bluuebunny/crossref_metadata_2025_split huggingface dataset:
The Product of
arcR
, the Sixth Gene of the
arc
Operon of
Lactobacillus sakei
, Is Essential for Expression of the Arginine Deiminase Pathway
Authors: Zúñiga Manuel , Miralles María del Carmen , Pérez-Martínez Gaspar 
Published: November 2002

ABSTRACT

Lactobacillus sakei
is a lactic acid bacterium commonly used as a starter culture for dry sausage production and can utilize arginine via the arginine deiminase pathway. The
arcABCTD
cluster of
L. sakei
has been characterized, and transcriptional studies have shown that its expression is subject to carbon catabolite repression and induction by arginine. Downstream of
arcD
an additional gene has been found; this gene,
arcR
, codes for a putative regulatory protein of the Crp/Fnr family. Transcriptional studies have shown that
arcR
is coordinately transcribed with the remaining
arc
genes, and therefore, these genes constitute the
arcABCTDR
operon. Northern analysis also showed a complex pattern of transcripts, suggesting that processing and partial termination may play a role in regulation of the expression of individual genes of the operon. Inactivation of
arcR
led to arrest of transcription of the operon, indicating that the ArcR protein is essential for expression of the
arc
genes. The availability of this mutant made it possible to study whether the ability to utilize arginine affects the growth of
L. sakei
in meat fermentations. Under our experimental conditions, expression of arginine deiminase does not confer an obvious advantage to
L. sakei
, since the wild type and an
arcR
mutant strain displayed similar dynamics of growth.
Flow Cytometric Assessment of Membrane Integrity of Ethanol-StressedOenococcus oeniCells
Authors: Graça da Silveira M. , Vitória San Romão M. , Loureiro-Dias Maria C. , Rombouts Frans M. , Abee Tjakko 
Published: November 2002

ABSTRACTThe practical application of commercial malolactic starter cultures ofOenococcus oenisurviving direct inoculation in wine requires insight into the mechanisms involved in ethanol toxicity and tolerance in this organism. Exposure to ethanol resulted in an increase in the permeability of the cytoplasmic membrane, enhancing passive proton influx and concomitant loss of intracellular material (absorbing at 260 nm). Cells grown in the presence of 8% (vol/vol) ethanol revealed adaptation to ethanol stress, since these cells showed higher retention of compounds absorbing at 260 nm. Moreover, for concentrations higher than 10% (vol/vol), lower rates of passive proton influx were observed in these ethanol-adapted cells, especially at pH 3.5. The effect of ethanol onO. oenicells was studied as the ability to efficiently retain carboxyfluorescein (cF) as an indicator of membrane integrity and enzyme activity and the uptake of propidium iodide (PI) to assess membrane damage. Flow cytometric analysis of both ethanol-adapted and nonadapted cells with a mixture of the two fluorescent dyes, cF and PI, revealed three main subpopulations of cells: cF-stained intact cells; cF- and PI-stained permeable cells, and PI-stained damaged cells. The subpopulation ofO. oenicells that maintained their membrane integrity, i.e., cells stained only with cF, was three times larger in the population grown in the presence of ethanol, reflecting the protective effect of ethanol adaptation. This information is of major importance in studies of microbial fermentations in order to assign bulk activities measured by classical methods to the very active cells that are effectively responsible for the observations.
Impact of Protozoan Grazing on Bacterial Community Structure in Soil Microcosms
Authors: Rønn Regin , McCaig Allison E. , Griffiths Bryan S. , Prosser James I. 
Published: November 2002

ABSTRACTThe influence of grazing by a mixed assemblage of soil protozoa (seven flagellates and one amoeba) on bacterial community structure was studied in soil microcosms amended with a particulate resource (sterile wheat roots) or a soluble resource (a solution of various organic compounds). Sterilized soil was reinoculated with mixed soil bacteria (obtained by filtering and dilution) or with bacteria and protozoa. Denaturing gradient gel electrophoresis (DGGE) of PCR amplifications of 16S rRNA gene fragments, as well as community level physiological profiling (Biolog plates), suggested that the mixed protozoan community had significant effects on the bacterial community structure. Excising and sequencing of bands from the DGGE gels indicated that high-G+C gram-positive bacteria closely related toArthrobacterspp. were favored by grazing, whereas the excised bands that decreased in intensity were related to gram-negative bacteria. The percentages of intensity found in bands related to high G+C gram positives increased from 4.5 and 12.6% in the ungrazed microcosms amended with roots and nutrient solution, respectively, to 19.3 and 32.9% in the grazed microcosms. Protozoa reduced the average bacterial cell size in microcosms amended with nutrient solution but not in the treatment amended with roots. Hence, size-selective feeding may explain some but not all of the changes in bacterial community structure. Five different protozoan isolates (Acanthamoebasp., two species ofCercomonas,Thaumatomonassp., andSpumellasp.) had different effects on the bacterial communities. This suggests that the composition of protozoan communities is important for the effect of protozoan grazing on bacterial communities.
Linking of Microorganisms to Phenanthrene Metabolism in Soil by Analysis of
13
C-Labeled Cell Lipids
Authors: Johnsen Anders R. , Winding Anne , Karlson Ulrich , Roslev Peter 
Published: November 2002

ABSTRACT

Phenanthrene-metabolizing soil microbial communities were characterized by examining mineralization of [
14
C]phenanthrene, by most-probable-number (MPN) counting, by 16S-23S spacer DNA analysis of the numerically dominant, culturable phenanthrene-degrading isolates, and by examining incorporation of [
13
C]phenanthrene-derived carbon into sterols and polar lipid fatty acids (PLFAs). An unpolluted agricultural soil, a roadside soil diffusely polluted with polycyclic aromatic hydrocarbons (PAHs), and two highly PAH-polluted soils from industrial sites were analyzed. Microbial phenanthrene degraders were not detected by MPN counting in the agricultural soil and the roadside soil. In the industrial soils, phenanthrene degraders constituted 0.04 and 3.6% of the total number of CFU. 16S-23S spacer DNA analysis followed by partial 16S DNA sequencing of representative isolates from one of the industrial soils showed that one-half of the isolates belonged to the genus
Sphingomonas
and the other half were closely related to an unclassified beta-proteobacterium. The
13
C-PLFA profiles of the two industrial soils were relatively similar and resembled the profiles of phenanthrene-degrading
Sphingomonas
reference strains and unclassified beta-proteobacterium isolates but did not match the profiles of
Pseudomonas
,
Mycobacterium
, or
Nocardia
reference strains. The
13
C-PLFA profiles of phenanthrene degraders in the agricultural soil and the roadside soil were different from each other and different from the profiles of the highly polluted industrial soils. Only in the roadside soil were 10me/12me18:0 PLFAs enriched in
13
C, suggesting that actinomycetes metabolized phenanthrene in this soil. The
13
C-PLFA profiles of the unpolluted agricultural soil did not resemble the profiles of any of the reference strains. In all of the soils investigated, no excess
13
C was recovered in the 18:2ω6,9 PLFA, suggesting that fungi did not contribute significantly to assimilation of [
13
C]phenanthrene.
Molecular Analysis of the Nitrate-Reducing Community from Unplanted and Maize-Planted Soils
Authors: Philippot Laurent , Piutti Séverine , Martin-Laurent Fabrice , Hallet Stéphanie , Germon Jean Claude 
Published: November 2002

ABSTRACT

Microorganisms that use nitrate as an alternative terminal electron acceptor play an important role in the global nitrogen cycle. The diversity of the nitrate-reducing community in soil and the influence of the maize roots on the structure of this community were studied. The
narG
gene encoding the membrane bound nitrate reductase was selected as a functional marker for the nitrate-reducing community. The use of
narG
is of special interest because the phylogeny of the
narG
gene closely reflects the 16S ribosomal DNA phylogeny. Therefore, targeting the
narG
gene provided for the first time a unique insight into the taxonomic composition of the nitrate-reducing community in planted and unplanted soils. The PCR-amplified
narG
fragments were cloned and analyzed by restriction fragment length polymorphism (RFLP). In all, 60 RFLP types represented by two or more clones were identified in addition to the 58 RFLP types represented by only one clone. At least one clone belonging to each RFLP type was then sequenced. Several of the obtained sequences were not related to the
narG
genes from cultivated bacteria, suggesting the existence of unidentified nitrate-reducing bacteria in the studied soil. However, environmental sequences were also related to NarG from many bacterial divisions, i.e.,
Actinobacteria
and α, β, and γ
Proteobacteria
. The presence of the plant roots resulted in a shift in the structure of the nitrate-reducing community between the unplanted and planted soils. Sequencing of RFLP types dominant in the rhizosphere or present only in the rhizosphere revealed that they are related to NarG from the
Actinobacteria
in an astonishingly high proportion.
Novel Food-Grade Plasmid Vector Based on Melibiose Fermentation for the Genetic Engineering of
Lactococcus lactis
Authors: Boucher Isabelle , Parrot Marc , Gaudreau Hélène , Champagne Claude P. , Vadeboncoeur Christian , Moineau Sylvain 
Published: November 2002

ABSTRACT

The α-galactosidase gene (
aga
) and a gene coding for a putative transcriptional regulator from the LacI/GalR family (
galR
) of
Lactococcus raffinolactis
ATCC 43920 were cloned and sequenced. When transferred into
Lactococcus lactis
and
Pediococcus acidilactici
strains,
aga
modified the sugar fermentation profile of the strains from melibiose negative (Mel
−
) to melibiose positive (Mel
+
). Analysis of
galA
mutants of
L. lactis
subsp.
cremoris
MG1363 indicated that the putative galactose permease GalA is also needed to obtain the Mel
+
phenotype. Consequently, GalA may also transport melibiose into this strain. We demonstrated that when
aga
was associated with the theta-type replicon of a natural
L. lactis
plasmid, it constituted the selectable marker of a cloning vector named pRAF800. Transcriptional analysis by reverse transcriptase PCR suggests that this vector is also suitable for gene expression. The α-galactosidase activity conferred by pRAF800 was monitored in an industrial strain grown in the presence of various carbon sources. The results indicated that the enzymatic activity was induced by galactose and melibiose, but not by glucose or lactose. The gene encoding the phage defense mechanism, AbiQ, was cloned into pRAF800, and the resulting clone (pRAF803) was transferred into an industrial
L. lactis
strain that became highly phage resistant. The measurements of various growth parameters indicated that cells were not affected by the presence of pRAF803. Moreover, the plasmid was highly stable in this strain even under starter production conditions. The
L. raffinolactis aga
gene represents the basis of a novel and convenient food-grade molecular tool for the genetic engineering of lactic acid bacteria.
Relatedness of Chromosomal and Plasmid DNAs of
Erwinia pyrifoliae
and
Erwinia amylovora
Authors: McGhee Gayle C. , Schnabel Elise L. , Maxson-Stein Kimberly , Jones Beatrix , Stromberg Verlyn K. , Lacy George H. , Jones Alan L. 
Published: November 2002

ABSTRACT

The plant pathogen
Erwinia pyrifoliae
has been classified as a separate species from
Erwinia amylovora
based in part on differences in molecular properties. In this study, these and other molecular properties were examined for
E. pyrifoliae
and for additional strains of
E. amylovora
, including strains from brambles (
Rubus
spp.). The nucleotide composition of the internal transcribed spacer (ITS) region was determined for six of the seven 16S-23S rRNA operons detected in these species with a 16S rRNA gene probe. Each species contained four operons with a tRNA
Glu
gene and two with tRNA
Ile
and tRNA
Ala
genes, and analysis of the operons from five strains of
E. amylovora
indicated a high degree of ITS variability among them. One tRNA
Glu
-containing operon from
E. pyrifoliae
Ep1/96 was identical to one in
E. amylovora
Ea110, but three tRNA
Glu
operons and two tRNA
Ile
and tRNA
Ala
operons from
E. pyrifoliae
contained unique nucleotide changes. When
groEL
sequences were used for species-specific identification,
E. pyrifoliae
and
E. amylovora
were the closest phylogenetic relatives among a set of 12 bacterial species. The placement of
E. pyrifoliae
distinct from
E. amylovora
corroborated molecular hybridization data indicating low DNA-DNA similarity between them. Determination of the nucleotide sequence of plasmid pEP36 from
E. pyrifoliae
Ep1/96 revealed a number of presumptive genes that matched genes previously found in pEA29 from
E. amylovora
and similar organization for the genes and origins of replication. Also, pEP36 and pEA29 were incompatible with clones containing the reciprocal origin regions. Finally, the ColE1-like plasmid pEP2.6 from strain Ep1/96 contained sequences found in small plasmids in
E. amylovora
strains IL-5 and IH3-1.
Aerobic Metabolism of 4-Hydroxybenzoic Acid in Archaea via an Unusual Pathway Involving an Intramolecular Migration (NIH Shift)
Authors: Fairley D. J. , Boyd D. R. , Sharma N. D. , Allen C. C. R. , Morgan P. , Larkin M. J. 
Published: November 2002

ABSTRACT

A novel haloarchaeal strain,
Haloarcula
sp. strain D1, grew aerobically on 4-hydroxybenzoic acid (4HBA) as a sole carbon and energy source and is the first member of the domain
Archaea
reported to do so. Unusually, D1 metabolized 4HBA via gentisic acid rather than via protocatechuic acid, hydroquinone, or catechol. Gentisate was detected in 4HBA-grown cultures, and gentisate 1,2-dioxygenase activity was induced in 4HBA-grown cells. Stoichiometric accumulation of gentisate from 4HBA was demonstrated in 4HBA-grown cell suspensions containing 2,2′-dipyridyl (which strongly inhibits gentisate 1,2-dioxygenase). To establish whether initial 1-hydroxylation of 4HBA with concomitant 1,2-carboxyl group migration to yield gentisate occurred, 2,6-dideutero-4HBA was synthesized and used as a substrate. Deuterated gentisate was recovered from cell suspensions and identified as 3-deutero-gentisate, using gas chromatography-mass spectrometry and proton nuclear magnetic resonance spectroscopy. This structural isomer would be expected only if a 1,2-carboxyl group migration had taken place, and it provides compelling evidence that the 4HBA pathway in
Haloarcula
sp. strain D1 involves a hydroxylation-induced intramolecular migration. To our knowledge, this is the first report of a pathway which involves such a transformation (called an NIH shift) in the domain
Archaea
.
Conspicuous Veils Formed by Vibrioid Bacteria on Sulfidic Marine Sediment
Authors: Thar Roland , Kühl Michael 
Published: November 2002

ABSTRACTWe describe the morphology and behavior of a hitherto unknown bacterial species that forms conspicuous veils (typical dimensions, 30 by 30 mm) on sulfidic marine sediment. The new bacteria were enriched on complex sulfidic medium within a benthic gradient chamber in oxygen-sulfide countergradients, but the bacteria have so far not been isolated in pure culture, and a detailed characterization of their metabolism is still lacking. The bacteria are colorless, gram-negative, and vibrioid-shaped (1.3- to 2.5- by 4- to 10-μm) cells that multiply by binary division and contain several spherical inclusions of poly-β-hydroxybutyric acid. The cells have bipolar polytrichous flagella and exhibit a unique swimming pattern, rotating and translating along their short axis. Free-swimming cells showed aerotaxis and aggregated at ca. 2 μM oxygen within opposing oxygen-sulfide gradients, where they were able to attach via a mucous stalk, forming a cohesive whitish veil at the oxic-anoxic interface. Bacteria attached to the veil kept rotating and adapted their stalk lengths dynamically to changing oxygen concentrations. The joint action of rotating bacteria on the veil induced a homogeneous water flow from the oxic water region toward the veil, whereby the oxygen uptake rate could be enhanced up to six times, as shown by model calculations. The veils showed a pronounced succession pattern. New veils were generated de novo within 24 h and had a homogeneous whitish translucent appearance. Bacterial competitors or eukaryotic predators were apparently kept away by the low oxygen concentration prevailing at the veil surface. Frequently, within 2 days the veil developed a honeycomb pattern of regularly spaced holes. After 4 days, most veils were colonized by grazing ciliates, leading to the fast disappearance of the new bacteria. Several-week-old veils finally developed into microbial mats consisting of green, purple, and colorless sulfur bacteria.
Genetic and Phenotypic Analysis of
Acinetobacter baumannii
Insertion Derivatives Generated with a Transposome System
Authors: Dorsey Caleb W. , Tomaras Andrew P. , Actis Luis A. 
Published: November 2002

ABSTRACT

Acinetobacter baumannii
is a metabolically versatile pathogen that causes severe infections in compromised patients. However, little is known about the genes and factors involved in its basic physiology and virulence properties. Insertion mutagenesis was used to initiate the identification and characterization of some of these factors and genes in the prototype strain 19606. The utilization of the pLOFKm suicide delivery vector, which harbors a suicide mini-Tn
10
derivative, proved to be unsuccessful for this purpose. The EZ::TN 〈R6Kγ
ori
/KAN-2〉 Tnp transposome system available from Epicentre was then used in conjunction with electroporation to generate isogenic insertional derivatives of
A. baumannii
19606. Replica plating showed that 2% of the colonies that grew after electroporation on agar plates without antibiotics also grew in the presence of 40 μg of kanamycin per ml. DNA hybridization proved that all of the kanamycin-resistant derivatives contained the EZ::TN 〈R6Kγ
ori
/KAN-2〉 insertion element, which was mapped to different genomic locations. Replica plating on Simmons citrate agar and microtiter plate-plastic tube assays identified growth- and biofilm-defective derivatives, respectively. The location of the insertion in several of these derivatives was determined by self-ligation of
Nde
I- or
Eco
RI-digested genomic DNA and electroporation of
Escherichia coli
TransforMax EC100D (
pir
+
). Sequence analysis of the recovered plasmids showed that some of the
A. baumannii
19606 growth-defective derivatives contain insertions within genes encoding activities required for the generation of energy and cell wall components and for the biosynthesis of amino acids and purines. A gene encoding a protein similar to the GacS sensor kinase was interrupted in four derivatives, while another had an insertion in a gene coding for a hypothetical sensor kinase.
A. baumannii
19606 derivatives with defective attachment or biofilm phenotypes had insertions within genes that appear to be part of a chaperone-usher transport system described for other bacteria. DNA hybridization experiments showed that the presence of strain 19606 genes encoding regulatory and attachment or biofilm functions is widespread among other
A. baumannii
clinical isolates.
Field Studies Using a Recombinant Mycoinsecticide (
Metarhizium anisopliae
) Reveal that It Is Rhizosphere Competent
Authors: Hu Gang , St. Leger Raymond J. 
Published: November 2002

ABSTRACT

In the summer of 2000, we released genetically altered insect-pathogenic fungi onto a plot of cabbages at a field site on the Upper Marlboro Research Station, Md. The transformed derivatives of
Metarhizium anisopliae
ARSEF 1080, designated GPMa and GMa, carried the
Aequorea victoria
green fluorescent protein (
gfp
) gene alone (GMa) or with additional protease genes (
Pr1
) (GPMa). The study (i) confirmed the utility of
gfp
for monitoring pathogen strains in field populations over time, (ii) demonstrated little dissemination of transgenic strains and produced no evidence of transmission by nontarget insects, (iii) found that recombinant fungi were genetically stable over 1 year under field conditions, and (iv) determined that deployment of the transgenic strains did not depress the culturable indigenous fungal microflora. The major point of the study was to monitor the fate (survivorship) of transformants under field conditions. In nonrhizosphere soil, the amount of GMa decreased from 10
5
propagules/g at depths of 0 to 2 cm to 10
3
propagules/g after several months. However, the densities of GMa remained at 10
5
propagules/g in the inner rhizosphere, demonstrating that rhizospheric soils are a potential reservoir for
M. anisopliae
. These results place a sharp focus on the biology of the soil/root interphase as a site where plants, insects, and pathogens interact to determine fungal biocontrol efficacy, cycling, and survival. However, the rhizospheric effect was less marked for GPMa, and overall it showed reduced persistence in soils than did GMa.
Bacterial Group II Introns in a Deep-Sea Hydrothermal Vent Environment
Authors: Podar Mircea , Mullineaux Lauren , Huang Hon-Ren , Perlman Philip S. , Sogin Mitchell L. 
Published: November 2002

ABSTRACT
Group II introns are catalytic RNAs and mobile retrotransposable elements known to be present in the genomes of some nonmarine bacteria and eukaryotic organelles. Here we report the discovery of group II introns in a bacterial mat sample collected from a deep-sea hydrothermal vent near 9°N on the East Pacific Rise. One of the introns was shown to self-splice in vitro. This is the first example of marine bacterial introns from molecular population structure studies of microorganisms that live in the proximity of hydrothermal vents. These types of mobile genetic elements may prove useful in improving our understanding of bacterial genome evolution and may serve as valuable markers in comparative studies of bacterial communities.
Xylanase and Acetyl Xylan Esterase Activities of XynA, a Key Subunit of the
Clostridium cellulovorans
Cellulosome for Xylan Degradation
Authors: Kosugi Akihiko , Murashima Koichiro , Doi Roy H. 
Published: November 2002

ABSTRACT

The
Clostridium cellulovorans xynA
gene encodes the cellulosomal endo-1,4-β-xylanase XynA, which consists of a family 11 glycoside hydrolase catalytic domain (CD), a dockerin domain, and a NodB domain. The recombinant acetyl xylan esterase (rNodB) encoded by the NodB domain exhibited broad substrate specificity and released acetate not only from acetylated xylan but also from other acetylated substrates. rNodB acted synergistically with the xylanase CD of XynA for hydrolysis of acetylated xylan. Immunological analyses revealed that XynA corresponds to a major xylanase in the cellulosomal fraction. These results indicate that XynA is a key enzymatic subunit for xylan degradation in
C. cellulovorans
.