bluuebunny/crossref_metadata_2025 huggingface dataset:
A medium for presumptive identification of Vibrio anguillarum
Authors: Alsina M , Martínez-Picado J , Jofre J , Blanch A R
Published: January 2020

A medium (VAM) for differentiation of Vibrio anguillarum is described. The presence of bile salts, the high pH, and the high NaCl concentration select mainly for Vibrio species. The high salinity and the ampicillin select for a fraction of Vibrio species, and sorbitol fermentation differentiates among those vibrios still able to grow. One hundred ninety-seven of 227 strains of V. anguillarum were identified with this medium. Only 3 of 66 strains of Vibrio that were not V. anguillarum or V. anguillarum-like were recognized with this medium, and any of 7 non-Vibrio strains related to fish diseases or Escherichia coli grew on the medium. It is our contention that the medium described here constitutes an efficient instrument for presumptive detection of V. anguillarum in pathological and environmental samples.
Inhibitory effects of enrichment media on the Accuprobe test for Listeria monocytogenes
Authors: Partis L , Newton K , Murby J , Wells R J
Published: January 2020

During an evaluation of the Accuprobe kit for the detection of Listeria monocytogenes, some of the enrichment media used were found to interfere with the test. Microscopic examination during the lysis step of the test revealed that media containing high salt greatly reduced or prevented cell lysis. This prevented the probe from binding to the cellular RNA, resulting in false-negative results.
Regulation of extracellular copper-binding proteins in copper-resistant and copper-sensitive mutants of Vibrio alginolyticus
Authors: Harwood V J , Gordon A S
Published: January 2020

Extracellular proteins of wild-type Vibrio alginolyticus were compared with those of copper-resistant and copper-sensitive mutants. One copper-resistant mutant (Cu40B3) constitutively produced an extracellular protein with the same apparent molecular mass (21 kDa) and chromatographic behavior as copper-binding protein (CuBP), a copper-induced supernatant protein which has been implicated in copper detoxification in wild-type V. alginolyticus. Copper-sensitive V. alginolyticus mutants displayed a range of alterations in supernatant protein profiles. CuBP was not detected in supernatants of one copper-sensitive mutant after cultures had been stressed with 50 microM copper. Increased resistance to copper was not induced by preincubation with subinhibitory levels of copper in the wild type or in the copper-resistant mutant Cu40B3. Copper-resistant mutants maintained the ability to grow on copper-amended agar after 10 or more subcultures on nonselective agar, demonstrating the stability of the phenotype. A derivative of Cu40B3 with wild-type sensitivity to copper which no longer constitutively expressed CuBP was isolated. The simultaneous loss of both constitutive CuBP production and copper resistance in Cu40B3 indicates that constitutive CuBP production is necessary for copper resistance in this mutant. These data support the hypothesis that the extracellular, ca. 20-kDa protein(s) of V. alginolyticus is an important factor in survival and growth of the organism at elevated copper concentrations. The range of phenotypes observed in copper-resistant and copper-sensitive V. alginolyticus indicate that altered sensitivity to copper was mediated by a variety of physiological changes.
Increased Nitrogenase-Dependent H
2
Photoproduction by
hup
Mutants of
Rhodospirillum rubrum
Authors: Kern Monika , Klipp Werner , Klemme Jobst-Heinrich
Published: January 2020

Transposon Tn
5
mutagenesis was used to isolate mutants of
Rhodospirillum rubrum
which lack uptake hydrogenase (Hup) activity. Three Tn
5
insertions mapped at different positions within the same 13-kb
Eco
RI fragment (fragment E1). Hybridization experiments revealed homology to the structural hydrogenase genes
hupSLM
from
Rhodobacter capsulatus
and
hupSL
from
Bradyrhizobium japonicum
in a 3.8-kb
Eco
RI-
Cla
I subfragment of fragment E1. It is suggested that this region contains at least some of the structural genes encoding the nickel-dependent uptake hydrogenase of
R. rubrum
. At a distance of about 4.5 kb from the fragment homologous to
hupSLM
, a region with homology to a DNA fragment carrying
hypDE
and
hoxXA
from
B. japonicum
was identified. Stable insertion and deletion mutations were generated in vitro and introduced into
R. rubrum
by homogenotization. In comparison with the wild type, the resulting
hup
mutants showed increased nitrogenase-dependent H
2
photoproduction. However, a mutation in a structural
hup
gene did not result in maximum H
2
production rates, indicating that the capacity to recycle H
2
was not completely lost. Highest H
2
production rates were obtained with a mutant carrying an insertion in a nonstructural
hup
-specific sequence and with a deletion mutant affected in both structural and nonstructural
hup
genes. Thus, besides the known Hup activity, a second, previously unknown Hup activity seems to be involved in H
2
recycling. A single regulatory or accessory gene might be responsible for both enzymes. In contrast to the nickel-dependent uptake hydrogenase, the second Hup activity seems to be resistant to the metal chelator EDTA.
Biochemical characterization of a glutaryl-7-aminocephalosporanic acid acylase from Pseudomonas strain BL072
Authors: Binder R , Brown J , Romancik G
Published: January 2020

Pseudomonas strain BL072 produces an acylase enzyme active in hydrolyzing glutaryl-7-aminocephalosporanic acid to 7-aminocephalosporanic acid. This acylase was purified by column chromatography and gel electrophoresis. The native acylase was composed of two subunits of approximately 65 and 24 kDa, though some heterogeneity was seen in both the native acylase and its small subunit. The isoelectric point of the acylase is approximately 8.5, and it has Km of 1.6 mM for glutaryl desacetoxy aminocephalosporanic acid. The acylase hydrolyzes the desacetoxy and desacetyl derivatives of glutaryl-7-aminocephalosporanic acid at rates similar to that of glutaryl-7-aminocephalosporanic acid. Cephalosporin C was hydrolyzed at a reduced rate. The pH optimum was found to be 8.0, and an activation energy of 9 kcal/mol (ca. 38 kJ/mol) was observed. The acylase has transacylase activity 10 times that of its hydrolytic activity. Eupergit C-immobilized acylase had a half-life of greater than 400 h.
Evolution of a Lytic Bacteriophage via DNA Acquisition from the
Lactococcus lactis
Chromosome
Authors: Moineau Sylvain , Pandian Sithian , Klaenhammer Todd R.
Published: January 2020

We discovered a phage-host interaction in which the lytic phage ul36, in response to pressure exerted by an abortive phage resistance mechanism, acquired a large DNA fragment from the chromosome of
Lactococcus lactis
NCK203 to form a new phage, ul37. Phage ul37 was characterized at morphological, phenotypic, and genotypic levels and was found to be a member of the P335 species. Although it exhibits a high level of DNA homology with ul36, phage ul37 is resistant to the abortive mechanism and has a longer tail, a different base plate, and apparently a different origin of replication. The chromosomal DNA implicated in the formation of new phage ul37 was disrupted by site-specific integration in NCK203. This strategy prevented the appearance of ul37 during subsequent infections with ul36.
Assimilation of Inorganic Nitrogen by Marine Invertebrates and Their Chemoautotrophic and Methanotrophic Symbionts
Authors: Lee Raymond W. , Childress James J.
Published: January 2020

Symbioses between marine invertebrates and their chemoautotrophic and methanotrophic symbionts are now known to exist in a variety of habitats where reduced chemical species are present. The utilization of chemical energy and reliance on C
1
compounds by these symbioses are well documented. Much less is known about their metabolism of nitrogen. Earlier work has shown that the tissues of organisms in these associations are depleted of
15
N compared with those of other marine organisms, indicating that local sources of nitrogen are assimilated and that novel mechanisms of nitrogen metabolism may be involved. Although these symbioses have access to rich sources of ammonium (NH
4
+
and NH
3
) and/or nitrate, several investigators have proposed that N
2
fixation may account for some of these isotope values. Here we report that [
15
N]ammonium and, to a lesser degree, [
15
N]nitrate are assimilated into organic compounds by
Solemya reidi
, a gutless clam containing S-oxidizing bacteria, and seep mussel Ia, an undescribed mytilid containing methanotrophic bacteria. In contrast,
Riftia pachyptila
, the giant hydrothermal vent tube worm symbiotic with S-oxidizing bacteria, assimilated nitrate but not exogenous ammonium. The rates of assimilation of these sources are sufficient to at least partially support C
1
compound metabolism. N
2
assimilation was not exhibited by the symbionts tested.
Molecular characterization of lactococcal bacteriophage Tuc2009 and identification and analysis of genes encoding lysin, a putative holin, and two structural proteins
Authors: Arendt E K , Daly C , Fitzgerald G F , van de Guchte M
Published: January 2020

Bacteriophage Tuc2009 is a temperate bacteriophage with a small isometric head and is isolated from Lactococcus lactis subsp. cremoris UC509. The phage genome is packaged by a headful mechanism, giving rise to circularly permuted molecules with terminal redundancy. The unit genome size is approximately 39 kb. A map of the phage genome on which several determinants could be localized was constructed: pac, the site of initiation of DNA packaging; lys (1,287 bp), specifying the phage lysin; S (267 bp), specifying a putative holin; and mp1 (522 bp) and mp2 (498 bp), each specifying one of the phage's structural proteins. lys, S, mp1, and mp2 were further characterized. lys and S are partially overlapping and appear to be part of one operon. The lysin shows homology to the lysins of the Streptococcus pneumoniae phages Cp-9, Cp-1, and Cp-7. The putative holin, which is thought to be involved in the release of lysin from the cytoplasm, contains two strongly hydrophobic presumptive transmembrane domains and a highly charged C-terminal domain.
Mode of Action of Nisin Z against
Listeria monocytogenes
Scott A Grown at High and Low Temperatures
Authors: Abee Tjakko , Rombouts Frank M. , Hugenholtz Jeroen , Guihard Gilles , Letellier Lucienne
Published: January 2020

Nisin Z, a natural nisin variant, was recently isolated from
Lactococcus lactis
subspecies
lactis
NIZO 22186. The gene for this lantibiotic, designated
nisZ
, has been cloned, and its nucleotide sequence was found to be identical to that of the precursor nisin gene with the exception of a single mutation resulting in the substitution of Asn-27 for His-27 in the mature polypeptide (J. W. M. Mulders, I. J. Boerrigter, H. S. Rollema, R. J. Siezen, and W. M. de Vos, Eur. J. Biochem. 201:581-584, 1991). A K
+
electrode was used to investigate the effect of various environmental parameters on the action of nisin Z against
Listeria monocytogenes
. Addition of nisin Z resulted in immediate loss of cell K
+
, depolarization of the cytoplasmic membrane, inhibition of respiratory activity, and hydrolysis and partial efflux of cellular ATP. The action of nisin Z was optimal at pH 6.0 and was significantly reduced by di- and trivalent cations. The lanthanide gadolinium (Gd
3+
) was an efficient inhibitor and prevented nisin Z activity completely at a concentration of 0.2 mM. Nisin Z-induced loss of cell K
+
was reduced at low temperatures, presumably as a result of the increased ordering of the lipid hydrocarbon chains in the cytoplasmic membrane. In cells grown at 30°C, the action of nisin Z was prevented below 7°C, whereas in cells grown at 4°C nisin Z was able to induce K
+
leakage at this low temperature.
Pyrrolnitrin Production by Biological Control Agent
Pseudomonas cepacia
B37w in Culture and in Colonized Wounds of Potatoes
Authors: Burkhead Karen D. , Schisler David A. , Slininger Patricia J.
Published: January 2020

Bacterial strain B37w (= NRRL B-14858), an isolate noteworthy because it inhibits the growth of the bioherbicide fungus
Colletotrichum truncatum
, was selected for further studies of bacterial antifungal properties. This isolate was identified as a
Pseudomonas cepacia
strain by performing carbohydrate utilization and fatty acid profile analyses, as well as other biochemical and physiological tests. Petri plate assays revealed that strain B37w exhibited antifungal activity against the potato dry rot fungus
Fusarium sambucinum.
Using bioautography, we correlated antifungal activity with production of a specific compound. Isolation from strain B37w and identification of the antifungal antibiotic pyrrolnitrin are described. A whole-potato assay revealed B37w's ability to colonize potato wounds. Wounded potatoes were inoculated with B37w, and pyrrolnitrin was detected in these potatoes by thin-layer chromatography-bioautography at a concentration on the order of nanograms per wound. We performed an assay in which we examined efficacy against
F. sambucinum
-incited potato dry rot and found that B37w inhibited disease development. This is the first report of
P. cepacia
or pyrrolnitrin activity against the economically important potato pathogen
F. sambucinum.
Use of
Lactobacillus plantarum
LPCO10, a Bacteriocin Producer, as a Starter Culture in Spanish-Style Green Olive Fermentations
Authors: Ruiz-Barba J. L. , Cathcart D. P. , Warner P. J. , Jiménez-Díaz R.
Published: January 2020

Bacteriocin-producing
Lactobacillus plantarum
LPCO10 and its non-bacteriocin-producing, bacteriocinimmune derivative,
L. plantarum
55-1, were evaluated separately for growth and persistence in natural Spanish-style green olive fermentations. Both strains were genetically marked and selectively enumerated using antibiotic-containing media. Plasmid profile and bacteriocin production (bac
+
) were used as additional markers. When olive brines were inoculated at 10
5
CFU/ml, the parent strain, LPCO10, proliferated to dominate the epiphytic microflora, sharing high population levels with other spontaneously occurring lactobacilli and persisting throughout the fermentation (12 weeks). In contrast, the derivative strain could not be isolated after 7 weeks. Stability of both plasmid profile and bac
+
(LPCO10 strain) or bac
-
(55-1 strain) phenotype was shown by
L. plantarum
LPCO10 and
L. plantarum
55-1 isolated throughout the fermentation. Bacteriocin activity could be found in the
L. plantarum
LPCO10-inoculated brines only after ammonium sulfate precipitation and concentration (20 times) of the final brine. Spontaneously occurring lactobacilli and lactic coccus populations, which were isolated from each of the fermenting brines studied during this investigation, were shown to be sensitive to the bacteriocins produced by
L. plantarum
LPCO10 when tested by the drop diffusion test. The declines in both pH and glucose levels throughout the fermentative process were similar in
L. plantarum
LPCO10- and in
L. plantarum
55-1-inoculated brines and were comparable to the declines in the uninoculated brines. However, the final concentration of lactic acid in
L. plantarum
LPCO10-inoculated brines was higher than in the
L. plantarum
55-1-inoculated brines and uninoculated brines. These results indicated that
L. plantarum
LPCO10 may be useful as a starter culture to control the lactic acid fermentation of Spanish-style green olives.
The DeLey-Doudoroff Pathway of Galactose Metabolism in
Azotobacter vinelandii
Authors: Wong T. Y. , Yao X.-T.
Published: January 2020

Azotobacter vinelandii
cell extracts reduced NAD
+
and oxidized
d
-galactose to galactonate that subsequently was converted to 2-keto-3-deoxy-galactonate. Further metabolism of 2-keto-3-deoxy-galactonate required the presence of ATP and resulted in the formation of pyruvate and glyceraldehyde 3-P. Radiorespirometry indicated a preferential release of CO
2
at the first carbon position of the
d
-galactose molecule. This suggested that
Azotobacter vinelandii
metabolizes
d
-galactose via the DeLey-Doudoroff pathway. The first enzyme of this pathway,
d
-galactose dehydrogenase, was partially characterized. It has a molecular weight of about 74,000 Da and an isoelectric point of 6.15. The pH optimum of the galactose dehydrogenase was about 9. The apparent

K
m

s for NAD
+
and
d
-galactose were 0.125 and 0.56 mM, respectively. Besides
d
-galactose, the active fraction of this galactose dehydrogenase also oxidized
l
-arabinose effectively. The electron acceptor for
d
-galactose or
l
-arabinose oxidation, NAD
+
, could not be replaced by NADP
+
. These substrate specificities were different from those reported in
Pseudomonas saccharophila, Pseudomonas fluorescens,
and
Rhizobium meliloti.
Characterization of Monoclonal Antibodies Specific for
Erwinia carotovora
subsp.
atroseptica
and Comparison of Serological Methods for Its Sensitive Detection on Potato Tubers
Authors: Gorris María Teresa , Alarcon Benito , Lopez María M. , Cambra Mariano
Published: January 2020

Seven monoclonal antibodies (MAbs) to
Erwinia carotovora
subsp.
atroseptica
have been produced. One, called 4G4, reacted with high specificity for serogroup I of
E. carotovora
subsp.
atroseptica
, the most common serogroup on potato tubers in different serological assays. Eighty-six strains belonging to different
E. carotovora
subsp.
atroseptica
serogroups were assayed. Some strains of serogroup XXII also reacted positively. No cross-reactions were observed against other species of plant pathogenic bacteria or 162 saprophytic bacteria from potato tubers. Only one strain of
E. chrysanthemi
from potato cross-reacted. A comparison of several serological techniques to detect
E. carotovora
subsp.
atroseptica
on potato tubers was performed with MAb 4G4 or polyclonal antibodies. The organism was extracted directly from potato peels of artificially inoculated tubers by soaking or selective enrichment under anaerobiosis in a medium with polypectate. MAb 4G4 was able to detect specifically 240
E. carotovora
subsp.
atroseptica
cells per ml by indirect immunofluorescence and immunofluorescence colony staining and after soaking by ELISA-DAS (double-antibody sandwich enzyme-linked immunosorbent assay) after enrichment. The same amount of cells was detected by using immunolectrotransfer with polyclonal antibodies, and
E. carotovora
subsp.
atroseptica
and subsp.
carotovora
were distinguished by the latter technique. ELISA-DAS using MAb 4G4 with an enrichment step also efficiently detected
E. carotovora
subsp.
atroseptica
in naturally infected tubers and plants.
Uncoupling of Bacterioplankton and Phytoplankton Production in Fresh Waters Is Affected by Inorganic Nutrient Limitation
Authors: Le Jianhua , Wehr John D. , Campbell Lori
Published: January 2020

Pelagic bacterial production is often positively correlated, or coupled, with primary production through utilization of autotrophically produced dissolved organic carbon. Recent studies indicate that inorganic N or P can directly limit both bacterial and phytoplanktonic growth. Our mesocosm experiments, with whole communities from mesotrophic Calder Lake, test whether this apparent bacterial-algal coupling may be the result of independent responses to limiting inorganic nutrients. In systems without N additions, numbers of bacteria but not phytoplankton increased 2- to 2.5-fold in response to P fertilization (0 to 2.0 μmol of P per liter); this resulted in uncoupled production patterns. In systems supplemented with 10 μmol of NH
4
NO
3
per liter, P addition resulted in up to threefold increases in bacteria and two- to fivefold increases in total phytoplankton biomass (close coupling). P limitation of pelagic bacteria occurred independently of phytoplankton dynamics, and regressions between bacterial abundance and phytoplankton chlorophyll
a
were nonsignificant in all systems without added N. We describe a useful and simple coupling index which predicts that shifts in phytoplankton and bacterioplankton growth will be unrelated (Δ bacteria/Δ phytoplankton → either + ∞ or - ∞) in systems with inorganic N/P (molar) ratios of <∼40. In systems with higher N/P ratios (>40), the coupling index will approach 1.0 and close coupling between bacteria and phytoplankton is predicted to occur.
Effect of a Pseudomonas rhamnolipid biosurfactant on cell hydrophobicity and biodegradation of octadecane
Authors: Zhang Y , Miller R M
Published: January 2020

In this study, the effect of a purified rhamnolipid biosurfactant on the hydrophobicity of octadecane-degrading cells was investigated to determine whether differences in rates of octadecane biodegradation resulting from the addition of rhamnolipid to four strains of Pseudomonas aeruginosa could be related to measured differences in hydrophobicity. Cell hydrophobicity was determined by a modified bacterial adherence to hydrocarbon (BATH) assay. Bacterial adherence to hydrocarbon quantitates the preference of cell surfaces for the aqueous phase or the aqueous-hexadecane interface in a two-phase system of water and hexadecane. On the basis of octadecane biodegradation in the absence of rhamnolipid, the four bacterial strains were divided into two groups: the fast degraders (ATCC 15442 and ATCC 27853), which had high cell hydrophobicities (74 and 55% adherence to hexadecane, respectively), and the slow degraders (ATCC 9027 and NRRL 3198), which had low cell hydrophobicities (27 and 40%, respectively). Although in all cases rhamnolipid increased the aqueous dispersion of octadecane at least 10(4)-fold, at low rhamnolipid concentrations (0.6 mM), biodegradation by all four strains was initially inhibited for at least 100 h relative to controls. At high rhamnolipid concentrations (6 mM), biodegradation by the fast degraders was slightly inhibited relative to controls, but the biodegradation by the slow degraders was enhanced relative to controls. Measurement of cell hydrophobicity showed that rhamnolipids increased the cell hydrophobicity of the slow degraders but had no effect on the cell hydrophobicity of the fast degraders. The rate at which the cells became hydrophobic was found to depend on the rhamnolipid concentration and was directly related to the rate of octadecane biodegradation.(ABSTRACT TRUNCATED AT 250 WORDS)
In vivo nuclear magnetic resonance study of the osmoregulation of phosphocholine-substituted beta-1,3;1,6 cyclic glucan and its associated carbon metabolism in Bradyrhizobium japonicum USDA 110
Authors: Pfeffer P E , Bécard G , Rolin D B , Uknalis J , Cooke P , Tu S
Published: January 2020

A phosphocholine-substituted beta-1,3;1,6 cyclic glucan (PCCG), an unusual cyclic oligosaccharide, has been isolated from Bradyrhizobium japonicum USDA 110 (D. B. Rolin, P. E. Pfeffer, S. F. Osman, B. S. Swergold, F. Kappler, and A. J. Benesi, Biochim. Biophys. Acta 1116:215-225, 1992). Data presented here suggest that PCCG synthesis is dependent on the carbon metabolism and that osmotic regulation of its biosynthesis parallels regulation of membrane-derived oligosaccharide biosynthesis observed in Escherichia coli (E. P. Kennedy, M. K. Rumley, H. Schulman, and L. M. G. van Golde, J. Biol. Chem. 251:4208-4213, 1976) and Agrobacterium tumefaciens (G. A. Cangelosi, G. Martinetti, and E. W. Nester, J. Bacteriol. 172:2172-2174, 1990). Growth of B. japonicum USDA 110 cells in the reference medium at relatively low osmotic pressures (LO) (65 mosmol/kg of H2O) caused a large accumulation of PCCG and unsubstituted beta-1,3;1,6 cyclic glucans (CG). Sucrose and polyethylene glycol, nonionic osmotica, reduce all growth rates and inhibit almost completely the production of PCCG at high osmotic pressures (HO) above 650 and 400 mosmol/kg of H2O), respectively. We used in vivo 13C nuclear magnetic resonance spectroscopy to identify the active osmolytes implicated in the osmoregulation process. The level of alpha,alpha-trehalose in B. japonicum cells grown in autoclaved or filter-sterilized solutions remained constant in HO (0.3 M sucrose or 250 g of polyethylene glycol 6000 per liter) medium. Significant amounts of glycogen and extracellular polysaccharides were produced only when glucose was present in the autoclaved HO 0.3 M sucrose media. The results of hypo- and hyperosmotic shocking of B. japonicum USDA 110 cells were monitored by using in vivo 31P and 13C nuclear magnetic resonance spectroscopy. The first observed osmoregulatory response of glycogen-containing cells undergoing hypoosmotic shock was release of P(i) into the medium. Within 7 h, reabsorption of P(i) was complete and production of PCCG was initiated. After 12 h, the PCCG content had increased by a factor of 7. Following the same treatment, cells containing little or no glycogen released trehalose and failed to produce PCCG. Thus the production of PCCG/CG in response to hypoosmotic shocking of stationary-phase cells was found to be directly linked to the interconversion of stored glycogen. Hyperosmotic shocking of LO-grown stationary-phase cells with sucrose had no effect on the content of previously synthesized CG/PCCG. The PCCG/CG content and its osmotically induced biosynthesis are discussed in terms of carbon metabolism and a possible role in hypoosmotic adaptation in B. japonicum USDA 110.
Efficacy of Chemical Disinfectants against Turbot Aquareovirus
Authors: Rivas Carmen , Bandín Isabel , Cepeda Catalina , Dopazo Carlos P.
Published: January 2020

The susceptibility of turbot aquareovirus to five chemical agents was examined. Treatment with 5 mg of malachite green per liter or 500 mg of iodine per liter resulted in a 90% reduction in virus titer within 1 h. Complete inactivation within 5 min was obtained with 2% formalin, 42.5% isopropanol, or 15 mg of free available chlorine per liter. Lower concentrations of chlorine were ineffective.
Microbial Transformation of Nitroaromatics in Surface Soils and Aquifer Materials
Authors: Bradley P. M. , Chapelle F. H. , Landmeyer J. E. , Schumacher J. G.
Published: January 2020

Microorganisms indigenous to surface soils and aquifer materials collected at a munitions-contaminated site transformed 2,4,6-trinitrotoluene (TNT), 2,4-dinitrotoluene (2,4-DNT), and 2,6-dinitrotoluene (2,6-DNT) to amino-nitro intermediates within 20 to 70 days. Carbon mineralization studies with both unlabeled (TNT, 2,4-DNT, and 2,6-DNT) and radiolabeled ([
14
C]TNT) substrates indicated that a significant fraction of these source compounds was degraded to CO
2
.
Purification and partial amino acid sequence of curvaticin FS47, a heat-stable bacteriocin produced by Lactobacillus curvatus FS47
Authors: Garver K I , Muriana P M
Published: January 2020

Curvaticin FS47, a bacteriocin produced by Lactobacillus curvatus FS47, is inhibitory to Listeria monocytogenes, as well as Lactobacillus, Pediococcus, Enterococcus, and Bacillus spp. The bacteriocin was purified by 40% ammonium sulfate precipitation, solid-phase extraction, and reversed-phase high-pressure liquid chromatography. Purified curvaticin FS47 was determined to be 4.07 kDa by mass spectrometry and was partially sequenced. Thirty-one N-terminal amino acids were identified; the curvaticin FS47 protein sequence did not show homology to the pediocin-like group of bacteriocins.
Analysis of fluorescent pseudomonads based on 23S ribosomal DNA sequences
Authors: Christensen H , Boye M , Poulsen L K , Rasmussen O F
Published: January 2020

The regions from positions 1391 to 1545 and 1620 to 1865 (Escherichia coli numbers) of the 23S ribosomal DNA sequences have been analyzed for a number of Pseudomonas fluorescens and P. putida isolates. Variability was observed only in three smaller regions from positions 1484 to 1508, 1531 to 1542, and 1714 to 1748, corresponding to helices 58, 59, and 63, respectively, where up to 53% dissimilarity was found. Sequence analysis did not allow clear distinctions among P. fluorescens biovars, P. chlororaphis, and P. putida biovar B.
Effect of Biocides on MS2 and K Coliphages
Authors: Maillard J.-Y. , Beggs T. S. , Day M. J. , Hudson R. A. , Russell A. D.
Published: January 2020

Several biocides commonly used in disinfection processes as antibacterial and antifungal agents were tested for activity against MS2 and K coliphages. MS2 was resistant to most biocides; only glutaraldehyde (0.5%) and peracetic acid (1%) achieved a 4-log
10
titer reduction in 20 min. In contrast, K phage was sensitive to most biocides, being resistant only to phenol (2%) and chlorhexidine (1%).
Identification and characterization of clustered genes for thermostable xylan-degrading enzymes, beta-xylosidase and xylanase, of Bacillus stearothermophilus 21
Authors: Baba T , Shinke R , Nanmori T
Published: January 2020

Bacillus stearothermophilus 21 is a gram-positive, facultative thermophilic aerobe that can utilize xylan as a sole source of carbon. We isolated this strain from soil, purified its extracellular xylanase and beta-xylosidase, and analyzed the two-step degradation of xylan by these enzymes (T. Nanmori, T. Watanabe, R. Shinke, A. Kohno, and Y. Kawamura, J. Bacteriol. 172:6669-6672, 1990). An Escherichia coli transformant carrying a 4.2-kbp chromosomal segment of this bacterium as a recombinant plasmid was isolated. It excreted active beta-xylosidase and xylanase into the culture medium. The plasmid was introduced into UV-sensitive E. coli CSR603, and its protein products were analyzed by the maxicell method. Proteins harboring beta-xylosidase and xylanase activities were identified, and their molecular masses were estimated by sodium dodecyl sulfate-polyarylamide gel electrophoresis to be 75 and 40 kDa, respectively. The values were identical to those of proteins prepared from cells of B. stearothermophilus 21. The genes for both enzymes were encoded in a 3.4-kbp PstI fragment derived from the 4.2-kbp chromosomal segment. The nucleotide sequence of the 4.2-kbp segment was accordingly determined. The beta-xylosidase gene (xylA) is located upstream of the xylanase gene (xynA) with a possible promoter and a Shine-Dalgarno sequence. The latter gene is preceded by two possible promoters and a Shine-Dalgarno sequence that are located within the 3'-terminal coding region of the former. The two genes thus appear to be, at least partly, expressed independently, which was experimentally confirmed in E. coli by deletion analysis.(ABSTRACT TRUNCATED AT 250 WORDS)
Desulfonation of Linear Alkylbenzenesulfonate Surfactants and Related Compounds by Bacteria
Authors: Kertesz Michael A. , Kölbener Pius , Stockinger Hermann , Beil Stefan , Cook Alasdair M.
Published: January 2020

Pseudomonas putida
S-313 (= DSM 6884) grew in sulfate-free medium when the sole sulfur source supplied was one of several arylsulfonates involved in the synthesis, application, or biodegradation of linear alkyl-benzenesulfonate (LAS) surfactants. 2-(4-Sulfophenyl)butyric acid, 4-
n
-butyl-1-methyl-6-sulfotetralin, and 4-toluenesulfonic acid were each completely utilized during growth, as were the model LAS 1-(4-sulfophenyl) octane and the arylsulfonate dyestuff Orange II. The product in each case was the corresponding phenol, which was identified by gas chromatography-mass spectrometry or
1
H nuclear magnetic resonance. Stoichiometric conversion of 4-toluenesulfonic acid to 4-cresol was observed. The molar growth yields observed were 2.4 to 2.8 kg of protein per mol of S, which were comparable to the yield for sulfate. Commercial LAS disappeared from growth medium inoculated with strain S-313, but negligible growth occurred; digestion of cells in alkali led to recovery of the LAS mixture, which seemingly sorbed to the cells. However, mixed culture L6 was readily obtained from batch enrichment cultures containing commercial LAS as a sole sulfur source and an inoculum from domestic sewage. Culture L6 desulfonated components of the LAS surfactant to the corresponding phenols, which were identified by gas chromatography-mass spectrometry. Compounds with shorter alkyl chains were desulfonated preferentially, as were the centrally substituted isomers. In the presence of 200 μM sulfate, culture L6 grew well and LAS disappeared, although this was due purely to sorption, as shown by digestion of the cells in alkali. Thus, under sulfate-limited conditions, LAS can be desulfonated directly.
Detection of Phanerochaete chrysosporium in soil by PCR and restriction enzyme analysis
Authors: Johnston C G , Aust S D
Published: January 2020

A nonradioactive method to detect Phanerochaete chrysosporium grown in a soil matrix was developed. This method involved DNA extraction, PCR amplification, and restriction enzyme analysis. Amplification of ligninase H8 DNA from pure cultures of P. chrysosporium was not as sensitive as amplification of the internal transcribed spacer (ITS) of the highly repetitive nuclear ribosomal DNA. Amplified ITS DNA was digested with restriction enzymes for analysis. The restriction enzyme pattern of PCR-amplified ITS DNA of P. chrysosporium was unique compared with those of unrelated fungi. Two strains of Phanerochaete chrysosporium and two strains of Phanerochaete sordida were indistinguishable by restriction enzyme analysis, while a third strain of P. chrysosporium had an unique pattern. These results were confirmed by sequence information and indicate that species designations of Phanerochaete spp. should be reexamined. The restriction enzyme pattern of DNA extracted and PCR amplified from P. chrysosporium grown in soil was identical to that from P. chrysosporium grown in pure culture. The ITS sequence was detected in 14 ng of the 100 micrograms of total DNA extracted from 1 g of soil.
Expression of superoxide dismutase in Listeria monocytogenes
Authors: Vasconcelos J A , Deneer H G
Published: January 2020

The nature and expression of superoxide dismutase (SOD; EC 1.15.1.1) in the gram-positive food-borne pathogen Listeria monocytogenes were examined. Metal depletion and reconstitution studies and resistance to H2O2 and potassium cyanide inactivation indicated that L. monocytogenes has a single SOD which utilizes manganese as a metal cofactor. The specific activity of SOD was unchanged in cells exposed to a heat shock at 42 degrees C or grown in the presence of paraquat-generated superoxide anion or of metal chelators in the medium. SOD levels increased, however, as the cells progressed through the logarithmic phase of growth and into the stationary phase. Furthermore, SOD activity decreased with decreasing growth temperatures and declined concurrently with decreased growth when higher concentrations of sodium chloride were added to the medium. Cells grown anaerobically possessed relatively high levels of SOD, although these levels were about 10 to 30% lower than those of aerobically grown bacteria. Different isolates of L. monocytogenes were found to produce approximately equivalent levels of SOD, although greater differences in SOD expression were seen among other species of Listeria. When compared with L. monocytogenes, for example, Listeria welshimeri typically produced about 30% greater SOD activity, whereas Listeria murrayi produced about 60% less total SOD activity. Although all species of Listeria produced a single Mn-type SOD, differences in the relative electrophoretic mobility of the native enzymes were noted. These data suggest that the single L. monocytogenes SOD enzyme is constitutively produced in response to many environmental factors and may also be responsive to the cellular growth rate.
Genetic diversity of Datisca cannabina-compatible Frankia strains as determined by sequence analysis of the PCR-amplified 16S rRNA gene
Authors: Mirza M S , Hameed S , Akkermans A D
Published: January 2020

The presence of Frankia strains in soil samples collected from northern areas of Pakistan was detected by inoculating Coriaria nepalensis and Datisca cannabina plants. The abundance of compatible Frankia strains in some areas was indicated by profuse nodulation of the host plants, whereas soil samples from other localities failed to result in nodulation. An oligonucleotide probe (COR/DAT) directed against the 16S rRNA gene of the endophytes of Coriaria and Datisca spp. that did not cross-react with the RNA gene of Frankia strains isolated from other hosts was developed. Genetic diversity among Frankia strains nodulating D. cannabina was determined by sequence analysis of the partial 16S rRNA gene amplified from nodules induced by soil samples from different localities by PCR. Four types of Frankia sequences and one non-Frankia sequence were detected by hybridization with a Frankia genus probe and the COR/DAT probe as well as by sequence analysis of the cloned PCR products.
Disinfection of human enteric viruses in water by copper and silver in combination with low levels of chlorine
Authors: Abad F X , Pintó R M , Diez J M , Bosch A
Published: January 2020

The efficacy of copper and silver ions, in combination with low levels of free chlorine (FC), was evaluated for the disinfection of hepatitis A virus (HAV), human rotavirus (HRV), human adenovirus, and poliovirus (PV) in water. HAV and HRV showed little inactivation in all conditions. PV showed more than a 4 log10 titer reduction in the presence of copper and silver combined with 0.5 mg of FC per liter or in the presence of 1 mg of FC per liter alone. Human adenovirus persisted longer than PV with the same treatments, although it persisted significantly less than HRV or HAV. The addition of 700 micrograms of copper and 70 micrograms of silver per liter did not enhance the inactivation rates after the exposure to 0.5 or 0.2 mg of FC per liter, although on some occasions it produced a level of inactivation similar to that induced by a higher dose of FC alone. Virus aggregates were observed in the presence of copper and silver ions, although not in the presence of FC alone. Our data indicate that the use of copper and silver ions in water systems may not provide a reliable alternative to high levels of FC for the disinfection of viral pathogens. Gene probe-based procedures were not adequate to monitor the presence of infectious HAV after disinfection. PV does not appear to be an adequate model viral strain to be used in disinfection studies. Bacteroides fragilis bacteriophages were consistently more resistant to disinfection than PV, suggesting that they would be more suitable indicators, although they survived significantly less than HAV or HRV.
Determination and Distribution of
cry
-Type Genes of
Bacillus thuringiensis
Isolates from Taiwan
Authors: Chak K.-F. , Chao D.-C. , Tseng M.-Y. , Kao S.-S. , Tuan S.-J. , Feng T.-Y.
Published: January 2020

Using PCR with a set of specific oligonucleotide primers to detect
cryI
-type genes, we were able to screen the
cry
-type genes of 225
Bacillus thuringiensis
soil isolates from Taiwan without much cost in time or labor. Some combinations of
cry
genes (the
cry
-type profile) in a single isolate were unique. We identified five distinct profiles of crystal genes from the
B. thuringiensis
soil isolates from Taiwan. The
cry
genes included
cryIA
(
a
),
cryIA
(
b
),
cryIA
(
c
),
cryIC, cryID
, and
cryIV
. Interestingly, 501
B. thuringiensis
isolates (93.5% of the total number that we identified) were isolated from areas at high altitudes. The profiles of
cry
-type genes were distinct in all isolation areas. The distribution of
cry
-type genes of our isolates therefore depended on geography. Using PCR footprinting to detect
cryIC
-type genes, we identified two distinct
cryIC
footprints from some of our isolates, indicating that these isolates may contain novel
cryIC
-type genes.
B. thuringiensis
isolates containing
cryIA
(
a
)-,
cryIA
(
b
)-, and
cryIA
(
c
)-type genes exhibited much greater activity against
Plutella xylostella
than did other isolates, indicating that multiple
cry
-type genes may be used as markers for the prediction of insecticidal activities.
Determination of
15
N Abundance in Nanogram Pools of NO
3
-
and NO
2
-
by Denitrification Bioassay and Mass Spectrometry
Authors: Højberg Ole , Johansen Henrik Saaby , Sørensen Jan
Published: January 2020

Suspensions of two strains of
Pseudomonas aeruginosa
(ON12 and ON12-1) were used to reduce NO
3
-
and NO
2
-
, respectively, to N
2
O. The evolved N
2
O was quantified by gas chromatography with electron capture detection, and the
15
N abundance was determined by mass spectrometry with a special inlet system and triple-collector detection. Sample gas containing unknown N
2
O pools as small as 0.5 ng of N was analyzed by use of a spike technique, in which a reference gas of N
2
O of natural
15
N abundance was added to obtain enough total N for the mass spectrometer. In NO
3
-
or NO
2
-
pools, the
15
N abundance could be determined in samples as small as approximately 3.5 ng of N. No cross-contamination took place between the NO
3
-
and NO
2
-
pools. The excellent separation of NO
3
-
and NO
2
-
pools, small sample size required, and low contamination risk during N
2
O analysis offer great advantages in isotope studies of inorganic N transformations by, e.g., nitrifying or denitrifying bacteria in the environment.
Accuracy, Reproducibility, and Interpretation of Fatty Acid Methyl Ester Profiles of Model Bacterial Communities
Authors: Haack Sheridan Kidd , Garchow Helen , Odelson David A. , Forney Larry J. , Klug Michael J.
Published: January 2020

We determined the accuracy and reproducibility of whole-community fatty acid methyl ester (FAME) analysis with two model bacterial communities differing in composition by using the Microbial ID, Inc. (MIDI), system. The biomass, taxonomic structure, and expected MIDI-FAME profiles under a variety of environmental conditions were known for these model communities a priori. Not all members of each community could be detected in the composite profile because of lack of fatty acid “signatures” in some isolates or because of variations (approximately fivefold) in fatty acid yield across taxa. MIDI-FAME profiles of replicate subsamples of a given community were similar in terms of fatty acid yield per unit of community dry weight and relative proportions of specific fatty acids. Principal-components analysis (PCA) of MIDI-FAME profiles resulted in a clear separation of the two different communities and a clustering of replicates of each community from two separate experiments on the first PCA axis. The first PCA axis accounted for 57.1% of the variance in the data and was correlated with fatty acids that varied significantly between communities and reflected the underlying community taxonomic structure. On the basis of our data, community fatty acid profiles can be used to assess the relative similarities and differences of microbial communities that differ in taxonomic composition. However, detailed interpretation of community fatty acid profiles in terms of biomass or community taxonomic composition must be viewed with caution until our knowledge of the quantitative and qualitative distribution of fatty acids over a wide variety of taxa and the effects of growth conditions on fatty acid profiles is more extensive.
Cloning of the pyruvate kinase gene (pyk) of Corynebacterium glutamicum and site-specific inactivation of pyk in a lysine-producing Corynebacterium lactofermentum strain
Authors: Gubler M , Jetten M , Lee S H , Sinskey A J
Published: January 2020

The pyruvate kinase gene pyk from Corynebacterium glutamicum was cloned by applying a combination of PCR, site-specific mutagenesis, and complementation. A 126-bp DNA fragment central to the C. glutamicum pyk gene was amplified from genomic DNA by PCR with degenerate oligonucleotides as primers. The cloned DNA fragment was used to inactivate the pyk gene in C. glutamicum by marker rescue mutagenesis via homologous recombination. The C. glutamicum pyk mutant obtained was unable to grow on minimal medium containing ribose as the sole carbon source. Complementation of this phenotype by a gene library resulted in the isolation of a 2.8-kb PstI-BamHI genomic DNA fragment harboring the C. glutamicum pyk gene. Multiple copies of plasmid-borne pyk caused a 20-fold increase of pyruvate kinase activity in C. glutamicum cell extracts. By using large internal fragments of the cloned C. glutamicum gene, pyk mutant derivatives of the lysine production strain Corynebacterium lactofermentum 21799 were generated by marker rescue mutagenesis. As determined in shake flask fermentations, lysine production in pyk mutants was 40% lower than that in the pyk+ parent strain, indicating that pyruvate kinase is essential for high-level lysine production. This finding questions an earlier hypothesis postulating that redirection of carbon flow at the phosphoenol pyruvate branch point of glycolysis through elimination of pyruvate kinase activity results in an increase of lysine production in C. glutamicum and its close relatives.
Pyranose Oxidase, a Major Source of H
2
O
2
during Wood Degradation by
Phanerochaete chrysosporium, Trametes versicolor,
and
Oudemansiella mucida
Authors: Daniel Geoffrey , Volc Jindrich , Kubatova Elena
Published: January 2020

The production of the H
2
O
2
-generating enzyme pyranose oxidase (POD) (EC 1.1.3.10) (synonym, glucose 2-oxidase), two ligninolytic peroxidases, and laccase in wood decayed by three white rot fungi was investigated by correlated biochemical, immunological, and transmission electron microscopic techniques. Enzyme activities were assayed in extracts from decayed birch wood blocks obtained by a novel extraction procedure. With the coupled peroxidase-chromogen (3-dimethylaminobenzoic acid plus 3-methyl-2-benzothiazolinone hydrazone hydrochloride) spectrophotometric assay, the highest POD activities were detected in wood blocks degraded for 4 months and were for
Phanerochaete chrysosporium
(149 mU g [dry weight] of decayed wood
-1
),
Trametes versicolor
(45 mU g
-1
), and
Oudemansiella mucida
(1.2 mU g
-1
), corresponding to wood dry weight losses of 74, 58, and 13%, respectively. Mn-dependent peroxidase activities in the same extracts were comparable to those of POD, while lignin peroxidase activity was below the detection limit for all fungi with the veratryl alcohol assay. Laccase activity was high with
T. versicolor
(422 mU g
-1
after 4 months), in trace levels with
O. mucida
, and undetectable in
P. chrysosporium
extracts. Evidence for C-2 specificity of POD was shown by thin-layer chromatography detection of 2-keto-
d
-glucose as the reaction product. By transmission electron microscopy-immunocytochemistry, POD was found to be preferentially localized in the hyphal periplasmic space of
P. chrysosporium
and
O. mucida
and associated with membranous materials in hyphae growing within the cell lumina or cell walls of partially and highly degraded birch fibers. An extracellular distribution of POD associated with slime coating wood cell walls was also noted. The periplasmic distribution in hyphae and extracellular location of POD are consistent with the reported ultrastructural distribution of H
2
O
2
-dependent Mn-dependent peroxidases. This fact and the dominant presence of POD and Mn-dependent peroxidase in extracts from degraded wood suggest a cooperative role of the two enzymes during white rot decay by the test fungi.