bluuebunny/crossref_metadata_2025 huggingface dataset:
Low-pH Adaptation and the Acid Tolerance Response of
Bifidobacterium longum
Biotype longum
Authors: Sánchez Borja , Champomier-Vergès Marie-Christine , Collado María del Carmen , Anglade Patricia , Baraige Fabienne , Sanz Yolanda , de los Reyes-Gavilán Clara G. , Margolles Abelardo , Zagorec Monique
Published: August 2007

Bifidobacteria are one of the main microbial inhabitants of the human colon. Usually administered in fermented dairy products as beneficial microorganisms, they have to overcome the acidic pH found in the stomach during the gastrointestinal transit to be able to colonize the lower parts of the intestine. The mechanisms underlying acid response and adaptation in
Bifidobacterium longum
biotype longum NCIMB 8809 and its acid-pH-resistant mutant
B. longum
biotype longum 8809dpH were studied. Comparison of protein maps, and protein identification by matrix-assisted laser desorption ionization-time of flight mass spectrometry analysis, allowed us to identify nine different proteins whose production largely changed in the mutant strain. Furthermore, the production of 47 proteins was modulated by pH in one or both strains. These included general stress response chaperones and proteins involved in transcription and translation as well as in carbohydrate and nitrogen metabolism, among others. Significant differences in the levels of metabolic end products and in the redox status of the cells were also detected between the wild-type strain and its acid-pH-resistant mutant in response to, or as a result of, adaptation to acid. Remarkably, the results of this work indicated that adaptation and response to low pH in
B. longum
biotype longum involve changes in the glycolytic flux and in the ability to regulate the internal pH. These changes were accompanied by a higher content of ammonium in the cytoplasm, likely coming from amino acid deamination, and a decrease of the bile salt hydrolase activity.
Microarray-Based Characterization of theListeria monocytogenesCold Regulon in Log- and Stationary-Phase Cells
Authors: Chan Yvonne C. , Raengpradub Sarita , Boor Kathryn J. , Wiedmann Martin
Published: August 2007

Whole-genome microarray experiments were performed to define theListeria monocytogenescold growth regulon and to identify genes differentially expressed during growth at 4 and 37°C. Microarray analysis using a stringent cutoff (adjustedP< 0.001; ≥2.0-fold change) revealed 105 and 170 genes that showed higher transcript levels in logarithmic- and stationary-phase cells, respectively, at 4°C than in cells grown at 37°C. A total of 74 and 102 genes showed lower transcript levels in logarithmic- and stationary-phase cells, respectively, grown at 4°C. Genes with higher transcript levels at 4°C in both stationary- and log-phase cells included genes encoding a two-component response regulator (lmo0287), a cold shock protein (cspL), and two RNA helicases (lmo0866 and lmo1722), whereas a number of genes encoding virulence factors and heat shock proteins showed lower transcript levels at 4°C. Selected genes that showed higher transcript levels at 4°C during both stationary and log phases were confirmed by quantitative reverse transcriptase PCR. Our data show that (i) a large number ofL. monocytogenesgenes are differentially expressed at 4 and 37°C, with more genes showing higher transcript levels than lower transcript levels at 4°C, (ii)L. monocytogenesgenes with higher transcript levels at 4°C include a number of genes and operons with previously reported or plausible roles in cold adaptation, and (iii)L. monocytogenesgenes with lower transcript levels at 4°C include a number of virulence and virulence-associated genes as well as some heat shock genes.
Inactivation of
Bacillus anthracis
Spores by Liquid Biocides in the Presence of Food Residue
Authors: Hilgren J. , Swanson K. M. J. , Diez-Gonzalez F. , Cords B.
Published: August 2007

Biocide inactivation of
Bacillus anthracis
spores in the presence of food residues after a 10-min treatment time was investigated. Spores of nonvirulent
Bacillus anthracis
strains 7702, ANR-1, and 9131 were mixed with water, flour paste, whole milk, or egg yolk emulsion and dried onto stainless-steel carriers. The carriers were exposed to various concentrations of peroxyacetic acid, sodium hypochlorite (NaOCl), or hydrogen peroxide (H
2
O
2
) for 10 min at 10, 20, or 30°C, after which time the survivors were quantified. The relationship between peroxyacetic acid concentration, H
2
O
2
concentration, and spore inactivation followed a sigmoid curve that was accurately described using a four-parameter logistic model. At 20°C, the minimum concentrations of peroxyacetic acid, H
2
O
2
, and NaOCl (as total available chlorine) predicted to inactivate 6 log
10
CFU of
B. anthracis
spores with no food residue present were 1.05, 23.0, and 0.78%, respectively. At 10°C, sodium hypochlorite at 5% total available chlorine did not inactivate more than 4 log
10
CFU. The presence of the food residues had only a minimal effect on peroxyacetic acid and H
2
O
2
sporicidal efficacy, but the efficacy of sodium hypochlorite was markedly inhibited by whole-milk and egg yolk residues. Sodium hypochlorite at 5% total available chlorine provided no greater than a 2-log
10
CFU reduction when spores were in the presence of egg yolk residue. This research provides new information regarding the usefulness of peroxygen biocides for
B. anthracis
spore inactivation when food residue is present. This work also provides guidance for adjusting decontamination procedures for food-soiled and cold surfaces.
Fine-Scale Genetic Diversity among
Burkholderia pseudomallei
Soil Isolates in Northeast Thailand
Authors: U'Ren Jana M. , Hornstra Heidie , Pearson Talima , Schupp James M. , Leadem Benjamin , Georgia Shalamar , Sermswan Rasana W. , Keim Paul
Published: August 2007

Burkholderia pseudomallei
soil isolates from northeast Thailand were genotyped using multiple-locus variable-number tandem repeat (VNTR) analysis (MLVA) and multilocus sequence typing (MLST). MLVA identified 19 genotypes within three clades, while MLST revealed two genotypes. These close genetic relationships imply a recent colonization followed by localized expansion, similar to what occurs in an outbreak situation.
Cryptosporidium
Genotypes in Wildlife from a New York Watershed
Authors: Feng Yaoyu , Alderisio Kerri A. , Yang Wenli , Blancero Lisa A. , Kuhne William G. , Nadareski Christopher A. , Reid Michael , Xiao Lihua
Published: August 2007

To identify the animal sources for
Cryptosporidium
contamination, we genotyped
Cryptosporidium
spp. in wildlife from the watershed of the New York City drinking water supply, using a small-subunit rRNA gene-based PCR-restriction fragment length polymorphism analysis and DNA sequencing. A total of 541 specimens from 38 species of wildlife were analyzed. One hundred and eleven (20.5%) of the wildlife specimens were PCR positive. Altogether, 21
Cryptosporidium
genotypes were found in wildlife samples, 11 of which were previously found in storm runoff in the watershed, and six of these 11 were from storm water genotypes of unknown animal origin. Four new genotypes were found, and the animal hosts for four storm water genotypes were expanded. With the exception of the cervine genotype, most genotypes were found in a limited number of animal species and have no major public health significance.
Comparison of Ergot Alkaloid Biosynthesis Gene Clusters in
Claviceps
Species Indicates Loss of Late Pathway Steps in Evolution of
C. fusiformis
Authors: Lorenz Nicole , Wilson Ella V. , Machado Caroline , Schardl Christopher L. , Tudzynski Paul
Published: August 2007

The grass parasites
Claviceps purpurea
and
Claviceps fusiformis
produce ergot alkaloids (EA) in planta and in submerged culture. Whereas EA synthesis (EAS) in
C. purpurea
proceeds via clavine intermediates to lysergic acid and the complex ergopeptines,
C. fusiformis
produces only agroclavine and elymoclavine. In
C. purpurea
the EAS gene (
EAS
) cluster includes
dmaW
(encoding the first pathway step),
cloA
(elymoclavine oxidation to lysergic acid), and the
lpsA/lpsB
genes (ergopeptine formation). We analyzed the corresponding
C. fusiformis EAS
cluster to investigate the evolutionary basis for chemotypic differences between the
Claviceps
species. Other than three peptide synthetase genes (
lpsC
and the tandem paralogues
lpsA1
and
lpsA2
), homologues of all
C. purpurea EAS
genes were identified in
C. fusiformis
, including homologues of
lpsB
and
cloA
, which in
C. purpurea
encode enzymes for steps after clavine synthesis. Rearrangement of the cluster was evident around
lpsB
, which is truncated in
C. fusiformis
. This and several frameshift mutations render Cf
lpsB
a pseudogene (Cf
lpsB

Ψ

). No obvious inactivating mutation was identified in Cf
cloA
. All
C. fusiformis EAS
genes, including Cf
lpsB

Ψ

and Cf
cloA
, were expressed in culture. Cross-complementation analyses demonstrated that Cf
cloA
and Cf
lpsB

Ψ

were expressed in
C. purpurea
but did not encode functional enzymes. In contrast, Cp
cloA
catalyzed lysergic acid biosynthesis in
C. fusiformis
, indicating that
C. fusiformis
terminates its EAS pathway at elymoclavine because the
cloA
gene product is inactive. We propose that the
C. fusiformis EAS
cluster evolved from a more complete cluster by loss of some
lps
genes and by rearrangements and mutations inactivating
lpsB
and
cloA
.
Zoo Animals as Reservoirs of Gram-Negative Bacteria Harboring Integrons and Antimicrobial Resistance Genes
Authors: Ahmed Ashraf M. , Motoi Yusuke , Sato Maiko , Maruyama Akito , Watanabe Hitoshi , Fukumoto Yukio , Shimamoto Tadashi
Published: August 2007

A total of 232 isolates of gram-negative bacteria were recovered from mammals, reptiles, and birds housed at Asa Zoological Park, Hiroshima prefecture, Japan. Forty-nine isolates (21.1%) showed multidrug resistance phenotypes and harbored at least one antimicrobial resistance gene. PCR and DNA sequencing identified class 1 and class 2 integrons and many β-lactamase-encoding genes, in addition to a novel AmpC β-lactamase gene,
bla
CMY-26
. Furthermore, the plasmid-mediated quinolone resistance genes
qnr
and
aac
(
6
′)-
Ib-cr
were also identified.
Fermentative Production ofl-Alanyl-l-Glutamine by a Metabolically EngineeredEscherichia coliStrain Expressingl-Amino Acid α-Ligase
Authors: Tabata Kazuhiko , Hashimoto Shin-ichi
Published: August 2007

In spite of its clinical and nutritional importance,l-alanyl-l-glutamine (Ala-Gln) has not been widely used due to the absence of an efficient manufacturing method. Here, we present a novel method for the fermentative production of Ala-Gln using anEscherichia colistrain expressingl-amino acid α-ligase (Lal), which catalyzes the formation of dipeptides by combining two amino acids in an ATP-dependent manner. Two metabolic manipulations were necessary for the production of Ala-Gln: reduction of dipeptide-degrading activity by combinatorial disruption of thedppandpepgenes and enhancement of the supply of substrate amino acids by deregulation of glutamine biosynthesis and overexpression of heterologousl-alanine dehydrogenase (Ald). Since expression of Lal was found to hamper cell growth, it was controlled using a stationary-phase-specific promoter. The final strain constructed was designated JKYPQ3 (pepA pepB pepD pepN dpp glnE glnB putA) containing pPE167 (lalandaldexpressed under the control of theuspApromoter) or pPE177 (lalandaldexpressed under the control of therpoHpromoter). Either strain produced more than 100 mM Ala-Gln extracellularly, in fed-batch cultivation on glucose-ammonium salt medium, without added alanine and glutamine. Because of the characteristics of Lal, no longer peptides (such as tripeptides) or dipeptides containingd-amino acids were formed.
Incubation of Environmental Samples in a Diffusion Chamber Increases the Diversity of Recovered Isolates
Authors: Bollmann Annette , Lewis Kim , Epstein Slava S.
Published: August 2007

The majority of microorganisms from natural environments cannot be grown in the laboratory. The diffusion-chamber-based approach is an alternative method that allows microorganisms to grow in their natural environment. An inoculum is sandwiched between semipermeable (0.03-μm-pore-size) membranes of the chamber, which is then returned to the source environment. The chamber allows for a free exchange of chemicals with the external milieu by diffusion while restricting the movement of cells. We used freshwater pond sediment to inoculate diffusion chambers and petri dishes. The diffusion chambers were incubated on top of the sediment for 4 weeks. Both chamber and petri dish cultivation resulted in the isolation of numerous representatives of
Alpha
-,
Beta
-, and
Gammaproteobacteria
;
Actinobacteria
;
Firmicutes
; and
Bacteroidetes
. However, the diffusion-chamber-based approach also led to the isolation of species from rarely cultivated groups, such as
Deltaproteobacteria, Verrucomicrobia, Spirochaetes
, and
Acidobacteria
. Material from the chambers was also transferred to new chambers in order to learn whether this will increase the recovery of isolates. Several isolates could be obtained only from material transferred through multiple diffusion chambers. This suggests that continuous cultivation in diffusion chambers adapts some microorganisms for growth under otherwise prohibitive in vitro conditions.
Isolation, Characterization, and Ecology of Sulfur-Respiring
Crenarchaea
Inhabiting Acid-Sulfate-Chloride-Containing Geothermal Springs in Yellowstone National Park
Authors: Boyd Eric S. , Jackson Robert A. , Encarnacion Gem , Zahn James A. , Beard Trevor , Leavitt William D. , Pi Yundan , Zhang Chuanlun L. , Pearson Ann , Geesey Gill G.
Published: August 2007

Elemental sulfur (S
0
) is associated with many geochemically diverse hot springs, yet little is known about the phylogeny, physiology, and ecology of the organisms involved in its cycling. Here we report the isolation, characterization, and ecology of two novel, S
0
-reducing
Crenarchaea
from an acid geothermal spring referred to as Dragon Spring. Isolate 18U65 grows optimally at 70 to 72°C and at pH 2.5 to 3.0, while isolate 18D70 grows optimally at 81°C and pH 3.0. Both isolates are chemoorganotrophs, dependent on complex peptide-containing carbon sources, S
0
, and anaerobic conditions for respiration-dependent growth. Glycerol dialkyl glycerol tetraethers (GDGTs) containing four to six cyclopentyl rings were present in the lipid fraction of isolates 18U65 and 18D70. Physiological characterization suggests that the isolates are adapted to the physicochemical conditions of Dragon Spring and can utilize the natural organic matter in the spring as a carbon and energy source. Quantitative PCR analysis of 16S rRNA genes associated with the S
0
flocs recovered from several acid geothermal springs using isolate-specific primers indicates that these two populations together represent 17 to 37% of the floc-associated DNA. The physiological characteristics of isolates 18U65 and 18D70 are consistent with their potential widespread distribution and putative role in the cycling of sulfur in acid geothermal springs throughout the Yellowstone National Park geothermal complex. Based on phenotypic and genetic characterization, the designations
Caldisphaera draconis
sp. nov. and
Acidilobus sulfurireducens
sp. nov. are proposed for isolates 18U65 and 18D70, respectively.
Genetic Screen for Regulatory Mutations in
Methanococcus maripaludis
and Its Use in Identification of Induction-Deficient Mutants of the Euryarchaeal Repressor NrpR
Authors: Lie Thomas J. , Leigh John A.
Published: August 2007

NrpR is an euryarchaeal transcriptional repressor of nitrogen assimilation genes. Previous studies with
Methanococcus maripaludis
demonstrated that NrpR binds to palindromic operator sequences, blocking transcription initiation. The metabolite 2-oxoglutarate, an indicator of cellular nitrogen deficiency, induces transcription by lowering the affinity of NrpR for operator DNA. In this report we build on existing genetic tools for
M. maripaludis
to develop a screen for change-of-function mutations in a transcriptional regulator and demonstrate the use of an X-Gal (5-bromo-4-chloro-3-indolyl-β-
d
-galactopyranoside) screen for strict anaerobes. We use the approach to address the primary structural requirements for the response of NrpR to 2-oxoglutarate.
nrpR
genes from the mesophilic
M. maripaludis
and the hyperthermophilic
Methanopyrus kandleri
were targeted for mutagenesis.
M. maripaludis nrpR
encodes a protein with two homologous NrpR domains while the
M. kandleri nrpR
homolog encodes a single NrpR domain. Random point mutagenesis and alanine replacement mutagenesis identified two amino acid residues of
M. kandleri
NrpR involved in induction of gene expression under nitrogen-deficient conditions and thus in the response to 2-oxoglutarate. Mutagenesis of the corresponding regions in either domain of
M. maripaludis
NrpR resulted in a similar effect, demonstrating a conserved structure-function relationship between the two repressors. The results indicate that in
M. maripaludis
, both NrpR domains participate in the 2-oxoglutarate response. The approach used here has wide adaptability to other regulatory systems in methanogenic
Archaea
and other strict anaerobes.
Inhibition of Fungal and Bacterial Plant Pathogens In Vitro and In Planta with Ultrashort Cationic Lipopeptides
Authors: Makovitzki Arik , Viterbo Ada , Brotman Yariv , Chet Ilan , Shai Yechiel
Published: August 2007

Plant diseases constitute an emerging threat to global food security. Many of the currently available antimicrobial agents for agriculture are highly toxic and nonbiodegradable and cause extended environmental pollution. Moreover, an increasing number of phytopathogens develop resistance to them. Recently, we have reported on a new family of ultrashort antimicrobial lipopeptides which are composed of only four amino acids linked to fatty acids (A. Makovitzki, D. Avrahami, and Y. Shai, Proc. Natl. Acad. Sci. USA 103:15997-16002, 2006). Here, we investigated the activities in vitro and in planta and the modes of action of these short lipopeptides against plant-pathogenic bacteria and fungi. They act rapidly, at low micromolar concentrations, on the membranes of the microorganisms via a lytic mechanism. In vitro microscopic analysis revealed wide-scale damage to the microorganism's membrane, in addition to inhibition of pathogen growth. In planta potent antifungal activity was demonstrated on cucumber fruits and leaves infected with the pathogen
Botrytis cinerea
as well as on corn leaves infected with
Cochliobolus heterostrophus
. Similarly, treatment with the lipopeptides of
Arabidopsis
leaves infected with the bacterial leaf pathogen
Pseudomonas syringae
efficiently and rapidly reduced the number of bacteria. Importantly, in contrast to what occurred with many native lipopeptides, no toxicity was observed on the plant tissues. These data suggest that the ultrashort lipopeptides could serve as native-like antimicrobial agents economically feasible for use in plant protection.
Seasonal Detection of Human Viruses and Coliphage in Newport Bay, California
Authors: Jiang Sunny C. , Chu Weiping , He Jian-Wen
Published: August 2007

Recent studies have shown that the fecal indicator bacteria (FIB) currently used to indicate water quality in the coastal environment may be inadequate to reflect human viral contamination. Coliphage was suggested as a better indicator of human viral pollution and was proposed by the U.S. EPA as an alternative indicator for fecal pollution in groundwater. In this study, we investigated the occurrence and distribution of FIB, F+ coliphage, and PCR-detectable human adenovirus and enterovirus for an entire year at 15 locations around the Newport Bay watershed, an important southern California estuary for water recreation and an ecological reserve. Peak concentrations and prevalences of FIB and F+ coliphage were associated with winter storms (wet weather). Human adenoviruses and enteroviruses, however, were detected by PCR in ∼5% of samples collected in the summer (dry weather) but only once in wet weather. These results demonstrated that FIB and coliphage have similar seasonal and freshwater-to-saltwater distribution patterns, while the detection of human viruses depends on a distribution pattern that is the opposite of that of FIB and coliphage. This research suggested that coliphage and FIB share similar environmental sources, while sources of human viruses in Newport Bay are perhaps different.
Cultivation-Independent Analysis of Fungal Genotypes in Soil by Using Simple Sequence Repeat Markers
Authors: Schwarzenbach Kaspar , Widmer Franco , Enkerli Jürg
Published: August 2007

Cultivation-independent analyses of fungi are used for community profiling as well as identification of specific strains in environmental samples. The objective of the present study was to adapt genotyping based on simple sequence repeat (SSR) marker detection for use in cultivation-independent monitoring of fungal species or strains in bulk soil DNA. As a model system, a fungal biocontrol agent (BCA) based on
Beauveria brongniartii
, for which six SSR markers have been developed, was used. Species specificity of SSR detection was verified with 15 fungal species. Real-time PCR was used to adjust for different detection sensitivities of the six SSR markers as well as for different template quantities. The limit for reliable detection per PCR assay was below 2 pg target DNA, corresponding to an estimated 45 genome copies of
B. brongniartii
. The cultivation-independent approach was compared to cultivation-dependent SSR analysis with soil samples from a
B. brongniartii
BCA-treated field plot. Results of the cultivation-independent method were consistent with cultivation-dependent genotyping and allowed for unambiguous identification and differentiation of the applied as well as indigenous strains in the samples. Due to the larger quantities of soil used for cultivation-dependent analysis, its sensitivity was higher, but cultivation-independent SSR genotyping was much faster. Therefore, cultivation-independent monitoring of
B. brongniartii
based on multiple SSR markers represents a rapid and strain-specific approach. This strategy may also be applicable to other fungal species or strains for which SSR markers have been developed.
Identification ofN-Acetylhexosamine 1-Kinase in the Complete Lacto-N-Biose I/Galacto-N-Biose Metabolic Pathway inBifidobacterium longum
Authors: Nishimoto Mamoru , Kitaoka Motomitsu
Published: August 2007

We have determined the functions of the enzymes encoded by thelnpB, lnpC, andlnpDgenes, located downstream of the lacto-N-biose phosphorylase gene (lnpA), inBifidobacterium longumJCM1217. ThelnpBgene encodes a novel kinase,N-acetylhexosamine 1-kinase, which producesN-acetylhexosamine 1-phosphate; thelnpCgene encodes UDP-glucose hexose 1-phosphate uridylyltransferase, which is also active onN-acetylhexosamine 1-phosphate; and thelnpDgene encodes a UDP-glucose 4-epimerase, which is active on both UDP-galactose and UDP-N-acetylgalactosamine. These results suggest that the gene operonlnpABCDencodes a previously undescribed lacto-N-biose I/galacto-N-biose metabolic pathway that is involved in the intestinal colonization of bifidobacteria and that utilizes lacto-N-biose I from human milk oligosaccharides or galacto-N-biose from mucin sugars.
Rhizobial Factors Required for Stem Nodule Maturation and Maintenance in
Sesbania rostrata-Azorhizobium caulinodans
ORS571 Symbiosis
Authors: Suzuki Shino , Aono Toshihiro , Lee Kyung-Bum , Suzuki Tadahiro , Liu Chi-Te , Miwa Hiroki , Wakao Seiji , Iki Taichiro , Oyaizu Hiroshi
Published: August 2007

The molecular and physiological mechanisms behind the maturation and maintenance of N
2
-fixing nodules during development of symbiosis between rhizobia and legumes still remain unclear, although the early events of symbiosis are relatively well understood.
Azorhizobium caulinodans
ORS571 is a microsymbiont of the tropical legume
Sesbania rostrata
, forming N
2
-fixing nodules not only on the roots but also on the stems. In this study, 10,080 transposon-inserted mutants of
A. caulinodans
ORS571 were individually inoculated onto the stems of
S. rostrata
, and those mutants that induced ineffective stem nodules, as displayed by halted development at various stages, were selected. From repeated observations on stem nodulation, 108 Tn
5
mutants were selected and categorized into seven nodulation types based on size and N
2
fixation activity. Tn
5
insertions of some mutants were found in the well-known nodulation, nitrogen fixation, and symbiosis-related genes, such as
nod, nif
, and
fix
, respectively, lipopolysaccharide synthesis-related genes, C
4
metabolism-related genes, and so on. However, other genes have not been reported to have roles in legume-rhizobium symbiosis. The list of newly identified symbiosis-related genes will present clues to aid in understanding the maturation and maintenance mechanisms of nodules.
mariner
-Based Transposon Mutagenesis of
Rickettsia prowazekii
Authors: Liu Zhi-Mei , Tucker Aimee M. , Driskell Lonnie O. , Wood David O.
Published: August 2007

Rickettsia prowazekii
, the causative agent of epidemic typhus, is an obligate intracellular bacterium that grows directly within the cytoplasm of its host cell, unbounded by a vacuolar membrane. The obligate intracytoplasmic nature of rickettsial growth places severe restrictions on the genetic analysis of this distinctive human pathogen. In order to expand the repertoire of genetic tools available for the study of this pathogen, we have employed the versatile
mariner
-based,
Himar1
transposon system to generate insertional mutants of
R. prowazekii
. A transposon containing the
R. prowazekii arr-2
rifampin resistance gene and a gene coding for a green fluorescent protein (GFP
UV
) was constructed and placed on a plasmid expressing the
Himar1
transposase. Electroporation of this plasmid into
R. prowazekii
resulted in numerous transpositions into the rickettsial genome. Transposon insertion sites were identified by rescue cloning, followed by DNA sequencing. Random transpositions integrating at TA sites in both gene coding and intergenic regions were identified. Individual rickettsial clones were isolated by the limiting-dilution technique. Using both fixed and live-cell techniques,
R. prowazekii
transformants expressing GFP
UV
were easily visible by fluorescence microscopy. Thus, a
mariner
-based system provides an additional mechanism for generating rickettsial mutants that can be screened using GFP
UV
fluorescence.
Severe Tryptophan Starvation Blocks Onset of Conventional Persistence and Reduces Reactivation of
Chlamydia trachomatis
Authors: Leonhardt Ralf M. , Lee Seung-Joon , Kavathas Paula B. , Cresswell Peter
Published: August 2007

The intracellular survival of the bacterial pathogen
Chlamydia trachomatis
depends on protein synthesis by the microbe soon after internalization. Pharmacologic inhibition of bacterial translation inhibits early trafficking of the parasitophorous vacuole (inclusion) to the microtubule-organizing center (MTOC) and promotes its fusion with lysosomes, which is normally blocked by
Chlamydia
. Depletion of cellular tryptophan pools by gamma interferon-inducible indoleamine-2,3-dioxygenase (IDO) is believed to be the major innate immune mechanism controlling
C. trachomatis
infection in human cells, an action to which the bacteria can respond by converting into a nonreplicating but highly reactivatable persistent state. However, whether severe IDO-mediated tryptophan starvation can be sufficient to fully arrest the chlamydial life cycle and thereby counteract the onset of persistence is unknown. Here we demonstrate that at low exogenous tryptophan concentrations a substantial fraction of
C. trachomatis
bacteria fail to traffic to the MTOC or to switch into the conventional persistent state in gamma interferon-induced human cells. The organisms stay scattered in the cell periphery, do not retain infectivity, and display only low transcriptional activity. Importantly, the rate at which these aberrant
Chlamydia
bacteria become reactivated upon replenishment of cellular tryptophan pools is substantially lower. Thus, severe tryptophan depletion in cells with high IDO activity affects chlamydial development more rigorously than previously described.
Inhibition of Staphylococcal Biofilm Formation by Nitrite
Authors: Schlag Steffen , Nerz Christiane , Birkenstock Timo A. , Altenberend Florian , Götz Friedrich
Published: August 2007

Several environmental stresses have been demonstrated to increase polysaccharide intercellular adhesin (PIA) synthesis and biofilm formation by the human pathogens
Staphylococcus aureus
and
Staphylococcus epidermidis
. In this study we characterized an adaptive response of
S. aureus
SA113 to nitrite-induced stress and show that it involves concomitant impairment of PIA synthesis and biofilm formation. Transcriptional analysis provided evidence that nitrite, either as the endogenous product of respiratory nitrate reduction or after external addition, causes repression of the
icaADBC
gene cluster, mediated likely by IcaR. Comparative microarray analysis revealed a global change in gene expression during growth in the presence of 5 mM sodium nitrite and indicated a response to oxidative and nitrosative stress. Many nitrite-induced genes are involved in DNA repair, detoxification of reactive oxygen and nitrogen species, and iron homeostasis. Moreover, preformed biofilms could be eradicated by the addition of nitrite, likely the result of the formation of toxic acidified nitrite derivatives. Nitrite-mediated inhibition of
S. aureus
biofilm formation was abrogated by the addition of nitric oxide (NO) scavengers, suggesting that NO is directly or indirectly involved. Nitrite also repressed biofilm formation of
S. epidermidis
RP62A.
A TolC-Like Protein Is Required for Heterocyst Development inAnabaenasp. Strain PCC 7120
Authors: Moslavac Suncana , Nicolaisen Kerstin , Mirus Oliver , Al Dehni Fadi , Pernil Rafael , Flores Enrique , Maldener Iris , Schleiff Enrico
Published: August 2007

The filamentous cyanobacteriumAnabaenasp. strain PCC 7120 forms heterocysts in a semiregular pattern when it is grown on N2as the sole nitrogen source. The transition from vegetative cells to heterocysts requires marked metabolic and morphological changes. We show that a trimeric pore-forming outer membrane β-barrel protein belonging to the TolC family, Alr2887, is up-regulated in developing heterocysts and is essential for diazotrophic growth. Mutants defective in Alr2887 did not form the specific glycolipid layer of the heterocyst cell wall, which is necessary to protect nitrogenase from external oxygen. Comparison of the glycolipid contents of wild-type and mutant cells indicated that the protein is not involved in the synthesis of glycolipids but might instead serve as an exporter for the glycolipid moieties or enzymes involved in glycolipid attachment. We propose that Alr2887, together with an ABC transporter like DevBCA, is part of a protein export system essential for assembly of the heterocyst glycolipid layer. We designate the alr2887 genehgdD(heterocystglycolipiddeposition protein).
Isolated Poly(3-Hydroxybutyrate) (PHB) Granules Are Complex Bacterial Organelles Catalyzing Formation of PHB from Acetyl Coenzyme A (CoA) and Degradation of PHB to Acetyl-CoA
Authors: Uchino Keiichi , Saito Terumi , Gebauer Birgit , Jendrossek Dieter
Published: August 2007

Poly(3-hydroxybutyrate) (PHB) granules isolated in native form (nPHB granules) from
Ralstonia eutropha
catalyzed formation of PHB from
14
C-labeled acetyl coenzyme A (CoA) in the presence of NADPH and concomitantly released CoA, revealing that PHB biosynthetic proteins (acetoacetyl-CoA thiolase, acetoacetyl-CoA reductase, and PHB synthase) are present and active in isolated nPHB granules in vitro. nPHB granules also catalyzed thiolytic cleavage of PHB in the presence of added CoA, resulting in synthesis of 3-hydroxybutyryl-CoA (3HB-CoA) from PHB. Synthesis of 3HB-CoA was also shown by incubation of artificial (protein-free) PHB with CoA and PhaZa1, confirming that PhaZa1 is a PHB depolymerase catalyzing the thiolysis reaction. Acetyl-CoA was the major product detectable after incubation of nPHB granules in the presence of NAD
+
, indicating that downstream mobilizing enzyme activities were also present and active in isolated nPHB granules. We propose that intracellular concentrations of key metabolites (CoA, acetyl-CoA, 3HB-CoA, NAD
+
/NADH) determine whether a cell accumulates or degrades PHB. Since the degradation product of PHB is 3HB-CoA, the cells do not waste energy by synthesis and degradation of PHB. Thus, our results explain the frequent finding of simultaneous synthesis and breakdown of PHB.
Involvement of the Detoxifying Enzyme Lactoylglutathione Lyase in
Streptococcus mutans
Aciduricity
Authors: Korithoski Bryan , Lévesque Céline M. , Cvitkovitch Dennis G.
Published: August 2007

Streptococcus mutans
, a normal inhabitant of dental plaque, is considered a primary etiological agent of dental caries. Its main virulence factors are acidogenicity and aciduricity, the abilities to produce acid and to survive and grow at low pH, respectively. Metabolic processes are finely regulated following acid exposure in
S. mutans
. Proteome analysis of
S. mutans
demonstrated that lactoylglutathione lyase (LGL) was up-regulated during acid challenge. The LGL enzyme catalyzes the conversion of toxic methylglyoxal, derived from glycolysis, to
S
-
d
-lactoylglutathione. Methylglyoxal inhibits the growth of cells in all types of organisms. The current study aimed to investigate the relationship between LGL and aciduricity and acidogenicity in
S. mutans
. An
S. mutans
isogenic mutant defective in
lgl
(LGLKO) was created, and its growth kinetics were characterized. Insertional inactivation of
lgl
resulted in an acid-sensitive phenotype. However, the glycolytic rate at pH 5.0 was greater for LGLKO than for
S. mutans
UA159 wild-type cells. LGL was involved in the detoxification of methylglyoxal, illustrated by the absence of enzyme activity in LGLKO and the hypersensitivity of LGLKO to methylglyoxal, compared with UA159 (MIC of 3.9 and 15.6 mM, respectively). Transcriptional analysis of
lgl
conducted by quantitative real-time PCR revealed that
lgl
was up-regulated (approximately sevenfold) during the exponential growth phase compared with that in the stationary growth phase. Gene expression studies conducted at low pH demonstrated that
lgl
was induced during acidic growth (∼3.5-fold) and following acid adaptation (∼2-fold).This study demonstrates that in
S. mutans
, LGL functions in the detoxification of methylglyoxal, resulting in increased aciduricity.
Epsilon-Toxin Plasmids of
Clostridium perfringens
Type D Are Conjugative
Authors: Hughes Meredith L. , Poon Rachael , Adams Vicki , Sayeed Sameera , Saputo Juliann , Uzal Francisco A. , McClane Bruce A. , Rood Julian I.
Published: August 2007

Isolates of
Clostridium perfringens
type D produce the potent epsilon-toxin (a CDC/U.S. Department of Agriculture overlap class B select agent) and are responsible for several economically significant enterotoxemias of domestic livestock. It is well established that the epsilon-toxin structural gene,
etx
, occurs on large plasmids. We show here that at least two of these plasmids are conjugative. The
etx
gene on these plasmids was insertionally inactivated using a chloramphenicol resistance cassette to phenotypically tag the plasmid. High-frequency conjugative transfer of the tagged plasmids into the
C. perfringens
type A strain JIR325 was demonstrated, and the resultant transconjugants were shown to act as donors in subsequent mating experiments. We also demonstrated the transfer of “unmarked” native ε-toxin plasmids into strain JIR325 by exploiting the high transfer frequency. The transconjugants isolated in these experiments expressed functional ε-toxin since their supernatants had cytopathic effects on MDCK cells and were toxic in mice. Using the widely accepted multiplex PCR approach for toxin genotyping, these type A-derived transconjugants were genotypically type D. These findings have significant implications for the
C. perfringens
typing system since it is based on the toxin profile of each strain. Our study demonstrated the fluid nature of the toxinotypes and their dependence upon the presence or absence of toxin plasmids, some of which have for the first time been shown to be conjugative.
TcpA, an FtsK/SpoIIIE Homolog, Is Essential for Transfer of the Conjugative Plasmid pCW3 inClostridium perfringens
Authors: Parsons Jennifer A. , Bannam Trudi L. , Devenish Rodney J. , Rood Julian I.
Published: August 2007

The conjugative tetracycline resistance plasmid pCW3 is the paradigm conjugative plasmid in the anaerobic gram-positive pathogenClostridium perfringens. Two closely related FtsK/SpoIIIE homologs, TcpA and TcpB, are encoded on pCW3, which is significant since FtsK domains are found in coupling proteins of gram-negative conjugation systems. To develop an understanding of the mechanism of conjugative transfer inC. perfringens, we determined the role of these proteins in the conjugation process. Mutation and complementation analysis was used to show that thetcpAgene was essential for the conjugative transfer of pCW3 and that thetcpBgene was not required for transfer. Furthermore, complementation of a pCW3ΔtcpAmutant with divergenttcpAhomologs provided experimental evidence that all of the known conjugative plasmids fromC. perfringensuse a similar transfer mechanism. Functional genetic analysis of the TcpA protein established the essential role in conjugative transfer of its Walker A and Walker B ATP-binding motifs and its FtsK-like RAAG motif. It is postulated that TcpA is the essential DNA translocase or coupling protein encoded by pCW3 and as such represents a key component of the unique conjugation process inC. perfringens.
Identification and Characterization of TriABC-OpmH, a Triclosan Efflux Pump of
Pseudomonas aeruginosa
Requiring Two Membrane Fusion Proteins
Authors: Mima Takehiko , Joshi Swati , Gomez-Escalada Margarita , Schweizer Herbert P.
Published: August 2007

Pseudomonas aeruginosa
achieves high-level (MIC > 1 mg/ml) triclosan resistance either by constitutive expression of MexAB-OprM, an efflux pump of the resistance nodulation cell division (RND) family, or expression of MexCD-OprJ, MexEF-OprN, and MexJK-OpmH in regulatory mutants. A triclosan-resistant target enzyme and perhaps other mechanisms probably act synergistically with efflux. To probe this notion, we exposed the susceptible Δ(
mexAB-oprM
) Δ(
mexCD-oprJ
) Δ(
mexEF-oprN
) Δ(
mexJK
) Δ(
mexXY
) strain PAO509 to increasing triclosan concentrations and derived a resistant strain, PAO509.5. This mutant overexpressed the PA0156-PA0157-PA0158 pump, which only effluxed triclosan, but not closely related compounds, antibiotics, and divalent cations, and was therefore renamed TriABC. Constitutive expression of the
triABC
operon was due to a single promoter-up mutation. Deletion of two adjacent genes,
pcaR
and
PA0159
, encoding transcriptional regulators had no effect on expression of this operon. TriABC is the only
P. aeruginosa
RND pump which contains two membrane fusion proteins, TriA and TriB, and both are required for efflux pump function. Probably owing to tight transcriptional coupling of the
triABC
genes, complementation of individual mutations was only partially achievable. Full complementation was only observed when a complete
triABC
operon was provided in
trans
, either in single or multiple copies. TriABC associated with OpmH, but not OprM, for assembly of a functional triclosan efflux pump. TriABC is the fifth RND pump in
P. aeruginosa
shown to efficiently efflux triclosan, supporting the notion that efflux is the primary mechanism responsible for this bacterium's high intrinsic and acquired triclosan resistance.
Corynebacterium diphtheriae
: Identification and Characterization of a Channel-Forming Protein in the Cell Wall
Authors: Schiffler Bettina , Barth Enrico , Daffé Mamadou , Benz Roland
Published: August 2007

The cell wall fraction of the gram-positive, nontoxic
Corynebacterium diphtheriae
strain C8
r
(−) Tox
−
(= ATCC 11913) contained a channel-forming protein, as judged from reconstitution experiments with artificial lipid bilayer experiments. The channel-forming protein was present in detergent-treated cell walls and in extracts of whole cells obtained using organic solvents. The protein had an apparent molecular mass of about 66 kDa as determined on Tricine-containing sodium dodecyl sulfate-polyacrylamide gel electrophoresis gels and consisted of subunits having a molecular mass of about 5 kDa. Single-channel experiments with the purified protein suggested that the protein formed channels with a single-channel conductance of 2.25 nS in 1 M KCl. Further single-channel analysis suggested that the cell wall channel is wide and water filled because it has only slight selectivity for cations over anions and its conductance followed the mobility sequence of cations and anions in the aqueous phase. Antibodies raised against PorA, the subunit of the cell wall channel of
Corynebacterium glutamicum
, detected both monomers and oligomers of the isolated protein, suggesting that there are highly conserved epitopes in the cell wall channels of
C. diphtheriae
and PorA. Localization of the protein on the cell surface was confirmed by an enzyme-linked immunosorbent assay. The prospective homology of PorA with the cell wall channel of
C. diphtheriae
was used to identify the cell wall channel gene,
cdporA
, in the known genome of
C. diphtheriae
. The gene and its flanking regions were cloned and sequenced. CdporA is a protein that is 43 amino acids long and does not have a leader sequence.
cdporA
was expressed in a
C. glutamicum
strain that lacked the major outer membrane channels PorA and PorH. Organic solvent extracts of the transformed cells formed in lipid bilayer membranes the same channels as the purified CdporA protein of
C. diphtheriae
formed, suggesting that the expressed protein is able to complement the PorA and PorH deficiency of the
C. glutamicum
strain. The study is the first report of a cell wall channel in a pathogenic
Corynebacterium
strain.
The Peptidyl-Prolyl Isomerase Activity of SlyD Is Not Required for Maturation of
Escherichia coli
Hydrogenase
Authors: Zhang Jie Wei , Leach Michael R. , Zamble Deborah B.
Published: August 2007

Escherichia coli
SlyD, which is involved in the biosynthesis of the metal cluster in the [NiFe]-hydrogenase enzymes, exhibits several activities including that of a peptidyl-prolyl isomerase (PPIase). Mutations that result in deficient PPIase activity do not produce corresponding decreases in the other activities of SlyD in vitro or in hydrogenase production levels in vivo.
New Locus Important for
Myxococcus
Social Motility and Development
Authors: Zhang Cui-ying , Cai Ke , Liu Hong , Zhang Yong , Pan Hong-wei , Wang Bing , Wu Zhi-hong , Hu Wei , Li Yue-zhong
Published: August 2007

The
mts
locus in salt-tolerant
Myxococcus fulvus
HW-1 was found to be critical for gliding motility, fruiting-body formation, and sporulation. The homologous genes in
Myxococcus xanthus
are also important for social motility and fruiting-body development. The
mts
genes were determined to be involved in cell-cell cohesion in both myxobacterial species.
Activation of
yeaR-yoaG
Operon Transcription by the Nitrate-Responsive Regulator NarL Is Independent of Oxygen- Responsive Regulator Fnr in
Escherichia coli
K-12
Authors: Lin Hsia-Yin , Bledsoe Peggy J. , Stewart Valley
Published: August 2007

The facultative aerobe
Escherichia coli
K-12 can use respiratory nitrate ammonification to generate energy during anaerobic growth. The toxic compound nitric oxide is a by-product of this metabolism. Previous transcript microarray studies identified the
yeaR-yoaG
operon, encoding proteins of unknown function, among genes whose transcription is induced in response to nitrate, nitrite, or nitric oxide. Nitrate and nitrite regulate anaerobic respiratory gene expression through the NarX-NarL and NarQ-NarP two-component systems. All known Nar-activated genes also require the oxygen-responsive Fnr transcription activator. However, previous studies indicated that
yeaR-yoaG
operon transcription does not require Fnr activation. Here, we report results from mutational analyses demonstrating that
yeaR
-
yoaG
operon transcription is activated by phospho-NarL protein independent of the Fnr protein. The phospho-NarL protein binding site is centered at position −43.5 with respect to the transcription initiation site. Expression from the
Shewanella oneidensis
MR-1
nnrS
gene promoter, cloned into
E. coli
, similarly was activated by phospho-NarL protein independent of the Fnr protein. Recently,
yeaR-yoaG
operon transcription was shown to be regulated by the nitric oxide-responsive NsrR repressor (N. Filenko et al., J. Bacteriol. 189:4410-4417, 2007). Our mutational analyses reveal the individual contributions of the Nar and NsrR regulators to overall
yeaR-yoaG
operon expression and document the NsrR operator centered at position −32. Thus, control of
yeaR-yoaG
operon transcription provides an example of overlapping regulation by nitrate and nitrite, acting through the Nar regulatory system, and nitric oxide, acting through the NsrR repressor.
Cobalt Targets Multiple Metabolic Processes in
Salmonella enterica
Authors: Thorgersen Michael P. , Downs Diana M.
Published: August 2007

Cobalt is essential for growth of
Salmonella enterica
and other organisms, yet this metal can be toxic when present in excess. Wild-type
Salmonella
exhibits several metabolic defects when grown in the presence of cobalt, some of which generate visible growth consequences. Work herein identifies sulfur assimilation, iron homeostasis, and Fe-S cluster metabolism as targets for cobalt toxicity. In each case it is proposed that cobalt exerts its effect by one of two mechanisms: direct competition with iron or indirectly through a mechanism that involves the status of reduced thiols in the cell. Cobalt toxicity results in decreased siroheme production, increased expression of the Fur regulon, and decreased activity of Fe-S cluster proteins. The consequences of reduced sulfite reductase activity in particular are exacerbated by the need for glutathione in cobalt resistance. Significantly, independent metabolic perturbations could be detected at cobalt concentrations below those required to generate a detectable growth defect.