bluuebunny/crossref_metadata_2025 huggingface dataset:
RNA Arbitrarily Primed PCR and Fourier Transform Infrared Spectroscopy Reveal Plasticity in the Acid Tolerance Response ofStreptococcus macedonicus
Authors: Papadimitriou Konstantinos , Boutou Effrossyni , Zoumpopoulou Georgia , Tarantilis Petros A. , Polissiou Moschos , Vorgias Constantinos E. , Tsakalidou Effie
Published: August 2008

We have previously reported that an acid tolerance response (ATR) can be induced inStreptococcus macedonicuscells at mid-log phase after autoacidification, transient exposure to acidic pH, or acid habituation, as well as at stationary phase. Here, we compared the transcriptional profiles of these epigenetic phenotypes, by RNA arbitrarily primed PCR (RAP-PCR), and their whole-cell chemical compositions, by Fourier transform infrared spectroscopy (FT-IR). RAP-PCR fingerprints revealed significant differences among the phenotypes, indicating that gene expression during the ATR is influenced not only by the growth phase but also by the treatments employed to induce the response. The genes coding for the mannose-specific IID component, the 1,2-diacylglycerol 3-glucosyltransferase, the 3-oxoacyl-acyl carrier protein, the large subunit of carbamoyl-phosphate synthase, and a hypothetical protein were found to be induced at least under some of the acid-adapting conditions. Furthermore, principal component analysis of the second-derivative-transformed FT-IR spectra segregatedS. macedonicusphenotypes individually in all spectral regions that are characteristic for major cellular constituents like the polysaccharides of the cell wall, fatty acids of the cell membrane, proteins, and other compounds that absorb in these regions. These findings provide evidence for major changes in cellular composition due to acid adaptation that were clearly different to some extent among the phenotypes. Overall, our data demonstrate the plasticity in the ATR ofS. macedonicus, which reflects the inherent ability of the bacterium to adjust the response to the distinctiveness of the imposed stress condition, probably to maximize its adaptability.
Changes in Bacterial Denitrifier Community Abundance over Time in an Agricultural Field and Their Relationship with Denitrification Activity
Authors: Dandie Catherine E. , Burton David L. , Zebarth Bernie J. , Henderson Sherri L. , Trevors Jack T. , Goyer Claudia
Published: August 2008

This study measured total bacterial and denitrifier community abundances over time in an agricultural soil cropped to potatoes (
Solanum tuberosum
L.) by using quantitative PCR. Samples were collected on 10 dates from spring to autumn and from three spatial locations: in the potato “hill” between plants (H), close to the plant (H
p
), and in the “furrow” (F). The denitrification rates, N
2
O emissions, and environmental parameters were also measured. Changes in denitrifier abundance over time and spatial location were small (1.7- to 2.7-fold for the
nirK
,
nosZ
, and
cnorB
B
guilds), whereas the
cnorB
P
community (
Pseudomonas mandelii
and closely related spp.) showed an ∼4.6-fold change. The seasonal patterns of denitrifier gene numbers varied with the specific community: lower
nosZ
gene numbers in April and May than in June and July, higher
cnorB
P
gene numbers in May and June than in March and April and September and November, higher
nirK
gene numbers in early spring than in late autumn, and no change in
cnorB
B
gene numbers. Gene numbers were higher for the H
p
than the H location for the
nosZ
and
nirK
communities and for the
cnorB
P
community on individual dates, presumably indicating an effect of the plant on denitrifier abundance. Higher
cnorB
P
gene numbers for the H location than the F location and for
nosZ
and
cnorB
B
on individual dates reflect the effect of spatial location on abundance. Denitrifier abundance changes were not related to any environmental parameter, although a weak relationship exists between
cnorB
P
gene numbers, extractable organic carbon values, and temperature. Denitrification and N
2
O emissions were mostly regulated by inorganic nitrogen availability and water-filled pore space but were uncoupled from denitrifier community abundances measured in this system.
Bacterial Community in the Crop of the Hoatzin, a Neotropical Folivorous Flying Bird
Authors: Godoy-Vitorino Filipa , Ley Ruth E. , Gao Zhan , Pei Zhiheng , Ortiz-Zuazaga Humberto , Pericchi Luis R. , Garcia-Amado Maria A. , Michelangeli Fabian , Blaser Martin J. , Gordon Jeffrey I. , Domínguez-Bello Maria G.
Published: August 2008

The hoatzin is unique among known avian species because of the fermentative function of its enlarged crop. A small-bodied flying foregut fermenter is a paradox, and this bird provides an interesting model to examine how diet selection and the gut microbiota contribute to maximizing digestive efficiency. Therefore, we characterized the bacterial population in the crop of six adult hoatzins captured from the wild. A total of 1,235 16S rRNA gene sequences were grouped into 580 phylotypes (67% of the pooled species richness sampled, based on Good's coverage estimator, with CACEand Chao1 estimates of 1,709 and 1,795 species-level [99% identity] operational taxonomic units, respectively). Members of 9 of the ∼75 known phyla inBacteriawere identified in this gut habitat; theFirmicuteswere dominant (67% of sequences, belonging to the classesClostridia,Mollicutes, andBacilli), followed by theBacteroidetes(30%, mostly in the orderBacteroidales),Proteobacteria(1.8%), andLentisphaerae,Verrucomicrobia, TM7,Spirochaetes,Actinobacteria, andAminanaerobia(all <0.1%). The novelty in this ecosystem is great; 94% of the phylotypes were unclassified at the “species” level and thus likely include novel cellulolytic lineages.
The Obligate Mutualist
Wigglesworthia glossinidia
Influences Reproduction, Digestion, and Immunity Processes of Its Host, the Tsetse Fly
Authors: Pais Roshan , Lohs Claudia , Wu Yineng , Wang Jingwen , Aksoy Serap
Published: August 2008

Tsetse flies (Diptera: Glossinidae) are vectors for trypanosome parasites, the agents of the deadly sleeping sickness disease in Africa. Tsetse also harbor two maternally transmitted enteric mutualist endosymbionts: the primary intracellular obligate
Wigglesworthia glossinidia
and the secondary commensal
Sodalis glossinidius
. Both endosymbionts are transmitted to the intrauterine progeny through the milk gland secretions of the viviparous female. We administered various antibiotics either continuously by per os supplementation of the host blood meal diet or discretely by hemocoelic injections into fertile females in an effort to selectively eliminate the symbionts to study their individual functions. A symbiont-specific PCR amplification assay and fluorescence in situ hybridization analysis were used to evaluate symbiont infection outcomes. Tetracycline and rifampin treatments eliminated all tsetse symbionts but reduced the fecundity of the treated females. Ampicillin treatments did not affect the intracellular
Wigglesworthia
localized in the bacteriome organ and retained female fecundity. The resulting progeny of ampicillin-treated females, however, lacked
Wigglesworthia
but still harbored the commensal
Sodalis
. Our results confirm the presence of two physiologically distinct
Wigglesworthia
populations: the bacteriome-localized
Wigglesworthia
involved with nutritional symbiosis and free-living
Wigglesworthia
in the milk gland organ responsible for maternal transmission to the progeny. We evaluated the reproductive fitness, longevity, digestion, and vectorial competence of flies that were devoid of
Wigglesworthia
. The absence of
Wigglesworthia
completely abolished the fertility of females but not that of males. Both the male and female
Wigglesworthia
-free adult progeny displayed longevity costs and were significantly compromised in their blood meal digestion ability. Finally, while the vectorial competence of the young newly hatched adults without
Wigglesworthia
was comparable to that of their wild-type counterparts, older flies displayed higher susceptibility to trypanosome infections, indicating a role for the mutualistic symbiosis in host immunobiology. The ability to rear adult tsetse that lack the obligate
Wigglesworthia
endosymbionts will now enable functional investigations into this ancient symbiosis.
Characterization of the Digestive-Tract Microbiota of
Hirudo orientalis
, a European Medicinal Leech
Authors: Laufer Alison S. , Siddall Mark E. , Graf Joerg
Published: August 2008

FDA-approved, postoperative use of leeches can lead to bacterial infections. In this study, we used culture-dependent and culture-independent approaches to characterize the digestive-tract microbiota of
Hirudo orientalis
. Surprisingly, two
Aeromonas
species,
A. veronii
and
A. jandaei
, were cultured. Uncultured
Rikenella
-like bacteria were most similar to isolates from
Hirudo verbana
.
Dynamics of Whole and Lysed Bacterial Cells during Parmigiano-Reggiano Cheese Production and Ripening
Authors: Gatti Monica , De Dea Lindner Juliano , De Lorentiis Angela , Bottari Benedetta , Santarelli Marcela , Bernini Valentina , Neviani Erasmo
Published: August 2008

Microbial succession during Parmigiano-Reggiano cheesemaking was monitored by length heterogeneity PCR (LH-PCR), considering the intact and lysed cells at different stages of cheese production and ripening. When starter species underwent autolysis, species coming from milk were able to grow. For the first time, the LH-PCR technique was applied to study a fermented food.
Insertional Inactivation of Branched-Chain α-Keto Acid Dehydrogenase in
Staphylococcus aureus
Leads to Decreased Branched-Chain Membrane Fatty Acid Content and Increased Susceptibility to Certain Stresses
Authors: Singh Vineet K. , Hattangady Dipti S. , Giotis Efstathios S. , Singh Atul K. , Chamberlain Neal R. , Stuart Melissa K. , Wilkinson Brian J.
Published: August 2008

Staphylococcus aureus
is a major community and nosocomial pathogen. Its ability to withstand multiple stress conditions and quickly develop resistance to antibiotics complicates the control of staphylococcal infections. Adaptation to lower temperatures is a key for the survival of bacterial species outside the host. Branched-chain α-keto acid dehydrogenase (BKD) is an enzyme complex that catalyzes the early stages of branched-chain fatty acid (BCFA) production. In this study, BKD was inactivated, resulting in reduced levels of BCFAs in the membrane of
S. aureus
. Growth of the BKD-inactivated mutant was progressively more impaired than that of wild-type
S. aureus
with decreasing temperature, to the point that the mutant could not grow at 12°C. The growth of the mutant was markedly stimulated by the inclusion of 2-methylbutyrate in the growth medium at all temperatures tested. 2-Methylbutyrate is a precursor of odd-numbered anteiso fatty acids and bypasses BKD. Interestingly, growth of wild-type
S. aureus
was also stimulated by including 2-methylbutyrate in the medium, especially at lower temperatures. The anteiso fatty acid content of the BKD-inactivated mutant was restored by the inclusion of 2-methylbutyrate in the medium. Fluorescence polarization measurements indicated that the membrane of the BKD-inactivated mutant was significantly less fluid than that of wild-type
S. aureus
. Consistent with this result, the mutant showed decreased toluene tolerance that could be increased by the inclusion of 2-methylbutyrate in the medium. The BKD-inactivated mutant was more susceptible to alkaline pH and oxidative stress conditions. Inactivation of the BKD enzyme complex in
S. aureus
also led to a reduction in adherence of the mutant to eukaryotic cells and its survival in a mouse host. In addition, the mutant offers a tool to study the role of membrane fluidity in the interaction of
S. aureus
with antimicrobial substances.
Removal of Antibiotic Resistance Gene-Carrying Plasmids from
Lactobacillus reuteri
ATCC 55730 and Characterization of the Resulting Daughter Strain,
L
.
reuteri
DSM 17938
Authors: Rosander Anna , Connolly Eamonn , Roos Stefan
Published: August 2008

The spread of antibiotic resistance in pathogens is primarily a consequence of the indiscriminate use of antibiotics, but there is concern that food-borne lactic acid bacteria may act as reservoirs of antibiotic resistance genes when distributed in large doses to the gastrointestinal tract.
Lactobacillus reuteri
ATCC 55730 is a commercially available probiotic strain which has been found to harbor potentially transferable resistance genes. The aims of this study were to define the location and nature of β-lactam, tetracycline, and lincosamide resistance determinants and, if they were found to be acquired, attempt to remove them from the strain by methods that do not genetically modify the organism before subsequently testing whether the probiotic characteristics were retained. No known β-lactam resistance genes was found, but penicillin-binding proteins from ATCC 55730, two additional resistant strains, and three sensitive strains of
L
.
reuteri
were sequenced and comparatively analyzed. The β-lactam resistance in ATCC 55730 is probably caused by a number of alterations in the corresponding genes and can be regarded as not transferable. The strain was found to harbor two plasmids carrying
tet
(W) tetracycline and
lnu
(A) lincosamide resistance genes, respectively. A new daughter strain,
L
.
reuteri
DSM 17938, was derived from ATCC 55730 by removal of the two plasmids, and it was shown to have lost the resistances associated with them. Direct comparison of the parent and daughter strains for a series of in vitro properties and in a human clinical trial confirmed the retained probiotic properties of the daughter strain.
The Presence of
icaADBC
Is Detrimental to the Colonization of Human Skin by
Staphylococcus epidermidis
Authors: Rogers K. L. , Rupp M. E. , Fey P. D.
Published: August 2008

Previous studies have demonstrated that
Staphylococcus epidermidis
isolates colonizing the skin of healthy humans do not typically encode
icaADBC
, the genes responsible for the production of polysaccharide intercellular adhesin or biofilms. It was therefore hypothesized that the presence of
icaADBC
was deleterious to the successful colonization of human skin by
S. epidermidis
. Using a human skin competition model, it was determined that the strong biofilm-producing
S. epidermidis
strain 1457 was outcompeted at 1, 3, and 10 days by an isogenic
icaADBC
mutant (1457
ica
::
dhfr
), suggesting a fitness cost for carriage of
icaADBC
.
Characterization of ApuB, an Extracellular Type II Amylopullulanase from
Bifidobacterium breve
UCC2003
Authors: O'Connell Motherway Mary , Fitzgerald Gerald F. , Neirynck Sabine , Ryan Sinead , Steidler Lothar , van Sinderen Douwe
Published: August 2008

The
apuB
gene of
Bifidobacterium breve
UCC2003 was shown to encode an extracellular amylopullulanase. ApuB is composed of a distinct N-terminally located α-amylase-containing domain which hydrolyzes α-1,4-glucosidic linkages in starch and related polysaccharides and a C-terminally located pullulanase-containing domain which hydrolyzes α-1,6 linkages in pullulan, allowing the classification of this enzyme as a bifunctional class II pullulanase. A knockout mutation of the
apuB
gene in
B. breve
UCC2003 rendered the resulting mutant incapable of growth in medium containing starch, amylopectin, glycogen, or pullulan as the sole carbon and energy source, confirming the crucial physiological role of this gene in starch metabolism.
Asexual Cephalosporin C Producer
Acremonium chrysogenum
Carries a Functional Mating Type Locus
Authors: Pöggeler Stefanie , Hoff Birgit , Kück Ulrich
Published: August 2008

Acremonium chrysogenum
, the fungal producer of the pharmaceutically relevant β-lactam antibiotic cephalosporin C, is classified as asexual because no direct observation of mating or meiosis has yet been reported. To assess the potential of
A. chrysogenum
for sexual reproduction, we screened an expressed sequence tag library from
A. chrysogenum
for the expression of mating type (
MAT
) genes, which are the key regulators of sexual reproduction. We identified two putative mating type genes that are homologues of the α-box domain gene,
MAT1-1-1
and
MAT1-1-2
, encoding an HPG domain protein defined by the presence of the three invariant amino acids histidine, proline, and glycine. In addition, cDNAs encoding a putative pheromone receptor and pheromone-processing enzymes, as well as components of a pheromone response pathway, were found. Moreover, the entire
A. chrysogenum MAT1-1
(Ac
MAT1-1
) gene and regions flanking the
MAT
region were obtained from a genomic cosmid library, and sequence analysis revealed that in addition to Ac
MAT1-1-1
and Ac
MAT1-1-2
, the Ac
MAT1-1
locus comprises a third mating type gene, Ac
MAT1-1-3
, encoding a high-mobility-group domain protein. The α-box domain sequence of Ac
MAT1-1-1
was used to determine the phylogenetic relationships of
A. chrysogenum
to other ascomycetes. To determine the functionality of the Ac
MAT1-1
locus, the entire
MAT
locus was transferred into a
MAT
deletion strain of the heterothallic ascomycete
Podospora anserina
(the PaΔMAT strain). After fertilization with a
P. anserina
MAT1-2 (MAT
+
) strain, the corresponding transformants developed fruiting bodies with mature ascospores. Thus, the results of our functional analysis of the Ac
MAT1-1
locus provide strong evidence to hypothesize a sexual cycle in
A. chrysogenum
.
Protozoal Digestion of Coat-Defective
Bacillus subtilis
Spores Produces “Rinds” Composed of Insoluble Coat Protein
Authors: Carroll Alicia Monroe , Plomp Marco , Malkin Alexander J. , Setlow Peter
Published: August 2008

The
Bacillus subtilis
spore coat is a multilayer, proteinaceous structure that consists of more than 50 proteins. Located on the surface of the spore, the coat provides resistance to potentially toxic molecules as well as to predation by the protozoan
Tetrahymena thermophila
. When coat-defective spores are fed to
Tetrahymena
, the spores are readily digested. However, a residue termed a “rind” that looks like coat material remains. As observed with a phase-contrast microscope, the rinds are spherical or hemispherical structures that appear to be devoid of internal contents. Atomic force microscopy and chemical analyses showed that (i) the rinds are composed of insoluble protein largely derived from both outer and inner spore coat layers, (ii) the amorphous layer of the outer coat is largely responsible for providing spore resistance to protozoal digestion, and (iii) the rinds and intact spores do not contain significant levels of silicon.
Binding Sequences for RdgB, a DNA Damage-Responsive Transcriptional Activator, and Temperature-Dependent Expression of Bacteriocin and Pectin Lyase Genes in
Pectobacterium carotovorum
subsp.
carotovorum
Authors: Yamada Kazuteru , Kaneko Jun , Kamio Yoshiyuki , Itoh Yoshifumi
Published: August 2008

Pectobacterium carotovorum
subsp.
carotovorum
strain Er simultaneously produces the phage tail-like bacteriocin carotovoricin (Ctv) and pectin lyase (Pnl) in response to DNA-damaging agents. The regulatory protein RdgB of the Mor/C family of proteins activates transcription of
pnl
through binding to the promoter. However, the optimal temperature for the synthesis of Ctv (23°C) differs from that for synthesis of Pnl (30°C), raising the question of whether RdgB directly activates
ctv
transcription. Here we report that RdgB directly regulates Ctv synthesis. Gel mobility shift assays demonstrated RdgB binding to the P
0
, P
1
, and P
2
promoters of the
ctv
operons, and DNase I footprinting determined RdgB-binding sequences (RdgB boxes) on these and on the
pnl
promoters. The RdgB box of the
pnl
promoter included a perfect 7-bp inverted repeat with high binding affinity to the regulator (
K

d

[dissociation constant] = 150 nM). In contrast, RdgB boxes of the
ctv
promoters contained an imperfect inverted repeat with two or three mismatches that consequently reduced binding affinity (
K

d

= 250 to 350 nM). Transcription of the
rdgB
and
ctv
genes was about doubled at 23°C compared with that at 30°C. In contrast, the amount of
pnl
transcription tripled at 30°C. Thus, the inverse synthesis of Ctv and Pnl as a function of temperature is apparently controlled at the transcriptional level, and reduced
rdgB
expression at 30°C obviously affected transcription from the
ctv
promoters with low-affinity RdgB boxes. Pathogenicity toward potato tubers was reduced in an
rdgB
knockout mutant, suggesting that the RdgAB system contributes to the pathogenicity of this bacterium, probably by activating
pnl
expression.
Bioaccumulation of Palladium by
Desulfovibrio fructosivorans
Wild-Type and Hydrogenase-Deficient Strains
Authors: Mikheenko I. P. , Rousset M. , Dementin S. , Macaskie L. E.
Published: August 2008

Wild-type
Desulfovibrio fructosivorans
and three hydrogenase-negative mutants reduced Pd(II) to Pd(0). The location of Pd(0) nanoparticles on the cytoplasmic membrane of the mutant retaining only cytoplasmic membrane-bound hydrogenase was strong evidence for the role of hydrogenases in Pd(0) deposition. Hydrogenase activity was retained at acidic pH, shown previously to favor Pd(0) deposition.
Cross-Species Hybridization with
Fusarium verticillioides
Microarrays Reveals New Insights into
Fusarium fujikuroi
Nitrogen Regulation and the Role of AreA and NMR
Authors: Schönig Birgit , Brown Daren W. , Oeser Birgitt , Tudzynski Bettina
Published: August 2008

In filamentous fungi, the GATA-type transcription factor AreA plays a major role in the transcriptional activation of genes needed to utilize poor nitrogen sources. In
Fusarium fujikuroi
, AreA also controls genes involved in the biosynthesis of gibberellins, a family of diterpenoid plant hormones. To identify more genes responding to nitrogen limitation or sufficiency in an AreA-dependent or -independent manner, we examined changes in gene expression of
F. fujikuroi
wild-type and Δ
areA
strains by use of a
Fusarium verticillioides
microarray representing ∼9,300 genes. Analysis of the array data revealed sets of genes significantly down- and upregulated in the
areA
mutant under both N starvation and N-sufficient conditions. Among the downregulated genes are those involved in nitrogen metabolism, e.g., those encoding glutamine synthetase and nitrogen permeases, but also those involved in secondary metabolism. Besides AreA-dependent genes, we found an even larger set of genes responding to N starvation and N-sufficient conditions in an AreA-independent manner. To study the impact of NMR on AreA activity, we examined the expression of several AreA target genes in the wild type and in
areA
and
nmr
deletion and overexpression mutants. We show that NMR interacts with AreA as expected but affects gene expression only in early growth stages. This is the first report on genome-wide expression studies examining the influence of AreA on nitrogen-responsive gene expression in a genome-wide manner in filamentous fungi.
PKC1
Is Essential for Protection against both Oxidative and Nitrosative Stresses, Cell Integrity, and Normal Manifestation of Virulence Factors in the Pathogenic Fungus
Cryptococcus neoformans
Authors: Gerik Kimberly J. , Bhimireddy Sujit R. , Ryerse Jan S. , Specht Charles A. , Lodge Jennifer K.
Published: August 2008

Cell wall integrity is crucial for fungal growth, survival, and pathogenesis. Responses to environmental stresses are mediated by the highly conserved Pkc1 protein and its downstream components. In this study, we demonstrate that both oxidative and nitrosative stresses activate the PKC1 cell integrity pathway in wild-type cells, as measured by phosphorylation of Mpk1, the terminal protein in the PKC1 phosphorylation cascade. Furthermore, deletion of
PKC1
shows that this gene is essential for defense against both oxidative and nitrosative stresses; however, other genes involved directly in the PKC1 pathway are dispensable for protection against these stresses. This suggests that Pkc1 may have multiple and alternative functions other than activating the mitogen-activated protein kinase cascade from a “top-down” approach. Deletion of
PKC1
also causes osmotic instability, temperature sensitivity, severe sensitivity to cell wall-inhibiting agents, and alterations in capsule and melanin. Furthermore, the vital cell wall components chitin and its deacetylated form chitosan appear to be mislocalized in a
pkc1Δ
strain, although this mutant contains wild-type levels of both of these polymers. These data indicate that loss of Pkc1 has pleiotropic effects because it is central to many functions either dependent on or independent of PKC1 pathway activation. Notably, this is the first time that Pkc1 has been implicated in protection against nitrosative stress in any organism.
The Gluconeogenic Enzyme Fructose-1,6-Bisphosphatase Is Dispensable for Growth of the Yeast
Yarrowia lipolytica
in Gluconeogenic Substrates
Authors: Jardón Raquel , Gancedo Carlos , Flores Carmen-Lisset
Published: August 2008

The genes encoding gluconeogenic enzymes in the nonconventional yeast
Yarrowia lipolytica
were found to be differentially regulated. The expression of
Y. lipolytica FBP1
(Yl
FBP1
) encoding the key enzyme fructose-1,6-bisphosphatase was not repressed by glucose in contrast with the situation in other yeasts; however, this sugar markedly repressed the expression of Yl
PCK1
, encoding phosphoenolpyruvate carboxykinase, and Yl
ICL1
, encoding isocitrate lyase. We constructed
Y. lipolytica
strains with two different disrupted versions of Yl
FBP1
and found that they grew much slower than the wild type in gluconeogenic carbon sources but that growth was not abolished as happens in most microorganisms. We attribute this growth to the existence of an alternative phosphatase with a high

K
m

(2.3 mM) for fructose-1,6-bisphosphate. The gene Yl
FBP1
restored fructose-1,6-bisphosphatase activity and growth in gluconeogenic carbon sources to a
Saccharomyces cerevisiae fbp1
mutant, but the introduction of the
FBP1
gene from
S. cerevisiae
in the Yl
fbp1
mutant did not produce fructose-1,6-bisphosphatase activity or growth complementation. Subcellular fractionation revealed the presence of fructose-1,6-bisphosphatase both in the cytoplasm and in the nucleus.
Role of Acetyl Coenzyme A Synthesis and Breakdown in Alternative Carbon Source Utilization in
Candida albicans
Authors: Carman Aaron J. , Vylkova Slavena , Lorenz Michael C.
Published: August 2008

Acetyl coenzyme A (acetyl-CoA) is the central intermediate of the pathways required to metabolize nonfermentable carbon sources. Three such pathways, i.e., gluconeogenesis, the glyoxylate cycle, and β-oxidation, are required for full virulence in the fungal pathogen
Candida albicans
. These processes are compartmentalized in the cytosol, mitochondria, and peroxosomes, necessitating transport of intermediates across intracellular membranes. Acetyl-CoA is trafficked in the form of acetate by the carnitine shuttle, and we hypothesized that the enzymes that convert acetyl-CoA to/from acetate, i.e., acetyl-CoA hydrolase (
ACH1
) and acetyl-CoA synthetase (
ACS1
and
ACS2
), would regulate alternative carbon utilization and virulence. We show that
C. albicans
strains depleted for
ACS2
are unviable in the presence of most carbon sources, including glucose, acetate, and ethanol; these strains metabolize only fatty acids and glycerol, a substantially more severe phenotype than that of
Saccharomyces cerevisiae acs2
mutants. In contrast, deletion of
ACS1
confers no phenotype, though it is highly induced in the presence of fatty acids, perhaps explaining why
acs2
mutants can utilize fatty acids. Strains lacking
ACH1
have a mild growth defect on some carbon sources but are fully virulent in a mouse model of disseminated candidiasis. Both
ACH1
and
ACS2
complement mutations in their
S. cerevisiae
homolog. Together, these results show that acetyl-CoA metabolism and transport are critical for growth of
C. albicans
on a wide variety of nutrients. Furthermore, the phenotypic differences between mutations in these highly conserved genes in
S. cerevisiae
and
C. albicans
support recent findings that significant functional divergence exists even in fundamental metabolic pathways between these related yeasts.
Identification and Characterization of Epp, the Secreted Processing Protease for the
Vibrio anguillarum
EmpA Metalloprotease
Authors: Varina Maureen , Denkin Steven M. , Staroscik Andrew M. , Nelson David R.
Published: August 2008

The zinc metalloprotease EmpA is a virulence factor for the fish pathogen
Vibrio anguillarum
. Previous studies demonstrated that EmpA is secreted as a 46-kDa proenzyme that is activated extracellularly by the removal of an ∼10-kDa propeptide. We hypothesized that a specific protease is responsible for processing secreted pro-EmpA into mature EmpA. To identify the protease responsible for processing pro-EmpA, a minitransposon mutagenesis (using mini-Tn
10
Km) clone bank of
V. anguillarum
was screened for reduced protease activity due to insertions in undescribed genes. One mutant with reduced protease activity was identified. The region containing the mini-Tn
10
Km was cloned, sequenced, and found to contain
epp
, an open reading frame encoding a putative protease. Further characterization of
epp
was done using strain M101, created by single-crossover insertional mutagenesis. Protease activity was absent in M101 cultures even when
empA
protease activity was induced by salmon gastrointestinal mucus. When the
epp
mutation was complemented with a wild-type copy of
epp
(M102), protease activity was restored. Western blot analysis of sterile filtered culture supernatants from wild-type (M93Sm) cells, M101 cells, and M102 cells revealed that only pro-EmpA was present in M101supernatants; both pro-EmpA and mature EmpA were detected in M93Sm and M102 supernatants. When sterile filtered culture supernatants from the
empA
mutant strain (M99) and M101 were mixed, protease activity was restored. Western blot analysis revealed that pro-EmpA in M101 culture supernatant was processed to mature EmpA only after mixing with M99 culture supernatant. These data show that Epp is the EmpA-processing protease.
Whole-Genome Transcriptional Profiling of
Bradyrhizobium japonicum
during Chemoautotrophic Growth
Authors: Franck William L. , Chang Woo-Suk , Qiu Jing , Sugawara Masayuki , Sadowsky Michael J. , Smith Stephanie A. , Stacey Gary
Published: August 2008

Bradyrhizobium japonicum
is a facultative chemoautotroph capable of utilizing hydrogen gas as an electron donor in a respiratory chain terminated by oxygen to provide energy for cellular processes and carbon dioxide assimilation via a reductive pentose phosphate pathway. A transcriptomic analysis of
B. japonicum
cultured chemoautotrophically identified 1,485 transcripts, representing 17.5% of the genome, as differentially expressed when compared to heterotrophic cultures. Genetic determinants required for hydrogen utilization and carbon fixation, including the uptake hydrogenase system and components of the Calvin-Benson-Bassham cycle, were strongly induced in chemoautotrophically cultured cells. A putative isocitrate lyase (
aceA
; blr2455) was among the most strongly upregulated genes, suggesting a role for the glyoxylate cycle during chemoautotrophic growth. Addition of arabinose to chemoautotrophic cultures of
B. japonicum
did not significantly alter transcript profiles. Furthermore, a subset of nitrogen fixation genes was moderately induced during chemoautotrophic growth. In order to specifically address the role of isocitrate lyase and nitrogenase in chemoautotrophic growth, we cultured
aceA
,
nifD
, and
nifH
mutants under chemoautotrophic conditions. Growth of each mutant was similar to that of the wild type, indicating that the glyoxylate bypass and nitrogenase activity are not essential components of chemoautotrophy in
B. japonicum
.
Cloning and Characterization of the DNA Region Responsible for Megacin A-216 Production in
Bacillus megaterium
216
Authors: Kiss Antal , Balikó Gabriella , Csorba Attila , Chuluunbaatar Tungalag , Medzihradszky Katalin F. , Alföldi Lajos
Published: August 2008

Upon induction,
Bacillus megaterium
216 produces the bacteriocin megacin A-216, which leads to lysis of the producer cell and kills
B. megaterium
and a few other bacterial species. The DNA region responsible for megacinogeny was cloned in
B. megaterium
. The nucleotide sequence of a 5,494-bp-long subfragment was determined, and the function of the genes on this fragment was studied by generating deletions and analyzing their effects on MegA phenotypes. An open reading frame (ORF) encoding a 293-amino-acid protein was identified as the gene (
megA
) coding for megacin A-216. BLAST searches detected sequence similarity between megacin A-216 and proteins with phospholipase A2 activity. Purified biologically active megacin A-216 preparations contained three proteins. Mass spectrometry analysis showed that the largest protein is the full-length translation product of the
megA
gene, whereas the two shorter proteins are fragments of the long protein created by cleavage between Gln-185 and Val-186. The molecular masses of the three polypeptides are 32,855, 21,018, and 11,855 Da, respectively. Comparison of different megacin preparations suggests that the intact chain as well as the two combined fragments can form biologically active megacin. An ORF located next to the
megA
gene and encoding a 91-amino-acid protein was shown to be responsible for the relative immunity displayed by the producer strain against megacin A-216. Besides the
megA
gene, at least two other genes, including a gene encoding a 188-amino-acid protein sharing high sequence similarity with RNA polymerase sigma factors, were shown to be required for induction of megacin A-216 expression.
Surface Spreading Motility Shown by a Group of Phylogenetically Related, Rapidly Growing Pigmented Mycobacteria Suggests that Motility Is a Common Property of Mycobacterial Species but Is Restricted to Smooth Colonies
Authors: Agustí Gemma , Astola Oihane , Rodríguez-Güell Elisabeth , Julián Esther , Luquin Marina
Published: August 2008

Motility in mycobacteria was described for the first time in 1999. It was reported that
Mycobacterium smegmatis
and
Mycobacterium avium
could spread on the surface of solid growth medium by a sliding mechanism and that the presence of cell wall glycopeptidolipids was essential for motility. We recently reported that
Mycobacterium vaccae
can also spread on growth medium surfaces; however, only smooth colonies presented this property. Smooth colonies of
M. vaccae
do not produce glycopeptidolipids but contain a saturated polyester that is absent in rough colonies. Here, we demonstrate that
Mycobacterium chubuense
,
Mycobacterium gilvum
,
Mycobacterium obuense
, and
Mycobacterium parafortuitum
, which are phylogenetically related to
M. vaccae
, are also motile. Such motility is restricted to smooth colonies, since natural rough mutants are nonmotile. Thin-layer chromatography analysis of the content of cell wall lipids confirmed the absence of glycopeptidolipids. However, compounds like the above-mentioned
M. vaccae
polyester were detected in all the strains but only in smooth colonies. Scanning electron microscopy showed great differences in the arrangement of the cells between smooth and rough colonies. The data obtained suggest that motility is a common property of environmental mycobacteria, and this capacity correlates with the smooth colonial morphotype. The species studied in this work do not contain glycopeptidolipids, so cell wall compounds or extracellular materials other than glycopeptidolipids are implicated in mycobacterial motility. Furthermore, both smooth motile and rough nonmotile variants formed biofilms on glass and polystyrene surfaces.
The Two-Component System PhoPR of
Clostridium acetobutylicum
Is Involved in Phosphate-Dependent Gene Regulation
Authors: Fiedler Tomas , Mix Maren , Meyer Uta , Mikkat Stefan , Glocker Michael O. , Bahl Hubert , Fischer Ralf-Jörg
Published: August 2008

The
phoPR
gene locus of
Clostridium acetobutylicum
ATCC 824 comprises two genes,
phoP
and
phoR
. Deduced proteins are predicted to represent a response regulator and sensor kinase of a phosphate-dependent two-component regulatory system. We analyzed the expression patterns of
phoPR
in P
i
-limited chemostat cultures and in response to P
i
pulses. A basic transcription level under high-phosphate conditions was shown, and a significant increase in mRNA transcript levels was found when external P
i
concentrations dropped below 0.3 mM. In two-dimensional gel electrophoresis experiments, a 2.5-fold increase in PhoP was observed under P
i
-limiting growth conditions compared to growth with an excess of P
i
. At least three different transcription start points for
phoP
were determined by primer extension analyses. Proteins PhoP and an N-terminally truncated *PhoR were individually expressed heterologously in
Escherichia coli
and purified. Autophosphorylation of *PhoR and phosphorylation of PhoP were shown in vitro. Electromobility shift assays proved that there was a specific binding of PhoP to the promoter region of the phosphate-regulated
pst
operon of
C. acetobutylicum
.
Carbonylated Proteins Are Detectable Only in a Degradation-Resistant Aggregate State in
Escherichia coli
Authors: Maisonneuve Etienne , Fraysse Laetitia , Lignon Sabrina , Capron Laure , Dukan Sam
Published: August 2008

Carbonylation is currently used as a marker for irreversible protein oxidative damage. Several studies indicate that carbonylated proteins are more prone to degradation than their nonoxidized counterparts. In this study, we observed that in
Escherichia coli
, more than 95% of the total carbonyl content consisted of insoluble protein and most were cytosolic proteins. We thereby demonstrate that, in vivo, carbonylated proteins are detectable mainly in an aggregate state. Finally, we show that detectable carbonylated proteins are not degraded in vivo. Here we propose that some carbonylated proteins escape degradation in vivo by forming carbonylated protein aggregates and thus becoming nondegradable. In light of these findings, we provide evidence that the accumulation of nondegradable carbonylated protein presented in an aggregate state contributes to the increases in carbonyl content observed during senescence.
Clp and Lon Proteases Occupy Distinct Subcellular Positions inBacillus subtilis
Authors: Simmons Lyle A. , Grossman Alan D. , Walker Graham C.
Published: August 2008

Among other functions, ATP-dependent proteases degrade misfolded proteins and remove several key regulatory proteins necessary to activate stress responses. InBacillus subtilis, ClpX, ClpE, and ClpC form homohexameric ATPases that couple to the ClpP peptidase. To understand where these peptidases and ATPases localize in living cells, each protein was fused to a fluorescent moiety. We found that ClpX-GFP (green fluorescent protein) and ClpP-GFP localized as focal assemblies in areas that were not occupied by the nucleoid. We found that the percentage of cells with ClpP-GFP foci increased following heat shock independently of protein synthesis. We determined that ClpE-YFP (yellow fluorescent protein) and ClpC-YFP formed foci coincident with nucleoid edges, usually near cell poles. Furthermore, we found that ClpQ-YFP (HslV) localized as small foci, usually positioned near the cell membrane. We found that ClpQ-YFP foci were dependent on the presence of the cognate hexameric ATPase ClpY (HslU). Moreover, we found that LonA-GFP is coincident with the nucleoid during normal growth and that LonA-GFP also localized to the forespore during development. We also investigated LonB-GFP and found that this protein localized to the forespore membrane early in development, followed by localization throughout the forespore later in development. Our comprehensive study has shown that inB. subtilisseveral ATP-fueled proteases occupy distinct subcellular locations. With these data, we suggest that substrate specificity could be determined, in part, by the spatial and temporal organization of proteases in vivo.
Role of the N-Terminal Domain of φC31 Integrase in
attB-attP
Synapsis
Authors: Rowley Paul A. , Smith Margaret C. M.
Published: August 2008

φC31 integrase is a serine recombinase containing an N-terminal domain (NTD) that provides catalytic activity and a large C-terminal domain that controls which pair of DNA substrates is able to synapse. We show here that substitutions in amino acid V129 in the NTD can lead to defects in synapsis and DNA cleavage, indicating that the NTD also has an important role in synapsis.
Role of the
Helicobacter hepaticus
Flagellar Sigma Factor FliA in Gene Regulation and Murine Colonization
Authors: Sterzenbach Torsten , Bartonickova Lucie , Behrens Wiebke , Brenneke Birgit , Schulze Jessika , Kops Friederike , Chin Elaine Y. , Katzowitsch Elena , Schauer David B. , Fox James G. , Suerbaum Sebastian , Josenhans Christine
Published: August 2008

The enterohepatic
Helicobacter
species
Helicobacter hepaticus
colonizes the murine intestinal and hepatobiliary tract and is associated with chronic intestinal inflammation, gall stone formation, hepatitis, and hepatocellular carcinoma. Thus far, the role of
H. hepaticus
motility and flagella in intestinal colonization is unknown. In other, closely related bacteria, late flagellar genes are mainly regulated by the sigma factor FliA (σ
28
). We investigated the function of the
H. hepaticus
FliA in gene regulation, flagellar biosynthesis, motility, and murine colonization. Competitive microarray analysis of the wild type versus an isogenic
fliA
mutant revealed that 11 genes were significantly more highly expressed in wild-type bacteria and 2 genes were significantly more highly expressed in the
fliA
mutant. Most of these were flagellar genes, but four novel FliA-regulated genes of unknown function were identified.
H. hepaticus
possesses two identical copies of the gene encoding the FliA-dependent major flagellin subunit FlaA (open reading frames HH1364 and HH1653). We characterized the phenotypes of mutants in which
fliA
or one or both copies of the
flaA
gene were knocked out.
flaA
_
1 flaA
_
2
double mutants and
fliA
mutants did not synthesize detectable amounts of FlaA and possessed severely truncated flagella. Also, both mutants were nonmotile and unable to colonize mice. Mutants with either
flaA
gene knocked out produced flagella morphologically similar to those of wild-type bacteria and expressed FlaA and FlaB.
flaA
_
1
mutants which had flagella but displayed reduced motility did not colonize mice, indicating that motility is required for intestinal colonization by
H. hepaticus
and that the presence of flagella alone is not sufficient.
Chemoreceptors in
Caulobacter crescentus
: Trimers of Receptor Dimers in a Partially Ordered Hexagonally Packed Array
Authors: Khursigara Cezar M. , Wu Xiongwu , Subramaniam Sriram
Published: August 2008

Chemoreceptor arrays are macromolecular complexes that form extended assemblies primarily at the poles of bacterial cells and mediate chemotaxis signal transduction, ultimately controlling cellular motility. We have used cryo-electron tomography to determine the spatial distribution and molecular architecture of signaling molecules that comprise chemoreceptor arrays in wild-type
Caulobacter crescentus
cells. We demonstrate that chemoreceptors are organized as trimers of receptor dimers, forming partially ordered hexagonally packed arrays of signaling complexes in the cytoplasmic membrane. This novel organization at the threshold between order and disorder suggests how chemoreceptors and associated molecules are arranged in signaling assemblies to respond dynamically in the activation and adaptation steps of bacterial chemotaxis.
The Use of Chromatin Immunoprecipitation to Define PpsR Binding Activity in
Rhodobacter sphaeroides
2.4.1
Authors: Bruscella Patrice , Eraso Jesus M. , Roh Jung Hyeob , Kaplan Samuel
Published: August 2008

The expression of genes involved in photosystem development in
Rhodobacter sphaeroides
is dependent upon three major regulatory networks: FnrL, the PrrBA (RegBA) two-component system, and the transcriptional repressor/antirepressor PpsR/AppA. Of the three regulators, PpsR appears to have the narrowest range of physiological effects, which are limited to effects on the structural and pigment biosynthetic activities involved in photosynthetic membrane function. Although a PrrA
−
mutant is unable to grow under photosynthetic conditions, when a
ppsR
mutation was present, photosynthetic growth occurred. An examination of the double mutant under anaerobic-dark-dimethyl sulfoxide conditions using microarray analysis revealed the existence of an “extended” PpsR regulon and new physiological roles. To characterize the PpsR regulon and to better ascertain the significance of degeneracy within the PpsR binding sequence in vivo, we adapted the chromatin immunoprecipitation technique to
R. sphaeroides
. We demonstrated that in vivo there was direct and significant binding by PpsR to newly identified genes involved in microaerobic respiration and periplasmic stress resistance, as well as to photosynthesis genes. The new members of the PpsR regulon are located outside the photosynthesis gene cluster and have degenerate PpsR binding sequences. The possible interaction under physiologic conditions with degenerate binding sequences in the presence of other biologically relevant molecules is discussed with respect to its importance in physiological processes and to the existence of complex phenotypes associated with regulatory mutants. This study further defines the DNA structure necessary for PpsR binding in situ.
Comprehensive Assessment of the Regulons Controlled by the FixLJ-FixK2-FixK1Cascade inBradyrhizobium japonicum
Authors: Mesa Socorro , Hauser Felix , Friberg Markus , Malaguti Emmanuelle , Fischer Hans-Martin , Hennecke Hauke
Published: August 2008

Symbiotic N2fixation inBradyrhizobium japonicumis controlled by a complex transcription factor network. Part of it is a hierarchically arranged cascade in which the two-component regulatory system FixLJ, in response to a moderate decrease in oxygen concentration, activates thefixK2gene. The FixK2protein then activates not only a number of genes essential for microoxic respiration in symbiosis (fixNOQPandfixGHIS) but also further regulatory genes (rpoN1,nnrR, andfixK1). The results of transcriptome analyses described here have led to a comprehensive and expanded definition of the FixJ, FixK2, and FixK1regulons, which, respectively, consist of 26, 204, and 29 genes specifically regulated in microoxically grown cells. Most of these genes are subject to positive control. Particular attention was addressed to the FixK2-dependent genes, which included a bioinformatics search for putative FixK2binding sites on DNA (FixK2boxes). Using an in vitro transcription assay with RNA polymerase holoenzyme and purified FixK2as the activator, we validated as direct targets eight new genes. Interestingly, the adjacent but divergently orientedfixK1andcycSgenes shared the same FixK2box for the activation of transcription in both directions. This recognition site may also be a direct target for the FixK1protein, because activation of thecycSpromoter required an intactfixK1gene and either microoxic or anoxic, denitrifying conditions. We present evidence thatcycScodes for ac-type cytochrome which is important, but not essential, for nitrate respiration. Two other, unexpected results emerged from this study: (i) specifically FixK1seemed to exert a negative control on genes that are normally activated by the N2fixation-specific transcription factor NifA, and (ii) a larger number of genes are expressed in a FixK2-dependent manner in endosymbiotic bacteroids than in culture-grown cells, pointing to a possible symbiosis-specific control.
Role of the
Campylobacter jejuni
Cj1461 DNA Methyltransferase in Regulating Virulence Characteristics
Authors: Kim Joo-Sung , Li Jiaqi , Barnes If H. A. , Baltzegar David A. , Pajaniappan Mohanasundari , Cullen Thomas W. , Trent M. Stephen , Burns Christopher M. , Thompson Stuart A.
Published: August 2008

Mutation of the cj1461 predicted methyltransferase gene reduced the motility of
Campylobacter jejuni
81-176. Electron microscopy revealed that the mutant strain had flagella but with aberrant structure. The Δcj1461 mutant was sevenfold more adherent to but 50-fold less invasive of INT-407 human epithelial cells than the wild type.