bluuebunny/crossref_metadata_2025 huggingface dataset:
Cloning and sequencing of agaA, a unique agarase 0107 gene from a marine bacterium, Vibrio sp. strain JT0107
Authors: Sugano Y , Matsumoto T , Kodama H , Noma M
Published: January 2020

An agarase gene (agaA) was cloned from genomic DNA of Vibrio sp. strain JT0107. An open reading frame of 2,985 nucleotides gave a primary translation product composed of the mature protein, agarase 0107 (975 amino acid residues, with a molecular weight of 105,271) and a signal peptide of 20 amino acid residues at the N terminus. Comparison of the deduced amino acid sequence of agarase 0107 with those of Streptomyces coelicolor and Pseudomonas atlantica suggests that these enzymes share two regions in common. The AgaA protein which was expressed in Escherichia coli had the agarase activity. Agarase 0107 hydrolyzes not only agarose but also neoagarotetraose [O-3,6-anhydro-alpha-L-galactopyranosyl (1-->3)-O-beta-D-galactopyranosyl(1-->4)-O-3,6-anhydro-alpha-L-galact opy ranosyl (1-->3)-D-galactose] to yield neoagarobiose [O-3,6-anhydro-alpha-L-galactopyranosyl(1-->3)-D-galactose]. This is a quite unique characteristic for a beta-agarase.
Natural occurrence of Fusarium mycotoxins (trichothecenes and zearalenone) in barley and corn in Korea
Authors: Kim J C , Kang H J , Lee D H , Lee Y W , Yoshizawa T
Published: January 2020

Barley is produced in four provinces, Chonbuk, Chonnam, Kyungbuk, and Kyungnam, and corn is mainly produced in the Kangwon province in Korea. The natural occurrence of Fusarium mycotoxins was surveyed in 39 barley and 46 corn samples from different areas. Five 8-ketotrichothecenes, namely deoxynivalenol (DON), nivalenol (NIV), 4-acetylnivalenol (4-ANIV), 3-acetyldeoxynivalenol (3-ADON), and 4,15-diacetylnivalenol (4,15-DANIV), and zearalenone (ZEA) were detected in barley. DON, NIV, and ZEA were the major contaminants in barley, with mean levels of 170, 1,011, and 287 ng/g, respectively. On the other hand, DON, 15-acetyldeoxynivalenol (15-ADON), NIV, 4-ANIV, 4,15-DANIV, and ZEA were detected in corn samples. DON and 15-ADON were the major contaminants in corn, with mean levels of 310 and 297 ng/g, respectively. The survey indicated that the natural occurrence of monoacetyl-DON and the ratios of NIV to DON in two cereals were different. In addition, this is the first report of the natural occurrence of 4,15-DANIV in cereals.
Heterogeneous Distribution of Microbial Activity in Methanogenic Aggregates: pH and Glucose Microprofiles
Authors: Lens Piet N. L. , De Beer Dirk , Cronenberg Carel C. H. , Houwen Frans P. , Ottengraf Simon P. P. , Verstraete Willy H.
Published: January 2020

Methanogenic aggregates, harvested from an upflow anaerobic sludge blanket reactor treating potato starch wastewater, were acclimatized to either glucose or a mixture of sugars and organic nitrogen compounds (i.e., diluted molasses). Both types of granules exhibited internal pH and substrate concentration gradients in mineral medium (pH 7.0, 30°C) as was measured with microelectrodes. Glucose-acclimatized granules suspended in a mineral medium lacking glucose exhibited a distinct internal pH decrease of about 1 U within the granule, suggesting strong metabolism by the acidogenic bacteria. Molasses-acclimatized and aged granules suspended in mineral medium did not exhibit such a pH decrease, suggesting the importance of the metabolic state of these acidogens. The pH gradient did not occur in deactivated granules and was not observable in strongly buffered media (mineral medium containing 33 mM phosphate or reactor liquid). When glucose (0.5 to 5.0 mM) was added to the mineral medium, granules exhibited a convex pH profile. Glucose consumption was located exclusively in the outer 200 to 300 μm of the aggregates (mean diameter = 1.5 mm). The addition of 20 mM 2-bromoethanesulfonic acid to the mineral medium indicated that the higher pH levels in the centre of the granule appeared to be related to the activity of methanogens. It is suggested that acidogenic activity occurs predominantly in the outer 200 to 300 μm of the aggregate and methanogenic activity occurs predominantly in the center of the investigated granules.
16S rRNA-targeted polymerase chain reaction and oligonucleotide hybridization to screen for Azoarcus spp., grass-associated diazotrophs
Authors: Hurek T , Burggraf S , Woese C R , Reinhold-Hurek B
Published: January 2020

Phylogenetic analyses after reverse transcriptase sequencing of 16S rRNA of nitrogen-fixing, grass-associated Azoarcus strains confirmed their affiliation to the beta subdivision of the Proteobacteria. Strains representing three different species formed a phylogenetically coherent unit related to Rhodocyclus purpureus, with actual percent similarities among the three sequences ranging from 93.1 to 97.3%. Within variable regions V2 and V5, we found stretches of sequences considerably conserved within the genus Azoarcus but differing from most other gram-negative bacteria, with the specificity being enhanced when different regions were combined. Genus-specific primers selected from both regions amplified fragments from all but one Azoarcus species in polymerase chain reactions (PCR) but not from any reference strain tested. Primers of lesser specificity generated fragments from members of all five Azoarcus species as well as from some reference strains. Those unspecific amplifications could be differentiated by oligonucleotide hybridization, detecting only fragments generated from Azoarcus strains except strain 6a3, which represents the same group which could not be detected by genus-specific PCR. Thus we propose the application of PCR amplification with 16S rRNA-targeted, genus-specific primers in combination with hybridization of a 16S rRNA-targeted oligonucleotide to PCR-generated fragments as diagnostic tests; this allows an initial screening for presence of members of the genus Azoarcus.
Transfer of neurotoxigenicity from Clostridium butyricum to a nontoxigenic Clostridium botulinum type E-like strain
Authors: Zhou Y , Sugiyama H , Johnson E A
Published: January 2020

Two Clostridium butyricum strains from infant botulism cases produce a toxic molecule very similar to C. botulinum type E neurotoxin. Chromosomal, plasmid, and bacteriophage DNAs of toxigenic and nontoxigenic strains of C. butyricum and C. botulinum type E were probed with (i) a synthesized 30-mer oligonucleotide encoding part of the L chain of type E botulinum toxin and (ii) the DNA of phages lysogenizing these cultures. The toxin gene probe hybridized to the chromosomal DNA of toxigenic strains but not to their plasmid DNA. All toxigenic and most nontoxigenic strains tested were lysogenized by a prophage on the chromosome. Prophages of toxigenic strains, irrespective of species, had related or identical DNAs which differed from the DNAs of prophages in nontoxigenic strains. The prophage of toxigenic strains was adjacent or close to the toxin gene on the chromosome. Phage DNAs purified from toxigenic strains did not hybridize with the toxin gene probe but could act as the template of the polymerase chain reaction to amplify the toxin gene. The toxin gene was not transferred between C. botulinum and C. butyricum (either direction) when different pairs of a possible gene donor and a recipient strain were grown as mixed cultures. Nontoxigenic C. butyricum or C. botulinum type E-like strains did not become toxigenic when grown in broth containing the phage induced from a toxigenic strain of the other species.(ABSTRACT TRUNCATED AT 250 WORDS)
Distribution of sulfate-reducing bacteria, O2, and H2S in photosynthetic biofilms determined by oligonucleotide probes and microelectrodes
Authors: Ramsing N B , Kühl M , Jørgensen B B
Published: January 2020

The vertical distribution of sulfate-reducing bacteria (SRB) in photosynthetic biofilms from the trickling filter of a sewage treatment plant was investigated with oligonucleotide probes binding to 16S rRNA. To demonstrate the effect of daylight and photosynthesis and thereby of increased oxygen penetration, we incubated two 4-mm-thick biofilm samples in darkness or exposed to light at natural intensity. Gradients of O2, H2S, and pH were examined with microelectrodes during incubation. The samples were subsequently frozen with liquid nitrogen and sliced on a cryomicrotome in 20-microns vertical slices. Fluorescent-dye-conjugated oligonucleotides were used as "phylogenetic" probes to identify single cells in the slices. Oligonucleotide sequences were selected which were complementary to short sequence elements (16 to 20 nucleotides) within the 16S rRNA of sulfate-reducing bacteria. The probes were labeled with fluorescein or rhodamine derivatives for subsequent visualization by epifluorescence microscopy. Five probes were synthesized for eukaryotes, eubacteria, SRB (including most species of the delta group of purple bacteria), Desulfobacter spp., and a nonhybridizing control. The SRB were unevenly distributed in the biofilm, being present in all states from single scattered cells to dense clusters of several thousand cells. To quantify the vertical distribution of SRB, we counted cells along vertical transects through the biofilm. This was done in a blind experiment to ascertain the reliability of the staining. A negative correlation between the vertical distribution of positively stained SRB cells and the measured O2 profiles was found. The distribution differed in light- and dark-incubated samples presumably because of the different extensions of the oxic surface layer. In both cases the SRB were largely restricted to anoxic layers.
Use of 5-cyano-2,3-ditolyl tetrazolium chloride for quantifying planktonic and sessile respiring bacteria in drinking water
Authors: Schaule G , Flemming H C , Ridgway H F
Published: January 2020

Direct microscopic quantification of respiring (i.e., viable) bacteria was performed for drinking water samples and biofilms grown on different opaque substrata. Water samples or biofilms developed in flowing drinking water were incubated with the vital redox dye 5-cyano-2,3-ditolyl tetrazolium chloride (CTC) and R2A medium. One hour of incubation with 0.5 mM CTC was sufficient to obtain intracellular reduction of CTC to the insoluble fluorescent formazan (CTF) product, which was indicative of cellular respiratory (i.e., electron transport) activity. This result was obtained with both planktonic and biofilm-associated cells. Planktonic bacteria were captured on 0.2-microns-pore-size polycarbonate membrane filters and examined by epifluorescence microscopy. Respiring cells containing CTF deposits were readily detected and quantified as red-fluorescing objects on a dark background. The number of CTC-reducing bacteria was consistently greater than the number of aerobic CFU determined on R2A medium. Approximately 1 to 10% of the total planktonic population (determined by counterstaining with 4,6-diamidino-2-phenylindole) were respirometrically active. The proportion of respiring bacteria in biofilms composed of drinking water microflora was greater, ranging from about 5 to 35%, depending on the substratum. Respiring cells were distributed more or less evenly in biofilms, as demonstrated by counterstaining with 4,6-diamidino-2-phenylindole. The amount of CTF deposited in single cells of Pseudomonas putida that formed monospecies biofilms was quantified by digital image analysis and used to indicate cumulative respiratory activity. These data indicated significant cell-to-cell variation in respiratory activity and reduced electron transport following a brief period of nutrient starvation.(ABSTRACT TRUNCATED AT 250 WORDS)
A soluble Bacillus cereus cytochrome P-450cin system catalyzes 1,4-cineole hydroxylations
Authors: Liu W , Rosazza J P
Published: January 2020

A cytochrome P-450-dependent monooxygenase system that catalyzes the stereospecific hydroxylation of the monoterpene substrate 1,4-cineole was demonstrated in cell-free preparations of Bacillus cereus UI-1477. 1,4-Cineole hydroxylations were catalyzed by a 100,000 x g (1-h)-centrifuging soluble, hexane-inducible enzyme that activated and incorporated molecular oxygen into hydroxylated products; required NADH; was inhibited by SKF-525A, imidazole, metyrapone, and octylamine; and displayed a 452-nm peak in the carbon monoxide difference absorption spectrum. The constant 7:1 ratio of endo/exo alcohol products formed when 1,4-cineole was hydroxylated by normal cells, hexane-induced cells, and cell extracts suggested that a single enzyme designated cytochrome P-450cin was responsible for both reactions.
Temporal expression of the major lignin peroxidase genes of Phanerochaete chrysosporium
Authors: Boominathan K , D'Souza T M , Naidu P S , Dosoretz C , Reddy C A
Published: January 2020

DNA probes specific for the genes encoding major lignin peroxidase (LIP) isozymes H2, H8, and H10 of Phanerochaete chrysosporium were constructed. These probes were used to study the temporal expression of the three lip genes in defined low-nitrogen medium. H2 gene transcripts were produced at high levels on days 4, 5, and 7 and at low levels on day 6, while the H8 gene transcripts peaked on day 4 and were produced in substantially lower amounts thereafter. H10 transcripts, on the other hand, peaked on day 4, dropped precipitously on day 5, and were barely detectable on days 6 and 7. There was no precise correlation between lip transcript and isozyme levels.
Effect of Medium Composition on the Denitrification of Nitrate by
Paracoccus denitrificans
Authors: Blaszczyk Mieczyslaw
Published: January 2020

The course of denitrification of nitrate in static cultures of
Paracoccus denitrificans
was studied. Reduction of nitrate to gaseous nitrogen without accumulation of nitrite because of parallel and balanced activities of nitrate and nitrite reductases was observed in nutrient broth. In minimal liquid cultures supplemented with either methanol, acetate, or ethanol as a sole carbon source, substantial amounts of nitrite (up to 70%) accumulated. The reduction in nitrite concentration began just after the transformation of nitrate to nitrite was completed. The addition of some growth factors to minimal media shortened the bacterial biomass doubling time. A correlation coefficient of 0.71 between the doubling time and the amount of accumulated nitrite in cultures was found. My results indicated that the type of denitrification carried out by
P. denitrificans
is not stable and depends on the nutritional composition of the culture medium.
Sodium Dodecyl Sulfate-Polyacrylamide Gel Electrophoresis Protein Banding Patterns among
Rhizobium leguminosarum
biovar phaseoli Strains Isolated from the Mexican Bean
Phaseolus coccineus
Authors: Arredondo-Peter R. , Escamilla E.
Published: January 2020

Several rhizobial strains were isolated from
Phaseolus coccineus
root nodules and were determined to be
Rhizobium leguminosarum
biovar phaseoli strains after reinfection of the same host plant. These strains were characterized by cultural procedures (growth on different carbon sources and intrinsic antibiotic resistance) and electrophoretic procedures (sodium dodecyl sulfate-polyacrylamide gel electrophoresis of total proteins). Our results showed that these rhizobia are very similar to each other, especially in their electrophoretic protein banding patterns, suggesting that they might belong to isolated populations.
Lipase and Esterase Activities of
Propionibacterium freudenreichii
subsp.
freudenreichii
Authors: Dupuis C. , Corre C. , Boyaval P.
Published: January 2020

The lipase and esterase activities of eight strains of dairy
Propionibacterium freudenreichii
subsp.
freudenreichii
were studied. A lipase activity was detected on whole cells and in the culture supernatant. The highest activity was expressed at 45°C and pH 6.8. An esterase activity was also detected in the culture medium. The electrophoresis of the intracellular fractions of the cells revealed from three to six different esterase activities. Two esterases were common to all the strains. The substrate specificity was dependent on each esterase, but no activity was revealed, in our experimental conditions, on ester substrates with a chain length longer than that of butyrate.
Decolorization of Azo, Triphenyl Methane, Heterocyclic, and Polymeric Dyes by Lignin Peroxidase Isoenzymes from
Phanerochaete chrysosporium
Authors: Ollikka Pauli , Alhonmäki Kirsi , Leppänen Veli-Matti , Glumoff Tuomo , Raijola Timo , Suominen Ilari
Published: January 2020

The ligninolytic enzyme system of
Phanerochaete chrysosporium
decolorizes several recalcitrant dyes. Three isolated lignin peroxidase isoenzymes (LiP 4.65, LiP 4.15, and LiP 3.85) were compared as decolorizers with the crude enzyme system from the culture medium. LiP 4.65 (H2), LiP 4.15 (H7), and LiP 3.85 (H8) were purified by chromatofocusing, and their kinetic parameters were found to be similar. Ten different types of dyes, including azo, triphenyl methane, heterocyclic, and polymeric dyes, were treated by the crude enzyme preparation. Most of the dyes lost over 75% of their color; only Congo red, Poly R-478, and Poly T-128 were decolorized less than the others, 54, 46, and 48%, respectively. Five different dyes were tested for decolorization by the three purified isoenzymes. The ability of the isoenzymes to decolorize the dyes in the presence of veratryl alcohol was generally comparable to that of the crude enzyme preparation, suggesting that lignin peroxidase plays a major role in the decolorization and that manganese peroxidase is not required to start the degradation of these dyes. In the absence of veratryl alcohol, the decolorization activity of the isoenzymes was in most cases dramatically reduced. However, LiP 3.85 was still able to decolorize 20% of methylene blue and methyl orange and as much as 60% of toluidine blue O, suggesting that at least some dyes can function as substrates for isoenzyme LiP 3.85 but not to the same extent for LiP 4.15 or LiP 4.65. Thus, the isoenzymes have different specificities towards dyes as substrates.
Stimulation of Ligninolytic Peroxidase Activity by Nitrogen Nutrients in the White Rot Fungus
Bjerkandera
sp. Strain BOS55
Authors: Kaal Erwin E. J. , de Jong Ed , Field Jim A.
Published: January 2020

Bjerkandera
sp. strain BOS55, a newly isolated wild-type white rot fungus, produced lignin peroxidase (LiP) in nitrogen (N)-sufficient glucose-peptone medium, whereas no LiP was detectable in N-limited medium. The production of LiP was induced by the peptide-containing components of this medium and also by soy bean protein. Furthermore, the production of manganese-dependent peroxidase was stimulated by organic N sources, although lower production was also evident in N-limited medium. Further research showed that the induction of LiP depended on the combination of pH and the type of N source. An amino acid mixture and ammonium induced LiP only at either pH 6 or 7.3, respectively. Peptone induced LiP activity at all pH values tested; however, the highest activity was observed at pH 7.3. The results presented here indicate that
Bjerkandera
spp. are distinct from the model white rot fungus,
Phanerochaete chrysosporium
, which produces ligninolytic peroxidases in response to N limitation.
Characterization of the Binding of Gallium, Platinum, and Uranium to
Pseudomonas fluorescens
by Small-Angle X-Ray Scattering and Transmission Electron Microscopy
Authors: Krueger Susan , Olson Gregory J. , Johnsonbaugh David , Beveridge T. J.
Published: January 2020

Small-angle X-ray scattering (SAXS) was used to determine the binding of Ga, U, and Pt to
Pseudomonas fluorescens
in aqueous buffer. Atomic absorption spectrophotometry was used to quantify the heavy metals during bulk analysis, whereas transmission electron microscopy of whole mounts and thin sections was used to determine the locations of the cell-bound metal precipitates, as well as their sizes and physical structures. Energy-dispersive X-ray spectroscopy confirmed the compositions and identities of the precipitates and helped show that they were associated primarily with the envelope layers of the bacteria. Unlike Ga and Pt, which were located only at the cell surface, U was also found intracellularly in ∼ 10% of the cells. This cytoplasmic location ultimately killed and lysed the cells. Surface-bound Ga and U were spread over the entire cell envelope (outer membrane-peptidoglycan-plasma membrane complex), whereas Pt was associated only with the lipopolysaccharide-rich, external face of the outer membrane. SAXS confirmed these data and showed that the bacteria were metal-enshrouded particles that were 1.0 to 1.5 μm in diameter. SAXS also provided a statistically significant representation of the bound metal precipitates, which ranged in size from 10 nm to 1 μm. The correlation between the microscopic data and the scattering data was extremely good. Since SAXS is performed in an aqueous milieu, it yields a more representative picture of the physical state of the metal bound to cell surfaces.
Impact of Chlorine and Heat on the Survival of
Hartmannella vermiformis
and Subsequent Growth of
Legionella pneumophila
Authors: Kuchta John M. , Navratil Jeannine S. , Shepherd Megan E. , Wadowsky Robert M. , Dowling John N. , States Stanley J. , Yee Robert B.
Published: January 2020

Hartmannella vermiformis
, a common amoebal inhabitant of potable-water systems, supports intracellular multiplication of
Legionella pneumophila
and is probably important in the transportation and amplification of legionellae within these systems. To provide a practical guide for decontamination of potable-water systems, we assessed the chlorine and heat resistance of
H. vermiformis. H. vermiformis
cysts and trophozoites were treated independently with chlorine at concentrations of 2.0 to 10.0 ppm for 30 min and then cocultured with
L. pneumophila.
Both cysts and trophozoites were sensitive to concentrations between 2.0 and 4.0 ppm and above (trophozoites somewhat more so than cysts), and 10.0 ppm was lethal to both forms. Hartmannellae treated with chlorine up to a concentration of 4.0 ppm supported the growth of legionellae. To determine whether heat would be an effective addendum to chlorine treatment of amoebae, hartmannellae were subjected to temperatures of 55 and 60°C for 30 min and alternatively to 50°C followed by treatment with chlorine at a concentration of 2 ppm. Fewer than 0.05% of the amoebae survived treatment at 55°C, and there were no survivors at 60°C. Pretreatment at 50°C appeared to make hartmannella cysts more susceptible to chlorine but did not further reduce the concentration of trophozoites.
Quantitative Reverse Sample Genome Probing of Microbial Communities and Its Application to Oil Field Production Waters
Authors: Voordouw Gerrit , Shen Yin , Harrington Clare S. , Telang Anita J. , Jack Thomas R. , Westlake Donald W. S.
Published: January 2020

This paper presents a protocol for quantitative analysis of microbial communities by reverse sample genome probing is presented in which (i) whole community DNA is isolated and labeled in the presence of a known amount of an added internal standard and (ii) the resulting spiked reverse genome probe is hybridized with a master filter on which denatured genomic DNAs from bacterial standards isolated from the target environment were spotted in large amounts (up to 1,500 ng) in order to improve detection sensitivity. This protocol allowed reproducible fingerprinting of the microbial community in oil field production waters at 19 sites from which water and biofilm samples were collected. It appeared that selected sulfate-reducing bacteria were significantly enhanced in biofilms covering the metal surfaces in contact with the production waters.
Effect of Manganese on Lignin Degradation by
Pleurotus ostreatus
during Solid-State Fermentation
Authors: Kerem Zohar , Hadar Yitzhak
Published: January 2020

Lignin degradation by
Pleurotus ostreatus
was studied under solid-state fermentation (SSF) in chemically defined medium containing various levels of Mn. Degradation of [
14
C]lignin prepared from cotton branches to soluble products, as well as its mineralization to
14
CO
2
, was enhanced by the addition of Mn. The effect of malonate on lignin mineralization was most marked during the first 10 days of SSF, in a treatment amended with 73 μM Mn. A high concentration of Mn (4.5 mM) caused inhibition of both fungal growth and mineralization rates during the first 2 weeks of incubation. Addition of malonate reversed this effect because of chelation of Mn. Mn was found to precipitate in all treatments, with or without the addition of malonate. α-Keto-γ-methiolbutyric acid cleavage to ethylene, an indication of
.
OH production, was observed as early as 3 days of incubation in all treatments.
Characterization and structural analysis of the laccase I gene from the newly isolated ligninolytic basidiomycete PM1 (CECT 2971)
Authors: Coll P M , Tabernero C , Santamaría R , Pérez P
Published: January 2020

We have isolated and characterized the cDNA and genomic DNA coding for a phenoloxidase, laccase I, previously purified from culture supernatant of the newly isolated ligninolytic basidiomycete PM1 (CECT 2971). A cDNA library from basidiomycete PM1 was constructed, and laccase-encoding cDNAs were identified by screening with antiserum raised against the purified enzyme. The lac1 gene coding for the laccase was identified in a partial genomic library by using the isolated cDNA as a probe. Nucleotide sequence determination of the full-length cDNA revealed an open reading frame of 1,551 bp encoding a polypeptide of 517 amino acid residues with a putative signal peptide of 21 amino acid residues. Ten small introns interrupted the genomic DNA. A single 1.8-kb transcript mRNA was detected by Northern (RNA) blot analysis, and its 5' end maps to a position 51 bp upstream from the site of initiation of protein synthesis. Eukaryotic regulatory sequences, CAAT and TATA, were observed in the 5' flanking region, which also contains sequences similar to those of copper-regulated proteins. Comparative analysis of the predicted amino acid sequence showed that basidiomycete PM1 laccase I had great similarity to the laccases from Coriolus versicolor, Coriolus hirsutus, and Phlebia radiata.
Uptake Hydrogenase (Hup) in Common Bean (
Phaseolus vulgaris
) Symbioses
Authors: Navarro Rosangela B. , Vargas Alvaro A. T. , Schröder Eduardo C. , van Berkum Peter
Published: January 2020

Strains of
Rhizobium
forming nitrogen-fixing symbioses with common bean were systematically examined for the presence of the uptake hydrogenase (
hup
) structural genes and expression of uptake hydrogenase (Hup) activity. DNA with homology to the
hup
structural genes of
Bradyrhizobium japonicum
was present in 100 of 248 strains examined.
Eco
RI fragments with molecular sizes of approximately 20.0 and 2.2 kb hybridized with an internal
Sac
I fragment, which contains part of both bradyrhizobial
hup
structural genes. The DNA with homology to the
hup
genes was located on pSym of one of the bean rhizobia. Hup activity was observed in bean symbioses with 13 of 30 strains containing DNA homologous with the
hup
structural genes. However, the Hup activity was not sufficient to eliminate hydrogen evolution from the nodules. Varying the host plant with two of the Hup
+
strains indicated that expression of Hup activity was host regulated, as has been reported with soybean, pea, and cowpea strains.
Isolation of a Degeneration-Resistant Mutant of
Clostridium acetobutylicum
NCIMB 8052
Authors: Kashket Eva R. , Cao Zhi-Yi
Published: January 2020

Unless periodically grown from germinated spores,
Clostridium acetobutylicum
tends to degenerate (that is, to spontaneously lose the capacity both to produce solvents and to develop into spores). To obtain mutants that are deficient in degeneration,
C. acetobutylicum
NCIMB 8052 was mated with
Enterococcus faecalis
BM4110 harboring transposon Tn
1545
. We developed a degeneration resistance assay based on a secondary effect of degeneration, the production of toxic levels of acetic and butyric acids. Erythromycin-resistant transconjugant clones were tested individually for longevity by repeated and timely subculturing. One long-lived mutant, A10, survived 18 ± 3 transfers (mean ± standard deviation;
n
= 20) before extinction, while the wild type (parental cells) survived 6.6 ± 1.5 transfers (
n
= 11). The three-fold difference in longevity is statistically significant. In a batch culture in a rich medium, the wild-type cells degenerated within 24 h after inoculation with 1% of an overnight culture derived from germinated spores. In contrast, A10 cells were able to switch to solventogenesis and to sporulate. In a minimal medium with greater buffering capacity, both cell types produced solvents and spores. Southern blots of
Eco
RI and
Hin
dIII restriction digests of A10 chromosomal DNA (but not parental DNA) showed that only one copy of Tn
1545
was inserted into the clostridial chromosome. Our findings are consistent with the hypothesis that there was an alteration at a regulatory locus that was effected by the insertion of the transposon.
Fermentation of lactose by yeast cells secreting recombinant fungal lactase
Authors: Ramakrishnan S , Hartley B S
Published: January 2020

Strains of Saccharomyces cerevisiae transformed with a yeast multicopy expression vector carrying the cDNA for Aspergillus niger secretory beta-galactosidase under the control of ADH1 promoter and terminator were studied for their fermentation properties on lactose (V. Kumar, S. Ramakrishnan, T. T. Teeri, J. K. C. Knowles, and B. S. Hartley, Biotechnology 10:82-85, 1992). Lactose was hydrolyzed extracellularly into glucose and galactose, and both sugars were utilized simultaneously. Diauxic growth patterns were not observed. However, a typical biphasic growth was observed on a mixture of glucose and galactose under aerobic and anaerobic conditions with transformants of a haploid S. cerevisiae strain, GRF167. Polyploid distiller's yeast (Mauri) transformants were selected simply on the basis of the cloned gene expression on X-Gal (5-bromo-4-chloro-3-indolyl-beta-D-galactopyranoside) plates. Rapid and complete lactose hydrolysis and higher ethanol (0.31 g/g of sugar) and biomass (0.24 g/g of sugar) production were observed with distiller's yeast grown under aerobic conditions. A constant proportion (10%) of the population retained the plasmid throughout the fermentation period (48 h). Nearly theoretical yields of ethanol were obtained under anaerobic conditions on lactose, glucose, galactose, and whey permeate media. However, the rate and the amount of lactose hydrolysis were lower under anaerobic than aerobic conditions. All lactose-grown cells expressed partial galactokinase activity.
Hydrophobic and Electrostatic Cell Surface Properties of Thermophilic Dairy Streptococci
Authors: van der Mei H. C. , de Vries J. , Busscher H. J.
Published: January 2020

Microbial adhesion to hydrocarbons (MATH) and microelectrophoresis were done in 10 mM potassium phosphate solutions to characterize the surfaces of thermophilic dairy streptococci, isolated from pasteurizers. Regardless of whether they were grown (in M17 broth) with lactose, sucrose, or glucose added, strains were relatively hydrophilic (showing low initial removal rates by hexadecane) and slightly negatively charged. A tendency exists for cells grown with sucrose added to be more hydrophilic than cells grown with glucose or lactose added. Also, the lowest isoelectric points, i.e., the pH values for which the zeta potentials are zero, were measured for strains with glucose added to the growth medium. The isoelectric points for the strains were all rather high, between pH 3 and 5, indicative of protein-rich surfaces, although X-ray photoelectron spectroscopy did not measure excessively large amounts of nitrogen on the cell surfaces. Both MATH and microelectrophoresis were done as a function of pH. Maxima in hydrophobicity were observed at certain pH values. Usually these pH values were in the range of the isoelectric points of the cells. Thus it appears that MATH measures an interplay of hydrophobicity and electrostatic interactions. MATH measures solely hydrophobicity only when electrostatic interactions are absent, i.e., close to the isoelectric points of the cells. Considering that these thermophilic streptococci are all rather hydrophilic, a possible pathway to prevent fouling in the pasteurization process might be to render the heat exchanger plates of the pasteurizer more hydrophobic.
Monitoring of Bacterial Sugars and Hydroxy Fatty Acids in Dust from Air Conditioners by Gas Chromatography-Mass Spectrometry
Authors: Fox Alvin , Rosario Rose Marie T. , Larsson Lennart
Published: January 2020

Bacterial levels in dust collected from hospital air-conditioning filters were examined by chemical analysis (without prior culture). The dust was analyzed by gas chromatography-mass spectrometry after hydrolysis and derivatization.
l
-Glycero-
d
-mannoheptose and hydroxy fatty acids (3-OH 12:0 and 3-OH 14:0) (primarily found in lipopolysaccharide) and muramic acid (a chemical marker for bacterial peptidoglycan) were present at higher levels in dust collected from filters primarily contacting outdoor (as opposed to indoor) air. The ratio of
l
-glycero-
d
-mannoheptose to muramic acid in dust (compared with those of a group of gram-positive and gram-negative bacteria) suggested that both dust types contained appreciable numbers of gram-negative bacteria. There is potential for the chemical assessment of the microbial content of airborne dust.
Minimal Requirements for Exponential Growth of
Lactococcus lactis
Authors: Jensen Peter Ruhdal , Hammer Karin
Published: January 2020

A minimal growth medium containing glucose, acetate, vitamins, and eight amino acids allowed for growth of
Lactococcus lactis
subsp.
lactis
, with a specific growth rate in batch culture of μ = 0.3 h
-1
. With 19 amino acids added, the growth rate increased to μ = 0.7 h
-1
and the exponential growth phase proceeded until high cell concentrations were reached. We show that morpholinepropanesulfonic acid (MOPS) is a suitable buffer for
L. lactis
and may be applied in high concentrations.
Isolation and characterization of Listeria monocytogenes-specific nucleotide sequences
Authors: Chen J , Brosch R , Luchansky J B
Published: January 2020

Subtracter probe hybridization was used to screen a partial genomic library of a clinical isolate of Listeria monocytogenes. Three clones that hybridized with genomic DNA from 174 strains of L. monocytogenes but not with genomic DNA from 32 strains representing other Listeria spp. were recovered. These data establish the utility of subtracter probe hybridization for recovering L. monocytogenes-specific sequences.
Virus inactivation by copper or iron ions alone and in the presence of peroxide
Authors: Sagripanti J L , Routson L B , Lytle C D
Published: January 2020

Cupric and ferric ions were able to inactivate five enveloped or nonenveloped, single- or double-stranded DNA or RNA viruses. The virucidal effect of these metals was enhanced by the addition of peroxide, particularly for copper(II). Under the conditions of our test, mixtures of copper(II) ions and peroxide were more efficient than glutaraldehyde in inactivating phi X174, T7, phi 6, Junin, and herpes simplex viruses. The substances described here should be able to inactivate most, if not all, viruses that have been found contaminating medical devices.
The Mode of Replication Is a Major Factor in Segregational Plasmid Instability in
Lactococcus lactis
Authors: Kiewiet Rense , Kok Jan , Seegers Jos F. M. L. , Venema Gerard , Bron Sierd
Published: January 2020

The effects of the rolling-circle and theta modes of replication on the maintenance of recombinant plasmids in
Lactococcus lactis
were studied. Heterologous
Escherichia coli
or bacteriophage λ DNA fragments of various sizes were inserted into vectors based on either the rolling-circle-type plasmid pWV01 or the theta-type plasmid pAMβ1. All pAMβ1 derivatives were stably maintained. pWV01 derivatives, however, showed size-dependent segregational instability, in particular when large DNA fragments were inserted. All recombinant pWV01 derivatives generated high-molecular-weight plasmid multimers (HMW) in amounts that were positively correlated with plasmid size and inversely correlated with the copy numbers of the monomeric plasmid forms. Formation of HMW or reductions in copy numbers were not observed with pAMβ1 derivatives. The results indicate that HMW formation and/or reduction in plasmid copy numbers is an important factor in the maintenance of pWV01 derivatives. It is concluded that theta-type plasmids are superior to rolling-circle-type plasmids for cloning in lactococci.
Inhibition of ruminal cellulose fermentation by extracts of the perennial legume cicer milkvetch (Astragalus cicer)
Authors: Weimer P J , Hatfield R D , Buxton D R
Published: January 2020

Cicer milkvetch (Astragalus cicer L.) is a perennial legume used as a pasture or rangeland plant for ruminants. A study was undertaken to determine whether reported variations in its ruminal digestibility may be related to the presence of an antinutritive material. In vitro fermentation of neutral detergent fiber (NDF) of cicer milkvetch by mixed rumen microflora was poorer than was the fermentation of NDF in alfalfa (Medicago sativa L.). Fermentation of cicer milkvetch NDF was improved by preextraction of the ground herbage with water for 3 h at 39 degrees C. Such water extracts selectively inhibited in vitro fermentation of pure cellulose by mixed ruminal microflora and by pure cultures of the ruminal bacteria Ruminococcus flavefaciens FD-1 and Fibrobacter succinogenes S85. Inhibition of the cellulose fermentation by mixed ruminal microflora was dependent upon the concentration of cicer milkvetch extract and was overcome upon prolonged incubation. Pure cultures exposed to the extract did not recover from inhibition, even after long incubation times, unless the inhibitory agent was removed (viz., by dilution of inhibited cultures into fresh medium). The extract did not affect the fermentation of cellobiose by R. flavefaciens but did cause some inhibition of cellobiose fermentation by F. succinogenes. Moreover, the extracts did not inhibit hydrolysis of crystalline cellulose, carboxymethyl cellulose, or p-nitrophenylcellobioside by supernatants of these pure cultures of cellulolytic bacteria or by a commercial cellulase preparation from the fungus Trichoderma reesei. The agent caused cellulose-adherent cells to detach from cellulose fibers, suggesting that the agent may act, at least in part, by disrupting the glycocalyx necessary for adherence to, and rapid digestion of, cellulose.
Xylosylation of Phenolic Hydroxyl Groups of the Monomeric Lignin Model Compounds 4-Methylguaiacol and Vanillyl Alcohol by
Coriolus versicolor
Authors: Kondo Ryuichiro , Yamagami Hikari , Sakai Kokki
Published: January 2020

When 4-methylguaiacol (MeG), a phenolic lignin model compound, was added to a culture that was inoculated with
Coriolus versicolor
, it was bioconverted into 2-methoxy-4-methylphenyl β-
d
-xyloside (MeG-Xyl). The phenolic hydroxyl group of vanillyl alcohol was much more extensively xylosylated than the alcoholic hydroxyl group. When a mixture of MeG and commercial UDP-xylose was incubated with cell extracts of mycelia, transformation of UDP-xylose into MeG-Xyl was observed. This result suggested that UDP-xylosyltransferase was involved in the xylosylation of phenolic hydroxyl groups of lignin model compounds.
Population genetics of human, animal, and environmental Yersinia strains
Authors: Dolina M , Peduzzi R
Published: January 2020

Multilocus enzyme electrophoresis was used to analyze 244 strains of nine Yersinia species isolated from the environment, animals, and humans at 18 genes encoding metabolic enzymes. All 18 enzymes were polymorphic. Among the 137 electrophoretic types (ETs) distinguished, the mean allelic diversity per locus was 0.531. Yersinia frederiksenii ETs were divided into three major clusters that were separated by a large genetic distance, and one ET was more closely related to Yersinia enterocolitica. Thus, strains classically identified as Y. frederiksenii may represent more than one species. Furthermore, two strains identified as Yersinia kristensenii proved to be more closely related to Yersinia mollaretii. Environmental strains formed independent groups. A very interesting ET consisting of as many as 61 isolates of Yersinia enterocolitica was detected, and the epidemiologic relevance of this ET is discussed. Human strains of Y. enterocolitica biotype 4 and Yersinia pseudotuberculosis were recognized as being closely related to animal strains of the same species. Therefore, animal strains of these two species may be considered potential human pathogens.
Methane Consumption in Temperate and Subarctic Forest Soils: Rates, Vertical Zonation, and Responses to Water and Nitrogen
Authors: Adamsen A. P. S. , King G. M.
Published: January 2020

Rates of methane consumption were measured in subarctic coniferous and temperate mixed-hardwood forest soils, using static chambers and intact soil cores. Rates at both sites were generally between 1 and 3 mg of CH
4
m
-2
day
-1
and decreased with increasing soil water contents above 20%. Addition of ammonium (1 μmol g of soil
-1
) strongly inhibited methane oxidation in the subarctic soils; a lesser inhibition was observed for temperate forest samples. The response to nitrogen additions occurred within a few hours and was probably due to physiological changes in the active methane-consuming populations. Methane consumption in soils from both sites was stratified vertically, with a pronounced subsurface maximum. This maximum was coincident with low levels of both nitrate and ammonium in the mixed-hardwood forest soil.
Ergosterol-to-Biomass Conversion Factors for Aquatic Hyphomycetes
Authors: Gessner Mark O. , Chauvet Eric
Published: January 2020

Fourteen strains of aquatic hyphomycete species that are common on decaying leaves in running waters were grown in liquid culture and analyzed for total ergosterol contents. Media included an aqueous extract from senescent alder leaves, a malt extract broth, and a glucose-mineral salt solution. Concentrations of ergosterol in fungal mycelium ranged from 2.3 to 11.5 mg/g of dry mass. The overall average was 5.5 mg/g. Differences among both species and growth media were highly significant but followed no systematic pattern. Stationary-phase mycelium had ergosterol contents 10 to 12% lower or higher than mycelium harvested during the growth phase, but these differences were only significant for one of four species examined. Availability of plant sterols in the growth medium had no clear effect on ergosterol concentrations in two species tested. To convert ergosterol contents determined in field samples to biomass values of aquatic hyphomycetes, a general multiplicative factor of 182 is proposed. More accurate estimates would be obtained with species-specific factors. Using these in combination with estimates of the proportion of the dominant species in a naturally established community on leaves resulted in biomass estimates that were typically 20% lower than those obtained with the general conversion factor. Improvements of estimates with species-specific factors may be limited, however, by intraspecific variability in fungal ergosterol content.
Use of the polymerase chain reaction and fluorescent-antibody methods for detecting viable but nonculturable Shigella dysenteriae type 1 in laboratory microcosms
Authors: Islam M S , Hasan M K , Miah M A , Sur G C , Felsenstein A , Venkatesan M , Sack R B , Albert M J
Published: January 2020

Epidemiological studies of shigellosis in Bangladesh have demonstrated that surface-water sources can act as foci of infection. Studies of laboratory microcosms have shown that shigellae become nonculturable but remain viable when exposed to environmental samples of water. The present study was carried out to detect viable but nonculturable Shigella dysenteriae 1 from laboratory microcosms by the polymerase chain reaction and the fluorescent-antibody techniques. S. dysenteriae 1 was inoculated into laboratory microcosms consisting of water samples collected from ponds, lakes, rivers, and drains in Bangladesh. The survival of S. dysenteriae in microcosms was assessed by viable counting on MacConkey agar. After 2 to 3 weeks, S. dysenteriae 1 became nonculturable but remained viable. After 6 weeks, this nonculturable but viable S. dysenteriae 1 was detected by both the polymerase chain reaction and the fluorescent-antibody methods. The viable but nonculturable state of S. dysenteriae 1 demonstrated in this study may be important for understanding the epidemiology of shigellosis.
Metabolism of tetralin (1,2,3,4-tetrahydronaphthalene) in Corynebacterium sp. strain C125
Authors: Sikkema J , de Bont J A
Published: January 2020

Corynebacterium sp. strain C125, originally isolated on o-xylene, was selected for its ability to grow on tetralin (1,2,3,4-tetrahydronaphthalene) as the sole source of carbon and energy. The catabolism of tetralin in Corynebacterium sp. strain C125 was shown to proceed via initial hydroxylation of the benzene nucleus at positions C-5 and C-6, resulting in the formation of the corresponding cis-dihydro diol. Subsequently, the dihydro diol was dehydrogenated by a NAD-dependent dehydrogenase to 5,6,7,8-tetrahydro-1,2-naphthalene diol. The aromatic ring was cleaved in the extradiol position by a catechol-2,3-dioxygenase. The ring fission product was subject to a hydrolytic attack, resulting in the formation of a carboxylic acid-substituted cyclohexanone. This is the first report of the catabolism of tetralin via degradation of the aromatic moiety.
Microelectrode Measurements of the Activity Distribution in Nitrifying Bacterial Aggregates
Authors: de Beer D. , van den Heuvel J. C. , Ottengraf S. P. P.
Published: January 2020

Microelectrodes for ammonium, oxygen, nitrate, and pH were used to study nitrifying aggregates grown in a fluidized-bed reactor. Local reactant fluxes and distribution of microbial activity could be determined from the microprofiles. The interfacial fluxes of the reactants closely reflected the stoichiometry of bacterial nitrification. Both ammonium consumption and nitrate production were localized in the outer shells, with a thickness of approximately 100 to 120 μm, of the aggregates. Under conditions in which ammonium and oxygen penetrated the whole aggregate, nitrification was restricted to this zone; oxygen was consumed in the central parts of the aggregates as well, probably because of oxidation of dead biomass. A sudden increase of the oxygen concentration to saturation (pure oxygen) was inhibitory to nitrification. The pH profiles showed acidification in the aggregates, but not to an inhibitory level. The distribution of activity was determined by the penetration depth of oxygen during aggregate development in the reactor. Mass transfer was significantly limited by the boundary layer surrounding the aggregates. Microelectrode measurements showed that the thickness of this layer was correlated with the diffusion coefficient of the species. Determination of the distribution of nitrifying activity required the use of ammonium or nitrate microelectrodes, whereas the use of oxygen microelectrodes alone would lead to erroneous results.