bluuebunny/crossref_metadata_2025_split huggingface dataset:
Molecular Diversity of
Renibacterium salmoninarum
Isolates Determined by Randomly Amplified Polymorphic DNA Analysis
Authors: Grayson T. Hilton , Atienzar Franck A. , Alexander Sarah M. , Cooper Lynne F. , Gilpin Martyn L. 
Published: April 2009

ABSTRACT

The molecular diversity among 60 isolates of
Renibacterium salmoninarum
which differ in place and date of isolation was investigated by using randomly amplified polymorphic DNA (RAPD) analysis. Isolates were grouped into 21 banding patterns which did not reflect the biological source. Four 16S-23S rRNA intergenic spacer (ITS1) sequence variations and two alleles of an exact tandem repeat locus, ETR-A, were the bases for formation of distinct groups within the RAPD clusters. This study provides evidence that the most common ITS1 sequence variant, SV1, possesses two copies of a 51-bp repeat unit at ETR-A and has been widely dispersed among countries which are associated with mainstream intensive salmonid culture.
Use of Sublimation To Prepare Solid Microbial Media with Water-Insoluble Substrates
Authors: Alley Jeremy F. , Brown Lewis R. 
Published: April 2009

ABSTRACT
A method was developed to deposit a visible layer of water-insoluble compounds via sublimation onto the surface of solid media. The compound is sublimed from a heated aluminum dish containing the compound onto the surface of an inverted, ice-cooled, inoculated agar petri dish. The method results in the deposition of a thin, even layer on the agar surface without the use of solvent. After incubation, clearing zones around colonies indicate the presence of compound-degrading microorganisms.
Homogeneity of Danish Environmental and Clinical Isolates of
Shewanella algae
Authors: Vogel Birte Fonnesbech , Holt Hanne Marie , Gerner-Smidt Peter , Bundvad Anemone , Søgaard Per , Gram Lone 
Published: April 2009

ABSTRACT

Danish isolates of
Shewanella algae
constituted by whole-cell protein profiling a very homogeneous group, and no clear distinction was seen between strains from the marine environment and strains of clinical origin. Although variation between all strains was observed by ribotyping and random amplified polymorphic DNA analysis, no clonal relationship between infective strains was found. From several patients, clonally identical strains of
S. algae
were reisolated up to 8 months after the primary isolation, indicating that the same strain may be able to maintain the infection.
Isolation and Characterization of
Campylobacter jejuni
subsp.
jejuni
from Macaroni Penguins (
Eudyptes chrysolophus
) in the Subantarctic Region
Authors: Broman T. , Bergström S. , On S. L. W. , Palmgren H. , McCafferty D. J. , Sellin M. , Olsen B. 
Published: April 2009

ABSTRACT

On Bird Island, South Georgia, albatrosses (
n
= 140), penguins (
n
= 100), and fur seals (
n
= 206) were sampled for
Campylobacter jejuni. C. jejuni
subsp.
jejuni
was recovered from three macaroni penguins (
Eudyptes chrysolophus
). These isolates, the first reported for the subantarctic region, showed low genetic diversity and high similarity to Northern Hemisphere
C. jejuni
isolates, possibly suggesting recent introduction to the area.
Multiregional Evaluation of the SimPlate Heterotrophic Plate Count Method Compared to the Standard Plate Count Agar Pour Plate Method in Water
Authors: Jackson R. Wayne , Osborne Karen , Barnes Gary , Jolliff Carol , Zamani Dianna , Roll Bruce , Stillings Amy , Herzog David , Cannon Shelly , Loveland Scott 
Published: April 2009

ABSTRACT

A new SimPlate heterotrophic plate count (HPC) method (IDEXX Laboratories, Westbrook, Maine) was compared with the pour plate method at 35°C for 48 h. Six laboratories tested a total of 632 water samples. The SimPlate HPC method was found to be equivalent to the pour plate method by regression analysis (
r
= 0.95;
y
= 0.99
X
+ 0.06).
Increased Production of Zeaxanthin and Other Pigments by Application of Genetic Engineering Techniques to
Synechocystis
sp. Strain PCC 6803
Authors: Lagarde Delphine , Beuf Laurent , Vermaas Wim 
Published: April 2009

ABSTRACT

The
psbAII
locus was used as an integration platform to overexpress genes involved in carotenoid biosynthesis in
Synechocystis
sp. strain PCC 6803 under the control of the strong
psbAII
promoter. The sequences of the genes encoding the yeast isopentenyl diphosphate isomerase (
ipi
) and the
Synechocystis
β-carotene hydroxylase (
crtR
) and the linked
Synechocystis
genes coding for phytoene desaturase and phytoene synthase (
crtP
and
crtB
, respectively) were introduced into
Synechocystis
, replacing the
psbAII
coding sequence. Expression of
ipi
,
crtR
, and
crtP
and
crtB
led to a large increase in the corresponding transcript levels in the mutant strains, showing that the
psbAII
promoter can be used to drive transcription and to overexpress various genes in
Synechocystis
. Overexpression of
crtP
and
crtB
led to a 50% increase in the myxoxanthophyll and zeaxanthin contents in the mutant strain, whereas the β-carotene and echinenone contents remained unchanged. Overexpression of
crtR
induced a 2.5-fold increase in zeaxanthin accumulation in the corresponding overexpressing mutant compared to that in the wild-type strain. In this mutant strain, zeaxanthin becomes the major pigment (more than half the total amount of carotenoid) and the β-carotene and echinenone amounts are reduced by a factor of 2. However, overexpression of
ipi
did not result in a change in the carotenoid content of the mutant. To further alter the carotenoid content of
Synechocystis
, the
crtO
gene, encoding β-carotene ketolase, which converts β-carotene to echinenone, was disrupted in the wild type and in the overexpressing strains so that they no longer produced echinenone. In this way, by a combination of overexpression and deletion of particular genes, the carotenoid content of cyanobacteria can be altered significantly.
Importance of
pfkA
for Rapid Growth of
Enterobacter cloacae
during Colonization of Crop Seeds
Authors: Roberts D. P. , Dery P. D. , Yucel I. , Buyer J. S. 
Published: April 2009

ABSTRACT

Enterobacter cloacae
A-11 is a prototrophic, glycolytic mutant of strain 501R3 with a single transposon insertion in
pfkA
. The populations of strain A-11 on cucumber and radish seeds were smaller than the populations of strain 501R3 in natural soil, but the populations of these two strains on pea, soybean, sunflower, and sweet corn seeds were similar (D. P. Roberts, P. D. Dery, I. Yucel, J. Buyer, M. A. Holtman, and D. Y. Kobayashi, Appl. Environ. Microbiol. 65:2513–2519, 1999). The net effect of the mutation in
pfkA
in vitro was a shift from rapid growth on certain carbohydrates detected in seed exudates to much slower growth on other carbohydrates, amino acids, and organic acids. The impact of the mutation in
pfkA
was greatest on the growth rate of
E. cloacae
on the seeds that released the smallest quantities of fructose, other carbohydrates, and amino acids. Corn, pea, soybean, and sunflower seeds released total amounts of carbohydrates and amino acids at rates that were approximately 10- to 100-fold greater than the rates observed with cucumber and radish seeds for the first 24 h after inhibition began. The growth rate of strain A-11 was significantly less (50% less) than the growth rate of strain 501R3 on radish seeds, and the growth rate of strain A-11 was too low to estimate on cucumber seeds in sterile sand for the first 24 h after inhibition began. The growth rate of strain A-11 was also significantly lower on soybean seeds, but it was only 17% lower than the growth rate of strain 501R3. The growth rates of strains 501R3 and A-11 were similar on pea, sunflower, and corn seeds in sterile sand for the first 30 h after imbibition began. Large reductions in the growth rates of strain A-11 on seeds were correlated with subsequent decreased levels of colonization of seeds compared to the levels of colonization of strain 501R3. The strain A-11 populations were significantly smaller than the strain 501R3 populations only on radish and cucumber seeds. The mutation in
pfkA
appears to decrease the level of colonization by
E. cloacae
for seeds that release small quantities of reduced carbon compounds by decreasing the size of the pool of compounds that support rapid growth by this bacterium.
Expression of
Alcaligenes eutrophus
Flavohemoprotein and Engineered
Vitreoscilla
Hemoglobin-Reductase Fusion Protein for Improved Hypoxic Growth of
Escherichia coli
Authors: Frey Alexander D. , Bailey James E. , Kallio Pauli T. 
Published: April 2009

ABSTRACT

Expression of the
vhb
gene encoding hemoglobin from
Vitreoscilla
sp. (VHb) in several organisms has been shown to improve microaerobic cell growth and enhance oxygen-dependent product formation. The amino-terminal hemoglobin domain of the flavohemoprotein (FHP) of the gram-negative hydrogen-oxidizing bacterium
Alcaligenes eutrophus
has 51% sequence homology with VHb. However, like other flavohemoglobins and unlike VHb, FHP possesses a second (carboxy-terminal) domain with NAD(P)H and flavin adenine dinucleotide (FAD) reductase activities. To examine whether the carboxy-terminal redox-active site of flavohemoproteins can be used to improve the positive effects of VHb in microaerobic
Escherichia coli
cells, we fused sequences encoding NAD(P)H, FAD, or NAD(P)H-FAD reductase activities of
A. eutrophus
in frame after the
vhb
gene. Similarly, the gene for FHP was modified, and expression cassettes encoding amino-terminal hemoglobin (FHPg), FHPg-FAD, FHPg-NAD, or FHP activities were constructed. Biochemically active heme proteins were produced from all of these constructions in
Escherichia coli
, as indicated by their ability to scavenge carbon monoxide. The presence of FHP or of VHb-FAD-NAD reductase increased the final cell density of transformed wild-type
E. coli
cells approximately 50 and 75%, respectively, for hypoxic fed-batch culture relative to the control synthesizing VHb. Approximately the same final optical densities were achieved with the
E. coli
strains expressing FHPg and VHb. The presence of VHb-FAD or FHPg-FAD increased the final cell density slightly relative to the VHb-expressing control under the same cultivation conditions. The expression of VHb-NAD or FHPg-NAD fusion proteins reduced the final cell densities approximately 20% relative to the VHb-expressing control. The VHb-FAD-NAD reductase-expressing strain was also able to synthesize 2.3-fold more recombinant β-lactamase relative to the VHb-expressing control.
A New Species of
Devosia
That Forms a Unique Nitrogen-Fixing Root-Nodule Symbiosis with the Aquatic Legume
Neptunia natans
(L.f.) Druce
Authors: Rivas Raul , Velázquez Encarna , Willems Anne , Vizcaíno Nieves , Subba-Rao Nanjappa S. , Mateos Pedro F. , Gillis Monique , Dazzo Frank B. , Martínez-Molina Eustoquio 
Published: April 2009

ABSTRACT

Rhizobia are the common bacterial symbionts that form nitrogen-fixing root nodules in legumes. However, recently other bacteria have been shown to nodulate and fix nitrogen symbiotically with these plants.
Neptunia natans
is an aquatic legume indigenous to tropical and subtropical regions and in African soils is nodulated by
Allorhizobium undicola.
This legume develops an unusual root-nodule symbiosis on floating stems in aquatic environments through a unique infection process. Here, we analyzed the low-molecular-weight RNA and 16S ribosomal DNA (rDNA) sequence of the same fast-growing isolates from India that were previously used to define the developmental morphology of the unique infection process in this symbiosis with
N. natans
and found that they are phylogenetically located in the genus
Devosia
, not
Allorhizobium
or
Rhizobium
. The 16S rDNA sequences of these two
Neptunia
-nodulating
Devosia
strains differ from the only species currently described in that genus,
Devosia riboflavina.
From the same isolated colonies, we also located their
nodD
and
nifH
genes involved in nodulation and nitrogen fixation on a plasmid of approximately 170 kb. Sequence analysis showed that their
nodD
and
nifH
genes are most closely related to
nodD
and
nifH
of
Rhizobium tropici
, suggesting that this newly described
Neptunia
-nodulating
Devosia
species may have acquired these symbiotic genes by horizontal transfer.
Membranes of Class IIa Bacteriocin-Resistant
Listeria monocytogenes
Cells Contain Increased Levels of Desaturated and Short-Acyl-Chain Phosphatidylglycerols
Authors: Vadyvaloo Viveka , Hastings John W. , van der Merwe Marthinus J. , Rautenbach Marina 
Published: April 2009

ABSTRACT

A major concern in the use of class IIa bacteriocins as food preservatives is the well-documented resistance development in target
Listeria
strains. We studied the relationship between leucocin A, a class IIa bacteriocin, and the composition of the major phospholipid, phosphatidylglycerol (PG), in membranes of both sensitive and resistant
L. monocytogenes
strains. Two wild-type strains,
L. monocytogenes
B73 and 412, two spontaneous mutants of
L. monocytogenes
B73 with intermediate resistance to leucocin A (±2.4 and ±4 times the 50% inhibitory concentrations [IC
50
] for sensitive strains), and two highly resistant mutants of each of the wild-type strains (>500 times the IC
50
for sensitive strains) were analyzed. Electrospray mass spectrometry analysis showed an increase in the ratios of unsaturated to saturated and short- to long-acyl-chain species of PG in all the resistant
L. monocytogenes
strains in our study, although their sensitivities to leucocin A were significantly different. This alteration in membrane phospholipids toward PGs containing shorter, unsaturated acyl chains suggests that resistant strains have cells with a more fluid membrane. The presence of this phenomenon in a strain (
L. monocytogenes
412P) which is resistant to both leucocin A and pediocin PA-1 may indicate a link between membrane composition and class IIa bacteriocin resistance in some
L. monocytogenes
strains. Treatment of strains with sterculic acid methyl ester (SME), a desaturase inhibitor, resulted in significant changes in the leucocin A sensitivity of the intermediate-resistance strains but no changes in the sensitivity of highly resistant strains. There was, however, a decrease in the amount of unsaturated and short-acyl-chain PGs after treatment with SME in one of the intermediate and both of the highly resistant strains, but the opposite effect was observed for the sensitive strains. It appears, therefore, that membrane adaptation may be part of a resistance mechanism but that several resistance mechanisms may contribute to a resistance phenotype and that levels of resistance vary according to the type of mechanisms present.
Characterization of the Adaptive Response to Trichloroethylene-Mediated Stresses in
Ralstonia pickettii
PKO1
Authors: Park Joonhong , Kukor Jerome J. , Abriola Linda M. 
Published: April 2009

ABSTRACT

In
Ralstonia pickettii
PKO1, a denitrifying toluene oxidizer that carries a toluene-3-monooxygenase (T3MO) pathway, the biodegradation of toluene and trichloroethylene (TCE) by the organism is induced by TCE at high concentrations. In this study, the effect of TCE preexposure was studied in the context of bacterial protective response to TCE-mediated toxicity in this organism. The results of TCE degradation experiments showed that cells induced by TCE at 110 mg/liter were more tolerant to TCE-mediated stress than were those induced by TCE at lower concentrations, indicating an ability of PKO1 to adapt to TCE-mediated stress. To characterize the bacterial protective response to TCE-mediated stress, the effect of TCE itself (solvent stress) was isolated from TCE degradation-dependent stress (toxic intermediate stress) in the subsequent chlorinated ethylene toxicity assays with both nondegradable tetrachloroethylene and degradable TCE. The results of the toxicity assays showed that TCE preexposure led to an increase in tolerance to TCE degradation-dependent stress rather than to solvent stress. The possibility that such tolerance was selected by TCE degradation-dependent stress during TCE preexposure was ruled out because a similar extent of tolerance was observed in cells that were induced by toluene, whose metabolism does not produce any toxic products. These findings suggest that the adaptation of TCE-induced cells to TCE degradation-dependent stress was caused by the combined effects of solvent stress response and T3MO pathway expression.
Increasing Acidification of Nonreplicating
Lactococcus lactis
Δ
thyA
Mutants by Incorporating ATPase Activity
Authors: Pedersen Martin B. , Koebmann Brian J. , Jensen Peter R. , Nilsson Dan 
Published: April 2009

ABSTRACT

Lactococcus lactis
MBP71 Δ
thyA
(thymidylate synthase) cannot synthesize dTTP de novo, and DNA replication is dependent on thymidine in the growth medium. In the nonreplicating state acidification by MBP71 was completely insensitive to bacteriophages (M. B. Pedersen, P. R. Jensen, T. Janzen, and D. Nilsson, Appl. Environ. Microbiol. 68:3010-3023, 2002). For nonreplicating MBP71 the biomass increased 3.3-fold over the first 3.5 h, and then the increase stopped. The rate of acidification increased 2.3-fold and then started to decrease. Shortly after inoculation the lactic acid flux was 60% of that of exponentially growing MBP71. However, when nonspecific ATPase activity was incorporated into MBP71, the lactic acid flux was restored to 100% but not above that point, indicating that control over the flux switched from ATP demand to ATP supply (i.e., to sugar transport and glycolysis). As determined by growing nonreplicating cells with high ATPase activity on various sugar sources, it appeared that glycolysis exerted the majority of the control. ATPase activity also stimulated the rate of acidification by nonreplicating MBP71 growing in milk, and pH 5.2 was reached 40% faster than it was without ATPase activity. We concluded that ATPase activity is a functional means of increasing acidification by nonreplicating
L. lactis
.
Postadaptational Resistance to Benzalkonium Chloride and Subsequent Physicochemical Modifications of
Listeria monocytogenes
Authors: To Monica S. , Favrin Stacy , Romanova Nadya , Griffiths Mansel W. 
Published: April 2009

ABSTRACT

Many studies have demonstrated that bacteria, including
Listeria monocytogenes
, are capable of adapting to disinfectants used in industrial settings after prolonged exposure to sublethal concentrations. However, the consequent alterations of the cell surface due to sanitizer adaptation of this pathogen are not fully understood. Two resistant and four sensitive
L. monocytogenes
strains from different sources were progressively subcultured with increasing sublethal concentrations of a surfactant, benzalkonium chloride (BC). To evaluate the effects of acquired tolerance to BC, parent and adapted strains were compared by using several morphological and physiological tests. Sensitive strains showed at least a fivefold increase in the MIC, while the MIC doubled for resistant strains after the adaptation period. The hydrophobicities of cells of parent and adapted strains were similar. Serological testing indicated that antigen types 1 and 4 were both present on the cell surface of adapted cells. The data suggest that efflux pumps are the major mechanism of adaptation in sensitive strains and are less important in originally resistant isolates. A different, unknown mechanism was responsible for the original tolerance of resistant isolates. In an originally resistant strain, there was a slight shift in the fatty acid profile after adaptation, whereas sensitive strains had similar profiles. Electron micrographs revealed morphological differences after adaptation. The changes in cell surface antigens, efflux pump utilization, and fatty acid profiles suggest that different mechanisms are used by resistant and sensitive strains for adaptation to BC.
Improved System for Protein Engineering of the Hydroxylase Component of Soluble Methane Monooxygenase
Authors: Smith Thomas J. , Slade Susan E. , Burton Nicolas P. , Murrell J. Colin , Dalton Howard 
Published: April 2009

ABSTRACT

Soluble methane monooxygenase (sMMO) of
Methylosinus
trichosporium
OB3b is a three-component oxygenase that catalyses the O
2
- and NAD(P)H-dependent oxygenation of methane and numerous other substrates. Despite substantial interest in the use of genetic techniques to study the mechanism of sMMO and manipulate its substrate specificity, directed mutagenesis of active-site residues was previously impossible because no suitable heterologous expression system had been found for expression in a highly active form of the hydroxylase component, which is an (αβγ)
2
complex containing the binuclear iron active site. A homologous expression system that enabled the expression of recombinant wild-type sMMO in a derivative of
M
.
trichosporium
OB3b from which the chromosomal copy of the sMMO-encoding operon had been partially deleted was previously reported. Here we report substantial development of this method to produce a system for the facile construction and expression of mutants of the hydroxylase component of sMMO. This new system has been used to investigate the functions of Cys 151 and Thr 213 of the α subunit, which are the only nonligating protonated side chains in the hydrophobic active site. Both residues were found to be critical for the stability and/or activity of sMMO, but neither was essential for oxygenation reactions. The T213S mutant was purified to >98% homogeneity. It had the same iron content as the wild type and had 72% wild-type activity toward toluene but only 17% wild-type activity toward propene; thus, its substrate profile was significantly altered. With these results, we have demonstrated proof of the principle for protein engineering of this uniquely versatile enzyme.
Mode of Action of the Antimicrobial Peptide Aureocin A53 from
Staphylococcus aureus
Authors: Netz Daili Jacqueline Aguilar , Bastos Maria do Carmo de Freire , Sahl Hans-Georg 
Published: April 2009

ABSTRACT

We investigated the mode of action of aureocin A53 on living bacterial cells and model membranes. Aureocin A53 acted bactericidally against
Staphylococcus simulans
22, with >90% of the cells killed within a few minutes. Cell death was followed by lysis, as indicated by a clearing of the cell suspension and Gram staining. Aureocin A53 rapidly dissipated the membrane potential and simultaneously stopped biosynthesis of DNA, polysaccharides, and protein. Aureocin A53 induced a rapid release of preaccumulated glutamate and Rb
+
. Experiments on model membranes demonstrated that aureocin A53 provoked significant leakage of carboxyfluorescein (CF) exclusively from acidic liposomes but only at relatively high concentrations (0.5 to 8 mol%). Thus, the bactericidal activity of aureocin A53 derives from membrane permeation via generalized membrane destruction rather than by formation of discrete pores within membranes. Tryptophan emission fluorescence spectroscopy demonstrated interaction of aureocin A53 with both acidic and neutral membranes, as indicated by similar blue shifts. Since there was no significant aureocin A53-induced CF leakage from neutral liposomes, its appears that the peptide does interact with neutral lipids without provoking membrane damage.