bluuebunny/crossref_metadata_2025_split huggingface dataset:
Characterization of Two New Glycosyl Hydrolases from the Lactic Acid Bacterium
Carnobacterium piscicola
Strain BA
Authors: Coombs Jonna , Brenchley Jean E. 
Published: July 2002

ABSTRACT

Three genes with homology to glycosyl hydrolases were detected on a DNA fragment cloned from a psychrophilic lactic acid bacterium isolate,
Carnobacterium piscicola
strain BA. A 2.2-kb region corresponding to an α-galactosidase gene,
agaA
, was followed by two genes in the same orientation,
bgaB
, encoding a 2-kb β-galactosidase, and
bgaC
, encoding a structurally distinct 1.76-kb β-galactosidase. This gene arrangement had not been observed in other lactic acid bacteria, including
Lactococcus lactis
, for which the genome sequence is known. To determine if these sequences encoded enzymes with α- and β-galactosidase activities, we subcloned the genes and examined the enzyme properties. The α-galactosidase, AgaA, hydrolyzes
para
-nitrophenyl-α-
d
-galactopyranoside and has optimal activity at 32 to 37°C. The β-galactosidase, BgaC, has an optimal activity at 40°C and a half-life of 15 min at 45°C. The regulation of these enzymes was tested in
C. piscicola
strain BA and activity on both α- and β-galactoside substrates decreased for cells grown with added glucose or lactose. Instead, an increase in activity on a phosphorylated β-galactoside substrate was found for the cells supplemented with lactose, suggesting that a phospho-galactosidase functions during lactose utilization. Thus, the two β-galactosidases may act synergistically with the α-galactosidase to degrade other polysaccharides available in the environment.
Benzoate Fermentation by the Anaerobic Bacterium
Syntrophus aciditrophicus
in the Absence of Hydrogen-Using Microorganisms
Authors: Elshahed Mostafa S. , McInerney Michael J. 
Published: July 2002

ABSTRACT

The anaerobic bacterium
Syntrophus aciditrophicus
metabolized benzoate in pure culture in the absence of hydrogen-utilizing partners or terminal electron acceptors. The pure culture of
S. aciditrophicus
produced approximately 0.5 mol of cyclohexane carboxylate and 1.5 mol of acetate per mol of benzoate, while a coculture of
S
.
aciditrophicus
with the hydrogen-using methanogen
Methanospirillum hungatei
produced 3 mol of acetate and 0.75 mol of methane per mol of benzoate. The growth yield of the
S. aciditrophicus
pure culture was 6.9 g (dry weight) per mol of benzoate metabolized, whereas the growth yield of the
S. aciditrophicus-M. hungatei
coculture was 11.8 g (dry weight) per mol of benzoate. Cyclohexane carboxylate was metabolized by
S. aciditrophicus
only in a coculture with a hydrogen user and was not metabolized by
S. aciditrophicus
pure cultures. Cyclohex-1-ene carboxylate was incompletely degraded by
S. aciditrophicus
pure cultures until a free energy change (Δ
G
′) of −9.2 kJ/mol was reached (−4.7 kJ/mol for the hydrogen-producing reaction). Cyclohex-1-ene carboxylate, pimelate, and glutarate transiently accumulated at micromolar levels during growth of an
S. aciditrophicus
pure culture with benzoate. High hydrogen (10.1 kPa) and acetate (60 mM) levels inhibited benzoate metabolism by
S. aciditrophicus
pure cultures. These results suggest that benzoate fermentation by
S. aciditrophicus
in the absence of hydrogen users proceeds via a dismutation reaction in which the reducing equivalents produced during oxidation of one benzoate molecule to acetate and carbon dioxide are used to reduce another benzoate molecule to cyclohexane carboxylate, which is not metabolized further. Benzoate fermentation to acetate, CO
2
, and cyclohexane carboxylate is thermodynamically favorable and can proceed at free energy values more positive than −20 kJ/mol, the postulated minimum free energy value for substrate metabolism.
Modification of a Rapid Method for the Identification of Gene-Specific Polymorphisms inCryptosporidiumparvumand Its Application to Clinical and Epidemiological Investigations
Authors: Elwin K. , Chalmers R. M. , Roberts R. , Guy E. C. , Casemore D. P. 
Published: July 2002

ABSTRACTThe application of genotyping to clinical isolates ofCryptosporidiumhas increased significantly our knowledge and understanding of the distribution and epidemiology of this parasite. However, some methods can be laborious and demand specialist technical expertise. PCR-restriction fragment length polymorphism (RFLP) techniques represent a more rapid and simple method of genotyping to support epidemiological and clinical investigations than conventional DNA analytical techniques. We describe a nested PCR-RFLP technique that identifies polymorphisms in theC.parvumthrombospondin-related adhesive protein gene locus; this method offers a sensitive and specific tool for the confirmation and investigation of disease associated withC.parvum. The potential of this enhanced method is demonstrated by its application to the confirmation and epidemiological investigation of an outbreak of cryptosporidiosis associated with a school visit to an open farm.
Synthesis of a Low-Molecular-Weight Form of Exopolysaccharide by
Bradyrhizobium japonicum
USDA 110
Authors: Louch Heather A. , Miller Karen J. 
Published: July 2002

ABSTRACT

A novel extracellular low-molecular-weight polysaccharide was detected as a contaminant within extracellular cyclic β-1,6-β-1,3-glucan preparations from
Bradyrhizobium japonicum
USDA 110 cultures. Compositional analysis, methylation analysis, and nuclear magnetic resonance analysis revealed that this low-molecular-weight polysaccharide was composed of the same pentasaccharide repeating unit previously described for the high-molecular-weight form of the exopolysaccharide (EPS) synthesized by
B. japonicum
strains. Mass spectrometry analysis indicated that the size of this low-molecular-weight form of EPS was consistent with a dimeric form of the pentasaccharide repeating unit.
Molecular Characterization of a Theta Replication Plasmid and Its Use for Development of a Two-Component Food-Grade Cloning System for
Lactococcus lactis
Authors: Émond Éric , Lavallée Richard , Drolet Geneviève , Moineau Sylvain , LaPointe Gisèle 
Published: July 2002

ABSTRACT

pCD4, a small, highly stable theta-replicating lactococcal plasmid, was used to develop a food-grade cloning system. Sequence analysis revealed five open reading frames and two putative
cis
-acting regions. None appears to code for undesirable phenotypes with regard to food applications. Functional analysis of the replication module showed that only the
cis
-acting
ori
region and the
repB
gene coding for the replication initiator protein were needed for the stable replication and maintenance of pCD4 derivatives in
Lactococcus lactis
. A two-component food-grade cloning system was derived from the pCD4 replicon. The vector pVEC1, which carries the functional pCD4 replicon, is entirely made up of
L. lactis
DNA and has no selection marker. The companion pCOM1 is a
repB
-deficient pCD4 derivative that carries an erythromycin resistance gene as a dominant selection marker. The pCOM1 construct can only replicate in
L. lactis
if
trans
complemented by the RepB initiator provided by pVEC1. Since only the cotransformants that carry both pVEC1 and pCOM1 can survive on plates containing erythromycin, pCOM1 can be used transiently to select cells that have acquired pVEC1. Due to the intrinsic incompatibility between these plasmids, pCOM1 can be readily cured from the cells grown on an antibiotic-free medium after the selection step. The system was used to introduce a phage resistance mechanism into the laboratory strain MG1363 of
L. lactis
and two industrial strains. The introduction of the antiphage barrier did not alter the wild-type plasmid profile of the industrial strains. The phenotype was stable after 100 generations and conferred an effective resistance phenotype against phages of the 936 and c2 species.
Overproduction of
l
-Lysine from Methanol by
Methylobacillus glycogenes
Derivatives Carrying a Plasmid with a Mutated
dapA
Gene
Authors: Motoyama Hiroaki , Yano Hiroshi , Terasaki Yoko , Anazawa Hideharu 
Published: July 2002

ABSTRACT

The
dapA
gene, encoding dihydrodipicolinate synthase (DDPS) partially desensitized to inhibition by
l
-lysine, was cloned from an
l
-threonine- and
l
-lysine-coproducing mutant of the obligate methylotroph
Methylobacillus glycogenes
DHL122 by complementation of the nutritional requirement of an
Escherichia coli dapA
mutant. Introduction of the
dapA
gene into DHL122 and AL119, which is the parent of DHL122 and an
l
-threonine producing mutant, elevated the specific activity of DDPS 20-fold and
l
-lysine production 2- to 3-fold with concomitant reduction of
l
-threonine in test tube cultures. AL119 containing the
dapA
gene produced 8 g of
l
-lysine per liter in a 5-liter jar fermentor from methanol as a substrate. Analysis of the nucleotide sequence of the
dapA
gene shows that it encodes a peptide with an
M
r
of 30,664 and that the encoded amino acid sequence is extensively homologous to those of other organisms. In order to study the mutation that occurred in DHL122, the
dapA
genes of the wild type and AL119 were cloned and sequenced. Comparison of the nucleotide sequences of the
dapA
genes revealed that the amino acid at residue 88 was F in DHL122 whereas it was L in the wild type and AL119, suggesting that this amino acid alteration that occurred in DHL122 caused the partial desensitization of DDPS to the inhibition by
l
-lysine. The similarity in the amino acid sequences of DDPS in
M. glycogenes
and other organisms suggests that the mutation of the
dapA
gene in DHL122 is located in the region concerned with interaction of the allosteric effector,
l
-lysine.
Role of Fatty Acid De Novo Biosynthesis in Polyhydroxyalkanoic Acid (PHA) and Rhamnolipid Synthesis by Pseudomonads: Establishment of the Transacylase (PhaG)-Mediated Pathway for PHA Biosynthesis in
Escherichia coli
Authors: Rehm Bernd H. A. , Mitsky Timothy A. , Steinbüchel Alexander 
Published: July 2002

ABSTRACT

Since
Pseudomonas aeruginosa
is capable of biosynthesis of polyhydroxyalkanoic acid (PHA) and rhamnolipids, which contain lipid moieties that are derived from fatty acid biosynthesis, we investigated various
fab
mutants from
P. aeruginosa
with respect to biosynthesis of PHAs and rhamnolipids. All isogenic
fabA, fabB, fabI, rhlG
, and
phaG
mutants from
P. aeruginosa
showed decreased PHA accumulation and rhamnolipid production. In the
phaG
(encoding transacylase) mutant rhamnolipid production was only slightly decreased. Expression of
phaG
from
Pseudomonas putida
and expression of the β-ketoacyl reductase gene
rhlG
from
P. aeruginosa
in these mutants indicated that PhaG catalyzes diversion of intermediates of fatty acid de novo biosynthesis towards PHA biosynthesis, whereas RhlG catalyzes diversion towards rhamnolipid biosynthesis. These data suggested that both biosynthesis pathways are competitive. In order to investigate whether PhaG is the only linking enzyme between fatty acid de novo biosynthesis and PHA biosynthesis, we generated five Tn
5
mutants of
P. putida
strongly impaired in PHA production from gluconate. All mutants were complemented by the
phaG
gene from
P. putida
, indicating that the transacylase-mediated PHA biosynthesis route represents the only metabolic link between fatty acid de novo biosynthesis and PHA biosynthesis in this bacterium. The transacylase-mediated PHA biosynthesis route from gluconate was established in recombinant
E. coli
, coexpressing the class II PHA synthase gene
phaC1
together with the
phaG
gene from
P. putida
, only when fatty acid de novo biosynthesis was partially inhibited by triclosan. The accumulated PHA contributed to 2 to 3% of cellular dry weight.
Species-Specific PCR for Identification of Common Contaminant Mollicutes in Cell Culture
Authors: Kong Fanrong , James Gregory , Gordon Susanna , Zelynski Anna , Gilbert Gwendolyn L. 
Published: July 2002

ABSTRACT

Mycoplasma arginini, M. fermentans, M. hyorhinis, M. orale
, and
Acholeplasma laidlawii
are the members of the class
Mollicutes
most commonly found in contaminated cell cultures. Previous studies have shown that the published PCR primer pairs designed to detect mollicutes in cell cultures are not entirely specific. The 16S rRNA gene, the 16S-23S rRNA intergenic spacer region, and the 5′ end of the 23S rRNA gene, as a whole, are promising targets for design of mollicute species-specific primer pairs. We analyzed the 16S rRNA genes, the 16S-23S rRNA intergenic spacer regions, and the 5′ end of the 23S rRNA genes of these mollicutes and developed PCR methods for species identification based on these regions. Using high melting temperatures, we developed a rapid-cycle PCR for detection and identification of contaminant mollicutes. Previously published, putative mollicute-specific primers amplified DNA from 73 contaminated cell lines, but the presence of mollicutes was confirmed by species-specific PCR in only 60. Sequences of the remaining 13 amplicons were identified as those of gram-positive bacterial species. Species-specific PCR primers are needed to confirm the presence of mollicutes in specimens and for identification, if required.
Characterization of a
Sinorhizobium
Isolate and Its Extracellular Polymer Implicated in Pollutant Transport in Soil
Authors: Janecka J. , Jenkins M. B. , Brackett N. S. , Lion L. W. , Ghiorse W. C. 
Published: July 2002

ABSTRACT

A bacterium isolated from soil (designated 9702-M4) synthesizes an extracellular polymer that facilitates the transport of such hydrophobic pollutants as polynuclear aromatic hydrocarbons, as well as the toxic metals lead and cadmium in soil. Biolog analysis, growth rate determinations, and percent G+C content identify 9702-M4 as a strain of
Sinorhizobium meliloti
. Sequence analysis of a 16S rDNA fragment gives 9702-M4 a phylogenetic designation most closely related to
Sinorhizobium fredii
. The extracellular polymer of isolate 9702-M4 is composed of both an extracellular polysaccharide (EPS) and a rough lipopolysaccharide. The EPS component is composed mainly of 4-glucose linkages with monomers of galactose, mannose, and glucuronic acid and has pyruval and acetyl constituents. The lipid fraction and the negative charge associated with carbonyl groups of the exopolymer are thought to account for the binding of polynuclear aromatic hydrocarbons and cationic metals.
The Microbial Community Structure of Drinking Water Biofilms Can Be Affected by Phosphorus Availability
Authors: Keinänen Minna M. , Korhonen Leena K. , Lehtola Markku J. , Miettinen Ilkka T. , Martikainen Pertti J. , Vartiainen Terttu , Suutari Merja H. 
Published: July 2002

ABSTRACT

Microbial communities in biofilms grown for 4 and 11 weeks under the flow of drinking water supplemented with 0, 1, 2, and 5 μg of phosphorus liter
−1
and in drinking and warm waters were compared by using phospholipid fatty acids (PLFAs) and lipopolysaccharide 3-hydroxy fatty acids (LPS 3-OH-FAs). Phosphate increased the proportion of PLFAs 16:1ω7
c
and 18:1ω7
c
and affected LPS 3-OH-FAs after 11 weeks of growth, indicating an increase in gram-negative bacteria and changes in their community structure. Differences in community structures between biofilms and drinking and warm waters can be assumed from PLFAs and LPS 3-OH-FAs, concomitantly with adaptive changes in fatty acid chain length, cyclization, and unsaturation.
Expanded Genetic Map of
Gibberella moniliformis
(
Fusarium verticillioides
)
Authors: Jurgenson James E. , Zeller Kurt A. , Leslie John F. 
Published: July 2002

ABSTRACT

Gibberella moniliformis
(
Fusarium verticillioides
) is primarily a pathogen of maize, but it can also cause disease in other crop species. This pathogenicity, as well as the contamination of food- and feedstuffs with the fumonisin mycotoxins, results in economically significant losses to both farmers and food processors. The dissection of important biological characters in this fungus has been hampered by the lack of a uniformly dense genetic map. The existing restriction fragment length polymorphism-based map contains significant gaps, making it difficult to routinely locate biologically important genes, such as those involved in pathogenicity or mycotoxin production, with precision. We utilized amplified fragment length polymorphisms (AFLPs) to saturate the existing genetic map and added 486 AFLP markers to the ∼150 markers on the existing map. The resulting map has an average marker interval of 3.9 map units and averages ∼21 kb/map unit. The additional markers expanded the map from 1,452 to 2,188 map units distributed across 12 chromosomes. The maximum distance between adjacent markers is 29 map units. We identified AFLP markers less than 1 map unit from the mating type (
MAT
) locus and 2.5 map units from the spore killer (
SK
) locus; eight AFLP markers map within 8.5 units of the
FUM1
(fumonisin biosynthetic) locus. The increased saturation of this map will facilitate further development of
G. moniliformis
as a model system for the genetic and population genetic studies of related, but less genetically tractable, plant pathogenic fungi.
Serotype-Cross-Reactive Immunoglobulin M Responses in Dengue Virus Infections Determined by Enzyme-Linked Immunosorbent Assay
Authors: Nawa Masaru , Yamada Ken-Ichiro , Takasaki Tomohiko , Akatsuka Toshitaka , Kurane Ichiro 
Published: July 2002

ABSTRACT
We developed immunoglobulin M (IgM) antibody capture enzyme-linked immunosorbent assays (ELISAs) with four monovalent dengue virus antigens. We attempted to determine whether IgM responses in dengue virus infections are serotype specific or serotype cross-reactive. Serum samples from 14 confirmed dengue cases were examined. In these 14 cases, which consisted of 12 Japanese and 2 non-Japanese patients, infecting dengue virus serotypes were defined by reverse transcription-PCR. Thirteen of the 14 cases were IgM positive in ELISA. IgM responses were serotype cross-reactive in these 13 cases but were highest against infecting dengue virus serotype in 9 of the 13 cases. These results indicate that IgM responses are generally dengue serotype cross-reactive but that IgM levels are highest against the infecting serotype in most dengue cases.
Procalcitonin Does Not Discriminate Infection from Inflammation after Allogeneic Bone Marrow Transplantation
Authors: Blijlevens N. M. A. , Donnelly J. P. , Meis J. F. G. M. , De Keizer M. H. , De Pauw B. E. 
Published: July 2002

ABSTRACT

Procalcitonin (PCT) is an early marker of bacterial infection but little is known about its value in neutropenic allogeneic bone marrow transplant (BMT) recipients. We collected plasma from 12 recipients of T-cell-depleted HLA-matched related BMT recipients who had been treated preemptively with meropenem from the day after BMT for at least 15 days. PCT and C-reactive protein (CRP) concentrations were determined on BMT days 1, 5, 8, 12, and 15, and their relationship to inflammatory events (IE), including mucositis, microbiologically and clinically defined infections, acute graft-versus-host disease (GVDH), and unexplained fever, was then determined. The PCT concentrations were all low and never exceeded 4 μg/liter, unlike CRP concentrations, which spanned the full range up to 350 mg/liter. All patients had mucositis, and there was no significant difference between PCT concentrations associated with mucositis alone and those associated with an additional IE on BMT days 1 to 12. However, on BMT day 15, the mean concentrations of PCT were 0.37 ± 0.05 μg/liter for the 10 patients that had an additional IE, compared with 0.11 ± 0.03 μg/liter for the 2 patients with mucositis only (
P
= 0.012), and GVHD rather than infection was involved in six cases. PCT was also not a sensitive marker of gram-positive bacteremia or pulmonary aspergillosis. Thus, PCT is of little value in discriminating infections from other inflammatory complications that occur following allogeneic BMT.
Assessment of Neutrophil Function in Patients with Septic Shock: Comparison of Methods
Authors: Wenisch C. , Fladerer P. , Patruta S. , Krause R. , Hörl W. 
Published: July 2002

ABSTRACTPatients with septic shock are shown to have decreased neutrophil phagocytic function by multiple assays, and their assessment by whole-blood assays (fluorescence-activated cell sorter analysis) correlates with assays requiring isolated neutrophils (microscopic and spectrophotometric assays). For patients with similar underlying conditions but without septic shock, this correlation does not occur.
Sacbrood Virus of the Honeybee (
Apis mellifera
): Rapid Identification and Phylogenetic Analysis Using Reverse Transcription-PCR
Authors: Grabensteiner Elvira , Ritter Wolfgang , Carter Michael J. , Davison Sean , Pechhacker Hermann , Kolodziejek Jolanta , Boecking Otto , Derakhshifar Irmgard , Moosbeckhofer Rudolf , Licek Elisabeth , Nowotny Norbert 
Published: July 2002

ABSTRACT

Sacbrood virus (SBV) infects larvae of the honeybee (
Apis mellifera
), resulting in failure to pupate and death. Until now, identification of viruses in honeybee infections has been based on traditional methods such as electron microscopy, immunodiffusion, and enzyme-linked immunosorbent assay. Culture cannot be used because no honeybee cell lines are available. These techniques are low in sensitivity and specificity. However, the complete nucleotide sequence of SBV has recently been determined, and with these data, we now report a reverse transcription-PCR (RT-PCR) test for the direct, rapid, and sensitive detection of these viruses. RT-PCR was used to target five different areas of the SBV genome using infected honeybees and larvae originating from geographically distinct regions. The RT-PCR assay proved to be a rapid, specific, and sensitive diagnostic tool for the direct detection of SBV nucleic acid in samples of infected honeybees and brood regardless of geographic origin. The amplification products were sequenced, and phylogenetic analysis suggested the existence of at least three distinct genotypes of SBV.
Real-Time PCR as a New Tool for Quantifying
Leishmania infantum
in Liver in Infected Mice
Authors: Bretagne Stéphane , Durand Rémy , Olivi Martine , Garin Jean-François , Sulahian Annie , Rivollet Danièle , Vidaud Michel , Deniau Michèle 
Published: July 2002

ABSTRACT

The parasitic loads of mouse livers experimentally infected with
Leishmania infantum
were determined using a double real-time quantitative PCR test targeted to the parasite DNA polymerase gene and to the mouse brain-derived neutrophic factor gene. The
Leishmania
DNA copy number was normalized to the number of mouse gene copies in order to quantify the former independently of liver weight. The correlation coefficient with the microtitration method was 0.66. This PCR assay can be considered for experimental pharmaceutical studies.
High Serum Interleukin-10 and Tumor Necrosis Factor Alpha Levels in Chronic Paracoccidioidomycosis
Authors: Fornari M. C. , Bava A. J. , Guereño M. T. , Berardi V. E. , Silaf M. R. , Negroni R. , Diez R. A. 
Published: July 2002

ABSTRACT

In patients with chronic paracoccidioidomycosis (
n
= 10), levels of tumor necrosis factor alpha, interleukin-10, and interleukin-2 in serum, measured by enzyme-linked immunosorbent assay (in picograms per milliliter, as mean ± standard error of the mean), were higher than in normal controls (
n
= 8): 186 ± 40 versus 40 ± 7 (
P
< 0.05), 203 ± 95 versus 20 ± 8 (
P
= 0.001), and 96.3 ± 78.57 versus 1.19 ± 1.19 (
P
= 0.045), respectively. Gamma interferon and interleukin-4 levels were similar in patients and controls.