bluuebunny/crossref_metadata_2025_split huggingface dataset:
Detection and Differentiation ofListeriaspp. by a Single Reaction Based on Multiplex PCR
Authors: Bubert Andreas , Hein Inge , Rauch Marcus , Lehner Angelika , Yoon ByoungSu , Goebel Werner , Wagner Martin 
Published: December 2019

ABSTRACTTheiapgene encodes the protein p60, which is common to allListeriaspecies. A previous comparison of the DNA sequences indicated conserved and species-specific gene portions. Based on these comparisons, a combination consisting of only five different primers that allows the specific detection and differentiation ofListeriaspecies with a single multiplex PCR and subsequent gel analysis was selected. One primer was derived from the conserved 3′ end and is specific for allListeriaspecies; the other four primers are specific forListeria monocytogenes,L. innocua,L. grayi, or the three grouped speciesL. ivanovii,L. seeligeri, andL. welshimeri, respectively. The PCR method, which also enables the simultaneous detection ofL. monocytogenesandL. innocua, was evaluated against conventional biotyping with 200 food hygiene-relevantListeriastrains. The results indicated the superiority of this technique. Thus, this novel type of multiplex PCR may be useful for rapidListeriaspecies confirmation and for identification ofListeriaspecies for strains isolated from different sources.
Prevention of Aerobic Spoilage of Maize Silage by a Genetically Modified Killer Yeast,Kluyveromyces lactis, Defective in the Ability To Grow on Lactic Acid
Authors: Kitamoto H. K. , Hasebe A. , Ohmomo S. , Suto E. G. , Muraki M. , Iimura Y. 
Published: December 2019

ABSTRACTIn this study, we propose a new process of adding a genetically modified killer yeast to improve the aerobic stability of silage. Previously constructedKluyveromyces lactiskiller strain PCK27, defective in growth on lactic acid due to disruption of the gene coding for phosphoenolpyruvate carboxykinase, a key enzyme for gluconeogenesis, inhibited the growth ofPichia anomalainoculated as an aerobic spoilage yeast and prevented a rise in pH in a model of silage fermentation. This suppressive effect of PCK27 was not only due to growth competition but also due to the killer protein produced. From these results, we concluded that strain PCK27 can be used as an additive to prolong the aerobic stability of maize silage. In the laboratory-scale experiment of maize silage, the addition of a killer yeast changed the yeast flora and significantly reduced aerobic spoilage.
A Phase Variant of
Azospirillum lipoferum
Lacks a Polar Flagellum and Constitutively Expresses Mechanosensing Lateral Flagella
Authors: Alexandre Gladys , Rohr René , Bally René 
Published: December 2019

ABSTRACT

Flagellation of a nonswimming variant of the mixed flagellated bacterium
Azospirillum lipoferum
4B was characterized by electron microscopy, and polyclonal antibodies were raised against polar and lateral flagellins. The variant cells lacked a polar flagellum due to a defect in flagellin synthesis and constitutively expressed lateral flagella. The variant cells were able to respond to conditions that restricted the rotation of lateral flagella by producing more lateral flagella, suggesting that the lateral flagella, as well as the polar flagellum, are mechanosensing.
Introduction of Peptidase Genes from
Lactobacillus delbrueckii
subsp.
lactis
into
Lactococcus lactis
and Controlled Expression
Authors: Wegmann U. , Klein J. R. , Drumm I. , Kuipers O. P. , Henrich B. 
Published: December 2019

ABSTRACT

Peptidases PepI, PepL, PepW, and PepG from
Lactobacillus delbrueckii
subsp.
lactis
, which have no counterparts in
Lactococcus lactis
, and peptidase PepQ were examined to determine their potential to confer new peptidolytic properties to lactococci. Controllable expression of the corresponding genes (
pep
genes) was achieved by constructing translational fusions with the promoter of the
nisA
gene (
P

nisA

). A suitable host strain, UKLc10, was constructed by chromosomal integration of the genes encoding the NisRK two-component system into the fivefold peptidase-deficient mutant IM16 of
L. lactis
. Recombinants of this strain were used to analyze growth, peptidase activities, peptide utilization, and intracellular protein cleavage products. After nisin induction of

P
nisA

::
pep
fusions, all of the peptidases were visible as distinct bands in protein gels. Despite the fact that identical transcription and translation signals were used to express the
pep
genes, the relative amounts of individual peptidases varied considerably. All of the peptidases exhibited activities in extracts of recombinant UKLc10 clones, but only PepL and PepG allowed the clones to utilize specific peptide substrates as sources of essential amino acids. In milk medium, induction of
pepG
and induction of
pepW
resulted in growth acceleration. The activities of all five peptidases during growth in milk medium were revealed by high-performance liquid chromatography analyses of intracellular amino acid and peptide pools.
Cloning, Expression, and Characterization of Cadmium and Manganese Uptake Genes from
Lactobacillus plantarum
Authors: Hao Zhiqi , Chen Shaolin , Wilson David B. 
Published: December 2019

ABSTRACT

An Mn
2+
and Cd
2+
uptake gene,
mntA
, was cloned from
Lactobacillus plantarum
ATCC 14917 into
Escherichia coli
. Its expression conferred on
E. coli
cells increased Cd
2+
sensitivity as well as energy-dependent Cd
2+
uptake activity. Both transcription and translation of
mntA
were induced by Mn
2+
starvation in
L. plantarum
, as indicated by reverse transcriptase PCR and immunoblotting. Two Cd
2+
uptake systems have been identified in
L. plantarum
: one is a high-affinity Mn
2+
and Cd
2+
uptake system that is expressed in Mn
2+
-starved cells, and the other is a nonsaturable Cd
2+
uptake system that is expressed in Cd
2+
-sufficient cells (Z. Hao, H. R. Reiske, and D. B. Wilson, Appl. Environ. Microbiol. 65:592–99, 1999). MntA was not detected in an Mn
2+
-dependent mutant of
L. plantarum
which had lost high-affinity Mn
2+
and Cd
2+
uptake activity. The results suggest that
mntA
is the gene encoding the high-affinity Mn
2+
and Cd
2+
transporter. On the basis of its predicted amino acid sequence, MntA belongs to the family of P-type cation-translocating ATPases. The topology and potential Mn
2+
- and Cd
2+
-binding sites of MntA are discussed. A second clone containing a low-affinity Cd
2+
transport system was also isolated.
Application of a DNA Hybridization–Hydrophobic-Grid Membrane Filter Method for Detection and Isolation of Verotoxigenic
Escherichia coli
Authors: Todd E. C. D. , Szabo R. A. , MacKenzie J. M. , Martin A. , Rahn K. , Gyles C. , Gao A. , Alves D. , Yee A. J. 
Published: December 2019

ABSTRACT

Verotoxigenic
Escherichia coli
(VTEC) strains were isolated from food and animal fecal samples by using PCR to screen for the presence of VTEC after broth enrichment and then filtering VTEC-positive cultures through hydrophobic-grid membrane filters (HGMFs) which were incubated on MacConkey agar. The filters were probed with a digoxigenin-labeled PCR product generated by amplification of a conserved verotoxin gene sequence. Replication of the growth on filters allowed probe-positive colonies to be picked. When ground beef samples were inoculated with VTEC strains, 100% of the strains were recovered, and the detection limit was 0.1 CFU per g. Similar results were obtained with seven types of artificially contaminated vegetables. A survey of 32 packages of vegetables and 23 samples of apple cider obtained at the retail level did not reveal the presence of VTEC. However, the intestinal fecal contents of a moose, 1 of 35 wild mammals and birds examined, contained
E. coli
O157:H7. The DNA hybridization-HGMF method was also used in a prevalence survey of 327 raw and 744 ready-to-eat products; VTEC strains were recovered from 4.9% of the raw products and 0.7% of the ready-to-eat products. No serotype O157:H7 strains were detected. This method is particularly suited for surveys in which low numbers of VTEC-positive samples are expected and isolates are required.
Inducible Amplification of Gene Copy Number and Heterologous Protein Production in the Yeast
Kluyveromyces lactis
Authors: Morlino Giovanni B. , Tizzani Lorenza , Fleer Reinhard , Frontali Laura , Bianchi Michele M. 
Published: December 2019

ABSTRACT

Heterologous protein production can be doubled by increasing the copy number of the corresponding heterologous gene. We constructed a host-vector system in the yeast
Kluyveromyces lactis
that was able to induce copy number amplification of pKD1 plasmid-based vectors upon expression of an integrated copy of the plasmid recombinase gene. We increased the production and secretion of two heterologous proteins, glucoamylase from the yeast
Arxula adeninivorans
and mammalian interleukin-1β, following gene dosage amplification when the heterologous genes were carried by pKD1-based vectors. The choice of the promoters for expression of the integrated recombinase gene and of the episomal heterologous genes are critical for the mitotic stability of the host-vector system.
Genetic Characterization of the Major Lactococcal Aromatic Aminotransferase and Its Involvement in Conversion of Amino Acids to Aroma Compounds
Authors: Rijnen Liesbeth , Bonneau Sophie , Yvon Mireille 
Published: December 2019

ABSTRACT

In lactococci, transamination is the first step of the enzymatic conversion of aromatic and branched-chain amino acids to aroma compounds. In previous work we purified and biochemically characterized the major aromatic aminotransferase (AraT) of a
Lactococcus lactis
subsp.
cremoris
strain. Here we characterized the corresponding gene and evaluated the role of AraT in the biosynthesis of amino acids and in the conversion of amino acids to aroma compounds. Amino acid sequence homologies with other aminotransferases showed that the enzyme belongs to a new subclass of the aminotransferase I subfamily γ; AraT is the best-characterized representative of this new aromatic-amino-acid-specific subclass. We demonstrated that AraT plays a major role in the conversion of aromatic amino acids to aroma compounds, since gene inactivation almost completely prevented the degradation of these amino acids. It is also highly involved in methionine and leucine conversion. AraT also has a major physiological role in the biosynthesis of phenylalanine and tyrosine, since gene inactivation weakly slowed down growth on medium without phenylalanine and highly affected growth on every medium without tyrosine. However, another biosynthesis aromatic aminotransferase is induced in the absence of phenylalanine in the culture medium.
Methanotroph Diversity in Landfill Soil: Isolation of Novel Type I and Type II Methanotrophs Whose Presence Was Suggested by Culture-Independent 16S Ribosomal DNA Analysis
Authors: Wise Mark G. , McArthur J Vaun , Shimkets Lawrence J. 
Published: December 2019

ABSTRACT

The diversity of the methanotrophic community in mildly acidic landfill cover soil was assessed by three methods: two culture-independent molecular approaches and a traditional culture-based approach. For the first of the molecular studies, two primer pairs specific for the 16S rRNA gene of validly published type I (including the former type X) and type II methanotrophs were identified and tested. These primers were used to amplify directly extracted soil DNA, and the products were used to construct type I and type II clone libraries. The second molecular approach, based on denaturing gradient gel electrophoresis (DGGE), provided profiles of the methanotrophic community members as distinguished by sequence differences in variable region 3 of the 16S ribosomal DNA. For the culturing studies, an extinction-dilution technique was employed to isolate slow-growing but numerically dominant strains. The key variables of the series of enrichment conditions were initial pH (4.8 versus 6.8), air/CH
4
/CO
2
headspace ratio (50:45:5 versus 90:9:1), and concentration of the medium (1× nitrate minimal salts [NMS] versus 0.2× NMS). Screening of the isolates showed that the nutrient-rich 1× NMS selected for type I methanotrophs, while the nutrient-poor 0.2× NMS tended to enrich for type II methanotrophs. Partial sequencing of the 16S rRNA gene from selected clones and isolates revealed some of the same novel sequence types. Phylogenetic analysis of the type I clone library suggested the presence of a new phylotype related to the
Methylobacter-Methylomicrobium
group, and this was confirmed by isolating two members of this cluster. The type II clone library also suggested the existence of a novel group of related species distinct from the validly published
Methylosinus
and
Methylocystis
genera, and two members of this cluster were also successfully cultured. Partial sequencing of the
pmoA
gene, which codes for the 27-kDa polypeptide of the particulate methane monooxygenase, reaffirmed the phylogenetic placement of the four isolates. Finally, not all of the bands separated by DGGE could be accounted for by the clones and isolates. This polyphasic assessment of community structure demonstrates that much diversity among the obligate methane oxidizers has yet to be formally described.
Cloning and Characterization of the
O
-Methyltransferase I Gene (
dmtA
) from
Aspergillus parasiticus
Associated with the Conversions of Demethylsterigmatocystin to Sterigmatocystin and Dihydrodemethylsterigmatocystin to Dihydrosterigmatocystin in Aflatoxin Biosynthesis
Authors: Motomura Marisa , Chihaya Naomi , Shinozawa Takao , Hamasaki Takashi , Yabe Kimiko 
Published: December 2019

ABSTRACT

O
-Methyltransferase I catalyzes both the conversion of demethylsterigmatocystin to sterigmatocystin and the conversion of dihydrodemethylsterigmatocystin to dihydrosterigmatocystin during aflatoxin biosynthesis. In this study, both genomic cloning and cDNA cloning of the gene encoding
O
-methyltransferase I were accomplished by using PCR strategies, such as conventional PCR based on the
N
-terminal amino acid sequence of the purified enzyme, 5′ and 3′ rapid amplification of cDNA ends PCR, and thermal asymmetric interlaced PCR (TAIL-PCR), and genes were sequenced by using
Aspergillus parasiticus
NIAH-26. A comparison of the genomic sequences with the cDNA of the
dmtA
region revealed that the coding region is interrupted by three short introns. The cDNA of the
dmtA
gene is 1,373 bp long and encodes a 386-amino-acid protein with a deduced molecular weight of 43,023, which is consistent with the molecular weight of the protein determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The C-terminal half of the deduced protein exhibits 76.3% identity with the coding region of the
Aspergillus nidulans
StcP protein, whereas the N-terminal half of
dmtA
exhibits 73.0% identity with the 5′ flanking region of the
stcP
gene, suggesting that translation of the
stcP
gene may start at a site upstream from methionine that is different from the site that has been suggested previously. Also, an examination of the 5′ and 3′ flanking regions of the
dmtA
gene in which TAIL-PCR was used demonstrated that the
dmtA
gene is located in the aflatoxin biosynthesis cluster between (and in the same orientation as) the
omtA
and
ord-2
genes. Northern blotting revealed that expression of the
dmtA
gene is influenced by both medium composition and culture temperature and that the pattern correlates with the patterns observed for other genes in the aflatoxin gene cluster. Furthermore, Southern blotting and PCR analyses of the
dmtA
gene showed that a
dmtA
homolog is present in
Aspergillus oryzae
SYS-2.
Relative Importance of Trophic Group Concentrations during Anaerobic Degradation of Volatile Fatty Acids
Authors: Voolapalli Ravi K. , Stuckey David C. 
Published: December 2019

ABSTRACT

Although obligate syntrophic reactions cannot proceed without hydrogenotrophs, it has been unclear from the literature whether potential improvements are achievable with higher concentrations of hydrogenotrophs. In this study, the relative importance of formate-/H
2
-utilizing and acetate-utilizing trophic groups in the anaerobic degradation of butyrate and propionate was assessed by adding various proportions of these enriched cultures to a mixed anaerobic seed inoculum. The improvement resulting from the additional acetate-utilizing cultures was much greater than with formate/H
2
utilizers. Furthermore, formate/H
2
utilizers did not improve propionate utilization significantly, suggesting the importance of optimum utilization of hydrogenotrophic capacity. During most of the volatile fatty acid (VFA) degradation period, the system responded with characteristic hydrogen levels to maintain the Gibbs free energy of oxidation approximately constant for both butyrate (−6 kJ) and propionate (−14 kJ). These free-energy values were independent of methanogenic activity, as well as the volume of the seed inoculum and the VFA concentrations present. By comparing the experimental results with kinetic and mass transfer models, it was postulated that the diffusional transfer of reducing equivalents was the major limiting factor for efficient VFA degradation. Therefore, for optimum utilization of the hydrogenotrophs, low acetate concentrations are vital to enable the system to respond with higher formate/H
2
levels, thus leading to improved transfer of reducing equivalents. Due to the small number of propionate utilizers (and hence their limited surface area) and low bulk liquid concentrations, the additional formate/H
2
utilizers were of minimal use for improving the degradation rate further. The butyrate degradation rates strongly correlated with the cumulative activity of hydrogenotrophs and acetotrophs over the experimental range studied, indicating the need to model obligate syntrophic reactions as a dependent function of methanogenic activity.
Leaching of Zinc Sulfide by
Thiobacillus ferrooxidans
: Bacterial Oxidation of the Sulfur Product Layer Increases the Rate of Zinc Sulfide Dissolution at High Concentrations of Ferrous Ions
Authors: Fowler T. A. , Crundwell F. K. 
Published: December 2019

ABSTRACT

This paper reports the results of leaching experiments conducted with and without
Thiobacillus ferrooxidans
at the same conditions in solution. The extent of leaching of ZnS with bacteria is significantly higher than that without bacteria at high concentrations of ferrous ions. A porous layer of elemental sulfur is present on the surfaces of the chemically leached particles, while no sulfur is present on the surfaces of the bacterially leached particles. The analysis of the data using the shrinking-core model shows that the chemical leaching of ZnS is limited by the diffusion of ferrous ions through the sulfur product layer at high concentrations of ferrous ions. The analysis of the data shows that diffusion through the product layer does not limit the rate of dissolution when bacteria are present. This suggests that the action of
T. ferrooxidans
in oxidizing the sulfur formed on the particle surface is to remove the barrier to diffusion by ferrous ions.
Initial Reactions in Anaerobic Alkane Degradation by a Sulfate Reducer, Strain AK-01
Authors: So Chi Ming , Young L. Y. 
Published: December 2019

ABSTRACT

An alkane-degrading, sulfate-reducing bacterial strain, AK-01, isolated from a petroleum-contaminated sediment was studied to elucidate its mechanism of alkane metabolism. Total cellular fatty acids of AK-01 were predominantly C even when it was grown on C-even alkanes and were predominantly C odd when grown on C-odd alkanes, suggesting that the bacterium anaerobically oxidizes alkanes to fatty acids. Among these fatty acids, some 2-, 4-, and 6-methylated fatty acids were specifically found only when AK-01 was grown on alkanes, and their chain lengths always correlated with those of the alkanes. When [1,2-
13
C
2
]hexadecane or perdeuterated pentadecane was used as the growth substrate,
13
C-labeled 2-Me-16:0, 4-Me-18:0, and 6-Me-20:0 fatty acids or deuterated 2-Me-15:0, 4-Me-17:0, and 6-Me-19:0 fatty acids were recovered, respectively, confirming that these monomethylated fatty acids were alkane derived. Examination of the
13
C-labeled 2-, 4-, and 6-methylated fatty acids by mass spectrometry showed that each of them contained two
13
C atoms, located at the methyl group and the adjacent carbon, thus indicating that the methyl group was the original terminal carbon of the [1,2-
13
C
2
]hexadecane. For perdeuterated pentadecane, the presence of three deuterium atoms, on the methyl group and its adjacent carbon, in each of the deuterated 2-, 4-, and 6-methylated fatty acids further supported the hypothesis that the methyl group was the terminal carbon of the alkane. Thus, exogenous carbon appears to be initially added to an alkane subterminally at the C-2 position such that the original terminal carbon of the alkane becomes a methyl group on the subsequently formed fatty acid. The carbon addition reaction, however, does not appear to be a direct carboxylation of inorganic bicarbonate. A pathway for anaerobic metabolism of alkanes by strain AK-01 is proposed.
The
bglA
Gene of
Aspergillus kawachii
Encodes Both Extracellular and Cell Wall-Bound β-Glucosidases
Authors: Iwashita Kazuhiro , Nagahara Tatsuya , Kimura Hitoshi , Takano Makoto , Shimoi Hitoshi , Ito Kiyoshi 
Published: December 2019

ABSTRACT

We cloned the genomic DNA and cDNA of
bglA
, which encodes β-glucosidase in
Aspergillus kawachii
, based on a partial amino acid sequence of purified cell wall-bound β-glucosidase CB-1. The nucleotide sequence of the cloned
bglA
gene revealed a 2,933-bp open reading frame with six introns that encodes an 860-amino-acid protein. Based on the deduced amino acid sequence, we concluded that the
bglA
gene encodes cell wall-bound β-glucosidase CB-1. The amino acid sequence exhibited high levels of homology with the amino acid sequences of fungal β-glucosidases classified in subfamily B. We expressed the
bglA
cDNA in
Saccharomyces cerevisiae
and detected the recombinant β-glucosidase in the periplasm fraction of the recombinant yeast.
A. kawachii
can produce two extracellular β-glucosidases (EX-1 and EX-2) in addition to the cell wall-bound β-glucosidase.
A. kawachii
in which the
bglA
gene was disrupted produced none of the three β-glucosidases, as determined by enzyme assays and a Western blot analysis. Thus, we concluded that the
bglA
gene encodes both extracellular and cell wall-bound β-glucosidases in
A. kawachii
.
Engineering of a Stable Whole-Cell Biocatalyst Capable of (
S
)-Styrene Oxide Formation for Continuous Two-Liquid-Phase Applications
Authors: Panke Sven , de Lorenzo Víctor , Kaiser Arnë , Witholt Bernard , Wubbolts Marcel G. 
Published: December 2019

ABSTRACT

Recombinant strains of
Pseudomonas putida
KT2440 carrying genetic expression cassettes with xylene oxygenase- and styrene monooxygenase-encoding genes on their chromosomes could be induced in shaking-flask experiments to specific activities that rivaled those of multicopy-plasmid-based
Escherichia coli
recombinants. Such strains maintained the introduced styrene oxidation activity in continuous two-liquid-phase cultures for at least 100 generations, although at a lower level than in the shaking-flask experiments. The data suggest that placement of target genes on the chromosome might be a suitable route for the construction of segregationally stable and highly active whole-cell biocatalysts.