bluuebunny/crossref_metadata_2025 huggingface dataset:
4-Methylumbelliferyl-β-
N
-Acetylglucosaminide Hydrolysis by a High-Affinity Enzyme, a Putative Marker of Protozoan Bacterivory
Authors: Vrba Jaroslav , Šimek Karel , Nedoma Jiř� , Hartman Petr
Published: January 2020

Hydrolysis of an artificial fluorogenic substrate, 4-methylumbelliferyl-β-
N
-acetylglucosaminide, has been studied in a monoculture predator-prey system with either a flagellate (
Bodo saltans
) or a ciliate (
Cyclidium
sp.) fed upon pure bacterial culture (
Aeromonas hydrophila
or
Alcaligenes xylosoxidans
).
Aeromonas hydrophila
produced a low-affinity β-
N
-acetylglucosaminidase-like enzyme (

K
m

, ≫100 μmol liter
-1
) but
Alcaligenes xylosoxidans
did not. Inoculation of both bacterial strains with bacterivorous protozoa induced the occurrence of another, high-affinity, β-
N
-acetylglucosaminidase-like enzyme (

K
m

, <0.5 μmol liter
-1
). The latter enzyme showed significant, close correlations with total grazing rates of both
B. saltans
(
r
2
= 0.96) and
Cyclidium
sp. (
r
2
= 0.89) estimated by using uptake of fluorescently labelled bacteria. Further significant correlations between several protozoan parameters and kinetic parameters of this enzyme suggest its likely protozoan origin. If both types of enzyme occurred together, they could be satisfactorily distinguished by using kinetic data analysis. Hence, measurements of β-
N
-acetylglucosaminidase-like activities might be promising to use to improve estimations of protozoan bacterivory.
Effects of temperature shift on acid and heat tolerance in Salmonella enteritidis phage type 4
Authors: Humphrey T J , Richardson N P , Statton K M , Rowbury R J
Published: January 2020

The transfer of cells of Salmonella enteritidis phage type 4 from 20 to 37-46 degrees C resulted in marked increases in acid and heat tolerance. The former was maximized within 5 to 15 min of the shift and was largely independent of protein synthesis. In contrast, induction of increased heat tolerance was slower, requiring more than 60 min to be completed, and was prevented by inhibition of protein synthesis. When cells were transferred to medium at temperatures between 47 and 50 degrees C, the kinetics of induction of heat tolerance were essentially the same as at the lower temperatures. In contrast, the cells became more acid sensitive. The results of these studies clearly show that although both acid and heat resistance can be enhanced by preexposure to high incubation temperatures, the mechanisms involved are different.
Substrate induction and metabolite accumulation during anaerobic toluene utilization by the denitrifying strain T1
Authors: Frazer A C , Ling W , Young L Y
Published: January 2020

The denitrifying strain T1 utilizes toluene anaerobically. We now report that anaerobic toluene degradation is inducible in strain T1. Fluoracetate treatment of cell suspensions inhibited both the rate of toluene metabolism and the formation of the toluene dead-end products benzylsuccinate and benzylfumarate, which is consistent with the pathway proposed by Evans et al. (Appl. Environ. Microbiol. 58:496-501, 1992). In addition, when either nitrate was limiting or fluoroacetate was added, benzoate was detected during toluene metabolism.
Utilization of Lactic Acid by
Fusarium oxysporum
var.
lini
: Regulation of Transport and Metabolism
Authors: Castro Ieso M. , Loureiro-Dias Maria C.
Published: January 2020

Lactic acid was transported in
Fusarium oxysporum
var.
lini
ATCC 10960 by a saturable transport system that had a half-saturation constant of 56.6 ± 7.5 μM and a maximum velocity of 0.61 ± 0.10 mmol h
-1
g
-1
(dry weight) at 26°C and pH 5.0. This transport system was inducible and was not expressed in the presence of a repressing substrate. Evidence is presented that the anionic form lactate
-
was taken up by the cells. Propionic, acetic, pyruvic, and bromoacetic acids but not succinic acid competitively inhibited the transport of lactic acid. Bromoacetic acid, which was not metabolized, was taken up to a steady-state level when intracellular and extracellular concentrations were identical, indicating that the transport system was not accumulative. The enzymatic activity that was physiologically more relevant in the metabolism of lactic acid was lactate: ferricytochrome
c
oxidase. This enzyme did not exhibit stereospecifity and was induced by lactic acid.
Cloning and nucleotide sequences of the homoserine dehydrogenase genes (hom) and the threonine synthase genes (thrC) of the gram-negative obligate methylotroph Methylobacillus glycogenes
Authors: Motoyama H , Maki K , Anazawa H , Ishino S , Teshiba S
Published: January 2020

We have cloned the homoserine dehydrogenase genes (hom) from the gram-negative obligate methylotrophs Methylobacillus glycogenes ATCC 21276 and ATCC 21371 by complementation of an Escherichia coli homoserine dehydrogenase-deficient mutant. The 4.15-kb DNA fragment cloned from M. glycogenes ATCC 21371 also complemented an E. coli threonine synthase-deficient mutant, suggesting the DNA fragment contained the thrC gene in addition to the hom gene. The homoserine dehydrogenases expressed in the E. coli recombinants were hardly inhibited by L-threonine, L-phenylalanine, or L-methionine. However, they became sensitive to the amino acids after storage at 4 degrees C for 4 days as in M. glycogenes. The structures of the homoserine dehydrogenases overexpressed in E. coli were thought to be different from those in M. glycogenes, probably in subunit numbers of the enzyme, and were thought to have converted to the correct structures during the storage. The nucleotide sequences of the hom and thrC genes were determined. The hom genes of M. glycogenes ATCC 21276 and ATCC 21371 encode peptides with M(r)s of 48,225 and 44,815, respectively. The thrC genes were located 50 bp downstream of the hom genes. The thrC gene of ATCC 21371 encodes a peptide with an M(r) of 52,111, and the gene product of ATCC 21276 was truncated. Northern (RNA) blot analysis suggests that the hom and thrC genes are organized in an operon. Significant homology between the predicted amino acid sequences of the hom and thrC genes and those from other microorganisms was found.
Stable Expression of
hom-1-thrB
in
Corynebacterium glutamicum
and Its Effect on the Carbon Flux to Threonine and Related Amino Acids
Authors: Reinscheid Dieter J. , Kronemeyer Wolfgang , Eggeling Lothar , Eikmanns Bernhard J. , Sahm Hermann
Published: January 2020

The
hom-1-thrB
operon encodes homoserine dehydrogenase resistant to feedback inhibition by L-threonine and homoserine kinase. Stable expression of this operon has not yet been attained in different
Corynebacterium glutamicum
strains. We studied the use of chromosomal integration and of a low-copy-number vector for moderate expression of the
hom-1-thrB
operon to enable an analysis of the physiological consequences of its expression in
C. glutamicum.
Strains carrying one, two, or three copies of
hom-1-thrB
were obtained. They showed proportionally increased enzyme activity of feedback-resistant homoserine dehydrogenase and of homoserine kinase. This phenotype was stably maintained in all recombinants for more than 70 generations. In a lysine-producing
C. glutamicum
strain which does not produce any threonine, expression of one copy of
hom-1-thrB
resulted in the secretion of 39 mM threonine. Additional copies resulted in a higher, although not proportional, accumulation of threonine (up to 69 mM). This indicates further limitations of threonine production. As the copy number of
hom-1-thrB
increased, increasing amounts of homoserine (up to 23 mM) and isoleucine (up to 34 mM) were secreted. Determination of the cytosolic concentration of the respective amino acids revealed an increase of intracellular threonine from 9 to 100 mM and of intracellular homoserine from 4 to 74 mM as the copy number of
hom-1-thrB
increased. These results suggest that threonine production with
C. glutamicum
is limited by the efflux system for this amino acid. Furthermore, the results show the successful use of moderate and stable
hom-1-thrB
expression for directing the carbon flux from aspartate to threonine.
Purification and Characterization of an
l
-Amino Amidase from
Mycobacterium neoaurum
ATCC 25795
Authors: Hermes H. F. M. , Tandler R. F. , Sonke T. , Dijkhuizen L. , Meijer E. M.
Published: January 2020

An
l
-amino amidase from
Mycobacterium neoaurum
ATCC 25795 responsible for the enantioselective resolution of
dl
-α-methyl valine amide was purified and characterized. The purification procedure included ammonium sulfate fractionation, gel filtration, and anion-exchange chromatography, which resulted in a homogeneous preparation of the enzyme with a native molecular mass of 136 kDa and a subunit molecular mass of 40 kDa. The purified enzyme displayed the highest activity at 50°C and at pH 8.0 and 9.5. The enzyme was strongly inhibited by the metal-chelating agent 1,10-phenanthroline, the disulfide-reducing agent dithiothreitol, and the cysteine proteinase inhibitor iodoacetamide. The purified amino amidase showed a unique
l
-enantioselective activity towards a broad range of both α-H- and α-alkyl-substituted amino acid amides, with the highest activity towards the cyclic amino acid amide
dl
-proline amide. No activity was measured with
dl
-mandelic acid amide nor with the dipeptide
l
-phenylalanine-
l
-leucine. The highest catalytic efficiency (
k
cat
/
K

m

ratio) was measured with
dl
-α-allyl alanine amide,
dl
-α-methyl phenylalanine amide, and
dl
-α-methyl leucine amide.
Aerobic and anaerobic degradation and mineralization of 14C-chitin by water column and sediment inocula of the York River estuary, Virginia
Authors: Boyer J N
Published: January 2020

Potential rates of chitin degradation (Cd) and mineralization (Cm) by estuarine water and sediment bacteria were measured as a function of inoculum source, temperature, and oxygen condition. In the water column inoculum, 88 to 93% of the particulate chitin was mineralized to CO2 with no apparent lag between degradation and mineralization. No measurable dissolved pool of radiolabel was found in the water column. For the sediment inocula, 70 to 90% of the chitin was degraded while only 55 to 65% was mineralized to CO2. 14C label recoveries in the dissolved pool were 19 to 21% for sand, 17 to 24% in aerobic mud, and 12 to 21% for the anaerobic mud. This uncoupling between degradation and mineralization occurred in all sediment inocula. More than 98% of the initial 14C-chitin was recovered in the three measured fractions. The highest Cd and Cm values, 30 and 27% day-1, occurred in the water column inoculum at 25 degrees C. The lowest Cd and Cm values were found in the aerobic and anaerobic mud inocula incubated at 15 degrees C. Significant differences in Cd and Cm values among water column and sediment inocula as well as between temperature treatments were evident. An increased incubation temperature resulted in shorter lag times before the onset of chitinoclastic bacterial growth, degradation, and mineralization and resulted in apparent Q10 values of 1.1 for water and 1.3 to 2.1 for sediment inocula. It is clear that chitin degradation and mineralization occur rapidly in the estuary and that water column bacteria may be more important in this process than previously acknowledged.
Evidence of a dormant but infective state of the fish pathogen Pasteurella piscicida in seawater and sediment
Authors: Magariños B , Romalde J L , Barja J L , Toranzo A E
Published: January 2020

The stability of Pasteurella piscicida strains in seawater and sediment microcosms at different temperatures (6 and 20 degrees C) was investigated during a 1-month period. Three strains of P. piscicida showed similar survival kinetics. By a standard plate count method they survived in water and sediment for only 6 to 12 days, depending on the strain and type of microcosm. During this starvation period, the metabolic activity of the cells was reduced by more than 80%. Culturable cells of each P. piscicida strain persisted better in sediment than in water, as well as at 20 degrees C compared to 6 degrees C. However, in all the microcosms, the acridine orange direct counts remained at about 10(5) cells per ml during the experimental period, which demonstrated that P. piscicida possesses a capacity to enter a viable but not culturable state. Moreover, dormant cells were always resuscitated by the addition of fresh medium to the microcosms, since we recovered numbers of culturable cells similar to the acridine orange direct counts. These resuscitated cells exhibited the same respiration rate as that seen prior to the start of the experiments. Although the biochemical, physiological, and serological characteristics; lipopolysaccharides; membrane proteins; and plasmid content of P. piscicida strains were unaffected during the starvation conditions, the dormant cells were smaller (dwarf cells) and had increased surface hydrophobicity. The starved cells maintained their infectivity and pathogenic potential for fish, with 50% lethal doses similar to those of the original strains.
Evaluation of Data Transformations and Validation of a Model for the Effect of Temperature on Bacterial Growth
Authors: Zwietering M. H. , Cuppers H. G. A. M. , de Wit J. C. , van 't Riet K.
Published: January 2020

The temperature of chilled foods is an important variable for controlling microbial growth in a production and distribution chain. Therefore, it is essential to model growth as a function of temperature in order to predict the number of organisms as a function of temperature and time. This article deals with the correct variance-stabilizing transformation of the growth parameters
A
(asymptotic level), μ (specific growth rate), and λ (lag time). This is of importance for the regression analysis of the data. A previously gathered data set and model for the effect of temperature on the growth of
Lactobacillus plantarum
(M. H. Zwietering, J. T. de Koos, B. E. Hasenack, J. C. de Wit, and K. van 't Riet, Appl. Environ. Microbiol. 57:1094-1101, 1991) is extended with new data. With the total data set (original and new data), a variance-stabilizing transformation is selected in order to determine which transformation should precede fitting. No transformation for the asymptote data, a square root for the growth rate, and a logarithmic transformation for the lag time were found to be appropriate. After these transformations, no significant correlation was found between the variance and the magnitude of the variable. Model corrections were made and model parameters were estimated by using the original data. With the new data, the models were validated by comparing the lack of fit of the models with the measurement error, using an
F
test. The predictions of the models for μ and λ were adequate. The model for
A
showed a systematic deviation, and therefore a new model for
A
is proposed.
Importance of cobalt for individual trophic groups in an anaerobic methanol-degrading consortium
Authors: Florencio L , Field J A , Lettinga G
Published: January 2020

Methanol is an important anaerobic substrate in industrial wastewater treatment and the natural environment. Previous studies indicate that cobalt greatly stimulates methane formation during anaerobic treatment of methanolic wastewaters. To evaluate the effect of cobalt in a mixed culture, a sludge with low background levels of cobalt was cultivated in an upflow anaerobic sludge blanket reactor. Specific inhibitors in batch assays were then utilized to study the effect of cobalt on the growth rate and activity of different microorganisms involved in the anaerobic degradation of methanol. Only methylotrophic methanogens and acetogens were stimulated by cobalt additions, while the other trophic groups utilizing downstream intermediates, H2-CO2 or acetate, were largely unaffected. The optimal concentration of cobalt for the growth and activity of methanol-utilizing methanogens and acetogens was 0.05 mg liter-1. The higher requirement of cobalt is presumably due to the previously reported production of unique corrinoid-containing enzymes (or coenzymes) by direct utilizers of methanol. This distinctly high requirement of cobalt by methylotrophs should be considered during methanolic wastewater treatment. Methylotroph methanogens presented a 60-fold-higher affinity for methanol than acetogens. This result in combination with the fact that acetogens grow slightly faster than methanogens under optimal cobalt conditions indicates that acetogens can outcompete methanogens only when reactor methanol and cobalt concentrations are high, provided enough inorganic carbon is available.
Nitrate Reduction in a Sulfate-Reducing Bacterium,
Desulfovibrio desulfuricans
, Isolated from Rice Paddy Soil: Sulfide Inhibition, Kinetics, and Regulation
Authors: Dalsgaard Tage , Bak Friedhelm
Published: January 2020

From the second-highest dilution in a most-probable-number dilution series with lactate and sulfate as substrates and rice paddy soil as the inoculum, a strain of
Desulfovibrio desulfuricans
was isolated. In addition to reducing sulfate, sulfite, and thiosulfate, the strain also reduced nitrate to ammonia. The latter process was studied in detail, since the ability to reduce nitrate was strongly influenced by the presence of sulfide. Sulfide inhibited both growth on nitrate and nitrate reduction. A 70% inhibition of the nitrate reduction rate was obtained at 127 μM sulfide, and growth was inhibited by 50% at approximately 320 μM sulfide and was not detectable above 700 μM sulfide. In contrast, sulfate reduction was not affected at concentrations of up to 5 mM. After growth with sulfate, an induction period of 2 to 4 days was needed before nitrate reduction started. When nitrate and sulfate were present simultaneously, only sulfate was reduced, except when sulfate was present at very low concentrations (4 μM). At higher sulfate concentrations (500 μM), nitrate reduction was temporarily halted. The affinity for nitrate uptake was extremely high (

K
m

= 0.05 μM) compared with that for sulfate uptake (

K
m

= 5 μM). Thus, at low nitrate concentrations this bacterium is favored relative to denitrifiers (

K
m

= 1.8 to 13.7 μM) or other nitrate ammonifiers (e.g.,
Clostridium
spp. [

K
m

= 500 μM]).
Characterization of an endoserine protease secreted by Arthrobacter aureus
Authors: Michotey V , Blanco C
Published: January 2020

A 22-kDa endoserine protease secreted by an Arthrobacter aureus strain was identified. The optimal temperature for this protease was 70 degrees C. Protease production was maximal at the end of the exponential growth phase, and the protease was induced by amino acids and peptides and repressed by carbon sources.
Inactivation of Phage MS2 by Iron-Aided Titanium Dioxide Photocatalysis
Authors: Sjogren Jon C. , Sierka Raymond A.
Published: January 2020

Near-UV photocatalytic disinfection was accomplished in aqueous titanium dioxide suspensions. A level of inactivation of phage MS2 of 90% increased to 99.9% after 2 μM ferrous sulfate was added. Hydroxyl radical oxidation, with Fenton reaction enhancement, is suspected to be primarily responsible for the viral degradation observed.
Pseudomonas putida
Strains Which Constitutively Overexpress Mercury Resistance for Biodetoxification of Organomercurial Pollutants
Authors: Horn Joanne M. , Brunke Maren , Deckwer W.-D. , Timmis Kenneth N.
Published: January 2020

Improved biocatalysts for mercury (Hg) remediation were generated by random mutagenesis of
Pseudomonas putida
with a minitransposon containing
merTPAB
, the structural genes specifying organomercury resistance. Subsequent selection for derivatives exhibiting elevated resistance levels to phenylmercury allowed the isolation of strains that constitutively express
merTPAB
at high levels, conferring the ability to cleave Hg from an organic moiety and reduce the freed Hg(II) to the less toxic elemental form, Hg
0
, at greater rates. Constitutive overexpression of
merTPAB
had no apparent effect on culture growth rates, even when Hg(II) was initially present at otherwise toxic concentrations. These properties were also combined with benzene and toluene catabolism, allowing detoxification of the metal component of phenyl mercuric acetate, as well as degradation of its aromatic moiety.
Detection of Salmonella spp. in oysters by PCR
Authors: Bej A K , Mahbubani M H , Boyce M J , Atlas R M
Published: January 2020

PCR DNA amplification of a region of the himA gene of Salmonella typhimurium specifically detected Salmonella spp. In oysters, 1 to 10 cells of Salmonella spp. were rapidly detected by the PCR following a pre-enrichment step to increase sensitivity and to ensure that detection was based on the presence of viable Salmonella spp.
Use of solid-phase PCR for enhanced detection of airborne microorganisms
Authors: Alvarez A J , Buttner M P , Toranzos G A , Dvorsky E A , Toro A , Heikes T B , Mertikas-Pifer L E , Stetzenbach L D
Published: January 2020

The solid-phase PCR (SP-PCR) was compared with a culture-based technique for the detection of aerosolized Escherichia coli DH1. Results with SP-PCR showed an increase in detection sensitivity over that of culture methods. Therefore, SP-PCR may be useful for the detection of airborne microorganisms which may be nonculturable because of aerosolization or sampling stress.
Cloning of Genes Involved in the Synthesis of Pyrrolnitrin from
Pseudomonas fluorescens
and Role of Pyrrolnitrin Synthesis in Biological Control of Plant Disease
Authors: Hill D. S. , Stein J. I. , Torkewitz N. R. , Morse A. M. , Howell C. R. , Pachlatko J. P. , Becker J. O. , Ligon J. M.
Published: January 2020

A soil isolate of
Pseudomonas fluorescens
(BL915) was shown to be an effective antagonist of
Rhizoctonia solani
-induced damping-off of cotton. Investigation of the biological basis of this antagonism revealed that the strain produces pyrrolnitrin, a secondary metabolite known to inhibit
R. solani
and other fungi. Mutants of strain BL915 that did not produce pyrrolnitrin and did not suppress damping-off of cotton by
R. solani
were generated by exposure to
N
-methyl-
N′
-nitro-
N
-nitrosoguanidine. A gene region that was capable of restoring pyrrolnitrin production to the non-pyrrolnitrin-producing mutants and of conferring this ability upon two other
P. fluorescens
strains not otherwise known to produce this compound or to be capable of suppressing damping-off caused by
R. solani
was isolated from strain BL915. The non-pyrrolnitrin-producing strains (mutants of BL915 and the other two
P. fluorescens
strains) which synthesized pyrrolnitrin after the introduction of the gene region from strain BL915 were also shown to be equal to strain BL915 in their ability to suppress
R. solani
-induced damping-off of cotton. These results indicate that we have isolated from
P. fluorescens
BL915 a gene(s) that has a role in the synthesis of pyrrolnitrin and that the production of this compound has a role in the ability of this strain to control damping-off of cotton by
R. solani.
Development of an amylolytic Lactobacillus plantarum silage strain expressing the Lactobacillus amylovorus alpha-amylase gene
Authors: Fitzsimons A , Hols P , Jore J , Leer R J , O'Connell M , Delcour J
Published: January 2020

An amylolytic Lactobacillus plantarum silage strain with the starch-degrading ability displayed by Lactobacillus amylovorus was developed. An active fragment of the gene coding for alpha-amylase production in L. amylovorus was cloned and integrated into the chromosome of the competitive inoculant strain L. plantarum Lp80 at the cbh locus. The alpha-amylase gene fragment was also introduced into L. plantarum Lp80 on an autoreplicative plasmid. Both constructions were also performed in the laboratory strain L. plantarum NCIB8826. All four recombinant strains secreted levels of amylase ranging from 23 to 69 U/liter, compared with 47 U/liter for L. amylovorus. Secretion levels were higher in L. plantarum NCIB8826 than in L. plantarum Lp80 derivatives and were higher in recombinant strains containing autoreplicative plasmids than in the corresponding integrants. The L. plantarum Lp80 derivative containing the L. amylovorus alpha-amylase gene fragment integrated into the host chromosome secreted alpha-amylase to a level comparable to that of L. amylovorus and was stable over 50 generations of growth under nonselective conditions. It grew to a higher cell density than either the parent strain or L. amylovorus in MRS medium containing a mixture of starch and glucose as the fermentable carbohydrate source. This recombinant alpha-amylolytic L. plantarum strain would therefore seem to have considerable potential as a silage inoculant for crops such as alfalfa, in which water-soluble carbohydrate levels are frequently low but starch is present as an alternative carbohydrate source.
Effects of Neem Leaf Volatiles on Submerged Cultures of Aflatoxigenic
Aspergillus parasiticus
Authors: Zeringue H. J. , Bhatnagar D.
Published: January 2020

Microbe-free compressed air was passed continuously for a 3-day test period through an enclosed system containing fresh neem leaves; the resultant emitted volatiles were passed over the surface of submerged liquid cultures of a wild-type aflatoxigenic isolate of
Aspergillus parasiticus
. Aflatoxin determinations for the fungal culture that received neem-derived volatiles, after a 3-day incubation period, resulted in a 90% overall reduction in aflatoxin production and a 51% reduction in fungal biomass when compared with cultures that did not receive neem volatiles. In a separate experiment but in a similarly enclosed system, volatiles from fresh neem leaves were collected on a small Tenax column and were thermally desorbed and cryogenically focused on a capillary gas chromatography column. The neem volatiles were subsequently separated and identified by gas chromatography-mass spectrometry. Sixty-eight compounds were identified by comparison of retention times and mass spectra with either authentic compounds or spectra from a computer-assisted library database of mass spectra. It was found that 10% of the total headspace volatiles were composed of C
3
to C
9
alkenals, which are toxic to aflatoxigenic
Aspergillus
spp., which could explain the bioactivity that resulted in reduced biomass in the neem-treated cultures.
Influence of preadsorbed milk proteins on adhesion of Listeria monocytogenes to hydrophobic and hydrophilic silica surfaces
Authors: al-Makhlafi H , McGuire J , Daeschel M
Published: January 2020

The adsorption of beta-lactoglobulin, bovine serum albumin, alpha-lactalbumin, and beta-casein for 8 h and beta-lactoglobulin and bovine serum albumin for 1 h at silanized silica surfaces of low and high hydrophobicity, followed by incubation in buffer and contact with Listeria monocytogenes, resulted in different numbers of cells adhered per unit of surface area. Adhesion to both surfaces was greatest when beta-lactoglobulin was present and was lowest when bovine serum albumin was present. Preadsorption of alpha-lactalbumin and beta-casein showed an intermediate effect on cell adhesion. Adsorption of beta-lactoglobulin for 1 h resulted in a generally lower number of cells adhered compared with the 8-h adsorption time, while the opposite result was observed with respect to bovine serum albumin. The adhesion data were explainable in terms of the relative rates of arrival to the surface and postadsorptive conformational change among the proteins, in addition to the extent of surface coverage in each case.
In vivo monitoring system for structure-function relationship analysis of the antibacterial peptide apidaecin
Authors: Taguchi S , Nakagawa K , Maeno M , Momose H
Published: January 2020

A unique antibacterial peptide derivative found in immune honeybee lymph, apidaecin 1b (AP1), was randomly mutagenized and characterized by a newly established system to analyze in vivo its structure-function relationship. Initially, a high-level expression host-vector system for AP1 in Escherichia coli was constructed by creating a fusion protein with the highly stable Streptomyces subtilisin inhibitor (SSI) molecule. Expression of the SSI-AP1 fusion protein was found to depend on the concentration of the transcriptional inducer isopropyl-beta-D-thio-galactopyranoside (IPTG) and to parallel the degree of growth inhibition of the transformant cells. Subsequently, apidaecin derivatives produced by localized random mutagenesis were screened with this IPTG concentration-controlled in vivo system by monitoring the growth inhibition patterns of the transformant cells. One mutant apidaecin (P9L) that had reduced activity was purified and isolated from the periplasmic fraction of an E. coli transformant. Its antibacterial activity was reduced to one-third of that of wild-type apidaecin. When considered together with the other mutations, it was concluded that several Pro residues, including that at the ninth position, are responsible for expression of the antibacterial action of apidaecin.
Pyrene Metabolism in
Crinipellis stipitaria
: Identification of
trans
-4,5-Dihydro-4,5-Dihydroxypyrene and 1-Pyrenylsulfate in Strain JK364
Authors: Lange Bettina , Kremer Stefan , Sterner Olov , Anke Heidrun
Published: January 2020

The isolation and identification of two novel metabolites in the fungal metabolism of pyrene are described. The plant-inhabiting basidiomycete
Crinipellis stipitaria
JK364 metabolized pyrene, a polycyclic aromatic hydrocarbon containing four rings, when grown in submerged cultures in a medium containing malt extract, glucose, and yeast extract. In experiments with [
14
C] pyrene, after 7 days of incubation 40% of the labeled substrate was converted into organic solvent-extractable metabolites. Metabolites isolated from cultures grown with pyrene were identified as 1-pyrenylsulfate and
trans
-4,5-dihydro-4,5-dihydroxypyrene. 1-Hydroxypyrene, the precursor of 1-pyrenylsulfate, was also detected. 1-Pyrenylsulfate was isolated from mycelial extracts, whereas
trans
-4,5-dihydro-4,5-dihydroxypyrene was recovered from the culture filtrate. Identification of the compounds was based on their UV spectra, mass spectra, and nuclear magnetic resonance spectra. This is the first report on the detoxification of a polycyclic aromatic hydrocarbon by a plant-inhabiting basidiomycete. The occurrence of 1-pyrenylsulfate and
trans
-4,5-dihydro-4,5-dihydroxypyrene among fungal metabolites of pyrene is also new.
The use of 16S rRNA-targeted oligonucleotide probes to study competition between ruminal fibrolytic bacteria: pure-culture studies with cellulose and alkaline peroxide-treated wheat straw
Authors: Odenyo A A , Mackie R I , Stahl D A , White B A
Published: January 2020

Specific oligonucleotide probes targeted to sites on the 16S rRNA of Ruminococcus albus 8, Ruminococcus flavefaciens FD-1, and Fibrobacter succinogenes S85 and a domain Bacteria probe were used to study bacterial interactions during the fermentation of cellulose and alkaline hydrogen peroxide-treated wheat straw in monocultures, dicultures, and tricultures. Results showed that R. albus 8 inhibited the growth of R. flavefaciens FD-1 when grown as a diculture with cellulose or alkaline hydrogen peroxide-treated wheat straw as the carbon source. In dicultures containing R. albus 8 and F. succinogenes S85 grown on cellulose or alkaline hydrogen peroxide-treated wheat straw, competition was not detected. R. flavefaciens FD-1 outcompeted F. succinogenes S85 when cellulose was used as the carbon source. In tricultures with cellulose as the carbon source, R. flavefaciens FD-1 was inhibited, R. albus 8 appeared to dominate during the early phase of degradation (12 to 48 h), while F. succinogenes S85 became predominant during the later phase of degradation (60 to 70 h). When alkaline hydrogen peroxide-treated wheat straw was used as a growth substrate, F. succinogenes S85 showed better growth than either R. albus 8 or R. flavefaciens FD-1. However, R. flavefaciens FD-1 was present in small numbers throughout the incubation period, unlike the growth patterns when cellulose was the carbon source.
Production of 10-Ketostearic Acid from Oleic Acid by
Flavobacterium
sp. Strain DS5 (NRRL B-14859)
Authors: Hou Ching T.
Published: January 2020

A microbial isolate,
Flavobacterium
sp. strain DS5, produces 10-ketostearic acid (10-KSA) from oleic acid in 85% yield. This is the first report on this type of reaction catalyzed by a
Flavobacterium
strain. The product was purified to give white, plate-like crystals melting at 79.2°C. The optimum time, pH, and temperature for the production of 10-KSA are 36 h, 7.5, and 30°C, respectively. A small amount of 10-hydroxystearic acid (10-HSA) (about 10% of the amount of the main product, 10-KSA) is also produced during the bioconversion. 10-KSA is not further metabolized by strain DS5 and accumulates in the medium. In contrast to growing cells, a resting-cell suspension of strain DS5 produces 10-HSA and 10-KSA in a ratio of 1:3. The crude cell extract obtained from ultrasonic disruption of the cells converts oleic acid mainly to 10-HSA (10-HSA/10-KSA ratio, 97:3). This result strongly suggested that oleic acid is converted to 10-KSA via 10-HSA. Enzymes catalyzing the hydration and secondary alcohol dehydrogenation are cell associated. Product 10-HSA from strain DS5 is 66% enantiomeric excess in the 10(
R
) form. The oleic acid conversion enzyme(s) reacts with unsaturated fatty acids in the order oleic acid > palmitoleic acid > linoleic acid > linolenic acid > γ-linolenic acid > myristoleic acid.
Production, Purification, and Properties of a Thermostable β-Glucosidase from a Color Variant Strain of
Aureobasidium pullulans
Authors: Saha Badal C. , Freer Shelby N. , Bothast Rodney J.
Published: January 2020

A color variant strain of
Aureobasidium pullulans
(NRRL Y-12974) produced β-glucosidase activity when grown in liquid culture on a variety of carbon sources, such as cellobiose, xylose, arabinose, lactose, sucrose, maltose, glucose, xylitol, xylan, cellulose, starch, and pullulan. An extracellular β-glucosidase was purified 129-fold to homogeneity from the cell-free culture broth of the organism grown on corn bran. The purification protocol included ammonium sulfate treatment, CM Bio-Gel A agarose column chromatography, and gel filtrations on Bio-Gel A-0.5m and Sephacryl S-200. The β-glucosidase was a glycoprotein with native molecular weight of 340,000 and was composed of two subunits with molecular weights of about 165,000. The enzyme displayed optimal activity at 75°C and pH 4.5 and had a specific activity of 315 μmol · min
-1
· mg of protein
-1
under these conditions. The purified β-glucosidase was active against
p
-nitrophenyl-β-
d
-glucoside, cellobiose, cellotriose, cellotetraose, cellopentaose, cellohexaose, and celloheptaose, with

K
m

values of 1.17, 1.00, 0.34, 0.36, 0.64, 0.68, and 1.65 mM, respectively. The enzyme activity was competitively inhibited by glucose (

K
i

= 5.65 mM), while fructose, arabinose, galactose, mannose, and xylose (each at 56 mM) and sucrose and lactose (each at 29 mM) were not inhibitory. The enzyme did not require a metal ion for activity, and its activity was not affected by
p
-chloromercuribenzoate (0.2 mM), EDTA (10 mM), or dithiothreitol (10 mM). Ethanol (7.5%, vol/vol) stimulated the initial enzyme activity by 15%. Glucose production was enhanced by 7.9% when microcrystalline cellulose (2%, wt/vol) was treated for 48 h with a commercial cellulase preparation (5 U/ml) that was supplemented with the purified β-glucosidase (0.21 U/ml) from
A. pullulans.
Sensitization of heat-treated Listeria monocytogenes to added lysozyme in milk
Authors: Kihm D J , Leyer G J , An G H , Johnson E A
Published: January 2020

Listeria monocytogenes was highly resistant to hen egg white lysozyme in whole milk but was sensitive in media and in phosphate buffer. Methods to sensitize the pathogen to lysozyme in milk were investigated. Treatment of whole milk by cation exchange to remove minerals, particularly Ca2+ and Mg2+, slightly promoted inactivation of L. monocytogenes by lysozyme at 4 degrees C over a period of 6 days. Heat treatment (62.5 degrees C for 15 s) strongly sensitized L. monocytogenes to lysozyme in demineralized milk and in MES [2-(N-morpholino)ethanesulfonic acid] buffer. Addition of Ca2+ or Mg2+ to the demineralized milk restored resistance to lysozyme. Cells were more rapidly heat inactivated at 55 degrees C in demineralized milk containing lysozyme, and addition of Ca2+ to the demineralized milk restored the resistance to heat. The results indicate that minerals or mineral-associated components protect L. monocytogenes from inactivation by lysozyme and heat in milk, probably by increasing cell surface stability. The heat treatment of foods containing added lysozyme can probably play a significant role in producing microbiologically safe foods.
Decreased susceptibility of melanized Cryptococcus neoformans to UV light
Authors: Wang Y , Casadevall A
Published: January 2020

Melanized Cryptococcus neoformans cells were less susceptible than nonmelanized cells to the fungicidal effects of UV light. Phenoloxidase-catalyzed production of melanin-like pigments may serve to protect the fungus against ionizing radiation.
A transcriptional reporter for in vivo promoter analysis in the archaeon Haloferax volcanii
Authors: Palmer J R , Daniels C J
Published: January 2020

We have used a modified intron-containing tRNA(Pro(UGG) gene (tRNA(ProM), derived from the Saccharomyces cerevisiae tRNA(Pro(UGG) gene, as a reporter to measure in vivo transcription from a halophilic archaeon promoter. Coupling of the yeast tRNA(ProM) gene to the Haloferax volcanii tRNA(Lys) promoter on the H. volcanii plasmid pWL201 led to the production of a single stable transcript that was readily quantitated by Northern (RNA) blot analysis. Comparison of tRNA(ProM) RNA production from constructs containing the wild-type tRNA(Lys) promoter and those containing mutant tRNA(Lys) promoters demonstrated that this assay system can be used to measure expression from strong and weak promoters.
Identification of a heroin esterase in Rhodococcus sp. strain H1
Authors: Cameron G W , Jordan K N , Holt P J , Baker P B , Lowe C R , Bruce N C
Published: January 2020

A strain of a Rhodococcus sp. (termed H1) capable of utilizing heroin as its sole carbon and energy source was isolated by selective enrichment. An inducible heroin esterase was partially purified and shown to catalyze the hydrolysis of both of the acetylester groups of heroin. The enzyme displays optimum activity at pH 8.5 and appears to be a trimer of identical subunits with an M(r) or 39,000 and a native M(r) of 120,000.
Evaluation of alternariol and alternariol methyl ether for mutagenic activity in Salmonella typhimurium
Authors: Davis V M , Stack M E
Published: January 2020

Alternariol and alternariol methyl ether were tested in the Ames Salmonella typhimurium assay, and both were shown, with and without metabolic activation, to be nonmutagenic to strains TA98 and TA100. The finding of other investigators that alternariol methyl ether is weakly mutagenic to TA98 without metabolic activation could have resulted from the presence of a small amount of one of the highly mutagenic altertoxins in the alternariol methyl ether originally tested.
Engineering of Escherichia coli central metabolism for aromatic metabolite production with near theoretical yield
Authors: Patnaik R , Liao J C
Published: January 2020

Escherichia coli and many other microorganisms synthesize aromatic amino acids through the condensation reaction between phosphoenolpyruvate (PEP) and erythrose 4-phosphate to form 3-deoxy-D-arabinoheptulosonate 7-phosphate (DAHP). It has been shown that overexpression of transketolase increases the production of DAHP in an aroB mutant strain (unable to further metabolize DAHP) with elevated DAHP synthase. However, the yield (percent conversion) of DAHP from glucose is still low. Stoichiometric analysis shows that many enzymes compete for intracellular PEP. In particular, the phosphotransferase system, responsible for glucose transport in E. coli, uses PEP as a phosphate donor and converts it to pyruvate, which is less likely to recycle back to PEP. This stoichiometric limitation greatly reduces the yield of aromatic metabolites. To relieve this limitation, we overexpressed PEP synthase in the presence of glucose and showed that it increased the final concentration and the yield of DAHP by almost twofold, to a near theoretical maximum. The PEP synthase effect is not observed without overproduced transketolase, suggesting that erythrose 4-phosphate is the first limiting metabolite. This result demonstrates the utility of pathway analysis and the limitation of central metabolites in the high-level overproduction of desired metabolites.
Evidence for microbial iron reduction in a landfill leachate-polluted aquifer (Vejen, Denmark)
Authors: Albrechtsen H J , Christensen T H
Published: January 2020

Aquifer sediment samples obtained from the anaerobic part of a landfill leachate plume in Vejen, Denmark, were suspended in groundwater or in an artificial medium and incubated. The strictly anaerobic suspensions were tested for reduction of ferric iron [Fe(III)] oxides, which was measured as an increase in the concentration of dissolved Fe(II). Iron reduction did not occur when the medium was inoculated with inactive sediment and when the organisms in the inoculated medium were killed by formaldehyde, by chloroform, or by pasteurization, whereas the level of iron reduction was significant when living bacteria were present. Mixed cultures were obtained from the sediment samples, and differences in apparent iron reduction rates among the different cultures were maintained during several transfers. In addition, iron reduction was observed in unamended incubation mixtures containing whole sediment and groundwater. Synthetic amorphous Fe(III) oxides, as well as naturally occurring sediment-bound Fe(III) oxides, could be reduced by the cultures. Together, our results provide evidence that iron-reducing bacteria are present and microbial iron reduction occurs in the polluted aquifer sediments which we studied.
A Structure-Activity Study with Aryl Acylamidases
Authors: Villarreal David T. , Turco Ronald F. , Konopka Allan
Published: January 2020

We examined the relationship between chemical structure and biodegradability of acylanilide herbicides by using a set of model compounds. Four bacterial isolates (one gram-negative and three gram-positive) that grew on acetanilide were used. These soil isolates cleaved the amide bond of acetanilide via an aryl acylamidase reaction, producing aniline and the organic acid acetate. A series of acetanilide analogs with alkyl substitutions on the nitrogen atom or the aromatic ring were tested for their ability to induce aryl acylamidase activity and act as substrates for the enzyme. The substrate range, in general, was limited to those analogs not disubstituted in the
ortho
position of the benzene ring or which did not contain an alkyl group on the nitrogen atom. These same N-substituted compounds did not induce enzyme activity either, whereas the
ortho
-substituted compounds could in some cases.
Methane Metabolism in a Temperate Swamp
Authors: Amaral J. A. , Knowles R.
Published: January 2020

Comparisons between in situ CH
4
concentration and potential factors controlling its net production were made in a temperate swamp. Seasonal measurements of water table level and depth profiles of pH, dissolved CH
4
, CO
2
, O
2
, SO
4
2-
, NO
3
-
, formate, acetate, propionate, and butyrate were made at two adjacent sites 1.5 to 2 m apart. Dissolved CH
4
was inversely correlated to O
2
and, in general, to NO
3
-
and SO
4
2-
, potential inhibitors of methanogenesis. At low water table levels (August 1992), maximal CH
4
(2 to 4 μM) occurred below 30 cm, whereas at high water table levels (October 1992) or under flooded conditions (May 1993), CH
4
maxima (4 to 55 μM) occurred in the top 10 to 20 cm. Higher CH
4
concentrations were likely supported by inputs of fresh organic matter from decaying leaf litter, as suggested by high acetate and propionate concentrations (25 to 100 μM) in one of the sites in fall and spring. Measurements of potential CH
4
production (and consumption) showed that the highest rates generally occurred in the top 10 cm of soil. Soil slurry incubations confirmed the importance of organic matter to CH
4
production but also showed that competition for substrates by nonmethanogenic microorganisms could greatly attenuate its effect.
Plant and algal interference in bacterial beta-D-galactosidase and beta-D-glucuronidase assays
Authors: Davies C M , Apte S C , Peterson S M , Stauber J L
Published: January 2020

Several commonly occurring freshwater and marine plants and algae were screened for beta-D-galactosidase and beta-D-glucuronidase activities by using a 60-min enzyme assay based on the hydrolysis by these enzymes of 4-methylumbelliferyl-beta-D-galactoside and 4-methylumbelliferyl- beta-glucuronide, respectively. All freshwater plant extracts tested showed beta-D-galactosidase activity several at relatively high levels, and a number also showed beta-D-glucuronidase activity. A number of the macroalgae showed no activity of either enzyme, but those showing beta-D-galactosidase activity also showed beta-D-glucuronidase activity. The majority of microalgae showed some beta-D-galactosidase activity, but few showed beta-D-glucuronidase activity. Further studies, using the commercial Colilert test and the marine water formulation of Colilert, revealed that 2 of 11 of the microalgal species and several of the plant extracts tested caused positive reactions. It was concluded that several plant extracts and algae could significantly interfere with the detection of coliform bacteria and Escherichia coli with the use of rapid assays, on the basis of their production of beta-D-galactosidase and beta-D-glucuronidase, respectively. The significance of the plant and algal interferences in tests such as Colilert is dependent on the levels of enzymes released under natural conditions, the dilution which they may undergo, and the numbers of algal cells present. This also applies to interferences in rapid enzyme assays.(ABSTRACT TRUNCATED AT 250 WORDS)
Inoculation of
Acacia mangium
with Alginate Beads Containing Selected
Bradyrhizobium
Strains under Field Conditions: Long-Term Effect on Plant Growth and Persistence of the Introduced Strains in Soil
Authors: Galiana Antoine , Prin Yves , Mallet Bernard , Gnahoua Guy-Modeste , Poitel Mireille , Diem Hoang Gia
Published: January 2020

The growth response of
Acacia mangium
Willd. to inoculation with selected
Bradyrhizobium
strains was investigated in two field trials in the Ivory Coast (West Africa). In the first trial (Anguededou), four provenances (i.e., trees originating from seeds harvested in different geographical areas) of
A. mangium
were inoculated with four
Bradyrhizobium
strains from different origins. Six months after being transplanted in the field, the heights of all inoculated trees showed a statistically significant increase of 9 to 26% compared with those of uninoculated trees, with the most effective strain being Aust 13c. After 19 months, the positive effect of inoculation on tree growth was confirmed. The effect of
A. mangium
provenance on tree growth was also highly significant. Trees from the Oriomo provenance of Papua New Guinea had a mean height that was 25% greater than those of other provenances. Analysis of variance showed a highly significant effect of interaction between strain and host provenance factors. Thus, most effective strain × provenance combinations could be proposed. Immunological identification of strains clearly showed that 90 to 100% of nodules from trees inoculated with three of the four
Bradyrhizobium
strains or from uninoculated trees contained exclusively Aust 13c 23 months after tree transplantation. This predominance of Aust 13c in nodules was still observed 42 months after tree transplantation. The second experiment (Port-Bouët), performed with a different soil, confirmed the long-term positive effect of Aust 13c on plant growth, its high competitive ability against indigenous strains, and its persistence in soil. Strain Aust 13c should thus be of great interest for inoculating
A. mangium
under a wide range of field conditions.