bluuebunny/crossref_metadata_2025 huggingface dataset:
An Improved Protocol for Quantification of Freshwater
Actinobacteria
by Fluorescence In Situ Hybridization
Authors: Sekar Raju , Pernthaler Annelie , Pernthaler Jakob , Warnecke Falk , Posch Thomas , Amann Rudolf
Published: May 2003

We tested a previously described protocol for fluorescence in situ hybridization of marine bacterioplankton with horseradish peroxidase-labeled rRNA-targeted oligonucleotide probes and catalyzed reporter deposition (CARD-FISH) in plankton samples from different lakes. The fraction of
Bacteria
detected by CARD-FISH was significantly lower than after FISH with fluorescently monolabeled probes. In particular, the abundances of aquatic
Actinobacteria
were significantly underestimated. We thus developed a combined fixation and permeabilization protocol for CARD-FISH of freshwater samples. Enzymatic pretreatment of fixed cells was optimized for the controlled digestion of gram-positive cell walls without causing overall cell loss. Incubations with high concentrations of lysozyme (10 mg ml
−1
) followed by achromopeptidase (60 U ml
−1
) successfully permeabilized cell walls of
Actinobacteria
for subsequent CARD-FISH both in enrichment cultures and environmental samples. Between 72 and >99% (mean, 86%) of all
Bacteria
could be visualized with the improved assay in surface waters of four lakes. For freshwater samples, our method is thus superior to the CARD-FISH protocol for marine
Bacteria
(mean, 55%) and to FISH with directly fluorochrome labeled probes (mean, 67%). Actinobacterial abundances in the studied systems, as detected by the optimized protocol, ranged from 32 to >55% (mean, 45%). Our findings confirm that members of this lineage are among the numerically most important
Bacteria
of freshwater picoplankton.
Identification of an Emergent and Atypical
Pseudomonas viridiflava
Lineage Causing Bacteriosis in Plants of Agronomic Importance in a Spanish Region
Authors: González Ana J. , Rodicio M. Rosario , Mendoza M. Carmen
Published: May 2003

Pseudomonas
strains with an atypical LOPAT profile (where LOPAT is a series of determinative tests: L, levan production; O, oxidase production; P, pectinolitic activity; A, arginine dihydrolase production; and T, tobacco hypersensibility) can be regarded as emergent pathogens in the Principality of Asturias (Spain), where they have been causing, since 1999, severe damage in at least three taxonomically unrelated orchard plants of agronomic importance: common bean (
Phaseolus vulgaris
), kiwifruit (
Actinidia deliciosa
), and lettuce (
Lactuca sativa
). These strains are mainly differentiated by production of yellowish mucoid material in hypersucrose medium, used for the levan test, and by a variable pectinolytic activity on different potato varieties. The atypical organisms were identified as
Pseudomonas viridiflava
based on their 16S rRNA sequences. Among them a certain intraspecies genetic heterogeneity was detected by randomly amplified polymorphic DNA (RAPD) typing. To differentiate between isolates of
P. viridiflava
and
Pseudomonas syringae
pathovars, a 16S ribosomal DNA restriction fragment length polymorphism method employing the restriction endonucleases
Sac
I and
Hin
fI was developed. This could be used as a means of reliable species determination after the usual phenotypical characterization, which includes the LOPAT tests.
Stable Carbon Isotope Fractionation by Sulfate-Reducing Bacteria
Authors: Londry Kathleen L. , Des Marais David J.
Published: May 2003

Biogeochemical transformations occurring in the anoxic zones of stratified sedimentary microbial communities can profoundly influence the isotopic and organic signatures preserved in the fossil record. Accordingly, we have determined carbon isotope discrimination that is associated with both heterotrophic and lithotrophic growth of pure cultures of sulfate-reducing bacteria (SRB). For heterotrophic-growth experiments, substrate consumption was monitored to completion. Sealed vessels containing SRB cultures were harvested at different time intervals, and δ
13
C values were determined for gaseous CO
2
, organic substrates, and products such as biomass. For three of the four SRB, carbon isotope effects between the substrates, acetate or lactate and CO
2
, and the cell biomass were small, ranging from 0 to 2‰. However, for
Desulfotomaculum acetoxidans
, the carbon incorporated into biomass was isotopically heavier than the available substrates by 8 to 9‰. SRB grown lithoautotrophically consumed less than 3% of the available CO
2
and exhibited substantial discrimination (calculated as isotope fractionation factors [α]), as follows: for
Desulfobacterium autotrophicum
, α values ranged from 1.0100 to 1.0123; for
Desulfobacter hydrogenophilus
, the α value was 0.0138, and for
Desulfotomaculum acetoxidans
, the α value was 1.0310. Mixotrophic growth of
Desulfovibrio desulfuricans
on acetate and CO
2
resulted in biomass with a δ
13
C composition intermediate to that of the substrates. The extent of fractionation depended on which enzymatic pathways were used, the direction in which the pathways operated, and the growth rate, but fractionation was not dependent on the growth phase. To the extent that environmental conditions affect the availability of organic substrates (e.g., acetate) and reducing power (e.g., H
2
), ecological forces can also influence carbon isotope discrimination by SRB.
Sequence versus Structure for the Direct Detection of 16S rRNA on Planar Oligonucleotide Microarrays
Authors: Chandler Darrell P. , Newton Gregory J. , Small Jonathan A. , Daly Don S.
Published: May 2003

A two-probe proximal chaperone detection system consisting of a species-specific capture probe for the microarray and a labeled, proximal chaperone probe for detection was recently described for direct detection of intact rRNAs from environmental samples on oligonucleotide arrays. In this study, we investigated the physical spacing and nucleotide mismatch tolerance between capture and proximal chaperone detector probes that are required to achieve species-specific 16S rRNA detection for the dissimilatory metal and sulfate reducer 16S rRNAs. Microarray specificity was deduced by analyzing signal intensities across replicate microarrays with a statistical analysis-of-variance model that accommodates well-to-well and slide-to-slide variations in microarray signal intensity. Chaperone detector probes located in immediate proximity to the capture probe resulted in detectable, nonspecific binding of nontarget rRNA, presumably due to base-stacking effects. Species-specific rRNA detection was achieved by using a 22-nt capture probe and a 15-nt detector probe separated by 10 to 14 nt along the primary sequence. Chaperone detector probes with up to three mismatched nucleotides still resulted in species-specific capture of 16S rRNAs. There was no obvious relationship between position or number of mismatches and within- or between-genus hybridization specificity. From these results, we conclude that relieving secondary structure is of principal concern for the successful capture and detection of 16S rRNAs on planar surfaces but that the sequence of the capture probe is more important than relieving secondary structure for achieving specific hybridization.
Antimicrobial Effects of Mustard Flour and Acetic Acid against
Escherichia coli
O157:H7,
Listeria monocytogenes
, and
Salmonella enterica
Serovar Typhimurium
Authors: Rhee Min-Suk , Lee Sun-Young , Dougherty Richard H. , Kang Dong-Hyun
Published: May 2003

This study was designed to investigate the individual and combined effects of mustard flour and acetic acid in the inactivation of food-borne pathogenic bacteria stored at 5 and 22°C. Samples were prepared to achieve various concentrations by the addition of acetic acid (0, 0.5, or 1%) along with mustard flour (0, 10, or 20%) and 2% sodium chloride (fixed amount). Acid-adapted three-strain mixtures of
Escherichia coli
O157:H7,
Listeria monocytogenes
, and
Salmonella enterica
serovar Typhimurium strains (10
6
to 10
7
CFU/ml) were inoculated separately into prepared mustard samples stored at 5 and 22°C, and samples were assayed periodically. The order of bacterial resistance, assessed by the time required for the nominated populations to be reduced to undetectable levels against prepared mustards at 5°C, was
S. enterica
serovar Typhimurium (1 day) <
E. coli
O157:H7 (3 days) <
L. monocytogenes
(9 days). The food-borne pathogens tested were reduced much more rapidly at 22°C than at 5°C. There was no synergistic effect with regard to the killing of the pathogens tested with the addition of 0.5% acetic acid to the mustard flour (10 or 20%). Mustard in combination with 0.5% acetic acid had less bactericidal activity against the pathogens tested than did mustard alone. The reduction of
E. coli
O157:H7 and
L. monocytogenes
among the combined treatments on the same storage day was generally differentiated as follows: control < mustard in combination with 0.5% acetic acid < mustard alone < mustard in combination with 1% acetic acid < acetic acid alone. Our study indicates that acidic products may limit microbial growth or survival and that the addition of small amounts of acetic acid (0.5%) to mustard can retard the reduction of
E. coli
O157:H7 and
L. monocytogenes
. These antagonistic effects may be changed if mustard is used alone or in combination with >1% acetic acid.
Characterization of Two Tetrachloroethene-Reducing, Acetate-Oxidizing Anaerobic Bacteria and Their Description asDesulfuromonas michiganensissp. nov
Authors: Sung Youlboong , Ritalahti Kirsti M. , Sanford Robert A. , Urbance John W. , Flynn Shannon J. , Tiedje James M. , Löffler Frank E.
Published: May 2003

Two tetrachlorethene (PCE)-dechlorinating populations, designated strains BB1 and BRS1, were isolated from pristine river sediment and chloroethene-contaminated aquifer material, respectively. PCE-to-cis-1,2-dichloroethene-dechlorinating activity could be transferred in defined basal salts medium with acetate as the electron donor and PCE as the electron acceptor. Taxonomic analysis based on 16S rRNA gene sequencing placed both isolates within theDesulfuromonascluster in the δ subdivision of theProteobacteria. PCE was dechlorinated at rates of at least 139 nmol min−1mg of protein−1at pH values between 7.0 and 7.5 and temperatures between 25 and 30°C. Dechlorination also occurred at 10°C. The electron donors that supported dechlorination included acetate, lactate, pyruvate, succinate, malate, and fumarate but not hydrogen, formate, ethanol, propionate, or sulfide. Growth occurred with malate or fumarate alone, whereas oxidation of the other electron donors depended strictly on the presence of fumarate, malate, ferric iron, sulfur, PCE, or TCE as an electron acceptor. Nitrate, sulfate, sulfite, thiosulfate, and other chlorinated compounds were not used as electron acceptors. Sulfite had a strong inhibitory effect on growth and dechlorination. Alternate electron acceptors (e.g., fumarate or ferric iron) did not inhibit PCE dechlorination and were consumed concomitantly. The putative fumarate, PCE, and ferric iron reductases were induced by their respective substrates and were not constitutively present. Sulfide was required for growth. Both strains tolerated high concentrations of PCE, and dechlorination occurred in the presence of free-phase PCE (dense non-aqueous-phase liquids). Repeated growth with acetate and fumarate as substrates yielded a BB1 variant that had lost the ability to dechlorinate PCE. Due to the 16S rRNA gene sequence differences with the closest relatives and the unique phenotypic characteristics, we propose that the new isolates are members of a new species,Desulfuromonas michiganensis, within theDesulfuromonascluster of theGeobacteraceae.
Enterolysin A, a Cell Wall-Degrading Bacteriocin fromEnterococcus faecalisLMG 2333
Authors: Nilsen Trine , Nes Ingolf F. , Holo Helge
Published: May 2003

A novel antimicrobial protein, designated enterolysin A, was purified from anEnterococcus faecalisLMG 2333 culture. Enterolysin A inhibits growth of selected enterococci, pediococci, lactococci, and lactobacilli. Antimicrobial activity was initially detected only on solid media, but by growing the bacteria in a fermentor under optimized production conditions (MRS broth with 4% [wt/vol] glucose, pH 6.5, and a temperature between 25 and 35°C), the bacteriocin activity was increased to 5,120 bacteriocin units ml−1. Enterolysin A production was regulated by pH, and activity was first detected in the transition between the logarithmic and stationary growth phases. Killing of sensitive bacteria by enterolysin A showed a dose-response behavior, and the bacteriocin has a bacteriolytic mode of action. Enterolysin A was purified, and the primary structure was determined by combined amino acid and DNA sequencing. This bacteriocin is translated as a 343-amino-acid preprotein with ansec-dependent signal peptide of 27 amino acids, which is followed by a sequence corresponding to the N-terminal part of the purified protein. Mature enterolysin A consists of 316 amino acids and has a calculated molecular weight of 34,501, and the theoretical pI is 9.24. The N terminus of enterolysin A is homologous to the catalytic domains of different cell wall-degrading proteins with modular structures. These include lysostaphin, ALE-1, zoocin A, and LytM, which are all endopeptidases belonging to the M37 protease family. The N-terminal part of enterolysin A is linked by a threonine-proline-rich region to a putative C-terminal recognition domain, which shows significant sequence identity to two bacteriophage lysins.
Thermophily in the
Geobacteraceae
:
Geothermobacter ehrlichii
gen. nov., sp. nov., a Novel Thermophilic Member of the
Geobacteraceae
from the “Bag City” Hydrothermal Vent
Authors: Kashefi Kazem , Holmes Dawn E. , Baross John A. , Lovley Derek R.
Published: May 2003

Little is known about the microbiology of the “Bag City” hydrothermal vent, which is part of a new eruption site on the Juan de Fuca Ridge and which is notable for its accumulation of polysaccharide on the sediment surface. A pure culture, designated strain SS015, was recovered from a vent fluid sample from the Bag City site through serial dilution in liquid medium with malate as the electron donor and Fe(III) oxide as the electron acceptor and then isolation of single colonies on solid Fe(III) oxide medium. The cells were gram-negative rods, about 0.5 μm by 1.2 to 1.5 μm, and motile and contained
c
-type cytochromes. Analysis of the 16S ribosomal DNA (rDNA) sequence of strain SS015 placed it in the family
Geobacteraceae
in the delta subclass of the
Proteobacteria
. Unlike previously described members of the
Geobacteraceae
, which are mesophiles, strain SS015 was a thermophile and grew at temperatures of between 35 and 65°C, with an optimum temperature of 55°C. Like many previously described members of the
Geobacteraceae
, strain SS015 grew with organic acids as the electron donors and Fe(III) or nitrate as the electron acceptor, with nitrate being reduced to ammonia. Strain SS015 was unique among the
Geobacteraceae
in its ability to use sugars, starch, or amino acids as electron donors for Fe(III) reduction. Under stress conditions, strain SS015 produced copious quantities of extracellular polysaccharide, providing a model for the microbial production of the polysaccharide accumulation at the Bag City site. The 16S rDNA sequence of strain SS015 was less than 94% similar to the sequences of previously described members of the
Geobacteraceae
; this fact, coupled with its unique physiological properties, suggests that strain SS015 represents a new genus in the family
Geobacteraceae
. The name
Geothermobacter ehrlichii
gen. nov., sp. nov., is proposed (ATCC BAA-635 and DSM 15274). Although strains of
Geobacteraceae
are known to be the predominant Fe(III)-reducing microorganisms in a variety of Fe(III)-reducing environments at moderate temperatures, strain SS015 represents the first described thermophilic member of the
Geobacteraceae
and thus extends the known environmental range of this family to hydrothermal environments.
Community-Level Physiological Profiling Performed with an Oxygen-Sensitive Fluorophore in a Microtiter Plate
Authors: Garland Jay L. , Roberts Michael S. , Levine Lanfang H. , Mills Aaron L.
Published: May 2003

Community-level physiological profiling based upon fluorometric detection of oxygen consumption was performed on hydroponic rhizosphere and salt marsh litter samples by using substrate levels as low as 50 ppm with incubation times between 5 and 24 h. The rate and extent of response were increased in samples acclimated to specific substrates and were reduced by limiting nitrogen availability in the wells.
Reductive Dehalogenation of Chlorobenzene Congeners in Cell Extracts of
Dehalococcoides
sp. Strain CBDB1
Authors: Hölscher Tina , Görisch Helmut , Adrian Lorenz
Published: May 2003

Enzymatic reductive dehalogenation of tri-, tetra-, penta-, and hexachlorobenzenes was demonstrated in cell extracts with low protein concentration (0.5 to 1 μg of protein/ml) derived from the chlorobenzene-respiring anaerobe
Dehalococcoides
sp. strain CBDB1. 1,2,3-trichlorobenzene dehalogenase activity was associated with the membrane fraction. Light-reversible inhibition by alkyl iodides indicated the presence of a corrinoid cofactor.
Endotoxin Inactivation in Water by Using Medium-Pressure UV Lamps
Authors: Anderson W. B. , Huck P. M. , Dixon D. G. , Mayfield C. I.
Published: May 2003

Deionized water was spiked with various concentrations of endotoxin and exposed to UV irradiation from medium-pressure UV lamps to assess endotoxin inactivation. It was found that endotoxin inactivation was proportional to the UV dose under the conditions examined. The inactivation rate was determined to be ∼0.55 endotoxin unit/ml per mJ/cm
2
of irradiation delivered.
Antimicrobial-Resistant
Campylobacter
Species from Retail Raw Meats
Authors: Ge Beilei , White David G. , McDermott Patrick F. , Girard Webb , Zhao Shaohua , Hubert Susannan , Meng Jianghong
Published: May 2003

The antimicrobial susceptibilities of 378
Campylobacter
isolates were determined. Resistance to tetracycline was the most common (82%), followed by resistance to doxycycline (77%), erythromycin (54%), nalidixic acid (41%), and ciprofloxacin (35%).
Campylobacter coli
displayed significantly higher rates of resistance to ciprofloxacin and erythromycin than
Campylobacter jejuni
, and
Campylobacter
isolates from turkey meat showed a greater resistance than those from chicken meat.
Habitat-Specific Diversity of
Borrelia burgdorferi
Sensu Lato in Europe, Exemplified by Data from Latvia
Authors: Etti Susanne , Hails Rosie , Schäfer Stefanie M. , De Michelis Simona , Sewell Henna-Sisko , Bormane Antra , Donaghy Michael , Kurtenbach Klaus
Published: May 2003

The distribution of
Borrelia burgdorferi
sensu lato genospecies in questing
Ixodes ricinus
ticks from ecologically distinct habitats in Latvia was analyzed. A significant variation in the frequency of the genospecies across sites was observed, pointing to the importance of the host community in the ecology of Lyme borreliosis.
Production of Native-Type
Streptoverticillium mobaraense
Transglutaminase in
Corynebacterium glutamicum
Authors: Date Masayo , Yokoyama Kei-ichi , Umezawa Yukiko , Matsui Hiroshi , Kikuchi Yoshimi
Published: May 2003

We previously observed secretion of active-form transglutaminase in
Corynebacterium glutamicum
by coexpressing the subtilisin-like protease SAM-P45 from
Streptomyces albogriseolus
to process the prodomain. However, the N-terminal amino acid sequence of the transglutaminase differed from that of the native
Streptoverticillium mobaraense
enzyme. In the present work we have used site-directed mutagenesis to generate an optimal SAM-P45 cleavage site in the C-terminal region of the prodomain. As a result, native-type transglutaminase was secreted.
Unexpected Thermal Destruction of Dried, Glass Bead-Immobilized Microorganisms as a Function of Water Activity
Authors: Laroche C. , Gervais P.
Published: May 2003

To help us understand the factors and mechanisms implicated in the death of microorganisms or their resistance to temperature in a low water activity environment, microorganisms were dried on the surface of glass beads before being subjected to high temperatures for a short period followed by rapid cooling. Two microorganisms were studied: the yeast
Saccharomyces cerevisiae
and the bacterium
Lactobacillus plantarum
. Experiments were carried out at 150, 200, and 250°C, with four durations of heat treatment and seven levels of initial water activity between 0.10 and 0.70. We observed an unexpected range of water activity, between 0.30 and 0.50, at which microorganisms were more resistant to the various treatments, with maximal viability at 0.35 for
L. plantarum
and 0.40 for
S. cerevisiae.
An Improved Cloning Vector for Construction of Gene Replacements in
Listeria monocytogenes
Authors: Li Guojie , Kathariou S.
Published: May 2003

Listeria monocytogenes
is a gram-positive, facultative intracellular bacterium implicated in severe food-borne illness (listeriosis) in humans. The construction of well-defined gene replacements in the genome of
L. monocytogenes
has been instrumental to several genetic studies of the virulence and other attributes of the organism. Construction of such mutations by currently available procedures, however, tends to be labor intensive, and gene replacement mutants are sometimes difficult to recover due to lack of direct selection for the construct. In this study we describe the construction and use of plasmid vector pGF-EM, which can be conjugatively transferred from
Escherichia coli
S17-1 to
L. monocytogenes
and which provides the genetic means for direct selection of gene replacements.
Discordant Phylogenies within the
rrn
Loci of Rhizobia
Authors: van Berkum Peter , Terefework Zewdu , Paulin Lars , Suomalainen Sini , Lindström Kristina , Eardly Bertrand D.
Published: May 2003

It is evident from complete genome sequencing results that lateral gene transfer and recombination are essential components in the evolutionary process of bacterial genomes. Since this has important implications for bacterial systematics, the primary objective of this study was to compare estimated evolutionary relationships among a representative set of α-
Proteobacteria
by sequencing analysis of three loci within their
rrn
operons. Tree topologies generated with 16S rRNA gene sequences were significantly different from corresponding trees assembled with 23S rRNA gene and internally transcribed space region sequences. Besides the incongruence in tree topologies, evidence that distinct segments along the 16S rRNA gene sequences of bacteria currently classified within the genera
Bradyrhizobium
,
Mesorhizobium
and
Sinorhizobium
have a reticulate evolutionary history was also obtained. Our data have important implications for bacterial taxonomy, because currently most taxonomic decisions are based on comparative 16S rRNA gene sequence analysis. Since phylogenetic placement based on 16S rRNA gene sequence divergence perhaps is questionable, we suggest that the proposals of bacterial nomenclature or changes in their taxonomy that have been made may not necessarily be warranted. Accordingly, a more conservative approach should be taken in the future, in which taxonomic decisions are based on the analysis of a wider variety of loci and comparative analytical methods are used to estimate phylogenetic relationships among the genomes under consideration.
Mutational Analysis of the Carboxy-Terminal (YGX)4Repeat Domain of CpsD, an Autophosphorylating Tyrosine Kinase Required for Capsule Biosynthesis inStreptococcus pneumoniae
Authors: Morona Judy K. , Morona Renato , Miller David C. , Paton James C.
Published: May 2003

InStreptococcus pneumoniae, CpsB, CpsC, and CpsD are essential for encapsulation, and mutants containing deletions ofcpsB,cpsC, orcpsDexhibit rough colony morphologies. CpsD is an autophosphorylating protein-tyrosine kinase, CpsC is required for CpsD tyrosine phosphorylation, and CpsB is a phosphotyrosine-protein phosphatase. We have previously shown that autophosphorylation of CpsD at tyrosine attenuates its activity and consequently reduces the level of encapsulation and negatively regulates CPS production. In this study, we further investigated the role of the carboxy-terminal (YGX)4repeat domain of CpsD in encapsulation. A CpsD truncation mutant in which the entire (YGX)4repeat domain was removed was indistinguishable from a strain in which the entirecpsDgene had been deleted, indicating that the carboxy-terminal (YGX)4tail is required for CpsD activity in capsular polysaccharide production. Double mutants having a single tyrosine residue at position 2, 3, or 4 in the (YGX)4repeat domain and lacking CpsB exhibited a rough colony morphology, indicating that in the absence of an active protein-tyrosine phosphatase, phosphorylation of just one of the tyrosine residues in the (YGX)4repeat was sufficient to inactivate CpsD. When various mutants in which CpsD had either one or combinations of two or three tyrosine residues in the (YGX)4repeat domain were examined, only those with three tyrosine residues in the (YGX)4repeat domain were indistinguishable from the wild-type strain. The mutants with either one or two tyrosine residues exhibited mucoid colony morphologies. Further analysis of the mucoid strains indicated that the mucoid phenotype was not due to overproduction of capsular polysaccharide, as these strains actually produced less capsular polysaccharide than the wild-type strain. Thus, the tyrosine residues in the (YGX)4repeat domain are essential for normal functioning of CpsD.
S
-Adenosylmethionine Transport in
Rickettsia prowazekii
Authors: Tucker Aimee M. , Winkler Herbert H. , Driskell Lonnie O. , Wood David O.
Published: May 2003

Rickettsia prowazekii
, the causative agent of epidemic typhus, is an obligate, intracellular, parasitic bacterium that grows within the cytoplasm of eucaryotic host cells. Rickettsiae exploit this intracellular environment by using transport systems for the compounds available in the host cell's cytoplasm. Analysis of the
R. prowazekii
Madrid E genome sequence revealed the presence of a mutation in the rickettsial
metK
gene, the gene encoding the enzyme responsible for the synthesis of
S
-adenosylmethionine (AdoMet). Since AdoMet is required for rickettsial processes, the apparent inability of this strain to synthesize AdoMet suggested the presence of a rickettsial AdoMet transporter. We have confirmed the presence of an AdoMet transporter in the rickettsiae which, to our knowledge, is the first bacterial AdoMet transporter identified. The influx of AdoMet into rickettsiae was a saturable process with a

K
T

of 2.3 μM. Transport was inhibited by
S
-adenosylethionine and
S
-adenosylhomocysteine but not by sinfungin or methionine. Transport was also inhibited by 2,4-dinitrophenol, suggesting an energy-linked transport mechanism, and by
N
-ethylmaleimide. AdoMet transporters with similar properties were also identified in the Breinl strain of
R. prowazekii
and in
Rickettsia typhi
. By screening
Escherichia coli
clone banks for AdoMet transport, the
R. prowazekii
gene coding for a transporter, RP076 (
sam
), was identified. AdoMet transport in
E. coli
containing the
R. prowazekii sam
gene exhibited kinetics similar to that seen in rickettsiae. The existence of a rickettsial transporter for AdoMet raises intriguing questions concerning the evolutionary relationship between the synthesis and transport of this essential metabolite.
Regulation of the Cellulosomal
celS
(
cel48A
) Gene of
Clostridium thermocellum
Is Growth Rate Dependent
Authors: Dror Tali W. , Morag Ely , Rolider Adi , Bayer Edward A. , Lamed Raphael , Shoham Yuval
Published: May 2003

Clostridium thermocellum
produces an extracellular multienzyme complex, termed cellulosome, that allows efficient solubilization of crystalline cellulose. One of the major enzymes in this complex is the CelS (Cel48A) exoglucanase. The regulation of CelS at the protein and transcriptional levels was studied using batch and continuous cultures. The results of sodium dodecyl sulfate-polyacrylamide gel electrophoresis and Western blot analyses indicated that the amount of CelS in the supernatant fluids of cellobiose-grown cultures is lower than that of cellulose-grown cultures. The transcriptional level of
celS
mRNA was determined quantitatively by RNase protection assays with batch and continuous cultures under carbon and nitrogen limitation. The amount of
celS
mRNA transcripts per cell was about 180 for cells grown under carbon limitation at growth rates of 0.04 to 0.21 h
−1
and 80 and 30 transcripts per cell for batch cultures at growth rates of 0.23 and 0.35 h
−1
, respectively. Under nitrogen limitation, the corresponding levels were 110, 40, and 30 transcripts/cell for growth rates of 0.07, 0.11, and 0.14 h
−1
, respectively. Two major transcriptional start sites were detected at positions −140 and −145 bp, upstream of the translational start site of the
celS
gene. The potential promoters exhibited homology to known sigma factors (i.e., σ
A
and σ
B
) of
Bacillus subtilis
. The relative activity of the two promoters remained constant under the conditions studied and was in agreement with the results of the RNase protection assay, in which the observed transcriptional activity was inversely proportional to the growth rate.
Synthesis of Catalytically Active Form III Ribulose 1,5-Bisphosphate Carboxylase/Oxygenase in Archaea
Authors: Finn Michael W. , Tabita F. Robert
Published: May 2003

Ribulose 1,5 bisphosphate carboxylase/oxygenase (RubisCO) catalyzes the biological reduction and assimilation of carbon dioxide gas to organic carbon; it is the key enzyme responsible for the bulk of organic matter found on earth. Until recently it was believed that there are only two forms of RubisCO, form I and form II. However, the recent completion of several genome-sequencing projects uncovered open reading frames resembling RubisCO in the third domain of life, the archaea. Previous work and homology comparisons suggest that these enzymes represent a third form of RubisCO, form III. While earlier work indicated that two structurally distinct recombinant archaeal RubisCO proteins catalyzed bona fide RubisCO reactions, it was not established that the
rbcL
genes of anaerobic archaea can be transcribed and translated to an active enzyme in the native organisms. In this report, it is shown not only that
Methanococcus jannaschii
,
Archaeoglobus fulgidus
,
Methanosarcina acetivorans
, and
Methanosarcina barkeri
possess open reading frames with the residues required for catalysis but also that the RubisCO protein from these archaea accumulates in an active form under normal growth conditions. In addition, the form III RubisCO gene (
rbcL
) from
M. acetivorans
was shown to complement RubisCO deletion strains of
Rhodobacter capsulatus
and
Rhodobacter sphaeroides
under both photoheterotrophic and photoautotrophic growth conditions. These studies thus indicate for the first time that archaeal form III RubisCO functions in a physiologically significant fashion to fix CO
2
. Furthermore, recombinant
M. jannaschii
,
M. acetivorans
, and
A. fulgidus
RubisCO possess unique properties with respect to quaternary structure, temperature optima, and activity in the presence of molecular oxygen compared to the previously described
Thermococcus kodakaraensis
and halophile proteins.
Repair System for Noncanonical Purines in
Escherichia coli
Authors: Burgis Nicholas E. , Brucker Jason J. , Cunningham Richard P.
Published: May 2003

Exposure of
Escherichia coli
strains deficient in molybdopterin biosynthesis (
moa
) to the purine base
N
-6-hydroxylaminopurine (HAP) is mutagenic and toxic. We show that
moa
mutants exposed to HAP also exhibit elevated mutagenesis, a hyperrecombination phenotype, and increased SOS induction. The
E. coli rdgB
gene encodes a protein homologous to a deoxyribonucleotide triphosphate pyrophosphatase from
Methanococcus jannaschii
that shows a preference for purine base analogs.
moa rdgB
mutants are extremely sensitive to killing by HAP and exhibit increased mutagenesis, recombination, and SOS induction upon HAP exposure. Disruption of the endonuclease V gene,
nfi
, rescues the HAP sensitivity displayed by
moa
and
moa rdgB
mutants and reduces the level of recombination and SOS induction, but it increases the level of mutagenesis. Our results suggest that endonuclease V incision of DNA containing HAP leads to increased recombination and SOS induction and even cell death. Double-strand break repair mutants display an increase in HAP sensitivity, which can be reversed by an
nfi
mutation. This suggests that cell killing may result from an increase in double-strand breaks generated when replication forks encounter endonuclease V-nicked DNA. We propose a pathway for the removal of HAP from purine pools, from deoxynucleotide triphosphate pools, and from DNA, and we suggest a general model for excluding purine base analogs from DNA. The system for HAP removal consists of a molybdoenzyme, thought to detoxify HAP, a deoxyribonucleotide triphosphate pyrophosphatase that removes noncanonical deoxyribonucleotide triphosphates from replication precursor pools, and an endonuclease that initiates the removal of HAP from DNA.
CodY Is a Nutritional Repressor of Flagellar Gene Expression inBacillus subtilis
Authors: Bergara F. , Ibarra C. , Iwamasa J. , Patarroyo J. C. , Aguilera R. , Márquez-Magaña L. M.
Published: May 2003

Expression of the σD-dependent flagellin gene,hag, is repressed by the CodY protein in nutrient-rich environments. Analysis of acodYmutant bearing ahag-lacZreporter suggests that the availability of amino acids in the environment is the specific signal that triggers this repression. Further,hag-lacZexpression appears to be sensitive to intracellular GTP levels, as demonstrated by increased expression upon addition of decoyinine. This result is consistent with the postulate that the availability of amino acids in the environment effects intracellular GTP levels through the stringent response. However, the levels ofhag-lacZmeasured upon the addition of subsets of amino acids suggest an additional mechanism(s). CodY is a DNA binding protein that could repress flagellin expression directly by binding to thehagpromoter region, or indirectly by binding to thefla/chepromoter region that governs expression of the σDtranscriptional activator required forhaggene expression. Using an electrophoretic mobility shift assay, we have demonstrated that purified CodY protein binds specifically to both thehagandfla/chepromoter fragments. Additionally, CodY acts as a nutritional repressor of transcription from thefla/chepromoter region that contains two functional promoters. CodY binds to both the σD- and σA-dependent promoters in this region, as demonstrated by DNase I footprint analyses. Footprint analyses of thehaggene demonstrated that CodY binds downstream of its σD-dependent promoter. Taken together, these results identify new members of the CodY regulon that encode motility functions inBacillus subtilisand are controlled by the σDalternate sigma factor.
Characterization of NorR Protein, a Multifunctional Regulator of
norA
Expression in
Staphylococcus aureus
Authors: Truong-Bolduc Que Chi , Zhang Xiamei , Hooper David C.
Published: May 2003

We characterized a
Staphylococcus aureus norA
gene expression regulator, NorR, initially identified from its binding to the
norA
promoter. The
norR
gene was 444 bp in length, located ∼7 kb upstream from the
norA
gene, and encoded a predicted 17.6-kDa protein. Overexpression of
norR
in wild-type
S. aureus
strain ISP794 led to a fourfold decrease in sensitivity to quinolones and ethidium bromide and an increase in the level of
norA
transcripts, suggesting that NorR acts as a positive regulator of
norA
expression. Overexpression of
norR
in
sarA
and
agr
mutants did not alter quinolone sensitivity or levels of
norA
transcription, indicating that the presence of these two global regulatory systems is necessary for NorR to affect the expression of
norA
. Insertion and disruption of
norR
in ISP794 increased resistance to quinolones by 4- to 16-fold but had no effect on
norA
transcription, suggesting that NorR acts as a repressor for another unidentified efflux pump or pumps. These mutants also exhibited an exaggerated clumping phenotype in liquid media, which was complemented fully by a plasmid-encoded
norR
gene. Collectively, these results indicate that NorR is a multifunctional regulator, affecting cell surface properties as well as the expression of NorA and likely other multidrug resistance efflux pumps.
Transcription Regulation Coupling of the DivergentargGandmetYPromoters inEscherichia coliK-12
Authors: Krin Evelyne , Laurent-Winter Christine , Bertin Philippe N. , Danchin Antoine , Kolb Annie
Published: May 2003

The cAMP-catabolite activator protein (CAP) complex is a pleiotropic regulator that regulates a vast number ofEscherichia coligenes, including those involved in carbon metabolism. We identified two new targets of this complex:argG, which encodes the arginosuccinate synthase involved in the arginine biosynthetic pathway, andmetY, which encodes one of the two methionine tRNA initiators, tRNAf2Met. The cAMP-CAP complex activatesargGtranscription and inhibitsmetYtranscription from the same DNA position. We also show that ArgR, the specific repressor of the arginine biosynthetic pathway, together with its arginine cofactor, acts on the regulation ofmetYmediated by CAP. The regulation of the two divergent promoters is thus simultaneously controlled not only by the cAMP-CAP complex, a global regulator, but also by a specific regulator of arginine metabolism, suggesting a previously unsuspected link between carbon metabolism and translation initiation.
Early Colonization Events in the Mutualistic Association between
Steinernema carpocapsae
Nematodes and
Xenorhabdus nematophila
Bacteria
Authors: Martens Eric C. , Heungens Kurt , Goodrich-Blair Heidi
Published: May 2003

The bacterium
Xenorhabdus nematophila
is a mutualist of the entomopathogenic nematode
Steinernema carpocapsae
. During its life cycle, the bacterium exists both separately from the nematode and as an intestinal resident of a nonfeeding nematode form, the infective juvenile (IJ). The progression of
X. nematophila
from an ex vivo existence to a specific and persistent colonization of IJs is a model to understand the mechanisms mediating the initiation and maintenance of benign host-microbe interactions. To help characterize this process, we constructed an
X. nematophila
strain that constitutively expresses green fluorescent protein, which allowed its presence to be monitored within IJs. Using this strain, we showed that few bacterial cells initiate colonization of an individual IJ and that these grow inside the lumen of the IJ intestine in a reproducible polyphasic pattern during colonization. In accordance with these two observations, we demonstrated that the final population of bacteria in a nematode is of predominantly monoclonal origin, suggesting that only one or two bacterial clones initiate or persist during colonization of an individual nematode. These data suggest that
X. nematophila
initiates IJ colonization by competing for limited colonization sites or resources within the nematode intestine. This report represents the first description of the biological interactions occurring between
X. nematophila
and
S. carpocapsae
during the early stages of the colonization process, provides insights into the physiology of
X. nematophila
in its host niche, and will facilitate interpretation of future data regarding the molecular events mediating this process.
Characterization of the
Xanthomonas axonopodis
pv. glycines Hrp Pathogenicity Island
Authors: Kim Jung-Gun , Park Byoung Keun , Yoo Chang-Hyuk , Jeon Eunkyung , Oh Jonghee , Hwang Ingyu
Published: May 2003

We sequenced an approximately 29-kb region from
Xanthomonas axonopodis
pv. glycines that contained the Hrp type III secretion system, and we characterized the genes in this region by Tn
3-gus
mutagenesis and gene expression analyses. From the region,
hrp
(hypersensitive response and pathogenicity) and
hrc
(
hrp
and conserved) genes, which encode type III secretion systems, and
hpa
(
hrp
-associated) genes were identified. The characteristics of the region, such as the presence of many virulence genes, low G+C content, and bordering tRNA genes, satisfied the criteria for a pathogenicity island (PAI) in a bacterium. The PAI was composed of nine
hrp
, nine
hrc
, and eight
hpa
genes with seven plant-inducible promoter boxes. The
hrp
and
hrc
mutants failed to elicit hypersensitive responses in pepper plants but induced hypersensitive responses in all tomato plants tested. The Hrp PAI of
X. axonopodis
pv. glycines resembled the Hrp PAIs of other
Xanthomonas
species, and the Hrp PAI core region was highly conserved. However, in contrast to the PAI of
Pseudomonas syringae
, the regions upstream and downstream from the Hrp PAI core region showed variability in the xanthomonads. In addition, we demonstrate that HpaG, which is located in the Hrp PAI region of
X. axonopodis
pv. glycines, is a response elicitor. Purified HpaG elicited hypersensitive responses at a concentration of 1.0 μM in pepper, tobacco, and
Arabidopsis thaliana
ecotype Cvi-0 by acting as a type III secreted effector protein. However, HpaG failed to elicit hypersensitive responses in tomato, Chinese cabbage, and
A. thaliana
ecotypes Col-0 and Ler. This is the first report to show that the harpin-like effector protein of
Xanthomonas
species exhibits elicitor activity.
Energy-Generating Enzymes ofBurkholderia cepaciaand Their Interactions with Macrophages
Authors: Punj Vasu , Sharma Rachna , Zaborina Olga , Chakrabarty A. M.
Published: May 2003

We previously demonstrated that several clinical and environmental isolates ofBurkholderia cepaciasecreted ATP-utilizing enzymes to the medium; the secretion of these enzymes by cystic fibrosis lung isolate strain 38 was shown to be greatly enhanced in the presence of α2-macroglobulin. Fractionation of the growth medium of cystic fibrosis isolate strain 71 belonging to genomovar I demonstrated the presence of two additional proteins, homologues ofPseudomonas aeruginosaazurin and cytochromec551, which are normally involved in electron transfer during denitrification. A Q-Sepharose column flowthrough fraction of the growth medium ofB. cepaciastrain 71 enriched with the azurin and cytochromec551homologues triggered apoptosis in macrophages and mast cells, leading to their death. Incubation of the Q-Sepharose column flowthrough fraction with antiazurin and anti-cytochromec551antibodies greatly reduced cell death. We cloned and hyperexpressed a gene fromB. cepaciastrain 71 that encodes the homologue ofP. aeruginosaazurin. Such azurin homologues were detected in the growth medium of several strains belonging to genomovars I, III, and VI but not in the growth medium of strains belonging to other genomovars. The growth medium of the strains that elaborated the azurin homologue had high cytotoxicity towards macrophages. Purified azurin homologue was shown to induce apoptosis in macrophages in a caspase-dependent manner and was localized in both the cytosol and nucleus when incubated with or microinjected into macrophages. This is an interesting example of the interaction of a bacterial protein normally involved in cellular energetics with macrophages to effect their cell death.
Genes of the GadX-GadW Regulon inEscherichia coli
Authors: Tucker Don L. , Tucker Nancy , Ma Zhuo , Foster John W. , Miranda Regina L. , Cohen Paul S. , Conway Tyrrell
Published: May 2003

Acid in the stomach is thought to be a barrier to bacterial colonization of the intestine.Escherichia coli, however, has three systems for acid resistance, which overcome this barrier. The most effective of these systems is dependent on transport and decarboxylation of glutamate. GadX regulates two genes that encode isoforms of glutamate decarboxylase critical to this system, but additional genes associated with the glutamate-dependent acid resistance system remained to be identified. ThegadXgene and a second downstreamaraC-like transcription factor gene,gadW, were mutated separately and in combination, and the gene expression profiles of the mutants were compared to those of the wild-type strain grown in neutral and acidified media under conditions favoring induction of glutamate-dependent acid resistance. Cluster and principal-component analyses identified 15 GadX-regulated, acid-inducible genes. Reverse transcriptase mapping demonstrated that these genes are organized in 10 operons. Analysis of the strain lacking GadX but possessing GadW confirmed that GadX is a transcriptional activator under acidic growth conditions. Analysis of the strain lacking GadW but possessing GadX indicated that GadW exerts negative control over three GadX target genes. The strain lacking both GadX and GadW was defective in acid induction of most but not all GadX target genes, consistent with the roles of GadW as an inhibitor of GadX-dependent activation of some genes and an activator of other genes. Resistance to acid was decreased under certain conditions in agadXmutant and even more so by combined mutation ofgadXandgadW. However, there was no defect in colonization of the streptomycin-treated mouse model by thegadXmutant in competition with the wild type, and thegadX gadWmutant was a better colonizer than the wild type. Thus,E. colicolonization of the mouse does not appear to require glutamate-dependent acid resistance.
Conservation of Plasmid Maintenance Functions between Linear and Circular Plasmids inBorrelia burgdorferi
Authors: Stewart Philip E. , Chaconas George , Rosa Patricia
Published: May 2003

The Lyme disease agentBorrelia burgdorferimaintains both linear and circular plasmids that appear to be essential for mammalian infection. Recent studies have characterized the circular plasmid regions that confer autonomous replication, but the genetic elements necessary for linear plasmid maintenance have not been experimentally identified. Two vectors derived from linear plasmids lp25 and lp28-1 were constructed and shown to replicate autonomously inB. burgdorferi. These vectors identify internal regions of linear plasmids necessary for autonomous replication inB. burgdorferi. Although derived from linear plasmids, the vectors are maintained in circular form inB. burgdorferi, indicating that plasmid maintenance functions are conserved, regardless of DNA form. Finally, derivatives of these vectors indicate that paralogous gene family 49 is apparently not required for either circular or linear plasmid replication.
Characterization of the Chlorate Reductase from
Pseudomonas chloritidismutans
Authors: Wolterink Arthur F. W. M. , Schiltz Emile , Hagedoorn Peter-Leon , Hagen Wilfred R. , Kengen Servé W. M. , Stams Alfons J. M.
Published: May 2003

A chlorate reductase has been purified from the chlorate-reducing strain
Pseudomonas chloritidismutans
. Comparison with the periplasmic (per)chlorate reductase of strain GR-1 showed that the cytoplasmic chlorate reductase of
P. chloritidismutans
reduced only chlorate and bromate. Differences were also found in N-terminal sequences, molecular weight, and subunit composition. Metal analysis and electron paramagnetic resonance measurements showed the presence of iron and molybdenum, which are also found in other dissimilatory oxyanion reductases.
Alpha-Toxin Is Required for Biofilm Formation by
Staphylococcus aureus
Authors: Caiazza Nicky C. , O'Toole G. A.
Published: May 2003

Staphylococcus aureus
is a common pathogen associated with nosocomial infections. It can persist in clinical settings and gain increased resistance to antimicrobial agents through biofilm formation. We have found that alpha-toxin, a secreted, multimeric, hemolytic toxin encoded by the
hla
gene, plays an integral role in biofilm formation. The
hla
mutant was unable to fully colonize plastic surfaces under both static and flow conditions. Based on microscopy studies, we propose that alpha-hemolysin is required for cell-to-cell interactions during biofilm formation.