bluuebunny/crossref_metadata_2025 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

Theiapgene 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

In 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

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

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

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

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

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

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

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

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

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

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

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

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

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.
Biophysical Characterization of Fungal Phytases (
myo
-Inositol Hexakisphosphate Phosphohydrolases): Molecular Size, Glycosylation Pattern, and Engineering of Proteolytic Resistance
Authors: Wyss Markus , Pasamontes Luis , Friedlein Arno , Rémy Roland , Tessier Michel , Kronenberger Alexandra , Middendorf Anke , Lehmann Martin , Schnoebelen Line , Röthlisberger Urs , Kusznir Eric , Wahl Guido , Müller Francis , Lahm Hans-Werner , Vogel Kurt , van Loon Adolphus P. G. M.
Published: December 2019

Phytases (
myo
-inositol hexakisphosphate phosphohydrolases) are found naturally in plants and microorganisms, particularly fungi. Interest in these enzymes has been stimulated by the fact that phytase supplements increase the availability of phosphorus in pig and poultry feed and thereby reduce environmental pollution due to excess phosphate excretion in areas where there is intensive livestock production. The wild-type phytases from six different fungi,
Aspergillus niger
,
Aspergillus terreus
,
Aspergillus fumigatus
,
Emericella nidulans
,
Myceliophthora thermophila
, and
Talaromyces thermophilus
, were overexpressed in either filamentous fungi or yeasts and purified, and their biophysical properties were compared with those of a phytase from
Escherichia coli
. All of the phytases examined are monomeric proteins. While
E. coli
phytase is a nonglycosylated enzyme, the glycosylation patterns of the fungal phytases proved to be highly variable, differing for individual phytases, for a given phytase produced in different expression systems, and for individual batches of a given phytase produced in a particular expression system. Whereas the extents of glycosylation were moderate when the fungal phytases were expressed in filamentous fungi, they were excessive when the phytases were expressed in yeasts. However, the different extents of glycosylation had no effect on the specific activity, the thermostability, or the refolding properties of individual phytases. When expressed in
A. niger
, several fungal phytases were susceptible to limited proteolysis by proteases present in the culture supernatant. N-terminal sequencing of the fragments revealed that cleavage invariably occurred at exposed loops on the surface of the molecule. Site-directed mutagenesis of
A. fumigatus
and
E. nidulans
phytases at the cleavage sites yielded mutants that were considerably more resistant to proteolytic attack. Therefore, engineering of exposed surface loops may be a strategy for improving phytase stability during feed processing and in the digestive tract.
Molecular Analysis of Ammonia-Oxidizing Bacteria of the β Subdivision of the Class
Proteobacteria
in Compost and Composted Materials
Authors: Kowalchuk George A. , Naoumenko Zinaida S. , Derikx Piet J. L. , Felske Andreas , Stephen John R. , Arkhipchenko Irina A.
Published: December 2019

Although the practice of composting animal wastes for use as biofertilizers has increased in recent years, little is known about the microorganisms responsible for the nitrogen transformations which occur in compost and during the composting process. Ammonia is the principle available nitrogenous compound in composting material, and the conversion of this compound to nitrite in the environment by chemolithotrophic ammonia-oxidizing bacteria is an essential step in nitrogen cycling. Therefore, the distribution of ammonia-oxidizing members of the β subdivision of the class
Proteobacteria
in a variety of composting materials was assessed by amplifying 16S ribosomal DNA (rDNA) and 16S rRNA by PCR and reverse transcriptase PCR (RT-PCR), respectively. The PCR and RT-PCR products were separated by denaturing gradient gel electrophoresis (DGGE) and were identified by hybridization with a hierarchical set of oligonucleotide probes designed to detect ammonia oxidizer-like sequence clusters in the genera
Nitrosospira
and
Nitrosomonas
. Ammonia oxidizer-like 16S rDNA was detected in almost all of the materials tested, including industrial and experimental composts, manure, and commercial biofertilizers. A comparison of the DGGE and hybridization results after specific PCR and RT-PCR suggested that not all of the different ammonia oxidizer groups detected in compost are equally active.
amoA
, the gene encoding the active-site-containing subunit of ammonia monooxygenase, was also targeted by PCR, and template concentrations were estimated by competitive PCR. Detection of ammonia-oxidizing bacteria in the composts tested suggested that such materials may not be biologically inert with respect to nitrification and that the fate of nitrogen during composting and compost storage may be affected by the presence of these organisms.
Attachment of the Yeast
Rhodosporidium toruloides
Is Mediated by Adhesives Localized at Sites of Bud Cell Development
Authors: Buck James W. , Andrews John H.
Published: December 2019

The basidiomycetous yeast
Rhodosporidium toruloides
(anamorph,
Rhodotorula glutinis
) is a common phylloplane epiphyte with biocontrol potential. To understand how
R. toruloides
adheres to plant surfaces, we obtained nonadherent fungal mutants after chemical mutagenesis with methane-sulfonic acid ethyl ester. Sixteen attachment-minus (Att
−
) mutants were identified by three methods: (i) screening capsule-minus colonies for loss of adhesive ability; (ii) enrichment for mutants unable to attach to polystyrene; and (iii) selection for reduced fluorescence of fluorescein isothiocyanate-concanavalin A (Con A)-stained cells by fluorescence-activated cell sorting. None of the 16 mutants attached to polystyrene or barley leaves. The lectin Con A eliminated adhesion in all of the wild-type isolates tested. Hapten competition assays indicated that Con A bound to mannose residues on the cell surface. Adhesion of wild-type
R. toruloides
was transient; nonadhesive cells subsequently became adhesive, with bud development. All Att
−
mutants and nonattaching wild-type cells lacked polar regions that stained intensely with fluorescein isothiocyanate-Con A and India ink. Lectin, enzyme, and chemical treatments showed that the polar regions consisted of alkali-soluble materials, including mannose residues. Tunicamycin treatment reduced wild-type adhesion, indicating that the mannose residues could be associated with glycoproteins. We concluded that compounds, including mannose residues, that are localized at sites of bud development mediate adhesion of
R. toruloides
to both polystyrene and barley leaf surfaces.
Use of the Integration Elements Encoded by the Temperate Lactococcal Bacteriophage TP901-1 To Obtain Chromosomal Single-Copy Transcriptional Fusions in
Lactococcus lactis
Authors: Brøndsted Lone , Hammer Karin
Published: December 2019

Previously we showed that only one phage-expressed protein (Orf1), a 425-bp region upstream of the
orf1
gene (presumably encoding a promoter), and the
attP
region are necessary and also sufficient for integration of the bacteriophage TP901-1 genome into the chromosome of
Lactococcus lactis
subsp.
cremoris
(B. Christiansen, L. Brøndsted, F. K. Vogensen, and K. Hammer, J. Bacteriol. 178:5164–5173, 1996). In this work, a further analysis of the phage-encoded elements involved in integration was performed. Here we demonstrate that even when the
orf1
gene is separated from the
attP
region, the Orf1 protein is able to promote site-specific integration of an
attP
-carrying plasmid into the
attB
site on the
L. lactis
subsp.
cremoris
chromosome. Furthermore, the first detailed deletion analysis of an
attP
region of a phage infecting lactic acid bacteria was carried out. We show that a fragment containing 56 bp of the
attP
region, including the core, is sufficient for the site-specific integration of a nonreplicating plasmid into the chromosome of
L. lactis
subsp.
cremoris
when the
orf1
gene is donated in
trans
. The functional 56-bp
attP
region of TP901-1 is substantially smaller than minimal
attP
regions identified for other phages. Based on the deletion analysis, several repeats located within the
attP
region seem to be necessary for site-specific integration of the temperate bacteriophage TP901-1. By use of the integrative elements (
attP
and
orf1
) expressed by the temperate lactococcal bacteriophage TP901-1, a system for obtaining stable chromosomal single-copy transcriptional fusions in
L. lactis
was constructed. Two promoter-reporter integration vectors containing the reporter gene
gusA
or
lacLM
, encoding β-glucuronidase or β-galactosidase, respectively, were constructed. Immediately upstream of both genes are found translational stop codons in all three reading frames as well as multiple restriction enzyme sites suitable for cloning of the promoter of interest. By transformation of
L. lactis
subsp.
cremoris
MG1363 containing the integrase gene on a replicating plasmid, the promoter-reporter integration vectors integrated with a high frequency site specifically into the chromosomal attachment site
attB
used by bacteriophage TP901-1.
Evaluation of Toxic Effects of Aeration and Trichloroethylene Oxidation on Methanotrophic Bacteria Grown with Different Nitrogen Sources
Authors: Chu Kung-Hui , Alvarez-Cohen Lisa
Published: December 2019

In this study we evaluated specific and nonspecific toxic effects of aeration and trichloroethylene (TCE) oxidation on methanotrophic bacteria grown with different nitrogen sources (nitrate, ammonia, and molecular nitrogen). The specific toxic effects, exerted directly on soluble methane monooxygenase (sMMO), were evaluated by comparing changes in methane uptake rates and naphthalene oxidation rates following aeration and/or TCE oxidation. Nonspecific toxic effects, defined as general cellular damage, were examined by using a combination of epifluorescent cellular stains to measure viable cell numbers based on respiratory activity and measuring formate oxidation activities following aeration and TCE transformation. Our results suggest that aeration damages predominantly sMMO rather than other general cellular components, whereas TCE oxidation exerts a broad range of toxic effects that damage both specific and nonspecific cellular functions. TCE oxidation caused sMMO-catalyzed activity and respiratory activity to decrease linearly with the amount of substrate degraded. Severe TCE oxidation toxicity resulted in total cessation of the methane, naphthalene, and formate oxidation activities and a 95% decrease in the respiratory activity of methanotrophs. The failure of cells to recover even after 7 days of incubation with methane suggests that cellular recovery following severe TCE product toxicity is not always possible. Our evidence suggests that generation of greater amounts of sMMO per cell due to nitrogen fixation may be responsible for enhanced TCE oxidation activities of nitrogen-fixing methanotrophs rather than enzymatic protection mechanisms associated with the nitrogenase enzymes.
Phylogenetic Differences between Particle-Associated and Planktonic Ammonia-Oxidizing Bacteria of the β Subdivision of the Class
Proteobacteria
in the Northwestern Mediterranean Sea
Authors: Phillips Carol J. , Smith Zena , Embley T. Martin , Prosser James I.
Published: December 2019

The aim of this study was to determine if there were differences between the types of ammonia-oxidizing bacteria of the β subdivision of the class
Proteobacteria
associated with particulate material and planktonic samples obtained from the northwestern Mediterranean Sea. A nested PCR procedure performed with ammonia oxidizer-selective primers was used to amplify 16S rRNA genes from extracted DNA. The results of partial and full-length sequence analyses of 16S rRNA genes suggested that different groups of ammonia-oxidizing bacteria were associated with the two sample types. The particle-associated sequences were predominantly related to
Nitrosomonas eutropha
, while the sequences obtained from the planktonic samples were related to a novel marine
Nitrosospira
group (cluster 1) for which there is no cultured representative yet. A number of oligonucleotide probes specific for different groups of ammonia oxidizers were used to estimate the relative abundance of sequence types in samples of clone libraries. The planktonic libraries contained lower proportions of ammonia oxidizer clones (0 to 26%) than the particulate material libraries (9 to 83%). Samples of the planktonic and particle-associated libraries showed that there were depth-related differences in the ammonia oxidizer populations, with the highest number of positive clones in the particle-associated sample occurring at a depth of 700 m. The greatest difference between planktonic and particle-associated populations occurred at a depth of 400 m, where only 4% of the clones in the planktonic library were identified as
Nitrosomonas
clones, while 96% of these clones were identified as clones that were related to the marine
Nitrosospira
species. Conversely, all ammonia oxidizer-positive clones obtained from the particle-associated library were members of the
Nitrosomonas
group. This is the first indication that
Nitrosomonas
species and
Nitrosospira
species may occupy at least two distinct environmental niches in marine environments. The occurrence of these groups in different niches may result from differences in physiological properties and, coupled with the different environmental conditions associated with these niches, may lead to significant differences in the nature and rates of nitrogen cycling in these environments.
Green Fluorescent Protein as a Reporter To Monitor Gene Expression and Food Colonization by
Aspergillus flavus
Authors: Du Wanglei , Huang Zhengyu , Flaherty Joseph E. , Wells Kevin , Payne Gary A.
Published: December 2019

Transformants of
Aspergillus flavus
containing the
Aequorea victoria gfp
gene fused to a viral promoter or the promoter region and 483 bp of the coding region of
A. flavus aflR
expressed green fluorescence detectable without a microscope or filters. Expression of green fluorescent protein fluorescence was correlated with resistance to aflatoxin accumulation in five corn genotypes inoculated with these transformants.
Phylogenetic Diversity of Symbiotic Methanogens Living in the Hindgut of the Lower Termite
Reticulitermes speratus
Analyzed by PCR and In Situ Hybridization
Authors: Shinzato Naoya , Matsumoto Tadao , Yamaoka Ikuo , Oshima Tairo , Yamagishi Akihiko
Published: December 2019

A phylogenetic analysis of the sequences of 60 clones of archaeal small-subunit rRNA genes amplified from the termite
Reticulitermes speratus
revealed that most of them (56 clones) clustered in the genus
Methanobrevibacter
. Three clones were classified in the order Thermoplasmales. The
Methanobrevibacter
-related symbionts were detected by in situ hybridization analysis.
Effect of Aromatic Compounds on Cellular Fatty Acid Composition of
Rhodococcus opacus
Authors: Tsitko Irina V. , Zaitsev Gennadi M. , Lobanok Anatoli G. , Salkinoja-Salonen Mirja S.
Published: December 2019

In cells of
Rhodococcus opacus
GM-14, GM-29, and 1CP, the contents of branched (10-methyl) fatty acids increased from 3% to 15 to 34% of the total fatty acids when the cells were grown on benzene, phenol, 4-chlorophenol, chlorobenzene, or toluene as the sole source of carbon and energy, in comparison with cells grown on fructose. In addition, the content of
trans
-hexadecenoic acid increased from 5% to 8 to 18% with phenol or chlorophenol as the carbon source. The 10-methyl branched fatty acid content of
R. opacus
GM-14 cells increased in a dose-related manner following exposure to phenol or toluene when toluene was not utilized as the growth substrate. The results suggest that 10-methyl branched fatty acids may participate in the adaptation of
R. opacus
to lipophilic aromatic compounds.
Involvement of Two Plasmids in the Degradation of Carbaryl by
Arthrobacter
sp. Strain RC100
Authors: Hayatsu Masahito , Hirano Motoko , Nagata Tadahiro
Published: December 2019

A bacterium capable of utilizing carbaryl (1-naphthyl
N
-methylcarbamate) as the sole carbon source was isolated from carbaryl-treated soil. This bacterium was characterized taxonomically as
Arthrobacter
and was designated strain RC100. RC100 hydrolyzes the
N
-methylcarbamate linkage to 1-naphthol, which was further metabolized via salicylate and gentisate. Strain RC100 harbored three plasmids (designated pRC1, pRC2, and pRC3). Mutants unable to degrade carbaryl arose at a high frequency after treating the culture with mitomycin C. All carbaryl-hydrolysis-deficient mutants (Cah
−
) lacked pRC1, and all 1-naphthol-utilization-deficient mutants (Nat
−
) lacked pRC2. The plasmid-free strain RC107 grew on gentisate as a carbon source. These two plasmids could be transferred to Cah
−
mutants or Nat
−
mutants by conjugation, resulting in the restoration of the Cah and Nah phenotypes.
Purification and Characterization of a Novel Peroxidase from
Geotrichum candidum
Dec 1 Involved in Decolorization of Dyes
Authors: Kim Seong Jun , Shoda Makoto
Published: December 2019

A peroxidase (DyP) involved in the decolorization of dyes and produced by the fungus strain
Geotrichum candidum
Dec 1 was purified. DyP, a glycoprotein, is glycosylated with
N
-acetylglucosamine and mannose (17%) and has a molecular mass of 60 kDa and an isoelectric point (pI) of 3.8. The absorption spectrum of DyP exhibited a Soret band at 406 nm corresponding to a hemoprotein, and its Na
2
S
2
O
4
-reduced form revealed a peak at 556 nm that indicates the presence of a protoheme as its prosthetic group. Nine of the 21 types of dyes that were decolorized by Dec 1 cells were decolorized by DyP; in particular, anthraquinone dyes were highly decolorized. DyP also oxidized 2,6-dimethoxyphenol and guaiacol but not veratryl alcohol. The optimal temperature for DyP activity was 30°C, and DyP activity was stable even after incubation at 50°C for 11 h.
Cell Surface-Associated Lipoteichoic Acid Acts as an Adhesion Factor for Attachment of
Lactobacillus johnsonii
La1 to Human Enterocyte-Like Caco-2 Cells
Authors: Granato Dominique , Perotti Fabienne , Masserey Isabelle , Rouvet Martine , Golliard Mireille , Servin Alain , Brassart Dominique
Published: December 2019

The influence of pH on the adhesion of two
Lactobacillus
strains to Caco-2 human intestinal cells was investigated. One strain,
Lactobacillus johnsonii
La1, was adherent at any pH between 4 and 7. The other one,
L. acidophilus
La10, did not attach to this cell line under the same experimental conditions. On the basis of these results, we used the monoclonal antibody technique as a tool to determine differences on the surface of these bacteria and to identify a factor for adhesion. Mice were immunized with live La1, and the hybridomas produced by fusion of spleen cells with ONS1 cells were screened for the production of antibodies specific for
L. johnsonii
La1. A set of these monoclonal antibodies was directed against a nonproteinaceous component of the
L. johnsonii
La1 surface. It was identified as lipoteichoic acid (LTA). This molecule was isolated, chemically characterized, and tested in adhesion experiments in the same system. The adhesion of
L. johnsonii
La1 to Caco-2 cells was inhibited in a concentration-dependent way by purified LTA as well as by
L. johnsonii
La1 culture supernatant that contained LTA. These results showed that the mechanism of adhesion of
L. johnsonii
La1 to human Caco-2 cells involves LTA.
Effects of Salinity and Temperature on Long-Term Survival of the Eel Pathogen
Vibrio vulnificus
Biotype 2 (Serovar E)
Authors: Marco-Noales Ester , Biosca Elena G. , Amaro Carmen
Published: December 2019

Vibrio vulnificus
biotype 2 (serovar E) is a primary eel pathogen. In this study, we performed long-term survival experiments to investigate whether the aquatic ecosystem can be a reservoir for this bacterium. We have used microcosms containing water of different salinities (ranging from 0.3 to 3.8%) maintained at three temperatures (12, 25, and 30°C). Temperature and salinity significantly affected long-term survival: (i) the optimal salinity for survival was 1.5%; (ii) lower salinities reduced survival, although they were nonlethal; and (ii) the optimal temperature for survival was dependent on the salinity (25°C for microcosms at 0.3 and 0.5% and 12°C for microcosms at 1.5 to 3.8%). In the absence of salts, culturability dropped to zero in a few days, without evidence of cellular lysis. Under optimal conditions of salinity and temperature, the bacterium was able to survive in the free-living form for at least 3 years. The presence of a capsule on the bacterial cell seemed to confer an advantage, since the long-term survival rate of opaque variants was significantly higher than that of translucent ones. Long-term-starved cells maintained their infectivity for eels (as determined by both intraperitoneal and immersion challenges) and mice. Examination under the microscope showed that (i) the capsule was maintained, (ii) the cell size decreased, (iii) the rod shape changed to coccuslike along the time of starvation, and (iv) membrane vesicles and extracellular material were occasionally produced. In conclusion,
V. vulnificus
biotype 2 follows a survival strategy similar to that of biotype 1 of this species in response to starvation conditions in water. Moreover, the aquatic ecosystem is one of its reservoirs.
Immunochemical Detection and Isolation of DNA from Metabolically Active Bacteria
Authors: Urbach Ena , Vergin Kevin L. , Giovannoni Stephen J.
Published: December 2019

Most techniques used to assay the growth of microbes in natural communities provide no information on the relationship between microbial productivity and community structure. To identify actively growing bacteria, we adapted a technique from immunocytochemistry to detect and selectively isolate DNA from bacteria incorporating bromodeoxyuridine (BrdU), a thymidine analog. In addition, we developed an immunocytochemical protocol to visualize BrdU-labeled microbial cells. Cultured bacteria and natural populations of aquatic bacterioplankton were pulse-labeled with exogenously supplied BrdU. Incorporation of BrdU into microbial DNA was demonstrated in DNA dot blots probed with anti-BrdU monoclonal antibodies and either peroxidase- or Texas red-conjugated secondary antibodies. BrdU-containing DNA was physically separated from unlabeled DNA by using antibody-coated paramagnetic beads, and the identities of bacteria contributing to both purified, BrdU-containing fractions and unfractionated, starting-material DNAs were determined by length heterogeneity PCR (LH-PCR) analysis. BrdU-containing DNA purified from a mixture of DNAs from labeled and unlabeled cultures showed >90-fold enrichment for the labeled bacterial taxon. The LH-PCR profile for BrdU-containing DNA from a labeled, natural microbial community differed from the profile for the community as a whole, demonstrating that BrdU was incorporated by a taxonomic subset of the community. Immunocytochemical detection of cells with BrdU-labeled DNA was accomplished by in situ probing with anti-BrdU monoclonal antibodies and Texas red-labeled secondary antibodies. Using this suite of techniques, microbial cells incorporating BrdU into their newly synthesized DNA can be quantified and the identities of these actively growing cells can be compared to the composition of the microbial community as a whole. Since not all strains tested could incorporate BrdU, these methods may be most useful when used to gain an understanding of the activities of specific species in the context of their microbial community.