bluuebunny/crossref_metadata_2025 huggingface dataset:
Evidence of Genomic Instability in
Campylobacter jejuni
Isolated from Poultry
Authors: Wassenaar Trudy M. , Geilhausen Barbara , Newell Diane G.
Published: December 2019

Poultry isolates of
Campylobacter jejuni
derived from a survey of meat processing batches were genotyped by pulsed-field gel electrophoresis (PFGE) of chromosomal DNA to establish the clonal relationships between single-colony isolates. In the majority of batches studied, one or two genotype patterns predominated. However, in one batch (batch A), 21 single-colony isolates gave 14 different PFGE genotypes. The banding patterns obtained with
Sma
I were sufficiently different to distinguish between genotypes, although the patterns also produced many common bands. The question of whether these isolates represented different clones or had a common clonal ancestry was addressed by additional genotypic and phenotypic methods. Restriction length polymorphism of PCR products obtained from the flagellin genes showed an identical flagellin genotype for all of these isolates. In contrast, unrelated control isolates resulted in different flagellin genotypes. Moreover, all 14 different PFGE genotypes of batch A had identical Penner serotypes and identical or similar biotypes and phage types. It was concluded that the isolates were of clonal origin and that the diversity in the PFGE banding patterns had most likely originated from genomic rearrangements. However, the PFGE genotypes were shown to be stable upon subculturing in vitro and after in vivo passage in chickens, and natural transformation between isogenic mutants carrying antibiotic markers did not occur in vivo in a chick colonization model. The possible mechanisms for the hypothesized genomic recombinations and the conditions that allow, induce, or select for such events are discussed.
Microbiology of a Nitrite-Oxidizing Bioreactor
Authors: Burrell Paul C. , Keller Jürg , Blackall Linda L.
Published: December 2019

The microbiology of the biomass from a nitrite-oxidizing sequencing batch reactor (NOSBR) fed with an inorganic salts solution and nitrite as the sole energy source that had been operating for 6 months was investigated by microscopy, by culture-dependent methods, and by molecular biological methods, and the seed sludge that was used to inoculate the NOSBR was investigated by molecular biological methods. The NOSBR sludge comprised a complex and diverse microbial community containing gram-negative and gram-positive rods, cocci, and filaments. By culture-dependent methods (i.e., micromanipulation and sample dilution and spread plate inoculation), 16 heterotrophs (6 gram positive and 10 gram negative) were identified in the NOSBR sludge (RC), but no autotrophs were isolated. 16S ribosomal DNA clone libraries of the two microbial communities revealed that the seed sludge (GC) comprised a complex microbial community dominated by
Proteobacteria
(29% beta subclass; 18% gamma subclass) and high G+C gram-positive bacteria (10%). Three clones (4%) were closely related to the autotrophic nitrite-oxidizer
Nitrospira moscoviensis
. The NOSBR sludge was overwhelmingly dominated by bacteria closely related to
N. moscoviensis
(89%). Two clone sequences were similar to those of the genus
Nitrobacter
. Near-complete insert sequences of eight RC and one GC
N. moscoviensis
clone were determined and phylogenetically analyzed. This is the first report of the presence of bacteria from the
Nitrospira
phylum in wastewater treatment systems, and it is hypothesized that these bacteria are the unknown nitrite oxidizers in these processes.
Ecophysiological and Phylogenetic Studies of
Nevskia ramosa
in Pure Culture
Authors: Stürmeyer Heike , Overmann Jörg , Babenzien Hans-Dietrich , Cypionka Heribert
Published: December 2019

During the last 100 years, the neuston bacterium
Nevskia ramosa
has been described several times. This bacterium forms conspicuous rosette-like microcolonies at the air-water interface. In this study, pure cultures of
Nevskia ramosa
were obtained for the first time, from a bog lake (strain Soe1, DSMZ 11499
T
) and a freshwater ditch (strain OL1, DSMZ 11500). The isolates showed special adaptations to life in the epineuston. They formed hydrophobic surface films with a dull appearance.
N. ramosa
is sensitive to UV radiation but revealed a very effective photorepair mechanism. Exposure to light at a wavelength of 350 nm after UV treatment raised the number of surviving cells by several orders of magnitude. The isolates grew with a broad range of organic substrates. Surface films were formed only in the absence of combined nitrogen; however, nitrogenase activity was not detected. It appears that during growth at the air-water interface the cells benefit from trapping ammonia from the air. The G+C content of the DNA was 67.8 and 69.0 mol% for strains Soe1 and OL1, respectively. The slight difference was confirmed by enterobacterial repetitive intergenic consensus PCR. The 16S rRNA sequences revealed 99.2% similarity. Thus, both isolates belong to the same species. The phylogenetic analysis indicated that
Nevskia
is a member of the gamma-subclass
Proteobacteria
that has no known close relatives.
Purification of Extracellular Cholesterol Oxidase with High Activity in the Presence of Organic Solvents from
Pseudomonas
sp. Strain ST-200
Authors: Doukyu Noriyuki , Aono Rikizo
Published: December 2019

Extracellular cholesterol oxidase of
Pseudomonas
sp. strain ST-200 was purified from the culture supernatant. This oxidase contained bound flavin and was categorized as a 3β-hydroxysteroid oxidase, converting 3β-hydroxyl groups to keto groups. The molecular mass was 60 kDa. The enzyme was stable at pH 4 to 11 and active at pH 5.0 to 8.5, showing optimal activity at pH 7 at 60°C. The Michaelis constant of the ST-200 cholesterol oxidase was lower than those of commercially available oxidases. The cholesterol oxidation rate was enhanced 3- to 3.5-fold in the presence of organic solvents, with log
P
ow
values (partition coefficients of the organic solvent between
n
-octanol and water), in the range of 2.1 to 4.2, compared with that in the absence of organic solvents.
Mercury Methylation by Interspecies Hydrogen and Acetate Transfer between Sulfidogens and Methanogens
Authors: Pak K.-R. , Bartha R.
Published: December 2019

Cocultures of
Desulfovibrio desulfuricans
and
Methanococcus maripaludis
grew on sulfate-free lactate medium while vigorously methylating Hg
2+
. Individually, neither bacterium could grow or methylate mercury in this medium. Similar synergistic growth of sulfidogens and methanogens may create favorable conditions for Hg
2+
methylation in low-sulfate anoxic freshwater sediments.
Isolation and Characterization of Pediocin AcH Chimeric Protein Mutants with Altered Bactericidal Activity
Authors: Miller Kurt W. , Schamber Robin , Osmanagaoglu Ozlem , Ray Bibek
Published: December 2019

A collection of pediocin AcH amino acid substitution mutants was generated by PCR random mutagenesis of DNA encoding the bacteriocin. Mutants were isolated by cloning mutagenized DNA into an
Escherichia coli malE
plasmid that directs the secretion of maltose binding protein-pediocin AcH chimeric proteins and by screening transformant colonies for bactericidal activity against
Lactobacillus plantarum
NCDO955 (K. W. Miller, R. Schamber, Y. Chen, and B. Ray, 1998. Appl. Environ. Microbiol. 64:14–20, 1998). In all, 17 substitution mutants were isolated at 14 of the 44 amino acids of pediocin AcH. Seven mutants (N5K, C9R, C14S, C14Y, G37E, G37R, and C44W) were completely inactive against the pediocin AcH-sensitive strains
L. plantarum
NCDO955,
Listeria innocua
Lin11,
Enterococcus faecalis
M1,
Pediococcus acidilactici
LB42, and
Leuconostoc mesenteroides
Ly. A C24S substitution mutant constructed by other means also was inactive against these bacteria. Nine other mutants (K1N, W18R, I26T, M31T, A34D, N41K, H42L, K43N, and K43E) retained from <1% to ∼60% of wild-type activity when assayed against
L. innocua
Lin11. One mutant, K11E, displayed ∼2.8-fold-higher activity against this indicator. About one half of the mutations mapped to amino acids that are conserved in the pediocin-like family of bacteriocins. All four cysteines were found to be required for activity, although only C9 and C14 are conserved among pediocin-like bacteriocins. Several basic amino acids as well as nonpolar amino acids located within the hydrophobic C-terminal region also were found to be important. The mutations are discussed in the context of structural models that have been proposed for the bacteriocin.
Influence of Reduced Water Activity on Lactose Metabolism byLactococcus lactissubsp.cremorisat Different pH Values
Authors: Liu S.-Q. , Asmundson R. V. , Gopal P. K. , Holland R. , Crow V. L.
Published: December 2019

The influence of reduced water activity (aw) on lactose metabolism byLactococcus lactissubsp.cremoris2254 and 2272 was studied at different pH values. In control incubations (aw, 0.99) with nongrowing cells in pH-controlled phosphate buffer, the levels of carbon recovered asl-(+)-lactate were 92% at pH 6.1 and 5.3 and 78% at pH 4.5. However, the levels of recovery decreased to ∼50% at all pH values tested when the awwas 0.88 (with glycerol as the humectant). When growing cells in broth controlled at pH 6.3 were used, a reduction in the awfrom 0.99 to 0.96 resulted in a decrease in the level of lactose carbon recovered asl-(+)-lactate from 100 to 71%. Low levels ofl-(+)-lactate carbon recovery (<50%) were also observed with cells resuspended in pH-uncontrolled reconstituted skim milk at awvalues of 0.99 and 0.87 and in young cheese curds. The missing lactose carbon could not be accounted for by acetate, ethanol, formate, acetaldehyde, or pyruvate. Attempts were made to determine where the missing lactose carbon was diverted to under the stress conditions used. Some of the missing lactose carbon was recovered as galactose (0.1 to 2.5 mM) in culture supernatants. Decreasing either the awor the pH resulted in increased galactose accumulation by nongrowing cells; adjusting both environmental factors together potentiated the effect. The sensitivities of the two lactococcal strains tested were different; strain 2272 was more prone to accumulate galactose under stress conditions. A methyl pentose(s) and additional galactose were found in acid-hydrolyzed supernatants from cultures containing both growing and nongrowing cells, indicating that a saccharide(s) rich in these components was formed by lactococci under low-awand low-pH stress conditions.
Fate of Residual Lignin during Delignification of Kraft Pulp by
Trametes versicolor
Authors: Reid Ian D.
Published: December 2019

The fungus
Trametes versicolor
can delignify and brighten kraft pulps. To better understand the mechanism of this biological bleaching and the by-products formed, I traced the transformation of pulp lignin during treatment with the fungus. Hardwood and softwood kraft pulps containing
14
C-labelled residual lignin were prepared by laboratory pulping of lignin-labelled aspen and spruce wood and then incubated with
T. versicolor
. After initially polymerizing the lignin, the fungus depolymerized it to alkali-extractable forms and then to soluble forms. Most of the labelled carbon accumulated in the water-soluble pool. The extractable and soluble products were oligomeric; single-ring aromatic products were not detected. The mineralization of the lignin carbon to CO
2
varied between experiments, up to 22% in the most vigorous cultures. The activities of the known enzymes laccase and manganese peroxidase did not account for all of the lignin degradation that took place in the
T. versicolor
cultures. This fungus may produce additional enzymes that could be useful in enzyme bleaching systems.
Low-Frequency Horizontal Transfer of an Element Containing the Chlorocatechol Degradation Genes from
Pseudomonas
sp. Strain B13 to
Pseudomonas putida
F1 and to Indigenous Bacteria in Laboratory-Scale Activated-Sludge Microcosms
Authors: Ravatn Roald , Zehnder Alexander J. B. , van der Meer Jan Roelof
Published: December 2019

The possibilities for low-frequency horizontal transfer of the self-transmissible chlorocatechol degradative genes (
clc
) from
Pseudomonas
sp. strain B13 were investigated in activated-sludge microcosms. When the
clc
genes were transferred into an appropriate recipient bacterium such as
Pseudomonas putida
F1, a new metabolic pathway for chlorobenzene degradation was formed by complementation which could be selected for by the addition of mono- or 1,4-dichlorobenzene (CB). Under optimized conditions with direct donor-recipient filter matings, very low transfer frequencies were observed (approximately 3.5 × 10
−8
per donor per 24 h). In contrast, in matings on agar plate surfaces, transconjugants started to appear after 8 to 10 days, and their numbers then increased during prolonged continuous incubation with CB. In activated-sludge microcosms, CB-degrading (CB
+
) transconjugants of strain F1 which had acquired the
clc
genes were detected but only when strain B13 cell densities of more than 10
5
CFU/ml could be maintained by the addition of its specific growth substrate, 3-chlorobenzoate (3CBA). The CB
+
transconjugants reached final cell densities of between 10
2
and 10
3
CFU/ml. When strain B13 was inoculated separately (without the designated recipient strain F1) into an activated-sludge microcosm, CB
+
transconjugants could not be detected. However, in this case a new 3CBA-degrading strain appeared which had acquired the
clc
genes from strain B13. The effects of selective substrates on the survival and growth of and gene transfer between bacteria degrading aromatic pollutants in a wastewater ecosystem are discussed.
Variants of Smooth
Salmonella enterica
Serovar Enteritidis That Grow to Higher Cell Density Than the Wild Type Are More Virulent
Authors: Guard-Petter Jean
Published: December 2019

Salmonella enterica
serovar Enteritidis that grows to a higher cell density (SE-HCD) than wild type while retaining O-chain lipopolysaccharide was isolated by transforming wild type serovar Enteritidis with the cell density sensor plasmid pSB402 and selecting for bioluminescence. A luminescent strain, SE-HCD, that emitted light in proportion with cell density and opacity through stationary phase was isolated. After a peak cell density of 1.5 × 10
11
CFU/ml was observed, luminescence decreased, although opacity continued to increase. Scanning electron microscopy revealed that changes in luminescence and opacity past peak cell density were associated with lysis of a swarming hyperflagellated coccobacillary cell type and emergence of a 10-to-30-fold-elongated rod cell type that lacked cell surface structures. Vigorous aeration was required to induce this dramatic cellular differentiation. The virulence of two isogenic variants with different patterns of light emission at an opacity of 0.2 after the culture was diluted 10-fold (1/10 OD) was assessed in animal models. Whereas SE-HCD1 killed 70% of 6-day-old chicks challenged subcutaneously, the same dose of SE-HCD2 did not kill any chicks. Conversely, subcutaneous challenge of hens with SE-HCD2 contaminated eggs five and seven times more often, respectively, than did SE-HCD1 or wild type serovar Enteritidis. Intravenous challenge with SE-HCD2 contaminated 22% of eggs versus 0.5% with wild type, depressed egg production for 4 weeks, and caused clinical signs of Gallinarum Disease (Fowl Typhoid) in hens. SE-HCD2 produced no contaminated eggs following oral infection, whereas wild type contaminated 1.3% of eggs. Thus, SE-HCD2 is better at contaminating eggs than wild type, but only by parenteral challenge. These results suggest that it may be possible to separate luminescent serovar Enteritidis into groups that infect different age groups and organs and contaminate eggs.
Structure of the β-Galactosidase Gene from
Thermus
sp. Strain T2: Expression in
Escherichia coli
and Purification in a Single Step of an Active Fusion Protein
Authors: Vian Alejandro , Carrascosa Alfonso V. , García José L. , Cortés Estrella
Published: December 2019

The nucleotide sequence of both the
bgaA
gene, coding for a thermostable β-galactosidase of
Thermus
sp. strain T2, and its flanking regions was determined. The deduced amino acid sequence of the enzyme predicts a polypeptide of 645 amino acids (
M
r
, 73,595). Comparative analysis of the open reading frames located in the flanking regions of the
bgaA
gene revealed that they might encode proteins involved in the transport and hydrolysis of sugars. The observed homology between the deduced amino acid sequences of BgaA and the β-galactosidase of
Bacillus stearothermophilus
allows us to classify the new enzyme within family 42 of glycosyl hydrolases. BgaA was overexpressed in its active form in
Escherichia coli
, but more interestingly, an active chimeric β-galactosidase was constructed by fusing the BgaA protein to the choline-binding domain of the major pneumococcal autolysin. This chimera illustrates a novel approach for producing an active and thermostable hybrid enzyme that can be purified in a single step by affinity chromatography on DEAE-cellulose, retaining the catalytic properties of the native enzyme. The chimeric enzyme showed a specific activity of 191,000 U/mg at 70°C and a

K
m

value of 1.6 mM with
o
-nitrophenyl-β-
d
-galactopyranoside as a substrate, and it retained 50% of its initial activity after 1 h of incubation at 70°C.
Hemoglobin Biosynthesis in
Vitreoscilla stercoraria
DW: Cloning, Expression, and Characterization of a New Homolog of a Bacterial Globin Gene
Authors: Joshi Meenal , Mande Shekhar , Dikshit Kanak L.
Published: December 2019

In the strictly aerobic, gram-negative bacterium
Vitreoscilla
strain C1, oxygen-limited growth conditions create a more than 50-fold increase in the expression of a homodimeric heme protein which was recognized as the first bacterial hemoglobin (Hb). The recently determined crystal structure of
Vitreoscilla
Hb has indicated that the heme pocket of microbial globins differs from that of eukaryotic Hbs. In an attempt to understand the diverse functions of Hb-like proteins in prokaryotes, we have cloned and characterized the gene (
vgb
) encoding an Hb-like protein from another strain of
Vitreoscilla
,
V. stercoraria
DW. Several silent changes were observed within the coding region of the
V. stercoraria vgb
gene. Apart from that,
V. stercoraria
Hb exhibited interesting differences between the A and E helices. Compared to its Hb counterpart from
Vitreoscilla
strain C1, the purified preparation of
V. stercoraria
Hb displays a slower autooxidation rate. The differences between
Vitreoscilla
Hb and
V. stercoraria
Hb were mapped onto the three-dimensional structure of
Vitreoscilla
Hb, which indicated that the four changes, namely, Ile7Val, Ile9Thr, Ile10Ser, and Leu62Val, present within the
V. stercoraria
Hb fall in the region where the A and E helices contact each other. Therefore, alteration in the relative orientation of the A and E helices and the corresponding conformational change in the heme binding pocket of
V. stercoraria
Hb can be correlated to its slower autooxidation rate. In sharp contrast to the oxygen-regulated biosynthesis of Hb in
Vitreoscilla
strain C1, production of Hb in
V. stercoraria
has been found to be low and independent of oxygen control, which is supported by the absence of a fumarate and nitrate reductase regulator box within the
V. stercoraria vgb
promoter region. Thus, the regulation mechanisms of the Hb-encoding gene appear to be quite different in the two closely related species of
Vitreoscilla
. The relatively slower autooxidation rate of
V. stercoraria
Hb, lack of oxygen sensitivity, and constitutive production of Hb suggest that it may have some other function(s) in the cellular physiology of
V. stercoraria
DW, together with facilitated oxygen transport, predicted for earlier reported
Vitreoscilla
Hb.
Effects of Manganese Peroxidase on Residual Lignin of Softwood Kraft Pulp
Authors: Reid Ian D. , Paice Michael G.
Published: December 2019

Manganese peroxidase treatment lowered the kappa number of kraft pulp and increased the alkali extractability of the residual lignin but did not directly solubilize it. This indicates that MnP partially oxidizes the lignin in the pulp but does not degrade it to soluble fragments.
Phosphoenolpyruvate Phosphomutase Activity in an
l
-Phosphonoalanine-Mineralizing Strain of
Burkholderia cepacia
Authors: Ternan Nigel G. , McGrath John W. , Quinn John P.
Published: December 2019

A strain of
Burkholderia cepacia
isolated by enrichment culture utilized
l
-2-amino-3-phosphonopropionic acid (phosphonoalanine) at concentrations up to 20 mM as a carbon, nitrogen, and phosphorus source in a phosphate-insensitive manner. Cells contained phosphoenolpyruvate phosphomutase activity, presumed to be responsible for cleavage of the C—P bond of phosphonopyruvate, the transamination product of
l
-phosphonoalanine; this was inducible in the presence of phosphonoalanine.
Kinetics of Acetate Oxidation by Two Sulfate Reducers Isolated from Anaerobic Granular Sludge
Authors: Elferink Stefanie J. W. H. Oude , Luppens Suzanne B. I. , Marcelis Christian L. M. , Stams Alfons J. M.
Published: December 2019

Kinetic parameters of acetate oxidation were determined for the sulfate reducers
Desulforhabdus amnigenus
and
Desulfobacca acetoxidans
. Based on these parameters, both sulfate reducers seem to be able to outcompete
Methanosaeta
spp. for acetate in acetate-fed anaerobic bioreactors. Mixed-substrate studies showed that
D. amnigenus
degraded acetate and hydrogen simultaneously but preferred lactate, propionate, and ethanol over acetate.
Impact on Arbuscular Mycorrhiza Formation of
Pseudomonas
Strains Used as Inoculants for Biocontrol of Soil-Borne Fungal Plant Pathogens
Authors: Barea J. M. , Andrade G. , Bianciotto V. , Dowling D. , Lohrke S. , Bonfante P. , O’Gara F. , Azcon-Aguilar C.
Published: December 2019

The arbuscular mycorrhizal symbiosis, a key component of agroecosystems, was assayed as a rhizosphere biosensor for evaluation of the impact of certain antifungal
Pseudomonas
inoculants used to control soil-borne plant pathogens. The following three
Pseudomonas
strains were tested: wild-type strain F113, which produces the antifungal compound 2,4-diacetylphloroglucinol (DAPG); strain F113G22, a DAPG-negative mutant of F113; and strain F113(pCU203), a DAPG overproducer. Wild-type strain F113 and mutant strain F113G22 stimulated both mycelial development from
Glomus mosseae
spores germinating in soil and tomato root colonization. Strain F113(pCU203) did not adversely affect
G. mosseae
performance. Mycelial development, but not spore germination, is sensitive to 10 μM DAPG, a concentration that might be present in the rhizosphere. The results of scanning electron and confocal microscopy demonstrated that strain F113 and its derivatives adhered to
G. mosseae
spores independent of the ability to produce DAPG.
Desulfurization of Dibenzothiophene and Diesel Oils by a Newly Isolated
Gordona
Strain, CYKS1
Authors: Rhee Sung-Keun , Chang Je Hwan , Chang Yong Keun , Chang Ho Nam
Published: December 2019

A dibenzothiophene (DBT)-desulfurizing bacterial strain was isolated and identified as
Gordona
strain CYKS1. Strain CYKS1 was found to transform DBT to 2-hydroxybiphenyl via the 4S pathway and to be able to also use organic sulfur compounds other than DBT as a sole sulfur source. Its desulfurization activity was susceptible to sulfate repression. Active resting cells for desulfurization could be prepared only in the early growth phase. When two types of diesel oils, middle distillate unit feed (MDUF) and light gas oil (LGO) containing various organic sulfur compounds including DBT, were treated with resting cells of strain CYKS1 for 12 h, the total sulfur content significantly decreased, from 0.15% (wt/wt) to 0.06% (wt/wt) for MDUF and from 0.3% (wt/wt) to 0.25% (wt/wt) for LGO. The newly isolated strain CYKS1 is considered to have good potential for application in the biodesulfurization of fossil fuels.
Structure-Activity Study of the Lantibiotic Mutacin II from
Streptococcus mutans
T8 by a Gene Replacement Strategy
Authors: Chen Ping , Novak Jan , Kirk Marion , Barnes Stephen , Qi FengXia , Caufield Page W.
Published: December 2019

Mutacin II, elaborated by group II
Streptococcus mutans
, is a ribosomally synthesized and posttranslationally modified polypeptide antibiotic containing unusual thioether and didehydro amino acids. To ascertain the role of specific amino acid residues in mutacin II antimicrobial activity, we developed a streptococcal expression system that facilitates the replacement of the
mutA
gene with a single copy of a mutated variant gene. As a result, variants of mutacin II can be designed and expressed. The system was tested by constructing the following mutant peptides: ΔN1, V7A, P9A, T10A, T10S, C15A, C26A, and C27A. All of these mutacin II variants except ΔN1 and T10A, which were not secreted, were isolated, and their identities were verified by mass spectrometry. Variants P9A, C15A, C26A, and C27A failed to exert antimicrobial activity. Because the P9A and T10A variants comprise the “hinge” region of mutacin II, these observations suggest that in addition to the thioether and didehydro amino acids, the hinge region is essential for biological activity and biosynthesis or export of the peptide. Tandem mass spectrometry of the N-terminal part of the wild-type molecule and its C15A variant confirmed that the threonine at position 10 is dehydrated and present as a didehydrobutyrine residue. This analysis of the active T10S variant further suggested that a didehydro amino acid at this position is specific for antimicrobial activity and that the biosynthetic machinery does not discriminate between threonine and serine. In contrast, the lack of production of mutacin variants with alanine substituted for threonine at position 10, as well as the deletion of asparagine at the N terminus (ΔN1), indicates that specific residues in the propeptide may be crucial for certain steps in the biosynthetic pathway of this lantibiotic.
Thermotoga neapolitana
Homotetrameric Xylose Isomerase Is Expressed as a Catalytically Active and Thermostable Dimer in
Escherichia coli
Authors: Hess J. Michael , Tchernajenko Vladimir , Vieille Claire , Zeikus J. Gregory , Kelly Robert M.
Published: December 2019

The
xylA
gene from
Thermotoga neapolitana
5068 was expressed in
Escherichia coli
. Gel filtration chromatography showed that the recombinant enzyme was both a homodimer and a homotetramer, with the dimer being the more abundant form. The purified native enzyme, however, has been shown to be exclusively tetrameric. The two enzyme forms had comparable stabilities when they were thermoinactivated at 95°C. Differential scanning calorimetry revealed thermal transitions at 99 and 109.5°C for both forms, with an additional shoulder at 91°C for the tetramer. These results suggest that the association of the subunits into the tetrameric form may have little impact on the stability and biocatalytic properties of the enzyme.
Atmospheric Methane Consumption by Forest Soils and Extracted Bacteria at Different pH Values
Authors: Amaral John A. , Ren Tie , Knowles Roger
Published: December 2019

The effect of pH on atmospheric methane (CH
4
) consumption was studied with slurries of forest soils and with bacteria extracted from the same soils. Soil samples were collected from a mixed hardwood stand in New Hampshire, from jackpine and aspen stands at the BOREAS (Boreal Ecosystem Atmosphere Study) site near Thompson, northern Manitoba, from sites in southern Québec, including a beech stand and a meadow, and from a site in Ontario (cultivated humisol). Consumption of atmospheric CH
4
(concentration, approximately 1.8 ppm) occurred at depths of >5 cm in both acidic (pH 4.5 to 5.2) and alkaline (pH 7.2 to 7.8) soils. In slurries of acidic soils, maximum activity occurred at different pH values (pH 4.0 to 6.5). Bacteria extracted from these soils by high-speed blending and density gradient centrifugation showed pH responses different from the pH responses of the slurries. In all cases, these bacteria had a methanotrophy pH optimum of 5.8 and exhibited no activity at pH 6.8 to 7.0, the pH optimum range for known methanotrophs. This difference in pH responses could be useful in modifying media currently used for isolation of these organisms. Methanotrophic activity was induced in previously non-CH
4
-consuming soils by preincubation with 5% (vol/vol) CH
4
(50,000 μl of CH
4
per liter) or by liquid enrichment with 20% CH
4
. The bacteria showed pH responses typical of known methanotrophs and not typical of preexisting consumers of ambient CH
4
. Furthermore, methanotrophs induced by high CH
4
levels were more readily extracted from soil than preexisting ambient CH
4
consumers were. In the alkaline soils, preexisting activity either was destroyed or resisted extraction by the procedure used. The results support the hypothesis that consumers of ambient CH
4
in soils are physiologically distinct from the known methanotrophs.
Heterologous Protein Secretion Directed by a Repressible Acid Phosphatase System of
Kluyveromyces lactis
: Characterization of Upstream Region-Activating Sequences in the
KIPHO5
Gene
Authors: Fermiñán Encarnación , Domínguez Angel
Published: December 2019

Transcription of the repressible acid phosphatase gene (
KIPHO5
) in
Kluyveromyces lactis
is strongly regulated in response to the level of inorganic phosphate (P
i
) present in the growth medium. We have begun a study of the promoter region of this gene in order to identify sequences involved in the phosphate control of
KIPHO5
expression and to design new expression-secretion systems in
K. lactis
. Deletion analysis and directed mutagenesis revealed two major identical upstream activating sequences (UAS) CACGTG at positions −430 (UAS1) and −192 (UAS2) relative to the ATG initiation codon. These sequences are identical to those described for
Saccharomyces cerevisiae
for the binding of Pho4p. Deletion or directed mutagenesis of either one or both UAS reduce
KIPHO5
expression by the same amount (approximately 80%). When fused to the coding region of trout growth hormone cDNA (
tGH-II
), the promoter and signal peptide-encoding region of the phosphate-repressible
KIPHO5
gene drives the expression of this gene and the secretion of the tGHII protein. Synthesis of tGHIIp in
K. lactis
transformants carrying this construct was found to be regulated by the P
i
present in the medium; derepression of heterologous protein expression can therefore be achieved by lowering the P
i
concentration.
Isolation and Characterization of Phenol-Degrading Denitrifying Bacteria
Authors: van Schie Paula M. , Young L. Y.
Published: December 2019

Phenol is a man-made as well as a naturally occurring aromatic compound and an important intermediate in the biodegradation of natural and industrial aromatic compounds. Whereas many microorganisms that are capable of aerobic phenol degradation have been isolated, only a few phenol-degrading anaerobic organisms have been described to date. In this study, three novel nitrate-reducing microorganisms that are capable of using phenol as a sole source of carbon were isolated and characterized. Phenol-degrading denitrifying pure cultures were obtained by enrichment culture from anaerobic sediments obtained from three different geographic locations, the East River in New York, N.Y., a Florida orange grove, and a rain forest in Costa Rica. The three strains were shown to be different from each other based on physiologic and metabolic properties. Even though analysis of membrane fatty acids did not result in identification of the organisms, the fatty acid profiles were found to be similar to those of
Azoarcus
species. Sequence analysis of 16S ribosomal DNA also indicated that the phenol-degrading isolates were closely related to members of the genus
Azoarcus
. The results of this study add three new members to the genus
Azoarcus
, which previously comprised only nitrogen-fixing species associated with plant roots and denitrifying toluene degraders.
Detection of Hemolysin Variants of Shiga Toxin-Producing
Escherichia coli
by PCR and Culture on VancomycinCefixime-Cefsulodin Blood Agar
Authors: Lehmacher Anselm , Meier Heidi , Aleksic Stojanka , Bockemühl Jochen
Published: December 2019

The presence of a hemolysin-encoding gene,
elyA
or
hlyA
, from Shiga toxin-producing
Escherichia coli
(STEC) was detected by PCR in each of 95 strains tested. PCR products of
elyA
from human STEC isolates of serovars frequently detected in Germany, such as O157:H−, O103:H2, O103:H−, O26:H11, and O26:H−, showed nucleotide sequences identical to previously reported ones for O157:H7 and O111:H− strains. Compared to them, four
elyA
amplicons derived from human isolates of rare STEC serovars showed identity of about 98% but lacked an
Alu
I restriction site. However, the nucleotide sequence of an amplicon derived from a porcine O138:K81:H− STEC strain was identical to the corresponding region of
hlyA
, encoding alpha-hemolysin, from
E. coli
. This
hlyA
amplicon showed 68% identity with the nucleotide sequence of the corresponding
elyA
fragment. It differed from the
elyA
PCR product in restriction fragments generated by
Alu
I,
Eco
RI, and
Mlu
I. Of the 95 representative STEC strains, 88 produced hemolysin on blood agar supplemented with vancomycin (30 mg/liter), cefixime (20 μg/liter), and cefsulodin (3 mg/liter) (BVCC). The lowest added numbers of two to six STEC CFU per g of stool or per ml of raw milk were detectable on BVCC plates after seeding of the preenrichment broth, modified tryptic soy broth (mTSB) supplemented with novobiocin (10 mg/liter), with 16 STEC strains. These strains represented the seven prevailing serovars diagnosed from German patients. However, with ground-beef samples, PCR was essential to identify the lowest added numbers of two to six STEC CFU among colonies of hemolyzing
Enterobacteriaceae
, such as
Serratia
spp. and alpha-hemolysin-producing
E. coli
. We conclude that preenrichment of stool and food samples in mTSB for 6 h followed by overnight culturing on BVCC is a simple method for the isolation and presumptive identification of STEC.
Construction and Use of an
ipb
DNA Module To Generate
Pseudomonas
Strains with Constitutive Trichloroethene and Isopropylbenzene Oxidation Activity
Authors: Berendes Frank , Sabarth Nicolas , Averhoff Beate , Gottschalk Gerhard
Published: December 2019

Pseudomonas
sp. strain JR1 exhibits trichloroethene (TCE) oxidation activity with isopropylbenzene (IPB) as the inducer substrate. We previously reported the genes encoding the first three enzymes of the IPB-degradative pathway (
ipbA1
,
ipbA2
,
ipbA3
,
ipbA4
,
ipbB
, and
ipbC
) and identified the initial IPB dioxygenase (IpbA1A2A3A4) as responsible for TCE cooxidation (U. Pflugmacher, B. Averhoff, and G. Gottschalk, Appl. Environ. Microbiol. 62:3967–3977, 1996). Primer extension analyses revealed multiple transcriptional start points located upstream of the translational initiation codon of
ipbA1
. The transcription from these start sites was found to be IPB dependent. Thirty-one base pairs upstream of the first transcriptional start point tandemly repeated DNA sequences overlapping the −35 region of a putative ς
70
promoter were found. These repeats exhibit significant sequence similarity to the operator-promoter region of the
xyl meta
operon in
Pseudomonas putida
, which is required for the binding of XylS, a regulatory protein of the XylS (also called AraC) family. These similarities suggest that the transcription of the IPB dioxygenase genes is modulated by a regulatory protein of the XylS/AraC family. The construction of an
ipb
DNA module devoid of this
ipb
operator-promoter region and the stable insertion of this DNA module into the genomes of different
Pseudomonas
strains resulted in pseudomonads with constitutive IPB and TCE oxidation activities. Constitutive TCE oxidation of two such
Pseudomonas
hybrid strains, JR1A::
ipb
and CBS-3::
ipb
, was found to be stable for more than 120 generations in antibiotic-free medium. Evaluation of constitutive TCE degradation rates revealed that continuous cultivation of strain JR1A::
ipb
resulted in a significant increase in rates of TCE degradation.
Small-Scale DNA Sample Preparation Method for Field PCR Detection of Microbial Cells and Spores in Soil
Authors: Kuske Cheryl R. , Banton Kaysie L. , Adorada Dante L. , Stark Peter C. , Hill Karen K. , Jackson Paul J.
Published: December 2019

Efficient, nonselective methods to obtain DNA from the environment are needed for rapid and thorough analysis of introduced microorganisms in environmental samples and for analysis of microbial community diversity in soil. A small-scale procedure to rapidly extract and purify DNA from soils was developed for in-the-field use. Amounts of DNA released from bacterial vegetative cells, bacterial endospores, and fungal conidia were compared by using hot-detergent treatment, freeze-thaw cycles, and bead mill homogenization. Combining a hot-detergent treatment with bead mill homogenization gave the highest DNA yields from all three microbial cell types and provided DNA from the broadest range of microbial groups in a natural soil community. Only the bead mill homogenization step was effective for DNA extraction from
Bacillus globigii
(
B. subtilis
subsp.
niger
) endospores or
Fusarium moniliforme
conidia. The hot-detergent–bead mill procedure was simplified and miniaturized. By using this procedure and small-scale, field-adapted purification and quantification procedures, DNA was prepared from four different soils seeded with
Pseudomonas putida
cells or
B. globigii
spores. In a New Mexico soil, seeded bacterial targets were detected with the same sensitivity as when assaying pure bacterial DNA (2 to 20 target gene copies in a PCR mixture). The detection limit of
P. putida
cells and
B. globigii
spores in different soils was affected by the amount of background DNA in the soil samples, the physical condition of the DNA, and the amount of DNA template used in the PCR.
Molecular Analysis of Bacterial Communities in a Three-Compartment Granular Activated Sludge System Indicates Community-Level Control by Incompatible Nitrification Processes
Authors: Holben William E. , Noto Kazuhiko , Sumino Tatsuo , Suwa Yuichi
Published: December 2019

Bacterial community structure and the predominant nitrifying activities and populations in each compartment of a three-compartment activated sludge system were determined. Each compartment was originally inoculated with the same activated sludge community entrapped in polyethylene glycol gel granules, and ammonium nitrogen was supplied to the system in an inorganic salts solution at a rate of 5.0 g of N liter of granular activated sludge
−1
day
−1
. After 150 days of operation, the system was found to comprise a series of sequential nitrifying reactions (K. Noto, T. Ogasawara, Y. Suwa, and T. Sumino, Water Res. 32:769–773, 1998), presumably mediated by different bacterial populations. Activity data showed that all NH
4
-N was completely oxidized in compartments one and two (approximately half in each), but no significant nitrite oxidation was observed in these compartments. In contrast, all available nitrite was oxidized to nitrate in compartment three. To study the microbial populations and communities in this system, total bacterial DNA isolated from each compartment was analyzed for community structure based on the G+C contents of the component populations. Compartment one showed dominant populations having 50 and 67% G+C contents. Compartment two was similar in structure to compartment one. The bacterial community in compartment three had dominant populations with 62 and 67% G+C contents and retained the 50% G+C content population only at a greatly diminished level. The 50% G+C content population from compartment one hybridized strongly with
amo
(ammonia monooxygenase) and
hao
(hydroxylamine oxidoreductase) gene probes from
Nitrosomonas europaea
. However, the 50% G+C content population from compartment two hybridized strongly with the
hao
probe but only weakly with the
amo
probe, suggesting that the predominant ammonia-oxidizing populations in compartments one and two might be different. Since different activities and populations come to dominate in each compartment from an identical inoculum, it appears that the nitrification processes may be somewhat incompatible, resulting in a series of sequential reactions and different communities in this three-compartment system.
Spatial and Temporal Variation of Phenanthrene-Degrading Bacteria in Intertidal Sediments
Authors: Berardesco Gina , Dyhrman Sonya , Gallagher Eugene , Shiaris Michael P.
Published: December 2019

Phenanthrene-degrading bacteria were isolated from a 1-m
2
intertidal sediment site in Boston Harbor. Samples were taken six times over 2 years. A total of 432 bacteria were isolated and characterized by biochemical testing. When clustered on the basis of phenotypic characteristics, the isolates could be separated into 68 groups at a similarity level of approximately 70%. Several groups (a total of 200 isolates) corresponded to well-characterized species belonging the genera
Vibrio
and
Pseudomonas
. Only 51 of the 437 isolates (<11.7% of the total) hybridized to a DNA probe that encodes the upper pathway of naphthalene and phenanthrene degradation in
Pseudomonas putida
NCIB 9816. A cluster analysis indicated that the species composition of the phenanthrene-degrading community changed significantly from sampling date to sampling date. At one sampling time, 12 6-mm-diameter core subsamples were taken within the 1-m
2
site to determine the spatial variability of the degrading communities. An analysis of molecular variance, performed with the phenotypic characteristics, indicated that only 6% of the variation occurred among the 12 subsamples, suggesting that the subsamples were almost identical in composition. We concluded that the communities of phenanthrene-degrading bacteria in the sediments are very diverse, that the community structure undergoes significant change with time but does not vary significantly on a spatial scale of centimeters, and that the predominant genes that encode phenanthrene degradation in the communities are not well-characterized.
Aerobic Mineralization of 2,6-Dichlorophenol by
Ralstonia
sp. Strain RK1
Authors: Steinle Patrick , Stucki Gerhard , Stettler Rolf , Hanselmann Kurt W.
Published: December 2019

A new aerobic bacterium was isolated from the sediment of a freshwater pond close to a contaminated site at Amponville (France). It was enriched in a fixed-bed reactor fed with 2,6-dichlorophenol (2,6-DCP) as the sole carbon and energy source at pH 7.5 and room temperature. The degradation of 2,6-DCP followed Monod kinetics at low initial concentrations. At concentrations above 300 μM (50 mg · liter
−1
), 2,6-DCP increasingly inhibited its own degradation. The base sequence of the 16S ribosomal DNA allowed us to assign the bacterium to the genus
Ralstonia
(formerly
Alcaligenes
). The substrate spectrum of the bacterium includes toluene, benzene, chlorobenzene, phenol, and all four
ortho
- and
para
-substituted mono- and dichlorophenol isomers. Substituents other than chlorine prevented degradation. The capacity to degrade 2,6-DCP was examined in two fixed-bed reactors. The microbial population grew on and completely mineralized 2,6-DCP at 2,6-DCP concentrations up to 740 μM in continuous reactor culture supplied with H
2
O
2
as an oxygen source. Lack of peroxide completely stopped further degradation of 2,6-DCP. Lowering the acid-neutralizing capacity of the medium to 1/10th the original capacity led to a decrease in the pH of the effluent from 7 to 6 and to a significant reduction in the degradation activity. A second fixed-bed reactor successfully removed low chlorophenol concentrations (20 to 26 μM) with hydraulic residence times of 8 to 30 min.
Capillary Electrophoresis Measurements of Electrophoretic Mobility for Colloidal Particles of Biological Interest
Authors: Glynn J. R. , Belongia B. M. , Arnold R. G. , Ogden K. L. , Baygents J. C.
Published: December 2019

The electrophoretic mobilities of three bacterial strains were investigated by capillary electrophoresis (CE) and were compared with results obtained by microelectrophoresis (ME). The CE measurements yielded bimodal electropherograms for two of the strains, thus illustrating for the first time that surface charge variations within a monoclonal population can be probed by CE. Intrapopulation variations were not detected by ME. The mobilities of three chemically distinct types of latex microspheres were also measured. Differences between the mean mobilities obtained by CE and ME were not statistically significant (
P
≤ 0.50); the standard deviations of the CE measurements were typically 2 to 10 times smaller than those obtained by comparable ME measurements. The reproducibility of CE permitted batch-to-batch mobility variations to be probed for the bacteria (one of the strains exhibited such variations), and aggregation was evident in one of the latex suspensions. These effects were not measurable with ME.
Effectiveness of SYTOX Green Stain for Bacterial Viability Assessment
Authors: Lebaron P. , Catala P. , Parthuisot N.
Published: December 2019

The effectiveness of SYTOX Green nucleic acid stain for measuring bacterial viability was tested on starved populations of
Escherichia coli
and
Salmonella typhimurium
. This stain underestimates the fraction of dead cells within starved populations containing cells with damaged nucleic acids or membranes. Its application to natural samples should be considered with caution.
Insecticidal Activity of
Bacillus laterosporus
Authors: Orlova Margarita V. , Smirnova Tatyana A. , Ganushkina Lyudmila A. , Yacubovich Victoria Y. , Azizbekyan Roudolf R.
Published: December 2019

The
Bacillus laterosporus
strains 921 and 615 were shown to have toxicity for larvae of the mosquitoes
Aedes aegypti
,
Anopheles stephensi
, and
Culex pipiens
. The larvicidal activity of
B. laterosporus
was associated with spores and crystalline inclusions. Purified
B. laterosporus
615 crystals were highly toxic for
Aedes aegypti
and
Anopheles stephensi
.
Microbial Community Changes in a Perturbed Agricultural Soil Investigated by Molecular and Physiological Approaches
Authors: Øvreås Lise , Jensen Sigmund , Daae Frida Lise , Torsvik Vigdis
Published: December 2019

Changes in soil microbial activity and diversity after incubation either with nitrogen or with a mixture of methane and air were examined. The perturbation by methane and air were characterized in detail and led to reduced diversity and enrichment of methanotrophs which were identified by denaturing gradient gel electrophoresis and 16S rRNA sequencing.