bluuebunny/crossref_metadata_2025 huggingface dataset:
d
-Ala:
d
-Ala Ligase Gene Flanking the
vanC
Cluster: Evidence for Presence of Three Ligase Genes in Vancomycin-Resistant
Enterococcus gallinarum
BM4174
Authors: Ambúr Ole-Herman , Reynolds Peter E. , Arias Cesar A.
Published: July 2002

An open reading frame located 230 nucleotides downstream from the stop codon of
vanS

c

and in the opposite direction relative to the other genes of the
vanC
cluster was identified in
Enterococcus gallinarum
BM4174. This gene (designated
ddl2
) encoded a protein of 343 amino acids that had significant predicted structural similarity to
d
-Ala:
d
-Ala ligases and displayed 33 and 35% amino acid identity to VanC-1 and the previously reported partial sequence of Ddl from
E. gallinarum
, respectively. Biochemical characterization by thin-layer chromatography confirmed that Ddl2 is a
d
-Ala:
d
-Ala ligase with no detectable
d
-Ala:
d
-Ser ligase activity. The vancomycin dependence of
Enterococcus faecalis
BM4320 (
ddl
mutant) was lost on electroporation of a plasmid construct expressing
ddl2
constitutively. The latter strain was able to grow in the absence of vancomycin, and peptidoglycan precursor analysis under the same conditions indicated the synthesis of pentapeptide[
d
-Ala] as the main precursor, confirming the activity of Ddl2 in vivo. Expression of
ddl
and
ddl2
in BM4174 was tested by reverse transcription-PCR: results suggested that both
d
-Ala:
d
-Ala ligases were expressed concomitantly. Our findings indicate that vancomycin-resistant
E. gallinarum
BM4174 is likely to express one
d
-Ala:
d
-Ser and two
d
-Ala:
d
-Ala ligase genes.
Oxidative Modifications of Kynostatin-272, a Potent Human Immunodeficiency Virus Type 1 Protease Inhibitor: Potential Mechanism for Altered Activity in Monocytes/Macrophages
Authors: Davis David A. , Read-Connole Elizabeth , Pearson Kara , Fales Henry M. , Newcomb Fonda M. , Moskovitz Jackob , Yarchoan Robert
Published: July 2002

Previous studies have indicated that human immunodeficiency virus type 1 (HIV-1) protease inhibitors (PIs) are less active at blocking viral replication in HIV-1 infected peripheral blood monocytes/macrophages (M/M) than in HIV-1-infected T cells. We explored the hypothesis that oxidative modification and/or metabolism of the PIs in M/M might account for this reduced potency. We first tested the susceptibility of several PIs (kynostatin-272 [KNI-272], saquinavir, indinavir, ritonavir, or JE-2147) to oxidation after exposure to hydrogen peroxide (H2O2): only KNI-272 was highly susceptible to oxidation. Treatment of KNI-272 with low millimolar concentrations of H2O2resulted in mono-oxidation of the sulfur in theS-methyl cysteine (methioalanine) moiety, as determined by reversed-phase high-performance liquid chromatography and mass spectrometry (RP-HPLC/MS). Higher concentrations of H2O2led to an additional oxidation of the sulfur in the thioproline moiety of KNI-272. None of the PIs were metabolized or oxidized when added to T cells and cultured for up to 12 days. However, when KNI-272 was added to M/M, the concentration of the original KNI-272 steadily decreased with a corresponding increase in the production of three KNI-272 metabolites as identified by RP-HPLC/MS. The structures of these metabolites were different from those produced by H2O2treatment. The two major products of M/M metabolism of KNI-272 were identified as isomeric forms of KNI-272 oxidized solely on the thioproline ring. Both metabolites had reduced capacities to inhibit HIV-1 protease activity when tested in a standard HIV-1 protease assay. These studies demonstrate that antiviral compounds can be susceptible to oxidative modification in M/M and that this can affect their antiviral potency.
1
H Nuclear Magnetic Resonance Study of Oxazolidinone Binding to Bacterial Ribosomes
Authors: Zhou Casey Chun , Swaney Steven M. , Shinabarger Dean L. , Stockman Brian J.
Published: July 2002

The oxazolidinones are a novel class of antibiotics that inhibit initiation of protein synthesis in bacteria. In order to investigate their novel mechanism of action, the interactions of several oxazolidinones with bacterial 70S ribosomes, 50S subunits, and 30S subunits have been characterized by
1
H nuclear magnetic resonance (NMR) line-broadening analyses and transferred nuclear Overhauser enhancement (TRNOE) experiments. PNU-177553 and PNU-100592 (eperezolid) and their corresponding enantiomers, PNU-184414 and PNU-107112, were studied. The dissociation constants were determined to be 94 ± 44 μM and 195 ± 40 μM for PNU-177553 and eperezolid, respectively. There was a ∼4-fold decrease in affinity for their corresponding enantiomers. The NMR-derived dissociation constants are consistent with their antibacterial activity. PNU-177553 and eperezolid were found to bind only to the 50S subunit, with similar affinity as to the 70S ribosome, and to have no affinity for the 30S subunit. Specific binding of PNU-177553 was further confirmed in TRNOE experiments in which positive NOEs observed for the small molecule alone were changed to negative NOEs in the presence of bacterial 70S ribosomes. The observed NOEs indicated that PNU-177553 did not adopt a significantly different conformation when bound to the 70S ribosome, compared to the extended conformation that exists when free in solution. Since this is likeliest the case for each of the four compounds included in this study, the A ring C5 side chain may be positioned in the proper orientation for antibacterial activity in PNU-177553 and eperezolid but not in their inactive enantiomers.
Pharmacodynamic Assessment of Clarithromycin in a Murine Model of Pneumococcal Pneumonia
Authors: Tessier Pamela R. , Kim Myo-Kyoung , Zhou Wen , Xuan Dawei , Li Chonghua , Ye Min , Nightingale Charles H. , Nicolau David P.
Published: July 2002

The pharmacodynamic profile of clarithromycin (CLR) was evaluated with a murine model of pneumonia. Eight
Streptococcus pneumoniae
isolates, including three macrolide-sensitive and five macrolide-resistant strains, were inoculated intratracheally into immunocompromised ICR mice as 10
8
-CFU bacterial suspensions. Orally administered CLR daily doses ranging from 5 to 600 mg/kg of body weight were given over 5 days, during which animal survival was monitored. The bacterial density in lung tissues was examined after 24 h of CLR treatment and in control groups. Pharmacokinetic analysis of CLR in mice demonstrated that the regimen of 150 mg/kg twice a day was representative of human pharmacokinetics and was used to compare the efficacy of CLR against sensitive and resistant
S. pneumoniae
strains. Immunocompetent CBA/J mice were also infected and treated as described above and evaluated for bacterial density and survival to assess the effect of the presence of leukocytes. All three pharmacodynamic parameters, the duration (percent) of the time that serum CLR concentrations remain above the MIC (%T>MIC), the ratio of the area under the concentration-time curve from 0 to 24 h (AUC
0-24
) to the MIC, and the ratio of the maximum concentration of drug in serum to the MIC, were found to be closely correlated to CLR bacterial efficacy (
P
< 0.001). Furthermore, all parameters had close correlation to bacterial density (
r
2
= 0.72 to 0.82), median survival (
r
2
= 0.93 to 0.94), and total percent survival (
r
2
= 0.91 to 0.92). These in vivo data suggest that the bacterial activity of CLR is closely correlated with all three parameters over a wide range of exposures and, as a consequence of parameter interdependency, AUC
0-24
/MIC is the most reasonable predictor of antibiotic efficacy. In this neutropenic pneumonia model, CLR was less efficacious against
S. pneumoniae
strains for which MICs were ≥4 μg/ml. However, the presence of leukocytes in the immunocompetent mice resulted in improved bactericidal activity, relative to that in the neutropenic animals, despite an MIC of 4 μg/ml.
Association of Metronidazole Resistance and Natural Competence in
Helicobacter pylori
Authors: Yeh Yu-Ching , Chang Kai-Chin , Yang Jyh-Chin , Fang Chi-Tai , Wang Jin-Town
Published: July 2002

To study whether the capability of horizontal DNA transfer is associated with metronidazole resistance in
Helicobacter pylori
, a total of 81 clinical isolates were tested for MICs of metronidazole (MTZ). The MIC assays were performed by using the E-test and reconfirmed by the agar dilution method. Natural competence assays were performed by transferring a chloramphenicol acetyltransferase cassette and a 23S rRNA gene from a clarithromycin-resistant strain (with an A-to-G mutation at nucleotide 2143) by using natural transformation. Of the 81 isolates, 65 (80.2%) were naturally competent while 16 were not. Among the 65 naturally competent strains, 39 (60%) were highly resistant to MTZ (MICs, >32 μg/ml) while only 2 of 16 (12.5%) noncompetent strains were highly MTZ resistant (
P
, <0.001). Therefore, there is an association between natural competence and MTZ resistance.
Internal Thiols and Reactive Oxygen Species in Candidacidal Activity Exerted by an N-Terminal Peptide of Human Lactoferrin
Authors: Lupetti Antonella , Paulusma-Annema Akke , Senesi Sonia , Campa Mario , van Dissel Jaap T. , Nibbering Peter H.
Published: July 2002

We previously showed that the energized mitochondrion and extracellular ATP are essential for the candidacidal activity of the N-terminal peptide of human lactoferrin, subsequently referred to as hLF(1-11). The present study focuses on the involvement of internal thiols and reactive oxygen species (ROS) in the candidacidal activity exerted by hLF(1-11). Our results reveal that hLF(1-11) reduced the internal thiol level ofCandida albicansby 20%. In agreement,N-acetyl-l-cysteine (NAC), which is a precursor of glutathione and an ROS scavenger, inhibited the candidacidal activity of hLF(1-11). In addition, azodicarboxylic acid bis(N,N-dimethylamide) (diamide), which oxidizes internal thiols, was candidacidal. Furthermore, hLF(1-11) increased the level of ROS production byC. albicansin a dose-dependent manner, and a correlation between ROS production and candidacidal activity was found. 6-Hydroxy-2,5,7,8-tetramethylchroman-2-carboxylic acid (trolox), which is an ROS scavenger, partially inhibited the hLF(1-11)-induced, but not the diamide-triggered, candidacidal activity. It is of interest that hLF(1-11) and diamide acted synergistically in killingC. albicansand in ROS production. In agreement, oxidized ATP, an irreversible inhibitor of extracellular ATP receptors, partially blocked the hLF(1-11)-induced, but not the diamide-triggered, candidacidal activity. Finally, the hLF(1-11)-induced activation of mitochondria was inhibited by NAC, indicating that internal thiols and ROS affect mitochondrial activity. Therefore, the candidacidal activity of hLF(1-11) involves both generation of ROS and reduction of internal thiols.
Use of Two-Dimensional Electrophoresis To Study Differential Protein Expression in Divercin V41-Resistant and Wild-Type Strains of
Listeria monocytogenes
Authors: Duffes Frederique , Jenoe Paul , Boyaval Patrick
Published: July 2002

The use of bacteriocins from food-grade lactic acid bacteria to fight against the food-borne pathogen
Listeria monocytogenes
has been gaining interest. However, the emergence of resistant cells is frequently reported when
Listeria
is exposed to such antibacterials. A two-dimensional electrophoresis study of whole-cell protein expression of
Listeria monocytogenes
variants sensitive or resistant to the action of a bacteriocin produced by
Carnobacterium divergens
V41, divercin V41, is reported in this paper. The resistant variant obtained from the sensitive strain of
L. monocytogenes
P was also resistant to piscicocins V1 and SF668, but remained sensitive to nisin. Its growth rate was 50% less than the sensitive strain, and the MIC for it was 10
4
times higher. No reversion of the resistance was observed after 20 successive cultures in the absence of divercin V41. Comparison of the protein patterns by two-dimensional gel electrophoresis analysis showed clear differences. In the resistant variant pattern, at least nine spots had disappeared and eight new ones were observed. One of the newly synthesized proteins was identified as a flagellin of
L. monocytogenes
. Direct interaction between flagellin and divercin V41 was not evidenced. Intracellular synthesis of flagellin is probably an indirect effect of a modification in transcriptional regulation with widespread effects through a sigma factor. An intense protein, only present in the sensitive strain, was identified as a non-heme iron-binding ferritin displaying strong similarities to Dps proteins. Common modifications in the transcriptional regulation for these two proteins are discussed.
Persistence of Selected
Spartina alterniflora
Rhizoplane Diazotrophs Exposed to Natural and Manipulated Environmental Variability
Authors: Bagwell Christopher E. , Lovell Charles R.
Published: July 2002

Rhizoplane-rhizosphere nitrogen-fixing microorganisms (diazotrophs) are thought to provide a major source of biologically available nitrogen in salt marshes dominated by
Spartina alterniflora
. Compositional and functional stability has been demonstrated for this important functional group; however, the quantitative responses of specific diazotroph populations to environmental variability have not been assessed. Changes in the relative abundances of selected rhizoplane diazotrophs in response to long-term fertilization were monitored quantitatively by reverse sample genome probing. Fertilization stimulated
Spartina
, with plant height nearly tripling after 1 year. Fertilization also resulted in significant changes in interstitial porewater parameters. Diazotrophic activity (acetylene reduction assay) was sensitive to the fertilization treatments and was inhibited in some plots on several sampling dates. However, inhibition was never consistent across all of the replicates within a treatment and activity always recovered. The rhizoplane diazotrophs were quite responsive to environmental variability and to experimental treatments, but none were displaced by either environmental variability or experimental treatments. All strains were detected consistently throughout this study, and extensive spatial heterogeneity in the distribution patterns of these organisms was observed. The physiological traits that differentiate the diazotroph populations presumably support competitiveness and niche specialization, resulting in the observed resilience of the diazotroph populations in the rhizosphere.
A Gene Encoding a Novel Multidomain β-1,4-Mannanase from
Caldibacillus cellulovorans
and Action of the Recombinant Enzyme on Kraft Pulp
Authors: Sunna Anwar , Gibbs Moreland D. , Chin Charles W. J. , Nelson Peter J. , Bergquist Peter L.
Published: July 2002

Genomic walking PCR was used to obtained a 4,567-bp nucleotide sequence from
Caldibacillus cellulovorans
. Analysis of this sequence revealed that there were three open reading frames, designated ORF1, ORF2, and ORF3. Incomplete ORF1 encoded a putative C-terminal cellulose-binding domain (CBD) homologous to members of CBD family IIIb, while putative ORF3 encoded a protein of unknown function. The putative ManA protein encoded by complete
manA
ORF2 was an enzyme with a novel multidomain structure and was composed of four domains in the following order: a putative N-terminal domain (D1) of unknown function, an internal CBD (D2), a β-mannanase catalytic domain (D3), and a C-terminal CBD (D4). All four domains were linked via proline-threonine-rich peptides. Both of the CBDs exhibited sequence similarity to family IIIb CBDs, while the mannanase catalytic domain exhibited homology to the family 5 glycosyl hydrolases. The purified recombinant enzyme ManAd3 expressed from the cloned catalytic domain (D3) exhibited optimum activity at 85°C and pH 6.0 and was extremely thermostable at 70°C. This enzyme exhibited high specificity with the substituted galactomannan locust bean gum, while more substituted galacto- and glucomannans were poorly hydrolyzed. Preliminary studies to determine the effect of the recombinant ManAd3 and a recombinant thermostable β-xylanase on oxygen-delignified
Pinus radiata
kraft pulp revealed that there was an increase in the brightness of the bleached pulp.
Growth of
Escherichia coli
O157:H7 in Bruised Apple (
Malus domestica
) Tissue as Influenced by Cultivar, Date of Harvest, and Source
Authors: Dingman Douglas W.
Published: July 2002

Four of five apple cultivars (Golden Delicious, Red Delicious, McIntosh, Macoun, and Melrose) inoculated with
Escherichia coli
O157:H7 promoted growth of the bacterium in bruised tissue independent of the date of harvest (i.e., degree of apple ripening) or the source of the apple (i.e., tree-picked or dropped fruit). Apple harvest for this study began 4 September 1998 and ended 9 October, with weekly sampling. Throughout this study, freshly picked (<2 days after harvest) McIntosh apples usually prevented the growth of
E. coli
O157:H7 for 2 days. Growth of
E. coli
O157:H7 did occur following 6 days of incubation in bruised McIntosh apple tissue. However, the maximum total cell number was approximately 80-fold less than the maximum total cell number recovered from Red Delicious apples. When fruit was stored for 1 month at 4°C prior to inoculation with
E. coli
O157:H7, all five cultivars supported growth of the bacterium. For each apple cultivar, the pH of bruised tissue was significantly higher and °Brix was significantly lower than the pH and °Brix of undamaged tissue regardless of the source. In freshly picked apples, changes in the pH did not occur over the harvest season. Bruised Golden Delicious, McIntosh, and Melrose apple tissue pHs were not significantly different (tree-picked or dropped), and the °Brix values of McIntosh, Macoun, and Melrose apple tissue were not significantly different. Single-cultivar preparations of cider did not support growth of
E. coli
, and the cell concentration of inoculated cider declined over an 11-day test period. The rate of decline in
E. coli
cell concentration in the McIntosh cider was greater than those in the other ciders tested. The findings of this study suggested that the presence of some factor besides, or in addition to, pH inhibited
E. coli
growth in McIntosh apples.
Importance of Cry1 δ-Endotoxin Domain II Loops for Binding Specificity in
Heliothis virescens
(L.)
Authors: Jurat-Fuentes Juan Luis , Adang Michael J.
Published: July 2002

We constructed a model for
Bacillus thuringiensis
Cry1 toxin binding to midgut membrane vesicles from
Heliothis virescens
. Brush border membrane vesicle binding assays were performed with five Cry1 toxins that share homologies in domain II loops. Cry1Ab, Cry1Ac, Cry1Ja, and Cry1Fa competed with
125
I-Cry1Aa, evidence that each toxin binds to the Cry1Aa binding site in
H. virescens
. Cry1Ac competed with high affinity (competition constant [
K
com
] = 1.1 nM) for
125
I-Cry1Ab binding sites. Cry1Aa, Cry1Fa, and Cry1Ja also competed for
125
I-Cry1Ab binding sites, though the
K
com
values ranged from 179 to 304 nM. Cry1Ab competed for
125
I-Cry1Ac binding sites (
K
com
= 73.6 nM) with higher affinity than Cry1Aa, Cry1Fa, or Cry1Ja. Neither Cry1Ea nor Cry2Aa competed with any of the
125
I-Cry1A toxins. Ligand blots prepared from membrane vesicles were probed with Cry1 toxins to expand the model of Cry1 receptors in
H. virescens
. Three Cry1A toxins, Cry1Fa, and Cry1Ja recognized 170- and 110-kDa proteins that are probably aminopeptidases. Cry1Ab and Cry1Ac, and to some extent Cry1Fa, also recognized a 130-kDa molecule. Our vesicle binding and ligand blotting results support a determinant role for domain II loops in Cry toxin specificity for
H. virescens
. The shared binding properties for these Cry1 toxins correlate with observed cross-resistance in
H. virescens.
Cloning, Nucleotide Sequencing, and Functional Analysis of a Novel, Mobile Cluster of Biodegradation Genes from
Pseudomonas aeruginosa
Strain JB2
Authors: Hickey W. J. , Sabat G. , Yuroff A. S. , Arment A. R. , Pérez-Lesher J.
Published: July 2002

We have identified in
Pseudomonas aeruginosa
strain JB2 a novel cluster of mobile genes encoding degradation of hydroxy- and halo-aromatic compounds. Nineteen open reading frames were located and, based on sequence similarities, were putatively identified as encoding a ring hydroxylating oxygenase (
hybABCD
), an ATP-binding cassette-type transporter, an extradiol ring-cleavage dioxygenase, transcriptional regulatory proteins, enzymes mediating chlorocatechol degradation, and transposition functions. Expression of
hybABCD
in
Escherichia coli
cells effected stoichiometric transformation of 2-hydroxybenzoate (salicylate) to 2,5-dihydroxybenzoate (gentisate). This activity was predicted from sequence similarity to functionally characterized genes,
nagAaGHAb
from
Ralstonia
sp. strain U2 (S. L. Fuenmayor, M. Wild, A. L. Boyes, and P. A. Williams, J. Bacteriol. 180:2522–2530, 1998), and is the second confirmed example of salicylate 5-hydroxylase activity effected by an oxygenase outside the flavoprotein group. Growth of strain JB2 or
Pseudomonas huttiensis
strain D1 (an organism that had acquired the 2-chlorobenzoate degradation phenotype from strain JB2) on benzoate yielded mutants that were unable to grow on salicylate or 2-chlorobenzoate and that had a deletion encompassing
hybABCD
and the region cloned downstream. The mutants' inability to grow on 2-chlorobenzoate suggested the loss of additional genes outside of, but contiguous with, the characterized region. Pulsed-field gel electrophoresis revealed a plasmid of >300 kb in strain D1, but no plasmids were detected in strain JB2. Hybridization analyses confirmed that the entire 26-kb region characterized here was acquired by strain D1 from strain JB2 and was located in the chromosome of both organisms. Further studies to delineate the element's boundaries and functional characteristics could provide new insights into the mechanisms underlying evolution of bacterial genomes in general and of catabolic pathways for anthropogenic pollutants in particular.
Physiological Ecology of
Clostridium glycolicum
RD-1, an Aerotolerant Acetogen Isolated from Sea Grass Roots
Authors: Küsel Kirsten , Karnholz Arno , Trinkwalter Tanja , Devereux Richard , Acker Georg , Drake Harold L.
Published: July 2002

An anaerobic, H
2
-utilizing bacterium, strain RD-1, was isolated from the highest growth-positive dilution series of a root homogenate prepared from the sea grass
Halodule wrightii
. Cells of RD-1 were gram-positive, spore-forming, motile rods that were linked by connecting filaments. Acetate was produced in stoichiometries indicative of an acetyl coenzyme A (acetyl-CoA) pathway-dependent metabolism when RD-1 utilized H
2
-CO
2
, formate, lactate, or pyruvate. Growth on sugars or ethylene glycol yielded acetate and ethanol as end products. RD-1 grew at the expense of glucose in the presence of low initial concentrations (up to 6% [vol/vol]) of O
2
in the headspace of static, horizontally incubated culture tubes; the concentration of O
2
decreased during growth in such cultures. Peroxidase, NADH oxidase, and superoxide dismutase activities were detected in the cytoplasmic fraction of cells grown in the presence of O
2
. In comparison to cultures incubated under strictly anoxic conditions, acetate production decreased, higher amounts of ethanol were produced, and lactate and H
2
became significant end products when RD-1 was grown on glucose in the presence of O
2
. Similarly, when RD-1 was grown on fructose in the presence of elevated salt concentrations, lower amounts of acetate and higher amounts of ethanol and H
2
were produced. When the concentration of O
2
in the headspace exceeded 1% (vol/vol), supplemental H
2
was not utilized. The 16S rRNA gene of RD-1 had a 99.7% sequence similarity to that of
Clostridium glycolicum
DSM 1288
T
, an organism characterized as a fermentative anaerobe. Comparative experiments with
C
.
glycolicum
DSM 1288
T
demonstrated that it had negligible H
2
- and formate-utilizing capacities. However, carbon monoxide dehydrogenase was detected in both RD-1 and
C
.
glycolicum
DSM 1288
T
. A 91.4% DNA-DNA hybridization between the genomic DNA of RD-1 and that of
C
.
glycolicum
DSM 1288
T
confirmed that RD-1 was a strain of
C
.
glycolicum
. These results indicate that (i) RD-1 metabolizes certain substrates via the acetyl-CoA pathway, (ii) RD-1 can tolerate and consume limited amounts of O
2
, (iii) oxic conditions favor the production of ethanol, lactate, and H
2
by RD-1, and (iv) the ability of RD-1 to cope with limited amounts of O
2
might contribute to its survival in a habitat subject to daily gradients of photosynthesis-derived O
2
.
An Altered
Pseudomonas
Diversity Is Recovered from Soil by Using Nutrient-Poor
Pseudomonas
-Selective Soil Extract Media
Authors: Aagot Nina , Nybroe Ole , Nielsen Preben , Johnsen Kaare
Published: July 2002

We designed five
Pseudomonas
-selective soil extract NAA media containing the selective properties of trimethoprim and sodium lauroyl sarcosine and 0 to 100% of the amount of Casamino Acids used in the classical
Pseudomonas
-selective Gould's S1 medium. All of the isolates were confirmed to be
Pseudomonas
by a
Pseudomonas
-specific OprF antibody and a
Pseudomonas
-specific PCR targeting 16S ribosomal DNA. The
Pseudomonas
isolates were characterized by classical physiological tests, repetitive extragenic palindromic-PCR, Fourier transform infrared spectroscopy, and carbon source utilization patterns. Several of these analyses showed that the amount of Casamino Acids significantly influenced the diversity of the recovered
Pseudomonas
isolates. Furthermore, the data suggested that specific
Pseudomonas
subpopulations were represented on the nutrient-poor media. The NAA 1:100 medium, containing ca. 15 mg of organic carbon per liter, consistently gave significantly higher
Pseudomonas
CFU counts than Gould's S1 when tested on four Danish soils. NAA 1:100 may, therefore, be a better medium than Gould's S1 for enumeration and isolation of
Pseudomonas
from the low-nutrient soil environment.
Surface Expression of the Conserved C Repeat Region of Streptococcal M6 Protein within the Pip Bacteriophage Receptor ofLactococcus lactis
Authors: Geller Bruce L. , Wade Nadine , Gilberts Thomas D. , Hruby Dennis E. , Johanson Ryan , Topisirovic Ljubisa
Published: July 2002

The C repeat region of the M6 protein (M6c) fromStreptococcus pyogeneswas expressed within the Pip bacteriophage receptor on the surface ofLactococcus lactis.M6c was also detected in the culture medium. Thepip-emm6callele was integrated into the chromosome and stably expressed without antibiotic selection. The level of cell-associated surface expression of PipM6c was 0.015% of total cellular protein. The amount of PipM6c on the cell surface was increased about 17-fold by expressingpip-emm6cfrom a high-copy-number plasmid. Replacing the nativepippromoter with stronger promoters isolated previously fromLactobacillus acidophilusincreased surface expression of PipM6c from the high-copy-number plasmid up to 27-fold. Concomitantly, the amount of PipM6c in the medium increased 113-fold. The amount of PipM6c did not vary greatly between exponential- and stationary-phase cultures. Western blots indicated that the full-length PipM6c protein and most of the numerous proteolytic products were found only on the cell surface, whereas only one proteolytic fragment was found in the culture medium.
16S Ribosomal DNA Terminal Restriction Fragment Pattern Analysis of Bacterial Communities in Feces of Rats Fed
Lactobacillus acidophilus
NCFM
Authors: Kaplan Christopher W. , Astaire Johanna C. , Sanders Mary Ellen , Reddy Bandaru S. , Kitts Christopher L.
Published: July 2002

16S ribosomal DNA terminal restriction fragment patterns from rat fecal samples were analyzed to track the dynamics of
Lactobacillus acidophilus
NCFM and discern bacterial populations that changed during feeding with NCFM.
Lactobacillus johnsonii
and
Ruminococcus flavefaciens
were tentatively identified as such bacterial populations. The presence of
L. johnsonii
was confirmed by isolation from feces.
Deep Desulfurization of Extensively Hydrodesulfurized Middle Distillate Oil by
Rhodococcus
sp. Strain ECRD-1
Authors: Grossman M. J. , Lee M. K. , Prince R. C. , Minak-Bernero V. , George G. N. , Pickering I. J.
Published: July 2002

Dibenzothiophene (DBT), and in particular substituted DBTs, are resistant to hydrodesulfurization (HDS) and can persist in fuels even after aggressive HDS treatment. Treatment by
Rhodococcus
sp. strain ECRD-1 of a middle distillate oil whose sulfur content was virtually all substituted DBTs produced extensive desulfurization and a sulfur level of 56 ppm.
Effects of Alkylphosphates and Nitrous Oxide on Microbial Degradation of Polycyclic Aromatic Hydrocarbons
Authors: Bogan Bill W. , Lahner Lisa M. , Trbovic Vesna , Szajkovics Ann M. , Paterek J. Robert
Published: July 2002

We conducted a series of liquid-culture experiments to begin to evaluate the abilities of gaseous sources of nitrogen and phosphorus to support biodegradation of polycyclic aromatic hydrocarbons (PAHs). Nutrients examined included nitrous oxide, as well as triethylphosphate (TEP) and tributylphosphate (TBP). Cultures were established using the indigenous microbial populations from one manufactured gas plant (MGP) site and one crude oil-contaminated drilling field site. Mineralization of phenanthrene was measured under alternative nutrient regimes and was compared to that seen with ammoniacal nitrogen and PO
4
. Parallel cultures were used to assess removal of a suite of three- to five-ring PAHs. In summary, the abilities of the different communities to degrade PAH when supplemented with N
2
O, TEP, and TBP were highly variable. For example, in the MGP soil, organic P sources, especially TBP, supported a considerably higher degree of removal of low-molecular-weight PAHs than did PO
4
; however, loss of high-molecular-weight compounds was impaired under these conditions. The disappearance of most PAHs was significantly less in the oil field soil when organophosphates were used. These results indicate that the utility of gaseous nutrients for PAH bioremediation in situ may be limited and will very likely have to be assessed on a case-by-case basis.
Cloning of a Phenol Oxidase Gene from
Acremonium murorum
and Its Expression in
Aspergillus awamori
Authors: Gouka Robin J. , van der Heiden Monique , Swarthoff Ton , Verrips C. Theo
Published: July 2002

Fungal multicopper oxidases have many potential industrial applications, since they perform reactions under mild conditions. We isolated a phenol oxidase from the fungus
Acremonium murorum
var.
murorum
that was capable of decolorizing plant chromophores (such as anthocyanins)
.
This enzyme is of interest in laundry-cleaning products because of its broad specificity for chromophores. We expressed an
A.
murorum
cDNA library in
Saccharomyces cerevisiae
and subsequently identified enzyme-producing yeast colonies based on their ability to decolor a plant chromophore. The cDNA sequence contained an open reading frame of 1,806 bp encoding an enzyme of 602 amino acids. The phenol oxidase was overproduced by
Aspergillus awamori
as a fusion protein with glucoamylase, cleaved in vivo
,
and purified from the culture broth by hydrophobic-interaction chromatography. The phenol oxidase is active at alkaline pH (the optimum for syringaldazine is pH 9) and high temperature (optimum, 60°C) and is fully stable for at least 1 h at 60°C under alkaline conditions. These characteristics and the high production level of 0.6 g of phenol oxidase per liter in shake flasks, which is equimolar with the glucoamylase protein levels, make this enzyme suitable for use in processes that occur under alkaline conditions, such as laundry cleaning.
Induction of
bphA
, Encoding Biphenyl Dioxygenase, in Two Polychlorinated Biphenyl-Degrading Bacteria, Psychrotolerant
Pseudomonas
Strain Cam-1 and Mesophilic
Burkholderia
Strain LB400
Authors: Master Emma R. , Mohn William W.
Published: July 2002

We investigated induction of biphenyl dioxygenase in the psychrotolerant polychlorinated biphenyl (PCB) degrader
Pseudomonas strain
Cam-1 and in the mesophilic PCB degrader
Burkholderia strain
LB400. Using a counterselectable gene replacement vector, we inserted a
lacZ
-Gm
r
fusion cassette between chromosomal genes encoding the large subunit (
bphA
) and small subunit (
bphE
) of biphenyl dioxygenase in Cam-1 and LB400, generating Cam-10 and LB400-1, respectively. Potential inducers of
bphA
were added to cell suspensions of Cam-10 and LB400-1 incubated at 30°C, and then beta-galactosidase activity was measured. Biphenyl induced beta-galactosidase activity in Cam-10 to a level approximately six times greater than the basal level in cells incubated with pyruvate. In contrast, the beta-galactosidase activities in LB400-1 incubated with biphenyl and in LB400-1 incubated with pyruvate were indistinguishable. At a concentration of 1 mM, most of the 40 potential inducers tested were inhibitory to induction by biphenyl of beta-galactosidase activity in Cam-10. The exceptions were naphthalene, salicylate, 2-chlorobiphenyl, and 4-chlorobiphenyl, which induced beta-galactosidase activity in Cam-10, although at levels that were no more than 30% of the levels induced by biphenyl. After incubation for 24 h at 7°C, biphenyl induced beta-galactosidase activity in Cam-10 to a level approximately four times greater than the basal level in cells incubated with pyruvate. The constitutive level of beta-galactosidase activity in LB400-1 grown at 15°C was approximately five times less than the level in LB400-1 grown at 30°C. Thus, there are substantial differences in the effects of physical and chemical environmental conditions on genetic regulation of PCB degradation in different bacteria.
Carbon Limitation Induces ς
S
-Dependent Gene Expression in
Pseudomonas fluorescens
in Soil
Authors: Koch Birgit , Worm Jakob , Jensen Linda E. , Højberg Ole , Nybroe Ole
Published: July 2002

Recent studies employing reporter gene technology indicate that the availabilities of the major nutrients nitrogen, phosphate, and iron to
Pseudomonas
are not severely limited in bulk soil. Indirect evidence has pointed to carbon limitation as a severe nutritional stress in this environment. We show that a plasmid (pGM115)-borne transcriptional fusion between the ς
S
-dependent
Escherichia coli
promoter
P

fic

and
lacZ
functions as a reliable reporter for carbon availability in
Pseudomonas fluorescens.
When
P. fluorescens
strain DF57(pGM115) was introduced into bulk soil, carbon-limiting conditions were indicated by citrate-repressible induction of β-galactosidase activity. To address carbon availability at the single-cell level, we developed an immunofluorescence double-staining procedure for individual DF57 cells expressing β-galactosidase from
P

fic

. Changes in cell size and expression of β-galactosidase were analyzed by flow cytometry. Cells extracted from soil microcosms reduced their size less than carbon-starved cells in pure culture and showed an increased tendency to aggregate. The single-cell analysis revealed that for cells residing in soil, the expression of β-galactosidase became heterogeneous and only a DF57 subpopulation appeared to be carbon limited. In soil amended with barley straw, limited nitrogen availability has been determined by use of the bioluminescent reporter strain
P. fluorescens
DF57-N3. We used strain DF57-N3(pGM115) as a double reporter for carbon and nitrogen limitation that allowed us to study the dynamics of carbon and nitrogen availabilities in more detail. In straw-amended soil β-galactosidase activity remained low, while nitrogen limitation-dependent bioluminescence appeared after a few days. Hence, nitrogen became limited under conditions where carbon resources were not completely exhausted.
Prevalence, Antigenic Specificity, and Bactericidal Activity of Poultry Anti-
Campylobacter
Maternal Antibodies
Authors: Sahin Orhan , Zhang Qijing , Meitzler Jerrel C. , Harr Brian S. , Morishita Teresa Y. , Mohan R.
Published: July 2002

Poultry are considered the major reservoir for
Campylobacter jejuni
, a leading bacterial cause of human food-borne diarrhea. To understand the ecology of
C. jejuni
and develop strategies to control
C. jejuni
infection in the animal reservoir, we initiated studies to examine the potential role of anti-
Campylobacter
maternal antibodies in protecting young broiler chickens from infection by
C. jejuni
. Using an enzyme-linked immunosorbent assay (ELISA), the prevalence of anti-
C. jejuni
antibodies in breeder chickens, egg yolks, and broilers from multiple flocks of different farms were examined. High levels of antibodies to the organism were detected in serum samples of breeder chickens and in egg yolk contents. To determine the dynamics of anti-
Campylobacter
maternal antibody transferred from yolks to hatchlings, serum samples collected from five broiler flocks at weekly intervals from 1 to 28 or 42 days of age were also examined by ELISA. Sera from the 1-day and 7-day-old chicks showed high titers of antibodies to
C. jejuni
. Thereafter, antibody titers decreased substantially and were not detected during the third and fourth weeks of age. The disappearance of anti-
Campylobacter
maternal antibodies during 3 to 4 weeks of age coincides with the appearance of
C. jejuni
infections observed in many broiler chicken flocks. As shown by immunoblotting, the maternally derived antibodies recognized multiple membrane proteins of
C. jejuni
ranging from 19 to 107 kDa. Moreover, in vitro serum bactericidal assays showed that anti-
Campylobacter
maternal antibodies were active in antibody-dependent complement-mediated killing of
C. jejuni
. Together, these results highlight the widespread presence of functional anti-
Campylobacter
antibodies in the poultry production system and provide a strong rationale for further investigation of the potential role of anti-
C. jejuni
maternal antibodies in protecting young chickens from infection by
C. jejuni
.
Cloning, Characterization, and Transcription of Three Laccase Genes from
Gaeumannomyces graminis
var.
tritici
, the Take-All Fungus
Authors: Litvintseva Anastasia P. , Henson Joan M.
Published: July 2002

Gaeumannomyces graminis
var.
tritici
, a filamentous ascomycete, is an important root pathogen of cereals that causes take-all disease and results in severe crop losses worldwide. Previously we identified a polyphenol oxidase (laccase) secreted by the fungus when induced with copper. Here we report cloning and partial characterization of three laccase genes (
LAC1
,
LAC2
, and
LAC3
) from
G. graminis
var.
tritici.
Predicted polypeptides encoded by these genes had 38 to 42% amino acid sequence identity and had conserved copper-binding sites characteristic of laccases. The sequence of the
LAC2
predicted polypeptide matched the N-terminal sequence of the secreted laccase that we purified in earlier studies. We also characterized expression patterns of these genes by reverse transcription-PCR.
LAC1
was transcribed constitutively, and transcription of
LAC2
was Cu inducible. All three genes were transcribed in planta; however, transcription of
LAC3
was observed only in planta or in the presence of host (wheat) plant homogenate.
Saccharomyces cerevisiae URH1
(Encoding Uridine-Cytidine
N
-Ribohydrolase): Functional Complementation by a Nucleoside Hydrolase from a Protozoan Parasite and by a Mammalian Uridine Phosphorylase
Authors: Mitterbauer Rudolf , Karl Thomas , Adam Gerhard
Published: July 2002

Nucleoside hydrolases catalyze the cleavage of N-glycosidic bonds in nucleosides, yielding ribose and the respective bases. While nucleoside hydrolase activity has not been detected in mammalian cells, many protozoan parasites rely on nucleoside hydrolase activity for salvage of purines and/or pyrimidines from their hosts. In contrast, uridine phosphorylase is the key enzyme of pyrimidine salvage in mammalian hosts and many other organisms. We show here that the open reading frame (ORF)
YDR400w
of
Saccharomyces cerevisiae
carries the gene encoding uridine hydrolase (
URH1
). Disruption of this gene in a conditionally pyrimidine-auxotrophic
S. cerevisiae
strain, which is also deficient in uridine kinase (
urk1
), leads to the inability of the mutant to utilize uridine as the sole source of pyrimidines. Protein extracts of strains overexpressing
YDR400w
show increased hydrolase activity only with uridine and cytidine, but no activity with inosine, adenosine, guanosine, and thymidine as substrates, demonstrating that ORF
YDR400w
encodes a uridine-cytidine
N
-ribohydrolase. Expression of a homologous cDNA from a protozoan parasite (
Crithidia fasciculata
) in a
ura3 urk1 urh1
mutant is sufficient to restore growth on uridine. Growth can also be restored by expression of a human uridine phosphorylase cDNA. Yeast strains expressing protozoan
N
-ribohydrolases or host phosphorylases could therefore become useful tools in drug screens for specific inhibitors.
Modeling Growth and Bacteriocin Production by
Lactobacillus amylovorus
DCE 471 in Response to Temperature and pH Values Used for Sourdough Fermentations
Authors: Messens Winy , Neysens Patricia , Vansieleghem Wim , Vanderhoeven Johan , De Vuyst Luc
Published: July 2002

The biokinetics of cell growth of
Lactobacillus amylovorus
DCE 471 and bacteriocin production by this strain were investigated as a function of the temperatures (28 to 44°C) and pH values (pH 4.2 to 6.4) that are characteristic of a sourdough fermentation process. The influence of temperature and pH on microbial behavior is described by using a successfully validated predictive model.
Application of Amplified Fragment Length Polymorphism Fingerprinting for Taxonomy and Identification of the Soft Rot Bacteria
Erwinia carotovora
and
Erwinia chrysanthemi
Authors: Avrova Anna O. , Hyman Lizbeth J. , Toth Rachel L. , Toth Ian K.
Published: July 2002

The soft rot bacteria
Erwinia carotovora
and
Erwinia chrysanthemi
are important pathogens of potato and other crops. However, the taxonomy of these pathogens, particularly at subspecies level, is unclear. An investigation using amplified fragment length polymorphism (AFLP) fingerprinting was undertaken to determine the taxonomic relationships within this group based on their genetic relatedness. Following cluster analysis on the similarity matrices derived from the AFLP gels, four clusters (clusters 1 to 4) resulted. Cluster 1 contained
Erwinia carotovora
subsp.
carotovora
(subclusters 1a and 1b) and
Erwinia carotovora
subsp.
odorifera
(subcluster 1c) strains, while cluster 2 contained
Erwinia carotovora
subsp.
atroseptica
(subcluster 2a) and
Erwinia carotovora
subsp.
betavasculorum
(subcluster 2b) strains. Clusters 3 and 4 contained
Erwinia carotovora
subsp.
wasabiae
and
E. chrysanthemi
strains, respectively. While
E. carotovora
subsp.
carotovora
and
E. chrysanthemi
showed a high level of molecular diversity (23 to 38% mean similarity),
E. carotovora
subsp.
odorifera
,
E. carotovora
subsp.
betavasculorum
,
E. carotovora
subsp.
atroseptica
, and
E. carotovora
subsp.
wasabiae
showed considerably less (56 to 76% mean similarity), which may reflect their limited geographical distributions and/or host ranges. The species- and subspecies-specific banding profiles generated from the AFLPs allowed rapid identification of unknown isolates and the potential for future development of diagnostics. AFLP fingerprinting was also found to be more differentiating than other techniques for typing the soft rot erwinias and was applicable to all strain types, including different serogroups.
Microheterogeneity in 16S Ribosomal DNA-Defined Bacterial Populations from a Stratified Planktonic Environment Is Related to Temporal Changes and to Ecological Adaptations
Authors: Casamayor Emilio O. , Pedrós-Alió Carlos , Muyzer Gerard , Amann Rudolf
Published: July 2002

Temporal changes of the bacterioplankton from a meromictic lake (Lake Vilar, Banyoles, Spain) were analyzed with four culture-independent techniques: epifluorescence microscopy, PCR-denaturing gradient gel electrophoresis (DGGE) fingerprinting, fluorescence in situ whole-cell hybridization and flow cytometry sorting. Microscopically, blooms of one cyanobacterium (
Synechococcus
sp.-like), one green sulfur bacterium (
Chlorobium phaeobacteroides
-like), and one purple sulfur bacterium (
Thiocystis minor
-like) were observed at different depths and times. DGGE retrieved these populations and, additionally, populations related to the
Cytophaga
-
Flavobacterium-Bacteroides
phylum as predominant community members. The analyses of partial 16S ribosomal DNA sequences from the DGGE fingerprints (550 bp analyzed) revealed higher genetic diversity than expected from microscopic observation for most of these groups. Thus, the sequences of two
Synechococcus
spp. (both had a similarity of 97% to
Synechococcus
sp. strain PCC6307 in 16S rRNA), two
Thiocystis
spp. (similarities to
Thiocystis minor
of 93 and 94%, respectively), and three
Cytophaga
spp. (similarities to
Cytophaga fermentans
of 88 and 89% and to
Cytophaga
sp. of 93%, respectively) were obtained. The two populations of
Synechococcus
exhibited different pigment compositions and temporal distributions and their 16S rRNA sequences were 97.3% similar. The two
Thiocystis
populations differed neither in pigment composition nor in morphology, but their 16S rRNA sequences were only 92.3% similar and they also showed different distributions over time. Finally, two of the
Cytophaga
spp. showed 96.2% similarity between the 16S rRNA sequences, but one of them was found to be mostly attached to particles and only in winter. Thus, the identity of the main populations changed over time, but the function of the microbial guilds was maintained. Our data showed that temporal shifts in the identity of the predominant population is a new explanation for the environmental 16S rRNA microdiversity retrieved from microbial assemblages and support the hypothesis that clusters of closely related 16S rRNA environmental sequences may actually represent numerous closely related, yet ecologically distinct, populations.
Cyanophycin Production in a Phycoerythrin-Containing Marine
Synechococcus
Strain of Unusual Phylogenetic Affinity
Authors: Wingard Lauren L. , Miller Scott R. , Sellker Jeanne M. L. , Stenn Erik , Allen Mary M. , Wood A. Michelle
Published: July 2002

Thirty-two strains of phycoerythrin-containing marine picocyanobacteria were screened for the capacity to produce cyanophycin, a nitrogen storage compound synthesized by some, but not all, cyanobacteria. We found that one of these strains,
Synechococcus
sp. strain G2.1 from the Arabian Sea, was able to synthesize cyanophycin. The cyanophycin extracted from the cells was composed of roughly equimolar amounts of arginine and aspartate (29 and 35 mol%, respectively), as well as a small amount of glutamate (15 mol%). Phylogenetic analysis, based on partial 16S ribosomal DNA (rDNA) sequence data, showed that
Synechococcus
sp. strain G2.1 formed a well-supported clade with several strains of filamentous cyanobacteria. It was not closely related to several other well-studied marine picocyanobacteria, including
Synechococcus
strains PCC7002, WH7805, and WH8018 and
Prochlorococcus
sp. strain MIT9312. This is the first report of cyanophycin production in a phycoerythrin-containing strain of marine or halotolerant
Synechococcus
, and its discovery highlights the diversity of this ecologically important functional group.