bluuebunny/crossref_metadata_2025 huggingface dataset:
Dual Labeling with Green Fluorescent Proteins for Confocal Microscopy
Authors: Cowan Stacie E. , Gilbert Eric , Khlebnikov Artem , Keasling J. D.
Published: April 2009

We report a dual labeling technique involving two green fluorescent protein (GFP) variants that is compatible with confocal microscopy. Two lasers were used to obtain images of (i) mixed cultures of cells, where one species contained GFPuv and another species contained GFPmut2 or GFPmut3, and (ii) a single species containing both GFPuv and GFPmut2 in the same cell. This method shows promise for monitoring gene expression and as a nondestructive and in situ technique for confocal microscopy of multispecies biofilms.
Molecular and Functional Analyses of the
metC
Gene of
Lactococcus lactis
, Encoding Cystathionine β-Lyase
Authors: Fernández María , van Doesburg Wim , Rutten Ger A. M. , Marugg Joey D. , Alting Arno C. , van Kranenburg Richard , Kuipers Oscar P.
Published: April 2009

The enzymatic degradation of amino acids in cheese is believed to generate aroma compounds and therefore to be essential for flavor development. Cystathionine β-lyase (CBL) can convert cystathionine to homocysteine but is also able to catalyze an α,γ elimination. With methionine as a substrate, it produces volatile sulfur compounds which are important for flavor formation in Gouda cheese. The
metC
gene, which encodes CBL, was cloned from the
Lactococcus lactis
model strain MG1363 and from strain B78, isolated from a cheese starter culture and known to have a high capacity to produce volatile compounds. The
metC
gene was found to be cotranscribed with a downstream
cysK
gene, which encodes a putative cysteine synthase. The MetC proteins of both strains were overproduced in strain MG1363 with the NICE (nisin-controlled expression) system, resulting in a >25-fold increase in cystathionine lyase activity. A disruption of the
metC
gene was achieved in strain MG1363. Determination of enzymatic activities in the overproducing and knockout strains revealed that MetC is essential for the degradation of cystathionine but that at least one lyase other than CBL contributes to methionine degradation via α,γ elimination to form volatile aroma compounds.
Phoma glomerataas a Mycoparasite of Powdery Mildew
Authors: Sullivan Raymond F. , White James F.
Published: April 2009

AmpelomycesandPhomaspecies are frequently confused with each other. Isolates previously attributed to the genusAmpelomyceswere shown to bePhomaisolates through studies of their morphology and life cycle and ribosomal DNA internal transcribed spacer region 1 sequence analysis.Phoma glomeratacan colonize and suppress development of powdery mildew on oak and may have utility as a mycoparasitic agent.
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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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.
Strain Variation in
Mycobacterium
marinum
Fish Isolates
Authors: Ucko M. , Colorni A. , Kvitt H. , Diamant A. , Zlotkin A. , Knibb W. R.
Published: April 2009

A molecular characterization of two
Mycobacterium
marinum
genes, 16S rRNA and
hsp65
, was carried out with a total of 21 isolates from various species of fish from both marine and freshwater environments of Israel, Europe, and the Far East. The nucleotide sequences of both genes revealed that all
M
.
marinum
isolates from fish in Israel belonged to two different strains, one infecting marine (cultured and wild) fish and the other infecting freshwater (cultured) fish. A restriction enzyme map based on the nucleotide sequences of both genes confirmed the divergence of the Israeli marine isolates from the freshwater isolates and differentiated the Israeli isolates from the foreign isolates, with the exception of one of three Greek isolates from marine fish which was identical to the Israeli marine isolates. The second isolate from Greece exhibited a single base alteration in the 16S rRNA sequence, whereas the third isolate was most likely a new
Mycobacterium
species. Isolates from Denmark and Thailand shared high sequence homology to complete identity with reference strain ATCC 927. Combined analysis of the two gene sequences increased the detection of intraspecific variations and was thus of importance in studying the taxonomy and epidemiology of this aquatic pathogen. Whether the Israeli
M
.
marinum
strain infecting marine fish is endemic to the Red Sea and found extremely susceptible hosts in the exotic species imported for aquaculture or rather was accidentally introduced with occasional imports of fingerlings from the Mediterranean Sea could not be determined.
Occurrence of Mycobacteria in Water Treatment Lines and in Water Distribution Systems
Authors: Le Dantec Corinne , Duguet Jean-Pierre , Montiel Antoine , Dumoutier Nadine , Dubrou Sylvie , Vincent Véronique
Published: April 2009

The frequency of recovery of atypical mycobacteria was estimated in two treatment plants providing drinking water to Paris, France, at some intermediate stages of treatment. The two plants use two different filtration processes, rapid and slow sand filtration. Our results suggest that slow sand filtration is more efficient for removing mycobacteria than rapid sand filtration. In addition, our results show that mycobacteria can colonize and grow on granular activated carbon and are able to enter distribution systems. We also investigated the frequency of recovery of mycobacteria in the water distribution system of Paris (outside buildings). The mycobacterial species isolated from the Paris drinking water distribution system are different from those isolated from the water leaving the treatment plants. Saprophytic mycobacteria (present in 41.3% of positive samples), potentially pathogenic mycobacteria (16.3%), and unidentifiable mycobacteria (54.8%) were isolated from 12 sites within the Paris water distribution system.
Mycobacterium gordonae
was preferentially recovered from treated surface water, whereas
Mycobacterium nonchromogenicum
was preferentially recovered from groundwater. No significant correlations were found among the presence of mycobacteria, the origin of water, and water temperature.
Molecular Analysis of Maltotriose Transport and Utilization bySaccharomycescerevisiae
Authors: Day Rachel E. , Rogers Peter J. , Dawes Ian W. , Higgins Vincent J.
Published: April 2009

Efficient fermentation of maltotriose is a desired property ofSaccharomycescerevisiaefor brewing. In a standard wort, maltotriose is the second most abundant sugar, and slower uptake leads to residual maltotriose in the finished product. The limiting factor of sugar metabolism is its transport, and there are conflicting reports on whether a specific maltotriose permease exists or whether the mechanisms responsible for maltose uptake also carry out maltotriose transport. In this study, radiolabeled maltotriose was used to show that overexpression of the maltose permease gene,MAL61, in an industrial yeast strain resulted in an increase in the rate of transport of maltotriose as well as maltose. A strain derived from W303-1A and lacking any maltose or maltotriose transporter but carrying a functional maltose transport activator (MAL63) was developed. By complementing this strain with permeases encoded byMAL31,MAL61, andAGT1, it was possible to measure their specific transport kinetics by using maltotriose and maltose. All three permeases were capable of high-affinity transport of maltotriose and of allowing growth of the strain on the sugar. Maltotriose utilization from the permease encoded byAGT1was regulated by the same genetic mechanisms as those involving the maltose transcriptional activator. Competition studies carried out with two industrial strains, one not containing any homologue ofAGT1, showed that maltose uptake and maltotriose uptake were competitive and that maltose was the preferred substrate. These results indicate that the presence of residual maltotriose in beer is not due to a genetic or physiological inability of yeast cells to utilize the sugar but rather to the lower affinity for maltotriose uptake in conjunction with deteriorating conditions present at the later stages of fermentation. Here we identify molecular mechanisms regulating the uptake of maltotriose and determine the role of each of the transporter genes in the cells.
Biodegradation of Hexahydro-1,3,5-Trinitro-1,3,5-Triazine and Its Mononitroso Derivative Hexahydro-1-Nitroso-3,5-Dinitro-1,3,5-Triazine by
Klebsiella pneumoniae
Strain SCZ-1 Isolated from an Anaerobic Sludge
Authors: Zhao Jian-Shen , Halasz Annamaria , Paquet Louise , Beaulieu Chantale , Hawari Jalal
Published: April 2009

In previous work, we found that an anaerobic sludge efficiently degraded hexahydro-1,3,5-trinitro-1,3,5-triazine (RDX), but the role of isolates in the degradation process was unknown. Recently, we isolated a facultatively anaerobic bacterium, identified as
Klebsiella pneumoniae
strain SCZ-1, using MIDI and the 16S rRNA method from this sludge and employed it to degrade RDX. Strain SCZ-1 degraded RDX to formaldehyde (HCHO), methanol (CH
3
OH) (12% of total C), carbon dioxide (CO
2
) (72% of total C), and nitrous oxide (N
2
O) (60% of total N) through intermediary formation of methylenedinitramine (O
2
NNHCH
2
NHNO
2
). Likewise, hexahydro-1-nitroso-3,5-dinitro-1,3,5-triazine (MNX) was degraded to HCHO, CH
3
OH, and N
2
O (16.5%) with a removal rate (0.39 μmol · h
−1
 · g [dry weight] of cells
−1
) similar to that of RDX (0.41 μmol · h
−1
 · g [dry weight] of cells
−1
) (biomass, 0.91 g [dry weight] of cells · liter
−1
). These findings suggested the possible involvement of a common initial reaction, possibly denitration, followed by ring cleavage and decomposition in water. The trace amounts of MNX detected during RDX degradation and the trace amounts of hexahydro-1,3-dinitroso-5-nitro-1,3,5-triazine detected during MNX degradation suggested that another minor degradation pathway was also present that reduced —NO
2
groups to the corresponding —NO groups.
RNA Stable Isotope Probing, a Novel Means of Linking Microbial Community Function to Phylogeny
Authors: Manefield Mike , Whiteley Andrew S. , Griffiths Robert I. , Bailey Mark J.
Published: April 2009

Identifying microorganisms responsible for recognized environmental processes remains a great challenge in contemporary microbial ecology. Only in the last few years have methodological innovations provided access to the relationship between the function of a microbial community and the phylogeny of the organisms accountable for it. In this study stable-isotope-labeled [
13
C]phenol was fed into a phenol-degrading community from an aerobic industrial bioreactor, and the
13
C-labeled RNA produced was used to identify the bacteria responsible for the process. Stable-isotope-labeled RNA was analyzed by equilibrium density centrifugation in concert with reverse transcription-PCR and denaturing gradient gel electrophoresis. In contradiction with findings from conventional methodologies, this unique approach revealed that phenol degradation in the microbial community under investigation is dominated by a member of the
Thauera
genus. Our results suggest that this organism is important for the function of this bioreactor.
Membrane-Bound ATPase Contributes to Hop Resistance ofLactobacillus brevis
Authors: Sakamoto Kanta , van Veen H. W. , Saito Hiromi , Kobayashi Hiroshi , Konings Wil N.
Published: April 2009

The activity of the membrane-bound H+-ATPase of the beer spoilage bacteriumLactobacillus brevisABBC45 increased upon adaptation to bacteriostatic hop compounds. The ATPase activity was optimal around pH 5.6 and increased up to fourfold whenL. breviswas exposed to 666 μM hop compounds. The extent of activation depended on the concentration of hop compounds and was maximal at the highest concentration tested. The ATPase activity was strongly inhibited byN,N′-dicyclohexylcarbodiimide, a known inhibitor of FoF1-ATPase. Western blots of membrane proteins ofL. breviswith antisera raised against the α- and β-subunits of FoF1-ATPase fromEnterococcus hiraeshowed that there was increased expression of the ATPase after hop adaptation. The expression levels, as well as the ATPase activity, decreased to the initial nonadapted levels when the hop-adapted cells were cultured further without hop compounds. These observations strongly indicate that proton pumping by the membrane-bound ATPase contributes considerably to the resistance ofL. brevisto hop compounds.
Efficacy of UV Irradiation in Inactivating
Cryptosporidium
parvum
Oocysts
Authors: Morita Shigemitsu , Namikoshi Atsushi , Hirata Tsuyoshi , Oguma Kumiko , Katayama Hiroyuki , Ohgaki Shinichiro , Motoyama Nobuyuki , Fujiwara Masahiro
Published: April 2009

To evaluate the effectiveness of UV irradiation in inactivating
Cryptosporidium parvum
oocysts, the animal infectivities and excystation abilities of oocysts that had been exposed to various UV doses were determined. Infectivity decreased exponentially as the UV dose increased, and the required dose for a 2-log
10
reduction in infectivity (99% inactivation) was approximately 1.0 mWs/cm
2
at 20°C. However,
C. parvum
oocysts exhibited high resistance to UV irradiation, requiring an extremely high dose of 230 mWs/cm
2
for a 2-log
10
reduction in excystation, which was used to assess viability. Moreover, the excystation ability exhibited only slight decreases at UV doses below 100 mWs/cm
2
. Thus, UV treatment resulted in oocysts that were able to excyst but not infect. The effects of temperature and UV intensity on the UV dose requirement were also studied. The results showed that for every 10°C reduction in water temperature, the increase in the UV irradiation dose required for a 2-log
10
reduction in infectivity was only 7%, and for every 10-fold increase in intensity, the dose increase was only 8%. In addition, the potential of oocysts to recover infectivity and to repair UV-induced injury (pyrimidine dimers) in DNA by photoreactivation and dark repair was investigated. There was no recovery in infectivity following treatment by fluorescent-light irradiation or storage in darkness. In contrast, UV-induced pyrimidine dimers in the DNA were apparently repaired by both photoreactivation and dark repair, as determined by endonuclease-sensitive site assay. However, the recovery rate was different in each process. Given these results, the effects of UV irradiation on
C. parvum
oocysts as determined by animal infectivity can conclusively be considered irreversible.
Low-Shear Modeled Microgravity Alters the
Salmonella enterica
Serovar Typhimurium Stress Response in an RpoS-Independent Manner
Authors: Wilson James W. , Ott C. Mark , Ramamurthy Rajee , Porwollik Steffen , McClelland Michael , Pierson Duane L. , Nickerson Cheryl A.
Published: April 2009

We have previously demonstrated that low-shear modeled microgravity (low-shear MMG) serves to enhance the virulence of a bacterial pathogen,
Salmonella enterica
serovar Typhimurium. The
Salmonella
response to low-shear MMG involves a signaling pathway that we have termed the low-shear MMG stimulon, though the identities of the low-shear MMG stimulon genes and regulatory factors are not known. RpoS is the primary sigma factor required for the expression of genes that are induced upon exposure to different environmental-stress signals and is essential for virulence in mice. Since low-shear MMG induces a
Salmonella
acid stress response and enhances
Salmonella
virulence, we reasoned that RpoS would be a likely regulator of the
Salmonella
low-shear MMG response. Our results demonstrate that low-shear MMG provides cross-resistance to several environmental stresses in both wild-type and isogenic
rpoS
mutant strains. Growth under low-shear MMG decreased the generation time of both strains in minimal medium and increased the ability of both strains to survive in J774 macrophages. Using DNA microarray analysis, we found no evidence of induction of the RpoS regulon by low-shear MMG but did find that other genes were altered in expression under these conditions in both the wild-type and
rpoS
mutant strains. Our results indicate that, under the conditions of these studies, RpoS is not required for transmission of the signal that induces the low-shear MMG stimulon. Moreover, our studies also indicate that low-shear MMG can be added to a short list of growth conditions that can serve to preadapt an
rpoS
mutant for resistance to multiple environmental stresses.
Pine Oil Cleaner-Resistant
Staphylococcus aureus
: Reduced Susceptibility to Vancomycin and Oxacillin and Involvement of SigB
Authors: Price Christopher T. D. , Singh Vineet K. , Jayaswal Radheshyam K. , Wilkinson Brian J. , Gustafson John E.
Published: April 2009

Mutants of
Staphylococcus aureus
strain COL resistant to a household pine oil cleaner (POC) were isolated on laboratory media containing POC.
S. aureus
mutants expressing the POC resistance (POC
r
) phenotype also demonstrate reduced susceptibility to the cell wall-active antibiotics vancomycin and oxacillin. The POC
r
phenotype is reliant on the
S. aureus
alternative transcription factor SigB, since inactivation of
sigB
abolished expression of elevated POC resistance and the reductions in vancomycin and oxacillin susceptibilities. The isolation of suppressor mutants of COL
sigB
::
kan
, which maintain the
sigB
::
kan
allele, indicates that the POC
r
phenotype can also be expressed to a lesser degree via a
sigB
-independent mechanism. These results bolster a growing body of reports suggesting that common disinfectants can select for bacteria with reduced susceptibilities to antibiotics. A series of in vitro-selected glycopeptide-intermediate
S. aureus
(GISA) isolates also expressed reductions in POC susceptibility compared to parent strains. Viewed collectively, our evidence suggests that mutations leading to the POC
r
phenotype may also be involved with the mechanism that leads to the GISA phenotype.
Food-Grade Delivery System for Controlled Gene Expression in
Lactococcus lactis
Authors: Henrich B. , Klein J. R. , Weber B. , Delorme C. , Renault P. , Wegmann U.
Published: April 2009

A food-grade system for the delivery of desired genes to
Lactococcus lactis
, their inducible expression, and their transfer to related strains was established. Based on the thermosensitive pG
+
host replicon, two types of plasmid vectors were constructed which contained sections of either the chromosomal
leu
operon of
L. lactis
or the
tel
operon from the lactococcal sex factor. Genes cloned into the
leu
or
tel
sequences of these vectors were delivered to the homologous regions of the chromosome or the sex factor through two single crossovers, leading to integration of the recombinant plasmids and subsequent excision of the vector portions. Inducible transcription of integrated genes was achieved by using the nisin-controlled expression (NICE) system. To establish the signal transduction genes
nisRK
in
L. lactis
, the vectors pLNG1363 (targeted to the chromosome) and pUK500 (targeted to the sex factor) were constructed. Fusions of six different peptidase genes (
pep
) from
Lactobacillus delbrueckii
with the nisin-inducible promoter

P
nisA

were delivered to the sex factor with derivatives of the vector pUK300. Food-grade recombinants of
L. lactis
were constructed which had the
nisRK
genes and individual

P
nisA

::
pep
fusions integrated either separately into the chromosome and the sex factor or simultaneously into the sex factor. With both types of recombinants, expression of

P
nisA

::
pep
fusions after induction with nisin was demonstrated. Depending on the loci used for integration of
nisRK
, variable induction rates were observed. Furthermore, an engineered sex factor carrying a

P
nisA

::
pepI
fusion was transfered by conjugation between two strains of
L. lactis
at a frequency of 4 × 10
−4
.
Inhibition of
Candida albicans
Biofilm Formation by Farnesol, a Quorum-Sensing Molecule
Authors: Ramage Gordon , Saville Stephen P. , Wickes Brian L. , López-Ribot José L.
Published: April 2009

Farnesol is a quorum-sensing molecule that inhibits filamentation in
Candida albicans
. Both filamentation and quorum sensing are deemed to be important factors in
C. albicans
biofilm development. Here we examined the effect of farnesol on
C. albicans
biofilm formation.
C. albicans
adherent cell populations (after 0, 1, 2, and 4 h of adherence) and preformed biofilms (24 h) were treated with various concentrations of farnesol (0, 3, 30, and 300 μM) and incubated at 37°C for 24 h. The extent and characteristics of biofilm formation were then assessed microscopically and with a semiquantitative colorimetric technique based on the use of 2,3-bis(2-methoxy-4-nitro-5-sulfo-phenyl)-2H-tetrazolium-5-carboxanilide. The results indicated that the effect of farnesol was dependent on the concentration of this compound and the initial adherence time, and preincubation with 300 μM farnesol completely inhibited biofilm formation. Supernatant media recovered from mature biofilms inhibited the ability of planktonic
C. albicans
to form filaments, indicating that a morphogenetic autoregulatory compound is produced in situ in biofilms. Northern blot analysis of RNA extracted from cells in biofilms indicated that the levels of expression of
HWP1
, encoding a hypha-specific wall protein, were decreased in farnesol-treated biofilms compared to the levels in controls. Our results indicate that farnesol acts as a naturally occurring quorum-sensing molecule which inhibits biofilm formation, and we discuss its potential for further development and use as a novel therapeutic agent.
Characterization of the
melA
Locus for α-Galactosidase in
Lactobacillus plantarum
Authors: Silvestroni Aurelio , Connes Cristelle , Sesma Fernando , de Giori Graciela Savoy , Piard Jean-Christophe
Published: April 2009

Alpha-galactosides are abundant sugars in legumes such as soy. Because of the lack of α-galactosidase (α-Gal) in the digestive tract, humans are unable to digest these sugars, which consequently induce flatulence. To develop the consumption of the otherwise highly nutritional soy products, the use of exogenous α-Gal is promising. In this framework, we characterized the
melA
gene for α-Gal in
Lactobacillus plantarum
. The
melA
gene encodes a cytoplasmic 84-kDa protein whose enzymatically active form occurs as oligomers. The
melA
gene was cloned and expressed in
Escherichia coli
, yielding an active α-Gal. We show that
melA
is transcribed from its own promoter, yielding a monocistronic mRNA, and that it is regulated at the transcriptional level, i.e., it is induced by melibiose but is not totally repressed by glucose. Posttranscriptional regulation by the carbon source could also occur. Upstream of
melA
, a putative galactoside transporter, designated RafP, was identified that shows high homology to LacS, the unique transporter for both α- and β-galactosides in
Streptococcus thermophilus. rafP
is also expressed as a monocistronic mRNA. Downstream of
melA
, the
lacL
and
lacM
genes were identified that encode a heterodimeric β-galactosidase. A putative
galM
gene identified in the same cluster suggests the presence of a galactose operon. These results indicate that the genes involved in galactoside catabolism are clustered in
L. plantarum
ATCC 8014. This first genetic characterization of
melA
and of its putative associated transporter,
rafP
, in a lactobacillus opens doors to various applications both in the manufacture of soy-derived products and in probiotic and nutraceutical issues.
Identification by PCR of
Fusarium culmorum
Strains Producing Large and Small Amounts of Deoxynivalenol
Authors: Bakan B. , Giraud-Delville C. , Pinson L. , Richard-Molard D. , Fournier E. , Brygoo Y.
Published: April 2009

Thirty deoxynivalenol-producing
F. culmorum
strains, isolated from wheat grains, were incubated in vitro and analyzed for trichothecene production. Seventeen strains produced more than 1 ppm of deoxynivalenol and acetyldeoxynivalenol and were considered high-deoxynivalenol-producing strains, whereas 13
F. culmorum
strains produced less than 0.07 ppm of trichothecenes and were considered low-deoxynivalenol-producing strains. For all strains, a 550-base portion of the trichodiene synthase gene (
tri5
) was amplified and sequenced. According to the
tri5
data, the
F. culmorum
strains tested clustered into two groups that correlated with in vitro deoxynivalenol production. For three high-producing and three low-producing
F. culmorum
strains, the
tri5-tri6
intergenic region was then sequenced, which confirmed the two separate clusters within the
F. culmorum
strains. According to the
tri5-tri6
sequence data, specific PCR primers were designed to allow differentiation of high-producing from low-producing
F. culmorum
strains.