bluuebunny/crossref_metadata_2025 huggingface dataset:
Leucine synthesis in Corynebacterium glutamicum: enzyme activities, structure of leuA, and effect of leuA inactivation on lysine synthesis
Authors: Pátek M , Krumbach K , Eggeling L , Sahm H
Published: January 2020

Enzymes and genes of the isopropylmalate pathway leading to leucine in Corynebacterium glutamicum were studied, and assays were performed to unravel their connection to lysine oversynthesis. The first enzyme of the pathway is inhibited by leucine (Ki = 0.4 mM), and all three enzyme activities of the isopropylmalate pathway are reduced upon addition of this amino acid to the growth medium. Three different DNA fragments were cloned, each resulting in an oversynthesis of one of the three enzymes. The leuA complementing fragment encoding the isopropylmalate synthase was sequenced. The leuA gene is 1,848 bp in size, encoding a polypeptide with an M(r) of 68,187. Upstream of leuA there is extensive hyphenated dyad symmetry and a putative leader peptide, which are features characteristic of attenuation control. In addition to leuA, the sequenced fragment contains an open reading frame with high coding probability whose disruption did not result in a detectable phenotype. Furthermore, the sequence revealed that this open reading frame separates leuA from lysC, which encodes the aspartate kinase initiating the synthesis of all amino acids of the aspartate family. The leuA gene was inactivated in three lysine-secreting strains by insertional mutagenesis. Fermentations were performed, and a roughly 50% higher lysine yield was obtained when appropriate leucine concentrations limiting for growth of the constructed strains were used.
Detection of mycoplasma contamination in cell cultures by a mycoplasma group-specific PCR
Authors: van Kuppeveld F J , Johansson K E , Galama J M , Kissing J , Bölske G , van der Logt J T , Melchers W J
Published: January 2020

The suitability of a 16S rRNA-based mycoplasma group-specific PCR for the detection of mycoplasma contamination in cell cultures was investigated. A total of 104 cell cultures were tested by using microbiological culture, DNA fluorochrome staining, DNA-rRNA hybridization, and PCR techniques. A comparison of the results obtained with these techniques revealed agreement for 95 cell cultures. Discrepant results, which were interpreted as false negative or false positive on the basis of a comparison with the results obtained with other methods, were observed with nine cell cultures. The microbiological culture technique produced false-negative results for four cell cultures. The hybridization technique produced false-negative results for two cell cultures, and for one of these cell cultures the DNA staining technique also produced a false-negative result. The PCR may have produced false-positive results for one cell culture. Ambiguous results were obtained with the remaining two cell cultures. Furthermore, the presence of contaminating bacteria interfered with the interpretation of the DNA staining results for 16 cell cultures. For the same reason the hybridization signals of nine cell cultures could not be interpreted. Our results demonstrate the drawbacks of each of the detection methods and the suitability of the PCR for the detection of mycoplasmas in cell cultures.
Modeling of Bacterial Growth with Shifts in Temperature
Authors: Zwietering M. H. , de Wit J. C. , Cuppers H. G. A. M. , van 't Riet K.
Published: January 2020

The temperature of chilled foods is an important variable for the shelf life of a product in a production and distribution chain. To predict the number of organisms as a function of temperature and time, it is essential to model the growth as a function of temperature. The temperature is often not constant in various stages of distribution. The objective of this research was to determine the effect of shifts in temperature. The suitability and usefulness of several models to describe the growth of
Lactobacillus plantarum
with fluctuating temperatures was evaluated. It can be assumed that temperature shifts within the lag phase can be handled by adding relative parts of the lag time to be completed and that temperature shifts within the exponential phase result in no lag phase. With these assumptions, the kinetic behavior of temperature shift experiments was reasonably well predicted, and this hypothesis was accepted statistically in 73% of the cases. Only shifts of temperature around the minimum temperature for growth showed very large deviations from the model prediction. The best results were obtained with the assumption that a temperature shift (within the lag phase as well as within the exponential phase) results in an additional lag phase. This hypothesis was accepted statistically in 93% of the cases. The length of the additional lag phase is one-fourth of the lag time normally found at the temperature after the shift.
Characterization of Microbial Community Structure in the Surface Sediment of Osaka Bay, Japan, by Phospholipid Fatty Acid Analysis
Authors: Rajendran Narasimmalu , Matsuda Osamu , Urushigawa Yoshikuni , Simidu Usio
Published: January 2020

Twenty-eight sediment samples collected from Osaka Bay, Japan, were analyzed for phospholipid ester-linked fatty acids (PLFA) to determine regional differences in microbial community structure of the bay. The abundance of three major groups of C
10
to C
19
PLFA (saturated, branched, and monounsaturated PLFA), which accounted for 84 to 97% of the total PLFA, indicated the predominance of prokaryotes in the sediment. The distribution of six clusters obtained by similarity analysis in the bay revealed a marked regional distribution in the PLFA profiles. Total PLFA concentrations (0.56 to 2.97 μg/g [dry weight] of the sediment) in sediments also showed marked variation among the stations, with higher concentrations of total PLFA in the central part of the bay. The biomass, calculated on the basis of total PLFA concentration, ranged from 0.25 × 10
8
to 1.35 × 10
8
cells per g (dry weight) of the sediment. The relative dominance of microbial groups in sediments was described by using the reported bacterial biomarker fatty acids. Very small amounts of the characteristic PLFA of microeukaryotes in sediments indicated the restricted distribution of microeukaryotes. By examining the distribution of clusters and groups of microorganisms in the bay, there were two characteristics of the distribution pattern: (i) the predominance of anaerobic bacteria and gram-positive prokaryotes, characterized by the high proportions of branched PLFA in the eastern and northeastern sides of the bay, where the reported concentrations of pollutants were also high, and (ii) the predominance of aerobic prokaryotes and eukaryotes, except for a few stations, in the western and southwestern sides of the bay, as evidenced by the large amounts of monounsaturated PLFA. Such significant regional differences in microbial community structure of the bay indicate shifts in microbial community structure.
Degradation of polycyclic aromatic hydrocarbons in the presence of synthetic surfactants
Authors: Tiehm A
Published: January 2020

The biodegradation of polycyclic aromatic hydrocarbons (PAH) often is limited by low water solubility and dissolution rate. Nonionic surfactants and sodium dodecyl sulfate increased the concentration of PAH in the water phase because of solubilization. The degradation of PAH was inhibited by sodium dodecyl sulfate because this surfactant was preferred as a growth substrate. Growth of mixed cultures with phenanthrene and fluoranthene solubilized by a nonionic surfactant prior to inoculation was exponential, indicating a high bioavailability of the solubilized hydrocarbons. Nonionic surfactants of the alkylethoxylate type and the alkylphenolethoxylate type with an average ethoxylate chain length of 9 to 12 monomers were toxic to a PAH-degrading Mycobacterium sp. and to several PAH-degrading mixed cultures. Toxicity of the surfactants decreased with increasing hydrophilicity, i.e., with increasing ethoxylate chain length. Nontoxic surfactants enhanced the degradation of fluorene, phenanthrene, anthracene, fluoranthene, and pyrene.
Significant Biogenesis of Chlorinated Aromatics by Fungi in Natural Environments
Authors: de Jong Ed , Field Jim A. , Spinnler Henri-Eric , Wijnberg Joannes B. P. A. , de Bont Jan A. M.
Published: January 2020

Common wood- and forest litter-degrading fungi produce chlorinated anisyl metabolites. These compounds, which are structurally related to xenobiotic chloroaromatics, occur at high concentrations of approximately 75 mg of chlorinated anisyl metabolites kg of wood
-1
or litter
-1
in the environment. The widespread ability among common fungi to produce large amounts of chlorinated aromatic compounds in the environment makes us conclude that these kinds of compounds can no longer be considered to originate mainly from anthropogenic sources.
Physiological Role of Chlorinated Aryl Alcohols Biosynthesized De Novo by the White Rot Fungus
Bjerkandera
sp. Strain BOS55
Authors: de Jong Ed , Cazemier Anne E. , Field Jim A. , de Bont Jan A. M.
Published: January 2020

The white rot fungus
Bjerkandera
sp. strain BOS55 produces veratryl, anisyl, 3-chloroanisyl, and 3,5-dichloroanisyl alcohol and the corresponding aldehydes de novo from glucose. All metabolites are produced simultaneously with the extracellular ligninolytic enzymes and have an important physiological function in the fungal ligninolytic system. Both mono- and dichlorinated anisyl alcohols are distinctly better substrates for the extracellular aryl alcohol oxidases than veratryl alcohol. The aldehydes formed are readily recycled by reduction by washed fungal mycelium, thus creating an extracellular H
2
O
2
production system regulated by intracellular enzymes. Lignin peroxidase does not oxidize the chlorinated anisyl alcohols either in the absence or in the presence of veratryl alcohol. It was therefore concluded that the chlorinated anisyl alcohols are well protected against the fungus's own aggressive ligninolytic enzymes. The relative amounts of veratryl alcohol and the chlorinated anisyl alcohols differ significantly according to the growth conditions, indicating that production of veratryl alcohol and the production of the (chlorinated) anisyl metabolites are independently regulated. We conclude that the chlorinated anisyl metabolites biosynthesized by the white rot fungus
Bjerkandera
sp. strain BOS55 can be purposefully produced for ecologically significant processes such as lignin degradation.
Isolation by genomic subtraction of DNA probes specific for Erwinia carotovora subsp. atroseptica
Authors: Darrasse A , Kotoujansky A , Bertheau Y
Published: January 2020

Erwinia carotovora subsp. atroseptica is a pathogen of potatoes in Europe because of its ability to induce blackleg symptoms early in the growing season. However, E. carotovora subsp. carotovora is not able to produce such severe symptoms under the same conditions. On the basis of the technique described by Straus and Ausubel (Proc. Natl. Acad. Sci. USA 87:1889-1893, 1990), we isolated DNA sequences of E. carotovora subsp. atroseptica 86.20 that were absent from the genomic DNA of E. carotovora subsp. carotovora CH26. Six DNA fragments ranging from ca. 180 to 400 bp were isolated, cloned, and sequenced. Each fragment was further hybridized with 130 microorganisms including 87 E. carotovora strains. One probe was specific for typical E. carotovora subsp. atroseptica strains, two probes hybridized with all E. carotovora subsp. atroseptica strains and with a few E. carotovora subsp. carotovora strains, and two probes recognized only a subset of E. carotovora subsp. atroseptica strains. The last probe was absent from the genomic DNA of E. carotovora subsp. carotovora CH26 but was present in the genomes of many strains, including those of other species and genera. This probe is homologous to the putP gene of Escherichia coli, which encodes a proline carrier. Further use of the probes is discussed.
Identification and characterization of a new plasmid carrying genes for degradation of 2,4-dichlorophenoxyacetate from Pseudomonas cepacia CSV90
Authors: Bhat M A , Tsuda M , Horiike K , Nozaki M , Vaidyanathan C S , Nakazawa T
Published: January 2020

Pseudomonas cepacia CSV90 is able to utilize 2,4-dichlorophenoxyacetate (2,4-D) and 2-methyl-4-chlorophenoxyacetate as sole sources of carbon and energy. Mutants of the strain CSV90 which had lost this ability appeared spontaneously on a nonselective medium. The wild-type strain harbored a 90-kb plasmid, pMAB1, whereas 2,4-D-negative mutants either lost the plasmid or had a 70-kb plasmid, pMAB2. The plasmid pMAB2 was found to have undergone a deletion of a 20-kb fragment of pMAB1. The plasmid-free mutants regained the ability to degrade 2,4-D after introduction of purified pMAB1 by electroporation. Cloning in Escherichia coli of a 10-kb BamHI fragment from pMAB1, the region absent in pMAB2, resulted in the expression of the gene tfdC encoding 3,5-dichlorocatechol 1,2-dioxygenase. After subcloning, the tfdC gene was located in a 1.6-kb HindIII fragment. The nucleotide sequence of the tfdC gene and the restriction map of its contiguous region are identical to those of the well-characterized 2,4-D-degradative plasmid pJP4 of Alcaligenes eutrophus, whereas the overall restriction maps of the two plasmids are different. The N-terminal 44-amino-acid sequence of the enzyme purified from the strain CSV90 confirmed the reading frame in the DNA sequence for tfdC and indicated that the initiation codon GUG is read as methionine instead of valine.
Characterization of the Lactobacillus helveticus CNRZ32 pepC gene
Authors: Fernández L , Bhowmik T , Steele J L
Published: January 2020

Sequence analysis of the aminopeptidase C gene (pepC) from Lactobacillus helveticus CNRZ32 identified a 1,332-nucleotide open reading frame coding for a polypeptide with motifs characteristic of cysteine proteinases. Homology to the pepC gene appears to be widely distributed among lactic acid bacteria.
PCR analysis of the cryI insecticidal crystal family genes from Bacillus thuringiensis
Authors: Ceron J , Covarrubias L , Quintero R , Ortiz A , Ortiz M , Aranda E , Lina L , Bravo A
Published: January 2020

A method allowing rapid and accurate identification of different subgroups within the insecticidal crystal CryI protein-producing family of Bacillus thuringiensis strains was established by using PCR technology. Thirteen highly homologous primers specific to regions within genes encoding seven different subgroups of B. thuringiensis CryI proteins were described. Differentiation among these strains was determined on the basis of the electrophoretic patterns of PCR products. B. thuringiensis strains, isolated from soil samples, were analyzed by PCR technology. Small amounts of bacterial lysates were assayed in two reaction mixtures containing six to eight primers. This method can be applied to rapidly detect the subgroups of CryI proteins that correspond with toxicity to various lepidopteran insects.
A bioassay based on recombinant DNA technology for determining selenium concentration
Authors: Reches M , Zhao C , Engelberg-Kulka H
Published: January 2020

The trace element selenium has recently attracted attention, particularly because (i) selenocysteine is involved in the active site of various prokaryotic and eukaryotic enzymes, some of which have a role in human health; (ii) selenocysteine incorporation into these proteins is coded by UGA codons; and (iii) as a result, selenocysteine is now considered to be the 21st amino acid in an expanded genetic code. Here, we built recombinant DNA constructs in which expression of the lac'Z gene is driven in Escherichia coli by UGA-directed selenocysteine incorporation. In this system, levels of beta-galactosidase activity are proportionally and specifically related to the presence and concentrations of several specific simple selenium derivatives. The system can thus be used as a sensitive bioassay for their determination. This bioassay is one of a few using recombinant DNA technology to provide a reporter for simple detection of a chemical trace element.
Identification of a Genetic Locus in
Pseudomonas aureofaciens
Involved in Fungal Inhibition
Authors: Carruthers F. L. , Conner A. J. , Mahanty H. K.
Published: January 2020

In iron-rich conditions,
Pseudomonas aureofaciens
PA147-2 produces an antibiotic-like compound that inhibits the growth of a plant fungal pathogen,
Aphanomyces euteiches.
To contribute to the potential use of PA147-2 as a biocontrol organism, we report the identification of a genetic locus important for antibiotic biosynthesis. Mutants defective for fungal inhibition (Af
-
) were generated by Tn
5
mutagenesis. Southern hybridization of total DNAs from three Af
-
mutants indicated that loss of fungal inhibition was due to a single Tn
5
insertion in each mutant. Restriction mapping of the mutation points showed that in two mutants the Tn
5
insertions were in the same 16.0-kb
Eco
RI fragment and were separated by 2.1 kb. A genomic library of PA147-2 was constructed and screened by using a region of DNA flanking the Tn
5
insertion in one mutant (PA109) as a probe to recover complementing cosmids. Three cosmids containing a 16.0-kb
Eco
RI fragment complementary to the two mutants were recovered. Allele replacement by homologous recombination with putative complementing cosmids restored one mutant to antifungal activity against
A. euteiches.
Southern analysis of the complemented mutants confirmed that allele replacement had occurred between cosmid DNA and Tn
5
. The wild-type 16.0-kb
Eco
RI fragment was cloned from the cosmid and complemented the two mutants to antifungal activity. An antifungal compound was isolated from PA147-2 grown on solid medium. Antifungal activity correlated to a peak on high-pressure liquid chromatography analysis. Under the same growth and extraction conditions, the antifungal activity seen in PA147-2 was absent in two Af
-
mutants. Furthermore, absence of an antifungal compound in each mutant correlated to the absence of the wild-type “antifungal” peak on high-pressure liquid chromatography analysis.
Construction from a single parent of baker's yeast strains with high freeze tolerance and fermentative activity in both lean and sweet doughs
Authors: Nakagawa S , Ouchi K
Published: January 2020

From a freeze-tolerant baker's yeast (Saccharomyces cerevisiae), 2,333 spore clones were obtained. To improve the leavening ability in lean dough of the parent strain, we selected 555 of the high-maltose-fermentative spore clones by using a method in which a soft agar solution containing maltose and bromocresol purple was overlaid on yeast colonies. By measuring the gassing power in the dough, we selected 66 spore clones with a good leavening ability in lean dough and a total of 694 hybrids were constructed by crossing them. Among these hybrids, we obtained 50 novel freeze-tolerant strains with good leavening ability in all lean, regular, and sweet doughs comparable to that of commercial baker's yeast. Hybrids with improved leavening ability or freeze tolerance compared with the parent yeast and commercial baker's yeasts were also obtained. These results suggest that hybridization between spore clones derived from a single parent strain is effective for improving the properties of baker's yeasts.
Role of Na+ in transport of Hg2+ and induction of the Tn21 mer operon
Authors: Selifonova O V , Barkay T
Published: January 2020

The effects of sodium ions on the uptake of Hg2+ and induction of the Tn21 mer operon were studied by using Escherichia coli HMS174 harboring the reporter plasmids pRB28 and pOS14. Plasmid pRB28 carries merRT', and pOS14 carries merRTPC of the mer operon, both cloned upstream of a promoterless luciferase gene cassette in pUCD615. The bioluminescent response to 1 microM Hg2+ was significantly inhibited in E. coli HMS174(pRB28) in minimal medium supplemented with sodium ions at 10 to 140 mM. After initial acceleration, light emission declined at 50 nM Hg2+ in the presence of Na+. The mer-lux assay with resting cells carrying pRB28 and 203Hg2+ uptake experiments showed increased induction and enhanced mercury uptake, respectively, in media supplemented with sodium ions. The presence of Na+ facilitated maintenance of bioluminescence in resting HMS174(pRB28) cells induced with 50 nM Hg2+. External K+ stimulated bioluminescent response in HMS174(pRB28) and HMS174(pOS14) grown in sodium phosphate minimal medium devoid of potassium ions. Sodium ions appear to facilitate mercury transport. We propose that sodium-coupled transport of mercuric ions can be one of the mechanisms for mercury uptake by E. coli and that the Na+ gradient may energize the transport of Hg2+.
Identification and purification of a protein that induces production of the Lactobacillus acidophilus bacteriocin lactacin B
Authors: Barefoot S F , Chen Y R , Hughes T A , Bodine A B , Shearer M Y , Hughes M D
Published: January 2020

Lactacin B is a heat-stable bacteriocin produced by Lactobacillus acidophilus N2 that is active against closely related lactobacilli, including Lactobacillus delbrueckii subsp. lactis (formerly Lactobacillus leichmannii) ATCC 4797. Pure producer cultures propagated in MRS broth (initial pH 6.5) contain no lactacin B; it is detected only in cultures maintained at pH 5.0 to 6.0 and produced optimally at pH 6.0 S. F. Barefoot and T. R. Klaenhammer, Antimicrob. Agents Chemother. 26:328-334, 1984). Associative growth of producer and indicator, L. delbrueckii subsp. lactis ATCC 4797, resulted in production of an inhibitor identical to lactacin B. Associative growth increased lactacin B production from nondetectable levels (< 100 activity units [AU]/ml) to between 3,200 and 6,400 AU/ml in MRS broth (initial pH 6.5) and resulted in early but equal production of lactacin B (approximately 25,600 AU/ml) in broth maintained at pH 6.0. Indicator cells, but not spent culture filtrates, induced lactacin B production. Indicator cells disrupted by a French pressure cell yielded cell-free filtrates containing inducing activity. Chromatofocusing and gel filtration high-performance liquid chromatography of cell-free filtrates yielded a protein with a pI of 4.1 and a molecular size of approximately 58 kDa that induced lactacin B production. Analytical isoelectric focusing yielded a single protein band. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis gels contained a 28-kDa protein suggesting a two-subunit structure. Protein sequencing identified an N-terminal serine and 18 additional amino acids. To our knowledge, there are not previous descriptions of proteins that induce bacteriocin production in lactic acid bacteria.
Methionine uptake and cytopathogenicity of viable but nonculturable Shigella dysenteriae type 1
Authors: Rahman I , Shahamat M , Kirchman P A , Russek-Cohen E , Colwell R R
Published: January 2020

A pathogenic strain of Shigella dysenteriae type 1 was selected for study to elucidate the physiology and potential pathogenicity of organisms in the viable but nonculturable (VBNC) state in the environment. Studies in our laboratory have shown that S. dysenteriae type 1 survives in laboratory microcosms in the VBNC state for long periods of time, i.e., more than 6 months. VBNC cells of S. dysenteriae type 1 were found to retain cytopathogenicity for cultured HeLa cells. To determine whether VBNC S. dysenteriae type 1 expressed protein after loss of culturability, 35S-labelled methionine was added to suspensions of VBNC cells. Total cellular proteins were extracted and examined by autoradiography. Results indicate that VBNC S. dysenteriae type 1 is capable of both active uptake of methionine and incorporation of methionine into protein. Amino acid uptake and protein synthesis substantiate the viability of cells of S. dysenteriae type 1 in the VBNC state, i.e., although the cells are unable to be cultured on laboratory media by standard bacteriological methods, the cells remain metabolically active. Furthermore, VBNC cells of S. dysenteriae type 1 may pose a potential public health hazard that has not yet been recognized.
Electroporation of Alcaligenes eutrophus with (mega) plasmids and genomic DNA fragments
Authors: Taghavi S , van der Lelie D , Mergeay M
Published: January 2020

Electroporation was used as a tool to explore the genetics of the heavy-metal-resistant strain Alcaligenes eutrophus CH34. A 12.9-kb A. eutrophus-Escherichia coli shuttle vector, pMOL850, was constructed to optimize electroporation conditions. This vector is derived from the E. coli plasmid pSUP202 and contains the replication region of the A. eutrophus megaplasmid pMOL28. Electroporation was used to transform A. eutrophus CH34 derivatives with megaplasmids (sizes up to 240 kb), and transformants were selected for resistance to heavy metals. Electroporation was also performed with endonuclease-digested genomic DNA. Transformation of markers affecting lysine biosynthesis (lysA194) and biosynthesis of the siderophore alcaligin E were observed. Transfer of the nonselected markers pheB332 and aro-333, linked to lysA194, confirmed the intervention of homologous recombination. However, during transformation of ale::Tn5-Tc, illegitimate recombination and transposition were also observed as an alternative for the inheritance of the Tn5-Tc markers.
Microbially Mediated Formation of Benzonaphthothiophenes from Benzo[
b
]thiophenes
Authors: Kropp Kevin G. , Gonçalves José A. , Andersson Jan T. , Fedorak Phillip M.
Published: January 2020

Studies of the microbial metabolism of benzo[
b
]thiophene (molecular weight 134) by three
Pseudomonas
isolates showed the formation of benzothiophene sulfoxide, benzothiophene sulfone, and a sulfur-containing metabolite with a molecular weight of 234. Desulfurization of the high-molecular-weight product with nickel boride gave 1-phenylnaphthalene, indicating that the metabolite was benzo[
b
]naphtho[1,2-
d
]thiophene. Similarly, the isolates were capable of producing the analogous dimethyl-substituted benzonaphthothiophenes from methylbenzothiophenes that had the methyl substitution on the benzene ring. The formation of benzo[
b
] naphtho[1,2-
d
]thiophene was also observed when a petroleum-degrading mixed culture was incubated with benzothiophene-supplemented Prudhoe Bay crude oil. Investigations into the mechanism of formation of these high-molecular-weight compounds showed that they resulted from an abiotic, Diels-Alder-type condensation of two molecules of the sulfoxide, which were microbially produced from the respective benzothiophene, with the subsequent loss of two atoms of hydrogen and oxygen and one atom of sulfur. The condensation products also formed from the sulfoxides of benzothiophene and methylbenzothiophenes when the sulfoxides were enzymatically synthesized by oxidation of the benzothiophene with horse heart cytochrome
c
and H
2
O
2
.
Degradation of methyl bromide by methanotrophic bacteria in cell suspensions and soils
Authors: Oremland R S , Miller L G , Culbertson C W , Connell T L , Jahnke L
Published: January 2020

Cell suspensions of Methylococcus capsulatus mineralized methyl bromide (MeBr), as evidence by its removal from the gas phase, the quantitative recovery of Br- in the spent medium, and the production of 14CO2 from [14C]MeBr. Methyl fluoride fluoride (MeF) inhibited oxidation of methane as well as that of [14C]MeBr. The rate of MeBr consumption by cells varied inversely with the supply of methane, which suggested a competitive relationship between these two substrates. However, MeBr did not support growth of the methanotroph. In soils exposed to high levels (10,000 ppm) of MeBr, methane oxidation was completely inhibited. At this concentration, MeBr removal rates were equivalent in killed and live controls, which indicated a chemical rather than biological removal reaction. At lower concentration (1,000 ppm) of MeBr, methanotrophs were active and MeBr consumption rates were 10-fold higher in live controls than in killed controls. Soils exposed to trace levels (10 ppm) of MeBr demonstrated complete consumption within 5 h of incubation, while controls inhibited with MeF or incubated without O2 had 50% lower removal rates. Aerobic soils oxidized [14C]MeBr to 14CO2, and MeF inhibited oxidation by 72%. Field experiments demonstrated slightly lower MeBr removal rates in chambers containing MeF than in chambers lacking MeF. Collectively, these results show that soil methanotrophic bacteria, as well as other microbes, can degrade MeBr present in the environment.
Metabolic activity of pathogenic bacteria during semicontinuous anaerobic digestion
Authors: Kearney T E , Larkin M J , Levett P N
Published: January 2020

In natural environments such as anaerobic digesters, bacteria are frequently subjected to the stress of nutrient fluxes because of the continual changes in the flow of nutrients, and to survive, they must be capable of adapting readily to nutrient changes. In this study, the metabolic activities of Escherichia coli, Salmonella typhimurium, Yersinia enterocolitica, Listeria monocytogenes, and Campylobacter jejuni were studied within culture bags (Versapor-200 filters, 0.22-microns pore size) in laboratory anaerobic digesters. The metabolic activity of these bacteria was indicated by their adenylate energy charge (EC) ratios and their ability to incorporate [3H]thymidine, which was related to the respective changes in viable numbers within the culture bags during anaerobic digestion. Fluctuations in the adenylate EC ratios, the uptake of [3H]thymidine, and the viable numbers of E. coli, S. typhimurium, Y. enterocolitica, and L. monocytogenes cells were probably due to constant changes in the amount of available nutrients within the anaerobic digesters. The viability of S. typhimurium increased quickly after a fresh supply of nutrients was added to the system as indicated by the uptake of [3H]thymidine and an increase in the adenylate EC ratios. The viable numbers of E. coli, S. typhimurium, Y. enterocolitica, and L. monocytogenes organisms declined rapidly from 10(7) to 10(8) CFU/ml to 10(3) to 10(4) CFU/ml and remained at this level for an indefinite period. The decimal reduction time calculated during the period of exponential decline ranged from 0.8 to 1.2 days for these bacteria. C. jejuni had the greatest mean decimal reduction time value (3.6 days).(ABSTRACT TRUNCATED AT 250 WORDS)
Quantification of
Frankia
Strains and Other Root-Associated Bacteria in Pure Cultures and in the Rhizosphere of Axenic Seedlings by High-Performance Liquid Chromatography-Based Muramic Acid Assay
Authors: Rönkkö R. , Pennanen T. , Smolander A. , Kitunen V. , Kortemaa H. , Haahtela K.
Published: January 2020

Application of a high-performance liquid chromatography-based muramic acid assay with precolumn fluorescence derivatization to quantification of root-associated bacteria was studied both in pure cultures and in the rhizosphere of axenic
Festuca rubra
seedlings. Quantities of muramic acid from acid-hydrolyzed cells of
Frankia
strains,
Streptomyces griseoviridis, Enterobacter agglomerans, Klebsiella pneumoniae, Pseudomonas
sp., and
Bacillus polymyxa
were mostly proportional to the respective cell protein and carbon quantities, but in some strains, culture age and particularly sporulation affected these ratios considerably. The muramic acid/cell protein ratio was generally 2 to 4 times higher in strains of the two actinomycete genera,
Frankia
and
Streptomyces
, than in the rest of the strains. Quantification of
Frankia
strains,
S. griseoviridis, E. agglomerans
, and
Pseudomonas
sp. was also attempted from the rhizosphere of
F. rubra
seedlings which had been inoculated with pure cultured bacteria and incubated briefly. It was possible to quantify
Frankia
cells by use of the muramic acid assay from both the root and the growth medium, whereas cells of the rest of the bacterial genera could only be detected in the medium. The detection limit for muramic acid was about 10 ng/ml hydrolysis volume, and from the
Festuca
rhizosphere, 28 to 63% of the muramic acid in the
Frankia
inoculum was recovered.
Survival of enteric viruses on environmental fomites
Authors: Abad F X , Pintó R M , Bosch A
Published: January 2020

The survival of human enteric viruses on several porous (paper and cotton cloth) and nonporous (aluminum, china, glazed tile, latex, and polystyrene) environmental surfaces has been evaluated. Viruses persisted for extended periods on several types of materials commonly found in institutions and domestic environments. The stability of the viruses was generally influenced by environmental factors such as relative humidity (RH), temperature, and the type of surface contaminated. Overall, hepatitis A virus (HAV) and human rotavirus (HRV) were more resistant to inactivation than enteric adenovirus (ADV) and poliovirus (PV). The resistance to the desiccation step appears to be of major significance in determining the survival of a virus dried on fomites. ADV and PV showed a pronounced decrease in titer at this stage, whereas HAV and HRV displayed little decay at the desiccation step. HAV and HRV persistence was not affected by the presence of fecal material. On nonporous surfaces, PV and ADV persisted better in the presence of feces. However, on porous fomites the presence of fecal material had a negative influence on the survival of PV and ADV. Except for HRV, greater virus survival was observed at 4 degrees than at 20 degrees C. PV and HAV survival was enhanced at high RH; the survival of the latter was enhanced at least for nonporous materials. When dried on porous materials, HRV also exhibited greater persistence at high RH. The survival of ADV was not affected by RH. The validity of using bacteriophages of Bacteroides fragilis as indicators of human viruses dried on fomites was evaluated.(ABSTRACT TRUNCATED AT 250 WORDS)
Membrane-permeabilizing activities of Bacillus thuringiensis coleopteran-active toxin CryIIIB2 and CryIIIB2 domain I peptide
Authors: Von Tersch M A , Slatin S L , Kulesza C A , English L H
Published: January 2020

Bacillus thuringiensis toxin CryIIIB2 exhibits activity against two agriculturally important pests, the Colorado potato beetle, Leptinotarsa decemlineata, and the Southern corn rootworm, Diabrotica undecimpunctata. CryIIIB2 shows significant structural similarity to Colorado potato beetle-active toxin CryIIIA, whose crystal structure has been determined elsewhere [J. Li, J. Carrol, and D. J. Ellar, Nature (London) 353:815-821, 1991]. A clone limited to the putative 7-alpha-helical bundle domain I peptide of CryIIIB2 was constructed by PCR. The truncated protein was expressed at high levels in Escherichia coli. Domain I peptide was isolated and compared with native CryIIIB2 toxin in promoting ion efflux from synthetic phospholipid vesicles and formation of ion channels in black lipid membranes. The results showed that CryIIIB2 domain I peptide is sufficient for ion channel formation and promotes ion efflux. Both native CryIIIB2 toxin and domain I peptide were inefficient channel-forming proteins that produced noisy ion channels of various conductance states. In ion efflux assays, native toxin promoted greater ion efflux from synthetic vesicles than did the truncated peptide.
Comparison of the Behavior of Epiphytic Fitness Mutants of
Pseudomonas syringae
under Controlled and Field Conditions
Authors: Beattie Gwyn A. , Lindow Steven E.
Published: January 2020

The epiphytic fitness of four Tn
5
mutants of
Pseudomonas syringae
that exhibited reduced epiphytic fitness in the laboratory was evaluated under field conditions. The mutants differed more from the parental strain under field conditions than under laboratory conditions in their survival immediately following inoculation onto bean leaves and in the size of the epiphytic populations that they established, demonstrating that their fitness was reduced more under field conditions than in the laboratory. Under both conditions, the four mutants exhibited distinctive behaviors. One mutant exhibited particularly large population decreases and short half-lives following inoculation but grew epiphytically at near-wild-type rates, while the others exhibited reduced survival only in the warmest, driest conditions tested and grew epiphytically at reduced rates or, in the case of one mutant, not at all. The presence of the parental strain, B728a, did not influence the survival or growth of three of the mutants under field conditions; however, one mutant, an auxotroph, established larger populations in the presence of B728a than in its absence, possibly because of cross-feeding by B728a in planta. Experiments with B728a demonstrated that established epiphytic populations survived exposure of leaves to dry conditions better than newly inoculated cells did and that epiphytic survival was not dependent on the cell density in the inoculum. Three of the mutants behaved similarly to two nonpathogenic strains of
P. syringae
, suggesting that the mutants may be altered in traits that are missing or poorly expressed in naturally occurring nonpathogenic epiphytes.
Binding of Insecticidal Crystal Proteins of
Bacillus thuringiensis
to the Midgut Brush Border of the Cabbage Looper,
Trichoplusia ni
(Hübner) (Lepidoptera: Noctuidae), and Selection for Resistance to One of the Crystal Proteins
Authors: Estada Ursula , Ferre Juan
Published: January 2020

The susceptibility of
Trichoplusia ni
larvae to several
Bacillus thuringiensis
insecticidal crystal proteins (ICPs) was tested. Neonatal larvae proved to be susceptible to solubilized trypsin-treated CryIA(a), CryIA(b), and CryIA(c) (50% lethal concentrations [LC
50
s], 570, 480, and 320 ng/cm
2
, respectively) but showed little susceptibility to CryIB and CryID (LC
50
s, 5,640 and 2,530 ng/cm
2
, respectively). The toxicity of ICPs was correlated to binding to the epithelial brush border of the midgut, as revealed by immunocytochemical staining with monoclonal antibodies. In vitro binding experiments with iodinated ICPs and brush border membrane vesicles indicated that CryIA(b) and CryIA(c) share the same high-affinity binding site, whereas CryIA(a) binds to a different one. The affinities of CryIA(b) and CryIA(c) for the binding site were similar (
K

d

= 3.6 and 4.7 nM, respectively), and the mean binding-site concentration was 0.71 pmol/mg of vesicle protein. Selection of a population with increasing concentrations of CryIA(b) produced 31-fold resistance in seven generations. The realized heritability (
h
2
) was 0.19. The increase of homozygosity (for resistance factors) as selection proceeded was reflected in the increase in the slopes of the dose-mortality curves. Resistance was specific for CryIA(b) and did not extend to CryIA(a) or even to CryIA(c). This result was not predicted by the binding-site model, in which CryIA(b) and CryIA(c) bind to the same high-affinity binding site. This result may suggest a more complicated relationship between in vitro binding of ICPs to specific sites in the epithelial membrane of the midgut and the in vivo toxic effect.
Glucose uptake by Listeria monocytogenes Scott A and inhibition by pediocin JD
Authors: Christensen D P , Hutkins R W
Published: January 2020

Glucose uptake by Listeria monocytogenes Scott A was inhibited by the bacteriocin pediocin JD and by the protonophore carbonyl cyanide m-chlorophenyhydrazone. Experiments with monensin, nigericin, chlorhexidine diacetate, dinitrophenol, and gramicidin, however, showed that glucose uptake could occur in the absence of a proton motive force. L. monocytogenes cell extracts phosphorylated glucose when phosphoenolpyruvate (PEP) was present in the assay mixture, and whole cells incubated with 2-deoxyglucose accumulated 2-deoxyglucose-6-phosphate, indicating the presence of a PEP-dependent phosphotransferase system in this organism. Glucose phosphorylation also occurred when ATP was present, suggesting that a proton motive force-mediated glucose transport system may also be present. We conclude that L. monocytogenes Scott A accumulates glucose by phosphotransferase and proton motive force-mediated systems, both of which are sensitive to pediocin JD.
Adsorption of Dissolved Organic Matter to the Inorganic Filter Substrate and Its Implications for
14
C Uptake Measurements
Authors: Maske Helmut , Garcia-Mendoza Ernesto
Published: January 2020

Inorganic carbon uptake rates for glass fiber-filtered samples are higher than those for membrane-filtered samples because of adsorption of dissolved organic matter to the filter substrate. Experimentally derived values for adsorption onto filters were as follows (relative units): GF/F filter, 1, quartz filter, 1.1, GF/C filter, 0.6; GN-6 Gelman filter, 0.1; Nuclepore and Poretics filter, 0.0; Anodisc filter, 0.4 to 1.9.
Analysis of the raw starch-binding domain by mutation of a glucoamylase from Aspergillus awamori var. kawachi expressed in Saccharomyces cerevisiae
Authors: Goto M , Semimaru T , Furukawa K , Hayashida S
Published: January 2020

Carboxy-terminal deletions were introduced into the raw starch-binding domain (A-515 to R-615) encoded by the gene for glucoamylase I (GAI) from Aspergillus awamori var. kawachi. Genes coding for proteins designated GA596 (A-1 to E-596), GA570 (A-1 to A-570), and GA559 (A-1 to N-559) were constructed and resulted in truncated proteins. All of the mutant genes were expressed heterologously in Saccharomyces cerevisiae. GA596 adsorbed to raw starch and digested it. GA570 and GA559 did not adsorb to raw starch or to an alpha-cyclodextrin-Sepharose CL-4B gel under our experimental conditions. However, GA570 was able to digest raw starch, and the digestion of raw starch by GA570 was inhibited by beta-cyclodextrin. Residue Trp-562 of GAI, which was suggested previously to contribute to formation of an inclusion complex with raw starch, was replaced by Leu (GAW562L), Phe (GAW562F), and Gly (GAW562G). GAW562L and GAW562F adsorbed to raw starch and an alpha-cyclodextrin gel, but GAW562G did not. Although GAW562L digested raw starch to the same extent as wild-type GAI (designated GAY), GAW562F and GAW562G exhibited less ability to digest raw starch. On the basis of our results, it appears that the sequence around Trp-562, PL(W-562)YVTVTLPA, is the minimal sequence necessary for digestion of raw starch and that hydrophobic residue Trp-562 contributes to formation of an inclusion complex. The sequence near Trp-589, which has abundant hydrogen bond-forming residues and the charged amino acid residues needed for stable adsorption to raw starch, probably assists in the formation of the inclusion complex.
Construction of phoE-caa, a novel PCR- and immunologically detectable marker gene for Pseudomonas putida
Authors: Zaat S A , Slegtenhorst-Eegdeman K , Tommassen J , Geli V , Wijffelman C A , Lugtenberg B J
Published: January 2020

In this paper we describe the construction and use in Pseudomonas putida WCS358 of phoE-caa, a novel hybrid marker gene, which allows monitoring both at the protein level by immunological methods and at the DNA level by PCR. The marker is based on the Escherichia coli outer membrane protein gene phoE and 75 bp of E. coli caa, which encode a nonbacteriocinic fragment of colicin A. This fragment contains an epitope which is recognized by monoclonal antibody (MAb) 1C11. As the epitope is contained in one of the cell surface-exposed loops of PhoE, whole cells of bacteria expressing the protein can be detected by using the MAb. The marker gene contains only E. coli sequences not coding for toxins and therefore can be considered environmentally safe. The hybrid PhoE-ColA protein was expressed in E. coli under conditions of phosphate starvation, and single cells could be detected by immunofluorescence microscopy with MAb 1C11. Using a wide-host-range vector the phoE-caa gene was introduced into P. putida WCS358. The gene appeared to be expressed under phosphate limitation in this species, and the gene product was present in the membrane fraction and reacted with MAb 1C11. The hybrid PhoE-ColA protein could be detected on whole cells of WCS358 mutant strains lacking (part of) the O-antigen of the lipopolysaccharide but not on wild-type WCS358 cells, unless these cells had previously been washed with 10 mM EDTA. In addition to immunodetection, the phoE-caa marker gene could be specifically detected by PCR with one primer directed to a part of the phoE sequence and a second primer that annealed to the caa insert.
Investigation of an Iron-Oxidizing Microbial Mat Community Located near Aarhus, Denmark: Laboratory Studies
Authors: Emerson David , Revsbech Niels Peter
Published: January 2020

We constructed a small flow chamber in which suboxic medium containing 60 to 120 μM FeCl
2
flowed up through a sample well into an aerated reservoir, thereby creating an suboxic-oxic interface similar to the physicochemical conditions that exist in natural iron seeps. When microbial mat material from the Marselisborg iron seep that contained up to 10
9
bacterial cells per cm
3
(D. Emerson and N. P. Revsbech, Appl. Environ. Microbiol. 60:4022-4031, 1994) was placed in the sample well of the chamber, essentially all of the Fe
2+
flowing through the sample well was oxidized at rates of up to 1,200 nmol of Fe
2+
oxidized per h per cm
3
of mat material. The oxidation rates of samples of the mat that were pasteurized prior to inoculation were only about 20 to 50% of the oxidation rates of unpasteurized samples. Sodium azide also significantly inhibited oxidation. These results suggest that at least 50% and up to 80% of the Fe oxidation in the chamber were actively mediated by the microbes in the mat. It also appeared that Fe stimulated the growth of the community since chambers fed with FeCl
2
accumulated masses of either filamentous or particulate growth, both in the sample well and attached to the walls of the chamber. Control chambers that did not receive FeCl
2
showed no sign of such growth. Furthermore, after 4 to 5 days the chambers fed with FeCl
2
contained 35 to 75% more protein than chambers not supplemented with FeCl
2
.
Leptothrix ochracea
and, to a lesser extent,
Gallionella
spp. were responsible for the filamentous growth, and the sheaths and stalks, respectively, of these two organisms harbored large numbers of Fe-encrusted, nonappendaged unicellular bacteria. In chambers where particulate growth predominated, the unicellular bacteria alone appeared to be the primary agents of iron oxidation. These results provide the first clear evidence that the “iron bacteria” commonly found associated with neutral-pH iron seeps are responsible for most of the iron oxidation and that the presence of ferrous iron appears to stimulate the growth of these organisms.
Comparison of Reactors for Oxygen-Sensitive Reactions: Reductive Dechlorination of Chlorophenols by Vitamin B
12s
Authors: Smith Mark H. , Woods Sandra L.
Published: January 2020

Serum bottles are frequently used for studies of reductive dechlorination by vitamin B
12
, but reducing conditions can be maintained only for several days. This time period is inadequate for evaluating the reductive dechlorination of some slow-reacting aromatic compounds. Sealed glass ampoules maintain reducing conditions for many months, but this method has the disadvantage of disallowing subsampling of the reaction mixture. A glass serum tube was modified for these experiments which not only maintained anoxic conditions for several days but also allowed subsamples to be removed during experiments. The modification was a restriction placed in the middle of the tube by heating in a flame, creating two chambers separated by a narrow neck. The lower chamber contained the oxygen-sensitive reaction mixture. The upper chamber, sealed with a septum and screw cap, was purged with purified nitrogen or argon introduced and vented through fused silica capillaries. Reductive dechlorination of chlorophenols by vitamin B
12
reduced with Ti(III) citrate was monitored in all three reactor types. Sealed ampoules maintained reducing conditions for up to 12 months. The two-chambered reactor maintained reducing conditions longer than the serum vials when frequent samples were taken.