bluuebunny/crossref_metadata_2025 huggingface dataset:
Plasmid-Mediated High-Level Gentamicin Resistance among Enteric Bacteria Isolated from Pet Turtles in Louisiana
Authors: Díaz María Alejandra , Cooper Richard Kent , Cloeckaert Axel , Siebeling Ronald John
Published: January 2006

The sale of small turtles is banned by the Food and Drug Administration from the U.S. market due to concerns about their excretion of
Salmonella
spp. To produce a safe pet for the export market, the Louisiana pet turtle industry uses gentamicin sulfate baths (1,000 μg/ml) to eradicate
Salmonella
spp. from turtle eggs. In 1999, we analyzed bacterial samples recovered from turtle farms and found that strains of
Salmonella enterica
subsp.
arizonae
and other bacteria, such as
Enterobacter cloacae
,
Citrobacter freundii
,
Pseudomonas aeruginosa
, and
Stenotrophomonas maltophilia
, were resistant to high concentrations of gentamicin (>2,000 μg/ml) and to other aminoglycosides. The goal of this study was to identify the gene(s) which contributes to the high-level gentamicin resistance phenotype observed in bacteria from environmental samples with turtle farming activity, particularly the salmonellae, and to estimate the incidence of such genes in these bacteria. R plasmids from gentamicin-resistant strains were transferred by conjugation and transformation to naive
Escherichia coli
cells. Cloning and sequencing of the gentamicin resistance determinants on these plasmids revealed the presence of the aminoglycoside acetyltransferase genes
aac(3)-IIa
and
aac(3)-VIa
; the latter was present as a gene cassette of a class 1 integron. Multiplex PCR assays showed that every gentamicin-resistant isolate carried one of these acetyltransferase genes. Pulsed-field gel electrophoresis and restriction enzyme digestion analysis of R plasmids carrying these genes revealed different restriction profiles and sizes, indicating a dissemination of the gentamicin resistance genes through mobile molecular elements. The data presented highlight the need to develop an alternate method for the eradication of
Salmonella
spp. from turtle eggs.
Inhibition of
Listeria monocytogenes
by Food-Borne Yeasts
Authors: Goerges Stefanie , Aigner Ulrike , Silakowski Barbara , Scherer Siegfried
Published: January 2006

Many bacteria are known to inhibit food pathogens, such as
Listeria monocytogenes
, by secreting a variety of bactericidal and bacteriostatic substances. In sharp contrast, it is unknown whether yeast has an inhibitory potential for the growth of pathogenic bacteria in food. A total of 404 yeasts were screened for inhibitory activity against five
Listeria monocytogenes
strains. Three hundred and four of these yeasts were isolated from smear-ripened cheeses. Most of the yeasts were identified by Fourier transform infrared spectroscopy. Using an agar-membrane screening assay, a fraction of approximately 4% of the 304 red smear cheese isolates clearly inhibited growth of
L. monocytogenes
. Furthermore, 14 out of these 304 cheese yeasts were cocultivated with
L. monocytogenes
WSLC 1364 on solid medium to test the antilisterial activity of yeast in direct cell contact with
Listeria
. All yeasts inhibited
L. monocytogenes
to a low degree, which is most probably due to competition for nutrients. However, one
Candida intermedia
strain was able to reduce the listerial cell count by 4 log units. Another four yeasts, assigned to
C. intermedia
(three strains) and
Kluyveromyces marxianus
(one strain), repressed growth of
L. monocytogenes
by 3 log units. Inhibition of
L. monocytogenes
was clearly pronounced in the cocultivation assay, which simulates the conditions and contamination rates present on smear cheese surfaces. We found no evidence that the unknown inhibitory molecule is able to diffuse through soft agar.
Cometabolism of Citrate and Glucose by
Enterococcus faecium
FAIR-E 198 in the Absence of Cellular Growth
Authors: Vaningelgem Frederik , Ghijsels Veerle , Tsakalidou Effie , De Vuyst Luc
Published: January 2006

Citrate metabolism by
Enterococcus faecium
FAIR-E 198, an isolate from Greek Feta cheese, was studied in modified MRS (mMRS) medium under different pH conditions and glucose and citrate concentrations. In the absence of glucose, this strain was able to metabolize citrate in a pH range from constant pH 5.0 to 7.0. At a constant pH 8.0, no citrate was metabolized, although growth took place. The main end products of citrate metabolism were acetate, formate, acetoin, and carbon dioxide, whereas ethanol and diacetyl were present in smaller amounts. In the presence of glucose, citrate was cometabolized, but it did not contribute to growth. Also, more acetate and less acetoin were formed compared to growth in mMRS medium and in the absence of glucose. Most of the citrate was consumed during the stationary phase, indicating that energy generated by citrate metabolism was used for maintenance. Experiments with cell-free fermented mMRS medium indicated that
E. faecium
FAIR-E 198 was able to metabolize another energy source present in the medium.
In Vivo Random Mutagenesis of
Bacillus subtilis
by Use of TnYLB-1, a
mariner
-Based Transposon
Authors: Le Breton Yoann , Mohapatra Nrusingh Prasad , Haldenwang W. G.
Published: January 2006

This report describes the construction and characterization of a
mariner
-based transposon system designed to be used in
Bacillus subtilis
, but potentially applicable to other gram-positive bacteria. Two pUC19-derived plasmids were created that contain the
mariner
-
Himar1
transposase gene, modified for expression in
B. subtilis
, under the control of either σ
A
- or σ
B
-dependent promoters. Both plasmids also contain a transposable element (TnYLB-1) consisting of a Kan
r
cassette bracketed by the
Himar1
-recognized inverse terminal repeats, as well as the temperature-sensitive replicon and Erm
r
gene of pE194ts. TnYLB-1 transposes into the
B. subtilis
chromosome with high frequency (10
−2
) from either plasmid. Southern hybridization analyses of 15 transposants and sequence analyses of the insertion sites of 10 of these are consistent with random transposition, requiring only a “TA” dinucleotide as the essential target in the recipient DNA. Two hundred transposants screened for sporulation proficiency and auxotrophy yielded five Spo
−
clones, three with insertions in known sporulation genes (
kinA
,
spoVT
, and
yqfD
) and two in genes (
ybaN
and
yubB
) with unknown functions. Two auxotrophic mutants were identified among the 200 transposants, one with an insertion in
lysA
and another in a gene (
yjzB
) whose function is unknown.
Construction of Improved Temperature-Sensitive and Mobilizable Vectors and Their Use for Constructing Mutations in the Adhesin-Encoding
acm
Gene of Poorly Transformable Clinical
Enterococcus faecium
Strains
Authors: Nallapareddy Sreedhar R. , Singh Kavindra V. , Murray Barbara E.
Published: January 2006

Inactivation by allelic exchange in clinical isolates of the emerging nosocomial pathogen
Enterococcus faecium
has been hindered by lack of efficient tools, and, in this study, transformation of clinical isolates was found to be particularly problematic. For this reason, a vector for allelic replacement (pTEX5500ts) was constructed that includes (i) the pWV01-based gram-positive
repAts
replication region, which is known to confer a high degree of temperature intolerance, (ii)
Escherichia coli oriR
from pUC18, (iii) two extended multiple-cloning sites located upstream and downstream of one of the marker genes for efficient cloning of flanking regions for double-crossover mutagenesis, (iv) transcriptional terminator sites to terminate undesired readthrough, and (v) a synthetic extended promoter region containing the
cat
gene for allelic exchange and a high-level gentamicin resistance gene,
aph
(
2
′′)
-Id
, to distinguish double-crossover recombination, both of which are functional in gram-positive and gram-negative backgrounds. To demonstrate the functionality of this vector, the vector was used to construct an
acm
(encoding an adhesin to collagen from
E. faecium
) deletion mutant of a poorly transformable multidrug-resistant
E. faecium
endocarditis isolate, TX0082. The
acm
-deleted strain, TX6051 (TX0082Δ
acm
), was shown to lack Acm on its surface, which resulted in the abolishment of the collagen adherence phenotype observed in TX0082. A mobilizable derivative (pTEX5501ts) that contains
oriT
of Tn
916
to facilitate conjugative transfer from the transformable
E. faecalis
strain JH2Sm::Tn
916
to
E. faecium
was also constructed. Using this vector, the
acm
gene of a nonelectroporable
E. faecium
wound isolate was successfully interrupted. Thus, pTEX5500ts and its mobilizable derivative demonstrated their roles as important tools by helping to create the first reported allelic replacement in
E. faecium
; the constructed this
acm
deletion mutant will be useful for assessing the role of
acm
in
E. faecium
pathogenesis using animal models.
Mercury Analysis of Acid- and Alkaline-Reduced Biological Samples: Identification of
meta
-Cinnabar as the Major Biotransformed Compound in Algae
Authors: Kelly David , Budd Kenneth , Lefebvre Daniel D.
Published: January 2006

The biotransformation of Hg
II
in pH-controlled and aerated algal cultures was investigated. Previous researchers have observed losses in Hg detection in vitro with the addition of cysteine under acid reduction conditions in the presence of SnCl
2
. They proposed that this was the effect of Hg-thiol complexing. The present study found that cysteine-Hg, protein and nonprotein thiol chelates, and nucleoside chelates of Hg were all fully detectable under acid reduction conditions without previous digestion. Furthermore, organic (R-Hg) mercury compounds could not be detected under either the acid or alkaline reduction conditions, and only β-HgS was detected under alkaline and not under acid SnCl
2
reduction conditions. The blue-green alga
Limnothrix planctonica
biotransformed the bulk of Hg
II
applied as HgCl
2
into a form with the analytical properties of β-HgS. Similar results were obtained for the eukaryotic alga
Selenastrum minutum
. No evidence for the synthesis of organomercurials such as CH
3
Hg
+
was obtained from analysis of either airstream or biomass samples under the aerobic conditions of the study. An analytical procedure that involved both acid and alkaline reduction was developed. It provides the first selective method for the determination of β-HgS in biological samples. Under aerobic conditions, Hg
II
is biotransformed mainly into β-HgS (
meta
-cinnabar), and this occurs in both prokaryotic and eukaryotic algae. This has important implications with respect to identification of mercury species and cycling in aquatic habitats.
Cloning and Expression of a Xylitol-4-Dehydrogenase Gene from
Pantoea ananatis
Authors: Aarnikunnas J. S. , Pihlajaniemi A. , Palva A. , Leisola M. , Nyyssölä A.
Published: January 2006

The
Pantoea ananatis
ATCC 43072 mutant strain is capable of growing with xylitol as the sole carbon source. The xylitol-4-dehydrogenase (XDH) catalyzing the oxidation of xylitol to
l
-xylulose was isolated from the cell extract of this strain. The N-terminal amino acid sequence of the purified protein was determined, and an oligonucleotide deduced from this peptide sequence was used to isolate the xylitol-4-dehydrogenase gene (
xdh
) from a
P. ananatis
gene library. Nucleotide sequence analysis revealed an open reading frame of 795 bp, encoding the xylitol-4-dehydrogenase, followed by a 5′ region of another open reading frame encoding an unknown protein. Results from a Northern analysis of total RNA isolated from
P. ananatis
ATCC 43072 suggested that
xdh
is transcribed as part of a polycistronic mRNA. Reverse transcription-PCR analysis of the transcript confirmed the operon structure and suggested that
xdh
was the first gene of the operon. Homology searches revealed that the predicted amino acid sequence of the
P. ananatis
XDH shared significant identity (38 to 51%) with members of the short-chain dehydrogenase/reductase family. The
P. ananatis xdh
gene was successfully overexpressed in
Escherichia coli
, XDH was purified to homogeneity, and some of its enzymatic properties were determined. The enzyme had a preference for NAD
+
as the cosubstrate, and in contrast to previous reports, the enzyme also showed a side activity for the
d
-form of xylulose. Xylitol was converted to
l
-xylulose with a high yield (>80%) by the resting recombinant cells, and the
l
-xylulose was secreted into the medium. No evidence of
d
-xylulose being synthesized by the recombinant cells was found.
Identification and Specific Detection of a Novel
Pseudomonadaceae
Cluster Associated with Soils from Winter Wheat Plots of a Long-Term Agricultural Field Experiment
Authors: Pesaro Manuel , Widmer Franco
Published: January 2006

The genus
Pseudomonas
(sensu stricto) represents a group of microorganisms directly involved in functions conferring plant health. We performed a study in the DOK long-term agricultural field experiment on the basis of previously published
Pseudomonas
-selective PCR primers in order to investigate the community structure of the microbial groups defined by the target range of these primers. Three different agricultural management systems, i.e., conventional, biodynamic, and bio-organic, along with mineral and unfertilized controls were investigated, with each system planted with either winter wheat or a grass-clover ley. Amplified small-subunit rRNA gene fragments were analyzed using the genetic profiling techniques restriction fragment length polymorphism (RFLP) and denaturing gradient gel electrophoresis (DGGE), revealing distinct differences between soils planted with winter wheat and grass clover but only minor differences between the management systems. Phylogenetic analyses of 59 clone sequences retrieved from bio-organic and unfertilized systems identified sequences related to
Pseudomonas fluorescens
and a novel cluster termed
Cellvibrio
-related
Pseudomonadaceae
(CRP). The CRP clones were exclusively isolated from winter wheat soil samples and were responsible for the crop-specific differences observed in RFLP and DGGE profiles. New primers were designed for the amplification of CRP targets directly from soil DNA, yielding strong signals exclusively for winter wheat soils. We concluded that crop-associated CRP exist in agricultural soils and that genetic profiling followed by specific probe design represents a valuable approach for identification as well as sensitive and rapid monitoring of novel microbial groups in the environment.
Introduction of Monochloramine into a Municipal Water System: Impact on Colonization of Buildings by
Legionella
spp
Authors: Moore Matthew R. , Pryor Marsha , Fields Barry , Lucas Claressa , Phelan Maureen , Besser Richard E.
Published: January 2006

Legionnaires' disease (LD) outbreaks are often traced to colonized potable water systems. We collected water samples from potable water systems of 96 buildings in Pinellas County, Florida, between January and April 2002, during a time when chlorine was the primary residual disinfectant, and from the same buildings between June and September 2002, immediately after monochloramine was introduced into the municipal water system. Samples were cultured for legionellae and amoebae using standard methods. We determined predictors of
Legionella
colonization of individual buildings and of individual sampling sites. During the chlorine phase, 19 (19.8%) buildings were colonized with legionellae in at least one sampling site. During the monochloramine phase, six (6.2%) buildings were colonized. In the chlorine phase, predictors of
Legionella
colonization included water source (source B compared to all others, adjusted odds ratio [aOR], 6.7; 95% confidence interval [CI], 2.0 to 23) and the presence of a system with continuously circulating hot water (aOR, 9.8; 95% CI, 1.9 to 51). In the monochloramine phase, there were no predictors of individual building colonization, although we observed a trend toward greater effectiveness of monochloramine in hotels and single-family homes than in county government buildings. The presence of amoebae predicted
Legionella
colonization at individual sampling sites in both phases (OR ranged from 15 to 46, depending on the phase and sampling site). The routine introduction of monochloramine into a municipal drinking water system appears to have reduced colonization by
Legionella
spp. in buildings served by the system. Monochloramine may hold promise as community-wide intervention for the prevention of LD.
Isolation of Poly-3-Hydroxybutyrate Metabolism Genes from Complex Microbial Communities by Phenotypic Complementation of Bacterial Mutants
Authors: Wang Chunxia , Meek David J. , Panchal Priya , Boruvka Natalie , Archibald Frederick S. , Driscoll Brian T. , Charles Trevor C.
Published: January 2006

The goal of this study was to initiate investigation of the genetics of bacterial poly-3-hydroxybutyrate (PHB) metabolism at the community level. We constructed metagenome libraries from activated sludge and soil microbial communities in the broad-host-range IncP cosmid pRK7813. Several unique clones were isolated from these libraries by functional heterologous complementation of a
Sinorhizobium meliloti bdhA
mutant, which is unable to grow on the PHB cycle intermediate
d
-3-hydroxybutyrate due to absence of the enzyme
d
-3-hydroxybutyrate dehydrogenase activity. Clones that conferred
d
-3-hydroxybutyrate utilization on
Escherichia coli
were also isolated. Although many of the
S. meliloti bdhA
mutant complementing clones restored
d
-3-hydroxybutyrate dehydrogenase activity to the mutant host, for some of the clones this activity was not detectable. This was also the case for almost all of the clones isolated in the
E. coli
selection. Further analysis was carried out on clones isolated in the
S. meliloti
complementation. Transposon mutagenesis to locate the complementing genes, followed by DNA sequence analysis of three of the genes, revealed coding sequences that were broadly divergent but lay within the diversity of known short-chain dehydrogenase/reductase encoding genes. In some cases, the amino acid sequence identity between pairs of deduced BdhA proteins was <35%, a level at which detection by nucleic acid hybridization based methods would probably not be successful.
Spirochaeta coccoides
sp. nov., a Novel Coccoid Spirochete from the Hindgut of the Termite
Neotermes castaneus
Authors: Dröge Stefan , Fröhlich Jürgen , Radek Renate , König Helmut
Published: January 2006

A novel spirochete strain, SPN1, was isolated from the hindgut contents of the termite
Neotermes castaneus
. The highest similarities (about 90%) of the strain SPN1 16S rRNA gene sequence are with spirochetes belonging to the genus
Spirochaeta
, and thus, the isolate could not be assigned to the so-called termite clusters of the treponemes or to a known species of the genus
Spirochaeta
. Therefore, it represents a novel species, which was named
Spirochaeta coccoides
. In contrast to all other known validly described spirochete species, strain SPN1 shows a coccoid morphology and is immotile. The isolated strain is obligately anaerobic and ferments different mono-, di-, and oligosaccharides by forming formate, acetate, and ethanol as the main fermentation end products. Furthermore, strain SPN1 is able to grow anaerobically with yeast extract as the sole carbon and energy source. The fastest growth was obtained at 30°C, the temperature at which the termites were also grown. The cells possess different enzymatic activities that are involved in the degradation of lignocellulose in the termite hindgut, such as β-
d
-glucosidase, α-
l
-arabinosidase, and β-
d
-xylosidase. Therefore, they may play an important role in the digestion of breakdown products from cellulose and hemicellulose in the termite gut.
Extensive Variation in Intracellular Symbiont Community Composition among Members of a Single Population of the Wood-Boring BivalveLyrodus pedicellatus(Bivalvia: Teredinidae)
Authors: Luyten Yvette A. , Thompson Janelle R. , Morrill Wendy , Polz Martin F. , Distel Daniel L.
Published: January 2006

Shipworms (wood-boring bivalves of the familyTeredinidae) harbor in their gills intracellular bacterial symbionts thought to produce enzymes that enable the host to consume cellulose as its primary carbon source. Recently, it was demonstrated that multiple genetically distinct symbiont populations coexist within one shipworm species,Lyrodus pedicellatus. Here we explore the extent to which symbiont communities vary among individuals of this species by quantitatively examining the diversity, abundance, and pattern of occurrence of symbiont ribotypes (unique 16S rRNA sequence types) among specimens drawn from a single laboratory-reared population. A total of 18 ribotypes were identified in two clone libraries generated from gill tissue of (i) a single specimen and (ii) four pooled specimens. Phylogenetic analysis assigned all of the ribotypes to a unique clade within the γ subgroup of proteobacteria which contained at least five well-supported internal clades (phylotypes). By competitive quantitative PCR and constant denaturant capillary electrophoresis, we estimated the number and abundance of symbiont phylotypes in gill samples of 13 individual shipworm specimens. Phylotype composition varied greatly; however, in all specimens the numerically dominant symbiont belonged to one of two nearly mutually exclusive phylotypes, each of which was detected with similar frequencies among specimens. A third phylotype, containing the culturable symbiontTeredinibacter turnerae, was identified in nearly all specimens, and two additional phylotypes were observed more sporadically. Such extensive variation in ribotype and phylotype composition among host specimens adds to a growing body of evidence that microbial endosymbiont populations may be both complex and dynamic and suggests that such genetic variation should be evaluated with regard to physiological and ecological differentiation.
Characterization of PhlG, a Hydrolase That Specifically Degrades the Antifungal Compound 2,4-Diacetylphloroglucinol in the Biocontrol Agent
Pseudomonas fluorescens
CHA0
Authors: Bottiglieri Mélanie , Keel Christoph
Published: January 2006

The potent antimicrobial compound 2,4-diacetylphloroglucinol (DAPG) is a major determinant of biocontrol activity of plant-beneficial
Pseudomonas fluorescens
CHA0 against root diseases caused by fungal pathogens. The DAPG biosynthetic locus harbors the
phlG
gene, the function of which has not been elucidated thus far. The
phlG
gene is located upstream of the
phlACBD
biosynthetic operon, between the
phlF
and
phlH
genes which encode pathway-specific regulators. In this study, we assigned a function to PhlG as a hydrolase specifically degrades DAPG to equimolar amounts of mildly toxic monoacetylphloroglucinol (MAPG) and acetate. DAPG added to cultures of a DAPG-negative Δ
phlA
mutant of strain CHA0 was completely degraded, and MAPG was temporarily accumulated. In contrast, DAPG was not degraded in cultures of a Δ
phlA
Δ
phlG
double mutant. To confirm the enzymatic nature of PhlG in vitro, the protein was histidine tagged, overexpressed in
Escherichia coli
, and purified by affinity chromatography. Purified PhlG had a molecular mass of about 40 kDa and catalyzed the degradation of DAPG to MAPG. The enzyme had a
k
cat
of 33 s
−1
and a

K
m

of 140 μM at 30°C and pH 7. The PhlG enzyme did not degrade other compounds with structures similar to DAPG, such as MAPG and triacetylphloroglucinol, suggesting strict substrate specificity. Interestingly, PhlG activity was strongly reduced by pyoluteorin, a further antifungal compound produced by the bacterium. Expression of
phlG
was not influenced by the substrate DAPG or the degradation product MAPG but was subject to positive control by the GacS/GacA two-component system and to negative control by the pathway-specific regulators PhlF and PhlH.
Use of
Bacillus thuringiensis
Toxins for Control of the Cotton Pest
Earias insulana
(Boisd.) (Lepidoptera: Noctuidae)
Authors: Ibargutxi María A. , Estela Anna , Ferré Juan , Caballero Primitivo
Published: January 2006

Thirteen of the most common lepidopteran-specific Cry proteins of
Bacillus thuringiensis
have been tested for their efficacy against newly hatched larvae of two populations of the spiny bollworm,
Earias insulana
. At a concentration of 100 μg of toxin per milliliter of artificial diet, six Cry toxins (Cry1Ca, Cry1Ea, Cry1Fa, Cry1Ja, Cry2Aa, and Cry2Ab) were not toxic at all. Cry1Aa, Cry1Ja, and Cry2Aa did not cause mortality but caused significant inhibition of growth. The other Cry toxins (Cry1Ab, Cry1Ac, Cry1Ba, Cry1Da, Cry1Ia, and Cry9Ca) were toxic to
E. insulana
larvae. The 50% lethal concentration values of these toxins ranged from 0.39 to 21.13 μg/ml (for Cry9Ca and Cry1Ia, respectively) for an
E. insulana
laboratory colony originating from Egypt and from 0.20 to 4.25 μg/ml (for Cry9Ca and Cry1Da, respectively) for a laboratory colony originating from Spain. The relative potencies of the toxins in the population from Egypt were highest for Cry9Ca and Cry1Ab, and they were both significantly more toxic than Cry1Ac and Cry1Ba, followed by Cry1Da and finally Cry1Ia. In the population from Spain, Cry9Ca was the most toxic, followed in decreasing order by Cry1Ac and Cry1Ba, and the least toxic was Cry1Da. Binding experiments were performed to test whether the toxic Cry proteins shared binding sites in this insect.
125
I-labeled Cry1Ac and Cry1Ab and biotinylated Cry1Ba, Cry1Ia, and Cry9Ca showed specific binding to the brush border membrane vesicles from
E. insulana
. Competition binding experiments among these toxins showed that only Cry1Ab and Cry1Ac competed for the same binding sites, indicating a high possibility that this insect may develop cross-resistance to Cry1Ab upon exposure to Cry1Ac transgenic cotton but not to the other toxins tested.
Characterization of Germination Receptors of
Bacillus cereus
ATCC 14579
Authors: Hornstra Luc M. , de Vries Ynte P. , Wells-Bennik Marjon H. J. , de Vos Willem M. , Abee Tjakko
Published: January 2006

Specific amino acids, purine ribonucleosides, or a combination of the two is required for efficient germination of endospores of
Bacillus cereus
ATCC 14579. A survey including 20 different amino acids showed that
l
-alanine,
l
-cysteine,
l
-threonine, and
l
-glutamine are capable of initiating the germination of endospores of
B. cereus
ATCC 14579. In addition, the purine ribonucleosides inosine and adenosine can trigger germination of the spores. Advanced annotation of the
B. cereus
ATCC 14579 genome revealed the presence of seven putative germination (
ger
) operons, termed
gerG
,
gerI
,
gerK
,
gerL
,
gerQ
,
gerR
, and
gerS
. To determine the role of the encoded putative receptors in nutrient-induced germination, disruption mutants were constructed by the insertion of pMUTIN4 into each of the seven operons. Four of the seven mutants were affected in the germination response to amino acids or purine ribonucleosides, whereas no phenotype could be attributed to the mutants with disrupted
gerK
,
gerL
, and
gerS
loci. The strain with a disrupted
gerR
operon was severely hampered in the ability to germinate: germination occurred in response to
l
-glutamine but not in the presence of any of the other amino acids tested. The
gerG
mutant showed significantly reduced
l
-glutamine-induced germination, which points to a role of this receptor in the
l
-glutamine germination signaling pathway.
gerR
,
gerI
, and
gerQ
mutants showed reduced germination rates in the presence of inosine, suggesting a role for these operons in ribonucleoside signaling. Efficient germination by the combination of
l
-glutamine and inosine was shown to involve the
gerG
and
gerI
operons, since the germination of mutants lacking either one of these receptors was significantly reduced. Germination triggered by the combination of
l
-phenylalanine and inosine was lost in the
gerI
mutant, indicating that both molecules are effective at the GerI receptor.
Antimicrobial Drug Resistance Genes Do Not Convey a Secondary Fitness Advantage to Calf-Adapted
Escherichia coli
Authors: Khachatryan Artashes R. , Hancock Dale D. , Besser Thomas E. , Call Douglas R.
Published: January 2006

Maintenance of antimicrobial drug resistance in bacteria can be influenced by factors unrelated to direct selection pressure such as close linkage to other selectively advantageous genes and secondary advantage conveyed by antimicrobial resistance genes in the absence of drug selection. Our previous trials at a dairy showed that the maintenance of the antimicrobial resistance genes is not influenced by specific antimicrobial selection and that the most prevalent antimicrobial resistance phenotype of
Escherichia coli
is specifically selected for in young calves. In this paper we examine the role of secondary advantages conveyed by antimicrobial resistance genes. We tested antimicrobial-susceptible null mutant strains for their ability to compete with their progenitor strains in vitro and in vivo. The null mutant strains were generated by selection for spontaneous loss of resistance genes in broth supplemented with fusaric acid or nickel chloride. On average, the null mutant strains were as competitive as the progenitor strains in vitro and in newborn calves (in vivo). Inoculation of newborn calves at the dairy with antimicrobial-susceptible strains of
E. coli
did not impact the prevalence of antimicrobial-resistant
E. coli
. Our results demonstrate that the antimicrobial resistance genes are not responsible for the greater fitness advantage of antimicrobial-resistant
E. coli
in calves, but the farm environment and the diet clearly exert critical selective pressures responsible for the maintenance of antimicrobial resistance genes. Our current hypothesis is that the antimicrobial resistance genes are linked to other genes responsible for differential fitness in dairy calves.
Mercury Methylation from Unexpected Sources: Molybdate-Inhibited Freshwater Sediments and an Iron-Reducing Bacterium
Authors: Fleming Emily J. , Mack E. Erin , Green Peter G. , Nelson Douglas C.
Published: January 2006

Methylmercury has been thought to be produced predominantly by sulfate-reducing bacteria in anoxic sediments. Here we show that in circumneutral pH sediments (Clear Lake, CA) application of a specific inhibitor of sulfate-reducing bacteria at appropriate concentrations typically inhibited less than one-half of all anaerobic methylation of added divalent mercury. This suggests that one or more additional groups of microbes are active methylators in these sediments impacted by a nearby abandoned mercury mine. From Clear Lake sediments, we isolated the iron-reducing bacterium
Geobacter
sp. strain CLFeRB, which can methylate mercury at a rate comparable to
Desulfobulbus propionicus
strain 1pr3, a sulfate-reducing bacterium known to be an active methylator. This is the first time that an iron-reducing bacterium has been shown to methylate mercury at environmentally significant rates. We suggest that mercury methylation by iron-reducing bacteria represents a previously unidentified and potentially significant source of this environmental toxin in iron-rich freshwater sediments.
Efficient and Stable Display of Functional Proteins on Bacterial Magnetic Particles Using Mms13 as a Novel Anchor Molecule
Authors: Yoshino Tomoko , Matsunaga Tadashi
Published: January 2006

Magnetic particles are increasingly used for various biomedical applications because they are easy to handle and separate from biological samples. In this work, a novel anchor molecule was used for targeted protein display onto magnetic nanoparticles. The magnetic bacterium
Magnetospirillum magneticum
AMB-1 synthesizes intracellular bacterial magnetic particles (BMPs) covered with a lipid bilayer membrane. In our recent research, an integral BMP membrane protein, Mms13, was isolated and used as an anchor molecule to display functional proteins onto BMPs. The anchoring properties of Mms13 were confirmed by luciferase fusion studies. The C terminus of Mms13 was shown to be expressed on the surface of BMPs, and Mms13 was bound to magnetite directly and tightly permitting stable localization of a large protein, luciferase (61 kDa), on BMPs. Consequently, luminescence intensity obtained from BMPs using Mms13 as an anchor molecule was >400 or 1,000 times higher than Mms16 or MagA, which previously were used as anchor molecules. Furthermore, the immunoglobulin G-binding domain of protein A (ZZ) was displayed uniformly on BMPs using Mms13, and antigen was detected by transmission electron microscopy using antibody-labeled gold nanoparticles on a single BMP displaying the ZZ-antibody complex. The results of this study demonstrated the utility of Mms13 as a molecular anchor, which will facilitate the assembly of other functional proteins onto BMPs in the near feature.
Aerobic Biodegradation of 2,2′-Dithiodibenzoic Acid Produced from Dibenzothiophene Metabolites
Authors: Young Rozlyn F. , Cheng Stephanie M. , Fedorak Phillip M.
Published: January 2006

Dibenzothiophene is a sulfur heterocycle found in crude oils and coal. The biodegradation of dibenzothiophene through the Kodama pathway by
Pseudomonas
sp. strain BT1d leads to the formation of three disulfides: 2-oxo-2-(2-thiophenyl)ethanoic acid disulfide, 2-oxo-2-(2-thiophenyl)ethanoic acid-2-benzoic acid disulfide, and 2,2′-dithiodibenzoic acid. When provided as the carbon and sulfur source in liquid medium, 2,2′-dithiodibenzoic acid was degraded by soil enrichment cultures. Two bacterial isolates, designated strains RM1 and RM6, degraded 2,2′-dithiodibenzoic acid when combined in the medium. Isolate RM6 was found to have an absolute requirement for vitamin B
12
, and it degraded 2,2′-dithiodibenzoic acid in pure culture when the medium was supplemented with this vitamin. Isolate RM6 also degraded 2,2′-dithiodibenzoic acid in medium containing sterilized supernatants from cultures of isolate RM1 grown on glucose or benzoate. Isolate RM6 was identified as a member of the genus
Variovorax
using the Biolog system and 16S rRNA gene analysis. Although the mechanism of disulfide metabolism could not be determined, benzoic acid was detected as a transient metabolite of 2,2′-dithiodibenzoic acid biodegradation by
Variovorax
sp. strain RM6. In pure culture, this isolate mineralized 2,2′-dithiodibenzoic acid, releasing 59% of the carbon as carbon dioxide and 88% of the sulfur as sulfate.
Putative ABC Transporter Responsible for Acetic Acid Resistance in
Acetobacter aceti
Authors: Nakano Shigeru , Fukaya Masahiro , Horinouchi Sueharu
Published: January 2006

Two-dimensional gel electrophoretic analysis of the membrane fraction of
Acetobacter aceti
revealed the presence of several proteins that were produced in response to acetic acid. A 60-kDa protein, named AatA, which was mostly induced by acetic acid, was prepared;
aatA
was cloned on the basis of its NH
2
-terminal amino acid sequence. AatA, consisting of 591 amino acids and containing ATP-binding cassette (ABC) sequences and ABC signature sequences, belonged to the ABC transporter superfamily. The
aatA
mutation with an insertion of the neomycin resistance gene within the
aatA
coding region showed reduced resistance to acetic acid, formic acid, propionic acid, and lactic acid, whereas the
aatA
mutation exerted no effects on resistance to various drugs, growth at low pH (adjusted with HCl), assimilation of acetic acid, or resistance to citric acid. Introduction of plasmid pABC101 containing
aatA
under the control of the
Escherichia coli lac
promoter into the
aatA
mutant restored the defect in acetic acid resistance. In addition, pABC101 conferred acetic acid resistance on
E. coli
. These findings showed that AatA was a putative ABC transporter conferring acetic acid resistance on the host cell. Southern blot analysis and subsequent nucleotide sequencing predicted the presence of
aatA
orthologues in a variety of acetic acid bacteria belonging to the genera
Acetobacter
and
Gluconacetobacter
. The fermentation with
A. aceti
containing
aatA
on a multicopy plasmid resulted in an increase in the final yield of acetic acid.
Protease Inhibitors Fail To Prevent Pore Formation by the Activated
Bacillus thuringiensis
Toxin Cry1Aa in Insect Brush Border Membrane Vesicles
Authors: Kirouac Martin , Vachon Vincent , Quievy Delphine , Schwartz Jean-Louis , Laprade Raynald
Published: January 2006

To investigate whether membrane proteases are involved in the activity of
Bacillus thuringiensis
insecticidal toxins, the rate of pore formation by trypsin-activated Cry1Aa was monitored in the presence of a variety of protease inhibitors with
Manduca sexta
midgut brush border membrane vesicles and by a light-scattering assay. Most of the inhibitors tested had no effect on the pore-forming ability of the toxin. However, phenylmethylsulfonyl fluoride, a serine protease inhibitor, promoted pore formation, although this stimulation only occurred at higher inhibitor concentrations than those commonly used to inhibit proteases. Among the metalloprotease inhibitors,
o
-phenanthroline had no significant effect; EDTA and EGTA reduced the rate of pore formation at pH 10.5, but only EDTA was inhibitory at pH 7.5. Neither chelator affected the properties of the pores already formed after incubation of the vesicles with the toxin. Taken together, these results indicate that, once activated, Cry1Aa is completely functional and does not require further proteolysis. The effect of EDTA and EGTA is probably better explained by their ability to chelate divalent cations that could be necessary for the stability of the toxin's receptors or involved elsewhere in the mechanism of pore formation.
Prevalence, Persistence, and Molecular Characterization of Glycopeptide-Resistant Enterococci in Norwegian Poultry and Poultry Farmers 3 to 8 Years after the Ban on Avoparcin
Authors: Sørum M. , Johnsen P. J. , Aasnes B. , Rosvoll T. , Kruse H. , Sundsfjord A. , Simonsen G. S.
Published: January 2006

Environmental reservoirs of glycopeptide-resistant enterococci (GRE) in Norway have been linked to former growth promoting use of the glycopeptide avoparcin in poultry production. We have examined the prevalence of fecal GRE in poultry and poultry farmers 3 to 8 years after the Norwegian avoparcin ban in 1995 and performed molecular analyses of the GRE population. Fecal samples from poultry farmers and their flocks on 29 previously avoparcin-exposed farms were collected on five occasions during the study period (1998 to 2003). All flocks (100%) were GRE positive in 1998. Throughout the study period, 78.5% of the poultry samples were GRE positive. Glycopeptide-resistant
Enterococcus faecium
(GREF) was isolated from 27.6% of the farmer samples in 1998 and from 27.8% of the samples collected between 1998 and 2003. The prevalence of fecal GRE in poultry declined significantly during the study period, but prevalence in samples from the farmers did not decline. PCR analysis revealed a specific Tn
1546
-plasmid junction fragment in 93.9% of
E. faecium
isolates. A putative postsegregation killing (PSK) system linked to Tn
1546
was detected in 97.1% of the isolates examined. Multilocus sequence typing of glycopeptide-susceptible (
n
= 10) and -resistant (
n
= 10)
E. faecium
isolates from humans (
n
= 10) and poultry (
n
= 10) on two farms displayed 17 different sequence types. The study confirms the continuing persistence of a widespread common plasmid-mediated
vanA
-pRE25-PSK element within a heterogeneous GRE population on Norwegian poultry farms 8 years after the avoparcin ban. Moreover, it suggests an important role of PSK systems in the maintenance of antimicrobial resistance determinants in reservoirs without apparent antimicrobial selection.
Stable Concentrated Emulsions of the 1-Monoglyceride of Capric Acid (Monocaprin) with Microbicidal Activities against the Food-Borne Bacteria
Campylobacter jejuni
,
Salmonella
spp., and
Escherichia coli
Authors: Thormar Halldor , Hilmarsson Hilmar , Bergsson Gudmundur
Published: January 2006

Of 11 fatty acids and monoglycerides tested against
Campylobacter jejuni
, the 1-monoglyceride of capric acid (monocaprin) was the most active in killing the bacterium. Various monocaprin-in-water emulsions were prepared which were stable after storage at room temperature for many months and which retained their microbicidal activity. A procedure was developed to manufacture up to 500 ml of 200 mM preconcentrated emulsions of monocaprin in tap water. The concentrates were clear and remained stable for at least 12 months. They were active against
C. jejuni
upon 160- to 200-fold dilution in tap water and caused a >6- to 7-log
10
reduction in viable bacterial count in 1 min at room temperature. The addition of 0.8% Tween 40 to the concentrates as an emulsifying agent did not change the microbicidal activity. Emulsions of monocaprin killed a variety of
Campylobacter
isolates from humans and poultry and also killed strains of
Campylobacter coli
and
Campylobacter lari
, indicating a broad anticampylobacter activity. Emulsions of 1.25 mM monocaprin in citrate-lactate buffer at pH 4 to 5 caused a >6- to 7-log
10
reduction in viable bacterial counts of
Salmonella
spp. and
Escherichia coli
in 10 min.
C. jejuni
was also more susceptible to monocaprin emulsions at low pH. The addition of 5 and 10 mM monocaprin emulsions to
Campylobacter
-spiked chicken feed significantly reduced the bacterial contamination. These results are discussed in view of the possible utilization of monocaprin emulsions in controlling the spread of food-borne bacteria from poultry to humans.
Sequence Analysis of Two Cryptic Plasmids from
Bifidobacterium longum
DJO10A and Construction of a Shuttle Cloning Vector
Authors: Lee Ju-Hoon , O’Sullivan Daniel J.
Published: January 2006

Bifidobacterium longum
DJO10A is a recent human isolate with probiotic characteristics and contains two plasmids, designated pDOJH10L and pDOJH10S. The complete sequences of both these plasmids have now been determined and consist of two circular DNA molecules of 10,073 and 3,661 bp, with G+C contents of 62.2% and 66.2%, respectively. Plasmid pDOJH10L is a cointegrate plasmid consisting of DNA regions exhibiting very high sequence identity to two other
B. longum
plasmids, pNAC2 (98%) and pKJ50 (96%), together with another region. Interestingly, the rolling circular replication (RCR) regions of both the pNAC2- and pKJ50-like plasmids were disrupted during the recombination event leading to a further recombination event to acquire a functional replicon. This consists of a new fused
rep
gene and an RCR-type
ori
consisting of a conserved DnaA box in an AT-rich region followed by four contiguous repeated sequences consistent with an iteron structure and an inverted repeat. The smaller pDOJH10S had no sequence similarity to any other characterized plasmid from bifidobacteria. In addition, it did not contain any features consistent with RCR, which is the replication mechanism proposed for all the bifidobacteria plasmids characterized to date. It did exhibit sequence similarity with several theta replication-related replication proteins from other gram-positive, high-G+C bacteria, with the closest match from a
Rhodococcus rhodochrous
plasmid, suggesting a theta mechanism of replication. S1 nuclease analysis of both plasmids in
B. longum
DJO10A revealed single-strand DNA intermediates for pDOJH10L, which is consistent for RCR, but none were detected for pDOJH10S. As the G+C content of pDOJH10S is similar to that of
Rhodococcus rhodochrous
(67%) and significantly higher than that of
B. longum
(60.1%), it may have been acquired through horizontal gene transfer from a
Rhodococcus
species, as both genera are members of the
Actinomycetes
and are intestinal inhabitants. An
Escherichia coli-B. longum
shuttle cloning vector was constructed from pDOJH10S and the
E. coli ori
region of p15A, a
lacZ
gene with a multiple cloning site of pUC18, and a chloramphenicol resistance gene (CAT) of pCI372 and was transformed successfully into
E. coli
and
B. longum
. It could not be introduced into lactic acid bacteria (
Lactococcus
and
Lactobacillus
), showing it was not very promiscuous. It was stably maintained in
B. longum
in the absence of antibiotic pressure for 92 generations, which is consistent with the segregational stability of theta-replicating plasmids in gram-positive bacteria. This is the first cloning vector for bifidobacteria that does not utilize RCR and should be useful for the stable introduction of heterologous genes into these dominant inhabitants of the large intestine.
Engineering the Monomer Composition of Polyhydroxyalkanoates Synthesized in
Saccharomyces cerevisiae
Authors: Zhang Bo , Carlson Ross , Srienc Friedrich
Published: January 2006

Polyhydroxyalkanoates (PHAs) have received considerable interest as renewable-resource-based, biodegradable, and biocompatible plastics with a wide range of potential applications. We have engineered the synthesis of PHA polymers composed of monomers ranging from 4 to 14 carbon atoms in either the cytosol or the peroxisome of
Saccharomyces cerevisiae
by harnessing intermediates of fatty acid metabolism. Cytosolic PHA production was supported by establishing in the cytosol critical β-oxidation chemistries which are found natively in peroxisomes. This platform was utilized to supply medium-chain (C
6
to C
14
) PHA precursors from both fatty acid degradation and synthesis to a cytosolically expressed medium-chain-length (mcl) polymerase from
Pseudomonas oleovorans
. Synthesis of short-chain-length PHAs (scl-PHAs) was established in the peroxisome of a wild-type yeast strain by targeting the
Ralstonia eutropha
scl polymerase to the peroxisome. This strain, harboring a peroxisomally targeted scl-PHA synthase, accumulated PHA up to approximately 7% of its cell dry weight. These results indicate (i) that
S. cerevisiae
expressing a cytosolic mcl-PHA polymerase or a peroxisomal scl-PHA synthase can use the 3-hydroxyacyl coenzyme A intermediates from fatty acid metabolism to synthesize PHAs and (ii) that fatty acid degradation is also possible in the cytosol as β-oxidation might not be confined only to the peroxisomes. Polymers of even-numbered, odd-numbered, or a combination of even- and odd-numbered monomers can be controlled by feeding the appropriate substrates. This ability should permit the rational design and synthesis of polymers with desired material properties.
Effect of Temperature and Light on Growth of and Photosynthesis bySynechococcusIsolates Typical of Those Predominating in the Octopus Spring Microbial Mat Community of Yellowstone National Park
Authors: Allewalt Jessica P. , Bateson Mary M. , Revsbech Niels Peter , Slack Kimberly , Ward David M.
Published: January 2006

Previous molecular analysis of the Octopus Spring cyanobacterial mat revealed numerous genetically distinct 16S rRNA sequences from predominantSynechococcuspopulations distantly related to the readily cultivated unicellular cyanobacteriumSynechococcus lividus. Patterns in genotype distribution relative to temperature and light conditions suggested that the organisms contributing these 16S rRNA sequences may fill distinct ecological niches. To test this hypothesis,Synechococcusisolates were cultivated using a dilution and filtration approach and then shown to be genetically relevant to natural mat populations by comparisons of similarities of 16S rRNA genes and 16S-23S internal transcribed spacer (ITS) regions. Most isolates were identical or nearly identical at both loci to predominant mat genotypes; others showed 1- to 2-nucleotide differences at the 16S rRNA locus and even greater difference in ITS sequences. Isolates with predominant mat genotypes had distinct temperature ranges and optima for growth that were consistent with their distributions in the mat. Isolates with genotypes not previously detected or known to be predominant in the mat exhibited temperature ranges and optima that were not representative of predominant mat populations and also grew more slowly. Temperature effects on photosynthesis did not reflect temperature relations for growth. However, the isolate with the highest temperature optimum and upper limit was capable of performing photosynthesis at a higher temperature than other isolates. Growth rate and photosynthetic responses provided evidence for light acclimation but evidence of, at best, only subtle light adaptation.