bluuebunny/crossref_metadata_2025 huggingface dataset:
Detection of Escherichia coli in sewage and sludge by polymerase chain reaction
Authors: Tsai Y L , Palmer C J , Sangermano L R
Published: January 2020

A method in which the polymerase chain reaction (PCR) was used was developed to amplify either a uidA gene fragment or a 16S rRNA gene fragment from Escherichia coli in sewage and sludge. Because of interference caused by humic acidlike substances, crude DNA extracts were purified with a Sephadex G-200 spun column before the PCR was begun. A Southern analysis in which a nonradioactive chemiluminescent method was used was performed to confirm the presence of PCR products. The sensitivity of detection for PCR products when the chemiluminescent method was used was determined to be 30 ag of E. coli genomic DNA template. In seeded sludge, the PCR amplified the target DNA from 80 E. coli cells per g of sludge and 50 Shigella dysenteriae cells per g of sludge. Because only 0.05 aliquot of a sludge extract was used for the PCR, we deduced that the PCR detected target DNA equivalent to the DNA of 2.5 to 4 cells in the extract. The PCR amplified the uidA fragment from diluted sewage influents and effluents containing E. coli cells. Therefore, the PCR performed with a chemiluminescent gene probe can be used to detect the presence of potentially pathogenic microorganisms in sewage and sludge. This technique can be expanded to permit direct detection of pathogenic microorganisms in water samples, thus leading to enhanced public health protection.
Evaluation of commercial presence-absence test kits for detection of total coliforms, Escherichia coli, and other indicator bacteria
Authors: Clark J A , el-Shaarawi A H
Published: January 2020

Evaluations of several commercial presence-absence (P-A) test kits were performed over a 6-month period in 1990 by using the Ontario Ministry of the Environment (MOE) P-A test for comparison. The general principles of the multiple-tube fermentation technique formed the basis for conducting the product evaluations. Each week, a surface water sample was diluted and inoculated into 25 99-ml dilution blanks for each of three dilutions. The inoculated dilution blanks from each dilution series were randomly sorted into sets of five. Three of these sets were inoculated into the P-A test kits or vice versa, as required. The other two sets were passed through membrane filters, and one set of five membrane filters was placed onto m-Endo agar LES to give replicate total coliform counts and the other set was placed onto m-TEC agar to give replicate fecal coliform results. A statistical analysis of the results was performed by a modified logistic transform method, which provided an improved way to compare binary data obtained from the different test kits. The comparative test results showed that three of the four commercial products tested gave very good levels of recovery and that the fourth commercial product gave only fair levels of recovery when the data were compared with the data from MOE P-A tests and membrane filter tests. P-A bottles showing positive results after 18 h of incubation that were subcultured immediately in ECMUG tubes frequently could be confirmed as containing total coliforms, fecal coliforms, or Escherichia coli after 6 h of incubation; thus, the total incubation time was only 24 h.(ABSTRACT TRUNCATED AT 250 WORDS)
Microgradients of Microbial Oxygen Consumption in a Barley Rhizosphere Model System
Authors: Højberg Ole , Sørensen Jan
Published: January 2020

A microelectrode technique was used to map the radial distribution of oxygen concentrations and oxygen consumption rates around single roots of 7-day-old barley seedlings. The seedlings were grown in gel-stabilized medium containing a nutrient solution, a soil extract, and an inert polymer. Oxygen consumption by microbial respiration in the rhizosphere (<5 mm from the root) and in bulk medium (>30 mm from the root) was determined by using Fick's laws of diffusion and an analytical approach with curve fitting to measured microprofiles of oxygen concentration. A marked increase of microbial respiration was observed in the inner 0- to 3-mm-thick, concentric zone around the root (rhizosphere). The volume-specific oxygen consumption rate (specific activity) was thus 30 to 60 times higher in the innermost 0 to 0.01 mm (rhizoplane) than in the bulk medium. The oxygen consumption rate in the root tissue was in turn 10 to 30 times higher than that in the rhizoplane. Both microbial respiration and oxygen uptake by the root varied between different roots. This was probably due to a between-root variation of the exudation rate for easily degradable carbon compounds supporting the microbial oxygen consumption.
Purification of a 40-kilodalton methyltransferase active in the aflatoxin biosynthetic pathway
Authors: Keller N P , Dischinger H C , Bhatnagar D , Cleveland T E , Ullah A H
Published: January 2020

The penultimate step in the aflatoxin biosynthetic pathway of the filamentous fungi Aspergillus flavus and A. parasiticus involves conversion of sterigmatocystin to O-methylsterigmatocystin. An S-adenosylmethionine-dependent methyltransferase that catalyzes this reaction was purified to homogeneity (> 90%) from 78-h-old mycelia of A. parasiticus SRRC 163. Purification of this soluble enzyme was carried out by five soft-gel chromatographic steps: cell debris remover treatment, QMA ACELL chromatography, hydroxylapatite-Ultrogel chromatography, DEAE-Spherodex chromatography, and Octyl Avidgel chromatography, followed by MA7Q high-performance liquid chromatography. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis of the protein peak from this step on silver staining identified a single band of approximately 40 kDa. This purified protein was distinct from the dimeric 168-kDa methyltransferase purified from the same fungal strain under identical growth conditions (D. Bhatnagar, A. H. J. Ullah, and T. E. Cleveland, Prep. Biochem. 18:321-349, 1988). The chromatographic behavior and N-terminal sequence of the 40-kDa enzyme were also distinct from those of the 168-kDa methyltransferase. The molar extinction coefficient of the 40-kDa enzyme at 278 nm was estimated to be 4.7 x 10(4) M-1 cm-1 in 50 mM potassium phosphate buffer (pH 7.5).
Regulation of the Periplasmic [Fe] Hydrogenase by Ferrous Iron in
Desulfovibrio vulgaris
(Hildenborough)
Authors: Bryant Richard D. , Van Ommen Kloeke Fintan , Laishley Edward J.
Published: January 2020

The periplasmic [Fe] hydrogenase from the sulfate-reducing bacterium
Desulfovibrio vulgaris
(Hildenborough) DSM 8303 was found to be regulated by ferrous iron availability. During growth with 5 ppm of iron, the enzyme derepressed and the specific activity increased approximately fourfold, whereas the presence of 100 ppm of ferrous iron repressed the enzyme. The repression-derepression phenomenon with ferrous iron was found to be operative when the cells were cultured under either hydrogen or nitrogen gas. This is the first reported case showing that the hydrogenase enzyme is regulated by iron, and the implications of this finding relative to the corrosion industry are discussed.
Effects of a lignin peroxidase-expressing recombinant, Streptomyces lividans TK23.1, on biogeochemical cycling and the numbers and activities of microorganisms in soil
Authors: Crawford D L , Doyle J D , Wang Z , Hendricks C W , Bentjen S A , Bolton H , Fredrickson J K , Bleakley B H
Published: January 2020

A recombinant actinomycete, Streptomyces lividans TK23.1, expressing a pIJ702-encoded extracellular lignin peroxidase gene cloned from the chromosome of Streptomyces viridosporus T7A, was released into soil in flask- and microcosm-scale studies to determine its effects on humification and elemental cycling and on the numbers, types, and activities of microorganisms native to the soil. Strain TK23.1 had been shown previously to transiently increase the rate of organic carbon mineralization in soil via an effect that was recombinant specific and particularly significant in nonsterile soils already possessing an active microflora. The results of this study confirmed the previous findings and showed that additional effects were measurable upon release of the recombinant strain TK23.1 into unamended soil and into soil amended with lignocellulose. In addition to a transient enhancement of carbon mineralization, the recombinant affected soil pH, the rate of incorporation of carbon into soil humus fractions, nitrogen cycling, the relative populations of some microbial groups, and also certain soil enzyme activities. Whereas the survival or persistence in soil of the recombinant TK23.1 strain and that of its parent, TK23, were similar, the observed effects on microbial numbers, types, and activities were recombinant specific and did not occur when the parental strain was released into soil. All of the measured effects were transient, generally lasting for only a few days. While the effects were statistically significant, their ecological significance appears to be minimal. This is the first report showing that a recombinant actinomycete can affect the microbial ecology of soil in ways that can be readily monitored by using a battery of microbiological, enzymological, and chemical assays.
Identification and characterization of the proBA operon of Streptococcus bovis
Authors: Campanile C , Forlani G , Basso A L , Marasco R , Ricca E , Sacco M , Ferrara L , De Felice M
Published: January 2020

A genomic DNA library of the rumen bacterium Streptococcus bovis was constructed in Escherichia coli, and recombinant plasmids able to complement proA and proB mutations of the host were found. Southern hybridization and restriction analysis showed that a 3.5-kb fragment of S. bovis DNA contained two genes, organized in an operon and coding for enzymes functionally similar to the glutamyl phosphate reductase-glutamyl kinase enzyme complex that in E. coli catalyzes the first step of proline biosynthesis. Complementation of the E. coli mutations was observed with the fragment inserted in both orientations, which suggested that the S. bovis proBA operon was transcribed from its own promoter. Genetic and biochemical data suggested that the proline biosynthetic pathway of S. bovis is similar to the one previously characterized for E. coli.
Rapid identification of Vibrio vulnificus on nonselective media with an alkaline phosphatase-labeled oligonucleotide probe
Authors: Wright A C , Miceli G A , Landry W L , Christy J B , Watkins W D , Morris J G
Published: January 2020

An oligonucleotide DNA probe (VVAP) was constructed from a portion of the Vibrio vulnificus cytolysin gene (hylA) sequence and labeled with alkaline phosphatase covalently linked to the DNA. Control and environmental isolates probed with VVAP showed an exact correlation with results obtained with a plasmid DNA probe (derived from pCVD702) previously described as having 100% specificity and sensitivity for this organism. Identification of V. vulnificus strains was confirmed independently by analysis of the cellular fatty acid composition and by API 20E. Naturally occurring V. vulnificus bacteria were detected without enrichment or selective media by VVAP in unseeded oyster homogenates and seawater collected from a single site in Chesapeake Bay during June at concentrations of 6 x 10(2) and 2 x 10(1) bacteria per ml, respectively. V. vulnificus bacteria were also enumerated by VVAP in oysters seeded with known concentrations of bacteria and plated on nonselective medium. The VVAP method provides a rapid, accurate means of identifying and enumerating V. vulnificus in seawater and oysters without the use of selective media or additional biochemical tests.
Comparison of methods for estimating the biomass of three food-borne fungi with different growth patterns
Authors: Schnürer J
Published: January 2020

To evaluate the effectiveness of steps taken to reduce the growth of molds in food and feed, methods that can accurately quantify the degree of fungal contamination of solid substrates are needed. In this study, the ergosterol assay has been evaluated by comparing the results of this assay with spore counts and hyphal length measurements made with a microscope and with CFU counts. Three fungi with different growth patterns during cultivation on a synthetic agar substrate were used in these experiments. For the nonsporulating Fusarium culmorum, there was good agreement between changes in hyphal length, CFU, and ergosterol content. Penicillium rugulosum and Rhizopus stolonifer produced many spores, and the production of spores coincided with large increases in CFU but not with increases in hyphal length or ergosterol content. Spores constituted between 3 and 5% of the total fungal mass. Changes in ergosterol level were closely related to changes in hyphal length. It was concluded that ergosterol level is a suitable marker for use in quantitatively monitoring fungal growth in solid substrates.
Karyotypic Variation within Clonal Lineages of the Rice Blast Fungus,
Magnaporthe grisea
Authors: Talbot Nicholas J. , Salch Yangkyo P. , Ma Margery , Hamer John E.
Published: January 2020

We have analyzed the karyotype of the rice blast fungus,
Magnaporthe grisea
, by using pulsed-filed gel electrophoresis. We tested whether the electrophoretic karyotype of an isolate was related to its pathotype, as determined by infection assays, or its genetic lineage, as determined by DNA fingerprinting. Highly reproducible electrophoretic karyotypes were obtained for a collection of U.S. and Chinese isolates representing a diverse collection of pathotypes and genetic lineages. Chromosomes ranged in size from 3 to 10 Mb. Although chromosome number was largely invariant, chromosome length polymorphisms were frequent. Minichromosomes were also found, although their presence was not ubiquitous. They ranged in number from 1 to 3 and in size from 470 kb to 2.2 Mb. Karyotypes were sufficiently variable as to obscure the obvious relatedness of isolates on the basis of pathogenicity assays or genetic lineage analysis by DNA fingerprinting. We documented that the electrophoretic karyotype of an isolate can change after prolonged serial transfer in culture and that this change did not alter the isolate's pathotype. The mechanisms bringing about karyotype variability involve deletions, translocations, and more complex rearrangements. We conclude that karyotypic variability in the rice blast fungus is a reflection of the lack of sexuality in wild populations which leads to the maintenance of neutral genomic rearrangements in clones of the fungus.
Development and use of a selective medium for isolation of Leuconostoc spp. from vegetables and dairy products
Authors: Benkerroum N , Misbah M , Sandine W E , Elaraki A T
Published: January 2020

A selective medium (LUSM medium) for the isolation of Leuconostoc spp. was developed. This medium contained 1.0% glucose, 1.0% Bacto Peptone (Difco), 0.5% yeast extract (BBL), 0.5% meat extract (Difco), 0.25% gelatin (Difco), 0.5% calcium lactate, 0.05% sorbic acid, 75 ppm of sodium azide (Sigma), 0.25% sodium acetate, 0.1% (vol/vol) Tween 80, 15% tomato juice, 30 micrograms of vancomycin (Sigma) per ml, 0.20 microgram of tetracycline (Serva) per ml, 0.5 mg of cysteine hydrochloride per ml, and 1.5% agar (Difco). LUSM medium was used successfully for isolation and enumeration of Leuconostoc spp. in dairy products and vegetables. Of 116 colony isolates obtained from fresh raw milk, curdled milk, or various vegetables, 115 were identified as members of the genus Leuconostoc. A total of 89 of these isolates were identified to species; 13.5% of the isolates were Leuconostoc cremoris, 7.9% were Leuconostoc mesenteroides subsp. mesenteroides, 11.2% were Leuconostoc mesenteroides subsp. dextranicum, 16.9% were Leuconostoc mesenteroides subsp. paramesenteroides, 10.1% were leuconostoc lactis, and 40.4% were Leuconostoc oenos. When we compared the counts obtained for two Leuconostoc strains, Leuconostoc dextranicum 181 and L. cremoris JLL8, on MRS agar and LUSM medium, we found no significant difference between the values obtained on the two media.
Cloning of the beta-amylase gene from Bacillus cereus and characteristics of the primary structure of the enzyme
Authors: Nanmori T , Nagai M , Shimizu Y , Shinke R , Mikami B
Published: January 2020

The gene encoding the beta-amylase of Bacillus cereus BQ10-S1 (SpoII) was cloned into Escherichia coli JM 109. A sequenced DNA fragment of 2,001 bp contains the beta-amylase gene. The N-terminal sequences (AVNGKG MNPDYKAYLMAPLKKI), the C-terminal sequences (SHTSSW), and the amino acid sequences of the five regions in the beta-amylase molecules were determined. The mature beta-amylase contains 514 amino acid residues with a molecular mass of 57,885 Da. The amino acid sequence homology with those of known beta-amylases was 52.7% for Bacillus polymyxa, 52.0% for Bacillus circulans, 43.4% for Clostridium thermosulfurogenes, 31.8% for Arabidopsis thaliana, 31.5% for barley, 29.9% for sweet potato, and 28.9% for soybean. Ten well-conserved regions were found between the N terminus and the area around residue 430, but the C-terminal region of 90 residues has no similarity with those of the plant beta-amylases. The homology search revealed that this C-terminal region has homology with C-terminal regions of the beta-amylase from C. thermosulfurogenes, some bacterial alpha-amylases, cyclodextrin glucanotransferase, and glucoamylase. Some of these sequences are known as the raw-starch-binding domain. These results suggest that B. cereus beta-amylase has an extra domain which has raw-starch-binding ability and that the domain has considerable sequence homology with those of other amylases or related enzymes from a wide variety of microorganisms.
Amounts of viable anaerobes, methanogens, and bacterial fermentation products in feces of rats fed high-fiber or fiber-free diets
Authors: Maczulak A E , Wolin M J , Miller T L
Published: January 2020

We investigated the impact of dietary fiber on the fecal output of microorganisms and microbial fermentation products of rats. Two groups of five male Wistar rats were fed high-fiber (HF) and fiber-free (FF) diets in the following order: (group 1) lab chow-->HF-->FF-->HF and (group 2) lab chow-->FF-->HF-->FF. Daily fecal output of total viable anaerobes was 71 times higher with the HF diet. Daily output of methanogens was 1.4 times higher for the HF diet than for the FF diet. Daily excretion of total fermentation acid products (acetate, propionate, butyrate, lactate, succinate, and formate) was 2.4 and 0.1 mmol for HF and FF diets, respectively. The ratios of acetate/propionate/butyrate were 69:21:10 for the HF diet and 92:7:1 for the FF diet. The results show that an HF diet significantly increases microbial growth in the colon and influences the proportions of organic acid products. The HF diet did not increase the ratio of methanogens to total anaerobes. We suggest that the contribution of host-derived substrates to colonic microbial growth and fermentation is insignificant.
A Method for Detection of Pseudobactin, the Siderophore Produced by a Plant-Growth-Promoting
Pseudomonas
Strain, in the Barley Rhizosphere
Authors: Buyer Jeffrey S. , Kratzke Marian G. , Sikora Lawrence J.
Published: January 2020

Detection in the rhizosphere of the siderophore produced by an inoculated microorganism is critical to determining the role of microbial iron chelators on plant growth promotion. We previously reported the development of monoclonal antibodies (MAb) to ferric pseudobactin, the siderophore of plant-growth-promoting
Pseudomonas
strain B10. One of these MAb reacted less strongly to pseudobactin than to ferric pseudobactin. The MAb reacted to Al(III), Cr(III), Cu(II), and Mn(II) complexes of pseudobactin at a level similar to the level at which it reacted to ferric pseudobactin and reacted less to the Zn(II) complex, but these metals would make up only a small fraction of chelated pseudobactin in soil on the basis of relative abundance of metals and relative binding constants. Fourteen-day-old barley plants grown in limed and autoclaved soil were inoculated with 10
9
CFU of
Pseudomonas
strain Sm1-3, a strain of
Pseudomonas
B10 Rif
r
Nal
r
selected for enhanced colonization, and sampled 3 days later. Extraction and analysis of the roots and surrounding soil using the MAb in an immunoassay indicated a concentration of 3.5 � 10
-10
mol of ferric pseudobacting g
-1
(wet weight). This is the first direct measurement of a pseudobactin siderophore in soil or rhizosphere samples.
Profiling of complex microbial populations by denaturing gradient gel electrophoresis analysis of polymerase chain reaction-amplified genes coding for 16S rRNA
Authors: Muyzer G , de Waal E C , Uitterlinden A G
Published: January 2020

We describe a new molecular approach to analyzing the genetic diversity of complex microbial populations. This technique is based on the separation of polymerase chain reaction-amplified fragments of genes coding for 16S rRNA, all the same length, by denaturing gradient gel electrophoresis (DGGE). DGGE analysis of different microbial communities demonstrated the presence of up to 10 distinguishable bands in the separation pattern, which were most likely derived from as many different species constituting these populations, and thereby generated a DGGE profile of the populations. We showed that it is possible to identify constituents which represent only 1% of the total population. With an oligonucleotide probe specific for the V3 region of 16S rRNA of sulfate-reducing bacteria, particular DNA fragments from some of the microbial populations could be identified by hybridization analysis. Analysis of the genomic DNA from a bacterial biofilm grown under aerobic conditions suggests that sulfate-reducing bacteria, despite their anaerobicity, were present in this environment. The results we obtained demonstrate that this technique will contribute to our understanding of the genetic diversity of uncharacterized microbial populations.
Transformation of 3- and 4-Picoline under Sulfate-Reducing Conditions
Authors: Kaiser J.-P. , Minard R. D. , Bollag J.-M.
Published: January 2020

A microbial population which transformed 3- and 4-picoline under sulfate-reducing conditions was isolated from a subsurface soil which had been previously exposed to different
N
-substituted aromatic compounds for several years. In the presence of sulfate, the microbial culture transformed 3- and 4-picoline (0.4 mM) within 30 days. From the amounts of ammonia released and of sulfide that were determined during the transformation of 3-picoline, it can be concluded that the parent compound was mineralized to carbon dioxide and ammonia. During the transformation of 4-picoline, a UV-absorbing intermediate accumulated in the culture medium. This metabolite was identified as 2-hydroxy-4-picoline by gas chromatography-mass spectrometry and nuclear magnetic resonance analysis, and its further transformation was detected only after an additional month of incubation. The small amount of sulfide produced during the oxidation of 4-picoline and the generation of the hydroxylated metabolite indicated that the initial step in the metabolic pathway of 4-picoline was a monohydroxylation at position 2 of the heterocyclic aromatic ring. The 3- and 4-picoline-degrading cultures could also transform benzoic acid; however, the other methylated pyridine derivatives, 2-picoline, dimethyl-pyridines, and trimethylpyridines, were not degraded.
Metabolic Activity and Population Dynamics of Rhizobia Introduced into Unamended and Bentonite-Amended Loamy Sand
Authors: Heijnen C. E. , Burgers S. L. G. E. , Veen J. A. Van
Published: January 2020

Respiration measurements showed that the cumulative amount of CO
2
respired by rhizobia introduced into sterile bentonite-amended loamy sand was significantly higher than it was in unamended loamy sand. The maintenance respiration of rhizobial cells was not influenced by the presence of bentonite clay. Carbon was used more efficiently during growth in bentonite-amended than in unamended loamy sand. The presence of bentonite clay increased the growth rate of rhizobia introduced into sterile soil. Survival studies performed in nonsterile bentonite-amended loamy sand showed that the use of high (10
10
cells per g of dry soil) rather than lower (10
4
to 10
7
cells per g of dry soil) inoculum densities increased the final survival levels of introduced rhizobia. In unamended loamy sand, the application of 10
10
or 10
7
cells per g of dry soil resulted in similar final survival levels. Pore shape and the continuity of the water-filled pore system were suggested to largely determine the colonization rate of protective microhabitats.
High-level cryIVD and cytA gene expression in Bacillus thuringiensis does not require the 20-kilodalton protein, and the coexpressed gene products are synergistic in their toxicity to mosquitoes
Authors: Chang C , Yu Y M , Dai S M , Law S K , Gill S S
Published: January 2020

Interactions among the 20-kDa protein gene and the cytA and cryIVD genes located in a 9.4-kb HindIII fragment were studied. A series of plasmids containing a combination of these different genes was constructed by using the Escherichia coli/Bacillus thuringiensis shuttle vector pHT3101. The plasmids were then used to transform an acrystalliferous strain, cryB, derived from B. thuringiensis subsp. kurstaki. The results from sodium dodecyl sulfate-polyacrylamide gel electrophoresis and immunoblot analyses suggest that although the 20-kDa protein is required for the efficient CytA protein production in E. coli, it is not required in B. thuringiensis. With or without the truncated 20-kDa protein gene, the CtyA and/or CryIVD proteins are produced and form parasporal inclusions in B. thuringiensis cells. However, more-efficient expression is obtained when a second protein, probably acting as a chaperonin, is present. In addition, the time course studies show that the CytA and CryIVD proteins are coordinately produced. Both the crude B. thuringiensis culture and purified inclusions from each recombinant B. thuringiensis strain are toxic to Culex quinquefasciatus larvae. The parasporal inclusions formed in B. thuringiensis cells are mosquitocidal, with CytA synergizing CryIVD toxicity.
Isolation and characterization of an extracellular glycosylated protein complex from Clostridium thermosaccharolyticum with pectin methylesterase and polygalacturonate hydrolase activity
Authors: Van Rijssel M , Gerwig G J , Hansen T A
Published: January 2020

An extracellular protein complex was isolated from the supernatant of a pectin-limited continuous culture of Clostridium thermosaccharolyticum Haren. The complex possessed both pectin methylesterase (EC 3.1.1.11) and exo-poly-alpha-galacturonate hydrolase (EC 3.2.1.82) activity and produced digalacturonate from the nonreducing end of the pectin chain. The protein consisted of 230- and 25-kDa subunits. The large subunit contained 10% (wt/wt) sugars (N-acetylgalactosamine and galactose). Under physiological conditions both activities acted in a coordinated manner: the ratio between methanol and digalacturonate released during degradation was constant and equal to the degree of esterification of the pectin used. Prolonged incubation of the enzyme with pectin led to a nondialyzable fraction that was enriched in neutral sugars, such as arabinose, rhamnose, and galactose; the high rhamnose/galacturonic acid ratio was indicative of hairy region-like structures. The smallest substrate utilized by the hydrolase was a tetragalacturonate. Vmax with oligogalacturonates increased with increasing chain length. The Km and Vmax for the polygalacturonate hydrolase with citrus pectate as a substrate were 0.8 g liter-1 and 180 mumol min-1 mg of protein-1, respectively. The Km and Vmax for the esterase with citrus pectin as a substrate were 1.2 g liter-1 and 440 mumol min-1 mg of protein-1, respectively. The temperature optima for the hydrolase and esterase were 70 and 60 degrees C, respectively. Both enzyme activities were stable for more than 1 h at 70 degrees C. The exo-polygalacturonate hydrolase of Clostridium thermosulfurogenes was partially purified while the methylesterase was also copurified.
Characterization of
Xanthomonas campestris
Pathovars by rRNA Gene Restriction Patterns
Authors: Berthier Yvette , Verdier Val�rie , Guesdon Jean-Luc , Chevrier Dani�le , Denis Jean-Baptiste , Decoux Guy , Lemattre Monique
Published: January 2020

Genomic DNA of 191 strains of the family
Pseudomonadaceae
, including 187 strains of the genus
Xanthomonas
, was cleaved by
Eco
RI endonuclease. After hybridization of Southern transfer blots with 2-acetylamino-fluorene-labelled
Escherichia coli
16+23S rRNA probe, 27 different patterns were obtained. The strains are clearly distinguishable at the genus, species, and pathovar levels. The variability of the rRNA gene restriction patterns was determined for four pathovars of
Xanthomonas campestris
species. The 16 strains of
X. campestris
pv. begoniae analyzed gave only one pattern. The variability of rRNA gene restriction patterns of
X. campestris
pv. manihotis strains could be related to ecotypes. In contrast, the variability of patterns observed for
X. campestris
pv. malvacearum was not correlated with pathogenicity or with the geographical origins of the strains. The highest degree of variability of DNA fingerprints was observed within
X. campestris
pv. dieffenbachiae, which is pathogenic to several hosts of the
Araceae
family. In this case, variability was related to both host plant and pathogenicity.
Selection of Pseudomonas sp. strain HBP1 Prp for metabolism of 2-propylphenol and elucidation of the degradative pathway
Authors: Kohler H P , van der Maarel M J , Kohler-Staub D
Published: January 2020

A mutant of Pseudomonas sp. strain HBP1, originally isolated on 2-hydroxybiphenyl, was selected for the ability to grow on 2-propylphenol as the sole carbon and energy source. In the mutant strain, which was designated as Pseudomonas sp. strain HBP1 Prp, the cellular induction mechanism involved in the synthesis of the NADH-dependent monooxygenase is changed. 2-Propylphenol, which is known to be a substrate of the monooxygenase, does not induce formation of the monooxygenase in the wild type but does have an induction effect in the mutant strain. Furthermore, in contrast to the wild type, mutant strain HBP1 Prp constitutively produces a small amount of monooxygenase and metapyrocatechase. The enzymes from strain HBP1 Prp catalyzing the first three steps in the degradation of 2-propylphenol--the NADH-dependent monooxygenase, the metapyrocatechase, and the meta fission product hydrolase--were partially purified, and their activities were measured. The product of the monooxygenase activity was identified by mass spectrometry as 3-propylcatechol. The metapyrocatechase used this compound as a substrate and produced a yellow meta fission product that was identified by mass spectrometry as 2-hydroxy-6-oxo-nona-2,4- dienoate. Butyrate could be detected as a product of the meta fission product hydrolase in crude cell extract of 2-propylphenol-grown cells, as well as an intermediate in culture broths during growth on 2-propylphenol. All three enzymes expressed highest activities for the metabolites of the degradation of 2-hydroxybiphenyl.
Viability and Isolation of Marine Bacteria by Dilution Culture: Theory, Procedures, and Initial Results
Authors: Button D. K. , Schut Frits , Quang Pham , Martin Ravonna , Robertson Betsy R.
Published: January 2020

Dilution culture, a method for growing the typical small bacteria from natural aquatic assemblages, has been developed. Each of 11 experimental trials of the technique was successful. Populations are measured, diluted to a small and known number of cells, inoculated into unamended sterilized seawater, and examined three times for the presence of 10
4
or more cells per ml over a 9-week interval. Mean viability for assemblage members is obtained from the frequency of growth, and many of the cultures produced are pure. Statistical formulations for determining viability and the frequency of pure culture production are derived. Formulations for associated errors are derived as well. Computer simulations of experiments agreed with computed values within the expected error, which verified the formulations. These led to strategies for optimizing viability determinations and pure culture production. Viabilities were usually between 2 and 60% and decreased with >5 mg of amino acids per liter as carbon. In view of difficulties in growing marine oligobacteria, these high values are noteworthy. Significant differences in population characteristics during growth, observed by high-resolution flow cytometry, suggested substantial population diversity. Growth of total populations as well as of cytometry-resolved subpopulations sometimes were truncated at levels of near 10
4
cells per ml, showing that viable cells could escape detection. Viability is therefore defined as the ability to grow to that population; true viabilities could be even higher. Doubling times, based on whole populations as well as individual subpopulations, were in the 1-day to 1-week range. Data were examined for changes in viability with dilution suggesting cell-cell interactions, but none could be confirmed. The frequency of pure culture production can be adjusted by inoculum size if the viability is known. These apparently pure cultures produced retained the size and apparent DNA-content characteristic of the bulk of the organisms in the parent seawater. Three cultures are now available, two of which have been carried for 3 years. The method is thus seen as a useful step for improving our understanding of typical aquatic organisms.
Comparison of fluorescent gentamicin-thallous-carbonate and KF streptococcal agars to enumerate enterococci and fecal streptococci in meats
Authors: Knudtson L M , Hartman P A
Published: January 2020

Two selective and differential media were compared for their abilities to enumerate enterococci and fecal streptococci in pork, beef, and poultry products. Counts obtained on KF streptococcal (KF) agar were compared with counts obtained on fluorescent gentamicin-thallous-carbonate (fGTC) agar. Reactions of 13 known enterococcal species were also observed. All 13 species of enterococci as well as Streptococcus bovis and Streptococcus equinus grew equally well on fGTC agar. KF streptococcal medium allowed growth of most species of enterococci but not S. bovis and S. equinus. Quantitative comparisons between the two media inoculated with pure cultures of known species of enterococci revealed equivalent plate counts following incubation. However, when meat samples were plated, counts on fGTC agar were consistently and significantly higher than counts on KF agar for all sample sources.
Effects of Physicochemical Factors and Bacterial Colony Morphotype on Association of
Vibrio vulnificus
with Hemocytes of
Crassostrea virginica
Authors: Harris-Young Linda , Tamplin Mark L. , Fisher William S. , Mason J. Walter
Published: January 2020

Vibrio vulnificus
is a naturally occurring marine bacterium that causes invasive disease of immunocompromised humans following the consumption of raw oysters. It is a component of the natural microbiota of Gulf Coast estuaries and has been found to inhabit tissues of oysters,
Crassostrea virginica
(Gmelin 1791). The interaction of
V. vulnificus
with oyster host defenses has not been reported in detail. We examined the interaction of
V. vulnificus
with phagocytic oyster hemocytes as a function of time, temperature, bacterial concentration, pretreatment with hemolymph, and
V. vulnificus
translucent and opaque colony morphotypes. Within these experimental parameters, the results showed that the association of
V. vulnificus
with hemocytes increased with time, temperature, and initial
V. vulnificus
/hemocyte ratio. Pretreatment of
V. vulnificus
with serum or an increased serum concentration did not enhance
V. vulnificus
-hemocyte associations, a result suggesting the absence of opsonic activity. More than 50% of hemocytes bound the translucent, avirulent morphotype, whereas 10 to 20% were associated with the opaque, virulent form, a result indicating that the degree of encapsulation was related to resistance to phagocytosis, as previously described for mammalian phagocytes. Understanding these cellular interactions may, in part, explain the persistence of
V. vulnificus
in oyster tissues and the ecology of
V. vulnificus
in estuarine environments.
Metabolism of
p
-Cresol by the Fungus
Aspergillus fumigatus
Authors: Jones Kerina H. , Trudgill Peter W. , Hopper David J.
Published: January 2020

The fungus
Aspergillus fumigatus
ATCC 28282 was shown to grow on
p
-cresol as its sole source of carbon and energy. A pathway for metabolism of this compound was proposed. This has protocatechuate as the ring-fission substrate with cleavage and metabolism by an
ortho
-fission pathway. The protocatechuate was formed by two alternative routes, either by initial attack on the methyl group, which is oxidized to carboxyl, followed by ring-hydroxylation, or by ring-hydroxylation as the first step with subsequent oxidation of 4-methylcatechol to the acid. The pathway was elucidated from several pieces of evidence. A number of compounds, including 4-hydroxybenzyl alcohol, 4-hydroxybenzaldehyde, 4-hydroxybenzoic acid, protocatechuic acid, protocatechualdehyde, and 4-methylcatechol, appeared transiently in the medium during growth on
p
-cresol. These compounds were oxidized without lag by
p
-cresol-grown cells but not by succinate-grown cells. Enzyme activities for most of the proposed steps were demonstrated in cell extracts after growth on
p
-cresol, and the products of these activities were identified. None of the activities were found in succinate-grown cells.
Degradation of Degradable Starch-Polyethylene Plastics in a Compost Environment
Authors: Johnson Kenneth E. , Pometto Anthony L. , Nikolov Zivko L.
Published: January 2020

The degradation performance of 11 types of commercially produced degradable starch-polyethylene plastic compost bags was evaluated in municipal yard waste compost sites at Iowa State University (Ames) and in Carroll, Dubuque, and Grinnell, Iowa. Masterbatches for plastic production were provided by Archer Daniels Midland Co. (Decatur, Ill.), St. Lawrence Starch Co. Ltd. (Mississauga, Ontario, Canada), and Fully Compounded Plastics (Decatur, Ill.). Bags differed in starch content (5 to 9%) and prooxidant additives (transition metals and a type of unsaturated vegetable oil). Chemical and photodegradation properties of each material were evaluated. Materials from St. Lawrence Starch Co. Ltd. and Fully Compounded Plastics photodegraded faster than did materials from Archer Daniels Midland Co., whereas all materials containing transition metals demonstrated rapid thermal oxidative degradation in 70°C-oven (dry) and high-temperature, high-humidity (steam chamber) treatments. Each compost site was seeded with test strips (200 to 800 of each type) taped together, which were recovered periodically over an 8- to 12-month period. At each sampling date, the compost row temperature was measured (65 to 95°C), the location of the recovered test strip was recorded (interior or exterior), and at least four strips were recovered for evaluation. Degradation was followed by measuring the change in polyethylene molecular weight distribution via high-temperature gel permeation chromatography. Our initial 8-month study indicated that materials recovered from the interior of the compost row demonstrated very little degradation, whereas materials recovered from the exterior degraded well. In the second-year study, however, degradation was observed in several plastic materials recovered from the interior of the compost row by month 5 at the Carroll site and almost every material by month 12 at the Grinnell site. The plastic bags collected from each community followed a similar degradation pattern. To our knowledge, this is the first scientific study demonstrating significant polyethylene degradation by these materials in a compost environment.
Isolation and Characterization of a Mo
6+
-Reducing Bacterium
Authors: Ghani Baharuddin , Takai Masataka , Hisham N. Zul , Kishimoto Noriaki , Ismail A. K. Mohamed , Tano Tatsuo , Sugio Tsuyoshi
Published: January 2020

A Mo
6+
-reducing bacterium (strain 48), which grew on medium supplemented with 200 mM Mo
6+
, was isolated from stream water obtained from Chengkau, Malaysia. The chemical properties of strain 48 conform to the characteristics of
Enterobacter cloacae
. Under anaerobic conditions in the glucose-yeast extract medium containing phosphate ion (2.9 mM) and Mo
6+
(10 mM), the bacterium reduced Mo
6+
to form molybdenum blue. Approximately 27% of Mo
6+
added to the medium was reduced after 28 h of cultivation. The reduction of Mo
6+
with glucose as an electron donor was strongly inhibited by iodoacetic acid, sodium fluoride, and sodium cyanide, suggesting an involvement of the glycolytic pathway and electron transport in Mo
6+
reduction. NADH and
N,N,N′,N′
-tetramethyl-
p
-phenylenediamine served as electron donors for Mo
6+
reduction. When NADH was used as an electron donor, at first cytochrome
b
in the cell extract was reduced, and then molybdenum blue was formed. Sodium cyanide strongly inhibited Mo
6+
reduction by NADH (5 mM) but not the reduction of cytochrome
b
in the cell extract, suggesting that the reduced component of the electron transport system after cytochrome
b
serves as an electron donor for Mo
6+
reduction. Both ferric and stannous ions strongly enhanced the activity of Mo
6+
reduction by NADH.
Exopolysaccharide production in biofilms: substratum activation of alginate gene expression by Pseudomonas aeruginosa
Authors: Davies D G , Chakrabarty A M , Geesey G G
Published: January 2020

Reporter gene technology was employed to detect the activity of an alginate promoter of Pseudomonas aeruginosa when the organism was grown as a biofilm on a Teflon mesh substratum and as planktonic cells in liquid medium. Alginate biosynthetic activity was determined with a mucoid cell line derived from a cystic fibrosis isolate and containing an alginate algC promoter fused to a lacZ reporter gene. Reporter activity was demonstrated with chromogenic and fluorogenic substrates for beta-galactosidase. Expression of algC was shown to be upregulated in biofilm cells compared with planktonic cells in liquid medium. Gene up-expression correlated with alginate biosynthesis as measured by Fourier transform infrared spectroscopy, uronic acid accumulation, and alginate-specific enzyme-linked immunosorbent assay. The algC promoter was shown to have maximum activity in planktonic cultures during the late lag and early log phases of the cell growth cycle. During a time course experiment, biofilm algC activity exceeded planktonic activity except during the period immediately following inoculation into fresh medium. In continuous-culture experiments, conversion of lacZ substrate was demonstrated microscopically in individual cells by epifluorescence microscopy.
Bacterial Florae in Larvae of the Lake Fly
Chironomus plumosus
Authors: Rouf M. A. , Rigney M. M.
Published: January 2020

Chironomus plumosus
midge larvae were collected from Lake Winnebago, Wisconsin, for 10 weeks in the summer of 1985 and 10 weeks in the summer of 1986 in order to determine their bacterial floras. Altogether, 18 genera and 29 species of bacteria were found and identified. Some spirillum-like bacteria which were not isolated in culture were found by electron microscopy. Scanning electron micrographs of sectioned larvae revealed bacteria throughout the intestines. Gram-positive organisms were most prevalent in the larvae in the early part of the summer, and gram-negative organisms were most prevalent during the later part. Larvae accumulate bacteria within their intestines.
Biodegradability and mechanical properties of poly-(beta-hydroxybutyrate-co-beta-hydroxyvalerate)-starch blends
Authors: Ramsay B A , Langlade V , Carreau P J , Ramsay J A
Published: January 2020

Wheat starch granules and poly-(beta-hydroxybutyrate-co-beta-hydroxyvalerate) [P(HB-co-HV), (19.1 mol% HV)] were blended at 160 degrees C. Increasing the starch content from 0 to 50% (wt/wt) decreased the tensile strength of P(HB-co-HV) from 18 MPa to 8 MPa and diminished flexibility as Young's modulus increased from 1,525 MPa to 2,498 MPa, but overall mechanical properties of the polymer remained in a useful range. A mixed microbial culture required more than 20 days to degrade 150-microns-thick samples of 100% P(HB-co-HV), whereas samples containing 50% (wt/wt) starch disappeared in fewer than 8 days. Starch granules degraded before P(HB-co-HV) did. Aerobic degradation proceeded more rapidly than anaerobic degradation.
Visual Reading Method for Detection of Bacterial Tannase
Authors: Osawa R. , Walsh T. P.
Published: January 2020

Tannase activity of bacteria capable of degrading tannin-protein complexes was determined by a newly developed visual reading method. The method is based on two phenomena: (i) the ability of tannase to hydrolyze methyl gallate to release free gallic acid and (ii) the green to brown coloration of gallic acid after prolonged exposure to oxygen in an alkaline condition. The method has been successfully used to detect the presence of tannase in the cultures of bacteria capable of degrading tannin-protein complexes.
Inhibitory effect of oolong tea polyphenols on glycosyltransferases of mutans Streptococci
Authors: Nakahara K , Kawabata S , Ono H , Ogura K , Tanaka T , Ooshima T , Hamada S
Published: January 2020

Oolong tea extract (OTE) was found to inhibit the water-insoluble glucan-synthesizing enzyme, glucosyltransferase I (GTase-I), of Streptococcus sobrinus 6715. The GTase-inhibitory substance in the OTE was purified successive adsorption chromatography on Diaion HP-21 and HP-20 columns; this was followed by further purification by Sephadex LH-20 column chromatography. A major fraction that inhibited GTase activity (fraction OTF10) was obtained, and the chemical analysis of OTF10 indicated that it was a novel polymeric polyphenol compound that had a molecular weight of approximately 2,000 and differed from other tea polyphenols. Catechins and all other low-molecular-weight polyphenols except theaflavin derived from balck tea did not show significant GTase-inhibitory activities. It was found that OTE amd PTF10 markedly inhibit GTase-I and yeast alpha-glucosidase, but not salivary alpha-amylase. Various GTases purified from S. sobrinus and Streptococcus mutans were examined for inhibition by OTE and OTF10. It was determined that S. sobrinus GTase-I and S. mutans cell-free GTase synthesizing water-soluble glucan were most susceptible to the inhibitory action of OTF10, while S. sobrinus GTase-Sa and S. mutans cell-associated GTase were moderately inhibited; no inhibition of S. sobrinus GTase-Sb was observed. Inhibition of a specific GTase or specific GTases of mutants streptococci resulted in decreased adherence of the growing cells of these organisms. The inhibitory effect of OTF10 on cellular adherence was significantly stronger than that of OTE.
Role of Campylobacter jejuni flagella as colonization factors for three-day-old chicks: analysis with flagellar mutants
Authors: Nachamkin I , Yang X H , Stern N J
Published: January 2020

Campylobacter jejuni, an important cause of human gastrointestinal infection, is a major food-borne pathogen in the United States and worldwide. Since poultry becomes colonized and/or contaminated during the early stages of production and is a major food-borne source for this organism, we studied the role of C. jejuni flagella on the ability of the bacterium to colonize the chicken gastrointestinal tract. Three-day-old chicks were orally challenged with a motile wild-type strain of C. jejuni IN9 or with flagellar mutants created from IN9 by disrupting the flagellin genes with a kanamycin resistance cassette by using shuttle mutagenesis (A. Labigne-Roussel, P. Courcoux, and L. Tompkins, J. Bacteriol. 170:1704-1708, 1988). One mutant, IN9-N3, lacked flagella and was nonmotile. The other, IN9-N7, produced a truncated flagellum and was partially motile. Three-day-old chicks were orally challenged with different doses of the wild-type strain and the two mutants. At challenge doses ranging from 3.0 x 10(4) to 6.6 x 10(8) CFU per chick, only the fully motile, wild-type strain colonized the chick ceca. Our results show that intact and motile flagella are important colonization factors for C. jejuni in chicks.