bluuebunny/crossref_metadata_2025_split huggingface dataset:
The Complete Amino Acid Substitutions at Position 131 That Are Positively Involved in Cold Adaptation of Subtilisin BPN′
Authors: Taguchi Seiichi , Komada Shingo , Momose Haruo 
Published: July 2002

ABSTRACT

To ascertain whether position 131 of a mesophilic protease, subtilisin BPN′, is a potential critical site for cold adaptation as screened by evolutionary engineering (S. Taguchi, A. Ozaki, and H. Momose, Appl. Environ. Microbiol. 64:492–495, 1998), a full set of subtilisin BPN′ mutants with mutations at position 131 was constructed by site-saturation mutagenesis. All mutated enzymes were measured for specific activity at 10°C by the quantitative titer microplate assay system using polyclonal antibody against subtilisin BPN′ and a synthetic chromogenic substrate. All the mutants exhibited proteolytic activities almost the same as or higher than that of the wild-type enzyme, suggesting that position 131 may be important for cold adaptation. In comparison with the wild type, purified mutants G131F, G131R, G131M, and G131W were found to acquire proteolytic activities (
k
cat
/

K
m

) at 10°C that were 150, 94, 84, and 50% higher, respectively. In particular, for the G131F mutant, temperature dependency in enzyme activity was shown by an increase in
k
cat
and a decrease in

K
m

. All of these amino acid substitution mutants, G131F, G131R, G131M, and G131W, acquired increased proteolytic activities at 10°C for three different synthetic peptide substrates but no increase in caseinolytic activity. Furthermore, they all conferred thermolability on the enzyme to differing extents in terms of the half-life of enzyme inactivation at 60°C. No significant correlation was found between the amino acids preferred for cold adaptation surveyed here and those present at position 131 of subtilisin of psychrophilic cells naturally occurring in cold environments. Based on these findings, position 131 is a contributor in artificial evolution for acquiring a cold-active character and may not be related to physiological requirements for subtilisin-producing cells living in cold environments. Therefore, saturation mutagenesis would be effective in achieving rapid improvement in protein properties via evolutionary engineering.
Characterization of Plasmid-Borne and Chromosome-Encoded Traits of
Agrobacterium
Biovar 1, 2, and 3 Strains from France
Authors: Ridé Michel , Ridé Suzanne , Petit Annik , Bollet Claude , Dessaux Yves , Gardan Louis 
Published: July 2002

ABSTRACT

We collected 111
Agrobacterium
isolates from galls of various origins (most of them from France) and analyzed both their plasmid-borne and chromosome-encoded traits. Phenotypic analysis of these strains allowed their classification in three phena which exactly matched the delineation of biovars 1, 2, and 3. A fourth phenon was identified which comprises three atypical strains. The phenotypic analysis has also allowed us to identify 12 additional characteristics which could be used to identify the three biovars of
Agrobacterium
. Our results also suggest that biovar 1 and 2 represent distinct species. Analysis of plasmid-borne traits confirmed that tartrate utilization is a common feature of biovar 3 strains (now named
Agrobacterium vitis
) and of
Agrobacterium
grapevine strains in general. Among pathogenic strains of
Agrobacterium
, several exhibited unusual opine synthesis and degradation patterns, and one strain of biovar 3 induced tumors containing vitopine and a novel opine-like molecule derived from putrescine. We have named this compound ridéopine.
Selective Enrichment with a Resuscitation Step for Isolation of Freeze-Injured
Escherichia coli
O157:H7 from Foods
Authors: Hara-Kudo Y. , Ikedo M. , Kodaka H. , Nakagawa H. , Goto K. , Masuda T. , Konuma H. , Kojima T. , Kumagai S. 
Published: July 2002

ABSTRACT

We studied injury of
Escherichia coli
O157:H7 cells in 11 food items during freeze storage and methods of isolating freeze-injured
E. coli
O157:H7 cells from foods. Food samples inoculated with
E. coli
O157:H7 were stored for 16 weeks at −20°C in a freezer. Noninjured and injured cells were counted by using tryptic soy agar and sorbitol MacConkey agar supplemented with cefixime and potassium tellurite. Large populations of
E. coli
O157:H7 cells were injured in salted cabbage, grated radish, seaweed, and tomato samples. In an experiment to detect
E. coli
O157:H7 in food samples artificially contaminated with freeze-injured
E. coli
O157:H7 cells, the organism was recovered most efficiently after the samples were incubated in modified
E. coli
broth without bile salts at 25°C for 2 h and then selectively enriched at 42°C for 18 h by adding bile salts and novobiocin. Our enrichment method was further evaluated by isolating
E. coli
O157:H7 from frozen foods inoculated with the organism prior to freezing. Two hours of resuscitation at 25°C in nonselective broth improved recovery of
E. coli
O157:H7 from frozen grated radishes and strawberries, demonstrating that the resuscitation step is very effective for isolating
E. coli
O157:H7 from frozen foods contaminated with injured
E. coli
O157:H7 cells.
Introduction of Raw Starch-Binding Domains into
Bacillus subtilis
α-Amylase by Fusion with the Starch-Binding Domain of
Bacillus
Cyclomaltodextrin Glucanotransferase
Authors: Ohdan Kohji , Kuriki Takashi , Takata Hiroki , Kaneko Hiroki , Okada Shigetaka 
Published: July 2002

ABSTRACT

We constructed two types of chimeric enzymes, Ch1 Amy and Ch2 Amy. Ch1 Amy consisted of a catalytic domain of
Bacillus subtilis
X-23 α-amylase (Ba-S) and the raw starch-binding domain (domain E) of
Bacillus
A2-5a cyclomaltodextrin glucanotransferase (A2-5a CGT). Ch2 Amy consisted of Ba-S and D (function unknown) plus E domains of A2-5a CGT. Ch1 Amy acquired raw starch-binding and -digesting abilities which were not present in the catalytic part (Ba-S). Furthermore, the specific activity of Ch1 Amy was almost identical when enzyme activity was evaluated on a molar basis. Although Ch2 Amy exhibited even higher raw starch-binding and -digesting abilities than Ch1 Amy, the specific activity was lower than that of Ba-S. We did not detect any differences in other enzymatic characteristics (amylolytic pattern, transglycosylation ability, effects of pH, and temperature on stability and activity) among Ba-S, Ch1 Amy, and Ch2 Amy.
Common Degradative Pathways of Morpholine, Thiomorpholine, and Piperidine by
Mycobacterium aurum
MO1: Evidence from
1
H-Nuclear Magnetic Resonance and Ionspray Mass Spectrometry Performed Directly on the Incubation Medium
Authors: Combourieu Bruno , Besse Pascale , Sancelme Martine , Godin Jean-Philippe , Monteil André , Veschambre Henri , Delort Anne-Marie 
Published: July 2002

ABSTRACT

In order to see if the biodegradative pathways for morpholine and thiomorpholine during degradation by
Mycobacterium aurum
MO1 could be generalized to other heterocyclic compounds, the degradation of piperidine by this strain was investigated by performing
1
H-nuclear magnetic resonance directly with the incubation medium. Ionspray mass spectrometry, performed without purification of the samples, was also used to confirm the structure of some metabolites during morpholine and thiomorpholine degradation. The results obtained with these two techniques suggested a general pathway for degradation of nitrogen heterocyclic compounds by
M. aurum
MO1. The first step of the degradative pathway is cleavage of the C—N bond; this leads formation of an intermediary amino acid, which is followed by deamination and oxidation of this amino acid into a diacid. Except in the case of thiodiglycolate obtained from thiomorpholine degradation, the dicarboxylates are completely mineralized by the bacterial cells. A comparison with previously published data showed that this pathway could be a general pathway for degradation by other strains of members of the genus
Mycobacterium.
Direct Detection of 16S rRNA in Soil Extracts by Using Oligonucleotide Microarrays
Authors: Small Jack , Call Douglas R. , Brockman Fred J. , Straub Timothy M. , Chandler Darrell P. 
Published: July 2002

ABSTRACT

We report on the development and validation of a simple microarray method for the direct detection of intact 16S rRNA from unpurified soil extracts. Total RNAs from
Geobacter chapellei
and
Desulfovibrio desulfuricans
were hybridized to an oligonucleotide array consisting of universal and species-specific 16S rRNA probes. PCR-amplified products from
Geobacter
and
Desulfovibrio
were easily and specifically detected under a range of hybridization times, temperatures, and buffers. However, reproducible, specific hybridization and detection of intact rRNA could be accomplished only by using a chaperone-detector probe strategy. With this knowledge, assay conditions were developed for rRNA detection using a 2-h hybridization time at room temperature. Hybridization specificity and signal intensity were enhanced using fragmented RNA. Formamide was required in the hybridization buffer in order to achieve species-specific detection of intact rRNA. With the chaperone detection strategy, we were able to specifically hybridize and detect
G. chapellei
16S rRNA directly from a total-RNA soil extract, without further purification or removal of soluble soil constituents. The detection sensitivity for
G. chapellei
16S rRNA in soil extracts was at least 0.5 μg of total RNA, representing approximately 7.5 × 10
6
Geobacter
cell equivalents of RNA. These results suggest that it is now possible to apply microarray technology to the direct detection of microorganisms in environmental samples, without using PCR.
Copper-Induced Inhibition of Growth of
Desulfovibrio desulfuricans
G20: Assessment of Its Toxicity and Correlation with Those of Zinc and Lead
Authors: Sani Rajesh Kumar , Peyton Brent M. , Brown Laura T. 
Published: July 2002

ABSTRACT

The toxicity of copper [Cu(II)] to sulfate-reducing bacteria (SRB) was studied by using
Desulfovibrio desulfuricans
G20 in a medium (MTM) developed specifically to test metal toxicity to SRB (R. K. Sani, G. Geesey, and B. M. Peyton, Adv. Environ. Res. 5:269–276, 2001). The effects of Cu(II) toxicity were observed in terms of inhibition in total cell protein, longer lag times, lower specific growth rates, and in some cases no measurable growth. At only 6 μM, Cu(II) reduced the maximum specific growth rate by 25% and the final cell protein concentration by 18% compared to the copper-free control. Inhibition by Cu(II) of cell yield and maximum specific growth rate increased with increasing concentrations. The Cu(II) concentration causing 50% inhibition in final cell protein was evaluated to be 16 μM. A Cu(II) concentration of 13.3 μM showed 50% inhibition in maximum specific growth rate. These results clearly show significant Cu(II) toxicity to SRB at concentrations that are 100 times lower than previously reported. No measurable growth was observed at 30 μM Cu(II) even after a prolonged incubation of 384 h. In contrast, Zn(II) and Pb(II), at 16 and 5 μM, increased lag times by 48 and 72 h, respectively, but yielded final cell protein concentrations equivalent to those of the zinc- and lead-free controls. Live/dead staining, based on membrane integrity, indicated that while Cu(II), Zn(II), and Pb(II) inhibited growth, these metals did not cause a loss of
D. desulfuricans
membrane integrity. The results show that
D. desulfuricans
in the presence of Cu(II) follows a growth pattern clearly different from the pattern followed in the presence of Zn(II) or Pb(II). It is therefore likely that Cu(II) toxicity proceeds by a mechanism different from that of Zn(II) or Pb(II) toxicity.
Stable-Isotope-Based Labeling of Styrene-Degrading Microorganisms in Biofilters
Authors: Alexandrino Maria , Knief Claudia , Lipski André 
Published: July 2002

ABSTRACTDeuterated styrene ([2H8]styrene) was used as a tracer in combination with phospholipid fatty acid (PLFA) analysis for characterization of styrene-degrading microbial populations of biofilters used for treatment of waste gases. Deuterated fatty acids were detected and quantified by gas chromatography-mass spectrometry. The method was evaluated with pure cultures of styrene-degrading bacteria and defined mixed cultures of styrene degraders and non-styrene-degrading organisms. Incubation of styrene degraders for 3 days with [2H8]styrene led to fatty acids consisting of up to 90% deuterated molecules. Mixed-culture experiments showed that specific labeling of styrene-degrading strains and only weak labeling of fatty acids of non-styrene-degrading organisms occurred after incubation with [2H8]styrene for up to 7 days. Analysis of actively degrading filter material from an experimental biofilter and a full-scale biofilter by this method showed that there were differences in the patterns of labeled fatty acids. For the experimental biofilter the fatty acids with largest amounts of labeled molecules were palmitic acid (16:0), 9,10-methylenehexadecanoic acid (17:0 cyclo9-10), and vaccenic acid (18:1cis11). These lipid markers indicated that styrene was degraded by organisms with aPseudomonas-like fatty acid profile. In contrast, the most intensively labeled fatty acids of the full-scale biofilter sample were palmitic acid andcis-11-hexadecenoic acid (16:1cis11), indicating that an unknown styrene-degrading taxon was present. Iso-, anteiso-, and 10-methyl-branched fatty acids showed no or weak labeling. Therefore, we found no indication that styrene was degraded by organisms with methyl-branched fatty fatty acids, such asXanthomonas,Bacillus,Streptomyces, orGordoniaspp.
Recovery and Phylogenetic Analysis of
nifH
Sequences from Diazotrophic Bacteria Associated with Dead Aboveground Biomass of
Spartina alterniflora
Authors: Lovell Charles R. , Friez Michael J. , Longshore John W. , Bagwell Christopher E. 
Published: July 2002

ABSTRACT

DNA was extracted from dry standing dead
Spartina alterniflora
stalks as well as dry
Spartina
wrack from the North Inlet (South Carolina) and Sapelo Island (Georgia) salt marshes. Partial
nifH
sequences were PCR amplified, the products were separated by denaturing gradient gel electrophoresis (DGGE), and the prominent DGGE bands were sequenced. Most sequences (109 of 121) clustered with those from α-
Proteobacteria
, and 4 were very similar (>99%) to that of
Azospirillum brasilense
. Seven sequences clustered with those from known γ-
Proteobacteria
and five with those from known anaerobic diazotrophs. The diazotroph assemblages associated with dead
Spartina
biomass in these two salt marshes were very similar, and relatively few major lineages were represented.
Sensitization of
Listeria monocytogenes
to Low pH, Organic Acids, and Osmotic Stress by Ethanol
Authors: Barker Clive , Park Simon F. 
Published: July 2002

ABSTRACT

The killing of
Listeria monocytogenes
following exposure to low pH, organic acids, and osmotic stress was enhanced by the addition of 5% (vol/vol) ethanol. At pH 3, for example, the presence of this agent stimulated killing by more than 3 log units in 40 min of exposure. The rate of cell death at pH 3.0 was dependent on the concentration of ethanol. Thus, while the presence 10% (vol/vol) ethanol at pH 3.0 stimulated killing by more than 3 log units in just 5 min, addition of 1.25% (vol/vol) ethanol resulted in less than 1 log unit of killing in 10 min. The ability of 5% (vol/vol) ethanol to stimulate killing at low pH and at elevated osmolarity was also dependent on the amplitude of the imposed stress, and an increase in the pH from 3.0 to 4.0 or a decrease in the sodium chloride concentration from 25 to 2.5% led to a marked reduction in the effectiveness of 5% (vol/vol) ethanol as an augmentative agent. Combinations of organic acids, low pH, and ethanol proved to be particularly effective bactericidal treatments; the most potent combination was pH 3.0, 50 mM formate, and 5 % (vol/vol) ethanol, which resulted in 5 log units of killing in just 4 min. Ethanol-enhanced killing correlated with damage to the bacterial cytoplasmic membrane.
Growth and Arginine Metabolism of the Wine Lactic Acid BacteriaLactobacillus buchneriandOenococcus oeniat Different pH Values and Arginine Concentrations
Authors: Mira de Orduña Ramón , Patchett Mark L. , Liu Shao-Quan , Pilone Gordon J. 
Published: July 2002

ABSTRACTDuring malolactic fermentation (MLF) in grape must and wine, heterofermentative lactic acid bacteria may degrade arginine, leading to the formation of ammonia and citrulline, among other substances. This is of concern because ammonia increases the pH and thus the risk of growth by spoilage bacteria, and citrulline is a precursor to the formation of carcinogenic ethyl carbamate (EC). Arginine metabolism and growth ofLactobacillus buchneriCUC-3 andOenococcus oenistrains MCW and Lo111 in wine were investigated. In contrast toL. buchneriCUC-3, both oenococci required a higher minimum pH for arginine degradation, and arginine utilization was delayed relative to the degradation of malic acid, the main aim of MLF. This allows the control of pH increase and citrulline formation from arginine metabolism by carrying out MLF with pure oenococcal cultures and inhibiting cell metabolism after malic acid depletion. MLF by arginine-degrading lactobacilli should be discouraged because arginine degradation may lead to the enhanced formation of acids from sugar degradation. A linear relationship was found between arginine degradation and citrulline excretion rates. From this data, strain-specific arginine-to-citrulline conversion ratios were calculated that ranged between 2.2 and 3.9% (wt/wt), and these ratios can be used to estimate the contribution of citrulline to the EC precursor pool from a given amount of initial arginine. Increasing arginine concentrations led to higher rates of growth ofL. buchneriCUC-3 but did not increase the growth yield of either oenococcus. These results suggest the use of non-arginine-degrading oenococci for inducing MLF.
Purification, Characterization, and Gene Cloning of Purine Nucleosidase from
Ochrobactrum anthropi
Authors: Ogawa Jun , Takeda Sou , Xie Sheng-Xue , Hatanaka Haruyo , Ashikari Toshihiko , Amachi Teruo , Shimizu Sakayu 
Published: July 2002

ABSTRACT

A bacterium,
Ochrobactrum anthropi
, produced a large amount of a nucleosidase when cultivated with purine nucleosides. The nucleosidase was purified to homogeneity. The enzyme has a molecular weight of about 170,000 and consists of four identical subunits. It specifically catalyzes the irreversible
N
-riboside hydrolysis of purine nucleosides, the

K
m

values being 11.8 to 56.3 μM. The optimal activity temperature and pH were 50°C and pH 4.5 to 6.5, respectively. Pyrimidine nucleosides, purine and pyrimidine nucleotides, NAD, NADP, and nicotinamide mononucleotide are not hydrolyzed by the enzyme. The purine nucleoside hydrolyzing activity of the enzyme was inhibited (mixed inhibition) by pyrimidine nucleosides, with

K
i

and

K
i

′ values of 0.455 to 11.2 μM. Metal ion chelators inhibited activity, and the addition of Zn
2+
or Co
2+
restored activity. A 1.5-kb DNA fragment, which contains the open reading frame encoding the nucleosidase, was cloned, sequenced, and expressed in
Escherichia coli
. The deduced 363-amino-acid sequence including a 22-residue leader peptide is in agreement with the enzyme molecular mass and the amino acid sequences of NH
2
-terminal and internal peptides, and the enzyme is homologous to known nucleosidases from protozoan parasites. The amino acid residues forming the catalytic site and involved in binding with metal ions are well conserved in these nucleosidases.
The
celA
Gene, Encoding a Glycosyl Hydrolase Family 3 β-Glucosidase in
Azospirillum irakense,
Is Required for Optimal Growth on Cellobiosides
Authors: Faure Denis , Henrissat Bernard , Ptacek David , Bekri My Ali , Vanderleyden Jos 
Published: July 2002

ABSTRACT

The CelA β-glucosidase of
Azospirillum irakense,
belonging to glycosyl hydrolase family 3 (GHF3), preferentially hydrolyzes cellobiose and releases glucose units from the C
3
, C
4
, and C
5
oligosaccharides. The growth of a Δ
celA
mutant on these cellobiosides was affected. In
A. irakense
, the GHF3 β-glucosidases appear to be functional alternatives for the GHF1 β-glucosidases in the assimilation of β-glucosides by other bacteria.
Identification and Disruption of the
proBA
Locus in
Listeria monocytogenes
: Role of Proline Biosynthesis in Salt Tolerance and Murine Infection
Authors: Sleator Roy D. , Gahan Cormac G. M. , Hill Colin 
Published: July 2002

ABSTRACT

Intracellular accumulation of the amino acid proline has previously been linked to the salt tolerance and virulence potential of a number of bacteria. Taking advantage of the
proBA
mutant
Escherichia coli
CSH26, we identified a listerial
proBA
operon coding for enzymes functionally similar to the glutamyl kinase (GK) and glutamylphosphate reductase (GPR) enzyme complex which catalyzes the first and second steps of proline biosynthesis in
E. coli
. The first gene of the operon,
proB
, is predicted to encode GK, a 276-residue protein with a calculated molecular mass of 30.03 kDa and pl of 5.2. Distal to the promoter and overlapping the 3′ end of
proB
by 17 bp is
proA
, which encodes GPR, a 415-residue protein with a calculated molecular mass of 45.50 kDa (pl 5.3). Using this information, we created a chromosomal deletion mutant by allelic exchange which is auxotrophic for proline. This mutant was used to assess the contribution of proline anabolism to osmotolerance and virulence. While inactivation of
proBA
had no significant effect on virulence in mouse assays (either perorally or intraperitoneally), growth at low (2 to 4% NaCl) and high (>6% NaCl) salt concentrations in complex media was significantly reduced in the absence of efficient proline synthesis. We conclude that while proline biosynthesis plays little, if any, role in the intracellular life cycle and infectious nature of
Listeria monocytogenes
, it can play an important role in survival in osmolyte-depleted environments of elevated osmolarity.
Laboratory-Scale Evidence for Lightning-Mediated Gene Transfer in Soil
Authors: Demanèche Sandrine , Bertolla Franck , Buret François , Nalin Renaud , Sailland Alain , Auriol Philippe , Vogel Timothy M. , Simonet Pascal 
Published: July 2002

ABSTRACT

Electrical fields and current can permeabilize bacterial membranes, allowing for the penetration of naked DNA. Given that the environment is subjected to regular thunderstorms and lightning discharges that induce enormous electrical perturbations, the possibility of natural electrotransformation of bacteria was investigated. We demonstrated with soil microcosm experiments that the transformation of added bacteria could be increased locally via lightning-mediated current injection. The incorporation of three genes coding for antibiotic resistance (plasmid pBR328) into the
Escherichia coli
strain DH10B recipient previously added to soil was observed only after the soil had been subjected to laboratory-scale lightning. Laboratory-scale lightning had an electrical field gradient (700 versus 600 kV m
−1
) and current density (2.5 versus 12.6 kA m
−2
) similar to those of full-scale lightning. Controls handled identically except for not being subjected to lightning produced no detectable antibiotic-resistant clones. In addition, simulated storm cloud electrical fields (in the absence of current) did not produce detectable clones (transformation detection limit, 10
−9
). Natural electrotransformation might be a mechanism involved in bacterial evolution.