bluuebunny/crossref_metadata_2025 huggingface dataset:
A method to detect enteroviruses in sewage sludge-amended soil using the PCR
Authors: Straub T M , Pepper I L , Abbaszadegan M , Gerba C P
Published: January 2020

PCR detection of seeded poliovirus type 1 in sludge-amended soil was made possible by utilizing Sephadex G-50 and Chelex-100 resins to remove compounds present in sludge-amended soil that may inhibit PCR. With this method, enteroviruses indigenous to an anerobically digested sludge were detected by PCR in 10 different soils amended with this sludge.
Inactivation of
ccmO
in
Synechococcus
sp. Strain PCC 7942 Results in a Mutant Requiring High Levels of CO
2
Authors: Marco E. , Martinez I. , Ronen-Tarazi M. , Orus M. I. , Kaplan A.
Published: January 2020

Inactivation of
ccmO
in
Synechococcus
sp. strain PCC 7942 resulted in a mutant which possesses aberrant carboxysomes and a normal inorganic carbon uptake capability but a reduced ability to photosynthetically utilize the internal inorganic carbon pool. Consequently, it exhibits low apparent photosynthetic affinity for extracellular inorganic carbon and demands high levels of CO
2
for growth.
Evaluation of Procedures for Isolation of Nontuberculous Mycobacteria from Soil and Water
Authors: Kamala T. , Paramasivan C. N. , Herbert Daniel , Venkatesan P. , Prabhakar R.
Published: January 2020

Six methods of decontamination each for the isolation of mycobacteria from soil and water were compared. On the basis of the results obtained, three of the six methods for soil and two of the six methods for water were further evaluated. For both soil and water samples, the method using 3% sodium lauryl sulfate in combination with 1% NaOH yielded more positives than the other methods.
Characterization and sequence of a Thermomonospora fusca xylanase
Authors: Irwin D , Jung E D , Wilson D B
Published: January 2020

TfxA is a thermostable xylanase produced by the thermophilic soil bacterium Thermomonospora fusca. The enzyme was purified to homogeneity from the culture supernatant of Streptomyces lividans transformed by plasmid pGG92, which carries the gene for TfxA, xynA. The molecular mass of TfxA by sodium dodecyl sulfate-polyacrylamide gel electrophoresis is 32 kDa. TfxA is extremely stable, retaining 96% of its activity after 18 h at 75 degrees C. It has a broad pH optimum around pH 7 and retains 80% of its maximum activity between pH 5 and 9. The native enzyme binds strongly to both cellulose and insoluble xylan even though it has no activity on cellulose. Treatment of TfxA with a T. fusca protease produced a 24-kDa catalytically active fragment that had the same N-terminal sequence as TfxA. The fragment does not bind to cellulose and binds weakly to xylan. The Vmax values for TfxA and the fragment are 600 and 540 mumol/min/mg, respectively, while the Kms are 1.1 and 2.3 mg of xylan per ml, respectively. The DNA sequence of the xynA gene was determined, and it contains an open reading frame that codes for a 42-amino-acid (42-aa) actinomycete signal peptide followed by the 32-kDa mature protein. There is a 21-aa Gly-Pro-rich region that separates the catalytic domain from an 86-aa C-terminal binding domain. The amino acid sequence of the catalytic domain of TfxA has from 40 to 72% identity with the sequence of 12 other xylanases from seven different organisms and belongs to family G.(ABSTRACT TRUNCATED AT 250 WORDS)
Bioavailability of Chlorocatechols in Naturally Contaminated Sediment Samples and of Chloroguaiacols Covalently Bound to C
2
-Guaiacyl Residues
Authors: Allard Ann-Sofie , Hynning Per-Åke , Remberger Mikael , Neilson Alasdair H.
Published: January 2020

Bacteria in anaerobic enrichment cultures that dechlorinated a range of chlorocatechols were used to examine the stability of endogenous chlorocatechols in a contaminated sediment sample and in interstitial water prepared from it. During incubation of the sediment sample for 450 days with or without added cells, there was a decrease in the concentration of solvent-extractable chlorocatechols but not in that of the total chlorocatechols, including sediment-associated components. In the presence of azide, the decrease in the concentrations of the former was eliminated or substantially decreased. Control experiments in which 3,4,5-trichlorocatechol was added to the sediment suspensions after 130 days showed that its dechlorination was accomplished not only by the added cells but also by the endemic microbial flora. It was concluded that the endogenous chlorocatechols in the sediment were not accessible to microorganisms with dechlorinating activity. On the other hand, microorganisms were apparently responsible for decreasing the solvent extractability of the chlorocatechols, and this effect decreased with increasing length of exposure time. Similar experiments carried out for 70 days with the sediment interstitial water showed that the chlorocatechols that were known to be associated with organic matter were also inaccessible to microbial dechlorination. Experiments with model compounds in which 4,5,6-trichloroguaiacol and tetrachloroguaiacol were covalently linked to C
2
-guaiacyl residues showed that these compounds were resistant to O demethylation or dechlorination during incubation with a culture having these activities. The only effect of microbial action was the quantitative reduction in 12 days of the C′1 keto group to an alcohol which was stable against further transformation for up to 65 days. The results of these experiments are consistent with the existence of chlorocatechols and chloroguaiacols in contaminated sediments and illustrate the cardinal significance of bioavailability in determining their recalcitrance to dechlorination and O demethylation, respectively. It is suggested that bioavailability is an important factor in determining the persistence of xenobiotics in natural ecosystems and that its omission represents a serious limitation in the interpretation of many laboratory experiments directed towards determining the persistence of xenobiotics in aquatic ecosystems.
Production of Bacteriolytic Enzymes by
Streptomyces globisporus
Regulated by Exogenous Bacterial Cell Walls
Authors: Brönneke Volker , Fiedler Franz
Published: January 2020

Mutanolysin biosynthesis and pigment production in
Streptomyces globisporus
ATCC 21553 were stimulated by adding bacterial cell walls to the medium. The increased bacteriolytic activity in the supernatant correlated with an increased de novo synthesis of mutanolysin and was between 4- and 20-fold higher than in cultures grown without bacterial cell walls. The increase in mutanolysin synthesis was brought about by enhanced transcription of the mutanolysin gene. The stimulation was only observed in medium which contained dextrin or starch as the carbon source. Glucose abolished the stimulation and also inhibited the low constitutive synthesis of mutanolysin. The induction of lytic activity was observed to require minimally 0.4 mg of bacterial cell walls per ml, whereas 0.6 mg of bacterial cell walls per ml yielded maximal lytic activity. Further supplements of bacterial cell walls did not result in enhanced lytic activity. The stimulation could be achieved independently of the phase of growth of the
Streptomyces
strain. Cultures grown in the presence of bacterial cell walls exhibited a higher growth yield. However, the accelerated growth was not the reason for the increased amount of mutanolysin produced. The growth of cultures with peptidoglycan monomers added to the medium instead of cell walls was similarly increased, but an effect on the biosynthesis of mutanolysin was not observed. All bacterial cell walls tested were capable of eliciting the stimulation of lytic activity, including cell walls of archaea, which contained pseudomurein.
Specificity of hydrolysis of bovine kappa-casein by cell envelope-associated proteinases from Lactococcus lactis strains
Authors: Reid J R , Coolbear T , Pillidge C J , Pritchard G G
Published: January 2020

The cell envelope-associated proteinases from Lactococcus lactis subsp. cremoris H2 (a PI-type proteinase-producing strain) and SK11 (a PIII-type proteinase-producing strain) both actively hydrolyze the kappa-casein component of bovine milk but with significant differences in the specificity of peptide bond hydrolysis. The peptide bonds Ala-23-Lys-24, Leu-32-Ser-33, Ala-71-Gln-72, Leu-79-Ser-80, Met-95-Ala-96, and Met-106-Ala-107 were cleaved by both proteinase types, although the relative rates of hydrolysis at some of these sites were quite different for the two proteinases. Small histidine-rich peptides were formed as early products of the action of the cell envelope-associated proteinases on kappa-casein, implicating this casein as a possible significant source of histidine, which is essential for starter growth. The major difference between the two proteinase types in their action on kappa-casein was in their ability to cleave bonds near the C-terminal end of the molecule. The bond Asn-160-Thr-161 and, to a lesser extent, the bond Glu-151-Val-152 were very rapidly cleaved by the PIII-type proteinase, whereas hydrolysis of these bonds by the PI-type proteinase was barely detectable (even after 24 h of digestion). Differential hydrolysis of kappa-casein at these sites by the two different proteinase types resulted in the formation of distinctive, high-M(r) products detectable by sodium dodecyl sulfate-polyacrylamide gel electrophoresis.(ABSTRACT TRUNCATED AT 250 WORDS)
A novel gene tag for identifying microorganisms released into the environment
Authors: Hwang I , Farrand S K
Published: January 2020

A novel method using a moc (mannityl opine catabolism) region from the Agrobacterium tumefaciens Ti plasmid pTi15955 was developed as a tag to identify genetically modified microorganisms released into the environment. Pseudomonas fluorescens 1855.344, a plant-growth-promoting rhizosphere bacterium, was chosen as the organism in which to develop and test the system. moc genes carried by pYDH208, a cosmid clone containing a 20-kb segment of the octopine-mannityl opine-type Ti plasmid, conferred on P. fluorescens strains the capacity to utilize mannopine and agropine (AGR) as a sole source of carbon and energy. Modified P. fluorescens strains containing moc or moc::nptII inserted into a chromosomal site were constructed by marker exchange. One such modified strain, PF5MT12, utilized AGR as a sole carbon source and contained detectable levels of mannopine cyclase, an easily assayable enzyme encoded by the moc region. Catabolism of AGR could be used to recover selectively the marked strain from mixed populations containing a large excess of closely related bacteria. Nucleic acid-based detection strategies were developed on the basis of the unique fusion region between Agrobacterium DNA and Pseudomonas DNA in strain PF5MT12. The specificity and sensitivity of detection of PF5MT12 were enhanced by amplifying the fused DNA region by using PCR. The target fragment could be detected at levels of sensitivity comparable to those of other described PCR-based gene tags, even in the presence of high levels of Agrobacterium, Pseudomonas, or Escherichia coli DNA. This gene tag strategy gives a method for direct selection and enumeration of the marked strain from mixtures containing a large excess of closely related bacteria and a sensitive and highly specific system for detection by PCR amplification of the target fragment even in the presence of large amounts of DNA from related or unrelated organisms.
In Vitro Bleaching of Hardwood Kraft Pulp by Extracellular Enzymes Excreted from White Rot Fungi in a Cultivation System Using a Membrane Filter
Authors: Kondo Ryuichiro , Kurashiki Kenji , Sakai Kokki
Published: January 2020

To clarify the role of excreted extracellular enzymes during long-term incubation in a pulp biobleaching system with white rot fungi, we developed a cultivation system in which a membrane filter is used; this membrane filter can prevent direct contact between hyphae and kraft pulp, but allows extracellular enzymes to attack the kraft pulp.
Phanerochaete sordida
YK-624 brightened the pulp 21.4 points to 54.0% brightness after a 5-day in vitro treatment; this value was significantly higher than the values obtained with
Phanerochaete chrysosporium
and
Coriolus versicolor
after a 7-day treatment. Our results indicate that cell-free, membrane-filtered components from the in vitro bleaching system are capable of delignifying unbleached kraft pulp. Obvious candidates for filterable reagents capable of delignifying and bleaching kraft pulp are peroxidase and phenoloxidase proteins. The level of secreted manganese peroxidase activity in the filterable components was substantial during strain YK-624 in vitro bleaching. A positive correlation between the level of manganese peroxidase and brightening of the pulp was observed.
Pressure Stabilization of Proteins from Extreme Thermophiles
Authors: Hei Derek J. , Clark Douglas S.
Published: January 2020

We describe the stabilization by pressure of enzymes, including a hydrogenase from
Methanococcus jannaschii
, an extremely thermophilic deep-sea methanogen. This is the first published report of proteins from thermophiles being stabilized by pressure. Inactivation studies of partially purified hydrogenases from an extreme thermophile (
Methanococcus igneus
), a moderate thermophile (
Methanococcus thermolithotrophicus
), and a mesophile (
Methanococcus maripaludis
), all from shallow marine sites, show that pressure stabilization is not unique to enzymes isolated from high-pressure environments. These studies suggest that pressure stabilization of an enzyme may be related to its thermophilicity. Further experiments comparing the effects of increased pressure on the stability of α-glucosidases from the hyperthermophile
Pyrococcus furiosus
and
Saccharomyces cerevisiae
support this possibility. We have also examined pressure effects on several highly homologous glyceraldehyde-3-phosphate dehydrogenases from mesophilic and thermophilic sources and a rubredoxin from
P. furiosus
. The results suggest that hydrophobic interactions, which have been implicated in the stabilization of many thermophilic proteins, contribute to the pressure stabilization of enzymes from thermophiles.
Phylogenetic Analysis of
Bradyrhizobium japonicum
and Photosynthetic Stem-Nodulating Bacteria from
Aeschynomene
Species Grown in Separated Geographical Regions
Authors: Wong Frank Y. K. , Stackebrandt Erko , Ladha J. K. , Fleischman Darrell E. , Date Richard A. , Fuerst John A.
Published: January 2020

Nearly complete and short partial 16S rRNA sequences were derived from PCR-amplified ribosomal DNAs of
Bradyrhizobium japonicum
USDA 136 and USDA 110 and five strains of bacteriochlorophyll-synthesizing bacteria isolated from stem nodules of
Aeschynomene indica
and other
Aeschynomene
species growing in different geographic regions, including India, The Philippines and North America. We confirmed that the five stem-nodulating strains examined synthesize bacteriochlorophyll
a
, and the absorption spectra of methanol-extracted cells contained a major absorbance peak at 770 nm. Strains isolated on different continents and from different
Aeschynomene
species were found to be phylogenetically homogeneous and exhibited levels of sequence similarity of more than 99%. The bacteriochlorophyll-synthesizing rhizobia,
Bradyrhizobium japonicum, Blastobacter denitrificans, Afipia felis
, and
Rhodopseudomonas palustris
exhibited levels of sequence similarity of 97% or greater and belong to a distinct line of descent within the alpha-2 subdivision of the
Proteobacteria
. Variable regions between positions 995 and 1045 provide potential target sites for design of a probe that is able to distinguish the photosynthetic rhizobia from closely related taxa.
Transfer and expression of degradative and antibiotic resistance plasmids in acidophilic bacteria
Authors: Quentmeier A , Friedrich C G
Published: January 2020

The genetic accessibility of selected acidophilic bacteria was investigated to evaluate their applicability to degrading pollutants in acidic environments. The IncP1 antibiotic resistance plasmids RP4 and pVK101 and the phenol degradation-encoding plasmid pPGH11 were transferred from neutrophilic bacteria into the extreme acidophilic eubacterium Acidiphilium cryptum at frequencies of 1.8 x 10(-2) to 9.8 x 10(-4) transconjugants per recipient cell. The IncQ antibiotic resistance plasmid pSUP106 was mobilizable to A. cryptum by triparental matings at a frequency of 10(-5) transconjugants per recipient cell. In the transconjugants, antibiotic resistances and the ability to degrade phenol were expressed. A. cryptum AC6 (pPGH11) grew with 2.5 mM phenol at a doubling time of 12 h and a yield of 0.52 g (dry cell weight) per g of phenol. A. cryptum harbored five native plasmids of 255 to 6.3 kb in size. Plasmids RP4 and pVK101 were transferred from Escherichia coli into Acidobacterium capsulatum at frequencies of 10(-3) and 2.3 x 10(-4) and to the facultative autotroph Thiobacillus acidophilus at frequencies of 1.1 x 10(-5) and 2.9 x 10(-6) transconjugants per recipient cell, respectively. Plasmid pPGH11 could not be transferred into the latter strains. T. acidophilus wild type contained six so far cryptic plasmids of 220 to 5 kb.
Use of gene probes to aid in recovery and identification of functionally dominant 2,4-dichlorophenoxyacetic acid-degrading populations in soil
Authors: Ka J O , Holben W E , Tiedje J M
Published: January 2020

The herbicide 2,4-dichlorophenoxyacetic acid (2,4-D) was applied to soils in microcosms, and degradation was monitored after each of five repeated additions. Total DNAs were isolated from soil bacterial communities after each 2,4-D treatment. The DNA samples were analyzed on slot blots and Southern blots by using a tfdA gene probe subcloned from plasmid pJP4 and a Spa probe derived from a different 2,4-D-degrading isolate, a Sphingomonas paucimobilis strain. 2,4-D applied to soil was quickly degraded by indigenous microbial populations. As determined by slot blot analyses of DNA from a Michigan soil, the increase in hybridization signal in response to 2,4-D treatments was greater with the Spa probe than with the tfdA probe. In contrast, the DNA from a Saskatchewan soil exhibited an increase in hybridization signal with the tfdA probe. This indicated that a population with 2,4-D-degradative gene sequences different from the tfdA gene sequence was dominant in the Michigan site, but not in the Saskatchewan site. A Southern blot analysis of DNA from Michigan soil showed that the dominant 2,4-D-degrading population was S. paucimobilis 1443. A less dominant 2,4-D-degrading population was detected with the tfdA probe; further analysis revealed that this population was a Pseudomonas pickettii 712. These gene probe analyses revealed that an important population carrying out 2,4-D degradation was not detected when the canonical tfdA gene probe was used. After a series of new strains were isolated, we identified a probe to detect and identify the dominant members of this new group.
Relative Role of Eukaryotic and Prokaryotic Microorganisms in Phenanthrene Transformation in Coastal Sediments
Authors: MacGillivray A. Ronald , Shiaris Michael P.
Published: January 2020

The relative role of eukaryotic versus prokaryotic microorganisms in phenanthrene transformation was measured in slurries of coastal sediment by two different approaches: detection of marker metabolites and use of selective inhibitors on phenanthrene biotransformation. Phenanthrene biotransformation was measured by polar metabolite formation and CO
2
evolution from [9-
14
C]phenanthrene. Radiolabeled metabolites were tentatively identified by high-performance liquid chromatography (HPLC) separation combined with UV/visible spectral analysis of HPLC peaks and comparison to authentic standards. Both yeasts and bacteria transformed phenanthrene in slurries of coastal sediment. Two products of phenanthrene oxidation by fungi, phenanthrene
trans
-3,4-dihydrodiol and 3-phenanthrol, were produced in yeast-inoculated sterile sediment. However, only products of phenanthrene oxidation typical of bacterial transformation, 1-hydroxy-2-naphthoic acid and phenanthrene
cis
-3,4-dihydrodiol, were isolated from slurries of coastal sediment with natural microbial populations. Phenanthrene
trans
-dihydrodiols or other products of fungal oxidation of phenanthrene were not detected in the slurry containing a natural microbial population. A predominant role for bacterial transformation of phenanthrene was also suggested from selective inhibitor experiments. Addition of streptomycin to slurries, at a concentration which suppressed bacterial viable counts and rates of [
methyl
-
3
H]thymidine uptake, completely inhibited phenanthrene transformation. Treatment with colchicine, at a concentration which suppressed yeast viable counts, depressed phenanthrene transformation by 40%, and this was likely due to nontarget inhibition of bacterial activity. The relative contribution of eukaryotic microorganisms to phenanthrene transformation in inoculated sterile sediment was estimated to be less than 3% of the total activity. We conclude that the predominant degraders of phenanthrene in muddy coastal sediments are bacteria and not eukaryotic microorganisms.
Determination of Diffusion Coefficients in Biofilms by Confocal Laser Microscopy
Authors: Lawrence J. R. , Wolfaardt G. M. , Korber D. R.
Published: January 2020

Microbial exopolymer may hinder the diffusion of nutrients, antibiotics, and other materials to the cell surface. Studies of diffusion in biofilms have been limited to indirect measurements. This study demonstrated the use of fluorescein and size-fractionated fluor-conjugated dextrans in conjunction with scanning confocal laser microscopy to directly monitor and determine diffusion coefficients within biofilms. The monitoring approaches were simple and, when combined with computerized image collection, allowed assembly of a data set suitable for calculation of one-dimensional diffusion coefficients for biofilm regions. With these techniques, it was shown that regional variability in the mobility of the dextrans occurred within mixed-species biofilms. Some regions exhibited rapid diffusion of all test molecules, while adjacent regions were only penetrated by the lower-molecular-weight compounds. The effective diffusion coefficients (

D
e

) determined in a mixed-species biofilm were a function of the molecular radius of the probe (i.e., fluorescein,

D
e

= 7.7 × 10
-8
cm
2
s
-1
; 4,000 molecular weight,

D
e

= 3.1 × 10
-8
cm
2
s
-1
; and 2,000,000 molecular weight,

D
e

= 0.7 × 10
-8
cm
2
s
-1
). These results demonstrated that diffusion in the biofilm was hindered relative to diffusion in the bulk solution. The study indicated that in situ monitoring by scanning laser microscopy is a useful approach for determining the mobility of fluorescently labeled molecules in biofilms, allowing image acquisition, appropriate scales of study, both
xy
and
xz
monitoring, and calculation of

D
e

values.
Identification, Biosynthesis, and Function of 1,3,4,6-Hexanetetracarboxylic Acid in
Methanobacterium thermoautotrophicum
ΔH
Authors: Gorkovenko Alexander , Roberts Mary F. , White Robert H.
Published: January 2020

An unusual compound, 1,3,4,6-hexanetetracarboxylic acid, was identified by
1
H and
13
C two-dimensional nuclear magnetic resonance spectroscopy and gas chromatography-mass spectrometry as one of the major components of the small-molecule pool in
Methanobacterium thermoautotrophicum
ΔH under optimal conditions of cell growth. Incorporation of
13
C- and
2
H-labeled acetates was consistent with the biosynthesis of this tetracarboxylic acid from α-ketoglutarate, two molecules of acetyl-coenzyme A, and one molecule of CO
2
, as established for the tetracarboxylic acid moiety of methanofuran.
13
CO
2
pulse-
12
CO
2
chase methodology was used to establish the turnover rate for this compound. In contrast to the two other major solutes in this bacterium, cyclic 2,3-diphosphoglycerate and glutamate, which are key metabolic intermediates, this free tetracarboxylic acid was metabolically inactive, with a half-life that exceeded the cell doubling time. Hence, this molecular pool cannot serve as a metabolic intermediate in cell biosynthesis. The functional role of free tetracarboxylate as a conservative part of a system that maintains high positive internal osmotic pressure in this bacterium is proposed.
Genetic Structure of
Rhizobium etli
biovar phaseoli Associated with Wild and Cultivated Bean Plants (
Phaseolus vulgaris
and
Phaseolus coccineus
) in Morelos, Mexico
Authors: Souza Valeria , Eguiarte Luis , Avila German , Cappello Renato , Gallardo Claudia , Montoya Javier , Piñero Daniel
Published: January 2020

The genetic structure of
Rhizobium etli
biovar phaseoli was determined for five populations in three different locations in the state of Morelos, Mexico, by using starch gel electrophoresis for five to nine polymorphic loci. Two populations were sampled during two different years from nodules of cultivated and wild common bean plants (
Phaseolus vulgaris
). The three other populations were associated with wild runner beans (
P. coccineus
) and sampled during 1988. The
Rhizobium
populations differ genetically both among sites and among populations within the same site in different years, as shown by differences in allelic frequencies, genetic differentiation analysis, and differences in electrotypes. The total genetic diversity for the five populations during 1988 was
H
= 0.487; there were also high levels of genetic variation within each population. We found the highest linkage disequilibrium in a global analysis for all the populations. At a local scale, we also found significant linkage disequilibrium in two populations, although the distribution of the
D′
suggest some recombination at a local scale. The other three rhizobium populations exhibit low linkage disequilibrium. A cluster analysis (UPGMA) of pairwise genetic distances showed that bacteria isolated from most wild
Phaseolus
spp. are grouped by population, whereas those obtained from cultivated
P. vulgaris
are very heterogeneous. The analysis of the genetic structure of
Rhizobium
strains may allow the identification of strains that are naturally well adapted to a wide range of different environments, which may be useful for agricultural purposes or as a starting point for developing improved
Rhizobium
strains.
Microbial Transformation of Ibuprofen by a
Nocardia
Species
Authors: Chen Yijun , Rosazza John P. N.
Published: January 2020

The carboxylic acid functional group of ibuprofen [α-methyl-4-(2-methylpropyl) benzene acetic acid] is reduced to the corresponding alcohol and subsequently esterified to the acetate derivative by cultures of
Nocardia
species strain NRRL 5646. The alcohol and ester microbial transformation products were isolated, and their structures were determined by
1
H and
13
C nuclear magnetic resonance spectroscopy and mass spectrometry. By derivatization of synthetic and microbiologically produced ibuprofen alcohols with
S
(+)-
O
-acetylmandelic acid, nuclear magnetic resonance analysis indicated that the carboxylic acid reductase of
Nocardia
sp. is
R
enantioselective, giving alcohol products with an enantiomeric excess of 61.2%. The
R
enantioselectivity of the carboxylic acid reductase enzyme system was confirmed by using cell extracts together with ATP and NADPH in the reduction of isomeric ibuprofens.
Characterization of a novel enantioselective halohydrin hydrogen-halide-lyase
Authors: Nakamura T , Nagasawa T , Yu F , Watanabe I , Yamada H
Published: January 2020

Enzymes Ia and Ib of Corynebacterium sp. strain N-1074 exhibit halohydrin hydrogen-halide-lyase (H-lyase) activity, catalyzing the interconversion of halohydrins to epoxides and hydrogen halide. H-lyase B produced in a recombinant Escherichia coli strain carrying one of the enzyme genes of Corynebacterium sp. strain N-1074 was purified and characterized. The purified enzyme catalyzed the transformation of prochiral 1,3-dichloro-2-propanol (DCP) into R-rich epichlorohydrin (ECH). The apparent Km values for DCP, ECH, and chloride were calculated to be 1.03, 5.00, and 4.00 mM, respectively. Maximum activity for the conversion of DCP to ECH was found at pH 8.0 to 9.0, and that for the reverse reaction was found at about pH 5.0. H-lyase B seemed to be identical to enzyme Ib of Corynebacterium sp. strain N-1074 from the comparison of the properties of each. The properties of H-lyase B and H-lyase A, which had been previously purified from another recombinant carrying its gene from Corynebacterium sp. strain N-1074, were also compared.
Effects of Temperature, Sulfide, and Food Abundance on Growth and Feeding of Anaerobic Ciliates
Authors: Massana Ramon , Stumm Claudius K. , Pedrós-Alió Carlos
Published: January 2020

The trophic role of ciliates in anaerobic food webs has not been assessed experimentally. In order to obtain basic information necessary to interpret field situations, we studied the effects of temperature, sulfide concentration, and food abundance on the growth and feeding activities of two anaerobic ciliates,
Plagiopyla nasuta
and
Metopus es.
The growth rate of
P. nasuta
increased with temperature from 8 to 18°C (
Q
10
= 2.0) and remained constant in the range between 18 and 24°C (0.22 day
-1
). Sulfide concentrations of between 0 and 1 mM did not affect the feeding activities, but concentrations greater than 2 mM were inhibitory. The functional response of
P. nasuta
feeding on fluorescently labeled heterotrophic and phototrophic bacteria was investigated. In both cases, the parameters of the functional response were almost identical when expressed in terms of biovolume: the maximal uptake rate (

U
m

) was 1,800 μm
3
ciliate
-1
h
-1
and the half-saturation constant for ingestion (
k
) was 1.5 × 10
7
μm
3
ml
-1
. The functional response of
M. es
feeding on heterotrophic bacteria was found to be similar to that of
P. nasuta.
These ciliates needed high bacterial abundances in order to maintain their growth (
k
of about 4 × 10
7
bacteria ml
-1
), implying that they will frequently be food limited in planktonic environments. Both the maximal uptake rates and the maximal clearance rates were comparable to those of aerobic ciliates. By combining the growth and feeding data, we estimated gross growth efficiencies of 12 and 13% for
P. nasuta
and
M. es
, respectively. These results indicate that the feeding rates of anaerobic ciliates are similar to those of aerobic ciliates. Their slower growth must, therefore, be due to the lower gross growth efficiency (likely due to anaerobic metabolism).
Enzymatic catalysis of mercury methylation by Desulfovibrio desulfuricans LS
Authors: Choi S C , Chase T , Bartha R
Published: January 2020

The recently defined role of methylcobalamin in Hg2+ methylation by Desulfovibrio desulfuricans LS enabled us to reexamine the question of whether the principal source of methylmercury is spontaneous transmethylation or an enzymatically catalyzed process. In cell extracts of D. desulfuricans LS, over 95% of the 57Co label was associated with macromolecules rather than with free cobalamin. Both gel filtration and electrophoresis of cell extracts identified a single corrinoid protein of 40 kDa in size. This finding, in combination with the previously reported light-reversible propyl iodide inhibition of the Hg2+ methylation process, led us to propose that this 40-kDa corrinoid protein is the in vivo methyl donor in D. desulfuricans LS. Under reducing conditions, cell extracts containing the corrinoid protein produced 14CH3Hg+ from Hg2+ and 5-14CH3-tetrahydrofolate with a maximum specific activity of 0.73 nmol min-1 mg of cell protein-1. The sequence of methyl transfer was from methyltetrahydrofolate to the corrinoid protein to Hg2+. The rate of methylation versus the Hg2+ concentration followed Michaelis-Menten kinetics, with an apparent Km of 0.87 mM HgCl2. The activity was oxygen sensitive, and Hg2+ methylation was optimal at 35 degrees C and pH 6.5. The observation of saturation kinetics and the 600-fold-higher rate of Hg2+ methylation (at pH 7.0) by cell extracts, compared with transmethylation by free methylcobalamin, proved that in vivo Hg2+ methylation is an enzymatically catalyzed process.
Regulation of Product Formation in
Bacteroides xylanolyticus
X5-1 by Interspecies Electron Transfer
Authors: Biesterveld Steven , Zehnder Alexander J. B. , Stams Alfons J. M.
Published: January 2020

Bacteroides xylanolyticus
X5-1 was grown in pure culture and in mixed culture with
Methanospirillum hungatei
JF-1 under xylose limitation in the chemostat. In the pure culture, ethanol, acetate, CO
2
, and hydrogen were the products. In the mixed culture, acetate, CO
2
, and presumably hydrogen were the only products formed by
B. xylanolyticus
X5-1. The biomass yield of
B. xylanolyticus
X5-1 increased because of cocultivation. In cell extracts of the pure culture, both NAD- and NADP-dependent acetaldehyde dehydrogenase and ethanol dehydrogenase activities were found. In cell extracts of the mixed culture, activities of these enzymes were not detected. Inhibition of methanogenesis in the mixed culture by the addition of bromoethanosulfonic acid (BES) resulted in an accumulation of H
2
, ethanol, and formate. Immediately after the addition of BES, NAD-dependent acetaldehyde dehydrogenase and ethanol dehydrogenase activities were detected. After a short lag phase, a NADP-dependent ethanol dehydrogenase was also detectable. The induction of acetaldehyde dehydrogenase and ethanol dehydrogenase was inhibited by chloramphenicol, suggesting de novo synthesis of these enzymes. These results are consistent with a model in which the shift in product formation caused by interspecies electron transfer is regulated at the level of enzyme synthesis.
Optimization of Novel Extracellular Polysaccharide Production by an
Enterobacter
sp, on Wood Hydrolysates
Authors: Meade Maura J. , Tanenbaum S. W. , Nakas J. P.
Published: January 2020

An environmental isolate identified as
Enterobacter cloacae
has been found to produce a highly viscous, anionic extracellular polysaccharide (EPS) from a weak mineral acid hydrolysate of hardwood. Production of this EPS has been optimized on the hydrolysate (initial pH, 6.3; NH
4
Cl amendment, 0.1%) so that crude yields approaching 9.83 g/liter were obtained. Although this EPS is polydisperse, its molecular mass as determined by gel exclusion chromatography centers at approximately 1,700 kDa. Solutions of this EPS have been examined rheologically under a variety of conditions and compare favorably with both xanthan and alginate.
Effects of Sainfoin (
Onobrychis viciifolia
Scop.) Condensed Tannins on Growth and Proteolysis by Four Strains of Ruminal Bacteria
Authors: Jones G. A. , McAllister T. A. , Muir A. D. , Cheng K.-J.
Published: January 2020

Sainfoin leaf condensed tannins inhibited growth and protease activity in
Butyrivibrio fibrisolvens
A38 and
Streptococcus bovis
45S1 but had little effect on
Prevotella ruminicola
B
1
4 or
Ruminobacter amylophilus
WP225. Tannins bound to cell coat polymers in all strains. Morphological changes in
B. fibrisolvens
and
S. bovis
implicated the cell wall as a target of tannin toxicity.
Identification and characterization of the alpha-acetolactate synthase gene from Lactococcus lactis subsp. lactis biovar diacetylactis
Authors: Marugg J D , Goelling D , Stahl U , Ledeboer A M , Toonen M Y , Verhue W M , Verrips C T
Published: January 2020

The conversion of 3-13C-labelled pyruvate in an acetoin-producing clone from a Lactococcus lactis subsp. lactis biovar diacetylactis strain DSM 20384 plasmid bank in Escherichia coli was studied by 13C nuclear magnetic resonance analysis. The results showed that alpha-acetolactate was the first metabolic product formed from pyruvate, whereas acetoin appeared at a much slower rate and reached only low concentrations. This alpha-acetolactate production shows that the cells express the gene for alpha-acetolactate synthase (als). Nucleotide sequence analysis identified an open reading frame encoding a protein of 554 amino acids. The deduced amino acid sequence exhibits extensive similarities to those of known alpha-acetolactate synthases from both prokaryotes and eukaryotes. The als gene is expressed on a monocistronic transcriptional unit, which is transcribed from a promoter located just upstream of the coding region.
Identification of Bifidobacterium strains by rRNA gene restriction patterns
Authors: Mangin I , Bourget N , Bouhnik Y , Bisetti N , Simonet J M , Decaris B
Published: January 2020

Total DNA from 21 collection or industrial Bifidobacterium strains was cleaved with various restriction endonucleases. Following electrophoresis, the fragments were subjected to Southern blot hybridization with a heterologous [alpha-32P]dCTP-labeled rDNA (genes coding for rRNA) 23S gene probe. The ribosomal patterns allowed all tested strains to be differentiated and previous classifications to be confirmed. The same method was used to characterize DNA from 121 Bifidobacterium isolates collected from the intestinal flora of five human volunteers after the induction of colonic bacterial imbalance by antibiotics and absorption of a resistant exogenous Bifidobacterium strain. Hybridizations with the ribosomal probe revealed 11 different ribosomal patterns in addition to that of the exogenous strain. They permitted the Bifidobacterium populations belonging to the dominant colonic flora to be monitored over time. This experiment revealed significant and sustained alterations of the endogenous intestinal flora; indeed, some strains were eliminated, while others, probably belonging to subdominant flora, replaced them. Furthermore, even 2 months after the end of antibiotic treatment, the colonic flora remained different from that observed before treatment. Finally, our results showed that antibiotherapy did not allow colonic colonization by the exogenous strain.
Growth and energetics of Leuconostoc oenos during cometabolism of glucose with citrate or fructose
Authors: Salou P , Loubiere P , Pareilleux A
Published: January 2020

The metabolic and energetic characterization of the growth of Leuconostoc oenos on glucose-citrate or glucose-fructose mixtures enables the potential role of this bacterium in the wine-making process to be ascertained. Moreover, mixotrophic conditions remain a suitable means for improving biomass productivities of malolactic starter cultures. When the malolactic bacterium L. oenos was grown in batch cultures on complex medium at pH 5.0 with glucose-citrate or glucose-fructose mixtures, enhancement of both the specific growth rate and biomass production yields was observed. While growth was possible on fructose as the sole source of energy, citrate alone did not allow subsequent biomass production. The metabolic interactions between the catabolic pathways of the glucose cosubstrates and the heterofermentation of hexoses led to an increased acetate yield as a result of modified NADH oxidation. However, the calculated global coenzyme regeneration showed that the reducing equivalent balance was never equilibrated. The stimulatory effects of these glucose cosubstrates on growth resulted from increased ATP synthesis by substrate-level phosphorylation via acetate kinase. While the energetic efficiency remained close to 10 g of biomass produced per mol of ATP, the increase in the specific growth rate and biomass production yields was directly related to the rate and yield of ATP generation.
Thermophilic Sulfate Reduction in Hydrothermal Sediment of Lake Tanganyika, East Africa
Authors: Elsgaard Lars , Prieur Daniel , Mukwaya Gashagaza M. , Jørgensen Bo B.
Published: January 2020

In environments with temperatures above 60°C, thermophilic prokaryotes are the only metabolically active life-forms. By using the
35
SO
4
2-
tracer technique, we studied the activity of sulfate-reducing microorganisms (SRM) in hot sediment from a hydrothermal vent site in the northern part of freshwater Lake Tanganyika (East Africa). Incubation of slurry samples at 8 to 90°C demonstrated meso- and thermophilic sulfate reduction with optimum temperatures of 34 to 45°C and 56 to 65°C, respectively, and with an upper temperature limit of 80°C. Sulfate reduction was stimulated at all temperatures by the addition of short-chain fatty acids and benzoate or complex substrates (yeast extract and peptone). A time course experiment showed that linear thermophilic sulfate consumption occurred after a lag phase (12 h) and indicated the presence of a large population of SRM in the hydrothermal sediment. Thermophilic sulfate reduction had a pH optimum of about 7 and was completely inhibited at pH 8.8 to 9.2. SRM could be enriched from hydrothermal chimney and sediment samples at 60 and 75°C. In lactate-grown enrichments, sulfide production occurred at up to 70 and 75°C, with optima at 63 and 71°C, respectively. Several sporulating thermophilic enrichments were morphologically similar to
Desulfotomaculum
spp. Dissimilatory sulfate reduction in the studied hydrothermal area of Lake Tanganyika apparently has an upper temperature limit of 80°C.
Effects of particulate materials and osmoprotectants on very-high-gravity ethanolic fermentation by Saccharomyces cerevisiae
Authors: Thomas K C , Hynes S H , Ingledew W M
Published: January 2020

The effects of osmoprotectants (such as glycine betaine and proline) and particulate materials on the fermentation of very high concentrations of glucose by the brewing strain Saccharomyces cerevisiae (uvarum) NCYC 1324 were studied. The yeast growing at 20 degrees C consumed only 15 g of the sugar per 100 ml from a minimal medium which initially contained 35% (wt/vol) glucose. Supplementing the medium with a mixture of glycine betaine, glycine, and proline increased the amount of sugar fermented to 30.5 g/100 ml. With such supplementation, the viability of the yeast cells was maintained above 80% throughout the fermentation, while it dropped to less than 12% in the unsupplemented controls. Among single additives, glycine was more effective than proline or glycine betaine. On incubating the cultures for 10 days, the viability decreased to only 55% with glycine, while it dropped to 36 and 27%, respectively, with glycine betaine and proline. It is suggested that glycine and proline, known to be poor nitrogen sources for growth, may serve directly or indirectly as osmoprotectants. Nutrients such as tryptone, yeast extract, and a mixture of purine and pyrimidine bases increased the sugar uptake and ethanol production but did not allow the population to maintain the high level of cell viability. While only 43% of the sugar was fermented in unsupplemented medium, the presence of particulate materials such as wheat bran, wheat mash insolubles, alumina, and soy flour increased sugar utilization to 68, 75, 81, and 82%, respectively.
Limited Genetic Diversity in the Endophytic Sugarcane Bacterium
Acetobacter diazotrophicus
Authors: Caballero-Mellado Jesus , Martinez-Romero Esperanza
Published: January 2020

Acetobacter diazotrophicus
isolates that originated from different sugarcane cultivars growing in diverse geographic regions of Mexico and Brazil were shown to have limited genetic diversity. Measurements of polymorphism in the electrophoretic mobilities of metabolic enzymes revealed that the mean genetic diversity per enzyme locus (among the four electrophoretic types distinguished) was 0.064. The results of the genetic analysis indicate that the genetic structure of
A. diazotrophicus
is clonal, with one largely predominant clone. Plasmids were present in 20 of 24 isolates, and the molecular sizes of the plasmids ranged from 2.0 to 170 kb. Two plasmids (a 20- to 24-kb plasmid detected in all 20 plasmid-containing isolates and a 170-kb plasmid observed in 14 isolates) were highly conserved among the isolates examined. Regardless of the presence of plasmids, all of the isolates shared a common pattern of
nif
structural gene organization on the chromosome.
Potential Role of Elicitins in the Interaction between
Phytophthora
Species and Tobacco
Authors: Kamoun Sophien , Young Mary , Förster Helga , Coffey Michael D. , Tyler Brett M.
Published: January 2020

The potential role of extracellular elicitor proteins (elicitins) from
Phytophthora
species as avirulence factors in the interaction between
Phytophthora
and tobacco was examined. A survey of 85
Phytophthora
isolates representing 14 species indicated that production of elicitin is almost ubiquitous except for isolates of
Phytophthora parasitica
from tobacco. The production of elicitins by isolates of
P. parasitica
correlated without exception with low or no virulence on tobacco. Genetic analysis was conducted by using a cross between two isolates of
P. parasitica
, segregating for production of elicitin and virulence on tobacco. Virulence assays of the progeny on tobacco confirmed the correlation between production of elicitin and low virulence.
Cultural and phylogenetic analysis of mixed microbial populations found in natural and commercial bioleaching environments
Authors: Goebel B M , Stackebrandt E
Published: January 2020

A range of autotrophic and heterotrophic enrichment cultures were established to determine the cultural bacterial diversity present in samples obtained from the acidic runoff of a chalcocite overburden heap and from laboratory-scale (1- to 4-liter) batch and continuous bioreactors which were being used for the commercial assessment of the bioleachability of zinc sulfide ore concentrates. Strains identified as Thiobacillus ferrooxidans, Thiobacillus thiooxidans, "Leptospirillum ferrooxidans," and Acidiphilium cryptum were isolated from both the natural site and the batch bioreactor, but only "L. ferrooxidans," a moderately thermophilic strain of T. thiooxidans, and a moderately thermophilic iron-oxidizing bacterium could be recovered from the continuous bioreactor running under steady-state conditions. Sequence analysis of the 16S rRNA genes of 33 representative strains revealed that all of the strains were closely related to strains which have been sequenced previously and also confirmed the phylogenetic diversity of bacteria present in bioleaching environments.
Biochemical and Molecular Characterization of
Obesumbacterium proteus
, a Common Contaminant of Brewing Yeasts
Authors: Prest Andrew G. , Hammond John R. M. , Stewart Gordon S. A. B.
Published: January 2020

We have evaluated the effectiveness of API 20E, Biolog testing, plasmid profiling, ribotyping, and enteric repetitive intergenic consensus (ERIC)-PCR to characterize, classify, and differentiate nine bacterial isolates of the common brewery contaminant
Obesumbacterium proteus.
Of the five typing techniques, Biolog testing, plasmid profiling, and ERIC-PCR provided the most differentiation, and API 20E testing and ribotyping were relatively indiscriminate. The molecular biology approach of ERIC-PCR offered the ideal combination of speed, simplicity, and discrimination in this study. Overall, the results are supportive of the view that
O. proteus
can be subdivided into two biogroups, biogroup 1, which has considerable biochemical and genetic homology to
Hafnia alvei
, and biogroup 2, which is relatively heterogeneous.
Evidence for Chlorine Migration during Oxidation of 2-Chlorobiphenyl by a Type II Methanotroph
Authors: Adriaens P.
Published: January 2020

Axenic methanotrophic culture strain CSC1, which was derived from a mixed-culture aquifer sample, oxidized 2-chlorobiphenyl to a hydroxylated chlorobiphenyl intermediate identified as 2-hydroxy-3-chlorobiphenyl. Minor amounts of another hydroxylated chlorobiphenyl isomer, assumed to be the nonmigrated product 3-hydroxy-2-chlorobiphenyl, were found in axenic culture incubation extracts. The occurrence of chlorine migration during oxidation indicates that the metabolite is formed via a concerted oxidation mechanism involving an arene oxide, which rearranges spontaneously to the hydroxylated intermediate via an NIH shift mechanism.