bluuebunny/crossref_metadata_2025 huggingface dataset:
Resistance Mechanisms in Clinical Isolates of
Candida albicans
Authors: White Theodore C. , Holleman Scott , Dy Francis , Mirels Laurence F. , Stevens David A.
Published: July 2002

Resistance to azole antifungals continues to be a significant problem in the common fungal pathogen
Candida albicans
. Many of the molecular mechanisms of resistance have been defined with matched sets of susceptible and resistant clinical isolates from the same strain. Mechanisms that have been identified include alterations in the gene encoding the target enzyme
ERG11
or overexpression of efflux pump genes including
CDR1
,
CDR2
, and
MDR1
. In the present study, a collection of unmatched clinical isolates of
C. albicans
was analyzed for the known molecular mechanisms of resistance by standard methods. The collection was assembled so that approximately half of the isolates were resistant to azole drugs. Extensive cross-resistance was observed for fluconazole, clotrimazole, itraconazole, and ketoconazole. Northern blotting analyses indicated that overexpression of
CDR1
and
CDR2
correlates with resistance, suggesting that the two genes may be coregulated.
MDR1
overexpression was observed infrequently in some resistant isolates. Overexpression of
FLU1
, an efflux pump gene related to
MDR1
, did not correlate with resistance, nor did overexpression of
ERG11
. Limited analysis of the
ERG11
gene sequence identified several point mutations in resistant isolates; these mutations have been described previously. Two of the most common point mutations in
ERG11
associated with resistance, D116E and E266D, were tested by restriction fragment length polymorphism analysis of the isolates from this collection. The results indicated that the two mutations occur frequently in different isolates of
C. albicans
and are not reliably associated with resistance. These analyses emphasize the diversity of mechanisms that result in a phenotype of azole resistance. They suggest that the resistance mechanisms identified in matched sets of susceptible and resistant isolates are not sufficient to explain resistance in a collection of unmatched clinical isolates and that additional mechanisms have yet to be discovered.
Helicobacter pylori
Primary Resistance to Metronidazole and Clarithromycin in Brazil
Authors: Prazeres Magalhães Paula , de Magalhães Queiroz Dulciene Maria , Campos Barbosa Daniela Vale , Aguiar Rocha Gifone , Nogueira Mendes Edilberto , Santos Adriana , Valle Corrêa Paulo Renato , Camargos Rocha Andreia Maria , Martins Teixeira Lúcia , Affonso de Oliveira Celso
Published: July 2002

Helicobacter pylori
resistance to metronidazole was detected in 107 (52.97%) of 202 strains. Twenty (9.85%) strains, 18 of them harboring 23S ribosomal DNA mutations, were resistant to clarithromycin. Metronidazole resistance was associated with female gender. Resistance to metronidazole and resistance to clarithromycin were associated. Increasing clarithromycin resistance rates were observed over time.
Bacterial Phosphating of Mild (Unalloyed) Steel
Authors: Volkland Hans-Peter , Harms Hauke , Müller Beat , Repphun Gernot , Wanner Oskar , Zehnder Alexander J. B.
Published: July 2002

Mild (unalloyed) steel electrodes were incubated in phosphate-buffered cultures of aerobic, biofilm-forming
Rhodococcus
sp. strain C125 and
Pseudomonas putida
mt2. A resulting surface reaction leading to the formation of a corrosion-inhibiting vivianite layer was accompanied by a characteristic electrochemical potential (E) curve. First, E increased slightly due to the interaction of phosphate with the iron oxides covering the steel surface. Subsequently, E decreased rapidly and after 1 day reached −510 mV, the potential of free iron, indicating the removal of the iron oxides. At this point, only scattered patches of bacteria covered the surface. A surface reaction, in which iron was released and vivianite precipitated, started. E remained at −510 mV for about 2 days, during which the vivianite layer grew steadily. Thereafter, E increased markedly to the initial value, and the release of iron stopped. Changes in E and formation of vivianite were results of bacterial activity, with oxygen consumption by the biofilm being the driving force. These findings indicate that biofilms may protect steel surfaces and might be used as an alternative method to combat corrosion.
Identification of Species and Sources of
Cryptosporidium
Oocysts in Storm Waters with a Small-Subunit rRNA-Based Diagnostic and Genotyping Tool
Authors: Xiao Lihua , Alderisio Kerri , Limor Josef , Royer Michael , Lal Altaf A.
Published: July 2002

The identification of
Cryptosporidium
oocysts in environmental samples is largely made by the use of an immunofluorescent assay. In this study, we have used a small-subunit rRNA-based PCR-restriction fragment length polymorphism technique to identify species and sources of
Cryptosporidium
oocysts present in 29 storm water samples collected from a stream in New York. A total of 12 genotypes were found in 27 positive samples; for 4 the species and probable origins were identified by sequence analysis, whereas the rest represent new genotypes from wildlife. Thus, this technique provides an alternative method for the detection and differentiation of
Cryptosporidium
parasites in environmental samples.
Identification of and Spatio-Temporal Differences between Microbial Assemblages from Two Neighboring Sulfurous Lakes: Comparison by Microscopy and Denaturing Gradient Gel Electrophoresis
Authors: Casamayor Emilio O. , Schäfer Hendrik , Bañeras Lluis , Pedrós-Alió Carlos , Muyzer Gerard
Published: July 2002

The microbial assemblages of Lake Cisó and Lake Vilar (Banyoles, northeast Spain) were analyzed in space and time by microscopy and by performing PCR-denaturing gradient gel electrophoresis (DGGE) and sequence analysis of 16S rRNA gene fragments. Samples obtained from different water depths and at two different times of the year (in the winter during holomixis and in the early spring during a phytoplankton bloom) were analyzed. Although the lakes have the same climatic conditions and the same water source, the limnological parameters were different, as were most of the morphologically distinguishable photosynthetic bacteria enumerated by microscopy. The phylogenetic affiliations of the predominant DGGE bands were inferred by performing a comparative 16S rRNA sequence analysis. Sequences obtained from Lake Cisó samples were related to gram-positive bacteria and to members of the division
Proteobacteria
. Sequences obtained from Lake Vilar samples were related to members of the
Cytophaga-Flavobacterium-Bacteroides
phylum and to cyanobacteria. Thus, we found that like the previously reported differences between morphologically distinct inhabitants of the two lakes, there were also differences among the community members whose morphologies did not differ conspicuously. The changes in the species composition from winter to spring were also marked. The two lakes both contained sequences belonging to phototrophic green sulfur bacteria, which is consistent with microscopic observations, but these sequences were different from the sequences of cultured strains previously isolated from the lakes. Euryarchaeal sequences (i.e., methanogen- and thermoplasma-related sequences) also were present in both lakes. These euryarchaeal group sequences dominated the archaeal sequences in Lake Cisó but not in Lake Vilar. In Lake Vilar, a new planktonic population related to the crenarchaeota produced the dominant archaeal band. The phylogenetic analysis indicated that new bacterial and archaeal lineages were present and that the microbial diversity of these assemblages was greater than previously known. We evaluated the correspondence between the abundances of several morphotypes and DGGE bands by comparing microscopy and sequencing results. Our data provide evidence that the sequences obtained from the DGGE fingerprints correspond to the microorganisms that are actually present at higher concentrations in the natural system.
The Function of Cytoplasmic Flavin Reductases in the Reduction of Azo Dyes by Bacteria
Authors: Russ Rainer , Rau Jörg , Stolz Andreas
Published: July 2002

A flavin reductase, which is naturally part of the ribonucleotide reductase complex of
Escherichia coli
, acted in cell extracts of recombinant
E. coli
strains under aerobic and anaerobic conditions as an “azo reductase.” The transfer of the recombinant plasmid, which resulted in the constitutive expression of high levels of activity of the flavin reductase, increased the reduction rate for different industrially relevant sulfonated azo dyes in vitro almost 100-fold. The flavin reductase gene (
fre
) was transferred to
Sphingomonas
sp. strain BN6, a bacterial strain able to degrade naphthalenesulfonates under aerobic conditions. The flavin reductase was also synthesized in significant amounts in the
Sphingomonas
strain. The reduction rates for the sulfonated azo compound amaranth were compared for whole cells and cell extracts from both recombinant strains,
E. coli
, and wild-type
Sphingomonas
sp. strain BN6. The whole cells showed less than 2% of the specific activities found with cell extracts. These results suggested that the cytoplasmic anaerobic “azo reductases,” which have been described repeatedly in in vitro systems, are presumably flavin reductases and that in vivo they have insignificant importance in the reduction of sulfonated azo compounds.
Identification of Nonpoint Sources of Fecal Pollution in Coastal Waters by Using Host-Specific 16S Ribosomal DNA Genetic Markers from Fecal Anaerobes
Authors: Bernhard Anne E. , Field Katharine G.
Published: July 2002

We describe a new PCR-based method for distinguishing human and cow fecal contamination in coastal waters without culturing indicator organisms, and we show that the method can be used to track bacterial marker sequences in complex environments. We identified two human-specific genetic markers and five cow-specific genetic markers in fecal samples by amplifying 16S ribosomal DNA (rDNA) fragments from members of the genus
Bifidobacterium
and the
Bacteroides-Prevotella
group and performing length heterogeneity PCR and terminal restriction fragment length polymorphism analyses. Host-specific patterns suggested that there are species composition differences in the
Bifidobacterium
and
Bacteroides-Prevotella
populations of human and cow feces. The patterns were highly reproducible among different hosts belonging to the same species. Additionally, all host-specific genetic markers were detected in water samples collected from areas frequently contaminated with fecal pollution. Ease of detection and longer survival in water made
Bacteroides-Prevotella
indicators better than
Bifidobacterium
indicators. Fecal 16S rDNA sequences corresponding to our
Bacteroides-Prevotella
markers comprised closely related gene clusters, none of which exactly matched previously published
Bacteroides
or
Prevotella
sequences. Our method detected host-specific markers in water at pollutant concentrations of 2.8 × 10
−5
to 2.8 × 10
−7
g (dry weight) of feces/liter and 6.8 × 10
−7
g (dry weight) of sewage/liter. Although our aim was to identify nonpoint sources of fecal contamination, the method described here should be widely applicable for monitoring spatial and temporal fluctuations in specific bacterial groups in natural environments.
Expanding the Known Diversity and Environmental Distribution of an Uncultured Phylogenetic Division of Bacteria
Authors: Dojka Michael A. , Harris J. Kirk , Pace Norman R.
Published: July 2002

Culture-independent molecular phylogenetic methods were used to explore the breadth of diversity and environmental distribution of members of the division-level “candidate” phylogenetic group WS6, recently discovered in a contaminated aquifer and with no cultivated representatives. A broad diversity of WS6-affiliated sequences were cloned from 7 of 12 environments investigated: mainly from anaerobic sediment environments. The number of sequences representing the WS6 candidate division was increased from 3 to 60 in this study. The extent of phylogenetic divergence (sequence difference) in this candidate division was found to be among the largest of any known bacterial division. This indicates that organisms representing the WS6 phylogenetic division offer a broad diversity of undiscovered biochemical and metabolic novelty. These results provide a framework for the further study of these evidently important kinds of organisms and tools, the sequences, with which to do so.
Phylogenetic Relationships of Butyrate-Producing Bacteria from the Human Gut
Authors: Barcenilla Adela , Pryde Susan E. , Martin Jennifer C. , Duncan Sylvia H. , Stewart Colin S. , Henderson Colin , Flint Harry J.
Published: July 2002

Butyrate is a preferred energy source for colonic epithelial cells and is thought to play an important role in maintaining colonic health in humans. In order to investigate the diversity and stability of butyrate-producing organisms of the colonic flora, anaerobic butyrate-producing bacteria were isolated from freshly voided human fecal samples from three healthy individuals: an infant, an adult omnivore, and an adult vegetarian. A second isolation was performed on the same three individuals 1 year later. Of a total of 313 bacterial isolates, 74 produced more than 2 mM butyrate in vitro. Butyrate-producing isolates were grouped by 16S ribosomal DNA (rDNA) PCR-restriction fragment length polymorphism analysis. The results indicate very little overlap between the predominant ribotypes of the three subjects; furthermore, the flora of each individual changed significantly between the two isolations. Complete sequences of 16S rDNAs were determined for 24 representative strains and subjected to phylogenetic analysis. Eighty percent of the butyrate-producing isolates fell within the XIVa cluster of gram-positive bacteria as defined by M. D. Collins et al. (Int. J. Syst. Bacteriol. 44:812–826, 1994) and A. Willems et al. (Int. J. Syst. Bacteriol. 46:195–199, 1996), with the most abundant group (10 of 24 or 42%) clustering with
Eubacterium rectale
,
Eubacterium ramulus
, and
Roseburia cecicola
. Fifty percent of the butyrate-producing isolates were net acetate consumers during growth, suggesting that they employ the butyryl coenzyme A-acetyl coenzyme A transferase pathway for butyrate production. In contrast, only 1% of the 239 non-butyrate-producing isolates consumed acetate.
Structural Changes and Interactions Involved in the Ca
2+
-Triggered Stabilization of the Cell-Bound Cell Envelope Proteinase in
Lactococcus lactis
subsp.
cremoris
SK11
Authors: Exterkate Fred A.
Published: July 2002

The cell-bound cell envelope proteinase (CEP) of the mesophilic cheese-starter organism
Lactococcus lactis
subsp.
cremoris
SK11 is protected from rapid thermal inactivation at 25°C by calcium bound to weak binding sites. The interactions with calcium are believed to trigger reversible structural rearrangements which are coupled with changes in specific activity (F. A. Exterkate and A. C. Alting, Appl. Env. Microbiol. 65:1390–1396, 1999). In order to determine the significance of the rearrangements for CEP stability and the nature of the interactions involved, the effects of the net charge present on the enzyme and of different neutral salts were studied with the stable Ca-loaded CEP, the unstable so-called “Ca-free” CEP and with the Ca-free CEP which was stabilized nonspecifically and essentially in its native conformation by the nonionic additive sucrose. The results suggest that strengthening of hydrophobic interactions is conducive to stabilization of the Ca-free CEP. On the other hand, a hydrophobic effect contributes significantly to the stability of the Ca-loaded CEP; a phased salting-in effect by a chaotropic salt suggests a complex inactivation process of this enzyme due to weakening of hydrophobic interactions and involving an intermediate enzyme species. Moreover, a Ca-triggered increase of a relatively significant hydrophobic effect in the sucrose-stabilized Ca-free CEP occurs. It is suggested that in the Ca-free CEP the absence of both local calcium-mediated backbone rigidification and neutralization of negative electrostatic potentials in the weak Ca-binding sites, and in addition the lack of significant hydrophobic stabilization, increase the relative effectiveness of electrostatic repulsive forces on the protein to an extent that causes the observed instability. The conditions in cheese seem to confer stability upon the cell-bound enzyme; its possible involvement in proteolysis throughout the ripening period is discussed.
Role of Volatile Fatty Acids in Development of the Cecal Microflora in Broiler Chickens during Growth
Authors: van der Wielen Paul W. J. J. , Biesterveld Steef , Notermans Servé , Hofstra Harm , Urlings Bert A. P. , van Knapen Frans
Published: July 2002

It is known that volatile fatty acids can inhibit growth of species of the family
Enterobacteriaceae
in vitro. However, whether these volatile fatty acids affect bacterial populations in the ceca of chickens is unknown. Therefore, a study was conducted to investigate if changes in volatile fatty acids in ceca of broiler chickens during growth affect bacterial populations. Results showed that members of the
Enterobacteriaceae
and enterococci are present in large numbers in 3-day-old broilers and start to decrease when broilers grow older. Lactobacilli are present in large numbers as well in 3-day-old broilers, but they remain stable during the growth of broilers. Acetate, butyrate, and propionate increase from undetectable levels in 1-day-old broilers to high concentrations in 15-day-old broilers, after which they stabilize. Significant negative correlations could be calculated between numbers of
Enterobacteriaceae
and concentrations of undissociated acetate, propionate, and butyrate. Furthermore, pure cultures of
Enterobacteriaceae
isolated from the ceca were grown in the presence of volatile fatty acids. Growth rates and maximal optical density decreased when these strains grew in the presence of increasing volatile fatty acid concentrations. It is concluded that volatile fatty acids are responsible for the reduction in numbers of
Enterobacteriaceae
in the ceca of broiler chickens during growth.
Bacillus anthracis
,
Bacillus cereus
, and
Bacillus thuringiensis
—One Species on the Basis of Genetic Evidence
Authors: Helgason Erlendur , Økstad Ole Andreas , Caugant Dominique A. , Johansen Henning A. , Fouet Agnes , Mock Michéle , Hegna Ida , Kolstø Anne-Brit
Published: July 2002

Bacillus anthracis
,
Bacillus cereus
, and
Bacillus thuringiensis
are members of the
Bacillus cereus
group of bacteria, demonstrating widely different phenotypes and pathological effects.
B. anthracis
causes the acute fatal disease anthrax and is a potential biological weapon due to its high toxicity.
B. thuringiensis
produces intracellular protein crystals toxic to a wide number of insect larvae and is the most commonly used biological pesticide worldwide.
B. cereus
is a probably ubiquitous soil bacterium and an opportunistic pathogen that is a common cause of food poisoning. In contrast to the differences in phenotypes, we show by multilocus enzyme electrophoresis and by sequence analysis of nine chromosomal genes that
B. anthracis
should be considered a lineage of
B. cereus
. This determination is not only a formal matter of taxonomy but may also have consequences with respect to virulence and the potential of horizontal gene transfer within the
B. cereus
group.
Archaeal Population Dynamics during Sequential Reduction Processes in Rice Field Soil
Authors: Lueders Tillmann , Friedrich Michael
Published: July 2002

The population dynamics of
Archaea
after flooding of an Italian rice field soil were studied over 17 days. Anoxically incubated rice field soil slurries exhibited a typical sequence of reduction processes characterized by reduction of nitrate, Fe
3+
, and sulfate prior to the initiation of methane production. Archaeal population dynamics were followed using a dual approach involving molecular sequence retrieval and fingerprinting of small-subunit (SSU) rRNA genes. We retrieved archaeal sequences from four clone libraries (30 each) constructed for different time points (days 0, 1, 8, and 17) after flooding of the soil. The clones could be assigned to known methanogens (i.e.,
Methanosarcinaceae
,
Methanosaetaceae
,
Methanomicrobiaceae
, and
Methanobacteriaceae
) and to novel euryarchaeotal (rice clusters I, II, and III) and crenarchaeotal (rice clusters IV and VI) lineages previously detected in anoxic rice field soil and on rice roots (R. Grosskopf, S. Stubner, and W. Liesack, Appl. Environ. Microbiol. 64:4983–4989, 1998). During the initiation of methanogenesis (days 0 to 17), we detected significant changes in the frequency of individual clones, especially of those affiliated with the
Methanosaetaceae
and
Methanobacteriaceae
. However, these findings could not be confirmed by terminal restriction fragment length polymorphism (T-RFLP) analysis of SSU rDNA amplicons. Most likely, the fluctuations in sequence composition of clone libraries resulted from cloning bias. Clonal SSU rRNA gene sequences were used to define operational taxonomic units (OTUs) for T-RFLP analysis, which were distinguished by group-specific
Taq
I restriction sites. Sequence analysis showed a high degree of conservation of
Taq
I restriction sites within the different archaeal lineages present in Italian rice field soil. Direct T-RFLP analysis of archaeal populations in rice field soil slurries revealed the presence of all archaeal lineages detected by cloning with a predominance of terminal restriction fragments characteristic of rice cluster I (389 bp),
Methanosaetaceae
(280 bp), and
Methanosarcinaceae
/rice cluster VI (182 bp). In general, the relative gene frequency of most detected OTUs remained rather constant over time during the first 17 days after flooding of the soil. Most minor OTUs (e.g.,
Methanomicrobiaceae
and rice cluster III) and
Methanosaetaceae
did not change in relative frequency. Rice cluster I (37 to 30%) and to a lesser extent rice cluster IV as well as
Methanobacteriaceae
decreased over time. Only the relative abundance of
Methanosarcinaceae
(182 bp) increased, roughly doubling from 15 to 29% of total archaeal gene frequency within the first 11 days, which was positively correlated to the dynamics of acetate and formate concentrations. Our results indicate that a functionally dynamic ecosystem, a rice field soil after flooding, was linked to a relatively stable archaeal community structure.
Variable Expressions of
Staphylococcus aureus
Bicomponent Leucotoxins Semiquantified by Competitive Reverse Transcription-PCR
Authors: Bronner Stéphane , Stoessel Patricia , Gravet Alain , Monteil Henri , Prévost Gilles
Published: July 2002

A competitive reverse transcription-PCR method was developed for the semiquantitation of the expression of genes encoding bicomponent leucotoxins of
Staphylococcus aureus
, e.g., Panton-Valentine leucocidin (
lukPV
), gamma-hemolysin (
hlgA
and
hlgCB
), and LukE-LukD (
lukED
). The optimization procedure included RNA preparation; reverse transcription; the use of various amounts of enzymes, antisense primer, and RNA; and the final amplification chain reaction. Reproducible results were obtained, with sensitivity for detection of cDNA within the range of 1 mRNA/10
4
CFU to 10
2
mRNA/CFU, depending on the gene. Both specific mRNAs were more significantly expressed at the late-exponential phase of growth. Expression was about 100-fold higher in yeast extract-Casamino Acids-pyruvate medium than in heart infusion medium. Expression of the widely distributed gamma-hemolysin locus in the NTCC 8178 strain was around 10-fold diminished compared with that in the ATCC 49775 strain. Because of the lower level of
hlgA
expression, the corresponding protein, which is generally not abundant in culture supernatant, should be investigated for its contribution to the leucotoxin-associated virulence. The
agr
,
sar
, and
agr sar
mutant strains revealed a great dependence with regard to leucotoxin expression on the global regulatory system in
S. aureus
, except that expression of
hlgA
was not affected in the
agr
mutant.
A Novel Gene Encoding Xanthan Lyase of
Paenibacillus alginolyticus
Strain XL-1
Authors: Ruijssenaars Harald J. , Hartmans Sybe , Verdoes Jan C.
Published: July 2002

Xanthan-modifying enzymes are powerful tools in studying structure-function relationships of this polysaccharide. One of these modifying enzymes is xanthan lyase, which removes the terminal side chain residue of xanthan. In this paper, the cloning and sequencing of the first xanthan lyase-encoding gene is described, i.e., the
xalA
gene, encoding pyruvated mannose-specific xanthan lyase of
Paenibacillus alginolyticus
XL-1. The
xalA
gene encoded a 100,823-Da protein, including a 36-amino-acid signal sequence. The 96,887-Da mature enzyme could be expressed functionally in
Escherichia coli
. Like the native enzyme, the recombinant enzyme showed no activity on depyruvated xanthan. Compared to production by
P. alginolyticus
, a 30-fold increase in volumetric productivity of soluble xanthan lyase was achieved by heterologous production in
E. coli
. The recombinant xanthan lyase was used to produce modified xanthan, which showed a dramatic loss of the capacity to form gels with locust bean gum.
Cellular Microcystin Content in N-Limited
Microcystis aeruginosa
Can Be Predicted from Growth Rate
Authors: Long Benedict M. , Jones Gary J. , Orr Philip T.
Published: July 2002

Cell quotas of microcystin (
Q
MCYST
; femtomoles of MCYST per cell), protein, and chlorophyll
a
(Chl
a
), cell dry weight, and cell volume were measured over a range of growth rates in N-limited chemostat cultures of the toxic cyanobacterium
Microcystis aeruginosa
MASH 01-A19. There was a positive linear relationship between
Q
MCYST
and specific growth rate (μ), from which we propose a generalized model that enables
Q
MCYST
at any nutrient-limited growth rate to be predicted based on a single batch culture experiment. The model predicts
Q
MCYST
from μ, μ
max
(maximum specific growth rate),
Q
MCYSTmax
(maximum cell quota), and
Q
MCYSTmin
(minimum cell quota). Under the conditions examined in this study, we predict a
Q
MCYSTmax
of 0.129 fmol cell
−1
at μ
max
and a
Q
MCYSTmin
of 0.050 fmol cell
−1
at μ = 0. Net MCYST production rate (
R
MCYST
) asymptotes to zero at μ = 0 and reaches a maximum of 0.155 fmol cell
−1
day
−1
at μ
max
. MCYST/dry weight ratio (milligrams per gram [dry weight]) increased linearly with μ, whereas the MCYST/protein ratio reached a maximum at intermediate μ. In contrast, the MCYST/Chl
a
ratio remained constant. Cell volume correlated negatively with μ, leading to an increase in intracellular MCYST concentration at high μ. Taken together, our results show that fast-growing cells of N-limited
M. aeruginosa
are smaller, are of lower mass, and have a higher intracellular MCYST quota and concentration than slow-growing cells. The data also highlight the importance of determining cell MCYST quotas, as potentially confusing interpretations can arise from determining MCYST content as a ratio to other cell components.
The Enterococcal Surface Protein, Esp, Is Involved in
Enterococcus faecalis
Biofilm Formation
Authors: Toledo-Arana Alejandro , Valle Jaione , Solano Cristina , Arrizubieta Marı́a Jesús , Cucarella Carme , Lamata Marta , Amorena Beatriz , Leiva José , Penadés José Rafael , Lasa Iñigo
Published: July 2002

The enterococcal surface protein, Esp, is a high-molecular-weight surface protein of unknown function whose frequency is significantly increased among infection-derived
Enterococcus faecalis
isolates. In this work, a global structural similarity was found between Bap, a biofilm-associated protein of
Staphylococcus aureus
, and Esp. Analysis of the relationship between the presence of the Esp-encoding gene (
esp
) and the biofilm formation capacity in
E. faecalis
demonstrated that the presence of the
esp
gene is highly associated (
P
< 0.0001) with the capacity of
E. faecalis
to form a biofilm on a polystyrene surface, since 93.5% of the
E. faecalis esp
-positive isolates were capable of forming a biofilm. Moreover, none of the
E. faecalis esp
-deficient isolates were biofilm producers. Depending on the
E. faecalis
isolate, insertional mutagenesis of
esp
caused either a complete loss of the biofilm formation phenotype or no apparent phenotypic defect. Complementation studies revealed that Esp expression in an
E. faecalis esp
-deficient strain promoted primary attachment and biofilm formation on polystyrene and polyvinyl chloride plastic from urine collection bags. Together, these results demonstrate that (i) biofilm formation capacity is widespread among clinical
E. faecalis
isolates, (ii) the biofilm formation capacity is restricted to the
E. faecalis
strains harboring
esp
, and (iii) Esp promotes primary attachment and biofilm formation of
E. faecalis
on abiotic surfaces.
Characterization of the Reduction of Selenate and Tellurite by Nitrate Reductases
Authors: Sabaty Monique , Avazeri Cécile , Pignol David , Vermeglio André
Published: July 2002

Preliminary studies showed that the periplasmic nitrate reductase (Nap) ofRhodobacter sphaeroidesand the membrane-bound nitrate reductases ofEscherichia coliare able to reduce selenate and tellurite in vitro with benzyl viologen as an electron donor. In the present study, we found that this is a general feature of denitrifiers. Both the periplasmic and membrane-bound nitrate reductases ofRalstonia eutropha, Paracoccus denitrificans, andParacoccus pantotrophuscan utilize potassium selenate and potassium tellurite as electron acceptors. In order to characterize these reactions, the periplasmic nitrate reductase ofR. sphaeroidesf. sp.denitrificansIL106 was histidine tagged and purified. TheVmaxandKmwere determined for nitrate, tellurite, and selenate. For nitrate, values of 39 μmol · min−1· mg−1and 0.12 mM were obtained forVmaxandKm, respectively, whereas theVmaxvalues for tellurite and selenate were 40- and 140-fold lower, respectively. These low activities can explain the observation that depletion of the nitrate reductase inR. sphaeroidesdoes not modify the MIC of tellurite for this organism.
Improvement and Optimization of Two Engineered Phage Resistance Mechanisms in
Lactococcus lactis
Authors: McGrath Stephen , Fitzgerald Gerald F. , van Sinderen Douwe
Published: July 2002

Homologous replication module genes were identified for four P335 type phages. DNA sequence analysis revealed that all four phages exhibited more than 90% DNA homology for at least two genes, designated

rep
2009

and
orf17
. One of these genes,

rep
2009

, codes for a putative replisome organizer protein and contains an assumed origin of phage DNA replication (

ori
2009

), which was identical for all four phages. DNA fragments representing the

ori
2009

sequence confer a phage-encoded resistance (Per) phenotype on lactococcal hosts when they are supplied on a high-copy-number vector. Furthermore, cloning multiple copies of the

ori
2009

sequence was found to increase the effectiveness of the Per phenotype conferred. A number of antisense plasmids targeting specific genes of the replication module were constructed. Two separate plasmids targeting

rep
2009

and
orf17
were found to efficiently inhibit proliferation of all four phages by interfering with intracellular phage DNA replication. These results represent two highly effective strategies for inhibiting bacteriophage proliferation, and they also identify a novel gene,
orf17
, which appears to be important for phage DNA replication. Furthermore, these results indicate that although the actual mechanisms of DNA replication are very similar, if not identical, for all four phages, expression of the replication genes is significantly different in each case.
Diversity of Sulfur Isotope Fractionations by Sulfate-Reducing Prokaryotes
Authors: Detmers Jan , Brüchert Volker , Habicht Kirsten S. , Kuever Jan
Published: July 2002

Batch culture experiments were performed with 32 different sulfate-reducing prokaryotes to explore the diversity in sulfur isotope fractionation during dissimilatory sulfate reduction by pure cultures. The selected strains reflect the phylogenetic and physiologic diversity of presently known sulfate reducers and cover a broad range of natural marine and freshwater habitats. Experimental conditions were designed to achieve optimum growth conditions with respect to electron donors, salinity, temperature, and pH. Under these optimized conditions, experimental fractionation factors ranged from 2.0 to 42.0‰. Salinity, incubation temperature, pH, and phylogeny had no systematic effect on the sulfur isotope fractionation. There was no correlation between isotope fractionation and sulfate reduction rate. The type of dissimilatory bisulfite reductase also had no effect on fractionation. Sulfate reducers that oxidized the carbon source completely to CO
2
showed greater fractionations than sulfate reducers that released acetate as the final product of carbon oxidation. Different metabolic pathways and variable regulation of sulfate transport across the cell membrane all potentially affect isotope fractionation. Previous models that explained fractionation only in terms of sulfate reduction rates appear to be oversimplified. The species-specific physiology of each sulfate reducer thus needs to be taken into account to understand the regulation of sulfur isotope fractionation during dissimilatory sulfate reduction.
Investigating Microbial (Micro)colony Heterogeneity by Vibrational Spectroscopy
Authors: Choo-Smith L.-P. , Maquelin K. , van Vreeswijk T. , Bruining H. A. , Puppels G. J. , Thi N. A. Ngo , Kirschner C. , Naumann D. , Ami D. , Villa A. M. , Orsini F. , Doglia S. M. , Lamfarraj H. , Sockalingum G. D. , Manfait M. , Allouch P. , Endtz H. P.
Published: July 2002

Fourier transform infrared and Raman microspectroscopy are currently being developed as new methods for the rapid identification of clinically relevant microorganisms. These methods involve measuring spectra from microcolonies which have been cultured for as little as 6 h, followed by the nonsubjective identification of microorganisms through the use of multivariate statistical analyses. To examine the biological heterogeneity of microorganism growth which is reflected in the spectra, measurements were acquired from various positions within (micro)colonies cultured for 6, 12, and 24 h. The studies reveal that there is little spectral variance in 6-h microcolonies. In contrast, the 12- and 24-h cultures exhibited a significant amount of heterogeneity. Hierarchical cluster analysis of the spectra from the various positions and depths reveals the presence of different layers in the colonies. Further analysis indicates that spectra acquired from the surface of the colonies exhibit higher levels of glycogen than do the deeper layers of the colony. Additionally, the spectra from the deeper layers present with higher RNA levels than the surface layers. Therefore, the 6-h colonies with their limited heterogeneity are more suitable for inclusion in a spectral database to be used for classification purposes. These results also demonstrate that vibrational spectroscopic techniques can be useful tools for studying the nature of colony development and biofilm formation.
Pathway of Propionate Oxidation by a Syntrophic Culture of
Smithella propionica
and
Methanospirillum hungatei
Authors: de Bok F. A. M. , Stams A. J. M. , Dijkema C. , Boone D. R.
Published: July 2002

The pathway of propionate conversion in a syntrophic coculture of
Smithella propionica
and
Methanospirillum hungatei
JF1 was investigated by
13
C-NMR spectroscopy. Cocultures produced acetate and butyrate from propionate. [3-
13
C]propionate was converted to [2-
13
C]acetate, with no [1-
13
C]acetate formed. Butyrate from [3-
13
C]propionate was labeled at the C2 and C4 positions in a ratio of about 1:1.5. Double-labeled propionate (2,3-
13
C) yielded not only double-labeled acetate but also single-labeled acetate at the C1 or C2 position. Most butyrate formed from [2,3-
13
C]propionate was also double labeled in either the C1 and C2 atoms or the C3 and C4 atoms in a ratio of about 1:1.5. Smaller amounts of single-labeled butyrate and other combinations were also produced. 1-
13
C-labeled propionate yielded both [1-
13
C]acetate and [2-
13
C]acetate. When
13
C-labeled bicarbonate was present, label was not incorporated into acetate, propionate, or butyrate. In each of the incubations described above,
13
C was never recovered in bicarbonate or methane. These results indicate that
S. propionica
does not degrade propionate via the methyl-malonyl-coenzyme A (CoA) pathway or any other of the known pathways, such as the acryloyl-CoA pathway or the reductive carboxylation pathway. Our results strongly suggest that propionate is dismutated to acetate and butyrate via a six-carbon intermediate.
Microbiological Analysis of Tube-Well Water in a Rural Area of Bangladesh
Authors: Islam M. S. , Siddika A. , Khan M. N. H. , Goldar M. M. , Sadique M. A. , Kabir A. N. M. H. , Huq Anwar , Colwell R. R.
Published: July 2002

Five tube-wells in Matlab, Bangladesh, were selected for analysis of selected biophysicochemical parameters. The results showed that all tube-well water samples contained zooplankton and bacteria. Results for some of the parameters were outside the accepted limits recommended by the World Health Organization for drinking water. It is concluded that water from tube-wells should be treated if used as drinking water.
Cloning, Sequencing, and Expression of the Cold-Inducible
hutU
Gene from the Antarctic Psychrotrophic Bacterium
Pseudomonas syringae
Authors: Janiyani Kamala L. , Ray M. K.
Published: July 2002

A promoter-fusion study with a Tn
5
-based promoter probe vector had earlier found that the
hutU
gene which encodes the enzyme urocanase for the histidine utilization pathway is upregulated at a lower temperature (4° C) in the Antarctic psychrotrophic bacterium
Pseudomonas syringae
. To examine the characteristics of the urocanase gene and its promoter elements from the psychrotroph, the complete
hutU
and its upstream region from
P. syringae
were cloned, sequenced, and analyzed in the present study. Northern blot and primer extension analyses suggested that the
hutU
gene is inducible upon a downshift of temperature (22 to 4°C) and that there is more than one transcription initiation site. One of the initiation sites was specific to the cells grown at 4°C, which was different from the common initiation sites observed at both 4 and 22°C. Although no typical promoter consensus sequences were observed in the flanking region of the transcription initiation sites, there was a characteristic CAAAA sequence at the −10 position of the promoters. Additionally, the location of the transcription and translation initiation sites suggested that the
hutU
mRNA contains a long 5′-untranslated region, a characteristic feature of many cold-inducible genes of mesophilic bacteria. A comparison of deduced amino acid sequences of urocanase from various bacteria, including the mesophilic and psychrotrophic
Pseudomonas
spp., suggests that there is a high degree of similarity between the enzymes. The enzyme sequence contains a signature motif (GXGX
2
GX
10
G) of the Rossmann fold for dinucleotide (NAD
+
) binding and two conserved cysteine residues in and around the active site. The psychrotrophic enzyme, however, has an extended N-terminal end.
Cloning, Sequence Analysis, and Expression in
Escherichia coli
of the Gene Encoding an α-Amino Acid Ester Hydrolase from
Acetobacter turbidans
Authors: Polderman-Tijmes Jolanda J. , Jekel Peter A. , de Vries Erik J. , van Merode Annet E. J. , Floris René , van der Laan Jan-Metske , Sonke Theo , Janssen Dick B.
Published: July 2002

The α-amino acid ester hydrolase from
Acetobacter turbidans
ATCC 9325 is capable of hydrolyzing and synthesizing β-lactam antibiotics, such as cephalexin and ampicillin. N-terminal amino acid sequencing of the purified α-amino acid ester hydrolase allowed cloning and genetic characterization of the corresponding gene from an
A. turbidans
genomic library. The gene, designated
aehA
, encodes a polypeptide with a molecular weight of 72,000. Comparison of the determined N-terminal sequence and the deduced amino acid sequence indicated the presence of an N-terminal leader sequence of 40 amino acids. The
aehA
gene was subcloned in the pET9 expression plasmid and expressed in
Escherichia coli
. The recombinant protein was purified and found to be dimeric with subunits of 70 kDa. A sequence similarity search revealed 26% identity with a glutaryl 7-ACA acylase precursor from
Bacillus laterosporus
, but no homology was found with other known penicillin or cephalosporin acylases. There was some similarity to serine proteases, including the conservation of the active site motif, GXSYXG. Together with database searches, this suggested that the α-amino acid ester hydrolase is a β-lactam antibiotic acylase that belongs to a class of hydrolases that is different from the Ntn hydrolase superfamily to which the well-characterized penicillin acylase from
E. coli
belongs. The α-amino acid ester hydrolase of
A. turbidans
represents a subclass of this new class of β-lactam antibiotic acylases.
Diversity of Nitrite Reductase (
nirK
and
nirS
) Gene Fragments in Forested Upland and Wetland Soils
Authors: Priemé Anders , Braker Gesche , Tiedje James M.
Published: July 2002

The genetic heterogeneity of nitrite reductase gene (
nirK
and
nirS
) fragments from denitrifying prokaryotes in forested upland and marsh soil was investigated using molecular methods.
nirK
gene fragments could be amplified from both soils, whereas
nirS
gene fragments could be amplified only from the marsh soil. PCR products were cloned and screened by restriction fragment length polymorphism (RFLP), and representative fragments were sequenced. The diversity of
nirK
clones was lower than the diversity of
nirS
clones. Among the 54 distinct
nirK
RFLP patterns identified in the two soils, only one pattern was found in both soils and in each soil two dominant groups comprised >35% of all clones. No dominance and few redundant patterns were seen among the
nirS
clones. Phylogenetic analysis of deduced amino acids grouped the
nirK
sequences into five major clusters, with one cluster encompassing most marsh clones and all upland clones. Only a few of the
nirK
clone sequences branched with those of known denitrifying bacteria. The
nirS
clones formed two major clusters with several subclusters, but all
nirS
clones showed less than 80% identity to
nirS
sequences from known denitrifying bacteria. Overall, the data indicated that the denitrifying communities in the two soils have many members and that the soils have a high richness of different
nir
genes, especially of the
nirS
gene, most of which have not yet been found in cultivated denitrifiers.
Transformation of Isopropylamine to
l
-Alaninol by
Pseudomonas
sp. Strain KIE171 Involves
N
-Glutamylated Intermediates
Authors: de Azevedo Wäsch Susana I. , van der Ploeg Jan R. , Maire Tere , Lebreton Alice , Kiener Andreas , Leisinger Thomas
Published: July 2002

Pseudomonas
sp. strain KIE171 was able to grow with isopropylamine or
l
-alaninol [
S
-(+)-2-amino-1-propanol] as the sole carbon source, but not with
d
-alaninol. To investigate the hypothesis that
l
-alaninol is an intermediate in the degradation of isopropylamine, two mini-Tn
5
mutants unable to utilize both isopropylamine and
l
-alaninol were isolated. Whereas mutant KIE171-BI transformed isopropylamine to
l
-alaninol, mutant KIE171-BII failed to do so. The two genes containing a transposon insertion were cloned, and the DNA regions flanking the insertions were sequenced. Two clusters, one comprising eight
ipu
(isopropylamine utilization) genes (
ipuABCDEFGH
) and the other encompassing two genes (
ipuI
and
orf259
), were identified. Comparisons of sequences of the deduced Ipu proteins and those in the database suggested that isopropylamine is transported into the cytoplasm by a putative permease, IpuG. The next step, the formation of γ-glutamyl-isopropylamide from isopropylamine, ATP, and
l
-glutamate, was shown to be catalyzed by IpuC, a γ-glutamylamide synthetase. γ-Glutamyl-isopropylamide is then subjected to stereospecific monooxygenation by the hypothetical four-component system IpuABDE, thereby yielding γ-glutamyl-
l
-alaninol [γ(
l
-glutamyl)-
l
-hydroxy-isopropylamide]. Enzymatic hydrolysis by a hydrolase, IpuF, was shown to finally liberate
l
-alaninol and to regenerate
l
-glutamate. No gene(s) encoding an enzyme for the next step in the degradation of isopropylamine was found in the
ipu
clusters. Presumably,
l
-alaninol is oxidized by an alcohol dehydrogenase to yield
l
-2-aminopropionaldehyde or it is deaminated by an ammonia lyase to propionaldehyde. Genetic evidence indicated that the aldehyde formed is then further oxidized by the hypothetical aldehyde dehydrogenases IpuI and IpuH to either
l
-alanine or propionic acid, compounds which can be processed by reactions of the intermediary metabolism.
Immobilization of Radionuclides and Heavy Metals through Anaerobic Bio-Oxidation of Fe(II)
Authors: Lack Joseph G. , Chaudhuri Swades K. , Kelly Shelly D. , Kemner Kenneth M. , O'Connor Susan M. , Coates John D.
Published: July 2002

Adsorption of heavy metals and radionuclides (HMR) onto iron and manganese oxides has long been recognized as an important reaction for the immobilization of these compounds. However, in environments containing elevated concentrations of these HMR the adsorptive capacity of the iron and manganese oxides may well be exceeded, and the HMR can migrate as soluble compounds in aqueous systems. Here we demonstrate the potential of a bioremediative strategy for HMR stabilization in reducing environments based on the recently described anaerobic nitrate-dependent Fe(II) oxidation by
Dechlorosoma
species. Bio-oxidation of 10 mM Fe(II) and precipitation of Fe(III) oxides by these organisms resulted in rapid adsorption and removal of 55 μM uranium and 81 μM cobalt from solution. The adsorptive capacity of the biogenic Fe(III) oxides was lower than that of abiotically produced Fe(III) oxides (100 μM for both metals), which may have been a result of steric hindrance by the microbial cells on the iron oxide surfaces. The binding capacity of the biogenic oxides for different heavy metals was indirectly correlated to the atomic radius of the bound element. X-ray absorption spectroscopy indicated that the uranium was bound to the biogenically produced Fe(III) oxides as U(VI) and that the U(VI) formed bidentate and tridentate inner-sphere complexes with the Fe(III) oxide surfaces.
Dechlorosoma suillum
oxidation was specific for Fe(II), and the organism did not enzymatically oxidize U(IV) or Co(II). Small amounts (less than 2.5 μM) of Cr(III) were reoxidized by
D. suillum
; however, this appeared to be inversely dependent on the initial concentration of the Cr(III). The results of this study demonstrate the potential of this novel approach for stabilization and immobilization of HMR in the environment.