bluuebunny/crossref_metadata_2025 huggingface dataset:
Cloning and Expression of the
Lactococcus lactis purDEK
Genes, Required for Growth in Milk
Authors: Nilsson Dan , Kilstrup Mogens
Published: December 2019

An operon containing the genes
purD
and
purE
and part of the
purK
gene was cloned from the facultative anaerobic gram-positive bacterium
Lactococcus lactis
by complementation of the
purD
mutation in
Escherichia coli
SØ609. The genes encode enzymes in the de novo pathway of purine nucleotides. The expression of the genes was regulated approximately 35-fold at the transcription level by the availability of purines in the growth medium. Deletion analysis of the nucleotide region upstream of
purD
indicated that a region of 145 bp is enough to give regulated expression of the reporter
lacLM
genes, which encode β-galactosidase. Deletion of a region 79 bp upstream of the transcription start point reduced the promoter activity 33-fold when incubated in a purine-free medium and to values below the detection limit when incubated in a purine-containing medium. No secondary transcription start points were mapped in or close to this region, indicating that a putative activator site and not a promoter was deleted or partly destroyed.
Construction of a Proline-Producing Mutant of the Extremely Thermophilic Eubacterium
Thermus thermophilus
HB27
Authors: Kosuge Takehide , Hoshino Takayuki
Published: December 2019

Growth of
Thermus thermophilus
HB27 was inhibited by a proline analog, 3,4-dehydroproline (DHP). This result suggested that the γ-glutamyl kinase (the product of the
proB
gene) was inhibited by feedback inhibition in
T. thermophilus
. DHP-resistant mutants were reported previously for
Escherichia coli
(A. M. Dandekar and S. L. Uratsu, J. Bacteriol. 170:5943–5945, 1988) and
Serratia marcescens
(K. Omori, S. Suzuki, Y. Imai, and S. Komatsubara, J. Gen. Microbiol. 138:693–699, 1992), and their mutated sites in the
proB
gene were identified. Comparison of the amino acid sequence of
T. thermophilus
γ-glutamyl kinase with those of
E. coli
and
S. marcescens
mutants revealed that the DHP resistance mutations occurred in the amino acids conserved among the three organisms. For eliminating the feedback inhibition, we first constructed a DHP-resistant mutant, TH401, by site-directed mutagenesis at the
proB
gene as reported for the proline-producing mutant of
S. marcescens
. The mutant, TH401, excreted about 1 mg of
l
-proline per liter at 70°C after 12 h of incubation. It was also suggested that
T. thermophilus
had a proline degradation and transport pathway since it was able to grow in minimal medium containing
l
-proline as sole nitrogen source. In order to disrupt the proline degradation or transport genes, TH401 was mutated by UV irradiation. Seven mutants unable to utilize
l
-proline for their growth were isolated. One of the mutants, TH4017, excreted about 2 mg of
l
-proline per liter in minimal medium at 70°C after 12 h of incubation.
Naturally Occurring Deletion Mutants Are Parasitic Genotypes in a Wild-Type Nucleopolyhedrovirus Population of
Spodoptera exigua
Authors: Muñoz Delia , Castillejo Juan I. , Caballero Primitivo
Published: December 2019

A wild-type nucleopolyhedrovirus (NPV) isolate from
Spodoptera exigua
from Florida (Se-US2) is a variant of the Se
M
NPV type strain since it has a unique DNA profile but is closely related to other known geographical isolates of Se
M
NPV. It consists of several genotypic variants, of which seven were identified in a Se-US2 virus stock by a modification of the in vivo cloning method developed by Smith and Crook (Virology 166:240–244, 1988). The US2A variant was the most prevalent genotype, and it was designated the prototype Se-US2 variant, while four of the variants (US2B, US2D, US2F, and US2H) were found at low frequency. US2C and US2E were also very abundant, and their diagnostic bands were easily observed in wild-type isolate restriction endonuclease patterns. The analysis of each variant, compared to the prototype US2A, showed that US2B and US2H presented minor differences, while US2D and US2F contained slightly larger insertions or deletions. Variants US2C and US2E contained major deletions of 21.1 and 14 kb, respectively, mapping at the same genomic region (between 14.5 and 30.2 map units [m.u.] and between 12.8 and 23 m.u., respectively). This is the first report of such deletion mutants in a natural baculovirus population. Variants US2A, US2B, US2D, US2F, and US2H were isolated as pure genotypes, but we failed to clone US2C and US2E in vivo. When these two variants appeared without apparent contamination with any other variant, they lost their pathogenicity for
Spodoptera exigua
larvae. A further biological characterization showed evidence that these two naturally occurring deletion mutants act as parasitic genotypes in the virus population. Bioassay data also demonstrated that pure US2A is significantly more pathogenic against second-instar
S. exigua
larvae than the wild-type isolate. The need for precise genotypic characterization of a baculovirus prior to its development as a bioinsecticide is discussed.
Cloning and High-Level Expression of α-Galactosidase cDNA fromPenicillium purpurogenum
Authors: Shibuya Hajime , Nagasaki Hiroaki , Kaneko Satoshi , Yoshida Shigeki , Park Gwi Gun , Kusakabe Isao , Kobayashi Hideyuki
Published: December 2019

The cDNA coding forPenicillium purpurogenumα-galactosidase (αGal) was cloned and sequenced. The deduced amino acid sequence of the α-Gal cDNA showed that the mature enzyme consisted of 419 amino acid residues with a molecular mass of 46,334 Da. The derived amino acid sequence of the enzyme showed similarity to eukaryotic αGals from plants, animals, yeasts, and filamentous fungi. The highest similarity observed (57% identity) was toTrichoderma reeseiAGLI. The cDNA was expressed inSaccharomyces cerevisiaeunder the control of the yeastGAL10promoter. Almost all of the enzyme produced was secreted into the culture medium, and the expression level reached was approximately 0.2 g/liter. The recombinant enzyme purified to homogeneity was highly glycosylated, showed slightly higher specific activity, and exhibited properties almost identical to those of the native enzyme fromP. purpurogenumin terms of the N-terminal amino acid sequence, thermoactivity, pH profile, and mode of action on galacto-oligosaccharides.
Generation of Polyclonal Antibodies of Predetermined Specificity against Pediocin PA-1
Authors: Martínez José M. , Martínez María I. , Suárez Ana M. , Herranz Carmen , Casaus Pilar , Cintas Luis M. , Rodríguez Juan M. , Hernández Pablo E.
Published: December 2019

Polyclonal antibodies of predetermined specificity for pediocin PA-1 (pedA1) have been generated by immunization of rabbits with a chemically synthesized C-terminal fragment of this bacteriocin (PH2) conjugated to the carrier protein keyhole limpet hemocyanin (KLH). The sensitivity and specificity of the PH2-KLH-generated antibodies were evaluated by the development of various enzyme-linked immunosorbent assays (ELISAs)—a noncompetitive indirect ELISA (NCI-ELISA), a competitive indirect ELISA (CI-ELISA), and a competitive direct ELISA (CD-ELISA)—and by immunodotting. All immunoassays indicated the existence of pedA1-specific antibodies with high relative affinities and adequate sensitivities in the sera of immunized animals. The limits of detection of pedA1 in MRS medium (Oxoid Ltd., Basingstoke, United Kingdom) were found to be 2.5 μg/ml by immunodotting and 1 μg/ml in the NCI-ELISA. However, the CI-ELISA enhanced the limit of detection of pedA1 to 0.025 μg/ml, while the amount of free pedA1 required for 50% binding inhibition was 10 μg/ml. Moreover, the CD-ELISA increased the affinity of the PH2-KLH-generated antibodies for pedA1; the limit of detection of pedA1 was less than 0.025 μg/ml, and the 50% binding inhibition value was reduced to 0.5 μg of pedA1/ml. All immunoassays and the slot dot assay detected the presence of pedA1 in the supernatant of the producing strainPediococcus acidilactici347, with no reactivity or negligible immunoreactivity with the supernatants of other lactic acid bacteria producing or not producing different bacteriocins. The approaches taken for the generation of antibodies and the development of immunoassays could prove useful for the generation and evaluation of antibodies of predetermined specificity for other bacteriocins of interest in the food industry.
Detection of
Ralstonia solanacearum
, Which Causes Brown Rot of Potato, by Fluorescent In Situ Hybridization with 23S rRNA-Targeted Probes
Authors: Wullings B. A. , Van Beuningen A. R. , Janse J. D. , Akkermans A. D. L.
Published: December 2019

During the past few years,
Ralstonia
(
Pseudomonas
)
solanacearum
race 3, biovar 2, was repeatedly found in potatoes in Western Europe. To detect this bacterium in potato tissue samples, we developed a method based on fluorescent in situ hybridization (FISH). The nearly complete genes encoding 23S rRNA of five
R. solanacearum
strains and one
Ralstonia pickettii
strain were PCR amplified, sequenced, and analyzed by sequence alignment. This resulted in the construction of an unrooted tree and supported previous conclusions based on 16S rRNA sequence comparison in which
R. solanacearum
strains are subdivided into two clusters. Based on the alignments, two specific probes, RSOLA and RSOLB, were designed for
R. solanacearum
and the closely related
Ralstonia syzygii
and blood disease bacterium. The specificity of the probes was demonstrated by dot blot hybridization with RNA extracted from 88 bacterial strains. Probe RSOLB was successfully applied in FISH detection with pure cultures and potato tissue samples, showing a strong fluorescent signal. Unexpectedly, probe RSOLA gave a less intense signal with target cells. Potato samples are currently screened by indirect immunofluorescence (IIF). By simultaneously applying IIF and the developed specific FISH, two independent targets for identification of
R. solanacearum
are combined, resulting in a rapid (1-day), accurate identification of the undesired pathogen. The significance of the method was validated by detecting the pathogen in soil and water samples and root tissue of the weed host
Solanum dulcamara
(bittersweet) in contaminated areas.
Engineering of a Single-Chain Variable-Fragment (scFv) Antibody Specific for the Stolbur Phytoplasma (Mollicute) and Its Expression in
Escherichia coli
and Tobacco Plants
Authors: Le Gall Fabrice , Bové Joseph-Marie , Garnier Monique
Published: December 2019

From a hybridoma cell line (2A10) producing an immunoglobulin G1 directed against the major membrane protein of the stolbur phytoplasma, we have engineered scFv (single-chain variable-fragment) antibodies from the variable heavy (VH) and light (VL) domains of the immunoglobulin. The scFv gene was cloned and expressed in
Escherichia coli
. The expressed protein of 30 kDa could be recovered from the periplasmic fraction of the bacterial cells and was shown to be fully functional toward its phytoplasmal antigen, since enzyme-linked immunosorbent assay or immunofluorescence (IF) detection of the stolbur phytoplasma antigen by the scFv was identical to that of the native immunoglobulin. The scFv gene was then cloned in plasmid pBG-dAb-BIN of
Agrobacterium tumefaciens
to transform tobacco plants. The transformed plants were screened by PCR and Northern blotting for the presence and expression of the transgene, respectively, and by IF for expression of the scFv. One transgenic tobacco line, 1A6, was selected for challenge inoculation with the stolbur phytoplasma. When grafted on a stolbur phytoplasma-infected tobacco rootstock, the transgenic tobacco shoots grew free of symptoms and flowered after 2 months, while normal tobacco shoots showed severe stolbur symptoms during the same period and eventually died.
Design of a Phage-Insensitive Lactococcal Dairy Starter via Sequential Transfer of Naturally Occurring Conjugative Plasmids
Authors: O’Sullivan David , Coffey Aidan , Fitzgerald Gerald F. , Hill Colin , Ross R. Paul
Published: December 2019

The plasmid-free
Lactococcus lactis
subsp.
cremoris
MG1614 is highly phage sensitive and lacks lactose fermenting ability (Lac) and primary casein degrading ability (Prt). Food grade gene transfer systems were used to sequentially superimpose different phage defense systems on this background, resulting in a gradual increase in resistance to bacteriophage in the derivatives. pLP712, encoding Lac and Prt, was then transferred to one of these hosts, into which plasmids encoding adsorption inhibition, restriction modification, and abortive infection had already been introduced. This resulted in a phage-resistant strain which was successfully used as a single-strain starter for cheddar cheese manufacture under industrial conditions.
A Polyphasic Approach To Study the Diversity and Vertical Distribution of Sulfur-Oxidizing
Thiomicrospira
Species in Coastal Sediments of the German Wadden Sea
Authors: Brinkhoff Thorsten , Santegoeds Cecilia M. , Sahm Kerstin , Kuever Jan , Muyzer Gerard
Published: December 2019

Recently, four
Thiomicrospira
strains were isolated from a coastal mud flat of the German Wadden Sea (T. Brinkhoff and G. Muyzer, Appl. Environ. Microbiol. 63:3789–3796, 1997). Here we describe the use of a polyphasic approach to investigate the functional role of these closely related bacteria. Microsensor measurements showed that there was oxygen penetration into the sediment to a depth of about 2.0 mm. The pH decreased from 8.15 in the overlaying water to a minimum value of 7.3 at a depth of 1.2 mm. Further down in the sediment the pH increased to about 7.8 and remained constant. Most-probable-number (MPN) counts of chemolithoautotrophic sulfur-oxidizing bacteria revealed nearly constant numbers along the vertical profile; the cell concentration ranged from 0.93 × 10
5
to 9.3 × 10
5
cells per g of sediment. A specific PCR was used to detect the presence of
Thiomicrospira
cells in the MPN count preparations and to determine their 16S rRNA sequences. The concentration of
Thiomicrospira
cells did not decrease with depth. It was found that
Thiomicrospira
strains were not dominant sulfur-oxidizing bacteria in this habitat. Denaturing gradient gel electrophoresis (DGGE) of PCR-amplified 16S ribosomal DNA fragments followed by hybridization analysis with a genus-specific oligonucleotide probe revealed the diversity of
Thiomicrospira
strains in the MPN cultures. Sequence analysis of the highest MPN dilutions in which the genus
Thiomicrospira
was detected revealed that there were four clusters of several closely related sequences. Only one of the 10
Thiomicrospira
sequences retrieved was related to sequences of known isolates from the same habitat. Slot blot hybridization of rRNA isolated from different sediment layers showed that, in contrast to the concentration of
Thiomicrospira
cells, the concentration of
Thiomicrospira
-specific rRNA decreased rapidly in the region below the oxic layer of the sediment. This study revealed the enormous sequence diversity of closely related microorganisms present in one habitat, which so far has been found only by sequencing molecular isolates. In addition, it showed that most of the
Thiomicrospira
populations in the sediment studied were quiescent.
In Vivo Transposon Mutagenesis in
Haemophilus influenzae
Authors: Kraiß Anita , Schlör Stefan , Reidl Joachim
Published: December 2019

In order to devise an in vivo insertion mutagenesis scheme for
Haemophilus influenzae
, a novel set of transposons has been constructed. These are Tn
10
-based minitransposons carried on pACYC184- and pACYC177-based replicons, which are suitable for in vivo transposition in
H. influenzae
. The transposon delivery system was designed to contain an
H. influenzae
-specific uptake signal sequence which facilitates DNA transformation into
H. influenzae
. The following mini-Tn
10
elements have been made suitable for specific tasks in
H. influenzae
: (i) Tn
10d-cat
, which can be used to generate chloramphenicol-selectable insertion mutations; (ii) Tn
10d-bla
, an ampicillin-selectable translational fusion system allowing the detection of membrane or secreted proteins; and (iii) Tn
10d-lacZcat
, a chloramphenicol-selectable
lacZ
transcriptional fusion system. For the rapid identification of the transposon insertions, a PCR fragment enrichment method was developed. This report demonstrates that this in vivo mutagenesis technique is a convenient tool for the analysis of biochemical and regulatory pathways in the human pathogen
H. influenzae
.
Molecular Cloning and Functional Expression in
Lactobacillus plantarum
80 of
xylT
, Encoding the
d
-Xylose–H
+
Symporter of
Lactobacillus brevis
Authors: Chaillou Stéphane , Bor Yeou-Cherng , Batt Carl A. , Postma Pieter W. , Pouwels Peter H.
Published: December 2019

A 3-kb region, located downstream of the
Lactobacillus brevis xylA
gene (encoding
d
-xylose isomerase), was cloned in
Escherichia coli
TG1. The sequence revealed two open reading frames which could code for the
d
-xylulose kinase gene (
xylB
) and another gene (
xylT
) encoding a protein of 457 amino acids with significant similarity to the
d
-xylose–H
+
symporters of
E. coli
, XylE (57%), and
Bacillus megaterium
, XylT (58%), to the
d
-xylose–Na
+
symporter of
Tetragenococcus halophila
, XylE (57%), and to the
l
-arabinose–H
+
symporter of
E. coli
, AraE (60%). The
L. brevis xylABT
genes showed an arrangement similar to that of the
B. megaterium xylABT
operon and the
T. halophila xylABE
operon. Southern hybridization performed with the
Lactobacillus pentosus xylR
gene (encoding the
d
-xylose repressor protein) as a probe revealed the existence of a
xylR
homologue in
L. brevis
which is not located with the
xyABT
locus. The existence of a functional XylR was further suggested by the presence of
xylO
sequences upstream of
xylA
and
xylT
and by the requirement of
d
-xylose for the induction of
d
-xylose isomerase,
d
-xylulose kinase, and
d
-xylose transport activities in
L. brevis
. When
L. brevis
was cultivated in a mixture of
d
-glucose and
d
-xylose, the
d
-xylose isomerase and
d
-xylulose kinase activities were reduced fourfold and the
d
-xylose transport activity was reduced by sixfold, suggesting catabolite repression by
d
-glucose of
d
-xylose assimilation. The
xylT
gene was functionally expressed in
Lactobacillus plantarum
80, a strain which lacks proton motive force-linked
d
-xylose transport activity. The role of the XylT protein was confirmed by the accumulation of
d
-xylose in
L. plantarum
80 cells, and this accumulation was dependent on the proton motive force generated by either malolactic fermentation or by the metabolism of
d
-glucose. The apparent affinity constant of XylT for
d
-xylose was approximately 215 μM, and the maximal initial velocity of transport was 35 nmol/min per mg (dry weight). Furthermore, of a number of sugars tested, only 6-deoxy-
d
-glucose inhibited the transport of
d
-xylose by XylT competitively, with a

K
i

of 220 μM.
Two-Component Anti-
Staphylococcus aureus
Lantibiotic Activity Produced by
Staphylococcus aureus
C55
Authors: Navaratna Maduwe A. D. B. , Sahl Hans-Georg , Tagg John R.
Published: December 2019

Staphylococcus aureus
C55 was shown to produce bacteriocin activity comprising three distinct peptide components, termed staphylococcins C55α, C55β, and C55γ. The three peptides were purified to homogeneity by a simple four-step purification procedure that consisted of ammonium sulfate precipitation followed by XAD-2 and reversed-phase (C
8
and C
18
) chromatography. The yield following C
8
chromatography was about 86%, with a more-than-300-fold increase in specific activity. When combined in approximately equimolar amounts, staphylococcins C55α and C55β acted synergistically to kill
S. aureus
or
Micrococcus luteus
but not
S. epidermidis
strains. The N-terminal amino acid sequences of all three peptides were obtained and staphylococcins C55α and C55β were shown to be lanthionine-containing (lantibiotic) molecules with molecular weights of 3,339 and 2,993, respectively. The C55γ peptide did not appear to be a lantibiotic, nor did it augment the inhibitory activities of staphylococcin C55α and/or C55β. Plasmids of 2.5 and 32.0 kb are present in strain C55, and following growth of this strain at elevated temperature (42°C), a large proportion of the progeny failed to produce strong bacteriocin activity and also lost the 32.0-kb plasmid. Protoplast transformation of these bacteria with purified 32-kb plasmid DNA regenerates the ability to produce the strong bacteriocin activity.
Characterization of the Prolyl Dipeptidyl Peptidase Gene (
dppIV
) from the Koji Mold
Aspergillus oryzae
Authors: Doumas Agnès , van den Broek Peter , Affolter Michael , Monod Michel
Published: December 2019

The koji mold
Aspergillus oryzae
secretes a prolyl dipeptidyl peptidase (DPPIV) when the fungus is cultivated in a medium containing wheat gluten as the sole nitrogen and carbon source (MMWG). We cloned and sequenced the DPPIV gene from an
A. oryzae
library by using the
A. fumigatus dppIV
gene as a probe. Reverse transcriptase PCR experiments showed that the
A. oryzae dppIV
gene consists of two exons, the first of which is only 6 bp long. The gene encodes an 87.2-kDa polypeptide chain which is secreted into the medium as a 95-kDa glycoprotein. Introduction of this gene into
A. oryzae
leads to overexpression of prolyl dipeptidyl peptidase activity, while disruption of the gene abolishes all prolyl dipeptidyl peptidase activity in MMWG. The
dppIV
null mutants did not exhibit any change in phenotype other than the absence of prolyl dipeptidyl peptidase activity, suggesting that this activity is not essential. This loss of activity diminished the number of dipeptides and increased the number of larger peptides present in the MMWG culture broth. These effects were reversed by the addition of purified, recombinant DPPIV from the methylotrophic yeast expression vector
Pichia pastoris
. Our results suggest that the DPPIV enzyme may be of importance in industrial hydrolysis of what gluten-based substrates, which are rich in Pro residues.
Metabolism of Plant Polysaccharides by
Leucoagaricus gongylophorus
, the Symbiotic Fungus of the Leaf-Cutting Ant
Atta sexdens
L
Authors: Gomes De Siqueira Célia , Bacci Maurício , Pagnocca Fernando Carlos , Bueno Odair Correa , Hebling Maria JoséAparecida
Published: December 2019

Atta sexdens
L. ants feed on the fungus they cultivate on cut leaves inside their nests. The fungus,
Leucoagaricus gongylophorus
, metabolizes plant polysaccharides, such as xylan, starch, pectin, and cellulose, mediating assimilation of these compounds by the ants. This metabolic integration may be an important part of the ant-fungus symbiosis, and it involves primarily xylan and starch, both of which support rapid fungal growth. Cellulose seems to be less important for symbiont nutrition, since it is poorly degraded and assimilated by the fungus. Pectin is rapidly degraded but slowly assimilated by
L. gongylophorus
, and its degradation may occur so that the fungus can more easily access other polysaccharides in the leaves.
Sinorhizobium fredii
and
Sinorhizobium meliloti
Produce Structurally Conserved Lipopolysaccharides and Strain-Specific K Antigens
Authors: Reuhs Bradley L. , Geller Daniel P. , Kim John S. , Fox Jennifer E. , Kolli V. S. Kumar , Pueppke Steven G.
Published: December 2019

Lipopolysaccharides (LPS) and capsular polysaccharides (K antigens) may influence the interaction of rhizobia with their specific hosts; therefore, we conducted a comparative analysis of
Sinorhizobium fredii
and
Sinorhizobium meliloti
, which are genetically related, yet symbiotically distinct, nitrogen-fixing microsymbionts of legumes. We found that both species typically produce strain-specific K antigens that consist of 3-deoxy-
d
-
manno
-2-octulosonic acid (Kdo), or other 1-carboxy-2-keto-3-deoxy sugars (such as sialic acid), and hexoses. The K antigens of each strain are distinguished by glycosyl composition, anomeric configuration, acetylation, and molecular weight distribution. One consistent difference between the K antigens of
S. fredii
and those of
S. meliloti
is the presence of
N
-acetyl groups in the polysaccharides of the latter. In contrast to the K antigens, the LPS of
Sinorhizobium
spp. are major common antigens. Rough (R) LPS is the predominant form of LPS produced by cultured cells, and some strains release almost no detectable smooth (S) LPS upon extraction.
Sinorhizobium
spp. are delineated into two major RLPS core serogroups, which do not correspond to species (i.e., host range). The O antigens of the SLPS, when present, have similar degrees of polymerization and appear to be structurally conserved throughout the genus. Interestingly, one strain was found to be distinct from all others:
S. fredii
HH303 produces a unique K antigen, which contains galacturonic acid and rhamnose, and the RLPS did not fall into either of the RLPS core serogroups. The results of this study indicate that the conserved S- and RLPS of
Sinorhizobium
spp. lack the structural information necessary to influence host specificity, whereas the variable K antigens may affect strain-cultivar interactions.
Characterization ofcryGenes in a MexicanBacillus thuringiensisStrain Collection
Authors: Bravo Alejandra , Sarabia Sergio , Lopez Lorena , Ontiveros Hernesto , Abarca Carolina , Ortiz Anabel , Ortiz Miriam , Lina Laura , Villalobos Francisco J. , Peña Guadalupe , Nuñez-Valdez María-Eugenia , Soberón Mario , Quintero Rodolfo
Published: December 2019

Mexico is located in a transition zone between the Nearctic and Neotropical biogeographical regions and contains a rich and unique biodiversity. A total of 496Bacillus thuringiensisstrains were isolated from 503 soil samples collected from the five macroregions of the country. The characterization of the strain collection provided useful information on the ecological patterns of distribution ofB. thuringiensisand opportunities for the selection of strains to develop novel bioinsecticidal products. The analysis of the strains was based on multiplex PCR with novel general and specific primers that could detect thecry1,cry3,cry5,cry7,cry8,cry9,cry11,cry12,cry13,cry14,cry21, andcytgenes. The proteins belonging to the Cry1 and Cry9 groups are toxic for lepidopteran insects. The Cry3, Cry7, and Cry8 proteins are active against coleopteran insects. The Cry5, Cry12, Cry13, and Cry14 proteins are nematocidal. The Cry11, Cry21, and Cyt proteins are toxic for dipteran insects. Six pairs of general primers are used in this method. Strains for which unique PCR product profiles were obtained with the general primers were further characterized by additional PCRs with specific primers. Strains containingcry1genes were the most abundant in our collection (49.5%). Thirty-three differentcry1-type profiles were identified.B. thuringiensisstrains harboringcry3genes represented 21.5% of the strains, and 7.9% of the strains containedcry11andcytgenes.cry7,cry8, andcry9genes were found in 0.6, 2.4, and 2.6% of the strains, respectively. No strains carryingcry5,cry12,cry13,cry14, orcry21genes were found. Finally, 14% of the strains did not give any PCR product and did not react with any polyclonal antisera. Our results indicate the presence of strains that may harbor potentially novel Cry proteins as well as strains with combinations of less frequently observedcrygenes.
Changes in Bacterial Species Composition in Enrichment Cultures with Various Dilutions of Inoculum as Monitored by Denaturing Gradient Gel Electrophoresis
Authors: Jackson Colin R. , Roden Eric E. , Churchill Perry F.
Published: December 2019

Denaturing gradient gel electrophoresis revealed changes in the bacterial species obtained from enrichment cultures with different inoculum dilutions. This inoculum dilution enrichment approach may facilitate the detection and isolation of a greater number of bacterial species than traditional enrichment techniques.
Transfer of Streptomycin Biosynthesis Gene Clusters within Streptomycetes Isolated from Soil
Authors: Egan Sharon , Wiener Pamela , Kallifidas Dimitrios , Wellington Elizabeth M. H.
Published: December 2019

Streptomyces
strains isolated from soil were found to possess various numbers of genes from the streptomycin biosynthesis cluster. The strains missing genes from the cluster also lacked the ability to produce streptomycin. Two of the isolates which contain only part of the cluster are apparently recipients of a gene transfer event. The implications for the role of gene transfer in antibiotic evolution are discussed.
Enzymatic Combustion of Aromatic and Aliphatic Compounds by Manganese Peroxidase from
Nematoloma frowardii
Authors: Hofrichter Martin , Scheibner Katrin , Schneegaß Ivonne , Fritsche Wolfgang
Published: December 2019

The direct involvement of manganese peroxidase (MnP) in the mineralization of natural and xenobiotic compounds was evaluated. A broad spectrum of aromatic substances were partially mineralized by the MnP system of the white rot fungus
Nematoloma frowardii
. The cell-free MnP system partially converted several aromatic compounds, including [U-
14
C]pentachlorophenol ([U-
14
C]PCP), [U-
14
C]catechol, [U-
14
C]tyrosine, [U-
14
C]tryptophan, [4,5,9,10-
14
C]pyrene, and [ring U-
14
C]2-amino-4,6-dinitrotoluene ([
14
C]2-AmDNT), to
14
CO
2
. Mineralization was dependent on the ratio of MnP activity to concentration of reduced glutathione (thiol-mediated oxidation), a finding which was demonstrated by using [
14
C]2-AmDNT as an example. At [
14
C]2-AmDNT concentrations ranging from 2 to 120 μM, the amount of released
14
CO
2
was directly proportional to the concentration of [
14
C]2-AmDNT. The formation of highly polar products was also observed with [
14
C]2-AmDNT and [U-
14
C]PCP; these products were probably low-molecular-weight carboxylic acids. Among the aliphatic compounds tested, glyoxalate was mineralized to the greatest extent. Eighty-six percent of the
14
COOH-glyoxalate and 9% of the
14
CHO-glyoxalate were converted to
14
CO
2
, indicating that decarboxylation reactions may be the final step in MnP-catalyzed mineralization. The extracellular enzymatic combustion catalyzed by MnP could represent an important pathway for the formation of carbon dioxide from recalcitrant xenobiotic compounds and may also have general significance in the overall biodegradation of resistant natural macromolecules, such as lignins and humic substances.
Significance of Viral Lysis and Flagellate Grazing as Factors Controlling Bacterioplankton Production in a Eutrophic Lake
Authors: Weinbauer Markus G. , Höfle Manfred G.
Published: December 2019

The effects of viral lysis and heterotrophic nanoflagellate (HNF) grazing on bacterial mortality were estimated in a eutrophic lake (Lake Plußsee in northern Germany) which was separated by a steep temperature and oxygen gradient into a warm and oxic epilimnion and a cold and anoxic hypolimnion. Two transmission electron microscopy-based methods (whole-cell examination and thin sections) were used to determine the frequency of visibly infected cells, and a model was used to estimate bacterial mortality due to viral lysis. Examination of thin sections also showed that between 20.2 and 29.2% (average, 26.1%) of the bacterial cells were empty (ghosts) and thus could not contribute to viral production. The most important finding was that the mechanism for regulating bacterial production shifted with depth from grazing control in the epilimnion to control due to viral lysis in the hypolimnion. We estimated that in the epilimnion viral lysis accounted on average for 8.4 to 41.8% of the summed mortality (calculated by determining the sum of the mortalities due to lysis and grazing), compared to 51.3 to 91.0% of the summed mortality in the metalimninon and 88.5 to 94.2% of the summed mortality in the hypolimnion. Estimates of summed mortality values indicated that bacterial production was controlled completely or almost completely in the epilimnion (summed mortality, 66.6 to 128.5%) and the hypolimnion (summed mortality, 43.4 to 103.3%), whereas in the metalimnion viral lysis and HNF grazing were not sufficient to control bacterial production (summed mortality, 22.4 to 56.7%). The estimated contribution of organic matter released by viral lysis of cells into the pool of dissolved organic matter (DOM) was low; however, since cell lysis products are very likely labile compared to the bulk DOM, they might stimulate bacterial production. The high mortality of bacterioplankton due to viral lysis in anoxic water indicates that a significant portion of bacterial production in the metalimnion and hypolimnion is cycled in the bacterium-virus-DOM loop. This finding has major implications for the fate and cycling of organic nutrients in lakes.
Adaptive Changes in Membrane Lipids of Barophilic Bacteria in Response to Changes in Growth Pressure
Authors: Yano Yutaka , Nakayama Akihiko , Ishihara Kenji , Saito Hiroaki
Published: December 2019

The lipid compositions of barophilic bacterial strains which contained docosahexaenoic acid (DHA [22:6n-3]) were examined, and the adaptive changes of these compositions were analyzed in response to growth pressure. In the facultatively barophilic strain 16C1, phosphatidylethanolamine (PE) and phosphatidylglycerol (PG) were major components which had the same fatty acid chains. However, in PE, monounsaturated fatty acids such as hexadecenoic acid were major components, and DHA accounted for only 3.7% of the total fatty acids, while in PG, DHA accounted for 29.6% of the total fatty acids. In response to an increase in growth pressure in strain 16C1, the amounts of saturated fatty acids in PE were reduced, and these decreases were mainly balanced by an increase in unsaturated fatty acids, including DHA. In PG, the decrease in saturated fatty acids was mainly balanced by an increase in DHA. Similar adaptive changes in fatty acid composition were observed in response to growth pressure in obligately barophilic strain 2D2. Furthermore, these adaptive changes in response were also observed in response to low temperature in strain 16C1. These results confirm that the general shift from saturated to unsaturated fatty acids including DHA is one of the adaptive changes in response to increases in pressure and suggest that DHA may play a role in maintaining the proper fluidity of membrane lipids under high pressure.
Physiological Characterization of a Bacterial Consortium Reductively Dechlorinating 1,2,3- and 1,2,4-Trichlorobenzene
Authors: Adrian Lorenz , Manz Werner , Szewzyk Ulrich , Görisch Helmut
Published: December 2019

A bacterial mixed culture reductively dechlorinating trichlorobenzenes was established in a defined, synthetic mineral medium without any complex additions and with pyruvate as the carbon and energy source. The culture was maintained over 39 consecutive transfers of small inocula into fresh media, enriching the dechlorinating activity. In situ probing with fluorescence-labeled rRNA-targeted oligonucleotide probes revealed that two major subpopulations within the microbial consortium were phylogenetically affiliated with a sublineage within the
Desulfovibrionaceae
and the gamma subclass of
Proteobacteria
. The bacterial consortium grew by fermentation of pyruvate, forming acetate, propionate, CO
2
, formate, and hydrogen. Acetate and propionate supported neither the reduction of trichlorobenzenes nor the reduction of sulfate when sulfate was present. Hydrogen and formate were used for sulfate reduction to sulfide. Sulfate strongly inhibited the reductive dechlorination of trichlorobenzenes. However, when sulfate was depleted in the medium due to sulfate reduction, dechlorination of trichlorobenzenes started. Similar results were obtained when sulfite was present in the cultures. Molybdate at a concentration of 1 mM strongly inhibited the dechlorination of trichlorobenzenes. Cultures supplied with molybdate plus sulfate did not reduce sulfate, but dechlorination of trichlorobenzenes occurred. Supplementation of electron-depleted cultures with various electron sources demonstrated that formate was used as a direct electron donor for reductive dechlorination, whereas hydrogen was not.
Determination of Complement-Mediated Killing of Bacteria by Viability Staining and Bioluminescence
Authors: Virta Marko , Lineri Sanna , Kankaanpää Pasi , Karp Matti , Peltonen Karita , Nuutila Jari , Lilius Esa-Matti
Published: December 2019

Complement-mediated killing of bacteria was monitored by flow cytometric, luminometric, and conventional plate counting methods. A flow cytometric determination of bacterial viability was carried out by using dual staining with a LIVE/DEAD BacLight bacterial viability kit. In addition to the viable cell population, several other populations emerged in the fluorescence histogram, and there was a dramatic decrease in the total cell count in the light-scattering histogram in the course of the complement reaction. To permit luminometric measurements,
Bacillus subtilis
and
Escherichia coli
were made bioluminescent by expressing an insect luciferase gene. Addition of substrate after the complement reaction resulted in bioluminescence, the level of which was a measure of the viable cell population. All three methods gave essentially the same killing rate, suggesting that the bacteriolytic activity of serum complement can be measured rapidly and conveniently by using viability stains or bioluminescence. In principle, any bacterial strain can be used for viability staining and flow cytometric analysis. For the bioluminescence measurements genetically engineered bacteria are needed, but the advantage is that it is possible to screen automatically a large number of samples.
Measurement of the Effects of Acetic Acid and Extracellular pH on Intracellular pH of Nonfermenting, Individual
Saccharomyces cerevisiae
Cells by Fluorescence Microscopy
Authors: Guldfeldt Lars Uhre , Arneborg Nils
Published: December 2019

The effects of acetic acid and extracellular pH (pH
ex
) on the intracellular pH (pH
i
) of nonfermenting, individual
Saccharomyces cerevisiae
cells were studied by using a new experimental setup comprising a fluorescence microscope and a perfusion system.
S. cerevisiae
cells grown in brewer’s wort to the stationary phase were stained with fluorescein diacetate and transferred to a perfusion chamber. The extracellular concentration of undissociated acetic acid at various pH
ex
values was controlled by perfusion with 2 g of total acetic acid per liter at pH
ex
3.5, 4.5, 5.6, and 6.5 through the chamber by using a high-precision pump. The pH
i
of individual
S. cerevisiae
cells during perfusion was measured by fluorescence microscopy and ratio imaging. Potential artifacts, such as fading and efflux of fluorescein, could be neglected within the experimental time used. At pH
ex
6.5, the pH
i
of individual
S. cerevisiae
cells decreased as the extracellular concentration of undissociated acetic acid increased from 0 to 0.035 g/liter, whereas at pH
ex
3.5, 4.5, and 5.6, the pH
i
of individual
S. cerevisiae
cells decreased as the extracellular concentration of undissociated acetic acid increased from 0 to 0.10 g/liter. At concentrations of undissociated acetic acid of more than 0.10 g/liter, the pH
i
remained constant. The decreases in pH
i
were dependent on the pH
ex
; i.e., the decreases in pH
i
at pH
ex
5.6 and 6.5 were significantly smaller than the decreases in pH
i
at pH
ex
3.5 and 4.5.
Intracellular Signal Triggered by Cholera Toxin in
Saccharomyces boulardii
and
Saccharomyces cerevisiae
Authors: Brandão Rogelio L. , Castro Ieso M. , Bambirra Eduardo A. , Amaral Sheila C. , Fietto Luciano G. , Tropia Maria José M. , Neves Maria José , Dos Santos Raquel G. , Gomes Newton C. M. , Nicoli Jacques R.
Published: December 2019

As is the case for
Saccharomyces boulardii, Saccharomyces cerevisiae
W303 protects Fisher rats against cholera toxin (CT). The addition of glucose or dinitrophenol to cells of
S. boulardii
grown on a nonfermentable carbon source activated trehalase in a manner similar to that observed for
S. cerevisiae
. The addition of CT to the same cells also resulted in trehalase activation. Experiments performed separately on the A and B subunits of CT showed that both are necessary for activation. Similarly, the addition of CT but not of its separate subunits led to a cyclic AMP (cAMP) signal in both
S. boulardii
and
S. cerevisiae
. These data suggest that trehalase stimulation by CT probably occurred through the cAMP-mediated protein phosphorylation cascade. The requirement of CT subunit B for both the cAMP signal and trehalase activation indicates the presence of a specific receptor on the yeasts able to bind to the toxin, a situation similar to that observed for mammalian cells. This hypothesis was reinforced by experiments with
125
I-labeled CT showing specific binding of the toxin to yeast cells. The adhesion of CT to a receptor on the yeast surface through the B subunit and internalization of the A subunit (necessary for the cAMP signal and trehalase activation) could be one more mechanism explaining protection against the toxin observed for rats treated with yeasts.
Fot
1 Insertions in the
Fusarium oxysporum
f. sp.
albedinis
Genome Provide Diagnostic PCR Targets for Detection of the Date Palm Pathogen
Authors: Fernandez Diana , Ouinten Mohamed , Tantaoui Abdelaziz , Geiger Jean-Paul , Daboussi Marie-Josée , Langin Thierry
Published: December 2019

Populations of
Fusarium oxysporum
f. sp.
albedinis
, the causal agent of Bayoud disease of date palm, are derivatives of a single clonal lineage and exhibit very similar
Fot
1 hybridization patterns. In order to develop a sensitive diagnostic tool for
F. oxysporum
f. sp.
albedinis
detection, we isolated several DNA clones containing a copy of the transposable element
Fot
1 from a genomic library of the date palm pathogen. Regions flanking the insertion sites were sequenced, and these sequences were used to design PCR primers that amplify the DNA regions at several
Fot
1 insertion sites. When tested on a large sample of
Fusarium
isolates, including 286
F. oxysporum
f. sp.
albedinis
isolates, 17 other special forms, nonpathogenic
F. oxysporum
isolates from palm grove soils, and 8 other
Fusarium
species, the primer pair TL3-FOA28 allowed amplification of a 400-bp fragment found only in
F. oxysporum
f. sp.
albedinis
. Sequence analysis showed that one of the
Fot
1 copies was truncated, lacking 182 bp at its 3′ terminus. The primer pair BI03-FOA1 amplified a 204-bp fragment which overlapped the
Fot
1 truncated copy and its 3′ site of insertion in the
F. oxysporum
f. sp.
albedinis
genome and identified 95% of the isolates. The primer pairs BIO3-FOA1 and TL3-FOA28 used in PCR assays thus provide a useful diagnostic tool for
F. oxysporum
f. sp.
albedinis
isolates.
Genomic Analysis of
Clostridium botulinum
Group II by Pulsed-Field Gel Electrophoresis
Authors: Hielm Sebastian , Björkroth Johanna , Hyytiä Eija , Korkeala Hannu
Published: December 2019

Pulsed-field gel electrophoresis (PFGE) was optimized for genomic analyses of
Clostridium botulinum
(nonproteolytic) group II. DNA degradation problems caused by extracellular DNases were overcome by fixation of cells with formaldehyde prior to isolation. A rapid (4-h) in situ DNA isolation method was also assessed and gave indistinguishable results. Genomic DNA from 21 strains of various geographical and temporal origins was digested with 15 rare-cutting restriction enzymes. Of these,
Apa
I,
Mlu
I,
Nru
I,
Sma
I, and
Xho
I gave the most revealing PFGE patterns, enabling strain differentiation. Twenty strains yielded PFGE patterns containing 13 pulsotypes. From summation of
Mlu
I,
Sma
I, and
Xho
I restriction fragments, the genome size of
C. botulinum
group II was estimated to be 3.6 to 4.1 Mb (mean ± standard deviation = 3,890 ± 170 kb). The results substantiate that after problems due to DNases are overcome, PFGE analysis will be a reproducible and highly discriminating epidemiological method for studying
C. botulinum
group II at the molecular level.
Differential Gene Expression in the Laccase Gene Family from Basidiomycete I-62 (CECT 20197)
Authors: Mansur Mariana , Suárez Teresa , González Aldo E.
Published: December 2019

A family of genes encoding laccases has recently been described for the basidiomycete I-62 (CECT 20197). Transcript levels of genes
lcc1
,
lcc2
, and
lcc3
were analyzed under four different culture conditions to study their expression patterns. Two of the laccase genes were clearly inducible by veratryl alcohol: the
lcc1
gene is inducible in early stages of growth, and the
lcc2
gene is also inducible but only when the organism reaches the stationary phase. Transcript levels for the third gene,
lcc3
, were uninduced by veratryl alcohol and repressed by glucose.
Anaerobic Benzene Degradation in Petroleum-Contaminated Aquifer Sediments after Inoculation with a Benzene-Oxidizing Enrichment
Authors: Weiner Jonathan M. , Lovley Derek R.
Published: December 2019

Sediments from the sulfate-reduction zone of a petroleum-contaminated aquifer, in which benzene persisted, were inoculated with a benzene-oxidizing, sulfate-reducing enrichment from aquatic sediments. Benzene was degraded, with apparent growth of the benzene-degrading population over time. These results suggest that the lack of benzene degradation in the sulfate-reduction zones of some aquifers may result from the failure of the appropriate benzene-degrading sulfate reducers to colonize the aquifers rather than from environmental conditions that are adverse for anaerobic benzene degradation.
Construction of a Sorbitol-Based Vector for Expression of Heterologous Proteins in
Saccharomyces cerevisiae
Authors: McGonigal T. , Bodelle P. , Schopp C. , Sarthy A. V.
Published: December 2019

A new inducible yeast expression vector, pXS7, was constructed by using the promoter and terminator sequences from the
Saccharomyces cerevisiae SOR1
gene, which codes for the sorbitol dehydrogenase protein. We cloned the coding sequence of the
Saccharomyces YEF3
gene in this vector and demonstrated an increase in YEF3 protein levels when cells were grown in the presence of the sugar sorbitol.
Purification and Characterization of Anti-
Listeria
Compounds Produced by
Geotrichum candidum
Authors: Dieuleveux V. , Van Der Pyl D. , Chataud J. , Gueguen M.
Published: December 2019

Geotrichum candidum
can produce and excrete compounds that inhibit
Listeria monocytogenes
. These were purified by ultrafiltration, centrifugal partition chromatography, thin-layer chromatography, gel filtration, and high-pressure liquid chromatography, and analyzed by liquid chromatography-mass spectrometry, infrared spectrometry, nuclear magnetic resonance spectrometry, and optical rotation. Two inhibitors were identified:
d
-3-phenyllactic acid and
d
-3-indollactic acid.