bluuebunny/crossref_metadata_2025_split huggingface dataset:
Gene Cloning, Transcriptional Analysis, Purification, and Characterization of Phenolic Acid Decarboxylase from
Bacillus subtilis
Authors: Cavin Jean-François , Dartois Véronique , Diviès Charles 
Published: December 2019

ABSTRACT

Bacillus subtilis
displays a substrate-inducible decarboxylating activity with the following three phenolic acids: ferulic,
p
-coumaric, and caffeic acids. Based on DNA sequence homologies between the
Bacillus pumilus
ferulate decarboxylase gene (
fdc
) (A. Zago, G. Degrassi, and C. V. Bruschi, Appl. Environ. Microbiol. 61:4484–4486, 1995) and the
Lactobacillus plantarum p
-coumarate decarboxylase gene (
pdc
) (J.-F. Cavin, L. Barthelmebs, and C. Diviès, Appl. Environ. Microbiol. 63:1939–1944, 1997), a DNA probe of about 300 nucleotides for the
L. plantarum pdc
gene was used to screen a
B. subtilis
genomic library in order to clone the corresponding gene in this bacterium. One clone was detected with this heterologous probe, and this clone exhibited phenolic acid decarboxylase (PAD) activity. The corresponding 5-kb insertion was partially sequenced and was found to contain a 528-bp open reading frame coding for a 161-amino-acid protein exhibiting 71 and 84% identity with the
pdc
- and
fdc
-encoded enzymes, respectively. The PAD gene (
pad
) is transcriptionally regulated by
p
-coumaric, ferulic, or caffeic acid; these three acids are the three substrates of PAD. The
pad
gene was overexpressed constitutively in
Escherichia coli
, and the stable purified enzyme was characterized. The difference in substrate specificity between this PAD and other PADs seems to be related to a few differences in the amino acid sequence. Therefore, this novel enzyme should facilitate identification of regions involved in catalysis and substrate specificity.
Fluorescent Pseudomonad Pyoverdines Bind and Oxidize Ferrous Ion
Authors: Xiao Rong , Kisaalita William S. 
Published: December 2019

ABSTRACT

Major pyoverdines from
Pseudomonas fluorescens
2-79 (Pf-B),
P. aeruginosa
ATCC 15692 (Pa-C), and
P. putida
ATCC 12633 (Pp-C) were examined by absorption and fluorescence spectroscopic techniques to investigate the interaction between ferrous ion and the pyoverdine ligand. At physiological pH, ferrous ion quenched the fluorescence of all three pyoverdines much faster than ferric ion did. Also, increased absorbance at 460 nm was observed to be much faster for Fe
2+
-pyoverdine than for Fe
3+
-pyoverdine. At pH 7.4, about 90% of Fe
3+
was bound by pyoverdine Pa-C after 24 h whereas Fe
2+
was bound by the pyoverdine completely in only 5 min. The possibility that Fe
2+
underwent rapid autoxidation before being bound by pyoverdine was considered unlikely, since the Fe
2+
concentration in pyoverdine-free samples remained constant over a 3-min period at pH 7.4. Incubating excess Fe
2+
with pyoverdine in the presence of 8-hydroxyquinoline, an Fe
3+
-specific chelating agent, resulted in the formation of a Fe
3+
-hydroxyquinoline complex, suggesting that the iron in the Fe
2+
-pyoverdine complex existed in the oxidized form. These results strongly suggested that pyoverdines bind and oxidize the ferrous ion.
Anaerobic Degradation of Hexadecan-2-one by a Microbial Enrichment Culture under Sulfate-Reducing Conditions
Authors: Hirschler Agnes , Rontani Jean-Francois , Raphel Danielle , Matheron Robert , Bertrand Jean-Claude 
Published: December 2019

ABSTRACT
A microbial enrichment culture from marine sediment was able to grow on hexadecan-2-one as the sole source of carbon and energy under sulfate-reducing conditions. Oxidation of the ketone involved carboxylation reactions and was coupled to sulfide production. This enrichment culture also grew on 6,10,14-trimethylpentadecan-2-one.
DNA Sequence Similarity between California Isolates of
Cryptosporidium parvum
Authors: Pereira Maria das Graças C. , Atwill Edward R. , Crawford Melissa R. , Lefebvre Rance B. 
Published: December 2019

ABSTRACT

We evaluated whether nucleic acid amplification with primers specific for
Cryptosporidium parvum
followed by automated DNA sequence analysis of the PCR amplicons could differentiate between California isolates of
C. parvum
obtained from livestock, humans, and feral pigs. Almost complete sequence identity existed among the livestock isolates and between the livestock and human isolates. DNA sequences from feral pig isolates differed from those from livestock and humans by 1.0 to 1.2%. The reference sequence obtained by Laxer et al. (M. A. Laxer, B. K. Timblin, and R. J. Patel, Am. J. Trop. Med. Hyg. 45:688–694, 1991.) differed from California isolates of
C. parvum
by 1.8 to 3.2%. These data suggest that DNA sequence analysis of the amplicon of Laxer et al. does not allow for differentiation between various strains of
C. parvum
or that our collection of isolates obtained from various hosts from across California was limited to one strain of
C. parvum.
A Gene System for Glucitol Transport and Metabolism in
Clostridium beijerinckii
NCIMB 8052
Authors: Tangney Martin , Brehm John K. , Minton Nigel P. , Mitchell Wilfrid J. 
Published: December 2019

ABSTRACT

The
gutD
gene of
Clostridium beijerinckii
NCIMB 8052 encoding glucitol 6-phosphate dehydrogenase was cloned on a 5.7-kbp chromosomal DNA fragment by complementing an
Escherichia coli gutD
mutant strain and selecting for growth on glucitol. Five open reading frames (ORFs) in the order
gutA1 gutA2 orfX gutB gutD
were identified in a 4.0-kbp region of the cloned DNA. The deduced products of four of these ORFs were homologous to components of the glucitol phosphotransferase system (PTS) and glucitol 6-phosphate dehydrogenase from
E. coli
, while the remaining ORF (
orfX
) encoded an enzyme which had similarities to members of a family of transaldolases. A strain in which
gutD
was inactivated by targeted integration lacked glucitol 6-phosphate dehydrogenase activity. The
gutA1
and
gutA2
genes encoded two polypeptides forming enzyme IIBC of the glucitol PTS comprising three domains in the order CBC. Domain IIA of the glucitol PTS was encoded by
gutB
. Glucitol phosphorylation assays in which soluble and membrane fractions of cells grown on glucose (which did not synthesize the glucitol PTS) or cells grown on glucitol were used confirmed that there is a separate, soluble, glucitol-specific PTS component, which is the product of the
gutB
gene. The
gut
genes were regulated at the level of transcription and were induced in the presence of glucitol. Cells grown in the presence of glucose and glucitol utilized glucose preferentially. Following depletion of glucose, the glucitol PTS and glucitol 6-phosphate dehydrogenase were synthesized, and glucitol was removed from the culture medium. RNA analysis showed that the
gut
genes were not expressed until glucose was depleted.
A Deficiency in Aspartate Biosynthesis in
Lactococcus lactis
subsp.
lactis
C2 Causes Slow Milk Coagulation
Authors: Wang Hua , Yu Weizhu , Coolbear Tim , O’Sullivan Dan , McKay Larry L. 
Published: December 2019

ABSTRACT

A mutant of fast milk-coagulating (Fmc
+
)
Lactococcus lactis
subsp.
lactis
C2, designated
L. lactis
KB4, was identified. Although possessing the known components essential for utilizing casein as a nitrogen source, which include functional proteinase (PrtP) activity and oligopeptide, di- and tripeptide, and amino acid transport systems, KB4 exhibited a slow milk coagulation (Fmc
−
) phenotype. When the amino acid requirements of
L. lactis
C2 were compared with those of KB4 by use of a chemically defined medium, it was found that KB4 was unable to grow in the absence of aspartic acid. This aspartic acid requirement could also be met by aspartate-containing peptides. The addition of aspartic acid to milk restored the Fmc
+
phenotype of KB4. KB4 was found to be defective in pyruvate carboxylase and thus was deficient in the ability to form oxaloacetate and hence aspartic acid from pyruvate and carbon dioxide. The results suggest that when lactococci are propagated in milk, aspartate derived from casein is unable to meet fully the nutritional demands of the lactococci, and they become dependent upon aspartate biosynthesis.
Phylogenetic Evidence for the Existence of Novel Thermophilic Bacteria in Hot Spring Sulfur-Turf Microbial Mats in Japan
Authors: Yamamoto Hiroyuki , Hiraishi Akira , Kato Kenji , Chiura Hiroshi X. , Maki Yonosuke , Shimizu Akira 
Published: December 2019

ABSTRACT

So-called sulfur-turf microbial mats, which are macroscopic white filaments or bundles consisting of large sausage-shaped bacteria and elemental sulfur particles, occur in sulfide-containing hot springs in Japan. However, no thermophiles from sulfur-turf mats have yet been isolated as cultivable strains. This study was undertaken to determine the phylogenetic positions of the sausage-shaped bacteria in sulfur-turf mats by direct cloning and sequencing of 16S rRNA genes amplified from the bulk DNAs of the mats. Common clones with 16S rDNA sequences with similarity levels of 94.8 to 99% were isolated from sulfur-turf mat samples from two geographically remote hot springs. Phylogenetic analysis showed that the phylotypes of the common clones formed a major cluster with members of the
Aquifex-Hydrogenobacter
complex, which represents the most deeply branching lineage of the domain bacteria. Furthermore, the bacteria of the sulfur-turf mat phylotypes formed a clade distinguishable from that of other members of the
Aquifex-Hydrogenobacter
complex at the order or subclass level. In situ hybridization with clone-specific probes for 16S rRNA revealed that the common phylotype of sulfur-turf mat bacteria is that of the predominant sausage-shaped bacteria.
Two New
Mycobacterium
Strains and Their Role in Toluene Degradation in a Contaminated Stream
Authors: Tay Stephen T.-L. , Hemond Harold F. , Polz Martin F. , Cavanaugh Colleen M. , Dejesus Indhira , Krumholz Lee R. 
Published: December 2019

ABSTRACT

Two toluene-degrading strains, T103 and T104, were isolated from rock surface biomass in a freshwater stream contaminated with toluene. The strains exhibit different capacities for degradation of toluene and other aromatic compounds and have characteristics of the genus
Mycobacterium
. Both are aerobic, rod-shaped, gram-positive, nonmotile, and acid-alcohol fast and produce yellow pigments. They have mainly straight-chain saturated and monounsaturated fatty acids with 10 to 20 carbon atoms and large amounts of tuberculostearic acid that are typical of mycobacteria. Fatty acid analyses indicate that T103 and T104 are different mycobacterial strains that are related at the subspecies level. Their identical 16S rDNA sequences are most similar to
Mycobacterium aurum
and
Mycobacterium komossense
, and they constitute a new species of fast-growing mycobacteria. Ecological studies reveal that toluene contamination has enriched for toluene-degrading bacteria in the epilithic microbial community. Strains T103 and T104 play only a small role in toluene degradation in the stream, although they are present in the habitat and can degrade toluene. Other microorganisms are consequently implicated in the biodegradation.
Comparison of Blue Nucleic Acid Dyes for Flow Cytometric Enumeration of Bacteria in Aquatic Systems
Authors: Lebaron Philippe , Parthuisot Nathalie , Catala Philippe 
Published: December 2019

ABSTRACT
Seven blue nucleic acid dyes from Molecular Probes Inc. (SYTO-9, SYTO-11, SYTO-13, SYTO-16, SYTO-BC, SYBR-I and SYBR-II) were compared with the DAPI (4′,6-diamidino-2-phenylindole) method for flow cytometric enumeration of live and fixed bacteria in aquatic systems. It was shown that SYBR-II and SYTO-9 are the most appropriate dyes for bacterial enumeration in nonsaline waters and can be applied to both live and dead bacteria. The fluorescence signal/noise ratio was improved when SYTO-9 was used to stain living bacteria in nonsaline waters. Inversely, SYBR-II is more appropriate than SYTO dyes for bacterial enumeration of unfixed and fixed seawater samples.
Sensitive and Rapid Detection of Viable
Giardia
Cysts and
Cryptosporidium parvum
Oocysts in Large-Volume Water Samples with Wound Fiberglass Cartridge Filters and Reverse Transcription-PCR
Authors: Kaucner Christine , Stinear Timothy 
Published: December 2019

ABSTRACT

We recently described a reverse transcription-PCR (RT-PCR) for detecting low numbers of viable
Cryptosporidium parvum
oocysts spiked into clarified environmental water concentrates. We have now modified the assay for direct analysis of primary sample concentrates with simultaneous detection of viable
C. parvum
oocysts,
Giardia
cysts, and a novel type of internal positive control (IPC). The IPC was designed to assess both efficiency of mRNA isolation and potential RT-PCR inhibition. Sensitivity testing showed that low numbers of organisms, in the range of a single viable cyst and oocyst, could be detected when spiked into 100-μl packed pellet volumes of concentrates from creek and river water samples. The RT-PCR was compared with an immunofluorescence (IF) assay by analyzing 29 nonspiked environmental water samples. Sample volumes of 20 to 1,500 liters were concentrated with a wound fiberglass cartridge filter. Frequency of detection for viable
Giardia
cysts increased from 24% by IF microscopy to 69% by RT-PCR. Viable
C. parvum
oocysts were detected only once by RT-PCR (3%) in contrast to detection of viable
Cryptosporidium
spp. in four samples by IF microscopy (14%), suggesting that
Cryptosporidium
species other than
C. parvum
were present in the water. This combination of the large-volume sampling method with RT-PCR represents a significant advance in terms of protozoan pathogen monitoring and in the wider application of PCR technology to this field of microbiology.
Use of Inducible Feedback-Resistant
N
-Acetylglutamate Synthetase (
argA
) Genes for Enhanced Arginine Biosynthesis by Genetically Engineered
Escherichia coli
K-12 Strains
Authors: Rajagopal B. S. , DePonte Joseph , Tuchman Mendel , Malamy Michael H. 
Published: December 2019

ABSTRACT

The goal of this work was to construct
Escherichia coli
strains capable of enhanced arginine production. The arginine biosynthetic capacity of previously engineered
E. coli
strains with a derepressed arginine regulon was limited by the availability of endogenous ornithine (M. Tuchman, B. S. Rajagopal, M. T. McCann, and M. H. Malamy, Appl. Environ. Microbiol. 63:33–38, 1997). Ornithine biosynthesis is limited due to feedback inhibition by arginine of
N
-acetylglutamate synthetase (NAGS), the product of the
argA
gene and the first enzyme in the pathway of arginine biosynthesis in
E. coli
. To circumvent this inhibition, the
argA
genes from
E. coli
mutants with feedback-resistant (fbr) NAGS were cloned into plasmids that contain “
arg
boxes,” which titrate the ArgR repressor protein, with or without the
E. coli carAB
genes encoding carbamyl phosphate synthetase and the
argI
gene for ornithine transcarbamylase. The free arginine production rates of “
arg
-derepressed”
E. coli
cells overexpressing plasmid-encoded
carAB
,
argI
, and fbr
argA
genes were 3- to 15-fold higher than that of an equivalent system overexpressing feedback-sensitive wild-type (wt)
argA
. The expression system with fbr
argA
produced 7- to 35-fold more arginine than a system overexpressing
carAB
and
argI
genes on a plasmid in a strain with a wt
argA
gene on the chromosome. The arginine biosynthetic capacity of
arg
-derepressed DH5α strains with plasmids containing only the fbr
argA
gene was similar to that of cells with plasmids also containing the
carAB
and
argI
genes. Plasmids containing wt or fbr
argA
were stably maintained under normal growth conditions for at least 18 generations. DNA sequencing identified different point mutations in each of the fbr
argA
mutants, specifically H15Y, Y19C, S54N, R58H, G287S, and Q432R.
Evidence of Genomic Instability in
Campylobacter jejuni
Isolated from Poultry
Authors: Wassenaar Trudy M. , Geilhausen Barbara , Newell Diane G. 
Published: December 2019

ABSTRACT

Poultry isolates of
Campylobacter jejuni
derived from a survey of meat processing batches were genotyped by pulsed-field gel electrophoresis (PFGE) of chromosomal DNA to establish the clonal relationships between single-colony isolates. In the majority of batches studied, one or two genotype patterns predominated. However, in one batch (batch A), 21 single-colony isolates gave 14 different PFGE genotypes. The banding patterns obtained with
Sma
I were sufficiently different to distinguish between genotypes, although the patterns also produced many common bands. The question of whether these isolates represented different clones or had a common clonal ancestry was addressed by additional genotypic and phenotypic methods. Restriction length polymorphism of PCR products obtained from the flagellin genes showed an identical flagellin genotype for all of these isolates. In contrast, unrelated control isolates resulted in different flagellin genotypes. Moreover, all 14 different PFGE genotypes of batch A had identical Penner serotypes and identical or similar biotypes and phage types. It was concluded that the isolates were of clonal origin and that the diversity in the PFGE banding patterns had most likely originated from genomic rearrangements. However, the PFGE genotypes were shown to be stable upon subculturing in vitro and after in vivo passage in chickens, and natural transformation between isogenic mutants carrying antibiotic markers did not occur in vivo in a chick colonization model. The possible mechanisms for the hypothesized genomic recombinations and the conditions that allow, induce, or select for such events are discussed.
Microbiology of a Nitrite-Oxidizing Bioreactor
Authors: Burrell Paul C. , Keller Jürg , Blackall Linda L. 
Published: December 2019

ABSTRACT

The microbiology of the biomass from a nitrite-oxidizing sequencing batch reactor (NOSBR) fed with an inorganic salts solution and nitrite as the sole energy source that had been operating for 6 months was investigated by microscopy, by culture-dependent methods, and by molecular biological methods, and the seed sludge that was used to inoculate the NOSBR was investigated by molecular biological methods. The NOSBR sludge comprised a complex and diverse microbial community containing gram-negative and gram-positive rods, cocci, and filaments. By culture-dependent methods (i.e., micromanipulation and sample dilution and spread plate inoculation), 16 heterotrophs (6 gram positive and 10 gram negative) were identified in the NOSBR sludge (RC), but no autotrophs were isolated. 16S ribosomal DNA clone libraries of the two microbial communities revealed that the seed sludge (GC) comprised a complex microbial community dominated by
Proteobacteria
(29% beta subclass; 18% gamma subclass) and high G+C gram-positive bacteria (10%). Three clones (4%) were closely related to the autotrophic nitrite-oxidizer
Nitrospira moscoviensis
. The NOSBR sludge was overwhelmingly dominated by bacteria closely related to
N. moscoviensis
(89%). Two clone sequences were similar to those of the genus
Nitrobacter
. Near-complete insert sequences of eight RC and one GC
N. moscoviensis
clone were determined and phylogenetically analyzed. This is the first report of the presence of bacteria from the
Nitrospira
phylum in wastewater treatment systems, and it is hypothesized that these bacteria are the unknown nitrite oxidizers in these processes.
Ecophysiological and Phylogenetic Studies of
Nevskia ramosa
in Pure Culture
Authors: Stürmeyer Heike , Overmann Jörg , Babenzien Hans-Dietrich , Cypionka Heribert 
Published: December 2019

ABSTRACT

During the last 100 years, the neuston bacterium
Nevskia ramosa
has been described several times. This bacterium forms conspicuous rosette-like microcolonies at the air-water interface. In this study, pure cultures of
Nevskia ramosa
were obtained for the first time, from a bog lake (strain Soe1, DSMZ 11499
T
) and a freshwater ditch (strain OL1, DSMZ 11500). The isolates showed special adaptations to life in the epineuston. They formed hydrophobic surface films with a dull appearance.
N. ramosa
is sensitive to UV radiation but revealed a very effective photorepair mechanism. Exposure to light at a wavelength of 350 nm after UV treatment raised the number of surviving cells by several orders of magnitude. The isolates grew with a broad range of organic substrates. Surface films were formed only in the absence of combined nitrogen; however, nitrogenase activity was not detected. It appears that during growth at the air-water interface the cells benefit from trapping ammonia from the air. The G+C content of the DNA was 67.8 and 69.0 mol% for strains Soe1 and OL1, respectively. The slight difference was confirmed by enterobacterial repetitive intergenic consensus PCR. The 16S rRNA sequences revealed 99.2% similarity. Thus, both isolates belong to the same species. The phylogenetic analysis indicated that
Nevskia
is a member of the gamma-subclass
Proteobacteria
that has no known close relatives.
Purification of Extracellular Cholesterol Oxidase with High Activity in the Presence of Organic Solvents from
Pseudomonas
sp. Strain ST-200
Authors: Doukyu Noriyuki , Aono Rikizo 
Published: December 2019

ABSTRACT

Extracellular cholesterol oxidase of
Pseudomonas
sp. strain ST-200 was purified from the culture supernatant. This oxidase contained bound flavin and was categorized as a 3β-hydroxysteroid oxidase, converting 3β-hydroxyl groups to keto groups. The molecular mass was 60 kDa. The enzyme was stable at pH 4 to 11 and active at pH 5.0 to 8.5, showing optimal activity at pH 7 at 60°C. The Michaelis constant of the ST-200 cholesterol oxidase was lower than those of commercially available oxidases. The cholesterol oxidation rate was enhanced 3- to 3.5-fold in the presence of organic solvents, with log
P
ow
values (partition coefficients of the organic solvent between
n
-octanol and water), in the range of 2.1 to 4.2, compared with that in the absence of organic solvents.
Mercury Methylation by Interspecies Hydrogen and Acetate Transfer between Sulfidogens and Methanogens
Authors: Pak K.-R. , Bartha R. 
Published: December 2019

ABSTRACT

Cocultures of
Desulfovibrio desulfuricans
and
Methanococcus maripaludis
grew on sulfate-free lactate medium while vigorously methylating Hg
2+
. Individually, neither bacterium could grow or methylate mercury in this medium. Similar synergistic growth of sulfidogens and methanogens may create favorable conditions for Hg
2+
methylation in low-sulfate anoxic freshwater sediments.