bluuebunny/crossref_metadata_2025_split huggingface dataset:
Phylogenetic Analysis of Particle-Attached and Free-Living Bacterial Communities in the Columbia River, Its Estuary, and the Adjacent Coastal Ocean
Authors: Crump Byron C. , Armbrust E. Virginia , Baross John A. 
Published: December 2019

ABSTRACT

The Columbia River estuary is a dynamic system in which estuarine turbidity maxima trap and extend the residence time of particles and particle-attached bacteria over those of the water and free-living bacteria. Particle-attached bacteria dominate bacterial activity in the estuary and are an important part of the estuarine food web. PCR-amplified 16S rRNA genes from particle-attached and free-living bacteria in the Columbia River, its estuary, and the adjacent coastal ocean were cloned, and 239 partial sequences were determined. A wide diversity was observed at the species level within at least six different bacterial phyla, including most subphyla of the class
Proteobacteria
. In the estuary, most particle-attached bacterial clones (75%) were related to members of the genus
Cytophaga
or of the α, γ, or δ subclass of the class
Proteobacteria
. These same clones, however, were rare in or absent from either the particle-attached or the free-living bacterial communities of the river and the coastal ocean. In contrast, about half (48%) of the free-living estuarine bacterial clones were similar to clones from the river or the coastal ocean. These free-living bacteria were related to groups of cosmopolitan freshwater bacteria (β-proteobacteria, gram-positive bacteria, and
Verrucomicrobium
spp.) and groups of marine organisms (gram-positive bacteria and α-proteobacteria [SAR11 and
Rhodobacter
spp.]). These results suggest that rapidly growing particle-attached bacteria develop into a uniquely adapted estuarine community and that free-living estuarine bacteria are similar to members of the river and the coastal ocean microbial communities. The high degree of diversity in the estuary is the result of the mixing of bacterial communities from the river, estuary, and coastal ocean.
Purification, Characterization, and Heterologous Expression in
Fusarium venenatum
of a Novel Serine Carboxypeptidase from
Aspergillus oryzae
Authors: Blinkovsky Alexander M. , Byun Tony , Brown Kimberly M. , Golightly Elizabeth J. 
Published: December 2019

ABSTRACT

A novel serine carboxypeptidase (EC
3.4.16.1
) was found in an
Aspergillus oryzae
fermentation broth and was purified to homogeneity. This enzyme has a molecular weight of ca. 67,000, as determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, and its specific activity is 21 U/mg for carbobenzoxy (Z)-Ala-Glu at pH 4.5 and 25°C. It has a ratio of bimolecular constants for Z-Ala-Lys and Z-Ala-Phe of 3.75. Optimal enzyme activity occurs at pH 4 to 4.5 and 58 to 60°C for Z-Ala-Ile. The N terminus of this carboxypeptidase is blocked. Internal fragments, obtained by cyanogen bromide digestion, were sequenced. PCR primers were then made based on the peptide sequence information, and the full-length gene sequence was obtained. An expression vector that contained the recombinant carboxypeptidase gene was used to transform a
Fusarium venenatum
host strain. The transformed strain of
F. venenatum
expressed an active recombinant carboxypeptidase. In
F. venenatum
, the recombinant carboxypeptidase produced two bands which had molecular weights greater than the molecular weight of the native carboxypeptidase from
A. oryzae
. Although the molecular weights of the native and recombinant enzymes differ, these enzymes have very similar kinetic parameters.
Genetic Structure of Natural Populations of
Escherichia coli
in Wild Hosts on Different Continents
Authors: Souza Valeria , Rocha Martha , Valera Aldo , Eguiarte Luis E. 
Published: December 2019

ABSTRACT

Current knowledge of genotypic and phenotypic diversity in the species
Escherichia coli
is based almost entirely on strains recovered from humans or zoo animals. In this study, we analyzed a collection of 202 strains obtained from 81 mammalian species representing 39 families and 14 orders in Australia and the Americas, as well as several reference strains; we also included a strain from a reptile and 10 from different families of birds collected in Mexico. The strains were characterized genotypically by multilocus enzyme electrophoresis (MLEE) and phenotypically by patterns of sugar utilization, antibiotic resistance, and plasmid profile. MLEE analysis yielded an estimated genetic diversity (H) of 0.682 for 11 loci. The observed genetic diversity in this sample is the greatest yet reported for
E. coli
. However, this genetic diversity is not randomly distributed; geographic effects and host taxonomic group accounted for most of the genetic differentiation. The genetic relationship among the strains showed that they are more associated by origin and host order than is expected by chance. In a dendrogram, the ancestral cluster includes primarily strains from Australia and ECOR strains from groups B and C. The most differentiated
E. coli
in our analysis are strains from Mexican carnivores and strains from humans, including those in the ECOR group A. The kinds and numbers of sugars utilized by the strains varied by host taxonomic group and country of origin. Strains isolated from bats were found to exploit the greatest range of sugars, while those from primates utilized the fewest. Toxins are more frequent in strains from rodents from both continents than in any other taxonomic group. Strains from Mexican wild mammals were, on average, as resistant to antibiotics as strains from humans in cities. On average, the Australian strains presented a lower antibiotic resistance than the Mexican strains. However, strains recovered from hosts in cities carried significantly more plasmids than did strains isolated from wild mammals. Previous studies have shown that natural populations of
E. coli
harbor an extensive genetic diversity that is organized in a limited number of clones. However, knowledge of this worldwide bacterium has been limited. Here, we suggest that the strains from a wide range of wild hosts from different regions of the world are organized in an ecotypic structure where adaptation to the host plays an important role in the population structure.
Heat-Induced Expression and Chemically Induced Expression of the
Escherichia coli
Stress Protein HtpG Are Affected by the Growth Environment
Authors: Mason C. Anthony , Dünner Janine , Indra Paul , Colangelo Teresa 
Published: December 2019

ABSTRACT

Differences in expression of the
Escherichia coli
stress protein HtpG were found following exposure of exponentially growing cells to heat or chemical shock when cells were grown under different environmental conditions. With an
htpG::lacZ
reporter system,
htpG
expression increased in cells grown in a complex medium (Luria-Bertani [LB] broth) following a temperature shock at 45°C. In contrast, no HtpG overexpression was detected in cells grown in a glucose minimal medium, despite a decrease in the growth rate. Similarly, in pyruvate-grown cells there was no heat shock induction of HtpG expression, eliminating the possibility that repression of HtpG in glucose-grown
E. coli
was due to catabolite repression. When 5 mM phenol was used as a chemical stress agent for cells growing in LB broth, expression of HtpG increased. However, when LB-grown cells were subjected to stress with 10 mM phenol and when both 5 and 10 mM phenol were added to glucose-grown cultures, repression of
htpG
expression was observed. 2-Chlorophenol stress resulted in overexpression of HtpG when cells were grown in complex medium but repression of HtpG synthesis when cells were grown in glucose. No induction of
htpG
expression was seen with 2,4-dichlorophenol in cells grown with either complex medium or glucose. The results suggest that, when a large pool of amino acids and proteins is available, as in complex medium, a much stronger stress response is observed. In contrast, when cells are grown in a simple glucose mineral medium,
htpG
expression either is unaffected or is even repressed by imposition of a stress condition. The results demonstrate the importance of considering differences in growth environment in order to better understand the nature of the response to an imposed stress condition.
Community Analysis of Biofilters Using Fluorescence In Situ Hybridization Including a New Probe for the
Xanthomonas
Branch of the Class
Proteobacteria
Authors: Friedrich Udo , Naismith Michèle M. , Altendorf Karlheinz , Lipski André 
Published: December 2019

ABSTRACT

Domain-, class-, and subclass-specific rRNA-targeted probes were applied to investigate the microbial communities of three industrial and three laboratory-scale biofilters. The set of probes also included a new probe (named XAN818) specific for the
Xanthomonas
branch of the class
Proteobacteria
; this probe is described in this study. The members of the
Xanthomonas
branch do not hybridize with previously developed rRNA-targeted oligonucleotide probes for the α-, β-, and γ-
Proteobacteria
. Bacteria of the
Xanthomonas
branch accounted for up to 4.5% of total direct counts obtained with 4′,6-diamidino-2-phenylindole. In biofilter samples, the relative abundance of these bacteria was similar to that of the γ-
Proteobacteria. Actinobacteria
(gram-positive bacteria with a high G+C DNA content) and α-
Proteobacteria
were the most dominant groups. Detection rates obtained with probe EUB338 varied between about 40 and 70%. For samples with high contents of gram-positive bacteria, these percentages were substantially improved when the calculations were corrected for the reduced permeability of gram-positive bacteria when formaldehyde was used as a fixative. The set of applied bacterial class- and subclass-specific probes yielded, on average, 58.5% (± a standard deviation of 23.0%) of the corrected eubacterial detection rates, thus indicating the necessity of additional probes for studies of biofilter communities. The
Xanthomonas
-specific probe presented here may serve as an efficient tool for identifying potential phytopathogens. In situ hybridization proved to be a practical tool for microbiological studies of biofiltration systems.
Cloning, Molecular Analysis, and Expression of the Polyhydroxyalkanoic Acid Synthase (
phaC
) Gene from
Chromobacterium violaceum
Authors: Kolibachuk Dana , Miller Andrea , Dennis Douglas 
Published: December 2019

ABSTRACT

The polyhydroxyalkanoic acid synthase gene from
Chromobacterium violaceum
(
phaC
Cv
) was cloned and characterized. A 6.3-kb
Bam
HI fragment was found to contain both
phaC
Cv
and the polyhydroxyalkanoic acid (PHA)-specific 3-ketothiolase (
phaA
Cv
).
Escherichia coli
strains harboring this fragment produced significant levels of PHA synthase and 3-ketothiolase, as judged by their activities. While
C. violaceum
accumulated poly(3-hydroxybutyrate) or poly(3-hydroxybutyrate-
co
-3-hydroxyvalerate) when grown on a fatty acid carbon source,
Klebsiella aerogenes
and
Ralstonia eutropha
(formerly
Alcaligenes eutrophus
), harboring
phaC
Cv
, accumulated the above-mentioned polymers and, additionally, poly(3-hydroxybutyrate-
co
-3-hydroxyhexanoate) when even-chain-length fatty acids were utilized as the carbon source. This finding suggests that the metabolic environments of these organisms are sufficiently different to alter the product range of the
C. violaceum
PHA synthase. Neither recombinant
E. coli
nor recombinant
Pseudomonas putida
harboring
phaC
Cv
accumulated significant levels of PHA. Sequence analysis of the
phaC
Cv
product shows homology with several PHA synthases, most notably a 48% identity with that of
Alcaligenes latus
(GenBank accession no. AAD10274).
Diel Rhythms in Ribulose-1,5-Bisphosphate Carboxylase/Oxygenase and Glutamine Synthetase Gene Expression in a Natural Population of Marine Picoplanktonic Cyanobacteria (
Synechococcus
spp.)
Authors: Wyman Michael 
Published: December 2019

ABSTRACT

Diel periodicity in the expression of key genes involved in carbon and nitrogen assimilation in marine
Synechococcus
spp. was investigated in a natural population growing in the surface waters of a cyclonic eddy in the northeast Atlantic Ocean.
Synechococcus
sp. cell concentrations within the upper mixed layer showed a net increase of three- to fourfold during the course of the experiment (13 to 22 July 1991), the population undergoing approximately one synchronous division per day. Consistent with the observed temporal pattern of phycoerythrin (CpeBA) biosynthesis, comparatively little variation was found in
cpeBA
mRNA abundance during either of the diel cycles investigated. In marked contrast, the relative abundance of transcripts originating from the genes encoding the large subunit of ribulose bisphosphate carboxylase/oxygenase (
rbcL
) and glutamine synthetase (
glnA
) showed considerable systematic temporal variation and oscillated during the course of each diel cycle in a reciprocal rhythm. Whereas activation of
rbcL
transcription was clearly not light dependent, expression of
glnA
appeared sensitive to endogenous changes in the physiological demands for nitrogen that arise as a natural consequence of temporal periodicity in photosynthetic carbon assimilation. The data presented support the hypothesis that a degree of temporal separation may exist between the most active periods of carbon and nitrogen assimilation in natural populations of marine
Synecoccoccus
spp.
Quantification of the Flavonoid-Degrading Bacterium
Eubacterium ramulus
in Human Fecal Samples with a Species-Specific Oligonucleotide Hybridization Probe
Authors: Simmering Rainer , Kleessen Brigitta , Blaut Michael 
Published: December 2019

ABSTRACT

To investigate the occurrence of the flavonoid-degrading bacterium
Eubacterium ramulus
in the human intestinal tract, an oligonucleotide probe designated S-S-E.ram-0997-a-A-18 was designed and validated, with over 90 bacterial strains representing the dominant described human fecal flora. Application of S-S-E.ram-0997-a-A-18 to fecal samples from 20 subjects indicated the presence of
E. ramulus
in each individual tested in numbers from 4.4 × 10
7
to 2.0 × 10
9
cells/g of fecal dry mass. Six fecal
E. ramulus
isolates were recognized by S-S-E.ram-0997-a-A-18 but exhibited different band patterns when analyzed by randomly amplified polymorphic DNA.
Bacterioplankton Compositions of Lakes and Oceans: a First Comparison Based on Fluorescence In Situ Hybridization
Authors: Glöckner Frank Oliver , Fuchs Bernhard M. , Amann Rudolf 
Published: December 2019

ABSTRACT

Fluorescence in situ hybridization (FISH) with rRNA-targeted oligonucleotide probes was used to investigate the phylogenetic composition of bacterioplankton communities in several freshwater and marine samples. An average of about 50% of the cells were detected by probes for the domains
Bacteria
and
Archaea
, and of these, about half could be identified at the subdomain level with a set of group-specific probes. Beta subclass proteobacteria constituted a dominant fraction in freshwater systems, accounting for 16% (range, 3 to 32%) of the cells, although they were essentially absent in the marine samples examined. Members of the
Cytophaga-Flavobacterium
cluster were the most abundant group detected in the marine systems, accounting for 18% (range, 2 to 72%) of the 4′,6-diamidino-2-phenylindole (DAPI) counts, and they were also important in freshwater systems (7%, range 0 to 18%). Furthermore, members of the alpha and gamma subclasses of
Proteobacteria
as well as members of the
Planctomycetales
were detected in both freshwater and marine water in abundances <7%.
Physicochemical Parameters for Growth of the Sea Ice Bacteria
Glaciecola punicea
ACAM 611
T
and
Gelidibacter
sp. Strain IC158
Authors: Nichols D. S. , Greenhill A. R. , Shadbolt C. T. , Ross T. , McMeekin T. A. 
Published: December 2019

ABSTRACT

The water activity and pH ranges for growth of
Glaciecola punicea
(a psychrophile) were extended when this organism was grown at suboptimal rather than optimal temperatures. No such extension was observed for
Gelidibacter
sp. strain IC158 (a psychrotolerant bacterium) at analogous temperatures. Salinity and pH may be primary physicochemical parameters controlling bacterial community development in sea ice.
Polyphosphate Kinase of
Acinetobacter
sp. Strain ADP1: Purification and Characterization of the Enzyme and Its Role during Changes in Extracellular Phosphate Levels
Authors: Trelstad Piper L. , Purdhani Pooja , Geißdörfer Walter , Hillen Wolfgang , Keasling J. D. 
Published: December 2019

ABSTRACT

Polyphosphate (polyP) is a ubiquitous biopolymer whose function and metabolism are incompletely understood. The polyphosphate kinase (PPK) of
Acinetobacter
sp. strain ADP1, an organism that accumulates large amounts of polyP, was purified to homogeneity and characterized. This enzyme, which adds the terminal phosphate from ATP to a growing chain of polyP, is a 79-kDa monomer. PPK is sensitive to magnesium concentrations, and optimum activity occurs in the presence of 3 mM MgCl
2
. The optimum pH was between pH 7 and 8, and significant reductions in activity occurred at lower pH values. The greatest activity occurred at 40°C. The half-saturation ATP concentration for PPK was 1 mM, and the maximum PPK activity was 28 nmol of polyP monomers per μg of protein per min. PPK was the primary, although not the sole, enzyme responsible for the production of polyP in
Acinetobacter
sp. strain ADP1. Under low-phosphate (P
i
) conditions, despite strong induction of the
ppk
gene, there was a decline in net polyP synthesis activity and there were near-zero levels of polyP in
Acinetobacter
sp. strain ADP1. Once excess phosphate was added to the P
i
-starved culture, both the polyP synthesis activity and the levels of polyP rose sharply. Increases in polyP-degrading activity, which appeared to be mainly due to a polyphosphatase and not to PPK working in reverse, were detected in cultures grown under low-P
i
conditions. This activity declined when phosphate was added.
Development and Characterization of a Gene Expression Reporter System for
Clostridium acetobutylicum
ATCC 824
Authors: Tummala Seshu B. , Welker Neil E. , Papoutsakis Eleftherios T. 
Published: December 2019

ABSTRACT

A gene expression reporter system (pHT3) for
Clostridium acetobutylicum
ATCC 824 was developed by using the
lacZ
gene from
Thermoanaerobacterium thermosulfurogenes
EM1 as the reporter gene. In order to test the reporter system, promoters of three key metabolic pathway genes,
ptb
(coding for phosphotransbutyrylase),
thl
(coding for thiolase), and
adc
(coding for acetoacetate decarboxylase), were cloned upstream of the reporter gene in pHT3 in order to construct vectors pHT4, pHT5, and pHTA, respectively. Detection of β-galactosidase activity in time course studies performed with strains ATCC 824(pHT4), ATCC 824(pHT5), and ATCC 824(pHTA) demonstrated that the reporter gene produced a functional β-galactosidase in
C. acetobutylicum
. In addition, time course studies revealed differences in the β-galactosidase specific activity profiles of strains ATCC 824(pHT4), ATCC 824(pHT5), and ATCC 824(pHTA), suggesting that the reporter system developed in this study is able to effectively distinguish between different promoters. The stability of the β-galactosidase produced by the reporter gene was also examined with strains ATCC 824(pHT4) and ATCC 824(pHT5) by using chloramphenicol treatment to inhibit protein synthesis. The data indicated that the β-galactosidase produced by the
lacZ
gene from
T. thermosulfurogenes
EM1 was stable in the exponential phase of growth. In pH-controlled fermentations of ATCC 824(pHT4), the kinetics of β-galactosidase formation from the
ptb
promoter and phosphotransbutyrylase formation from its own autologous promoter were found to be similar.
Generalized Transduction of Small
Yersinia enterocolitica
Plasmids
Authors: Hertwig Stefan , Popp Andreas , Freytag Barbara , Lurz Rudi , Appel Bernd 
Published: December 2019

ABSTRACT

To study phage-mediated gene transfer in
Yersinia
, the ability of
Yersinia
phages to transduce naturally occurring plasmids was investigated. The transduction experiments were performed with a temperate phage isolated from a pathogenic
Yersinia enterocolitica
strain and phage mixtures isolated from sewage. Small plasmids (4.3 and 5.8 kb) were transduced at a frequency of 10
−5
to 10
−7
/PFU. However, we could not detect the transduction of any indigenous virulence plasmid (ca. 72 kb) in pathogenic
Yersinia
strains. Transductants obtained by infection with the temperate phage were lysogenic and harbored the phage genome in their chromosomes.
Purification of Mutacin III from Group III
Streptococcus mutans
UA787 and Genetic Analyses of Mutacin III Biosynthesis Genes
Authors: Qi Fengxia , Chen Ping , Caufield Page W. 
Published: December 2019

ABSTRACT

Previously, members of our group reported the isolation and characterization of mutacin II from
Streptococcus mutans
T8 and the genetic analyses of the mutacin II biosynthesis genes (J. Novak, P. W. Caufield, and E. J. Miller, J. Bacteriol. 176:4316–4320, 1994; F. Qi, P. Chen, and P. W. Caufield, Appl. Environ. Microbiol. 65:652–658, 1999; P. Chen, F. Qi, J. Novak, and P. W. Caufield, Appl. Environ. Microbiol. 65:1356–1360, 1999). In this study, we cloned and sequenced the mutacin III biosynthesis gene locus from a group III strain of
S. mutans
, UA787. DNA sequence analysis revealed eight open reading frames, which we designated
mutR
,
-A
,
-A′
,
-B
,
-C
,
-D
,
-P
, and
-T
. MutR bears strong homology with MutR of mutacin II, while MutA, -B, -C, -D, -P, and -T are counterparts of proteins in the lantibiotic epidermin group. MutA′ has 60% amino acid identity with MutA and therefore appears to be a duplicate of MutA. Insertional inactivation demonstrated that
mutA
is an essential gene for mutacin III production, while
mutA′
is not required. Mutacin III was purified to homogeneity by using reverse-phase high-pressure liquid chromatography. N-terminal peptide sequencing of the purified mutacin III determined
mutA
to be the structural gene for prepromutacin III. The molecular mass of the purified peptide was measured by laser disorption mass spectrophotometry and found to be 2,266.43 Da, consistent with our supposition that mutacin III has posttranslational modifications similar to those of the lantibiotic epidermin.
An α-
l
-Arabinofuranosidase from
Trichoderma reesei
Containing a Noncatalytic Xylan-Binding Domain
Authors: Nogawa Masahiro , Yatsui Kenji , Tomioka Akiko , Okada Hirofumi , Morikawa Yasushi 
Published: December 2019

ABSTRACT

l
-Sorbose, an excellent cellulase and xylanase inducer from
Trichoderma reesei
PC-3-7, also induced α-
l
-arabinofuranosidase (α-AF) activity. An α-AF induced by
l
-sorbose was purified to homogeneity, and its molecular mass was revealed to be 35 kDa (AF35), which was not consistent with that of the previously reported α-AF. Another species, with a molecular mass of 53 kDa (AF53), which is identical to that of the reported α-AF, was obtained by a different purification procedure. Acid treatment of the ammonium sulfate-precipitated fraction at pH 3.0 in the purification steps or pepsin treatment of the purified AF53 reduced the molecular mass to 35 kDa. Both purified enzymes have the same enzymological properties, such as pH and temperature effects on activity and kinetic parameters for
p
-nitrophenyl-α-
l
-arabinofuranoside (pNPA). Moreover, the N-terminal amino acid sequences of these enzymes were identical with that of the reported α-AF. Therefore, it is obvious that AF35 results from the proteolytic cleavage of the C-terminal region of AF53. Although AF35 and AF53 showed the same catalytic constant with pNPA, the former showed drastically reduced specific activity against oat spelt xylan compared to the latter. Furthermore, AF53 was bound to xylan rather than to crystalline cellulose (Avicel), but AF35 could not be bound to any of the glycans. These results suggest that AF53 is a modular glycanase, which consists of an N-terminal catalytic domain and a C-terminal noncatalytic xylan-binding domain.