bluuebunny/crossref_metadata_2025_split huggingface dataset:
Key Aromatic-Ring-Cleaving Enzyme, Protocatechuate 3,4-Dioxygenase, in the Ecologically Important Marine
Roseobacter
Lineage
Authors: Buchan Alison , Collier Lauren S. , Neidle Ellen L. , Moran Mary Ann 
Published: July 2002

ABSTRACT

Aromatic compound degradation in six bacteria representing an ecologically important marine taxon of the α-proteobacteria was investigated. Initial screens suggested that isolates in the
Roseobacter
lineage can degrade aromatic compounds via the β-ketoadipate pathway, a catabolic route that has been well characterized in soil microbes. Six
Roseobacter
isolates were screened for the presence of protocatechuate 3,4-dioxygenase, a key enzyme in the β-ketoadipate pathway. All six isolates were capable of growth on at least three of the eight aromatic monomers presented (anthranilate, benzoate,
p
-hydroxybenzoate, salicylate, vanillate, ferulate, protocatechuate, and coumarate). Four of the
Roseobacter
group isolates had inducible protocatechuate 3,4-dioxygenase activity in cell extracts when grown on
p
-hydroxybenzoate. The
pcaGH
genes encoding this ring cleavage enzyme were cloned and sequenced from two isolates,
Sagittula stellata
E-37 and isolate Y3F, and in both cases the genes could be expressed in
Escherichia coli
to yield dioxygenase activity. Additional genes involved in the protocatechuate branch of the β-ketoadipate pathway (
pcaC
,
pcaQ
, and
pobA
) were found to cluster with
pcaGH
in these two isolates. Pairwise sequence analysis of the
pca
genes revealed greater similarity between the two
Roseobacter
group isolates than between genes from either
Roseobacter
strain and soil bacteria. A degenerate PCR primer set targeting a conserved region within PcaH successfully amplified a fragment of
pcaH
from two additional
Roseobacter
group isolates, and Southern hybridization indicated the presence of
pcaH
in the remaining two isolates. This evidence of protocatechuate 3,4-dioxygenase and the β-ketoadipate pathway was found in all six
Roseobacter
isolates, suggesting widespread abilities to degrade aromatic compounds in this marine lineage.
Exogenous Isolation of Antibiotic Resistance Plasmids from Piggery Manure Slurries Reveals a High Prevalence and Diversity of IncQ-Like Plasmids
Authors: Smalla Kornelia , Heuer Holger , Götz Antje , Niemeyer Dagmar , Krögerrecklenfort Ellen , Tietze Erhard 
Published: July 2002

ABSTRACT

Antibiotic resistance plasmids were exogenously isolated in biparental matings with piggery manure bacteria as plasmid donors in
Escherichia coli
CV601 and
Pseudomonas putida
UWC1 recipients. Surprisingly, IncQ-like plasmids were detected by dot blot hybridization with an IncQ
oriV
probe in several
P. putida
UWC1 transconjugants. The capture of IncQ-like plasmids in biparental matings indicates not only their high prevalence in manure slurries but also the presence of efficiently mobilizing plasmids. In order to elucidate unusual hybridization data (weak or no hybridization with IncQ
repB
or IncQ
oriT
probes) four IncQ-like plasmids (pIE1107, pIE1115, pIE1120, and pIE1130), each representing a different
Eco
RV restriction pattern, were selected for a more thorough plasmid characterization after transfer into
E. coli
K-12 strain DH5α by transformation. The characterization of the IncQ-like plasmids revealed an astonishingly high diversity with regard to phenotypic and genotypic properties. Four different multiple antibiotic resistance patterns were found to be conferred by the IncQ-like plasmids. The plasmids could be mobilized by the RP4 derivative pTH10 into
Acinetobacter
sp.,
Ralstonia eutropha
,
Agrobacterium tumefaciens
, and
P. putida
, but they showed diverse patterns of stability under nonselective growth conditions in different host backgrounds. Incompatibility testing and PCR analysis clearly revealed at least two different types of IncQ-like plasmids. PCR amplification of total DNA extracted directly from different manure samples and other environments indicated the prevalence of both types of IncQ plasmids in manure, sewage, and farm soil. These findings suggest that IncQ plasmids play an important role in disseminating antibiotic resistance genes.
Monitoring Bacterial Transport by Stable Isotope Enrichment of Cells
Authors: Holben William E. , Ostrom Peggy H. 
Published: July 2002

ABSTRACT

Understanding the transport and behavior of bacteria in the environment has broad implications in diverse areas, ranging from agriculture to groundwater quality, risk assessment, and bioremediation. The ability to reliably track and enumerate specific bacterial populations in the context of native communities and environments is key to developing this understanding. We report a novel bacterial tracking approach, based on altering the stable carbon isotope value (δ
13
C) of bacterial cells, which provides specific and sensitive detection and quantification of those cells in environmental samples. This approach was applied to the study of bacterial transport in saturated porous media. The transport of introduced organisms was indicated by mass spectrometric analysis of groundwater samples, where the presence of
13
C-enriched bacteria resulted in increased δ
13
C values of the samples, allowing specific and sensitive detection and enumeration of the bacteria of interest. We demonstrate the ability to produce highly
13
C-enriched bacteria, present data indicating that results obtained with this approach accurately represent intact introduced bacteria, and include field data on the use of this stable isotope approach to monitor in situ bacterial transport. This detection strategy allows sensitive detection of an introduced, unmodified bacterial strain in the presence of the indigenous bacterial community, including itself in its unenriched form.
Dynamics of Bacterial Community Composition and Activity during a Mesocosm Diatom Bloom
Authors: Riemann Lasse , Steward Grieg F. , Azam Farooq 
Published: July 2002

ABSTRACTBacterial community composition, enzymatic activities, and carbon dynamics were examined during diatom blooms in four 200-liter laboratory seawater mesocosms. The objective was to determine whether the dramatic shifts in growth rates and ectoenzyme activities, which are commonly observed during the course of phytoplankton blooms and their subsequent demise, could result from shifts in bacterial community composition. Nutrient enrichment of metazoan-free seawater resulted in diatom blooms dominated by aThalassiosirasp., which peaked 9 days after enrichment (≈24 μg of chlorophyllaliter−1). At this time bacterial abundance abruptly decreased from 2.8 × 106to 0.75 × 106ml−1, and an analysis of bacterial community composition, by denaturing gradient gel electrophoresis (DGGE) of PCR-amplified 16S rRNA gene fragments, revealed the disappearance of three dominant phylotypes. Increased viral and flagellate abundances suggested that both lysis and grazing could have played a role in the observed phylotype-specific mortality. Subsequently, new phylotypes appeared and bacterial production, abundance, and enzyme activities shifted from being predominantly associated with the <1.0-μm size fraction towards the >1.0-μm size fraction, indicating a pronounced microbial colonization of particles. Sequencing of DGGE bands suggested that the observed rapid and extensive colonization of particulate matter was mainly by specialized α-Proteobacteria- andCytophagales-related phylotypes. These particle-associated bacteria had high growth rates as well as high cell-specific aminopeptidase, β-glucosidase, and lipase activities. Rate measurements as well as bacterial population dynamics were almost identical among the mesocosms indicating that the observed bacterial community dynamics were systematic and repeatable responses to the manipulated conditions.
Transformation of
Acinetobacter
sp. Strain BD413(pFG4Δ
nptII
) with Transgenic Plant DNA in Soil Microcosms and Effects of Kanamycin on Selection of Transformants
Authors: Nielsen Kaare M. , van Elsas Jan D. , Smalla Kornelia 
Published: July 2002

ABSTRACT

Here we show that horizontal transfer of DNA, extracted from transgenic sugar beets, to bacteria, based on homologous recombination, can occur in soil. Restoration of a 317-bp-deleted
nptII
gene in
Acinetobacter
sp. strain BD413(pFG4) cells incubated in sterile soil microcosms was detected after addition of nutrients and transgenic plant DNA encoding a functional
nptII
gene conferring bacterial kanamycin resistance. Selective effects of the addition of kanamycin on the population dynamics of
Acinetobacter
sp. cells in soil were found, and high concentrations of kanamycin reduced the CFU of
Acinetobacter
sp. cells from 10
9
CFU/g of soil to below detection. In contrast to a chromosomal
nptII
-encoded kanamycin resistance, the pFG4-generated resistance was found to be unstable over a 31-day incubation period in vitro.
Bioconversion of Ferulic Acid into Vanillic Acid by Means of a Vanillate-Negative Mutant of
Pseudomonas fluorescens
Strain BF13
Authors: Civolani Claudio , Barghini Paolo , Roncetti Anna Rita , Ruzzi Maurizio , Schiesser Alma 
Published: July 2002

ABSTRACT

From a ferulic-acid-degrading
Pseudomonas fluorescens
strain (BF13), we have isolated a transposon mutant, which retained the ability to bioconvert ferulic acid into vanillic acid but lost the ability to further degrade the latter acid. The mutant, BF13-97, was very stable, and therefore it was suitable to be used as a biocatalyst for the preparative synthesis of vanillic acid from ferulic acid. By use of resting cells we determined the effect on the bioconversion rate of several parameters, such as the addition of nutritional factors, the concentration of the biomass, and the carbon source on which the biomass was grown. The optimal yield of vanillic acid was obtained with cells pregrown on M9 medium containing
p
-coumaric acid (0.1% [wt/vol]) as a sole carbon source and yeast extract (0.001% [wt/vol]) as a source of nutritional factors. Under these conditions, 1 mg (wet weight) of biomass produced 0.23 mg of vanillic acid per h. The genomic region of BF13-97 flanking the transposon's site of insertion was cloned and sequenced revealing two open reading frames of 1,062 (
vanA
) and 954 (
vanB
) bp, respectively. The
van
genes are organized in a cluster and encode the subunits of the vanillate-
O
-demethylase, which catalyzes the first step of the vanillate catabolism. Amino acid sequences deduced from
vanA
and
vanB
genes were shown to have high identity with known VanAs and VanBs from
Pseudomonas
and
Acinetobacter
spp. Highly conserved regions known to exist in class IA oxygenases were also found in the vanillate-
O
-demethylase components from
P. fluorescens
BF13. The terminal oxygenase VanA is characterized by a conserved Rieske-type [2Fe-2S]
R
ligand center. The reductase VanB contains a plant-type ferredoxin [2Fe-2S]
Fd
, flavin mononucleotide, and NAD-ribose binding domains which are located in its C-terminal and N-terminal halves, respectively. Transfer of wild-type
vanAB
genes to BF13-97 complemented this mutant, which recovered its ability to grow on either vanillic or ferulic acid.
Sequence Analysis and Initial Characterization of Two Isozymes of Hydroxylaminobenzene Mutase from
Pseudomonas pseudoalcaligenes
JS45
Authors: Davis John K. , Paoli George C. , He Zhongqi , Nadeau Lloyd J. , Somerville Charles C. , Spain Jim C. 
Published: July 2002

ABSTRACT

Pseudomonas pseudoalcaligenes
JS45 grows on nitrobenzene by a partially reductive pathway in which the intermediate hydroxylaminobenzene is enzymatically rearranged to 2-aminophenol by hydroxylaminobenzene mutase (HAB mutase). The properties of the enzyme, the reaction mechanism, and the evolutionary origin of the gene(s) encoding the enzyme are unknown. In this study, two open reading frames (
habA
and
habB
), each encoding an HAB mutase enzyme, were cloned from a
P. pseudoalcaligenes
JS45 genomic library and sequenced. The open reading frames encoding HabA and HabB are separated by 2.5 kb and are divergently transcribed. The deduced amino acid sequences of HabA and HabB are 44% identical. The HAB mutase specific activities in crude extracts of
Escherichia coli
clones synthesizing either HabA or HabB were similar to the specific activities of extracts of strain JS45 grown on nitrobenzene. HAB mutase activity in
E. coli
extracts containing HabB withstood heating at 85°C for 10 min, but extracts containing HabA were inactivated when they were heated at temperatures above 60°C. HAB mutase activity in extracts of
P. pseudoalcaligenes
JS45 grown on nitrobenzene exhibited intermediate temperature stability. Although both the
habA
gene and the
habB
gene conferred HAB mutase activity when they were separately cloned and expressed in
E. coli
, reverse transcriptase PCR analysis indicated that only
habA
is transcribed in
P. pseudoalcaligenes
JS45. A mutant strain derived from strain JS45 in which the
habA
gene was disrupted was unable to grow on nitrobenzene, which provided physiological evidence that HabA is involved in the degradation of nitrobenzene. A strain in which
habB
was disrupted grew on nitrobenzene. Gene Rv3078 of
Mycobacterium tuberculosis
H37Rv encodes a protein whose deduced amino acid sequence is 52% identical to the HabB amino acid sequence.
E. coli
containing
M. tuberculosis
gene Rv3078 cloned into pUC18 exhibited low levels of HAB mutase activity. Sequences that exhibit similarity to transposable element sequences are present between
habA
and
habB
, as well as downstream of
habB
, which suggests that horizontal gene transfer resulted in acquisition of one or both of the
hab
genes.
Duplication of a Truncated Paralog of the Family B DNA Polymerase Gene
Aa-polB
in the
Agrocybe aegerita
Mitochondrial Genome
Authors: Barroso Gerard , Bois Frederic , Labarère Jacques 
Published: July 2002

ABSTRACT

The
Agrocybe aegerita
mitochondrial genome contains a truncated family B DNA polymerase gene (
Aa-polB P1
) whose nucleotide sequence is 86% identical to the previously described and potentially functional
Aa-polB
gene. A tRNA
Met
gene occurs at the 3′ end of the
Aa-polB P1
gene. The
Aa-polB P1
gene could result from reverse transcription of an
Aa-polB
mRNA primed by a tRNA
Met
followed by the integration of the cDNA after recombination at the mitochondrial tRNA locus. Two naturally occurring alleles of
Aa-polB P1
carry one or two copies of the disrupted sequence. In strains with two copies of
Aa-polB P1
, these copies are inverted relative to one another and separated by a short sequence carrying the tRNA
Met
gene. Both
A. aegerita
mitochondrial family B DNA polymerases were found to be related to other family B DNA polymerases (36 to 53% amino acid similarity), including the three enzymes of the archaebacterium
Sulfolobus solfataricus
. If mitochondria originated from a fusion between a
Clostridium
-like eubacterium and a
Sulfolobus
-like archaebacterium, then the
A. aegerita
family B DNA polymerase genes could be remnants of the archaebacterial genes.
Purification and Characterization of Two Different α-l-Rhamnosidases, RhaA and RhaB, fromAspergillus aculeatus
Authors: Manzanares Paloma , van den Broeck Hetty C. , de Graaff Leo H. , Visser Jaap 
Published: July 2002

ABSTRACTTwo proteins exhibiting α-l-rhamnosidase activity, RhaA and RhaB, were identified upon fractionation and purification of a culture filtrate fromAspergillus aculeatusgrown on hesperidin. Both proteins were shown to be N glycosylated and had molecular masses of 92 and 85 kDa, of which approximately 24 and 15%, respectively, were contributed by carbohydrate. RhaA and RhaB, optimally active at pH 4.5 to 5, showedKmandVmaxvalues of 2.8 mM and 24 U/mg (RhaA) and 0.30 mM and 14 U/mg (RhaB) when tested forp-nitrophenyl-α-l-rhamnopyranoside. Both enzymes were able to hydrolyze α-1,2 and α-1,6 linkages to β-d-glucosides. Using polyclonal antibodies, the corresponding cDNA of both α-l-rhamnosidases,rhaAandrhaB, was cloned. On the basis of the amino acid sequences derived from the cDNA clones, both proteins are highly homologous (60% identity).
Two Distinct Hemolytic Activities in
Xenorhabdus nematophila
Are Active against Immunocompetent Insect Cells
Authors: Brillard Julien , Ribeiro Carlos , Boemare Noël , Brehélin Michel , Givaudan Alain 
Published: July 2002

ABSTRACT

Xenorhabdus
spp. and
Photorhabdus
spp. are major insect bacterial pathogens symbiotically associated with nematodes. These bacteria are transported by their nematode hosts into the hemocoel of the insect prey, where they proliferate within hemolymph. In this work we report that wild strains belonging to different species of both genera are able to produce hemolysin activity on blood agar plates. Using a hemocyte monolayer bioassay, cytolytic activity against immunocompetent cells from the hemolymph of
Spodoptera littoralis
(Lepidoptera: Noctuidae) was found only in supernatants of
Xenorhabdus
; none was detected in supernatants of various strains of
Photorhabdus
. During in vitro bacterial growth of
Xenorhabdus nematophila
F1, two successive bursts of cytolytic activity were detected. The first extracellular cytolytic activity occurred when bacterial cells reached the stationary phase. It also displayed a hemolytic activity on sheep red blood cells, and it was heat labile. Among insect hemocyte types, granulocytes were the preferred target. Lysis of hemocytes by necrosis was preceded by a dramatic vacuolization of the cells. In contrast the second burst of cytolytic activity occurred late during stationary phase and caused hemolysis of rabbit red blood cells, and insect plasmatocytes were the preferred target. This second activity is heat resistant and produced shrinkage and necrosis of hemocytes. Insertional inactivation of
flhD
gene in
X. nematophila
leads to the loss of hemolysis activity on sheep red blood cells and an attenuated virulence phenotype in
S. littoralis
(A. Givaudan and A. Lanois, J. Bacteriol. 182:107–115, 2000). This mutant was unable to produce the early cytolytic activity, but it always displayed the late cytolytic effect, preferably active on plasmatocytes. Thus,
X. nematophila
produced two independent cytolytic activities against different insect cell targets known for their major role in cellular immunity.
Differentiation of Two Bovine Lentiviruses by a Monoclonal Antibody on the Basis of Epitope Specificity
Authors: Zheng Ling , Zhang Shucheng , Wood Charles , Kapil Sanjay , Wilcox Graham E. , Loughin Thomas A. , Minocha H. C. 
Published: July 2002

ABSTRACT

Bovine immunodeficiency virus (BIV) and Jembrana disease virus (JDV) are bovine lentiviruses that are closely related genetically. A recombinant fusion protein containing the capsid protein of BIV expressed in
Escherichia coli
was used to immunize mice and produce monoclonal antibodies. Six hybridomas specific for BIV capsid protein were identified, and one antibody, designated 10H1, was characterized further. Competitive binding assays were performed to analyze the topography of antigenic determinants by enzyme-linked immunosorbent assay and demonstrated the existence of at least three distinct antigenic determinants on capsid protein. The monoclonal antibody reacted specifically with both BIV capsid and the recombinant fusion protein in Western immunoblot analyses. However, it did not react with the recombinant capsid fusion protein of JDV, indicating that BIV contains at least one unique epitope in the capsid protein that is absent in JDV. Further mapping of the epitope by chemical cleavage analysis identified that the epitope is located at the 6.4-kDa N terminus of the 29-kDa capsid protein. This monoclonal antibody assay will be valuable for distinguishing the two closely related lentiviruses by Western blotting.
Serum S100B Levels in Patients with Lupus Erythematosus: Preliminary Observation
Authors: Portela Luis V. C. , Brenol João C. T. , Walz Roger , Bianchin Marino , Tort Adriano B. L. , Canabarro Ubirajara P. , Beheregaray Simone , Marasca João A. , Xavier Ricardo M. , Neto Eurico C. , Gonçalves Carlos A. , Souza Diogo O. 
Published: July 2002

ABSTRACT
S100B is an astrocytic calcium-binding protein which has been proposed as a biochemical marker of brain damage or dysfunction in acute and chronic diseases. We investigated whether serum S100B levels could be related to systemic lupus erythematosus (SLE) activity. Patients were grouped as having inactive SLE (ISLE), active SLE without central nervous system (CNS) involvement (ASLE), or active SLE with unequivocal neurologic or psychiatric manifestation (NPSLE). The control group consisted of age- and sex-matched healthy blood donors. S100B levels were determined using a luminescence immunoassay. All SLE groups had higher levels of serum S100B than the control group. Among the SLE groups, significantly higher levels of serum S100B protein were found in the NPSLE group than in the ISLE and ASLE groups, and there was no significant difference in S100B levels between the ISLE and ASLE groups. These preliminary results point to a putative relevance of serum S100B protein levels in SLE patients, specifically concerning CNS involvement present in this disease.
Host Cellular Immune Response to Pneumococcal Lung Infection in Mice
Authors: Kadioglu Aras , Gingles Neill A. , Grattan Kate , Kerr Alison , Mitchell Tim J. , Andrew Peter W. 
Published: July 2002

ABSTRACTAlthough there is substantial evidence that pneumolysin is an important virulence factor in pneumococcal pneumonia, relatively little is known about how it influences cellular infiltration into the lungs. We investigated how the inability of mutant pneumococci to produce pneumolysin altered the pattern of inflammation and cellular infiltration into the lungs. The effect on bacterial growth in the lungs also was assessed. There were three phases of growth of wild-type bacteria in the lungs: a decline followed by a rapid increase and then stasis or decline. The absence of pneumolysin was associated with a more rapid early decline and then a much slower increase in numbers. The pattern of inflammatory-cell accumulation also had distinct stages, and the timing of these stages was influenced by the presence of pneumolysin. Neutrophils began to accumulate about 12 to 16 h after infection with wild-type pneumococci. This accumulation occurred after the early decline in pneumococcal numbers but coincided with the period of rapid growth. Following infection with pneumococci unable to make pneumolysin, neutrophil influx was slower and less intense. Coincident with the third stage of pneumococcal growth was an accumulation of T and B lymphocytes at the sites of inflammation, but the accumulation was not associated with an increase in the total number of lymphocytes in the lungs. Lymphocyte accumulation in the absence of pneumolysin occurred but was delayed.
Mutations in the Extracellular Protein Secretion Pathway Genes (
eps
) Interfere with Rugose Polysaccharide Production in and Motility of
Vibrio cholerae
Authors: Ali Afsar , Johnson Judith A. , Franco Augusto A. , Metzger Daniel J. , Connell Terry D. , Morris J. Glenn , Sozhamannan Shanmuga 
Published: July 2002

ABSTRACT

Vibrio cholerae
is the causal organism of the diarrheal disease cholera. The rugose variant of
V. cholerae
is associated with the secretion of an exopolysaccharide. The rugose polysaccharide has been shown to confer increased resistance to a variety of agents, such as chlorine, bioacids, and oxidative and osmotic stresses. It also promotes biofilm formation, thereby increasing the survival of the bacteria in the aquatic environments. Here we show that the extracellular protein secretion system (gene designated
eps
) is involved directly or indirectly in the production of rugose polysaccharide. A Tn
phoA
insertion in
epsD
gene of the
eps
operon abolished the production of rugose polysaccharide, reduced the secretion of cholera toxin and hemolysin, and resulted in a nonmotile phenotype. We have constructed defined mutations of the
epsD
and
epsE
genes that affected these phenotypes and complemented these defects by plasmid clones of the respective wild-type genes. These results suggest a major role for the
eps
system in pathogenesis and environmental survival of
V. cholerae
.