bluuebunny/crossref_metadata_2025 huggingface dataset:
Cloning and Characterization of a
Rhizobium leguminosarum
Gene Encoding a Bacteriocin with Similarities to RTX Toxins
Authors: Oresnik Ivan J. , Twelker Sunny , Hynes Michael F.
Published: December 2019

A 3-kb region containing the determinant for bacteriocin activity from
Rhizobium leguminosarum
248 was isolated and characterized by Tn
5
insertional mutagenesis and DNA sequencing. Southern hybridizations showed that this bacteriocin was encoded on the plasmid pRL1JI and that homologous loci were not found in other unrelated
R. leguminosarum
strains. Tn
5
insertional mutagenesis showed that mutations in the C-terminal half of the bacteriocin open reading frame apparently did not abolish bacteriocin activity. Analysis of the deduced amino acid sequence revealed that, similarly to RTX proteins (such as hemolysin and leukotoxin), this protein contains a characteristic nonapeptide repeated up to 18 times within the protein. In addition, a novel 19- to 25-amino-acid motif that occurred every 130 amino acids was detected. Bacteriocin bioactivity was correlated with the presence of a protein of approximately 100 kDa in the culture supernatants, and the bacteriocin bioactivity demonstrated a calcium dependence in both
R. leguminosarum
and
Sinorhizobium meliloti
. A mutant of strain 248 unable to produce this bacteriocin was found to have a statistically significant reduction in competitiveness for nodule occupancy compared to two test strains in coinoculation assays. However, this strain was unable to compete any more successfully with a third test strain, 3841, than was wild-type 248.
Indication that the Nitrogen Source Influences Both Amount and Size of Exopolysaccharides Produced by
Streptococcus thermophilus
LY03 and Modelling of the Bacterial Growth and Exopolysaccharide Production in a Complex Medium
Authors: Degeest Bart , De Vuyst Luc
Published: December 2019

Streptococcus thermophilus
LY03 is a yogurt strain producing the same exopolysaccharide material in both milk and MRS broth. Actually, two types of exopolysaccharides are produced simultaneously. The two exopolysaccharides are identical in monomer composition (galactose and glucose in a 4:1 ratio) but differ in molecular size. Gel permeation chromatography revealed a high-molecular-mass exopolysaccharide (1.8 × 10
6
) and a low-molecular-mass exopolysaccharide (4.1 × 10
5
). Both exopolysaccharides can be isolated from the fermentation broth separately. The proportion in which they are produced is strongly dependent on the carbon/nitrogen ratio of the fermentation broth. A shift from a high-molecular-mass exopolysaccharide to a low-molecular-mass exopolysaccharide was observed with increasing initial complex nitrogen concentrations. All necessary biokinetic parameters to study the kinetics of
S. thermophilus
LY03 fermentations were obtained from a mathematical model which describes both
S. thermophilus
LY03 growth and exopolysaccharide production and degradation. The model is valid with various initial complex nitrogen concentrations and can be applied to simulate exopolysaccharide production in a milk medium.
Solubilization of Phosphates and Micronutrients by the Plant-Growth-Promoting and Biocontrol Fungus
Trichoderma harzianum
Rifai 1295-22
Authors: Altomare C. , Norvell W. A. , Björkman T. , Harman G. E.
Published: December 2019

We investigated the capability of the plant-growth-promoting and biocontrol fungus
Trichoderma harzianum
Rifai 1295-22 (T-22) to solubilize in vitro some insoluble or sparingly soluble minerals via three possible mechanisms: acidification of the medium, production of chelating metabolites, and redox activity. T-22 was able to solubilize MnO
2
, metallic zinc, and rock phosphate (mostly calcium phosphate) in a liquid sucrose-yeast extract medium, as determined by inductively coupled plasma emission spectroscopy. Acidification was not the major mechanism of solubilization since the pH of cultures never fell below 5.0 and in cultures containing MnO
2
the pH rose from 6.8 to 7.4. Organic acids were not detected by high-performance thin-layer chromatography in the culture filtrates. Fe
2
O
3
, MnO
2
, Zn, and rock phosphate were also solubilized by cell-free culture filtrates. The chelating activity of T-22 culture filtrates was determined by a method based on measurement of the equilibrium concentration of the chrome azurol S complex in the presence of other chelating substances. A size exclusion chromatographic separation of the components of the culture filtrates indicated the presence of a complexed form of Fe but no chelation of Mn. In liquid culture,
T. harzianum
T-22 also produced diffusible metabolites capable of reducing Fe(III) and Cu(II), as determined by the formation of Fe(II)-Na
2
-bathophenanthrolinedisulfonic acid and Cu(I)-Na
2
-2,9-dimethyl-4,7-diphenyl-1,10-phenanthrolinedisulfonic acid complexes. This is the first report of the ability of a
Trichoderma
strain to solubilize insoluble or sparingly soluble minerals. This activity may explain, at least partially, the ability of T-22 to increase plant growth. Solubilization of metal oxides by
Trichoderma
involves both chelation and reduction. Both of these mechanisms also play a role in biocontrol of plant pathogens, and they may be part of a multiple-component action exerted by T-22 to achieve effective biocontrol under a variety of environmental conditions.
Biochemical and Structural Characterization of the Glucan and Fructan Exopolysaccharides Synthesized by the
Lactobacillus reuteri
Wild-Type Strain and by Mutant Strains
Authors: Van Geel-Schutten G. H. , Faber E. J. , Smit E. , Bonting K. , Smith M. R. , Ten Brink B. , Kamerling J. P. , Vliegenthart J. F. G. , Dijkhuizen L.
Published: December 2019

Lactobacillus reuteri
LB 121 cells growing on sucrose synthesize large amounts of a glucan (
d
-glucose) and a fructan (
d
-fructose) with molecular masses of 3,500 and 150 kDa, respectively. Methylation studies and
13
C or
1
H nuclear magnetic resonance analysis showed that the glucan has a unique structure consisting of terminal, 4-substituted, 6-substituted, and 4,6-disubstituted α-glucose in a molar ratio of 1.1:2.7:1.5:1.0. The fructan was identified as a (2→6)-β-
d
-fructofuranan or levan, the first example of levan synthesis by a
Lactobacillus
species. Strain LB 121 possesses glucansucrase and levansucrase enzymes that occur in a cell-associated and a cell-free state after growth on sucrose, raffinose, or maltose but remain cell associated during growth on glucose. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis of sucrose culture supernatants, followed by staining of gels for polysaccharide synthesizing activity with sucrose as a substrate, revealed the presence of a single glucansucrase protein of 146 kDa. Growth of strain LB 121 in chemostat cultures resulted in rapid accumulation of spontaneous exopolysaccharide-negative mutants that had lost both glucansucrase and levansucrase (e.g., strain K-24). Mutants lacking all levansucrase activity specifically emerged following a pH shiftdown (e.g., strain 35-5). Strain 35-5 still possessed glucansucrase and synthesized wild-type glucan.
Production of Cry11A and Cry11Ba Toxins in
Bacillus sphaericus
Confers Toxicity towards
Aedes aegypti
and Resistant
Culex
Populations
Authors: Servant Pascale , Rosso Marie-Laure , Hamon Sylviane , Poncet Sandrine , Delécluse Armelle , Rapoport Georges
Published: December 2019

Cry11A from
Bacillus thuringiensis
subsp.
israelensis
and Cry11Ba from
Bacillus thuringiensis
subsp.
jegathesan
were introduced, separately and in combination, into the chromosome of
Bacillus sphaericus
2297 by in vivo recombination. Two loci on the
B. sphaericus
chromosome were chosen as target sites for recombination: the binary toxin locus and the gene encoding the 36-kDa protease that may be responsible for the cleavage of the Mtx protein. Disruption of the protease gene did not increase the larvicidal activity of the recombinant strain against
Aedes aegypti
and
Culex pipiens
. Synthesis of the Cry11A and Cry11Ba toxins made the recombinant strains toxic to
A. aegypti
larvae to which the parental strain was not toxic. The strain containing Cry11Ba was more toxic than strains containing the added Cry11A or both Cry11A and Cry11Ba. The production of the two toxins together with the binary toxin did not significantly increase the toxicity of the recombinant strain to susceptible
C. pipiens
larvae. However, the production of Cry11A and/or Cry11Ba partially overcame the resistance of
C. pipiens
SPHAE and
Culex quinquefasciatus
GeoR to
B. sphaericus
strain 2297.
Formation of Hyodeoxycholic Acid from Muricholic Acid and Hyocholic Acid by an Unidentified Gram-Positive Rod Termed HDCA-1 Isolated from Rat Intestinal Microflora
Authors: Eyssen H. J. , De Pauw G. , Van Eldere J.
Published: December 2019

From the rat intestinal microflora we isolated a gram-positive rod, termed HDCA-1, that is a member of a not previously described genomic species and that is able to transform the 3α,6β,7β-trihydroxy bile acid β-muricholic acid into hyodeoxycholic acid (3α,6α-dihydroxy acid) by dehydroxylation of the 7β-hydroxy group and epimerization of the 6β-hydroxy group into a 6α-hydroxy group. Other bile acids that were also transformed into hyodeoxycholic acid were hyocholic acid (3α,6α,7α-trihydroxy acid), α-muricholic acid (3α,6β,7α-trihydroxy acid), and ω-muricholic acid (3α,6α,7β-trihydroxy acid). The strain HDCA-1 could not be grown unless a nonconjugated 7-hydroxylated bile acid and an unidentified growth factor produced by a
Ruminococcus productus
strain that was also isolated from the intestinal microflora were added to the culture medium. Germfree rats selectively associated with the strain HDCA-1 plus a bile acid-deconjugating strain and the growth factor-producing
R. productus
strain converted β-muricholic acid almost completely into hyodeoxycholic acid.
Seasonal Dynamics of Bacterioplankton Community Structure in a Eutrophic Lake as Determined by 5S rRNA Analysis
Authors: Höfle Manfred G. , Haas Heike , Dominik Katja
Published: December 2019

Community structure of bacterioplankton was studied during the major growth season for phytoplankton (April to October) in the epilimnion of a temperate eutrophic lake (Lake Plußsee, northern Germany) by using comparative 5S rRNA analysis. Estimates of the relative abundances of single taxonomic groups were made on the basis of the amounts of single 5S rRNA bands obtained after high-resolution electrophoresis of RNA directly from the bacterioplankton. Full-sequence analysis of single environmental 5S rRNAs enabled the identification of single taxonomic groups of bacteria. Comparison of partial 5S rRNA sequences allowed the detection of changes of single taxa over time. Overall, the whole bacterioplankton community showed two to eight abundant (>4% of the total 5S rRNA) taxa. A distinctive seasonal succession was observed in the taxonomic structure of this pelagic community. A rather-stable community structure, with seven to eight different taxonomic units, was observed beginning in April during the spring phytoplankton bloom. A strong reduction in this diversity occurred at the beginning of the clear-water phase (early May), when only two to four abundant taxa were observed, with one taxon dominating (up to 72% of the total 5S rRNA). The community structure during summer stagnation (June and July) was characterized by frequent changes of different dominating taxa. During late summer, a dinoflagellate bloom (Ceratium hirudinella) occurred, withComamonas acidovorans(β-subclass of the classProteobacteria) becoming the dominant bacterial species (average abundance of 43% of the total 5S rRNA). Finally, the seasonal dynamics of the community structure of bacterioplankton were compared with the abundances of other major groups of the aquatic food web, such as phyto- and zooplankton, revealing that strong grazing pressure by zooplankton can reduce microbial diversity substantially in pelagic environments.
Phosphate Stress in Cultures and Field Populations of the Dinoflagellate
Prorocentrum minimum
Detected by a Single-Cell Alkaline Phosphatase Assay
Authors: Dyhrman Sonya T. , Palenik Brian
Published: December 2019

Alkaline phosphatase activity is a common marker of phosphate stress in many phytoplankton, but it has been difficult to attribute alkaline phosphatase activity to specific organisms or groups of phytoplankton in the field with traditional biochemical procedures. A new alkaline phosphatase substrate, ELF-97 (enzyme-labeled fluorescence), shows promise in this regard. When a phosphate group is cleaved from the ELF-97 reagent, the remaining molecule precipitates near the site of enzyme activity, thus fluorescently tagging cells with alkaline phosphatase activity. We characterized ELF-97 labeling in axenic cultures of a common dinoflagellate,
Prorocentrum minimum
, in order to understand ELF-97 labeling dynamics when phosphate nutrition varies. Enzyme activity, as detected by ELF-97 labeling, appears to be induced in late-log- or early-stationary-phase cultures if cells are grown in low-phosphate media and is lost when phosphate-stressed cells are refed with phosphate. ELF-97 appears to label an inducible intracellular alkaline phosphatase in
P. minimum
based on confocal microscopy studies. This may limit the use of this reagent to organisms that lack high levels of constitutive intracellular phosphatases. After laboratory cultures were characterized, ELF-97 was used to assay field populations of
P. minimum
in Narragansett Bay during two 1-week periods, and 12 to 100% of the
P. minimum
cells were labeled. The level of cell labeling was reduced by 3 days of incubation with added inorganic phosphate. Our results indicate that ELF-97 is an excellent new tool for monitoring phytoplankton phosphate stress in the environment when the data are supported by appropriate laboratory studies.
Electrophoretic Mobilities of
Escherichia coli
O157:H7 and Wild-Type
Escherichia coli
Strains
Authors: Lytle Darren A. , Rice Eugene W. , Johnson Clifford H. , Fox Kim R.
Published: December 2019

The electrophoretic mobilities (EPMs) of a number of
Escherichia coli
O157:H7 and wild-type
E. coli
strains were measured. The effects of pH and ionic strength on the EPMs were investigated. The EPMs of
E. coli
O157:H7 strains differed from those of wild-type strains. As the suspension pH decreased, the EPMs of both types of strains increased.
Key Physiology of Anaerobic Ammonium Oxidation
Authors: Strous Marc , Kuenen J. Gijs , Jetten Mike S. M.
Published: December 2019

The physiology of anaerobic ammonium oxidizing (anammox) aggregates grown in a sequencing batch reactor was investigated quantitatively. The physiological pH and temperature ranges were 6.7 to 8.3 and 20 to 43°C, respectively. The affinity constants for the substrates ammonium and nitrite were each less than 0.1 mg of nitrogen per liter. The anammox process was completely inhibited by nitrite concentrations higher than 0.1 g of nitrogen per liter. Addition of trace amounts of either of the anammox intermediates (1.4 mg of nitrogen per liter of hydrazine or 0.7 mg of nitrogen per liter of hydroxylamine) restored activity completely.
Identification of Mimotope Peptides Which Bind to the Mycotoxin Deoxynivalenol-Specific Monoclonal Antibody
Authors: Yuan Qiaoping , Pestka James J. , Hespenheide Brandon M. , Kuhn Leslie A. , Linz John E. , Hart L. Patrick
Published: December 2019

Monoclonal antibody 6F5 (mAb 6F5), which recognizes the mycotoxin deoxynivalenol (DON) (vomitoxin), was used to select for peptides that mimic the mycotoxin by employing a library of filamentous phages that have random 7-mer peptides on their surfaces. Two phage clones selected from the random peptide phage-displayed library coded for the amino acid sequences SWGPFPF and SWGPLPF. These clones were designated DONPEP.2 and DONPEP.12, respectively. The results of a competitive enzyme-linked immunosorbent assay (ELISA) suggested that the two phage displayed peptides bound to mAb 6F5 specifically at the DON binding site. The amino acid sequence of DONPEP.2 plus a structurally flexible linker at the C terminus (SWGPFPFGGGSC) was synthesized and tested to determine its ability to bind to mAb 6F5. This synthetic peptide (designated peptide C430) and DON competed with each other for mAb 6F5 binding. When translationally fused with bacterial alkaline phosphatase, DONPEP.2 bound specifically to mAb 6F5, while the fusion protein retained alkaline phosphatase activity. The potential of using DONPEP.2 as an immunochemical reagent in a DON immunoassay was evaluated with a DON-spiked wheat extract. When peptide C430 was conjugated to bovine serum albumin, it elicited antibody specific to peptide C430 but not to DON in both mice and rabbits. In an in vitro translation system containing rabbit reticulocyte lysate, synthetic peptide C430 did not inhibit protein synthesis but did show antagonism toward DON-induced protein synthesis inhibition. These data suggest that the peptides selected in this study bind to mAb 6F5 and that peptide C430 binds to ribosomes at the same sites as DON.
Abundance and Diversity of
Archaea
in Heavy-Metal-Contaminated Soils
Authors: Sandaa Ruth-Anne , Enger Øivind , Torsvik Vigdis
Published: December 2019

The impact of heavy-metal contamination on archaean communities was studied in soils amended with sewage sludge contaminated with heavy metals to varying extents. Fluorescent in situ hybridization showed a decrease in the percentage of
Archaea
from 1.3% ± 0.3% of 4′,6-diamidino-2-phenylindole-stained cells in untreated soil to below the detection limit in soils amended with heavy metals. A comparison of the archaean communities of the different plots by denaturing gradient gel electrophoresis revealed differences in the structure of the archaean communities in soils with increasing heavy-metal contamination. Analysis of cloned 16S ribosomal DNA showed close similarities to a unique and globally distributed lineage of the kingdom
Crenarchaeota
that is phylogenetically distinct from currently characterized crenarchaeotal species.
Anaerobic Mineralization of Quaternary Carbon Atoms: Isolation of Denitrifying Bacteria on Dimethylmalonate
Authors: Kniemeyer Olaf , Probian Christina , Rosselló-Mora Ramon , Harder Jens
Published: December 2019

The microbial capacity to degrade simple organic compounds with quaternary carbon atoms was demonstrated by enrichment and isolation of five denitrifying strains on dimethylmalonate as the sole electron donor and carbon source. Quantitative growth experiments showed a complete mineralization of dimethylmalonate. According to phylogenetic analysis of the complete 16S rRNA genes, two strains isolated from activated sewage sludge were related to the genus
Paracoccus
within the α-
Proteobacteria
(98.0 and 98.2% 16S rRNA gene similarity to
Paracoccus denitrificans
T
), and three strains isolated from freshwater ditches were affiliated with the β-
Proteobacteria
(97.4 and 98.3% 16S rRNA gene similarity to
Herbaspirillum seropedicae
T
and
Acidovorax facilis
T
, respectively). Most-probable-number determinations for denitrifying populations in sewage sludge yielded 4.6 × 10
4
dimethylmalonate-utilizing cells ml
−1
, representing up to 0.4% of the total culturable nitrate-reducing population.
Genetic Diversity of
Fusarium oxysporum
Strains from Common Bean Fields in Spain
Authors: Alves-Santos Fernando M. , Benito Ernesto P. , Eslava Arturo P. , Díaz-Mínguez JoséMaría
Published: December 2019

Fusarium wilt is an endemic disease in El Barco de Avila (Castilla y León, west-central Spain), where high-quality common bean cultivars have been cultured for the last century. We used intergenic spacer (IGS) region polymorphism of ribosomal DNA, electrophoretic karyotype patterns, and vegetative compatibility and pathogenicity analyses to assess the genetic diversity within
Fusarium oxysporum
isolates recovered from common bean plants growing in fields around El Barco de Avila. Ninety-six vegetative compatibility groups (VCGs) were found among 128 isolates analyzed; most of these VCGs contained only a single isolate. The strains belonging to pathogenic VCGs and the most abundant nonpathogenic VCGs were further examined for polymorphisms in the IGS region and electrophoretic karyotype patterns. Isolates belonging to the same VCG exhibited the same IGS haplotype and very similar electrophoretic karyotype patterns. These findings are consistent with the hypothesis that VCGs represent clonal lineages that rarely, if ever, reproduce sexually. The
F. oxysporum
f. sp.
phaseoli
strains recovered had the same IGS haplotype and similar electrophoretic karyotype patterns, different from those found for
F. oxysporum
f. sp.
phaseoli
from the Americas, and were assigned to three new VCGs (VCGs 0166, 0167, and 0168). Based on our results, we do not consider the strains belonging to
F. oxysporum
f. sp.
phaseoli
to be a monophyletic group within
F. oxysporum
, as there is no correlation between pathogenicity and VCG, IGS restriction fragment length polymorphism, or electrophoretic karyotype.
Genetic Diversity within
Cryptosporidium parvum
and Related
Cryptosporidium
Species
Authors: Xiao Lihua , Morgan Una M. , Limor Josef , Escalante Ananias , Arrowood Michael , Shulaw William , Thompson R. C. A. , Fayer Ronald , Lal Altaf A.
Published: December 2019

To assess the genetic diversity in
Cryptosporidium parvum
, we have sequenced the small subunit (SSU) rRNA gene of seven
Cryptosporidium
spp., various isolates of
C. parvum
from eight hosts, and a
Cryptosporidium
isolate from a desert monitor. Phylogenetic analysis of the SSU rRNA sequences confirmed the multispecies nature of the genus
Cryptosporidium
, with at least four distinct species (
C. parvum
,
C. baileyi
,
C. muris
, and
C. serpentis
). Other species previously defined by biologic characteristics, including
C. wrairi
,
C. meleagridis
, and
C. felis
, and the desert monitor isolate, clustered together or within
C. parvum
. Extensive genetic diversities were present among
C. parvum
isolates from humans, calves, pigs, dogs, mice, ferrets, marsupials, and a monkey. In general, specific genotypes were associated with specific host species. A PCR-restriction fragment length polymorphism technique previously developed by us could differentiate most
Cryptosporidium
spp. and
C. parvum
genotypes, but sequence analysis of the PCR product was needed to differentiate
C. wrairi
and
C. meleagridis
from some of the
C. parvum
genotypes. These results indicate a need for revision in the taxonomy and assessment of the zoonotic potential of some animal
C. parvum
isolates.
Culture-Independent Identification of Microorganisms That Respond to Specified Stimuli
Authors: Borneman James
Published: December 2019

A new approach that permits culture-independent identification of microorganisms that respond to specified stimuli was developed. This approach was illustrated by examination of microorganisms that grew in response to various nutrient supplements added to soil. A thymidine nucleotide analog, bromodeoxyuridine (BrdU), and supplements were added to soil and incubated for 3 days. DNA was extracted from the soil, and the newly synthesized DNA was isolated by immunocapture of the BrdU-labeled DNA. The unique perspective this approach offers was demonstrated by comparing the microbial community structures obtained from total soil DNA and the BrdU-labeled fraction in an rRNA gene (rDNA) analysis. The traditional total DNA analysis revealed no notable differences between the treatments, whereas the BrdU-labeled DNA showed significantly different banding patterns between the nutrient supplement treatments and compared with total DNA banding patterns. PCR primers were developed to specifically amplify the intergenic region of an rDNA sequence unique to the BrdU analysis of a phosphate supplement treatment. Amplification of DNA from all treatments using these primers showed that it was unique to the phosphate treatment and that it was present in both the total DNA and BrdU-labeled DNA fractions. This result demonstrates the promise of this new strategy, because it was able to permit identification of a sequence from a phosphate-responsive organism that was not discernable in the traditional total DNA community structure analysis.
Differentiation of
Fusarium subglutinans
f. sp.
pini
by Histone Gene Sequence Data
Authors: Steenkamp E. T. , Wingfield B. D. , Coutinho T. A. , Wingfield M. J. , Marasas W. F. O.
Published: December 2019

Fusarium subglutinans
f. sp.
pini
(=
F. circinatum
) is a pathogen of pine and is one of eight mating populations (i.e., biological species) in the
Gibberella fujikuroi
species complex. This species complex includes
F. thapsinum
,
F. moniliforme
(=
F. verticillioides
),
F. nygamai
, and
F. proliferatum
, as well as
F. subglutinans
associated with sugarcane, maize, mango, and pineapple. Differentiating these forms of
F. subglutinans
usually requires pathogenicity tests, which are often time-consuming and inconclusive. Our objective was to develop a technique to differentiate isolates of
F. subglutinans
f. sp.
pini
from other isolates identified as
F. subglutinans
. We sequenced the histone H3 gene from a representative set of
Fusarium
isolates. The H3 gene sequence was conserved and contained two introns in all the isolates studied. From both the intron and the exon sequence data, we developed a PCR-restriction fragment length polymorphism technique that reliably distinguishes
F. subglutinans
f. sp.
pini
from the other biological species in the
G. fujikuroi
species complex.
Purification and Properties of an Esterase from the Yeast
Saccharomyces cerevisiae
and Identification of the Encoding Gene
Authors: Degrassi Giuliano , Uotila Lasse , Klima Raffaella , Venturi Vittorio
Published: December 2019

We purified an intracellular esterase that can function as an
S
-formylglutathione hydrolase from the yeast
Saccharomyces cerevisiae
. Its molecular mass was 40 kDa, as determined by gel filtration and sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The isoelectric point was 5.0 by isoelectric focusing. The enzyme activity was optimal at 50°C and pH 7.0. The corresponding gene,
YJLO68C
, was identified by its N-terminal amino acid sequence and is not essential for cell viability. Null mutants have reduced esterase activities and grow slowly in the presence of formaldehyde. This enzyme may be involved in the detoxification of formaldehyde, which can be metabolized to
S
-formylglutathione by
S. cerevisiae
.
Optimization of Terminal-Restriction Fragment Length Polymorphism Analysis for Complex Marine Bacterioplankton Communities and Comparison with Denaturing Gradient Gel Electrophoresis
Authors: Moeseneder Markus M. , Arrieta Jesús M. , Muyzer Gerard , Winter Christian , Herndl Gerhard J.
Published: December 2019

The potential of terminal-restriction fragment length polymorphism (T-RFLP) and the detection of operational taxonomic units (OTUs) by capillary electrophoresis (CE) to characterize marine bacterioplankton communities was compared with that of denaturing gradient gel electrophoresis (DGGE). A protocol has been developed to optimize the separation and detection of OTUs between 20 and 1,632 bp by using CE and laser-induced fluorescence detection. Additionally, we compared T-RFLP fingerprinting to DGGE optimized for detection of less abundant OTUs. Similar results were obtained with both fingerprinting techniques, although the T-RFLP approach and CE detection of OTUs was more sensitive, as indicated by the higher number of OTUs detected. We tested the T-RFLP fingerprinting technique on complex marine bacterial communities by using the 16S rRNA gene and 16S rRNA as templates for PCR. Samples from the Northern and Middle Adriatic Sea and from the South and North Aegean Sea were compared. Distinct clusters were identifiable for different sampling sites. Thus, this technique is useful for rapid evaluation of the biogeographical distribution and relationships of bacterioplankton communities.
Characterization of Muscle Sarcoplasmic and Myofibrillar Protein Hydrolysis Caused by
Lactobacillus plantarum
Authors: Fadda Silvina , Sanz Yolanda , Vignolo Graciela , Aristoy M.-Concepción , Oliver Guillermo , Toldrá Fidel
Published: December 2019

Strains of
Lactobacillus plantarum
originally isolated from sausages were screened for proteinase and aminopeptidase activities toward synthetic substrates; on the basis of that screening,
L. plantarum
CRL 681 was selected for further assays on muscle proteins. The activities of whole cells, cell extracts (CE), and a combination of both on sarcoplasmic and myofibrillar protein extracts were determined by protein, peptide, and free-amino-acid analyses. Proteinase from whole cells initiated the hydrolysis of sarcoplasmic proteins. The addition of CE intensified the proteolysis. Whole cells generated hydrophilic peptides from both sarcoplasmic and myofibrillar proteins. Other peptides of a hydrophobic nature resulted from the combination of whole cells and CE. The action of both enzymatic sources on myofibrillar proteins caused maximal increases in lysine, arginine, and leucine, while the action of those on sarcoplasmic proteins mainly released alanine. In general, pronounced hydrolysis of muscle proteins required enzyme activities from whole cells in addition to those supplied by CE.
Transport and Utilization of Hexoses and Pentoses in the Halotolerant Yeast
Debaryomyces hansenii
Authors: Nobre Alexandra , Lucas Cândida , Leão Cecília
Published: December 2019

Debaryomyces hansenii
is a yeast species that is known for its halotolerance. This organism has seldom been mentioned as a pentose consumer. In the present work, a strain of this species was investigated with respect to the utilization of pentoses and hexoses in mixtures and as single carbon sources. Growth parameters were calculated for batch aerobic cultures containing pentoses, hexoses, and mixtures of both types of sugars. Growth on pentoses was slower than growth on hexoses, but the values obtained for biomass yields were very similar with the two types of sugars. Furthermore, when mixtures of two sugars were used, a preference for one carbon source did not inhibit consumption of the other. Glucose and xylose were transported by cells grown on glucose via a specific low-affinity facilitated diffusion system. Cells derepressed by growth on xylose had two distinct high-affinity transport systems for glucose and xylose. The sensitivity of labeled glucose and xylose transport to dissipation of the transmembrane proton gradient by the protonophore carbonyl cyanide
m
-chlorophenylhydrazone allowed us to consider these transport systems as proton symports, although the cells displayed sugar-associated proton uptake exclusively in the presence of NaCl or KCl. When the
V
max
values of transport systems for glucose and xylose were compared with glucose- and xylose-specific consumption rates during growth on either sugar, it appeared that transport did not limit the growth rate.
Colonization Pattern of the Biocontrol Strain
Pseudomonas chlororaphis
MA 342 on Barley Seeds Visualized by Using Green Fluorescent Protein
Authors: Tombolini Riccardo , van der Gaag Dirk Jan , Gerhardson Berndt , Jansson Janet K.
Published: December 2019

Pseudomonas chlororaphis
MA 342 is a potent biocontrol agent that can be used against several seed-borne diseases of cereal crops, including net blotch of barley caused by the fungus
Drechslera teres
. In this study, strain MA 342 was tagged with the
gfp
gene (encoding the green fluorescent protein) in order to study the fate of cells after seed inoculation. The
gfp
-tagged strain, MA 342G2, had the same biocontrol efficacy as the wild type when it was applied at high cell concentrations to seeds but was less effective at lower cell concentrations. By comparing cell counts determined by microscopy to the number of CFU, we found that the number of culturable cells was significantly lower than the total number of bacteria on seeds which were inoculated and dried for 20 h. Confocal microscopy and epifluorescence stereomicroscopy were used to determine the pattern of MA 342G2 colonization and cell aggregation on barley seeds. Immediately after inoculation of seeds, bacteria were found mainly under the seed glume, and there was no particular aggregation pattern. However, after the seeds were sown, irregularly distributed areas of bacterial aggregation were found, which reflected epiphytic colonization of glume cells. There was a trend towards bacterial aggregation near the embryo but never within the embryo. Bacterial aggregates were regularly found in the groove of each seed formed by the base of the coleoptile and the scutellum. Based on these results, we suggest that MA 342 colocalizes with the pathogen
D. teres
, which facilitates the action of the fungistatic compound(s) produced by this strain.
The Branched-Chain Dodecylbenzene Sulfonate Degradation Pathway of
Pseudomonas aeruginosa
W51D Involves a Novel Route for Degradation of the Surfactant Lateral Alkyl Chain
Authors: Campos-García Jesús , Esteve Abraham , Vázquez-Duhalt Rafael , Ramos Juán Luis , Soberón-Chávez Gloria
Published: December 2019

Pseudomonas aeruginosa
W51D is able to grow by using branched-chain dodecylbenzene sulfonates (B-DBS) or the terpenic alcohol citronellol as a sole source of carbon. A mutant derived from this strain (W51M1) is unable to degrade citronellol but still grows on B-DBS, showing that the citronellol degradation route is not the main pathway involved in the degradation of the surfactant alkyl moiety. The structures of the main B-DBS isomers and of some intermediates were identified by gas chromatography-mass spectrometric analysis, and a possible catabolic route is proposed.
Improved Method for Purification of Bacterial DNA from Bovine Milk for Detection of
Brucella
spp. by PCR
Authors: Romero C. , Lopez-Goñi I.
Published: December 2019

Different methods of extraction of bacterial DNA from bovine milk to improve the direct detection of
Brucella
by PCR were evaluated. We found that the use of a lysis buffer with high concentrations of Tris, EDTA, and NaCl, high concentrations of sodium dodecyl sulfate and proteinase K, and high temperatures of incubation was necessary for the efficient extraction of
Brucella
DNA. The limit of detection by PCR was 5 to 50
Brucella
CFU/ml of milk.
Comparison and Recovery of
Escherichia coli
and Thermotolerant Coliforms in Water with a Chromogenic Medium Incubated at 41 and 44.5°C
Authors: Alonso Jose L. , Soriano Adela , Carbajo Oscar , Amoros Inmaculada , Garelick Hemda
Published: December 2019

This study compared the performance of a commercial chromogenic medium, CHROMagarECC (CECC), and CECC supplemented with sodium pyruvate (CECCP) with the membrane filtration lauryl sulfate-based medium (mLSA) for enumeration of
Escherichia coli
and non-
E. coli
thermotolerant coliforms (KEC). To establish that we could recover the maximum KEC and
E. coli
population, we compared two incubation temperature regimens, 41 and 44.5°C. Statistical analysis by the Fisher test of data did not demonstrate any statistically significant differences (
P
= 0.05) in the enumeration of
E. coli
for the different media (CECC and CECCP) and incubation temperatures. Variance analysis of data performed on KEC counts showed significant differences (
P
= 0.01) between KEC counts at 41 and 44.5°C on both CECC and CECCP. Analysis of variance demonstrated statistically significant differences (
P
= 0.05) in the enumeration of total thermotolerant coliforms (TTCs) on CECC and CECCP compared with mLSA. Target colonies were confirmed to be
E. coli
at a rate of 91.5% and KEC of likely fecal origin at a rate of 77.4% when using CECCP incubated at 41°C. The results of this study showed that CECCP agar incubated at 41°C is efficient for the simultaneous enumeration of
E. coli
and KEC from river and marine waters.
Production of a New
d
-Amino Acid Oxidase from the Fungus
Fusarium oxysporum
Authors: Gabler Matthias , Fischer Lutz
Published: December 2019

The fungus
Fusarium oxysporum
produced a
d
-amino acid oxidase (EC
1.4.3.3
) in a medium containing glucose as the carbon and energy source and ammonium sulfate as the nitrogen source. The specific
d
-amino acid oxidase activity was increased up to 12.5-fold with various
d
-amino acids or their corresponding derivatives as inducers. The best inducers were
d
-alanine (2.7 μkat/g of dry biomass) and
d
-3-aminobutyric acid (2.6 μkat/g of dry biomass). The addition of zinc ions was necessary to permit the induction of peroxisomal
d
-amino acid oxidase. Bioreactor cultivations were performed on a 50-liter scale, yielding a volumetric
d
-amino acid oxidase activity of 17 μkat liter
−1
with
d
-alanine as an inducer. Under oxygen limitation, the volumetric activity was increased threefold to 54 μkat liter
−1
(3,240 U liter
−1
).
Development of an In Vitro Bioassay for
Clostridium botulinum
Type B Neurotoxin in Foods That Is More Sensitive than the Mouse Bioassay
Authors: Wictome Matthew , Newton Kirsti , Jameson Karen , Hallis Bassam , Dunnigan Paul , Mackay Eric , Clarke Sally , Taylor Richard , Gaze Joy , Foster Keith , Shone Clifford
Published: December 2019

A novel, in vitro bioassay for detection of the botulinum type B neurotoxin in a range of media was developed. The assay is amplified by the enzymic activity of the neurotoxin’s light chain and includes the following three stages: first, a small, monoclonal antibody-based immunoaffinity column captures the toxin; second, a peptide substrate is cleaved by using the endopeptidase activity of the type B neurotoxin; and finally, a modified enzyme-linked immunoassay system detects the peptide cleavage products. The assay is highly specific for type B neurotoxin and is capable of detecting type B toxin at a concentration of 5 pg ml
−1
(0.5 mouse 50% lethal dose ml
−1
) in approximately 5 h. The format of the test was found to be suitable for detecting botulinum type B toxin in a range of foodstuffs with a sensitivity that exceeds the sensitivity of the mouse assay. Using highly specific monoclonal antibodies as the capture phase, we found that the endopeptidase assay was capable of differentiating between the type B neurotoxins produced by proteolytic and nonproteolytic strains of
Clostridium botulinum
type B.
Distribution and Diversity of Sulfur-Oxidizing
Thiomicrospira
spp. at a Shallow-Water Hydrothermal Vent in the Aegean Sea (Milos, Greece)
Authors: Brinkhoff Thorsten , Sievert Stefan M. , Kuever Jan , Muyzer Gerard
Published: December 2019

A shallow-water hydrothermal vent system in the Aegean Sea close to the island of Milos (Greece) was chosen to study the diversity and distribution of the chemolithoautotrophic sulfur-oxidizing bacterium
Thiomicrospira
. Cell numbers in samples from different regions around a solitary vent decreased toward the center of the vent (horizontal distribution), as well as with depth (vertical distribution), corresponding to an increase in temperature (from ca. 25 to 60°C) and a decrease in pH (from ca. pH 7 to 5).
Thiomicrospira
was one of the most abundant culturable sulfur oxidizers and was even dominant in one region. Phylogenetic analysis of
Thiomicrospira
spp. present in the highest most-probable-number (MPN) dilutions revealed that most of the obtained sequences grouped in two new closely related clusters within the
Thiomicrospira
branch. Two different new isolates, i.e., Milos-T1 and Milos-T2, were obtained from high-dilution (10
−5
) enrichments. Phylogenetic analysis indicated that isolate Milos-T1 is related to the recently described
Thiomicrospira kuenenii
and
Hydrogenovibrio marinus
, whereas isolate Milos-T2 grouped with the MPN sequences of cluster 2. The predominance of strain Milos-T2 was indicated by its identification in several environmental samples by hybridization analysis of denaturing gradient gel electrophoresis (DGGE) patterns and by sequencing of one of the corresponding bands, i.e., ML-1, from the DGGE gel. The results shown in this paper support earlier indications that
Thiomicrospira
species are important members of hydrothermal vent communities.
Enzymatic Formation of G-Group Aflatoxins and Biosynthetic Relationship between G- and B-Group Aflatoxins
Authors: Yabe Kimiko , Nakamura Miki , Hamasaki Takashi
Published: December 2019

We detected biosynthetic activity for aflatoxins G
1
and G
2
in cell extracts of
Aspergillus parasiticus
NIAH-26. We found that in the presence of NADPH, aflatoxins G
1
and G
2
were produced from
O
-methylsterigmatocystin and dihydro-
O
-methylsterigmatocystin, respectively. No G-group aflatoxins were produced from aflatoxin B
1
, aflatoxin B
2
, 5-methoxysterigmatocystin, dimethoxysterigmatocystin, or sterigmatin, confirming that B-group aflatoxins are not the precursors of G-group aflatoxins and that G- and B-group aflatoxins are independently produced from the same substrates (
O
-methylsterigmatocystin and dihydro-
O
-methylsterigmatocystin). In competition experiments in which the cell-free system was used, formation of aflatoxin G
2
from dihydro-
O
-methylsterigmatocystin was suppressed when
O
-methylsterigmatocystin was added to the reaction mixture, whereas aflatoxin G
1
was newly formed. This result indicates that the same enzymes can catalyze the formation of aflatoxins G
1
and G
2
. Inhibition of G-group aflatoxin formation by methyrapone, SKF-525A, or imidazole indicated that a cytochrome P-450 monooxygenase may be involved in the formation of G-group aflatoxins. Both the microsome fraction and a cytosol protein with a native mass of 220 kDa were necessary for the formation of G-group aflatoxins. Due to instability of the microsome fraction, G-group aflatoxin formation was less stable than B-group aflatoxin formation. The
ordA
gene product, which may catalyze the formation of B-group aflatoxins, also may be required for G-group aflatoxin biosynthesis. We concluded that at least three reactions, catalyzed by the
ordA
gene product, an unstable microsome enzyme, and a 220-kDa cytosol protein, are involved in the enzymatic formation of G-group aflatoxins from either
O
-methylsterigmatocystin or dihydro-
O
-methylsterigmatocystin.
Occurrence of
Shewanella algae
in Danish Coastal Water and Effects of Water Temperature and Culture Conditions on Its Survival
Authors: Gram Lone , Bundvad Anemone , Melchiorsen Jette , Johansen Charlotte , Fonnesbech Vogel Birte
Published: December 2019

The marine bacterium
Shewanella algae
, which was identified as the cause of human cases of bacteremia and ear infections in Denmark in the summers of 1994 and 1995, was detected in seawater only during the months (July, August, September, and October) when the water temperature was above 13°C. The bacterium is a typical mesophilic organism, and model experiments were conducted to elucidate the fate of the organism under cold and nutrient-limited conditions. The culturable count of
S. algae
decreased rapidly from 10
7
CFU/ml to 10
1
CFU/ml in approximately 1 month when cells grown at 20 to 37°C were exposed to cold (2°C) seawater. In contrast, the culturable count of cells exposed to warmer seawater (10 to 25°C) remained constant. Allowing the bacterium a transition period in seawater at 20°C before exposure to the 2°C seawater resulted in 100% survival over a period of 1 to 2 months. The cold protection offered by this transition (starvation) probably explains the ability of the organism to persist in Danish seawater despite very low (0 to 1°C) winter water temperatures. The culturable counts of samples kept at 2°C increased to 10
5
to 10
7
CFU/ml at room temperature. Most probable number analysis showed this result to be due to regrowth rather than resuscitation. It was hypothesized that
S. algae
would survive cold exposure better if in the biofilm state; however, culturable counts from
S. algae
biofilms decreased as rapidly as did counts of planktonic cells.
A Most-Probable-Number Assay for Enumeration of Infectious
Cryptosporidium parvum
Oocysts
Authors: Slifko Theresa R. , Huffman Debra E. , Rose Joan B.
Published: December 2019

Cryptosporidium
is globally established as a contaminant of drinking and recreational waters. A previously described cell culture infectivity assay capable of detecting infectious oocysts was adapted to quantify viable oocysts through sporozoite invasion and clustering of foci. Eight experiments were performed by using oocysts less than 4 months of age to inoculate host HCT-8 cell monolayers. Oocysts were diluted in a standard 5- or 10-fold multiple dilution format, levels of infection and clustering were determined, and the most probable number (MPN) of infectious oocysts in the stock suspension was calculated. The MPN was compared to the initial oocyst inoculum to determine the level of correlation. For oocysts less than 30 days of age, the correlation coefficient (
r
) was 0.9726 (0.9306 to 0.9893;
n
= 20). A two-tailed
P
value (alpha = 0.05) indicated that
P
was less than 0.0001. This strong correlation suggests that the MPN can be used to effectively enumerate infectious oocysts in a cell culture system. Age affected the degree of oocyst infectivity. Oocyst infectivity was tested by the focus detection method (FDM)-MPN assay and in BALB/c mice before and after treatment with pulsed white light (PureBrite). The FDM-MPN assay and animal infectivity assays both demonstrated more than a 4 log
10
inactivation. Municipal water systems and a host of other water testing organizations could utilize the FDM-MPN assay for routine survival and disinfection studies.
Long-Chain Polyphosphate Causes Cell Lysis and Inhibits
Bacillus cereus
Septum Formation, Which Is Dependent on Divalent Cations
Authors: Maier Simon K. , Scherer Siegfried , Loessner Martin J.
Published: December 2019

We investigated the cellular mechanisms that led to growth inhibition, morphological changes, and lysis of
Bacillus cereus
WSBC 10030 when it was challenged with a long-chain polyphosphate (polyP). At a concentration of 0.1% or higher, polyP had a bacteriocidal effect on log-phase cells, in which it induced rapid lysis and reductions in viable cell counts of up to 3 log units. The cellular debris consisted of empty cell wall cylinders and polar caps, suggesting that polyP-induced lysis was spatially specific. This activity was strictly dependent on active growth and cell division, since polyP failed to induce lysis in cells treated with chloramphenicol and in stationary-phase cells, which were, however, bacteriostatically inhibited by polyP. Similar observations were made with
B. cereus
spores; 0.1% polyP inhibited spore germination and outgrowth, and a higher concentration (1.0%) was even sporocidal. Supplemental divalent metal ions (Mg
2+
and Ca
2+
) could almost completely block and reverse the antimicrobial activity of polyP; i.e., they could immediately stop lysis and reinitiate rapid cell division and multiplication. Interestingly, a sublethal polyP concentration (0.05%) led to the formation of elongated cells (average length, 70 μm) after 4 h of incubation. While DNA replication and chromosome segregation were undisturbed, electron microscopy revealed a complete lack of septum formation within the filaments. Exposure to divalent cations resulted in instantaneous formation and growth of ring-shaped edges of invaginating septal walls. After approximately 30 min, septation was complete, and cell division resumed. We frequently observed a minicell-like phenotype and other septation defects, which were probably due to hyperdivision activity after cation supplementation. We propose that polyP may have an effect on the ubiquitous bacterial cell division protein FtsZ, whose GTPase activity is known to be strictly dependent on divalent metal ions. It is tempting to speculate that polyP, because of its metal ion-chelating nature, indirectly blocks the dynamic formation (polymerization) of the Z ring, which would explain the aseptate phenotype.