittent occlusion: a study using dogs
Authors: Ruseler-van Embden J G , Schouten W R , van Lieshout L M , Auwerda H J
Published: January 2020

Bacterial flora, activities of 10 potential mucus- and dietary polysaccharide-degrading enzymes, blood group antigenicity of the intestinal glycoproteins, and proteolytic activity in the output from experimentally colectomized dogs with conventional ileostomies and dogs with valveless ileal reservoirs (pouches) were determined. The ileostomies of dogs with conventional surgery (group II) and with pouches (group III) were occluded intermittently during a 6-week period. The duration of occlusion was progressively increased. Group I, five dogs with conventional ileostomies, served as a control group. After occlusion of the ileal pouch for 7 h, total numbers of bacteria increased threefold, glycosidase activity increased fivefold, and blood group antigenicity of the intestinal glycoproteins, which was high in the output from the nonoccluded pouch, was no longer detectable. Proteolytic activity was not influenced by occlusion of the pouch. Significantly lower numbers of bacteria, only minor glycosidase activity, high blood group antigenicities of the intestinal glycoproteins, and higher proteolytic activity were found in ileostomy effluents from groups I and II. Histopathological examination showed chronic inflammation and changes in crypt-villus ratio in all dogs with ileal reservoirs; the ileal mucosa from the dogs with conventional ileostomies did not show any abnormalities. Consequences of the flora-related enzyme activities for the ileal mucosa are discussed.
Generation of antibodies reactive with fumonisins B1, B2, and B3 by using cholera toxin as the carrier-adjuvant
Authors: Azcona-Olivera J I , Abouzied M M , Plattner R D , Norred W P , Pestka J J
Published: January 2020

Murine polyclonal antibodies reactive with fumonisins B1, B2, and B3 were produced after a novel immunization procedure with cholera toxin as both a hapten carrier protein and adjuvant. Immunization of mice with two 7.5-micrograms doses of fumonisin B1-cholera toxin conjugate without adjuvant resulted in the production of fumonisin B1-specific antibodies in all mice within 15 days when intraperitoneal, subcutaneous, and intravenous routes were used. In contrast, conventional immunization procedures with fumonisin B1-bovine serum albumin conjugates with and without Freund's adjuvant were largely ineffective. Fumonisin antibodies could be readily mass-produced in ascites fluid by using cholera toxin as a carrier-adjuvant. A competitive indirect enzyme-linked immunosorbent assay (ELISA) was devised whereby immobilized fumonisin B1-ovalbumin and free fumonisin B1 competed for antibody binding. The detection limit for fumonisin B1 in the ELISA was 100 ng/ml. The antiserum cross-reacted with fumonisins B2 and B3 but not with the hydrolyzed backbone of fumonisin B1 and tricarballylic acid. Concentrations of fumonisins B1, B2, and B3 required for 50% binding inhibition were 260, 300, and 650 ng/ml, respectively. These polyclonal antibodies should find wide usage in the ELISA for fumonisins in foods, feeds, and tissues.
Degradation of 2-methylbenzoic acid by Pseudomonas cepacia MB2
Authors: Higson F K , Focht D D
Published: January 2020

We report the isolation of Pseudomonas cepacia MB2, believed to be the first microorganism to utilize 2-methylbenzoic acid as the sole carbon source. Its growth range included all mono- and dimethylbenzoates (with the exception of 2,5- and 2,6-dimethylbenzoates) and 3-chloro-2-methylbenzoate (but not 4- or 5-chloro-2-methylbenzoate) but not chlorobenzoates lacking a methyl group. 2-Chlorobenzoate, 3-chlorobenzoate, and 2,3-, 2,4-, and 3,4-dichlorobenzoates inhibited growth of MB2 on 2-methylbenzoate as a result of cometabolism to the corresponding chlorinated catechols which blocked the key enzyme catechol 2,3-dioxygenase. A metapyrocatechase-negative mutant, MB2-G5, showed accumulation of dimethylcatechols from 2,3- and 3,4-dimethylbenzoates, and phenols were detected in resting-cell transformation extracts bearing the same substitution pattern as the original substrate, presumably following thermal degradation of the intermediate dihydrodiol. 2-Methylphenol was also found in extracts of the mutant cells with 2-methylbenzoate. These observations suggested a major route of methylbenzoate metabolism to be dioxygenation to a carboxy-hydrodiol which then forms a catechol derivative. In addition, the methyl group of 2-methylbenzoate was oxidized to isobenzofuranone (by cells of MB2-G5) and to phthalate (by cells of a separate mutant that could not utilize phthalate, MB2-D2). This pathway also generated a chlorinated isobenzofuranone from 3-chloro-2-methylbenzoate.
Construction of Bordetella pertussis strains that overproduce genetically inactivated pertussis toxin
Authors: Zealey G R , Loosmore S M , Yacoob R K , Cockle S A , Herbert A B , Miller L D , Mackay N J , Klein M H
Published: January 2020

Nontoxic analogs of pertussis toxin (PT), produced by in vitro mutagenesis of the tox operon, are immunogenic and protective against infection by Bordetella pertussis. The moderate levels of PT production by B. pertussis, however, make it the limiting antigen in the formulation of multicomponent, acellular, recombinant whooping cough vaccines. To increase production of the highly detoxified Lys9Gly129 PT analog by B. pertussis, additional copies of the mutated tox operon were integrated into the bacterial chromosome at the tox or fha locus by unmarked allelic exchange. Recombinant strains produced in this way secreted elevated levels of the PT analog proportional to gene dosage. The strains were stable during 10-liter fermentations, and yields of up to 80 mg of PT analog per liter were obtained under production-scale conditions. The nontoxic analog was purified and shown to be indistinguishable from material obtained from a B. pertussis strain that contained only a single copy of the toxLys9Gly129 operon. Such strains are therefore suitable for large-scale, industrial production of an acellular whooping cough vaccine containing a genetically detoxified PT analog.
Interactions between marine bacteria and dissolved-phase and beached hydrocarbons after the Exxon Valdez oil spill
Authors: Button D K , Robertson B R , McIntosh D , Jüttner F
Published: January 2020

Turnover times for toluene in Resurrection Bay after the Exxon Valdez grounding were determined to be decades, longer than expected considering that dissolved hydrocarbons were anticipated to drift with the current and stimulate development of additional hydrocarbon-utilizing capacity among the microflora in that downcurrent location. These turnover times were based on the recovery of 14CO2 from added [14C]toluene that was oxidized. The concentrations of toluene there, 0.1 to 0.2 microgram/liter, were similar to prespill values. Oxidation rates appeared to be enhanced upstream near islands in the wake of the wind-blown slick, and even more within the slick itself. Specific affinities of the water column bacteria for toluene were computed with the help of biomass data, as measured by high-resolution flow cytometry. They were a very low 0.3 to 3 liters/g of cells.h-1, indicating limited capacity to utilize this hydrocarbon. Since current-driven mixing rates exceeded those of oxidation, dissolved spill components such as toluene should enter the world-ocean pool of hydrocarbons rather than biooxidize in place. Some of the floating oil slick washed ashore and permeated a coarse gravel beach. A bacterial biomass of 2 to 14 mg/kg appeared in apparent response to the new carbon and energy source. This biomass was computed from that of the organisms and associated naphthalene oxidation activity washed from the gravel compared with the original suspension. These sediment organisms were very small at approximately 0.06 microns 3 in volume, low in DNA at approximately 5.5 g per cell, and unlike the aquatic bacteria obtained by enrichment culture but quite similar to the oligobacteria in the water column.(ABSTRACT TRUNCATED AT 250 WORDS)
Survival of
Bacillus licheniformis
in Seawater Model Ecosystems
Authors: Nybroe Ole , Christoffersen Kirsten , Riemann Bo
Published: January 2020

The fate of
Bacillus licheniformis
DSM 13 was monitored after introduction into laboratory microcosms and mesocosms established in the Knebel Vig estuary, Denmark. The model organism was detected by a combination of immunofluorescence microscopy and nonselective plating followed by colony blotting. This allowed simultaneous quantification of intact cells and culturable cells.
B. licheniformis
DSM 13 adapted poorly to the conditions in filtered (0.2-μm-pore-size filter) seawater. Results from additional microcosm studies using natural seawater demonstrated that protozoan grazing also was important in regulating the population of the introduced model organism. In experiments using mesocosms,
B. licheniformis
DSM 13 also showed a rapid die-off. The introduction of the organism led to increased nutrient levels and to increased growth of both autotrophic and heterotrophic components of the plankton community compared with those of control enclosures. Thereby, a more intensive predation impact on the bacterioplankton community was induced. The combination of microcosm and mesocosm experiments provides a scenario in which the influence of single biotic and abiotic factors on survival of introduced organisms can be tested and in which the effect of the introduction on ecosystem structure and function can be evaluated. This test concept might prove useful in risk assessment of genetically modified microorganisms.
Metabolism of benzonitrile and butyronitrile by Klebsiella pneumoniae
Authors: Nawaz M S , Heinze T M , Cerniglia C E
Published: January 2020

A strain of Klebsiella pneumoniae that used aliphatic nitriles as the sole source of nitrogen was adapted to benzonitrile as the sole source of carbon and nitrogen. Gas chromatographic and mass spectral analyses of culture filtrates indicated that K. pneumoniae metabolized 8.4 mM benzonitrile to 4.0 mM benzoic acid and 2.7 mM ammonia. In addition, butyronitrile was metabolized to butyramide and ammonia. The isolate also degraded mixtures of benzonitrile and aliphatic nitriles. Cell extracts contained nitrile hydratase and amidase activities. The enzyme activities were higher with butyronitrile and butyramide than with benzonitrile and benzamide, and amidase activities were twofold higher than nitrile hydratase activities. K. pneumoniae appears promising for the bioremediation of sites contaminated with aliphatic and aromatic nitriles.
Accumulation of Copper and Other Metals by Copper-Resistant Plant-Pathogenic and Saprophytic Pseudomonads
Authors: Cooksey Donald A. , Azad Hamid R.
Published: January 2020

Copper-resistant strains of
Pseudomonas syringae
carrying the
cop
operon produce periplasmic copper-binding proteins, and this sequestration outside the cytoplasm has been proposed as a resistance mechanism. In this study, strain PS61 of
P. syringae
carrying the cloned
cop
operon accumulated more total cellular copper than without the operon. Several other copper-resistant pseudomonads with homology to
cop
were isolated from plants, and these bacteria also accumulated copper. Two highly resistant species accumulated up to 115 to 120 mg of copper per g (dry weight) of cells.
P. putida
08891 was more resistant to several metals than
P. syringae
pv. tomato PT23, but this increased resistance was not correlated with an increased accumulation of metals other than copper. Several metals were accumulated by both PT23 and
P. putida
, but when copper was added to induce the
cop
operon, there was generally no increase of accumulation of the other metals, suggesting that the
cop
operon does not contribute to accumulation of these other metals. The exceptions were aluminum for PT23 and iron for
P. putida
, which accumulated to higher levels when copper was added to the cultures. The results of this study support the role of copper sequestration in the copper resistance mechanism of
P. syringae
and suggest that this mechanism is common to several copper-resistant
Pseudomonas
species found on plants to which antimicrobial copper compounds are applied for plant disease control.
Localization of Peptidases in Lactococci
Authors: Tan Paris S. T. , Chapot-Chartier Marie-Pierre , Pos Klaas M. , Rousseau Micheline , Boquien Clair-Yves , Gripon Jean-Claude , Konings Wil N.
Published: January 2020

The localization of two aminopeptidases, an X-prolyl-dipeptidyl aminopeptidase, an endopeptidase, and a tripeptidase in
Lactococcus lactis
was studied. Polyclonal antibodies raised against each purified peptidase are specific and do not cross-react with other peptidases. Experiments were performed by immunoblotting after cell fractionation and by electron microscopy of immunogold-labeled peptidases. All peptidases were found to be intracellular. However, immunogold studies showed a peripheral labeling of the X-prolyl-dipeptidyl aminopeptidase, the tripeptidase, and the endopeptidase. This peripheral location was further supported by the detection of these three enzymes in cell membrane fractions in which none of the two aminopeptidases was present.
Classification of virulent and temperate bacteriophages of Listeria spp. on the basis of morphology and protein analysis
Authors: Zink R , Loessner M J
Published: January 2020

A set of 22 phages of Listeria species (listeriaphages) (21 temperate and 1 virulent) were compared on the basis of morphology and protein composition. All 22 phages had icosahedral heads and exhibited either contractile or noncontractile tails. They represented two different morphotypes. Twenty phages belonged to the Siphoviridae family and could be differentiated only on the basis of tail length. Accordingly, they could be assigned to previously defined listeriaphage species. Two other phages were classified as members of the Myoviridae family, one of which (A511) should be regarded as a new species. It was found to be substantially different from all other known listeriaphages. All phages exhibited typical protein profiles, which were determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and subsequent laser densitometrical analysis of the gels. It was then possible to distinguish eight protein subgroups on the basis of unique protein patterns. This classification corresponds well to the previous groupings based on host range. Most of the phages revealed two or three major proteins ranging from 21 to 24 kDa and 30 to 36 kDa. In addition, at least 10 minor proteins could be observed for each phage. Our results indicate that the major proteins are structural proteins of the capsid and tail, and the protein band ranging from 30 to 35 kDa could clearly be assigned to the proteins of the phage capsid.
Influence of elevated temperature on starch hydrolysis by enterotoxin-positive and enterotoxin-negative strains of Clostridium perfringens type A
Authors: García-Alvarado J S , Rodriguez M A , Labbé R G
Published: January 2020

Enterotoxin-positive (Ent+) and enterotoxin-negative (Ent-) strains of Clostridium perfringens were cultured in Duncan-Strong sporulation medium containing starch at 37 and 46 degrees C. At 37 degrees C, all strains degraded starch and sporulated well. However, only Ent- strains could hydrolyze starch, grow extensively, and sporulate at 46 degrees C. Growth, sporulation, and starch hydrolysis by Ent+ strains at 46 degrees C were equivalent to those obtained at 37 degrees C when alpha-amylase was added to the cultures during growth. The total amount of extracellular plus intracellular amylase in cultures of Ent+ strains was significantly less in cells incubated at 46 degrees C than in cells incubated at 37 degrees C. These results contradict an earlier report that Ent+ strains cannot sporulate well near their optimal growth temperature (R. G. Labbe and C. L. Duncan, Can. J. Microbiol. 20:1493-1501, 1974) and suggest that synthesis of alpha-amylase in Ent+ strains is regulated by temperature.
Cloning and nucleotide sequence of the gene coding for citrate synthase from a thermotolerant Bacillus sp
Authors: Schendel F J , August P R , Anderson C R , Hanson R S , Flickinger M C
Published: January 2020

The structural gene coding for citrate synthase from the gram-positive soil isolate Bacillus sp. strain C4 (ATCC 55182) capable of secreting acetic acid at pH 5.0 to 7.0 in the presence of dolime has been cloned from a genomic library by complementation of an Escherichia coli auxotrophic mutant lacking citrate synthase. The nucleotide sequence of the entire 3.1-kb HindIII fragment has been determined, and one major open reading frame was found coding for citrate synthase (ctsA). Citrate synthase from Bacillus sp. strain C4 was found to be a dimer (Mr, 84,500) with a subunit with an Mr of 42,000. The N-terminal sequence was found to be identical with that predicted from the gene sequence. The kinetics were best fit to a bisubstrate enzyme with an ordered mechanism. Bacillus sp. strain C4 citrate synthase was not activated by potassium chloride and was not inhibited by NADH, ATP, ADP, or AMP at levels up to 1 mM. The predicted amino acid sequence was compared with that of the E. coli, Acinetobacter anitratum, Pseudomonas aeruginosa, Rickettsia prowazekii, porcine heart, and Saccharomyces cerevisiae cytoplasmic and mitochondrial enzymes.
Bacterial Growth in the Cold: Evidence for an Enhanced Substrate Requirement
Authors: Wiebe W. J. , Sheldon W. M. , Pomeroy L. R.
Published: January 2020

Growth responses and biovolume changes for four facultatively psychrophilic bacterial isolates from Conception Bay, Newfoundland, and the Arctic Ocean were examined at temperatures from - 1.5 to 35°C, with substrate concentrations of 0.15, 1.5, and 1,500 mg of proteose peptone-yeast extract per liter. For two cultures, growth in 0.1, 1.0, and 1,000 mg of proline per liter was also examined. At 10 to 15°C and above, growth rates showed no marked effect of substrate concentration, while at - 1.5 and 0°C, there was an increasing requirement for organic nutrients, with generation times in low-nutrient media that were two to three times longer than in high-nutrient media. Biovolume showed a clear dependence on substrate concentration and quality; the largest cells were in the highest-nutrient media. Biovolume was also affected by temperature; the largest cells were found at the lowest temperatures. These data have implications for both food web structure and carbon flow in cold waters and for the effects of global climate change, since the change in growth rate is most dramatic at the lowest temperatures.
Influence of a supplemental carbon source on anaerobic dechlorination of pentachlorophenol in granular sludge
Authors: Hendriksen H V , Larsen S , Ahring B K
Published: January 2020

Anaerobic dechlorination of pentachlorophenol (PCP) was studied in two upflow anaerobic sludge blanket reactors. One reactor received glucose (0.9 g liter-1) as an additional carbon source; the other one served as a control. The concentration of PCP in the medium was 4.5 and 3.0 mg liter-1 in the experimental and control reactors, respectively. The reactors were inoculated with granular sludge previously grown on sugar-containing wastewater. After 10 months of continuous operation, the removal of PCP was 99% in the glucose-amended reactor, whereas the removal in the control reactor varied between 32 and 77%. Furthermore, 94% of the PCP was completely dechlorinated in the glucose reactor compared with a maximum of 20% in the control reactor. In the same period, the amount of biomass in the glucose reactor had increased by approximately 150% compared with that in the control reactor, where no growth of the sludge bed occurred. Batch culture activity tests showed that the addition of glucose had a stimulatory effect on the dechlorination rate of PCP per gram of volatile solids. This indicated that the better performance of the glucose-amended reactor was due to a higher concentration of biomass and a direct stimulatory effect of glucose on the dechlorination rate. The pattern of dechlorination of PCP showed that an initial para cleavage was followed by two ortho cleavages.
Influence of oxidation state on iron binding by Bacillus licheniformis capsule
Authors: McLean R J , Beauchemin D , Beveridge T J
Published: January 2020

We examined ferric (Fe3+) and ferrous (Fe2+) iron binding by the anionic gamma-glutamyl capsule polymer of Bacillus licheniformis ATCC 9945. The addition of FeCl3 to B. licheniformis capsule under aerobic conditions resulted in flocculation due to the capsule-induced formation of amorphous, rust-colored ferrihydrite. Significant binding of iron, which could be attributed to binding by both the anionic capsule and the ferrihydrite precipitate, occurred. In contrast, the addition of FeCl2 to B. licheniformis capsule under anaerobic conditions resulted in significantly less iron being bound and no color change or flocculation occurring. Capsule-bound ferric iron could be partially released upon addition of several reducing agents. From these observations, it can be concluded that the oxidation state of iron significantly influences its tendency to be bound by anionic bacterial polymers such as capsules.
Development of plasmid cloning vectors for Thermus thermophilus HB8: expression of a heterologous, plasmid-borne kanamycin nucleotidyltransferase gene
Authors: Mather M W , Fee J A
Published: January 2020

While several Thermus genes have been cloned and T. thermophilus has been shown to be transformable, molecular genetic studies of these thermophiles have been hampered by the absence of selectable cloning vectors. We have constructed a selectable plasmid by random insertion of a heterologous gene encoding a thermostable kanamycin nucleotidyltransferase activity into a cryptic, multicopy plasmid from T. thermophilus HB8. This plasmid should serve as a suitable starting point for the development of a gene expression system for T. thermophilus.
Existence of a Hydrogen Sulfide:Ferric Ion Oxidoreductase in Iron-Oxidizing Bacteria
Authors: Sugio Tsuyoshi , White Kathy J. , Shute Elizabeth , Choate Donna , Blake Robert C.
Published: January 2020

The existence of a hydrogen sulfide:ferric ion oxidoreductase, which catalyzes the oxidation of elemental sulfur with ferric ions as an electron acceptor to produce ferrous and sulfite ions, was assayed with washed intact cells and cell extracts of various kinds of iron-oxidizing bacteria, such as
Thiobacillus ferrooxidans
13598, 13661, 14119, 19859, 21834, 23270, and 33020 from the American Type Culture Collection,
Leptospirillum ferrooxidans
2705 and 2391 from the Deutsche Sammlung von Mikroorganismen,
L. ferrooxidans
BKM-6-1339 and P3A, and moderately thermophilic iron-oxidizing bacterial strains BC1, TH3, and Alv. It was found that hydrogen sulfide:ferric ion oxidoreductase activity comparable to that of
T. ferrooxidans
AP19-3 was present in all iron-oxidizing bacteria tested, suggesting a wide distribution of this enzyme in iron-oxidizing bacteria.
Survival, stress resistance, and alterations in protein expression in the marine vibrio sp. strain S14 during starvation for different individual nutrients
Authors: Nyström T , Olsson R M , Kjelleberg S
Published: January 2020

The response of the marine Vibrio sp. strain S14 to starvation for carbon, nitrogen, or phosphorus and to simultaneous depletion of all these nutrients (multiple-nutrient starvation) was examined with respect to survival, stress resistance, quantitative and qualitative alterations in protein and RNA synthesis, and the induction of the stringent control. Of the conditions tested, carbon starvation and multiple-nutrient starvation both promoted long-term starvation resistance and a rapid induction of the stringent control, as deduced from the kinetics of RNA synthesis. Carbon- and multiple-nutrient-starved cells were also found to become increasingly resistant to heat, UV, near-UV, and CdCl2 stress. Nitrogen- and phosphorus-starved cells demonstrated a poor ability to survive in the presence of carbon and did not develop a marked resistance to the stresses examined. The carbon, nitrogen, and phosphorus starvation stimulons consisted of about 20 proteins each, while simultaneous starvation for all the nutrients elicited an increased synthesis of 42 polypeptides. Nine common proteins were found to be induced regardless of the starvation condition used and were tentatively termed general starvation proteins. It was also demonstrated that the total number of proteins induced in response to multiple-nutrient starvation was not a predictable sum of the different individual starvation stimulons. Multiple-nutrient starvation induced 14 proteins which were not detected at increased levels of expression in response to individual starvation conditions. Furthermore, four out of five phosphorus starvation-specific polypeptides were not induced during simultaneous starvation for phosphorus, nitrogen, and carbon. The results are discussed in light of the physiological alterations previously described for Vibrio sp. strain S14 cells starved for carbon, nitrogen, and phosphorus simultaneously.
Partial characterization of a DNA restriction endonuclease from Ruminococcus flavefaciens FD-1 and its inhibition by site-specific adenine methylation
Authors: Morrison M , Mackie R I , White B A
Published: January 2020

The principal DNA restriction-modification system of the cellulolytic ruminal bacterium Ruminococcus flavefaciens FD-1 is described. The restriction endonuclease RflFI could be separated from cell extracts by phosphocellulose and heparin-sepharose chromatography. Restriction enzyme digests utilizing RflFI alone or in combination with SalI, a restriction enzyme isolated from Streptomyces albus G, showed that the DNA sequence recognized by RflFI either overlapped or was the same as that recognized by SalI. DNA sequence analysis confirmed that RflFI was identical in activity to SalI, with the recognition sequence being 5'-GTCGAC-3' and cleavage occurring between G and T. Adenine methylation within this sequence can be catalyzed in vitro by TaqI methylase, and this inhibited the cleavage of plasmid DNA molecules by RflFI and SalI. Chromosomal DNA from R. flavefaciens FD-1 is also methylated within this DNA sequence because neither restriction endonuclease could degrade this DNA substrate. These findings provide a means to protect plasmid molecules from degradation prior to gene transfer experiments with R. flavefaciens FD-1.
Evaluation of
Leptospirillum ferrooxidans
for Leaching
Authors: Sand Wolfgang , Rohde Katrin , Sobotke Birgit , Zenneck Claus
Published: January 2020

The importance of
Leptospirillum ferrooxidans
for leach processes has been evaluated by studying the lithotrophic flora of three mine biotopes and a heap leaching operation, by percolation experiments with inoculated, sterilized ore, and by morphological, physiological, and genetic investigations of pure and mixed cultures of
L. ferrooxidans, Thiobacillus ferrooxidans
, and
Thiobacillus thiooxidans.
In biotopes of 20°C or above,
Leptospirillum
-like bacteria are as abundant as
T. ferrooxidans.
Leptospirilli represent at least one-half of the ferrous-iron-oxidizing population. Percolation experiments confirmed this result. Leptospirilli were as numerous as
T. ferrooxidans.
At reduced temperatures, the generation times of leptospirilli increase more so than those of
T. ferrooxidans.
At 14°C,
Leptospirillum
grows slowly and
T. ferrooxidans
dominates the population. Physiological investigations indicate that
L. ferrooxidans
is a strict chemolithoautotroph, metabolizing only ferrous iron and pyrite. Even an addition of 0.05% (wt/vol) yeast extract inhibited its growth. The maximum ferrous-iron-oxidizing activity of
L. ferrooxidans
amounts to about 40% of the activity of
T. ferrooxidans.
After growth on sulfidic ore, both species exhibit reduced iron-oxidizing activities,
L. ferrooxidans
exhibiting one-third and
T. ferrooxidans
exhibiting one-seventh of their maximum activities. Surprisingly, the absolute values are similar. For indirect leaching,
L. ferrooxidans
is as important as
T. ferrooxidans.
This was confirmed by the results of percolation experiments.
L. ferrooxidans
together with
T. thiooxidans
mobilized metals at least as well as
T. ferrooxidans
did. The best results were obtained with a mixed culture of all three species.
Molecular cloning of the isocitrate dehydrogenase gene of an extreme thermophile, Thermus thermophilus HB8
Authors: Miyazaki K , Eguchi H , Yamagishi A , Wakagi T , Oshima T
Published: January 2020

The gene coding for isocitrate dehydrogenase of an extreme thermophile, Thermus thermophilus HB8, was cloned and sequenced. This gene consists of a single open reading frame of 1,485 bp preceded by a Shine-Dalgarno ribosome binding site. Promoter- and terminatorlike sequences were detected upstream and downstream of the open reading frame, respectively. The G + C content of the coding region was 65.6%, and that of the third nucleotide of the codons was 90.3%. On the basis of the deduced amino acid sequence, the Mr of the monomeric enzyme was calculated as 54,189, an Mr which is similar to that of the purified protein determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. A comparison of the amino acid sequence of the T. thermophilus enzyme with that of the Escherichia coli enzyme showed (i) a 37% overall similarity; (ii) the conservation of the Ser residue, which is known to be phosphorylated in the E. coli enzyme, and of the surrounding sequence; and (iii) the presence of 141 extra residues at the C terminus of the T. thermophilus enzyme. T. thermophilus isocitrate dehydrogenase showed a high sequence homology (33% of the amino acid sequence is identical) to isopropylmalate dehydrogenase from the same organism and was suggested to have evolved from a common ancestral enzyme.
A Hydrogen-Oxidizing, Fe(III)-Reducing Microorganism from the Great Bay Estuary, New Hampshire
Authors: Caccavo Frank , Blakemore Richard P. , Lovley Derek R.
Published: January 2020

A dissimilatory Fe(III)- and Mn(IV)-reducing bacterium was isolated from bottom sediments of the Great Bay estuary, New Hampshire. The isolate was a facultatively anaerobic gram-negative rod which did not appear to fit into any previously described genus. It was temporarily designated strain BrY. BrY grew anaerobically in a defined medium with hydrogen or lactate as the electron donor and Fe(III) as the electron acceptor. BrY required citrate, fumarate, or malate as a carbon source for growth on H
2
and Fe(III). With Fe(III) as the sole electron acceptor, BrY metabolized hydrogen to a minimum threshold at least 60-fold lower than the threshold reported for pure cultures of sulfate reducers. This finding supports the hypothesis that when Fe(III) is available, Fe(III) reducers can outcompete sulfate reducers for electron donors. Lactate was incompletely oxidized to acetate and carbon dioxide with Fe(III) as the electron acceptor. Lactate oxidation was also coupled to the reduction of Mn(IV), U(VI), fumarate, thiosulfate, or trimethylamine
n
-oxide under anaerobic conditions. BrY provides a model for how enzymatic metal reduction by respiratory metal-reducing microorganisms has the potential to contribute to the mobilization of iron and trace metals and to the immobilization of uranium in sediments of Great Bay Estuary.
Biochemical and Electron Microscopic Studies of the
Streptomyces reticuli
Cellulase (Avicelase) in Its Mycelium-Associated and Extracellular Forms
Authors: Schlochtermeier André , Niemeyer Frank , Schrempf Hildgund
Published: January 2020

Streptomyces reticuli
is able to grow efficiently with crystalline cellulose (Avicel) as the sole carbon source. Cultivation in the presence of the nonionic detergent Tween 80 at a concentration of 0.1% led to a 10-fold increase in extracellular cellulolytic activity. Under these conditions, one single 82-kDa cellulase (Avicelase) capable of degrading crystalline and soluble cellulose as well as cellodextrins and
p
-nitrophenylcellobioside was purified to apparent homogeneity by a procedure which consisted of two consecutive anion-exchange chromatographies followed by chromatofocusing. Aggregation, which was a major problem during protein purification, could be avoided by including Triton X-100 at a concentration of 0.1% in every chromatographic step. The Avicelase was identified in extracellular and mycelium-associated forms, the latter of which could be released efficiently by nonionic detergents. In addition, a 42-kDa truncated form retaining cellulolytic activity was identified which had been generated from the 82-kDa enzyme by a protease. Antibodies raised against the mycelium-associated Avicelase reacted with the 42-kDa derivative and the extracellular form. The mycelial association of the enzyme was confirmed by immunofluorescence and immunoelectron microscopies.
Purification and Characterization of a Keratinase from a Feather-Degrading
Bacillus licheniformis
Strain
Authors: Lin Xiang , Lee Chung-Ginn , Casale Ellen S. , Shih Jason C. H.
Published: January 2020

A keratinase was isolated from the culture medium of feather-degrading
Bacillus licheniformis
PWD-1 by use of an assay of the hydrolysis of azokeratin. Membrane ultrafiltration and carboxymethyl cellulose ion-exchange and Sephadex G-75 gel chromatographies were used to purify the enzyme. The specific activity of the purified keratinase relative to that in the original medium was approximately 70-fold. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis analysis and Sephadex G-75 chromatography indicated that the purified keratinase is monomeric and has a molecular mass of 33 kDa. The optimum pH and the pI were determined to be 7.5 and 7.25, respectively. Under standard assay conditions, the apparent temperature optimum was 50°C. The enzyme is stable when stored at −20°C. The purified keratinase hydrolyzes a broad range of substrates and displays higher proteolytic activity than most proteases. In practical applications, keratinase is a useful enzyme for promoting the hydrolysis of feather keratin and improving the digestibility of feather meal.
Expression and transfer of engineered catabolic pathways harbored by Pseudomonas spp. introduced into activated sludge microcosms
Authors: Nüsslein K , Maris D , Timmis K , Dwyer D F
Published: January 2020

Two genetically engineered microorganisms (GEMs), Pseudomonas sp. strain B13 FR1(pFRC20P) (FR120) and Pseudomonas putida KT2440(pWWO-EB62) (EB62), were introduced into activated sludge microcosms that had the level of aeration, nutrient makeup, and microbial community structure of activated sludge reactors. FR120 contains an experimentally assembled ortho cleavage route for simultaneous degradation of 3-chlorobenzoate (3CB) and 4-methyl benzoate (4MB); EB62 contains a derivative TOL plasmid-encoded degradative pathway for toluene experimentally evolved so that it additionally processes 4-ethyl benzoate (4EB). Experiments assessed survival of the GEMs, their ability to degrade target substrates, and lateral transfer of plasmid-encoded recombinant DNA. GEMs added at initial densities of 10(6) to 10(7) bacteria per ml of activated sludge declined to stable population densities of 10(4) to 10(5) bacteria per ml. FR120 degraded combinations of 3CB and 4MB (1 mM each) following 3 days of adaptation in the microcosms. Indigenous microorganisms required an 8-day adaptation period before degradation of 4MB was observed; 3CB was degraded only after the concentration of 4MB was much reduced. The indigenous microbial community was killed when both compounds were present at concentrations of 4.0 mM. However, in parallel microcosms containing FR120, the microbial community maintained a normal density of viable cells. Indigenous microbes readily degraded 4EB (2 mM), and EB62 did not significantly increase the observed rate of degradation. In filter matings, transfer of pFRC20P, which specifies mobilization but not transfer functions, from FR120 to P. putida UWC1 was not detectable (< 10(-7) transconjugants per donor cell).(ABSTRACT TRUNCATED AT 250 WORDS)
Use of an oligonucleotide probe to detect Vibrio parahaemolyticus in artificially contaminated oysters
Authors: Lee C , Chen L H , Liu M L , Su Y C
Published: January 2020

A 26-mer oligonucleotide specific to Vibrio parahaemolyticus was synthesized from a 1,275-bp thermostable direct hemolysin (tdh) gene. This oligonucleotide probe specifically reacted with DNA from 89 of 95 V. parahaemolyticus isolates but not with DNA from other vibrios or other enteric and nonenteric organisms (n = 48). The probe hybridized with Southern blots of 0.5-kb HindIII-restricted chromosomal DNA fragments from all but five V. parahaemolyticus test isolates. The probe could be used to directly identify V. parahaemolyticus in artificially contaminated food without an isolation step.
Stabilization of botulinum toxin type A during lyophilization
Authors: Goodnough M C , Johnson E A
Published: January 2020

Botulinum toxin for medical use is diluted to very low concentrations (nanograms per milliliter); when it is preserved by lyophilization, considerable loss of activity can occur. In the present study, conditions that gave > 90% recovery of the toxicity after lyophilization of solutions containing 20 to 1,000 mouse 50% lethal doses per ml were found. Toxicity was recovered upon drying 0.1 ml of toxin solution when the pH was maintained below 7 and bovine or human serum albumins were used as stabilizers. Various other substances tested with albumin, including glucose, sucrose, trehalose, mannitol, glycine, and cellibiose, did not increase recovery on drying.
Discrimination of Listeria monocytogenes from other Listeria species by ligase chain reaction
Authors: Wiedmann M , Czajka J , Barany F , Batt C A
Published: January 2020

A ligase chain reaction assay based on a single-base-pair difference in the V9 region of the 16S rRNA gene (16S rDNA) was developed to distinguish between Listeria monocytogenes and other Listeria species. For this purpose, two pairs of primers were designed, with one primer of each pair being radioactively labeled. The ligated product was separated from the primers by denaturing polyacrylamide gel electrophoresis and then detected by autoradiography. To achieve a higher sensitivity, the 16S rDNA was initially amplified by polymerase chain reaction prior to the ligase chain reaction. The ligase chain reaction was tested on 19 different Listeria species and strains and proved to be a highly specific diagnostic method for the detection of L. monocytogenes.