bluuebunny/crossref_metadata_2025 huggingface dataset:
Production of Sulfur Flavors by Ten Strains of
Geotrichum candidum
Authors: Berger Celine , Khan Jeffrey A. , Molimard Pascal , Martin Nathalie , Spinnler Henry E.
Published: December 2019

Ten strains of
Geotrichum candidum
were studied on a liquid cheese model medium for the production of sulfur compounds which contribute to the aroma of cheeses. The volatile components produced by each cultured strain were extracted by dynamic headspace extractions, separated and quantified by gas chromatography (GC), and identified by GC-mass spectrometry. It was shown that four strains of this microorganism produced significant quantities of
S
-methyl thioacetate,
S
-methyl thiopropionate,
S
-methyl thiobutanoate,
S
-methyl thio
iso
butanoate,
S
-methyl thio
iso
valerate, and
S
-methyl thiohexanoate. This is the first example of the production of these compounds by a fungus. In addition, dimethyldisulfide, dimethyltrisulfide, dimethylsulfide, and methanethiol, which are more commonly associated with the development of cheese flavor in bacterial cultures, were also produced by
G. candidum
in various yields, depending on the strain selected. The potential application of these strains in cultured microbial associations to produce modified cheeses with more desirable organoleptic properties is discussed.
Requirement for Phosphoglucose Isomerase of
Xanthomonas campestris
in Pathogenesis of Citrus Canker
Authors: Tung Shu Yun , Kuo Tsong Teh
Published: December 2019

A mutant (XT906) of
Xanthomonas campestris
pv. citri, the causal agent of citrus canker, was induced by insertion of the transposon Tn
5tac
1 and isolated. This mutant did not grow or elicit canker disease in citrus leaves but was still able to induce a hypersensitive response in a nonhost plant (the common bean). The mutant was also unable to grow on minimal medium containing fructose or glycerol as the sole carbon source. A 2.5-kb fragment of wild-type DNA that complemented the mutant phenotype of XT906 was isolated. Sequence analysis revealed that this DNA fragment encoded a protein of 562 amino acids that shows homology to phosphoglucose isomerase (PGI). Enzyme activity assay confirmed that the encoded protein possesses PGI activity. Analysis of the activity of the promoter of the
pgi
gene revealed that it was inhibited by growth in complex medium but induced by culture in plant extract. These results demonstrate that PGI is required for pathogenicity of
X. campestris
pv. citri.
Anastomosis Formation and Nuclear and Protoplasmic Exchange in Arbuscular Mycorrhizal Fungi
Authors: Giovannetti Manuela , Azzolini Dario , Citernesi Anna Silvia
Published: December 2019

We observed anastomosis between hyphae originating from the same spore and from different spores of the same isolate of the arbuscular mycorrhizal fungi
Glomus mosseae
,
Glomus caledonium
, and
Glomus intraradices
. The percentage of contacts leading to anastomosis ranged from 35 to 69% in hyphae from the same germling and from 34 to 90% in hyphae from different germlings. The number of anastomoses ranged from 0.6 to 1.3 per cm (length) of hyphae in mycelia originating from the same spore. No anastomoses were observed between hyphae from the same or different germlings of
Gigaspora rosea
and
Scutellospora castanea
; no interspecific or intergeneric hyphal fusions were observed. We monitored anastomosis formation with time-lapse and video-enhanced light microscopy. We observed complete fusion of hyphal walls and the migration of a mass of particles in both directions within the hyphal bridges. In hyphal bridges of
G. caledonium
, light-opaque particles moved at the speed of 1.8 ± 0.06 μm/s. We observed nuclear migration between hyphae of the same germling and between hyphae belonging to different germlings of the same isolate of three
Glomus
species. Our work suggests that genetic exchange may occur through intermingling of nuclei during anastomosis formation and opens the way to studies of vegetative compatibility in natural populations of arbuscular mycorrhizal fungi.
Growth of Facultatively Heterofermentative Lactobacilli on Starter Cell Suspensions
Authors: Rapposch S. , Eliskases-Lechner F. , Ginzinger W.
Published: December 2019

The growth of facultatively heterofermentative lactobacilli (FHL) on cell suspensions of the homofermentative
Lactobacillus helveticus
was investigated. Osmotic lysis of
L. helveticus
led to a significant increase of ribose. It decreased steadily in parallel with the growth of FHL, strongly suggesting that the bacteria used ribose as a growth substrate.
Hemolytic Activity and Siderophore Production in Different
Aeromonas
Species Isolated from Fish
Authors: Santos Jesús A. , González César J. , Otero Andrés , García-López María-Luisa
Published: December 2019

The hemolytic activity and siderophore production of several strains of motile aeromonads were determined. The hemolytic activity of
Aeromonas caviae
and
Aeromonas eucrenophila
was enhanced after trypsinization of the samples. The enhancement of hemolysis was observed in strains that carried an aerolysin-like gene, detected by a PCR procedure. Siderophore production was demonstrated in all but one strain of
Aeromonas jandaei
. No apparent relationship was observed between the presence of plasmid DNA and hemolysis or siderophore production.
Analysis of
nifH
Gene Pool Complexity in Soil and Litter at a Douglas Fir Forest Site in the Oregon Cascade Mountain Range
Authors: Widmer F. , Shaffer B. T. , Porteous L. A. , Seidler R. J.
Published: December 2019

Nitrogen-fixing microbial populations in a Douglas fir forest on the western slope of the Oregon Cascade Mountain Range were analyzed. The complexity of the
nifH
gene pool (
nifH
is the marker gene which encodes nitrogenase reductase) was assessed by performing nested PCR with bulk DNA extracted from plant litter and soil. The restriction fragment length polymorphisms (RFLPs) of PCR products obtained from litter were reproducibly different than the RFLPs of PCR products obtained from the underlying soil. The characteristic differences were found during the entire sampling period between May and September. RFLP analyses of cloned
nifH
PCR products also revealed characteristic patterns for each sample type. Among 42
nifH
clones obtained from a forest litter library nine different RFLP patterns were found, and among 64
nifH
clones obtained from forest soil libraries 13 different patterns were found. Only two of the patterns were found in both the litter and the soil, indicating that there were major differences between the nitrogen-fixing microbial populations. A sequence analysis of clones representing the 20 distinct patterns revealed that 19 of the patterns had a proteobacterial origin. All of the
nifH
sequences obtained from the Douglas fir forest litter localized in a distinct phylogenetic cluster characterized by the
nifH
sequences of members of the genera
Rhizobium
,
Sinorhizobium
, and
Azospirillum
. The
nifH
sequences obtained from soil were found in two additional clusters, one characterized by sequences of members of the genera
Bradyrhizobium
,
Azorhizobium
,
Herbaspirillum
, and
Thiobacillus
and the other, represented by a single
nifH
clone, located between the gram-positive bacteria and the cyanobacteria. Our results revealed the distinctness of the nitrogen-fixing microbial populations in litter and soil in a Douglas fir forest; the differences may be related to special requirements for degradation and mineralization processes in the plant litter.
1,4-Benzoquinone Reductase from
Phanerochaete chrysosporium
: cDNA Cloning and Regulation of Expression
Authors: Akileswaran Lakshmi , Brock Barry J. , Cereghino Joan Lin , Gold Michael H.
Published: December 2019

A cDNA clone encoding a quinone reductase (QR) from the white rot basidiomycete
Phanerochaete chrysosporium
was isolated and sequenced. The cDNA consisted of 1,007 nucleotides and a poly(A) tail and encoded a deduced protein containing 271 amino acids. The experimentally determined eight-amino-acid N-terminal sequence of the purified QR protein from
P. chrysosporium
matched amino acids 72 to 79 of the predicted translation product of the cDNA. The
M
r
of the predicted translation product, beginning with Pro-72, was essentially identical to the experimentally determined
M
r
of one monomer of the QR dimer, and this finding suggested that QR is synthesized as a proenzyme. The results of in vitro transcription-translation experiments suggested that QR is synthesized as a proenzyme with a 71-amino-acid leader sequence. This leader sequence contains two potential KEX2 cleavage sites and numerous potential cleavage sites for dipeptidyl aminopeptidase. The QR activity in cultures of
P. chrysosporium
increased following the addition of 2-dimethoxybenzoquinone, vanillic acid, or several other aromatic compounds. An immunoblot analysis indicated that induction resulted in an increase in the amount of QR protein, and a Northern blot analysis indicated that this regulation occurs at the level of the
qr
mRNA.
Genotype Analysis of
Escherichia coli
Strains Isolated from Children and Chickens Living in Close Contact
Authors: Kariuki S. , Gilks C. , Kimari J. , Obanda A. , Muyodi J. , Waiyaki P. , Hart C. A.
Published: December 2019

Escherichia coli
isolates from rectal swabs from 62 chickens and stools from 42 children living in close contact with chickens on the same farms in Kiambu district, Kenya, were compared for their genetic relatedness. Antibiotic susceptibility profiles broadly categorized isolates from the children and from the chickens into two separate clusters: the majority (144; 85.5%) of the
E. coli
isolates from children were multidrug resistant, while the majority (216; 87.1%) of the
E. coli
isolates from chickens were either fully susceptible or resistant only to tetracycline. Sixty- and 100- to 110-MDA plasmids were found to encode the transferable resistance to co-trimoxazole and tetracycline.
Hin
dIII restriction endonuclease digestion of the 60- and 100- to 110-MDA plasmids produced four distinct patterns for isolates from children and three distinct patterns for isolates from chickens.
Xba
I digestion of genomic DNA followed by pulsed-field gel electrophoresis (PFGE) analysis produced 14 distinct clusters. There were six distinct PFGE clusters among the isolates from children, while among the isolates from chickens there were seven distinct clusters. Only one PFGE cluster contained isolates from both children and chickens, with the isolates displaying an approximately 60% coefficient of similarity. This study showed that although several different genotypes of
E. coli
were isolated from children and chickens from the same farms, the
E. coli
strains from these two sources were distinct.
Ethanol Synthesis by Genetic Engineering in Cyanobacteria
Authors: Deng Ming-De , Coleman John R.
Published: December 2019

Cyanobacteria are autotrophic prokaryotes which carry out oxygenic photosynthesis and accumulate glycogen as the major form of stored carbon. In this research, we introduced new genes into a cyanobacterium in order to create a novel pathway for fixed carbon utilization which results in the synthesis of ethanol. The coding sequences of pyruvate decarboxylase (
pdc
) and alcohol dehydrogenase II (
adh
) from the bacterium
Zymomonas mobilis
were cloned into the shuttle vector pCB4 and then used to transform the cyanobacterium
Synechococcus
sp. strain PCC 7942. Under control of the promoter from the
rbcLS
operon encoding the cyanobacterial ribulose-1,5-bisphosphate carboxylase/oxygenase, the
pdc
and
adh
genes were expressed at high levels, as demonstrated by Western blotting and enzyme activity analyses. The transformed cyanobacterium synthesized ethanol, which diffused from the cells into the culture medium. As cyanobacteria have simple growth requirements and use light, CO
2
, and inorganic elements efficiently, production of ethanol by cyanobacteria is a potential system for bioconversion of solar energy and CO
2
into a valuable resource.
Genetic Evidence That High Noninduced Maltase and Maltose Permease Activities, Governed by
MALx3
-Encoded Transcriptional Regulators, Determine Efficiency of Gas Production by Baker’s Yeast in Unsugared Dough
Authors: Higgins Vincent J. , Braidwood Mark , Bell Phil , Bissinger Peter , Dawes Ian W. , Attfield Paul V.
Published: December 2019

Strain selection and improvement in the baker’s yeast industry have aimed to increase the speed of maltose fermentation in order to increase the leavening activity of industrial baking yeast. We identified two groups of baker’s strains of
Saccharomyces cerevisiae
that can be distinguished by the mode of regulation of maltose utilization. One group (nonlagging strains), characterized by rapid maltose fermentation, had at least 12-fold more maltase and 130-fold-higher maltose permease activities than maltose-lagging strains in the absence of inducing sugar (maltose) and repressing sugar (glucose). Increasing the noninduced maltase activity of a lagging strain 13-fold led to an increase in CO
2
production in unsugared dough. This increase in CO
2
production also was seen when the maltose permease activity was increased 55-fold. Only when maltase and maltose permease activities were increased in concert was CO
2
production by a lagging strain similar to that of a nonlagging strain. The noninduced activities of maltase and maltose permease constitute the largest determinant of whether a strain displays a nonlagging or a lagging phenotype and are dependent upon the
MALx3
allele. Previous strategies for strain improvement have targeted glucose derepression of maltase and maltose permease expression. Our results suggest that increasing noninduced maltase and maltose permease levels is an important target for improved maltose metabolism in unsugared dough.
Diversity of Arbuscular Mycorrhizal Fungus Populations in Heavy-Metal-Contaminated Soils
Authors: Del Val C. , Barea J. M. , Azcón-Aguilar C.
Published: December 2019

High concentrations of heavy metals have been shown to adversely affect the size, diversity, and activity of microbial populations in soil. The aim of this work was to determine how the diversity of arbuscular mycorrhizal (AM) fungi is affected by the addition of sewage-amended sludge containing heavy metals in a long-term experiment. Due to the reduced number of indigenous AM fungal (AMF) propagules in the experimental soils, several host plants with different life cycles were used to multiply indigenous fungi. Six AMF ecotypes were found in the experimental soils, showing consistent differences with regard to their tolerance to the presence of heavy metals. AMF ecotypes ranged from very sensitive to the presence of metals to relatively tolerant to high rates of heavy metals in soil. Total AMF spore numbers decreased with increasing amounts of heavy metals in the soil. However, species richness and diversity as measured by the Shannon-Wiener index increased in soils receiving intermediate rates of sludge contamination but decreased in soils receiving the highest rate of heavy-metal-contaminated sludge. Relative densities of most AMF species were also significantly influenced by soil treatments. Host plant species exerted a selective influence on AMF population size and diversity. We conclude based on the results of this study that size and diversity of AMF populations were modified in metal-polluted soils, even in those with metal concentrations that were below the upper limits accepted by the European Union for agricultural soils.
Enhancement of Solar Water Pasteurization with Reflectors
Authors: Safapour Negar , Metcalf Robert H.
Published: December 2019

A simple and reliable method that could be used in developing countries to pasteurize milk and water with solar energy is described. A cardboard reflector directs sunshine onto a black jar, heating water to pasteurizing temperatures in several hours. A reusable water pasteurization indicator verifies that pasteurization temperatures have been reached.
Isolation of Fecal Coliform Bacteria from the Diamondback Terrapin (
Malaclemys terrapin centrata
)
Authors: Harwood Valerie J. , Butler Joseph , Parrish Danny , Wagner Victoria
Published: December 2019

Total and fecal coliform bacteria were isolated from the cloaca and feces of the estuarine diamondback terrapin. The majority of samples contained fecal coliforms.
Escherichia coli
was the predominant fecal coliform species isolated, and members of the genus
Salmonella
were isolated from 2 of 39 terrapins. Fecal coliform numbers are used to regulate shellfish harvests, and diamondback terrapins inhabit the brackish-water habitats where oyster beds are found; therefore, these findings have implications for the efficacy of current regulatory parameters in shellfishing waters.
Degradation of Chloronitrobenzenes by a Coculture of
Pseudomonas putida
and a
Rhodococcus
sp
Authors: Park Hee-Sung , Lim Sung-Jin , Chang Young Keun , Livingston Andrew G. , Kim Hak-Sung
Published: December 2019

A single microorganism able to mineralize chloronitrobenzenes (CNBs) has not been reported, and degradation of CNBs by coculture of two microbial strains was attempted.
Pseudomonas putida
HS12 was first isolated by analogue enrichment culture using nitrobenzene (NB) as the substrate, and this strain was observed to possess a partial reductive pathway for the degradation of NB. From high-performance liquid chromatography-mass spectrometry and
1
H nuclear magnetic resonance analyses, NB-grown cells of
P. putida
HS12 were found to convert 3- and 4-CNBs to the corresponding 5- and 4-chloro-2-hydroxyacetanilides, respectively, by partial reduction and subsequent acetylation. For the degradation of CNBs,
Rhodococcus
sp. strain HS51, which degrades 4- and 5-chloro-2-hydroxyacetanilides, was isolated and combined with
P. putida
HS12 to give a coculture. This coculture was confirmed to mineralize 3- and 4-CNBs in the presence of an additional carbon source. A degradation pathway for 3- and 4-CNBs by the two isolated strains was also proposed.
Enhanced Bioaccumulation of Heavy Metal Ions by Bacterial Cells Due to Surface Display of Short Metal Binding Peptides
Authors: Kotrba Pavel , Dolečková Lucie , de Lorenzo Víctor , Ruml Tomas
Published: December 2019

Metal binding peptides of sequences Gly-His-His-Pro-His-Gly (named HP) and Gly-Cys-Gly-Cys-Pro-Cys-Gly-Cys-Gly (named CP) were genetically engineered into LamB protein and expressed in
Escherichia coli
. The Cd
2+
-to-HP and Cd
2+
-to-CP stoichiometries of peptides were 1:1 and 3:1, respectively. Hybrid LamB proteins were found to be properly folded in the outer membrane of
E. coli
. Isolated cell envelopes of
E. coli
bearing newly added metal binding peptides showed an up to 1.8-fold increase in Cd
2+
binding capacity. The bioaccumulation of Cd
2+
, Cu
2+
, and Zn
2+
by
E. coli
was evaluated. Surface display of CP multiplied the ability of
E. coli
to bind Cd
2+
from growth medium fourfold. Display of HP peptide did not contribute to an increase in the accumulation of Cu
2+
and Zn
2+
. However, Cu
2+
ceased contribution of HP for Cd
2+
accumulation, probably due to the strong binding of Cu
2+
to HP. Thus, considering the cooperation of cell structures with inserted peptides, the relative affinities of metal binding peptide and, for example, the cell wall to metal ion should be taken into account in the rational design of peptide sequences possessing specificity for a particular metal.
Comparison of Flagellin Genes from Clinical and Environmental
Pseudomonas aeruginosa
Isolates
Authors: Morgan J. Alun W. , Bellingham Nessa F. , Winstanley Craig , Ousley Margaret A. , Hart C. Anthony , Saunders Jon R.
Published: December 2019

Pseudomonas aeruginosa
, an important opportunistic pathogen, was isolated from environmental samples and compared to clinically derived strains. While
P. aeruginosa
was isolated readily from an experimental mushroom-growing unit, it was found only rarely in other environmental samples. A flagellin gene PCR-restriction fragment length polymorphism analysis of the isolates revealed that environmental and clinical
P. aeruginosa
strains are not readily distinguishable. The variation in the central regions of the flagellin genes of seven of the isolates was investigated further. The strains used included two strains with type a genes (998 bp), four strains with type b genes (1,258 bp), and one strain, K979, with a novel flagellin gene (2,199 bp). The route by which flagellin gene variation has occurred in
P. aeruginosa
is discussed.
Metabolic Engineering of a 1,2-Propanediol Pathway in
Escherichia coli
Authors: Altaras Nedim E. , Cameron Douglas C.
Published: December 2019

1,2-Propanediol (1,2-PD) is a major commodity chemical that is currently derived from propylene, a nonrenewable resource. A goal of our research is to develop fermentation routes to 1,2-PD from renewable resources. Here we report the production of enantiomerically pure
R
-1,2-PD from glucose in
Escherichia coli
expressing NADH-linked glycerol dehydrogenase genes (
E. coli gldA
or
Klebsiella pneumoniae dhaD
). We also show that
E. coli
overexpressing the
E. coli
methylglyoxal synthase gene (
mgs
) produced 1,2-PD. The expression of either glycerol dehydrogenase or methylglyoxal synthase resulted in the anaerobic production of approximately 0.25 g of 1,2-PD per liter.
R
-1,2-PD production was further improved to 0.7 g of 1,2-PD per liter when methylglyoxal synthase and glycerol dehydrogenase (
gldA
) were coexpressed. In vitro studies indicated that the route to
R
-1,2-PD involved the reduction of methylglyoxal to
R
-lactaldehyde by the recombinant glycerol dehydrogenase and the reduction of
R
-lactaldehyde to
R
-1,2-PD by a native
E. coli
activity. We expect that
R
-1,2-PD production can be significantly improved through further metabolic and bioprocess engineering.
Organization of the Gene Cluster for Biosynthesis of Penicillin in
Penicillium nalgiovense
and Antibiotic Production in Cured Dry Sausages
Authors: Laich Federico , Fierro Francisco , Cardoza Rosa Elena , Martin Juan F.
Published: December 2019

Several fungal isolates obtained from two cured meat products from Spain were identified as
Penicillium nalgiovense
by their morphological features and by DNA fingerprinting. All
P. nalgiovense
isolates showed antibiotic activity in agar diffusion assays, and their penicillin production in liquid complex medium ranged from 6 to 38 μg · ml
−1
. We constructed a restriction map of the penicillin gene cluster of
P. nalgiovense
and found that the organization of the penicillin biosynthetic genes (
pcbAB
,
pcbC
, and
penDE
) is the same as in
Penicillium chrysogenum
and
Aspergillus nidulans
. The
pcbAB
gene is located in an orientation opposite that of the
pcbC
and
penDE
genes in all three species. Significant amounts of penicillin were found in situ in the casing and the outer layer of salami meat during early stages of the curing process, coinciding with fungal colonization, but no penicillin was detected in the cured salami. The antibiotic produced in situ was sensitive to penicillinase.
Identification of Epichloë Endophytes In Planta by a Microsatellite-Based PCR Fingerprinting Assay with Automated Analysis
Authors: Moon Christina D. , Tapper Brian A. , Scott Barry
Published: December 2019

Epichloë endophytes are a group of filamentous fungi that include both sexual (Epichloë) and asexual (Neotyphodium) species. As a group they are genetically diverse and form both antagonistic and mutualistic associations with temperate grasses. We report here on the development of a microsatellite-based PCR system for fingerprinting this group of fungi with template isolated from either culture or infected plant material. M13mp19 partial genomic libraries were constructed for size-fractionated genomic DNA from two endophyte strains. These libraries were screened with a mixture of DIG-labeled dinucleotide and trinucleotide repeat probes. Positive clones were sequenced, and nine unique microsatellite loci were identified. An additional microsatellite was serendipitously identified in the 3′ untranscribed region of the 3-hydroxy-3-methylglutaryl coenzyme A (HMG CoA) reductase gene fromN. loliiLp19. Primers were designed for each locus and a panel of endophytes, from different taxonomic groupings, was screened to determine the degree of polymorphism. On the basis of these results a multiplex assay was developed for strain identification with fluorescently labeled primers for five of these loci. Using this system the size of the products amplified can be precisely determined by automated analysis, and an allele profile for each strain can be readily generated. The assay was shown to resolve endophyte groupings to the level of known isozyme phenotype groupings. In a blind test the assay was used successfully to identify a set of endophytes in planta. A reference database of allele sizes has been established for the panel of endophytes examined, and this will be expanded as new strains are analyzed.
A Randomly Amplified Polymorphic DNA Marker Specific for the
Bacillus cereus
Group Is Diagnostic for
Bacillus anthracis
Authors: Daffonchio Daniele , Borin Sara , Frova Giuseppe , Gallo Romina , Mori Elena , Fani Renato , Sorlini Claudia
Published: December 2019

Aiming to develop a DNA marker specific for
Bacillus anthracis
and able to discriminate this species from
Bacillus cereus
,
Bacillus thuringiensis
, and
Bacillus mycoides
, we applied the randomly amplified polymorphic DNA (RAPD) fingerprinting technique to a collection of 101 strains of the genus
Bacillus
, including 61 strains of the
B. cereus
group. An 838-bp RAPD marker (SG-850) specific for
B. cereus
,
B. thuringiensis
,
B. anthracis
, and
B. mycoides
was identified. This fragment included a putative (366-nucleotide) open reading frame highly homologous to the
ypuA
gene of
Bacillus subtilis
. The restriction analysis of the SG-850 fragment with
Alu
I distinguished
B. anthracis
from the other species of the
B. cereus
group.
Thermus aquaticus
ATCC 33923 Amylomaltase Gene Cloning and Expression and Enzyme Characterization: Production of Cycloamylose
Authors: Terada Yoshinobu , Fujii Kazutoshi , Takaha Takeshi , Okada Shigetaka
Published: December 2019

The amylomaltase gene of the thermophilic bacterium
Thermus aquaticus
ATCC 33923 was cloned and sequenced. The open reading frame of this gene consisted of 1,503 nucleotides and encoded a polypeptide that was 500 amino acids long and had a calculated molecular mass of 57,221 Da. The deduced amino acid sequence of the amylomaltase exhibited a high level of homology with the amino acid sequence of potato disproportionating enzyme (D-enzyme) (41%) but a low level of homology with the amino acid sequence of the
Escherichia coli
amylomaltase (19%). The amylomaltase gene was overexpressed in
E. coli
, and the enzyme was purified. This enzyme exhibited maximum activity at 75°C in a 10-min reaction with maltotriose and was stable at temperatures up to 85°C. When the enzyme acted on amylose, it catalyzed an intramolecular transglycosylation (cyclization) reaction which produced cyclic α-1,4-glucan (cycloamylose), like potato D-enzyme. The yield of cycloamylose produced from synthetic amylose with an average molecular mass of 110 kDa was 84%. However, the minimum degree of polymerization (DP) of the cycloamylose produced by
T. aquaticus
enzyme was 22, whereas the minimum DP of the cycloamylose produced by potato D-enzyme was 17. The
T. aquaticus
enzyme also catalyzed intermolecular transglycosylation of maltooligosaccharides. A detailed analysis of the activity of
T. aquaticus
ATCC 33923 amylomaltase with maltooligosaccharides indicated that the catalytic properties of this enzyme differ from those of
E. coli
amylomaltase and the plant D-enzyme.
Mechanism of Peroxidase Inactivation in Liquid Cultures of the Ligninolytic Fungus
Pleurotus pulmonarius
Authors: Böckle Brigitte , Martínez María Jesús , Guillén Francisco , Martínez Ángel T.
Published: December 2019

It has recently been reported that
Pleurotus pulmonarius
secretes a versatile peroxidase that oxidizes Mn
2+
, as well as different phenolic and nonphenolic aromatic compounds; this enzyme has also been detected in other
Pleurotus
species and in
Bjerkandera
species. During culture production of the enzyme, the activity of the main peak was as high as 1,000 U/liter (measured on the basis of the Mn
3+
-tartrate formation) but this peak was very ephemeral due to enzyme instability (up to 80% of the activity was lost within 15 h). In culture filtrates inactivation was even faster; all peroxidase activity was lost within a few hours. Using different inhibitor compounds, we found that proteases were not responsible for the decrease in peroxidase activity. Peroxidase instability coincided with an increase in the H
2
O
2
concentration, which reached 200 μM when filtrates were incubated for several hours. It also coincided with the onset of biosynthesis of anisylic compounds and a decrease in the pH of the culture. Anisyl alcohol is the natural substrate of the enzyme aryl-alcohol oxidase, the main source of extracellular H
2
O
2
in
Pleurotus
cultures, and addition of anisyl alcohol to filtrates containing stable peroxidase activity resulted in rapid inactivation. A decrease in the culture pH could also dramatically affect the stability of the
P. pulmonarius
peroxidase, as shown by using pH values ranging from 6 to 3.25, which resulted in an increase in the level of inactivation by 10 μM H
2
O
2
from 5 to 80% after 1 h. Moreover, stabilization of the enzyme was observed after addition of catalase, Mn
2+
, or some phenols or after dialysis of the culture filtrate. We concluded that extracellular H
2
O
2
produced by the fungus during oxidation of aromatic metabolites is responsible for inactivation of the peroxidase and that the enzyme can protect itself in the presence of different reducing substrates.
Identification of Aerobically and Anaerobically Induced Genes in
Enterococcus faecalis
by Random Arbitrarily Primed PCR
Authors: Shepard Brett D. , Gilmore Michael S.
Published: December 2019

Enterococci have emerged among the leading causes of nosocomial infection. With the goal of analyzing enterococcal genes differentially expressed in environments related to commensal or environmental colonization and infection sites, we adapted and optimized a method more commonly used in the study of eukaryotic gene expression, random arbitrarily primed PCR (RAP-PCR). The RAP-PCR method was systematically optimized, allowing the technique to be used in a highly reproducible manner with gram-positive bacterial RNA. In the present study, aerobiosis was chosen as a variable for the induction of changes in gene expression by
Enterococcus faecalis
. Aerobically and anaerobically induced genes were detected and identified to the sequence level, and differential gene expression was confirmed by quantitative, specifically primed RT-PCR. Differentially expressed genes included several sharing identity with those of other organisms related to oxygen metabolism, as well as hypothetical genes lacking identity to known genes.
Estimation of Bacterial Cell Numbers in Humic Acid-Rich Salt Marsh Sediments with Probes Directed to 16S Ribosomal DNA
Authors: Edgcomb Virginia P. , McDonald John H. , Devereux Richard , Smith David W.
Published: December 2019

The feasibility of using probes directed towards ribosomal DNAs (rDNAs) as a quantitative approach to estimating cell numbers was examined and applied to study the structure of a bacterial community in humic acid-rich salt marsh sediments. Hybridizations were performed with membrane-bound nucleic acids by using seven group-specific DNA oligonucleotide probes complementary to 16S rRNA coding regions. These included a general eubacterial probe and probes encompassing most members of the gram-negative, mesophilic sulfate-reducing bacteria (SRB). DNA was extracted from sediment samples, and contaminating materials were removed by a series of steps. Efficiency of DNA extraction was 48% based on the recovery of tritiated plasmid DNA added to samples prior to extraction. Reproducibility of the extraction procedure was demonstrated by hybridizations to replicate samples. Numbers of target cells in samples were estimated by comparing the amount of hybridization to extracted DNA obtained with each probe to that obtained with a standard curve of genomic DNA for reference strains included on the same membrane. In June, numbers of SRB detected with an SRB-specific probe ranged from 6.0 × 10
7
to 2.5 × 10
9
(average, 1.1 × 10
9
± 5.2 × 10
8
) cells g of sediment
−1
. In September, numbers of SRB detected ranged from 5.4 × 10
8
to 7.3 × 10
9
(average, 2.5 × 10
9
± 1.5 × 10
9
) cells g of sediment
−1
. The capability of using rDNA probes to estimate cell numbers by hybridization to DNA extracted from complex matrices permits initiation of detailed studies on community composition and changes in communities based on cell numbers in formerly intractable environments.
β-Tubulin mRNA as a Marker of
Cryptosporidium parvum
Oocyst Viability
Authors: Widmer Giovanni , Orbacz Elizabeth A. , Tzipori Saul
Published: December 2019

Determining the viability of waterborne
Cryptosporidium parvum
oocysts remains a technical challenge. rRNA and mRNA were evaluated in a reverse transcription (RT)-PCR assay as potential markers of oocyst viability. The rationale for this approach is the rapid turnover and postmortem decay of cellular RNA. The β-tubulin mRNA and an anonymous mRNA transcript were chosen as potential markers because they are the only mRNA species in
C. parvum
known to possess introns. This feature facilitated the distinction between genuine RT-PCR products and PCR products originating from copurifying DNA. Prolonged incubation at room temperature of initially viable oocysts resulted in a gradual decrease in mRNA levels, which correlated with the loss of oocyst infectivity to neonatal mice. In contrast, oocysts stored at 4°C for over 39 weeks maintained their infectivity and displayed no decrease in the level of β-tubulin RT-PCR product. The postmortem decay of two mRNA species demonstrates that RT-PCR analysis can provide information on the viability of
C. parvum
oocysts. The methodological similarity between PCR detection and RT-PCR viability analysis could facilitate the development of a combined detection and viability assay.
Cloning and Partial Characterization of Endopolygalacturonase Genes fromBotrytis cinerea
Authors: Wubben J. P. , Mulder W. , ten Have A. , van Kan J. A. L. , Visser J.
Published: December 2019

Botrytis cinereais a plant-pathogenic fungus infecting over 200 different plant species. We use a molecular genetic approach to study the process of pectin degradation by the fungus. Recently, we described the cloning and characterization of an endopolygalacturonase (endoPG) gene fromB. cinerea(Bcpg1) which is required for full virulence. Here we describe the cloning and characterization of five additional endoPG-encoding genes fromB. cinereaSAS56. The identity at the amino acid level between the six endoPGs ofB. cinereavaried from 34 to 73%. Phylogenetic analysis, by using a group of 35 related fungal endoPGs and as an outgroup one plant PG, resulted in the identification of five monophyletic groups of closely related proteins. The endoPG proteins fromB. cinereaSAS56 could be assigned to three different monophyletic groups. DNA blot analysis revealed the presence of the complete endoPG gene family in other strains ofB. cinerea, as well as in otherBotrytisspecies. Differential gene expression of the gene family members was found in mycelium grown in liquid culture with either glucose or polygalacturonic acid as the carbon source.
Counting and Size Classification of Active Soil Bacteria by Fluorescence In Situ Hybridization with an rRNA Oligonucleotide Probe
Authors: Christensen Henrik , Hansen Michael , Sørensen Jan
Published: December 2019

A fluorescence in situ hybridization (FISH) technique based on binding of a rhodamine-labelled oligonucleotide probe to 16S rRNA was used to estimate the numbers of ribosome-rich bacteria in soil samples. Such bacteria, which have high cellular rRNA contents, were assumed to be active (and growing) in the soil. Hybridization to an rRNA probe, EUB338, for the domain
Bacteria
was performed with a soil slurry, and this was followed by collection of the bacteria by membrane filtration (pore size, 0.2 μm). A nonsense probe, NONEUB338 (which has a nucleotide sequence complementary to the nucleotide sequence of probe EUB338), was used as a control for nonspecific staining. Counting and size classification into groups of small, medium, and large bacteria were performed by fluorescence microscopy. To compensate for a difference in the relative staining intensities of the probes and for binding by the rhodamine part of the probe, control experiments in which excess unlabelled probe was added were performed. This resulted in lower counts with EUB338 but not with NONEUB338, indicating that nonspecific staining was due to binding of rhodamine to the bacteria. A value of 4.8 × 10
8
active bacteria per g of dry soil was obtained for bulk soil incubated for 2 days with 0.3% glucose. In comparison, a value of 3.8 × 10
8
active bacteria per g of dry soil was obtained for soil which had been air dried and subsequently rewetted. In both soils, the majority (68 to 77%) of actively growing bacteria were members of the smallest size class (cell width, 0.25 to 0.5 μm), but the active (and growing) bacteria still represented only approximately 5% of the total bacterial population determined by DAPI (4′,6-diamidino-2-phenylindole) staining. The FISH technique in which slurry hybridization is used holds great promise for use with phylogenetic probes and for automatic counting of soil bacteria.
Semiautomated Metabolic Staining Assay for
Bacillus cereus
Emetic Toxin
Authors: Finlay W. J. J. , Logan N. A. , Sutherland A. D.
Published: December 2019

This paper describes a specific, sensitive, semiautomated, and quantitative Hep-2 cell culture-based 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay for
Bacillus cereus
emetic toxin. Of nine
Bacillus
,
Brevibacillus
, and
Paenibacillus
species assessed for emetic toxin production, only
B. cereus
was cytotoxic.
Expression of Two Major Chitinase Genes of
Trichoderma atroviride
(
T. harzianum
P1) Is Triggered by Different Regulatory Signals
Authors: Mach Robert L. , Peterbauer Clemens K. , Payer Kathrin , Jaksits Sylvia , Woo Sheridan L. , Zeilinger Susanne , Kullnig Cornelia M. , Lorito Matteo , Kubicek Christian P.
Published: December 2019

Regulation of the expression of the two major chitinase genes,
ech42
(encoding the CHIT42 endochitinase) and
nag1
(encoding the CHIT73
N
-acetyl-β-
d
-glucosaminidase), of the chitinolytic system of the mycoparasitic biocontrol fungus
Trichoderma atroviride
(=
Trichoderma harzianum
P1) was investigated by using a reporter system based on the
Aspergillus niger
glucose oxidase. Strains harboring fusions of the
ech42
or
nag1
5′ upstream noncoding sequences with the
A. niger goxA
gene displayed a glucose oxidase activity pattern that was consistent under various conditions with expression of the native
ech42
and
nag1
genes, as assayed by Northern analysis. The expression product of
goxA
in the mutants was completely secreted into the medium, detectable on Western blots, and quantifiable by enzyme-linked immunosorbent assay.
nag1
gene expression was triggered during growth on fungal (
Botrytis cinerea
) cell walls and on the chitin degradation product
N
-acetylglucosamine.
N
-Acetylglucosamine, di-
N
-acetylchitobiose, or tri-
N
-acetylchitotriose also induced
nag1
gene expression when added to mycelia pregrown on different carbon sources.
ech42
expression was also observed during growth on fungal cell walls but, in contrast, was not triggered by addition of chitooligomers to pregrown mycelia. Significant
ech42
expression was observed after prolonged carbon starvation, independent of the use of glucose or glycerol as a carbon source, suggesting that relief of carbon catabolite repression was not involved in induction during starvation. In addition,
ech42
gene transcription was triggered by physiological stress, such as low temperature, high osmotic pressure, or the addition of ethanol. Four copies of a putative stress response element (CCCCT) were found in the
ech42
promoter.
d
-Pantothenate Synthesis in
Corynebacterium glutamicum
and Use of
panBC
and Genes Encoding
l
-Valine Synthesis for
d
-Pantothenate Overproduction
Authors: Sahm Hermann , Eggeling Lothar
Published: December 2019

d
-Pantothenate is synthesized via four enzymes from ketoisovalerate, which is an intermediate of branched-chain amino acid synthesis. We quantified three of these enzyme activities in
Corynebacterium glutamicum
and determined specific activities ranging from 0.00014 to 0.001 μmol/min mg (protein)
−1
. The genes encoding the ketopantoatehydroxymethyl transferase and the pantothenate synthetase were cloned, sequenced, and functionally characterized. These studies suggest that
panBC
constitutes an operon. By using
panC
, an assay system was developed to quantify
d
-pantothenate. The wild type of
C. glutamicum
was found to accumulate 9 μg of this vitamin per liter. A strain was constructed (i) to abolish
l
-isoleucine synthesis, (ii) to result in increased ketoisovalerate formation, and (iii) to enable its further conversion to
d
-pantothenate. The best resulting strain has
ilvA
deleted from its chromosome and has two plasmids to overexpress genes of ketoisovalerate (
ilvBNCD
) and
d
-pantothenate (
panBC
) synthesis. With this strain a
d
-pantothenate accumulation of up to 1 g/liter is achieved, which is a 10
5
-fold increase in concentration compared to that of the original wild-type strain. From the series of strains analyzed it follows that an increased ketoisovalerate availability is mandatory to direct the metabolite flux into the
d
-pantothenate-specific part of the pathway and that the availability of β-alanine is essential for
d
-pantothenate formation.
Study of the Response of a Biofilm Bacterial Community to UV Radiation
Authors: Elasri Mohamed O. , Miller Robert V.
Published: December 2019

We have developed a bioluminescent whole-cell biosensor that can be incorporated into biofilm ecosystems. RM4440 is a
Pseudomonas aeruginosa
FRD1 derivative that carries a plasmid-based
recA-luxCDABE
fusion. We immobilized RM4440 in an alginate matrix to simulate a biofilm, and we studied its response to UV radiation damage. The biofilm showed a protective property by physical shielding against UV C, UV B, and UV A. Absorption of UV light by the alginate matrix translated into a higher survival rate than observed with planktonic cells at similar input fluences. UV A was shown to be effectively blocked by the biofilm matrix and to have no detectable effects on cells contained in the biofilm. However, in the presence of photosensitizers (i.e., psoralen), UV A was effective in inducing light production and cell death. RM4440 has proved to be a useful tool to study microbial communities in a noninvasive manner.
A New Sensitive Bioassay for Determination of Microbially Available Phosphorus in Water
Authors: Lehtola Markku J. , Miettinen Ilkka T. , Vartiainen Terttu , Martikainen Pertti J.
Published: December 2019

The content of assimilable organic carbon has been proposed to control the growth of microbes in drinking water. However, recent results have shown that there are regions where it is predominantly phosphorus which determines the extent of microbial growth in drinking waters. Even a very low concentration of phosphorus (below 1 μg of P liter
−1
) can promote extensive microbial growth. We present here a new sensitive method to determine microbially available phosphorus concentrations in water down to 0.08 μg of P liter
−1
. The method is a bioassay in which the analysis of phosphorus in a water sample is based on maximum growth of
Pseudomonas fluorescens
P17 when the energy supply and inorganic nutrients, with the exception of phosphorus, do not limit bacterial growth. Maximum growth (CFU) in the water sample is related to the concentration of phosphorus with the factor 373,200 ± 9,400 CFU/μg of PO
4
-P. A linear relationship was found between cell growth and phosphorus concentration between 0.05 to 10 μg of PO
4
-P liter
−1
. The content of microbially available phosphorus in Finnish drinking waters varied from 0.1 to 10.2 μg of P liter
−1
(median, 0.60 μg of P liter
−1
).
Pullulanase Type I from
Fervidobacterium pennavorans
Ven5: Cloning, Sequencing, and Expression of the Gene and Biochemical Characterization of the Recombinant Enzyme
Authors: Bertoldo Costanzo , Duffner Fiona , Jorgensen Per L. , Antranikian Garabed
Published: December 2019

The gene encoding the type I pullulanase from the extremely thermophilic anaerobic bacterium
Fervidobacterium pennavorans
Ven5 was cloned and sequenced in
Escherichia coli
. The
pulA
gene from
F. pennavorans
Ven5 had 50.1% pairwise amino acid identity with
pulA
from the anaerobic hyperthermophile
Thermotoga maritima
and contained the four regions conserved among all amylolytic enzymes. The pullulanase gene (
pulA
) encodes a protein of 849 amino acids with a 28-residue signal peptide. The
pulA
gene was subcloned without its signal sequence and overexpressed in
E. coli
under the control of the
trc
promoter. This clone,
E. coli
FD748, produced two proteins (93 and 83 kDa) with pullulanase activity. A second start site, identified 118 amino acids downstream from the ATG start site, with a Shine-Dalgarno-like sequence (GGAGG) and TTG translation initiation codon was mutated to produce only the 93-kDa protein. The recombinant purified pullulanases (rPulAs) were optimally active at pH 6 and 80°C and had a half-life of 2 h at 80°C. The rPulAs hydrolyzed α-1,6 glycosidic linkages of pullulan, starch, amylopectin, glycogen, α-β-limited dextrin. Interestingly, amylose, which contains only α-1,4 glycosidic linkages, was not hydrolyzed by rPulAs. According to these results, the enzyme is classified as a debranching enzyme, pullulanase type I. The extraordinary high substrate specificity of rPulA together with its thermal stability makes this enzyme a good candidate for biotechnological applications in the starch-processing industry.
Mutational Analysis of the Role of HPr in
Listeria monocytogenes
Authors: Christensen Douglas P. , Benson Andrew K. , Hutkins Robert W.
Published: December 2019

The regulatory role of HPr, a protein of the phosphotransferase system (PTS), was investigated in
Listeria monocytogenes
. By constructing mutations in the conserved histidine 15 and serine 46 residues of HPr, we were able to examine how HPr regulates PTS activity. The results indicated that histidine 15 was phosphorylated in a phosphoenolpyruvate (PEP)-dependent manner and was essential for PTS activity. Serine 46 was phosphorylated in an ATP-dependent manner by a membrane-associated kinase. ATP-dependent phosphorylation of serine 46 was significantly enhanced in the presence of fructose 1,6-diphosphate and resulted in a reduction of PTS activity. The presence of a charge at position 15 did not inhibit ATP-dependent phosphorylation of serine 46, a finding unique to gram-positive PEP-dependent PTSs studied to this point. Finally, HPr phosphorylated at serine 46 does not appear to possess self-phosphatase activity, suggesting a specific phosphatase protein may be essential for the recycling of HPr to its active form.