bluuebunny/crossref_metadata_2025 huggingface dataset:
Transgene-Induced Silencing of the Zoosporogenesis-Specific
NIFC
Gene Cluster of
Phytophthora infestans
Involves Chromatin Alterations
Authors: Judelson Howard S. , Tani Shuji
Published: May 2007

Clustered within the genome of the oomycete phytopathogen
Phytophthora infestans
are four genes encoding spore-specific nuclear LIM interactor-interacting factors (NIF proteins, a type of transcriptional regulator) that are moderately conserved in DNA sequence.
NIFC1
,
NIFC2
, and
NIFC3
are zoosporogenesis-induced and grouped within 4 kb, and 20 kb away resides a sporulation-induced form,
NIFS
. To test the function of the
NIFC
family, plasmids expressing full-length hairpin constructs of
NIFC1
or
NIFC2
were stably transformed into
P. infestans.
This triggered silencing of the cognate gene in about one-third of transformants, and all three
NIFC
genes were usually cosilenced. However,
NIFS
escaped silencing despite its high sequence similarity to the
NIFC
genes. Silencing of the three
NIFC
genes impaired zoospore cyst germination by 60% but did not affect other aspects of the life cycle. Silencing was transcriptional based on nuclear run-on assays and associated with tighter chromatin packing based on nuclease accessibility experiments. The chromatin alterations extended a few hundred nucleotides beyond the boundaries of the transcribed region of the
NIFC
cluster and were not associated with increased DNA methylation. A plasmid expressing a short hairpin RNA having sequence similarity only to
NIFC1
silenced both that gene and an adjacent member of the gene cluster, likely due to the expansion of a heterochromatic domain from the targeted locus. These data help illuminate the mechanism of silencing in
Phytophthora
and suggest that caution should be used when interpreting silencing experiments involving closely spaced genes.
The
Fusarium verticillioides FUM
Gene Cluster Encodes a Zn(II)2Cys6 Protein That Affects
FUM
Gene Expression and Fumonisin Production
Authors: Brown Daren W. , Butchko Robert A. E. , Busman Mark , Proctor Robert H.
Published: May 2007

Fumonisins are mycotoxins produced by some
Fusarium
species and can contaminate maize or maize products. Ingestion of fumonisins is associated with diseases, including cancer and neural tube defects, in humans and animals. In fungi, genes involved in the synthesis of mycotoxins and other secondary metabolites are often located adjacent to each other in gene clusters. Such genes can encode structural enzymes, regulatory proteins, and/or proteins that provide self-protection. The fumonisin biosynthetic gene cluster includes 16 genes, none of which appear to play a role in regulation. In this study, we identified a previously undescribed gene (
FUM21
) located adjacent to the fumonisin polyketide synthase gene,
FUM1
. The presence of a Zn(II)2Cys6 DNA-binding domain in the predicted protein suggested that
FUM21
was involved in transcriptional regulation.
FUM21
deletion (Δ
fum21
) mutants produce little to no fumonisin in cracked maize cultures but some
FUM1
and
FUM8
transcripts in a liquid GYAM medium. Complementation of a Δ
fum21
mutant with a wild-type copy of the gene restored fumonisin production. Analysis of
FUM21
cDNAs identified four alternative splice forms (ASFs), and microarray analysis indicated the ASFs were differentially expressed. Based on these data, we present a model for how
FUM21
ASFs may regulate fumonisin biosynthesis.
Dual Regulation of the
Bacillus subtilis
Regulon Comprising the
lmrAB
and
yxaGH
Operons and
yxaF
Gene by Two Transcriptional Repressors, LmrA and YxaF, in Response to Flavonoids
Authors: Hirooka Kazutake , Kunikane Satoshi , Matsuoka Hiroshi , Yoshida Ken-Ichi , Kumamoto Kanako , Tojo Shigeo , Fujita Yasutaro
Published: May 2007

Bacillus subtilis
LmrA is known to be a repressor that regulates the
lmrAB
and
yxaGH
operons;
lmrB
and
yxaG
encode a multidrug resistance pump and quercetin 2,3-dioxygenase, respectively. DNase I footprinting analysis revealed that LmrA and YxaF, which are paralogous to each other, bind specifically to almost the same
cis
sequences, LmrA/YxaF boxes, located in the promoter regions of the
lmrAB
operon, the
yxaF
gene, and the
yxaGH
operon for their repression and containing a consensus sequence of AWTATAtagaNYGgTCTA, where W, Y, and N stand for A or T, C or T, and any base, respectively (three-out-of-four match [in lowercase type]). Gel retardation analysis indicated that out of the eight flavonoids tested, quercetin, fisetin, and catechin are most inhibitory for LmrA to DNA binding, whereas quercetin, fisetin, tamarixetin, and galangin are most inhibitory for YxaF. Also, YxaF bound most tightly to the tandem LmrA/YxaF boxes in the
yxaGH
promoter region. The
lacZ
fusion experiments essentially supported the above-mentioned in vitro results, except that galangin did not activate the
lmrAB
and
yxaGH
promoters, probably due to its poor incorporation into cells. Thus, the LmrA/YxaF regulon presumably comprising the
lmrAB
operon, the
yxaF
gene, and the
yxaGH
operon is induced in response to certain flavonoids. The in vivo experiments to examine the regulation of the synthesis of the reporter β-galactosidase and quercetin 2,3-dioxgenase as well as that of multidrug resistance suggested that LmrA represses the
lmrAB
and
yxaGH
operons but that YxaF represses
yxaGH
more preferentially.
Delineation of the
Salmonella enterica
Serovar Typhimurium HilA Regulon through Genome-Wide Location and Transcript Analysis
Authors: Thijs Inge M. V. , De Keersmaecker Sigrid C. J. , Fadda Abeer , Engelen Kristof , Zhao Hui , McClelland Michael , Marchal Kathleen , Vanderleyden Jos
Published: May 2007

The
Salmonella enterica
serovar Typhimurium HilA protein is the key regulator for the invasion of epithelial cells. By a combination of genome-wide location and transcript analysis, the HilA-dependent regulon has been delineated. Under invasion-inducing conditions, HilA binds to most of the known target genes and a number of new target genes. The
sopB
,
sopE
, and
sopA
genes, encoding effector proteins secreted by the type III secretion system on
Salmonella
pathogenicity island 1 (SPI-1), were identified as being both bound by HilA and differentially regulated in an HilA mutant. This suggests a cooperative role for HilA and InvF in the regulation of SPI-1-secreted effectors. Also,
siiA
, the first gene of SPI-4, is both bound by HilA and differentially regulated in an HilA mutant, thus linking this pathogenicity island to the invasion key regulator. Finally, the interactions of HilA with the SPI-2 secretion system gene
ssaH
and the flagellar gene
flhD
imply a repressor function for HilA under invasion-inducing conditions.
Deletion and Substitution Analysis of theEscherichia coliTonB Q160 Region
Authors: Vakharia-Rao Hema , Kastead Kyle A. , Savenkova Marina I. , Bulathsinghala Charles M. , Postle Kathleen
Published: May 2007

The active transport of iron siderophores and vitamin B12across the outer membrane (OM) ofEscherichia colirequires OM transporters and the potential energy of the cytoplasmic membrane (CM) proton gradient and CM proteins TonB, ExbB, and ExbD. A region at the amino terminus of the transporter, called the TonB box, directly interacts with TonB Q160 region residues. R158 and R166 in the TonB Q160 region were proposed to play important roles in cocrystal structures of the TonB carboxy terminus with OM transporters BtuB and FhuA. In contrast to predictions based on the crystal structures, none of the single, double, or triple alanyl substitutions at arginyl residues significantly decreased TonB activity. Even the quadruple R154A R158A R166A R171A mutant TonB still retained 30% of wild-type activity. Up to five residues centered on TonB Q160 could be deleted without inactivating TonB or preventing its association with the OM. TonB mutant proteins with nested deletions of 7, 9, or 11 residues centered on TonB Q160 were inactive and appeared never to have associated with the OM. Because the 7-residue-deletion mutant protein (TonBΔ7, lacking residues S157 to Y163) could still form disulfide-linked dimers when combined with W213C or F202C in the TonB carboxy terminus, the TonBΔ7 deletion did not prevent necessary energy-dependent conformational changes that occur in the CM. Thus, it appeared that initial contact with the OM is made through TonB residues S157 to Y163. It is hypothesized that the TonB Q160 region may be part of a large disordered region required to span the periplasm and contact an OM transporter.
Convergent Peripheral Pathways Catalyze Initial Glucose Catabolism inPseudomonas putida: Genomic and Flux Analysis
Authors: del Castillo Teresa , Ramos Juan L. , Rodríguez-Herva José J. , Fuhrer Tobias , Sauer Uwe , Duque Estrella
Published: May 2007

In this study, we show that glucose catabolism inPseudomonas putidaoccurs through the simultaneous operation of three pathways that converge at the level of 6-phosphogluconate, which is metabolized by the Edd and Eda Entner/Doudoroff enzymes to central metabolites. When glucose enters the periplasmic space through specific OprB porins, it can either be internalized into the cytoplasm or be oxidized to gluconate. Glucose is transported to the cytoplasm in a process mediated by an ABC uptake system encoded by open reading frames PP1015 to PP1018 and is then phosphorylated by glucokinase (encoded by theglkgene) and converted by glucose-6-phosphate dehydrogenase (encoded by thezwfgenes) to 6-phosphogluconate. Gluconate in the periplasm can be transported into the cytoplasm and subsequently phosphorylated by gluconokinase to 6-phosphogluconate or oxidized to 2-ketogluconate, which is transported to the cytoplasm, and subsequently phosphorylated and reduced to 6-phosphogluconate. In the wild-type strain, glucose was consumed at a rate of around 6 mmol g−1h−1, which allowed a growth rate of 0.58 h−1and a biomass yield of 0.44 g/g carbon used. Flux analysis of13C-labeled glucose revealed that, in the Krebs cycle, most of the oxalacetate fraction was produced by the pyruvate shunt rather than by the direct oxidation of malate by malate dehydrogenase. Enzymatic and microarray assays revealed that the enzymes, regulators, and transport systems of the three peripheral glucose pathways were induced in response to glucose in the outer medium. We generated a series of isogenic mutants in one or more of the steps of all three pathways and found that, although all three functioned simultaneously, the glucokinase pathway and the 2-ketogluconate loop were quantitatively more important than the direct phosphorylation of gluconate. In physical terms, glucose catabolism genes were organized in a series of clusters scattered along the chromosome. Within each of the clusters, genes encoding porins, transporters, enzymes, and regulators formed operons, suggesting that genes in each cluster coevolved. Theglkgene encoding glucokinase was located in an operon with theeddgene, whereas thezwf-1gene, encoding glucose-6-phosphate dehydrogenase, formed an operon with theedagene. Therefore, the enzymes of the glucokinase pathway and those of the Entner-Doudoroff pathway are physically linked and induced simultaneously. It can therefore be concluded that the glucokinase pathway is a sine qua non condition forP. putidato grow with glucose.
Silencing Essential Protein Secretion in
Mycobacterium smegmatis
by Using Tetracycline Repressors
Authors: Guo Xinzheng V. , Monteleone Mercedes , Klotzsche Marcus , Kamionka Annette , Hillen Wolfgang , Braunstein Miriam , Ehrt Sabine , Schnappinger Dirk
Published: May 2007

Many processes that are essential for mycobacterial growth are poorly understood. To facilitate genetic analyses of such processes in mycobacteria, we and others have developed regulated expression systems that are repressed by a tetracycline repressor (TetR) and induced with tetracyclines, permitting the construction of conditional mutants of essential genes. A disadvantage of these systems is that tetracyclines function as transcriptional inducers and have to be removed to initiate gene silencing. Recently, reverse TetR mutants were identified that require tetracyclines as corepressors. Here, we report that one of these mutants, TetR r1.7, allows efficient repression of
lacZ
expression in
Mycobacterium smegmatis
in the presence but not the absence of anhydrotetracycline (atc). TetR and TetR r1.7 also allowed efficient silencing of the essential
secA1
gene, as demonstrated by inhibition of the growth of a conditional mutant and dose-dependent depletion of the SecA1 protein after the removal or addition, respectively, of atc. The kinetics of SecA1 depletion were similar with TetR and TetR r1.7. To test whether silencing of
secA1
could help identify substrates of the general secretion pathway, we analyzed the main porin of
M. smegmatis
, MspA. This showed that the amount of cell envelope-associated MspA decreased more than 90-fold after
secA1
silencing. We thus demonstrated that TetR r1.7 allows the construction of conditional mycobacterial mutants in which the expression of an essential gene can be efficiently silenced by the addition of atc and that gene silencing permits the identification of candidate substrates of mycobacterial secretion systems.
Mutagenesis and Molecular Modeling Reveal Three Key Extracellular Loops of the Membrane Receptor HasR That Are Involved in Hemophore HasA Binding
Authors: Barjon Clément , Wecker Karine , Izadi-Pruneyre Nadia , Delepelaire Philippe
Published: May 2007

On the basis of the three-dimensional model of the heme/hemophore TonB-dependent outer membrane receptor HasR, mutants with six-residue deletions in the 11 putative extracellular loops were generated. Although all mutants continued to be active TonB-dependent heme transporters, mutations in three loops abolished hemophore HasA binding both in vivo and in vitro.
The Two Carboxylases of
Corynebacterium glutamicum
Essential for Fatty Acid and Mycolic Acid Synthesis
Authors: Gande Roland , Dover Lynn G. , Krumbach Karin , Besra Gurdyal S. , Sahm Hermann , Oikawa Tadao , Eggeling Lothar
Published: May 2007

The suborder
Corynebacterianeae
comprises bacteria like
Mycobacterium tuberculosis
and
Corynebacterium glutamicum
, and these bacteria contain in addition to the linear fatty acids, unique α-branched β-hydroxy fatty acids, called mycolic acids. Whereas acetyl-coenzyme A (CoA) carboxylase activity is required to provide malonyl-CoA for fatty acid synthesis, a new type of carboxylase is apparently additionally present in these bacteria. It activates the α-carbon of a linear fatty acid by carboxylation, thus enabling its decarboxylative condensation with a second fatty acid to afford mycolic acid synthesis. We now show that the acetyl-CoA carboxylase of
C. glutamicum
consists of the biotinylated α-subunit AccBC, the β-subunit AccD1, and the small peptide AccE of 8.9 kDa, forming an active complex of approximately 812,000 Da. The carboxylase involved in mycolic acid synthesis is made up of the two highly similar β-subunits AccD2 and AccD3 and of AccBC and AccE, the latter two identical to the subunits of the acetyl-CoA carboxylase complex. Since AccD2 and AccD3 orthologues are present in all
Corynebacterianeae
, these polypeptides are vital for mycolic acid synthesis forming the unique hydrophobic outer layer of these bacteria, and we speculate that the two β-subunits present serve to lend specificity to this unique large multienzyme complex.
A Robust Species Tree for the
Alphaproteobacteria
Authors: Williams Kelly P. , Sobral Bruno W. , Dickerman Allan W.
Published: May 2007

The branching order and coherence of the alphaproteobacterial orders have not been well established, and not all studies have agreed that mitochondria arose from within the
Rickettsiales
. A species tree for 72 alphaproteobacteria was produced from a concatenation of alignments for 104 well-behaved protein families. Coherence was upheld for four of the five orders with current standing that were represented here by more than one species. However, the family
Hyphomonadaceae
was split from the other
Rhodobacterales
, forming an expanded group with
Caulobacterales
that also included
Parvularcula
. The three earliest-branching alphaproteobacterial orders were the
Rickettsiales
, followed by the
Rhodospirillales
and then the
Sphingomonadales
. The principal uncertainty is whether the expanded
Caulobacterales
group is more closely associated with the
Rhodobacterales
or the
Rhizobiales
. The mitochondrial branch was placed within the
Rickettsiales
as a sister to the combined
Anaplasmataceae
and
Rickettsiaceae
, all subtended by the
Pelagibacter
branch.
Pelagibacter
genes will serve as useful additions to the bacterial outgroup in future evolutionary studies of mitochondrial genes, including those that have transferred to the eukaryotic nucleus.
Differential Regulation of
ponA
and
pilMNOPQ
Expression by the MtrR Transcriptional Regulatory Protein in
Neisseria gonorrhoeae
Authors: Folster Jason P. , Dhulipala Vijaya , Nicholas Robert A. , Shafer William M.
Published: May 2007

Neisseria gonorrhoeae
utilizes the
mtrCDE
-encoded efflux pump system to resist not only host-derived, hydrophobic antimicrobials that bathe mucosal surfaces, which likely aids in its ability to colonize and infect numerous sites within the human host, but also antibiotics that have been used clinically to treat infections. Recently, overexpression of the MtrC-MtrD-MtrE efflux pump was shown to be critically involved in the capacity of gonococci to develop chromosomally mediated resistance to penicillin G, which for over 40 years was used to treat gonococcal infections. Mutations in either the promoter or the coding sequence of the
mtrR
gene, which encodes a repressor of the efflux pump operon, decrease gonococcal susceptibility to penicillin. We now describe the capacity of MtrR to directly or indirectly influence the expression of two other loci that are involved in gonococcal susceptibility to penicillin:
ponA
, which encodes penicillin-binding protein 1 (PBP 1), and the
pilMNOPQ
operon, which encodes components of the type IV pilus secretion system, with PilQ acting as a channel for entry for penicillin. We determined that MtrR increases the expression of
ponA
directly or indirectly, resulting in increased levels of PBP 1, while repressing the expression of the divergently transcribed
pilM
gene, the first gene in the
pilMNOPQ
operon. Taken together with other studies, the results presented herein indicate that transcriptional regulation of gonococcal genes by MtrR is centrally involved in determining levels of gonococcal susceptibility to penicillin and provides a framework for understanding how resistance developed over the years.
Differential Expression of the Three Multicopper Oxidases from
Myxococcus xanthus
Authors: Sánchez-Sutil María Celestina , Gómez-Santos Nuria , Moraleda-Muñoz Aurelio , Martins Lígia O. , Pérez Juana , Muñoz-Dorado José
Published: May 2007

Myxococcus xanthus
is a soil bacterium that undergoes a unique life cycle among the prokaryotes upon starvation, which includes the formation of macroscopic structures, the fruiting bodies, and the differentiation of vegetative rods into coccoid myxospores. This peculiarity offers the opportunity to study the copper response in this bacterium in two different stages. In fact,
M. xanthus
vegetative rods exhibit 15-fold-greater resistance against copper than developing cells. However, cells preadapted to this metal reach the same levels of resistance during both stages. Analysis of the
M. xanthus
genome reveals that many of the genes involved in copper resistance are redundant, three of which encode proteins of the multicopper oxidase family (MCO). Each MCO gene exhibits a different expression profile in response to external copper addition. Promoters of
cuoA
and
cuoB
respond to Cu(II) ions during growth and development; however, they show a 10-fold-increased copper sensitivity during development. The promoter of
cuoC
shows copper-independent induction upon starvation, but it is copper up-regulated during growth. Phenotypic analyses of deletion mutants reveal that CuoB is involved in the primary copper-adaptive response; CuoA and CuoC are necessary for the maintenance of copper tolerance; and CuoC is required for normal development. These roles seem to be carried out through cuprous oxidase activity.
Proteomic Identification of a Novel
Anaplasma phagocytophilum
DNA Binding Protein That Regulates a Putative Transcription Factor
Authors: Wang Xueqi , Kikuchi Takane , Rikihisa Yasuko
Published: May 2007

Anaplasma phagocytophilum
, the etiologic agent of human granulocytic anaplasmosis, is an obligatory intracellular bacterium. Little is known about the gene regulatory mechanisms for this bacterium. A gene encoding a putative transcription factor,
tr1
, upstream of three tandem genes encoding outer membrane proteins, including the major outer membrane protein P44, is driven by a strong promoter. In the present study, gel mobility shift assays revealed the presence of
A. phagocytophilum
proteins that interact with the promoter region of
tr1
. These proteins interacting with the
tr1
promoter region were purified by biotin-labeled DNA affinity chromatography from a large amount of host cell-free bacteria. Mass spectrometry identified the major protein as an
A. phagocytophilum
12.5-kDa hypothetical protein, which was named ApxR. In a DNase I protection assay, recombinant ApxR (rApxR) bound cooperatively to four 24- or 25-bp sites within 235 bp upstream of
tr1
: regions III and IV proximal to
tr1
had higher affinity than regions I and II did. Deletion assays showed that regions III and IV were essential for rApxR binding, whereas regions I and II upstream of regions III and IV were not. The primary
cis
-acting region was region IV, since region IV alone was sufficient for rApxR to strongly transactivate the downstream gene in a
lacZ
reporter assay. Addition of regions I, II, and III did not enhance transactivation. These results show that ApxR is a novel transcriptional regulator that directly regulates
tr1
.
Predicted Glycosyl Transferase Genes Located outside the HEP Island Are Required for Formation of Heterocyst Envelope Polysaccharide in
Anabaena
sp. Strain PCC 7120
Authors: Wang Yu , Lechno-Yossef Sigal , Gong Yangmin , Fan Qing , Wolk C. Peter , Xu Xudong
Published: May 2007

During maturation, heterocysts form an envelope layer of polysaccharide, called heterocyst envelope polysaccharide (HEP), whose synthesis depends on a cluster of genes, the HEP island, and on an additional, distant gene,
hepB
, or a gene immediately downstream from
hepB
. We show that HEP formation depends upon the predicted glycosyl transferase genes all4160 at a third locus and alr3699, which is adjacent to
hepB
and is cotranscribed with it. Mutations in the histidine kinase genes
hepN
and
hepK
appear to silence the promoter of
hepB
and incompletely down-regulate all4160.
Global Gene Expression Patterns ofNostoc punctiformein Steady-State Dinitrogen-Grown Heterocyst-Containing Cultures and at Single Time Points during the Differentiation of Akinetes and Hormogonia
Authors: Campbell Elsie L. , Summers Michael L. , Christman Harry , Martin Miriam E. , Meeks John C.
Published: May 2007

The vegetative cells of the filamentous cyanobacteriumNostoc punctiformecan differentiate into three mutually exclusive cell types: nitrogen-fixing heterocysts, spore-like akinetes, and motile hormogomium filaments. A DNA microarray consisting of 6,893N. punctiformegenes was used to identify the global transcription patterns at single time points in the three developmental states, compared to those in ammonium-grown time zero cultures. Analysis of ammonium-grown cultures yielded a transcriptome of 2,935 genes, which is nearly twice the size of a soluble proteome. The NH4+-grown transcriptome was enriched in genes encoding core metabolic functions. A steady-state N2-grown (heterocyst-containing) culture showed differential transcription of 495 genes, 373 of which were up-regulated. The majority of the up-regulated genes were predicted from studies of heterocyst differentiation and N2fixation; other genes are candidates for more detailed genetic analysis. Three days into the developmental process, akinetes showed a similar number of differentially expressed genes (497 genes), which were equally up- and down-regulated. The down-regulated genes were enriched in core metabolic functions, consistent with entry into a nongrowth state. There were relatively few adaptive genes up-regulated in 3-day akinetes, and there was little overlap with putative heterocyst developmental genes. There were 1,827 differentially transcribed genes in 24-h hormogonia, which was nearly fivefold greater than the number in akinete-forming or N2-fixing cultures. The majority of the up-regulated adaptive genes were genes encoding proteins for signal transduction and transcriptional regulation, which is characteristic of a motile filament that is poised to sense and respond to the environment. The greatest fraction of the 883 down-regulated genes was involved in core metabolism, also consistent with entry into a nongrowth state. The differentiation of heterocysts (steady state, N2grown), akinetes, and hormogonia appears to involve the up-regulation of genes distinct for each state.
The Copper-Inducible
cin
Operon Encodes an Unusual Methionine-Rich Azurin-Like Protein and a Pre-Q
0
Reductase in
Pseudomonas putida
KT2440
Authors: Quaranta Davide , McCarty Reid , Bandarian Vahe , Rensing Christopher
Published: May 2007

The genome sequences of several pseudomonads have revealed a gene cluster containing genes for a two-component heavy metal histidine sensor kinase and response regulator upstream of
cinA
and
cinQ
, which we show herein to encode a copper-containing azurin-like protein and a pre-Q
0
reductase, respectively. In the presence of copper,
Pseudomonas putida
KT2440 produces the CinA and CinQ proteins from a bicistronic mRNA. UV-visible spectra of CinA show features at 439, 581, and 719 nm, which is typical of the plastocyanin family of proteins. The redox potential of the protein was shown to be 456 ± 4 mV by voltametric titrations. Surprisingly, CinQ is a pyridine nucleotide-dependent nitrile oxidoreductase that catalyzes the conversion of pre-Q
0
to pre-Q
1
in the nucleoside queuosine biosynthetic pathway. Gene disruptions of
cinA
and
cinQ
did not lead to a significant increase in the copper sensitivity of
P. putida
KT2440 under the conditions tested. Possible roles of CinA and CinQ to help pseudomonads adapt and survive under prolonged copper stress are discussed.
The Extracytoplasmic Function-Type Sigma Factor SigM of
Corynebacterium glutamicum
ATCC 13032 Is Involved in Transcription of Disulfide Stress-Related Genes
Authors: Nakunst Diana , Larisch Christof , Hüser Andrea T. , Tauch Andreas , Pühler Alfred , Kalinowski Jörn
Published: May 2007

The gene for the extracytoplasmic function (ECF) sigma factor SigM was deleted from the chromosome of the gram-positive soil bacterium
Corynebacterium glutamicum
to elucidate the role of the SigM protein in the regulation of gene expression. Comparative DNA microarray hybridizations of the
C. glutamicum
wild type and
sigM
-deficient mutant
C. glutamicum
DN1 revealed 23 genes with enhanced expression in the
sigM
-proficient strain, encoding functions in the assembly of iron-sulfur clusters (
suf
operon), thioredoxin reductase (
trxB
), thioredoxins (
trxC
,
trxB1
), chaperones (
groES
,
groEL
,
clpB
), and proteins involved in the heat shock response (
hspR
,
dnaJ
,
grpE
). Deletion of the
sigM
gene rendered the
C. glutamicum
cells more sensitive to heat, cold, and the presence of the thiol oxidant diamide. Transcription of the
sigM
gene increased under different stress conditions, including heat shock, cold shock, and disulfide stress caused by diamide treatment, suggesting a regulatory role for SigM under thiol-oxidative stress conditions. Stress-responsive promoters were determined upstream of the
suf
operon and of the
trxB
,
trxC
, and
trxB1
genes. The deduced SigM consensus promoter is characterized by the −35 hexamer gGGAAT and the −10 hexamer YGTTGR. Transcription of the
sigM
gene is apparently controlled by the ECF sigma factor SigH, since a
sigH
mutant was unable to enhance the expression of
sigM
and the SigM regulon under thiol-oxidative stress conditions. A typical SigH-responsive promoter was mapped upstream of the
sigM
gene. The ECF sigma factor SigM is apparently part of a regulatory cascade, and its transcription is controlled by SigH under conditions of thiol-oxidative stress.
Mutational Analysis of theompAPromoter fromFlavobacterium johnsoniae
Authors: Chen Shicheng , Bagdasarian Michael , Kaufman Michael G. , Bates Adam K. , Walker Edward D.
Published: May 2007

Sequences that mediate the initiation of transcription inFlavobacteriumspecies are not well known. The majority of identifiedFlavobacteriumpromoter elements show homology to those of other members of the phylumBacteroidetes, but not of proteobacteria, and they function poorly inEscherichia coli. In order to analyze theFlavobacteriumpromoter structure systematically, we investigated the −33 consensus element, −7 consensus element, and spacer length of theFlavobacterium ompApromoter by measuring the effects of site-directed mutations on promoter activity. The nonconserved sequences in the spacer region and in regions close to the consensus motifs were randomized in order to determine their importance for promoter activity. Most of the base substitutions in these regions caused large decreases in promoter activity. The optimal −33/−7 motifs (TTTG/TANNTTTG) were identical toBacteroides fragilisσABfrconsensus −33/−7 promoter elements but lacked similarity to theE. coliσ70promoter elements. The length of the spacer separating the −33 and −7 motifs of theompApromoter also had a pronounced effect on promoter activity, with 19 bp being optimal. In addition to the consensus promoter elements and spacer length, the GC content of the core promoter sequences had a pronounced effect onFlavobacteriumpromoter activity. This information was used to conduct a scan of theFlavobacterium johnsoniaeandB. fragilisgenomes for putative promoters, resulting in 188 hits inB. fragilisand 109 hits inF. johnsoniae.
An Antibiotic-Inducible Cell Wall-Associated Protein That Protects
Bacillus subtilis
from Autolysis
Authors: Salzberg Letal I. , Helmann John D.
Published: May 2007

In
Bacillus subtilis
, antibiotics that impair cell wall synthesis induce a characteristic stress response including the σ
W
and σ
M
regulons and the previously uncharacterized
yoeB
gene. Here we demonstrate that YoeB is a cell wall-associated protein with weak sequence similarity to a noncatalytic domain of class B penicillin-binding proteins. A
yoeB
-null mutant exhibits an increased rate of autolysis in response to cell wall-targeting antibiotics or nutrient depletion. This phenotype does not appear to be correlated with gross alterations in peptidoglycan structure or levels of autolysins. Promoter dissection experiments define a minimal region necessary for antibiotic-mediated induction of
yoeB
, and this region is highly conserved preceding
yoeB
homologs in close relatives of
B. subtilis
. These results support a model in which induction of YoeB in response to cell envelope stress decreases the activity of autolysins and thereby reduces the rate of antibiotic-dependent cell death.
Contribution of the Mannan Backbone of Cryptococcal Glucuronoxylomannan and a Glycolytic Enzyme ofStaphylococcus aureusto Contact-Mediated Killing ofCryptococcus neoformans
Authors: Ikeda Reiko , Saito Fumito , Matsuo Miki , Kurokawa Kenji , Sekimizu Kazuhisa , Yamaguchi Masashi , Kawamoto Susumu
Published: May 2007

The fungal pathogenCryptococcus neoformansis killed by the bacteriumStaphylococcus aureus, and the killing is inhibited by soluble capsular polysaccharides. To investigate the mechanism of killing, cells in coculture were examined by scanning and transmission electron microscopy.S. aureusattached to the capsule ofC. neoformans, and the ultrastructure of the attachedC. neoformanscells was characteristic of dead cells. To identify the molecules that contributed to the fungal-bacterial interaction, we treated each with NaIO4or protease. Treatment ofC. neoformanswith NaIO4promoted adherence. It was inferred that cleavage of xylose and glucuronic acid side chains of glucuronoxylomannan (GXM) allowedS. aureusto recognize mannose residues in the backbone, which resisted periodate oxidation. On the other hand, treatment ofS. aureuswith protease decreased adherence, suggesting that protein contributed to attachment inS. aureus. In confirmation, side chain-cleaved polysaccharide or defined α-(1→3)-mannan inhibited the killing at lower concentrations than native GXM did. Also, these polysaccharides reduced the adherence of the two species and induced clumping of pureS. aureuscells. α-(1→3)-Mannooligosaccharides with a degree of polymerization (DP) of ≥3 induced cluster formation ofS. aureusin a dose-dependent manner. Surface plasmon resonance analyses showed interaction of GXM and surface protein fromS. aureus; the interaction was inhibited by oligosaccharides with a DP of ≥3. Conformations of α-(1→3) oligosaccharides were predicted. The three-dimensional structures of mannooligosaccharides larger than triose appeared curved and could be imagined to be recognized by a hypothetical staphylococcal lectin. Native polyacrylamide gel electrophoresis of staphylococcal protein followed by electroblotting, enzyme-linked immunolectin assay, protein staining, and N-terminal amino acid sequencing suggested that the candidate protein was triosephosphate isomerase (TPI). The enzymatic activities were confirmed by using whole cells ofS. aureus. TPI point mutants ofS. aureusdecreased the ability to interact withC. neoformans. Thus, TPI onS. aureusadheres to the capsule ofC. neoformansby recognizing the structure of mannotriose units in the backbone of GXM; we suggest that this contact is required for killing ofC. neoformans.
Role of Peptidoglycan Amidases in the Development and Morphology of the Division Septum in
Escherichia coli
Authors: Priyadarshini Richa , de Pedro Miguel A. , Young Kevin D.
Published: May 2007

Escherichia coli
contains multiple peptidoglycan-specific hydrolases, but their physiological purposes are poorly understood. Several mutants lacking combinations of hydrolases grow as chains of unseparated cells, indicating that these enzymes help cleave the septum to separate daughter cells after cell division. Here, we confirm previous observations that in the absence of two or more amidases, thickened and dark bands, which we term septal peptidoglycan (SP) rings, appear at division sites in isolated sacculi. The formation of SP rings depends on active cell division, and they apparently represent a cell division structure that accumulates because septal synthesis and hydrolysis are uncoupled. Even though septal constriction was incomplete, SP rings exhibited two properties of mature cell poles: they behaved as though composed of inert peptidoglycan, and they attracted the IcsA protein. Despite not being separated by a completed peptidoglycan wall, adjacent cells in these chains were often compartmentalized by the inner membrane, indicating that cytokinesis could occur in the absence of invagination of the entire cell envelope. Finally, deletion of penicillin-binding protein 5 from amidase mutants exacerbated the formation of twisted chains, producing numerous cells having septa with abnormal placements and geometries. The results suggest that the amidases are necessary for continued peptidoglycan synthesis during cell division, that their activities help create a septum having the appropriate geometry, and that they may contribute to the development of inert peptidoglycan.
Release of the Lipopolysaccharide Deacylase PagL from Latency Compensates for a Lack of Lipopolysaccharide Aminoarabinose Modification-Dependent Resistance to the Antimicrobial Peptide Polymyxin B inSalmonella enterica
Authors: Kawasaki Kiyoshi , China Kotaro , Nishijima Masahiro
Published: May 2007

Salmonella entericamodifies its lipopolysaccharide (LPS), including the lipid A portion, to adapt to its environments. The lipid A 3-O-deacylase PagL exhibits latency; deacylation of lipid A is not usually observed in vivo despite the expression of PagL, which is under the control of a two-component regulatory system, PhoP-PhoQ. In contrast, PagL is released from latency inpmrAandpmrEmutants, both of which are deficient in aminoarabinose-modified lipid A, although the biological significance of this is not clear. The attachment of aminoarabinose to lipid A decreases the net anionic charge at the membrane's surface and reduces electrostatic repulsion between neighboring LPS molecules, leading to increases in bacterial resistance to cationic antimicrobial peptides, including polymyxin B. Here we examined the effects of the release of PagL from latency on resistance to polymyxin B. ThepmrA pagLandpmrE pagLdouble mutants were more susceptible to polymyxin B than were the parentalpmrAandpmrEmutants, respectively. Furthermore, introduction of the PagL expression plasmid into thepmrA pagLdouble mutant increased the resistance to polymyxin B. In addition, PagL-dependent deacylation of lipid A was observed in a mutant in which lipid A could not be modified with phosphoethanolamine, which partly contributes to the PmrA-dependent resistance to polymyxin B. These results, taken together, suggest that the release of PagL from latency compensates for the loss of resistance to polymyxin B that is due to a lack of other modifications to LPS.
Additive Effects of SbcCD and PolX Deficiencies in the In Vivo Repair of DNA Double-Strand Breaks inDeinococcus radiodurans
Authors: Bentchikou Esma , Servant Pascale , Coste Geneviève , Sommer Suzanne
Published: May 2007

Orthologs of proteins SbcD (Mre11) and SbcC (Rad50) exist in all kingdoms of life and are involved in a wide variety of DNA repair and maintenance functions, including homologous recombination and nonhomologous end joining. Here, we have inactivated thesbcCand/orsbcDgenes ofDeinococcus radiodurans, a highly radioresistant bacterium able to mend hundreds of radiation-induced DNA double-strand breaks (DSB). Mutants devoid of the SbcC and/or SbcD proteins displayed reduced survival and presented a delay in kinetics of DSB repair and cell division following γ-irradiation. It has been recently reported thatD. radioduransDNA polymerase X (PolX) possesses a structure-modulated 3′-to-5′ exonuclease activity reminiscent of specific nuclease activities displayed by the SbcCD complex fromEscherichia coli. We constructed a double mutant devoid of SbcCD and PolX proteins. The double-mutant ΔsbcCDΔpolXDr(whereDrindicatesD. radiodurans) bacteria are much more sensitive to γ-irradiation than the single mutants, suggesting that the deinococcal SbcCD and PolX proteins may play important complementary roles in processing damaged DNA ends. We propose that they are part of a backup repair system acting to rescue cells containing DNA lesions that are excessively numerous or difficult to repair.
Inspiratory muscle training: a simple cost-effective treatment for inspiratory stridor: Table 1
Authors: Dickinson John , Whyte Greg , McConnell Alison
Published: May 2007

This case study describes the support given to a British elite athlete in the build up to the 2004 Athens Olympic Games. The athlete had complained of breathing symptoms during high intensity training that led to a reduction in performance and premature cessation of training. Following a negative eucapnic voluntary hyperpnoea challenge and observation during high intensity exercise, the athlete was diagnosed with inspiratory stridor. Inspiratory muscle training (IMT) was implemented to attenuate the inspiratory stridor. Following an 11-week IMT programme, the athlete had a 31% increase in mouth inspiratory pressure and a reduction in recovery between high intensity sprints. The athlete reported a precipitous fall in symptoms and was able to complete high intensity training without symptoms. This case shows that IMT is a suitable cost-effective intervention for athletes who present with inspiratory stridor.
Lung abscess in a professional rugby player: an illustration of overtraining syndrome?
Authors: Castinel Bernard H , Adam Philippe , Prat Christophe , Mourlanette Pierre
Published: May 2007

As in other endurance sports, the intensity of training sessions and the pace of competition has significantly increased since rugby union became a professional sport. The case history is presented of a professional rugby player who was diagnosed with septicaemia and a lung abscess following an infected wound to the ear. The symptoms only resolved after a large dose of antibiotics and 3 months of rest. It is hypothesised that this may be an example of overtraining syndrome, but complementary blood analyses would be necessary to confirm this. The case underlines the importance of clinically assessing the individual capacity of players to recover, in order to prevent overtraining and to maintain a high level of performance during the whole season.
Prognostic significance of p21WAF1/CIP1, p27Kip1, p53 and E-cadherin expression in gastric cancer
Authors: Gamboa-Dominguez Armando , Seidl Stefan , Reyes-Gutierrez Edgardo , Hermannstädter Christine , Quintanilla-Martinez Leticia , Busch Raymonde , Höfler Heinz , Fend Falko , Luber Birgit
Published: May 2007

Background:Gastric carcinoma is characterised by numerous genetic and epigenetic alterations that influence cell cycle progression, apoptosis and DNA repair. These alterations include down-regulation of the cyclin-dependent kinase (CDK) inhibitors p21WAF1/CIP1and p27Kip1, and mutations of the tumour suppressor protein p53 and the cell adhesion molecule E-cadherin. Combined evaluation of the prognostic significance of these alterations has not been reported in Mexican Mestizo patients.

Aims:To evaluate p21WAF1/CIP1, p27Kip1, p53 and E-cadherin protein expression, including mutant E-cadherin variants with deletion of exon 8 (del 8) or 9 (del 9), in gastric cancer from Mexican patients.

Methods:Immunohistochemistry for the above-mentioned markers, including mutation-specific E-cadherin antibodies, was carried out in 69 gastric carcinomas; expression levels were correlated with histotype, tumour stage and prognosis.

Results:Expression of p21WAF1/CIP1alone or in combination with p27Kip1or in the absence of p53 was associated with favourable prognosis. Staining ofdel 8anddel 9E-cadherin was found exclusively in patients negative for p53 and positive for p21WAF1/CIP1, suggesting that the p21WAF1/CIP1regulatory function of p53 was intact.

Conclusion:Combined evaluation of the prognostic significance of cell cycle regulators and E-cadherin should be performed. Even though patients negative for p53 and positive for p21WAF1/CIP1have a favourable prognosis, it may have a negative influence on prognosis if they acquire in addition E-cadherin mutations which have been shown previously to be associated with poor survival.
Progressive derailment of cell cycle regulators in endometrial carcinogenesis
Authors: Horrée N , van Diest P J , van der Groep P , Sie-Go D M D S , Heintz A P M
Published: May 2007

Background:

Derailments of the control mechanisms of the cell cycle can initiate carcinogenesis, and play a role in progression to cancer.

Aim:

To explore the expression of cell cycle proteins in normal, premalignant and malignant endometrial lesions representing the morphologically well defined stepwise model of human endometrial carcinogenesis

Methods:

Observational study. Paraffin-embedded specimens from inactive endometrium (n = 16), endometrial hyperplasia (n = 23) and endometrioid endometrial carcinoma (n = 39) were stained immunohistochemically for cyclin A, cyclin B1, cyclin D1, cyclin E, cdk2, p16, p21, p27, p53 and Ki67(MIB-1)). Differences in expression between the tissues, and correlation with classical prognostic factors for the carcinomas were analysed.

Results:

Expression of cyclin A and Ki67 gradually increased from normal through hyperplasia to carcinoma, indicating that proliferation increases over the carcinogenetic spectrum. cdk2, p16 and p21 gradually increased from normal through hyperplasia to carcinoma, indicating their potential importance in both early and late carcinogenesis. Cyclin D1, cyclin E and p53 especially increased and p27 decreased from hyperplasia to carcinoma, underlining their role in late carcinogenesis. In cancers, expression of cyclin A, p53 and Ki67 was positively correlated to grade, and cyclin A was positively correlated with cdk2, p21, Ki67, cyclin E and p53.

Conclusion:

During (endometrioid) endometrial carcinogenesis, there is increasing proliferation paralleled by progressive derailment of cyclin B1, cyclin D1, cyclin E, p16, p21, p27, p53, and cdk2, indicating the importance of these cell cycle regulators in endometrial carcinogenesis.
A growing burden: the pathogenesis, investigation and management of non-alcoholic fatty liver disease
Authors: Riley P , O’Donohue J , Crook M
Published: May 2007

Non-alcoholic fatty liver disease (NAFLD) is the most common hepatic disorder in western countries, and its incidence is increasing. This review outlines the significant health burden posed by NAFLD and discusses what is presently known about its pathogenesis, including the roles of the metabolic syndrome, obesity, insulin resistance, hepatic steatosis, reactive oxygen species, inflammatory cytokines and adipocytokines. The way in which NAFLD is clinically diagnosed is described, and areas of uncertainty surrounding its investigation are identified, before discussing the relative merits of the limited treatment options available and looking ahead to potential therapeutic strategies for the future.
Immune factors involved in the cervical immune response in the HIV/HPV co-infection
Authors: Nicol A F , Nuovo G J , Salomão-Estevez A , Grinsztein B , Tristão A , Russomano F , Lapa e Silva J R , Oliveira M P , Pirmez C
Published: May 2007

Aims:

Immune factors influencing the progression of cervical intraepithelial neoplasia (CIN) to cancer remain poorly defined. This study investigates the expression of RANTES, MIP1α, COX1, COX2, STAT3, TGFβRI, IL10R, TNFαRII and TLR4 in the cervical immune response in HIV/HPV (human papillomavirus) co-infected women.

Methods:

Cervical biopsies of 36 patients were assayed by immunohistochemistry, and the Ventana Benchmark System was used for HIV-nef detection.

Results:

Cervices from HIV-positive patients exhibited nef in cells mainly around blood vessels, and showed a decreased expression of all the immune factors tested except IL10R and STAT3, while RANTES (5.54 cells/mm2) was highly expressed in comparison with controls (1.41 cells/mm2, p = 0.028). COX1 was decreased in the HIV/HPV- (0.32 cells/mm2, p = 0.017) and HPV-infected patients (0.21 cells/mm2, p = 0.015) compared with controls (3.28 cells/mm2).

Conclusions:

It is suggested that RANTES in HIV/HPV co-infection may influence the development of CIN leading to progression to cervical cancer.