bluuebunny/crossref_metadata_2025_split huggingface dataset:
Characterization and Heterologous Expression of the Genes Encoding Enterocin A Production, Immunity, and Regulation in
Enterococcus faecium
DPC1146
Authors: O’Keeffe Triona , Hill Colin , Ross R. Paul 
Published: December 2019

ABSTRACT

Enterocin A is a small, heat-stable, antilisterial bacteriocin produced by
Enterococcus faecium
DPC1146. The sequence of a 10,879-bp chromosomal region containing at least 12 open reading frames (ORFs), 7 of which are predicted to play a role in enterocin biosynthesis, is presented. The genes
entA
,
entI
, and
entF
encode the enterocin A prepeptide, the putative immunity protein, and the induction factor prepeptide, respectively. The deduced proteins EntK and EntR resemble the histidine kinase and response regulator proteins of two-component signal transducing systems of the AgrC-AgrA type. The predicted proteins EntT and EntD are homologous to ABC (ATP-binding cassette) transporters and accessory factors, respectively, of several other bacteriocin systems and to proteins implicated in the signal-sequence-independent export of
Escherichia coli
hemolysin A. Immediately downstream of the
entT
and
entD
genes are two ORFs, the product of one of which, ORF4, is very similar to the product of the
yteI
gene of
Bacillus subtilis
and to
E. coli
protease IV, a signal peptide peptidase known to be involved in outer membrane lipoprotein export. Another potential bacteriocin is encoded in the opposite direction to the other genes in the enterocin cluster. This putative bacteriocin-like peptide is similar to LafX, one of the components of the lactacin F complex. A deletion which included one of two direct repeats upstream of the
entA
gene abolished enterocin A activity, immunity, and ability to induce bacteriocin production. Transposon insertion upstream of the
entF
gene also had the same effect, but this mutant could be complemented by exogenously supplied induction factor. The putative EntI peptide was shown to be involved in the immunity to enterocin A. Cloning of a 10.5-kb amplicon comprising all predicted ORFs and regulatory regions resulted in heterologous production of enterocin A and induction factor in
Enterococcus faecalis
, while a four-gene construct (
entAITD
) under the control of a constitutive promoter resulted in heterologous enterocin A production in both
E. faecalis
and
Lactococcus lactis
.
Identification of Four Phage Resistance Plasmids from
Lactococcus lactis
subsp.
cremoris
HO2
Authors: Forde Amanda , Daly Charles , Fitzgerald Gerald F. 
Published: December 2019

ABSTRACT

The bacteriophage-host sensitivity patterns of 16 strains of
Lactococcus lactis
originally isolated from a mixed strain Cheddar cheese starter culture were determined. Using phages obtained from cheese factory whey, four of the strains were found to be highly phage resistant. One of these isolates,
Lactococcus lactis
subsp.
cremoris
HO2, was studied in detail to determine the mechanisms responsible for the phage insensitivity phenotypes. Conjugal transfer of plasmid DNA from strain HO2 allowed a function to be assigned to four of its six plasmids. A 46-kb molecule, designated pCI646, was found to harbor the lactose utilization genes, while this and plasmids of 58 kb (pCI658), 42 kb (pCI642), and 4.5 kb (pCI605) were shown to be responsible for the phage resistance phenotypes observed against the small isometric-headed phage φ712 (936 phage species) and the prolate-headed phage φc2 (c2 species). pCI658 was found to mediate an adsorption-blocking mechanism and was also responsible for the fluffy pellet phenotype of cells containing the molecule. pCI642 and pCI605 were both shown to be required for the operation of a restriction-modification system.
Degradation of Ciprofloxacin by Basidiomycetes and Identification of Metabolites Generated by the Brown Rot Fungus
Gloeophyllum striatum
Authors: Wetzstein Heinz-Georg , Stadler Marc , Tichy Hans-Volker , Dalhoff Axel , Karl Wolfgang 
Published: December 2019

ABSTRACT

Ciprofloxacin (CIP), a fluoroquinolone antibacterial drug, is widely used in the treatment of serious infections in humans. Its degradation by basidiomycetous fungi was studied by monitoring
14
CO
2
production from [
14
C]CIP in liquid cultures. Sixteen species inhabiting wood, soil, humus, or animal dung produced up to 35%
14
CO
2
during 8 weeks of incubation. Despite some low rates of
14
CO
2
formation, all species tested had reduced the antibacterial activity of CIP in supernatants to between 0 and 33% after 13 weeks.
Gloeophyllum striatum
was used to identify the metabolites formed from CIP. After 8 weeks, mycelia had produced 17 and 10%
14
CO
2
from C-4 and the piperazinyl moiety, respectively, although more than half of CIP (applied at 10 ppm) had been transformed into metabolites already after 90 h. The structures of 11 metabolites were elucidated by high-performance liquid chromatography combined with electrospray ionization mass spectrometry and
1
H nuclear magnetic resonance spectroscopy. They fell into four categories as follows: (i) monohydroxylated congeners, (ii) dihydroxylated congeners, (iii) an isatin-type compound, proving elimination of C-2, and (iv) metabolites indicating both elimination and degradation of the piperazinyl moiety. A metabolic scheme previously described for enrofloxacin degradation could be confirmed and extended. A new type of metabolite, 6-defluoro-6-hydroxy-deethylene-CIP, provided confirmatory evidence for the proposed network of congeners. This may result from sequential hydroxylation of CIP and its congeners by hydroxyl radicals. Our findings reveal for the first time the widespread potential for CIP degradation among basidiomycetes inhabiting various environments, including agricultural soils and animal dung.
Variation in Resistance of Natural Isolates of
Escherichia coli
O157 to High Hydrostatic Pressure, Mild Heat, and Other Stresses
Authors: Benito Amparo , Ventoura Georgia , Casadei Maria , Robinson Tobin , Mackey Bernard 
Published: December 2019

ABSTRACT

Strains of
Escherichia coli
O157 isolated from patients with clinical cases of food-borne illness and other sources exhibited wide differences in resistance to high hydrostatic pressure. The most pressure-resistant strains were also more resistant to mild heat than other strains. Strain C9490, a representative pressure-resistant strain, was also more resistant to acid, oxidative, and osmotic stresses than the pressure-sensitive strain NCTC 12079. Most of these differences in resistance were observed only in stationary-phase cells, the only exception being acid resistance, where differences were also apparent in the exponential phase. Membrane damage in pressure-treated cells was revealed by increased uptake of the fluorescent dyes ethidium bromide and propidium iodide. When strains were exposed to the same pressure for different lengths of time, the pressure-sensitive strains took up stain sooner than the more resistant strain, which suggested that the differences in resistance may be related to susceptibility to membrane damage. Our results emphasize the importance of including stress-resistant strains of
E. coli
O157 when the efficacy of a novel or mild food preservation treatment is tested.
Selection of
clc
,
cba
, and
fcb
Chlorobenzoate-Catabolic Genotypes from Groundwater and Surface Waters Adjacent to the Hyde Park, Niagara Falls, Chemical Landfill
Authors: Peel Michelle C. , Wyndham R. Campbell 
Published: December 2019

ABSTRACT

The frequency of isolation of three nonhomologous chlorobenzoate catabolic genotypes (
clc
,
cba
, and
fcb
) was determined for 464 isolates from freshwater sediments and groundwater in the vicinity of the Hyde Park industrial landfill site in the Niagara watershed. Samples were collected from both contaminated and noncontaminated sites during spring, summer, and fall and enriched at 4, 22, or 32°C with micromolar to millimolar concentrations of chlorobenzoates and 3-chlorobiphenyl (M. C. Peel and R. C. Wyndham, Microb. Ecol: 33:59–68, 1997). Hybridization at moderate stringency to restriction-digested genomic DNA with DNA probes revealed the chlorocatechol 1,2-dioxygenase operon (
clcABD
), the 3-chlorobenzoate 3,4-(4,5)-dioxygenase operon (
cbaABC
), and the 4-chlorobenzoate dehalogenase (
fcbB
) gene in isolates enriched from all contaminated sites in the vicinity of the industrial landfill. Nevertheless, the known genes were found in less than 10% of the isolates from the contaminated sites, indicating a high level of genetic diversity in the microbial community. The known genotypes were not enriched from the noncontaminated control sites nearby. The
clc
,
cba
, and
fcb
isolates were distributed across five phenotypically distinct groups based on Biolog carbon source utilization, with the breadth of the host range decreasing in the order
clc > cba > fcb
. Restriction fragment length polymorphism (RFLP) patterns showed that the
cba
genes were conserved in all isolates whereas the
clc
and
fcb
genes exhibited variation in RFLP patterns. These observations are consistent with the recent spread of the
cba
genes by horizontal transfer as part of transposon Tn
5271
in response to contaminant exposure at Hyde Park. Consistent with this hypothesis, IS
1071
, the flanking element in Tn
5271
, was found in all isolates that carried the
cba
genes. Interestingly, IS
1071
was also found in a high proportion of isolates from Hyde Park carrying the
clc
and
fcb
genes, as well as in type strains carrying the
clcABD
operon and the biphenyl (
bph
) catabolic genes.
Modes of Action of Acarbose Hydrolysis and Transglycosylation Catalyzed by a Thermostable Maltogenic Amylase, the Gene for Which Was Cloned from a
Thermus
Strain
Authors: Kim Tae-Jip , Kim Myo-Jeong , Kim Byung-Cheon , Kim Jae-Cherl , Cheong Tae-Kyou , Kim Jung-Wan , Park Kwan-Hwa 
Published: December 2019

ABSTRACT

A maltogenic amylase gene was cloned in
Escherichia coli
from a gram-negative thermophilic bacterium,
Thermus
strain IM6501. The gene encoded an enzyme (ThMA) with a molecular mass of 68 kDa which was expressed by the expression vector p6xHis119. The optimal temperature of ThMA was 60°C, which was higher than those of other maltogenic amylases reported so far. Thermal inactivation kinetic analysis of ThMA indicated that it was stabilized in the presence of 10 mM EDTA. ThMA harbored both hydrolysis and transglycosylation activities. It hydrolyzed β-cyclodextrin and starch mainly to maltose and pullulan to panose. ThMA not only hydrolyzed acarbose, an amylase inhibitor, to glucose and pseudotrisaccharide (PTS) but also transferred PTS to 17 sugar acceptors, including glucose, fructose, maltose, cellobiose, etc. Structural analysis of acarbose transfer products by using methylation, thin-layer chromatography, high-performance ion chromatography, and nuclear magnetic resonance indicated that PTS was transferred primarily to the C-6 of the acceptors and at lower degrees to the C-3 and/or C-4. The transglycosylation of sugar to
methyl
-α-
d
-glucopyranoside by forming an α-(1,3)-glycosidic linkage was demonstrated for the first time by using acarbose and ThMA. Kinetic analysis of the acarbose transfer products showed that the C-4 transfer product formed most rapidly but readily hydrolyzed, while the C-6 transfer product was stable and accumulated in the reaction mixture as the main product.
Phenotypic Expression of PCR-Generated Random Mutations in a
Pseudomonas putida
Gene after Its Introduction into an
Acinetobacter
Chromosome by Natural Transformation
Authors: Kok Ruben G. , Young David M. , Ornston L. Nicholas 
Published: December 2019

ABSTRACT

Localized sets of random point mutations generated by PCR amplification can be transferred efficiently to the chromosome of
Acinetobacter
ADP1 (also known as strain BD413) by natural transformation. The technique does not require cloning of PCR fragments in plasmids: PCR-amplified DNA fragments are internalized by cells and directly incorporated into their genomes by homologous recombination. Previously such procedures for random mutagenesis could be applied only to
Acinetobacter
genes affording the selection of mutant phenotypes. Here we describe the construction of a vector and recipient that allow for mutagenesis, recovery, and expression of heterologous genes that may lack a positive selection. The plasmid carries an
Acinetobacter
chromosomal segment interrupted by a multiple cloning site next to a kanamycin resistance marker. The insertion of heterologous DNA into the multiple cloning site prepares the insert as a target for PCR mutagenesis. PCR amplifies the kanamycin resistance marker and a flanking region of
Acinetobacter
DNA along with the insert of heterologous DNA. Nucleotide sequence identity between the flanking regions and corresponding chromosomal segments in an engineered
Acinetobacter
recipient allows homologous recombination of the PCR-amplified DNA fragments into a specific chromosomal docking site from which they can be expressed. The recipient strain contains only a portion of the kanamycin resistance gene, so donor DNA containing both this gene and the mutagenized insert can be selected by demanding growth of recombinants in the presence of kanamycin. The effectiveness of the technique was demonstrated with the relatively GC-rich
Pseudomonas putida xylE
gene. After only one round of PCR amplification (35 cycles), donor DNA produced transformants of which up to 30% carried a defective
xylE
gene after growth at 37°C. Of recombinant clones that failed to express
xylE
at 37°C, about 10% expressed the gene when grown at 22°C. The techniques described here could be adapted to prepare colonies with an altered function in any gene for which either a selection or a suitable phenotypic screen exists.
Diversity of Nitrous Oxide Reductase (
nosZ
) Genes in Continental Shelf Sediments
Authors: Scala David J. , Kerkhof Lee J. 
Published: December 2019

ABSTRACT

Diversity of the nitrous oxide reductase (
nosZ
) gene was examined in sediments obtained from the Atlantic Ocean and Pacific Ocean continental shelves. Approximately 1,100 bp of the
nosZ
gene were amplified via PCR, using
nosZ
gene-specific primers. Thirty-seven unique copies of the
nosZ
gene from these marine environments were characterized, increasing the
nosZ
sequence database fourfold. The average DNA similarity for comparisons between all 49 variants of the
nosZ
gene was 64% ± 10%. Alignment of the derived amino acid sequences confirmed the conservation of important structural motifs. A highly conserved region is proposed as the copper binding, catalytic site (Cu
Z
) of the mature protein. Phylogenetic analysis demonstrated three major clusters of
nosZ
genes, with little overlap between environmental and culture-based groups. Finally, the two non-culture-based gene clusters generally corresponded to sampling location, implying that denitrifier communities may be restricted geographically.
Monounsaturated but Not Polyunsaturated Fatty Acids Are Required for Growth of the Deep-Sea Bacterium
Photobacterium profundum
SS9 at High Pressure and Low Temperature
Authors: Allen Eric E. , Facciotti Daniel , Bartlett Douglas H. 
Published: December 2019

ABSTRACT

There is considerable evidence correlating the production of increased proportions of membrane unsaturated fatty acids (UFAs) with bacterial growth at low temperatures or high pressures. In order to assess the importance of UFAs to microbial growth under these conditions, the effects of conditions altering UFA levels in the psychrotolerant piezophilic deep-sea bacterium
Photobacterium profundum
SS9 were investigated. The fatty acids produced by
P. profundum
SS9 grown at various temperatures and pressures were characterized, and differences in fatty acid composition as a function of phase growth, and between inner and outer membranes, were noted.
P. profundum
SS9 was found to exhibit enhanced proportions of both monounsaturated (MUFAs) and polyunsaturated (PUFAs) fatty acids when grown at a decreased temperature or elevated pressure. Treatment of cells with cerulenin inhibited MUFA but not PUFA synthesis and led to a decreased growth rate and yield at low temperature and high pressure. In addition, oleic acid-auxotrophic mutants were isolated. One of these mutants, strain EA3, was deficient in the production of MUFAs and was both low-temperature sensitive and high-pressure sensitive in the absence of exogenous 18:1 fatty acid. Another mutant, strain EA2, produced little MUFA but elevated levels of the PUFA species eicosapentaenoic acid (EPA; 20:5
n
-3). This mutant grew slowly but was not low-temperature sensitive or high-pressure sensitive. Finally, reverse genetics was employed to construct a mutant unable to produce EPA. This mutant, strain EA10, was also not low-temperature sensitive or high-pressure sensitive. The significance of these results to the understanding of the role of UFAs in growth under low-temperature or high-pressure conditions is discussed.
Wide Distribution and Diversity of Members of the Bacterial Kingdom
Acidobacterium
in the Environment
Authors: Barns Susan M. , Takala Shannon L. , Kuske Cheryl R. 
Published: December 2019

ABSTRACT

To assess the distribution and diversity of members of the recently identified bacterial kingdom
Acidobacterium
, members of this kingdom present in 43 environmental samples were surveyed by PCR amplification. A primer designed to amplify rRNA gene sequences (ribosomal DNAs [rDNAs]) from most known members of the kingdom was used to interrogate bulk DNA extracted from the samples. Positive PCR results were obtained with all temperate soil and sediment samples tested, as well as some hot spring samples, indicating that members of this kingdom are very widespread in terrestrial environments. PCR primers specific for four phylogenetic subgroups within the kingdom were used in similar surveys. All four subgroups were detected in most neutral soils and some sediments, while only two of the groups were seen in most low-pH environments. The combined use of these primers allowed identification of a novel lineage within the kingdom in a hot spring environment. Phylogenetic analysis of rDNA sequences from our survey and the literature outlines at least six major subgroups within the kingdom. Taken together, these data suggest that members of the
Acidobacterium
kingdom are as genetically and metabolically diverse, environmentally widespread and perhaps as ecologically important as the well-known
Proteobacteria
and gram-positive bacterial kingdoms.
Combined Microautoradiography–16S rRNA Probe Technique for Determination of Radioisotope Uptake by Specific Microbial Cell Types In Situ
Authors: Ouverney Cleber C. , Fuhrman Jed A. 
Published: December 2019

ABSTRACT

We propose a novel method for studying the function of specific microbial groups in situ. Since natural microbial communities are dynamic both in composition and in activities, we argue that the microbial “black box” should not be regarded as homogeneous. Our technique breaks down this black box with group-specific fluorescent 16S rRNA probes and simultaneously determines
3
H-substrate uptake by each of the subgroups present via microautoradiography (MAR). Total direct counting, fluorescent in situ hybridization, and MAR are combined on a single slide to determine (i) the percentages of different subgroups in a community, (ii) the percentage of total cells in a community that take up a radioactively labeled substance, and (iii) the distribution of uptake within each subgroup. The method was verified with pure cultures. In addition, in situ uptake by members of the α subdivision of the class
Proteobacteria
(α-Proteobacteria) and of the
Cytophaga-Flavobacterium
group obtained off the California coast and labeled with fluorescent oligonucleotide probes for these subgroups showed that not only do these organisms account for a large portion of the picoplankton community in the sample examined (∼60% of the universal probe-labeled cells and ∼50% of the total direct counts), but they also are significant in the uptake of dissolved amino acids in situ. Nearly 90% of the total cells and 80% of the cells belonging to the α-Proteobacteria and
Cytophaga-Flavobacterium
groups were detectable as active organisms in amino acid uptake tests. We suggest a name for our triple-labeling technique, substrate-tracking autoradiographic fluorescent in situ hybridization (STARFISH), which should aid in the “dissection” of microbial communities by type and function.
cumA
, a Gene Encoding a Multicopper Oxidase, Is Involved in Mn
2+
Oxidation in
Pseudomonas putida
GB-1
Authors: Brouwers Geert-Jan , de Vrind Johannes P. M. , Corstjens Paul L. A. M. , Cornelis Pierre , Baysse Christine , de Vrind-de Jong Elisabeth W. 
Published: December 2019

ABSTRACT

Pseudomonas putida
GB-1-002 catalyzes the oxidation of Mn
2+
. Nucleotide sequence analysis of the transposon insertion site of a nonoxidizing mutant revealed a gene (designated
cumA
) encoding a protein homologous to multicopper oxidases. Addition of Cu
2+
increased the Mn
2+
-oxidizing activity of the
P. putida
wild type by a factor of approximately 5. The growth rates of the wild type and the mutant were not affected by added Cu
2+
. A second open reading frame (designated
cumB
) is located downstream from
cumA
. Both
cumA
and
cumB
probably are part of a single operon. The translation product of
cumB
was homologous (level of identity, 45%) to that of
orf74
of
Bradyrhizobium japonicum
. A mutation in
orf74
resulted in an extended lag phase and lower cell densities. Similar growth-related observations were made for the
cumA
mutant, suggesting that the
cumA
mutation may have a polar effect on
cumB
. This was confirmed by site-specific gene replacement in
cumB
. The
cumB
mutation did not affect the Mn
2+
-oxidizing ability of the organism but resulted in decreased growth. In summary, our data indicate that the multicopper oxidase CumA is involved in the oxidation of Mn
2+
and that CumB is required for optimal growth of
P. putida
GB-1-002.
Formation of Bound Residues during Microbial Degradation of [
14
C]Anthracene in Soil
Authors: Kästner M. , Streibich S. , Beyrer M. , Richnow H. H. , Fritsche W. 
Published: December 2019

ABSTRACT

Carbon partitioning and residue formation during microbial degradation of polycyclic aromatic hydrocarbons (PAH) in soil and soil-compost mixtures were examined by using [
14
C]anthracenes labeled at different positions. In native soil 43.8% of [9-
14
C]anthracene was mineralized by the autochthonous microflora and 45.4% was transformed into bound residues within 176 days. Addition of compost increased the metabolism (67.2% of the anthracene was mineralized) and decreased the residue formation (20.7% of the anthracene was transformed). Thus, the higher organic carbon content after compost was added did not increase the level of residue formation. [
14
C]anthracene labeled at position 1,2,3,4,4a,5a was metabolized more rapidly and resulted in formation of higher levels of residues (28.5%) by the soil-compost mixture than [
14
C]anthracene radiolabeled at position C-9 (20.7%). Two phases of residue formation were observed in the experiments. In the first phase the original compound was sequestered in the soil, as indicated by its limited extractability. In the second phase metabolites were incorporated into humic substances after microbial degradation of the PAH (biogenic residue formation). PAH metabolites undergo oxidative coupling to phenolic compounds to form nonhydrolyzable humic substance-like macromolecules. We found indications that monomeric educts are coupled by C-C- or either bonds. Hydrolyzable ester bonds or sorption of the parent compounds plays a minor role in residue formation. Moreover, experiments performed with
14
CO
2
revealed that residues may arise from CO
2
in the soil in amounts typical for anthracene biodegradation. The extent of residue formation depends on the metabolic capacity of the soil microflora and the characteristics of the soil. The position of the
14
C label is another important factor which controls mineralization and residue formation from metabolized compounds.