bluuebunny/crossref_metadata_2025_split huggingface dataset:
Flagellar Formation in C-Ring-Defective Mutants by Overproduction of FliI, the ATPase Specific for Flagellar Type III Secretion
Authors: Konishi Manabu , Kanbe Masaomi , McMurry Jonathan L. , Aizawa Shin-Ichi 
Published: August 2009

ABSTRACT

The flagellar cytoplasmic ring (C ring), which consists of three proteins, FliG, FliM, and FliN, is located on the cytoplasmic side of the flagellum. The C ring is a multifunctional structure necessary for flagellar protein secretion, torque generation, and switching of the rotational direction of the motor. The deletion of any one of the
fliG
,
fliM
, and
fliN
genes results in a Fla
−
phenotype. Here, we show that the overproduction of the flagellum-specific ATPase FliI overcomes the inability of basal bodies with partial C-ring structures to produce complete flagella. Flagella made upon FliI overproduction were paralyzed, indicating that an intact C ring is essential for motor function. In FliN- or FliM-deficient mutants, flagellum production was about 10% of the wild-type level, while it was only a few percent in FliG-deficient mutants, suggesting that the size of partial C rings affects the extent of flagellation. For flagella made in C-ring mutants, the hook length varied considerably, with many being markedly shorter or longer than that of the wild type. The broad distribution of hook lengths suggests that defective C rings cannot control the hook length as tightly as the wild type even though FliK and FlhB are both intact.
Homologues of the
Bacillus subtilis
SpoVB Protein Are Involved in Cell Wall Metabolism
Authors: Vasudevan Pradeep , McElligott Jessica , Attkisson Christa , Betteken Michael , Popham David L. 
Published: August 2009

ABSTRACT

Members of the COG2244 protein family are integral membrane proteins involved in synthesis of a variety of extracellular polymers. In several cases, these proteins have been suggested to move lipid-linked oligomers across the membrane or, in the case of
Escherichia coli
MviN, to flip the lipid II peptidoglycan precursor.
Bacillus subtilis
SpoVB was the first member of this family implicated in peptidoglycan synthesis and is required for spore cortex polymerization. Three other COG2244 members with high similarity to SpoVB are encoded within the
B. subtilis
genome. Mutant strains lacking any or all of these genes (
yabM
,
ykvU
, and
ytgP
) in addition to
spoVB
are viable and produce apparently normal peptidoglycan, indicating that their function is not essential in
B. subtilis
. Phenotypic changes associated with loss of two of these genes suggest that they function in peptidoglycan synthesis. Mutants lacking YtgP produce long cells and chains of cells, suggesting a role in cell division. Mutants lacking YabM exhibit sensitivity to moenomycin, an antibiotic that blocks peptidoglycan polymerization by class A penicillin-binding proteins. This result suggests that YabM may function in a previously observed alternate pathway for peptidoglycan strand synthesis.
Homotaurine Metabolized to 3-Sulfopropanoate in
Cupriavidus necator
H16: Enzymes and Genes in a Patchwork Pathway
Authors: Mayer Jutta , Cook Alasdair M. 
Published: August 2009

ABSTRACT

Homotaurine (3-aminopropanesulfonate), a natural product and an analogue of GABA (4-aminobutyrate), was found to be a sole source of nitrogen for
Cupriavidus necator
(
Ralstonia eutropha
) H16, whose genome sequence is known. Homotaurine nitrogen was assimilated into cell material, and the quantitative fate of the organosulfonate was sulfopropanoate, which was recovered in the growth medium. The first scalar reaction was shown to be inducible homotaurine:2-oxoglutarate aminotransferase, which released 3-sulfopropanal from homotaurine. This aminotransferase was purified to homogeneity and characterized. Peptide mass fingerprinting yielded locus tag H16_B0981, which was annotated
gabT
, for GABA transaminase (EC 2.6.1.19). Inducible, NAD(P)
+
-coupled 3-sulfopropanal dehydrogenase, which yielded 3-sulfopropanoate from 3-sulfopropanal, was also purified and characterized. Peptide mass fingerprinting yielded locus tag H16_B0982, which was annotated
gabD1
, for succinate-semialdehyde dehydrogenase (EC 1.2.1.16). GabT and GabD1 were each induced during growth with GABA, and cotranscription of
gabTD
was observed. In other organisms, regulator GabC or GabR is encoded contiguous with
gabTD
: candidate GabR′ was found in strain H16 and in many other organisms. An orthologue of the GABA permease (GabP), established in
Escherichia coli
, is present at H16_B1890, and it was transcribed constitutively. We presume that GabR′PTD are responsible for the inducible metabolism of homotaurine to intracellular 3-sulfopropanoate. The nature of the exporter of this highly charged compound was unclear until we realized from the sodium dodecyl sulfate-polyacrylamide gel electrophoresis data that sulfoacetaldehyde acetyltransferase (EC 2.3.3.15; H16_B1872) was strongly induced during growth with homotaurine and inferred that the sulfite exporter encoded at the end of the gene cluster (H16_B1874) has a broad substrate range that includes 3-sulfopropanoate.
Identification of the Site-Specific DNA Invertase Responsible for the Phase Variation of SusC/SusD Family Outer Membrane Proteins in
Bacteroides fragilis
Authors: Nakayama-Imaohji Haruyuki , Hirakawa Hideki , Ichimura Minoru , Wakimoto Shin , Kuhara Satoru , Hayashi Tetsuya , Kuwahara Tomomi 
Published: August 2009

ABSTRACT

The human gut microbe
Bacteroides fragilis
can alter the expression of its surface molecules, such as capsular polysaccharides and SusC/SusD family outer membrane proteins, through reversible DNA inversions. We demonstrate here that DNA inversions at 12 invertible regions, including three gene clusters for SusC/SusD family proteins, were controlled by a single tyrosine site-specific recombinase (Tsr0667) encoded by BF0667 in
B. fragilis
strain YCH46. Genetic disruption of BF0667 diminished or attenuated shufflon-type DNA inversions at all three
susC
/
susD
genes clusters, as well as simple DNA inversions at nine other loci, most of which colocalized with
susC/susD
family genes. The inverted repeat sequences found within the Tsr0667-regulated invertible regions shared the consensus motif sequence AGTYYYN
4
GDACT. Tsr0667 specifically mediated the DNA inversions of 10 of the 12 regions, even under an
Escherichia coli
background when the invertible regions were exposed to BF0667 in
E. coli
cells. Thus, Tsr0667 is an additional globally acting DNA invertase in
B. fragilis
, which probably involves the selective expression of SusC/SusD family outer membrane proteins.
CebR as a Master Regulator for Cellulose/Cellooligosaccharide Catabolism Affects Morphological Development in
Streptomyces griseus
Authors: Marushima Kazuya , Ohnishi Yasuo , Horinouchi Sueharu 
Published: August 2009

ABSTRACT

Streptomyces griseus
mutants exhibiting deficient glucose repression of β-galactosidase activity on lactose-containing minimal medium supplemented with a high concentration of glucose were isolated. One of these mutants had a 12-bp deletion in
cebR
, which encodes a LacI/GalR family regulator. Disruption of
cebR
in the wild-type strain caused the same phenotype as the mutant, indicating that CebR is required for glucose repression of β-galactosidase activity. Recombinant CebR protein bound to a 14-bp inverted-repeat sequence (designated the CebR box) present in the promoter regions of
cebR
and the putative cellobiose utilization operon,
cebEFG-bglC
. The DNA-binding activity of CebR was impaired by cellooligosaccharides, including cellobiose, cellotriose, cellotetraose, cellopentaose, and cellohexaose. In agreement with this observation, transcription from the
cebE
and
cebR
promoters was greatly enhanced by the addition of cellobiose to the medium. Seven other genes containing one or two CebR boxes in their upstream regions were found in the
S. griseus
genome. Five of these genes encode putative secreted proteins: two cellulases, a cellulose-binding protein, a pectate lyase, and a protein of unknown function. These five genes and
cebEFG-bglC
were transcribed at levels 4 to 130 times higher in the Δ
cebR
mutant than in the wild-type strain, as determined by quantitative reverse transcription-PCR. These findings indicate that CebR is a master regulator of cellulose/cellooligosaccharide catabolism. Unexpectedly, the Δ
cebR
mutant formed very few aerial hyphae on lactose-containing medium, demonstrating a link between carbon source utilization and morphological development.
Characterization of the FtsZ-Interacting Septal Proteins SepF and Ftn6 in the Spherical-Celled Cyanobacterium
Synechocystis
Strain PCC 6803
Authors: Marbouty Martial , Saguez Cyril , Cassier-Chauvat Corinne , Chauvat Franck 
Published: August 2009

ABSTRACT

Assembly of the tubulin-like cytoskeletal protein FtsZ into a ring structure at midcell establishes the location of the nascent division sites in prokaryotes. However, it is not yet known how the assembly and contraction of the Z ring are regulated, especially in cyanobacteria, the environmentally crucial organisms for which only one FtsZ partner protein, ZipN, has been described so far. Here, we characterized SepF and Ftn6, two novel septal proteins, in the spherical-celled strain
Synechocystis
PCC 6803. Both proteins were found to be indispensable to
Synechocystis
sp. strain PCC 6803. The depletion of both SepF and Ftn6 resulted in delayed cytokinesis and the generation of giant cells but did not prevent FtsZ polymerization, as shown by the visualization of green fluorescent protein (GFP)-tagged FtsZ polymers. These GFP-tagged Z-ring-like structures often appeared to be abnormal, because these reporter cells respond to the depletion of either SepF or Ftn6 with an increased abundance of total, natural, and GFP-tagged FtsZ proteins. In agreement with their septal localization, we found that both SepF and Ftn6 interact physically with FtsZ. Finally, we showed that SepF, but not Ftn6, stimulates the formation and/or stability of FtsZ polymers in vitro.
Stress-Induced β-Lactam Antibiotic Resistance Mutation and Sequences of Stationary-Phase Mutations in the
Escherichia coli
Chromosome
Authors: Petrosino Joseph F. , Galhardo Rodrigo S. , Morales Liza D. , Rosenberg Susan M. 
Published: August 2009

ABSTRACT

In some enterobacterial pathogens, but not in
Escherichia coli
, loss-of-function mutations are a common route to clinically relevant β-lactam antibiotic resistance. We previously constructed an assay system for studying enterobacterial β-lactam resistance mutations using the well-developed genetics of
E. coli
by integrating enterobacterial
ampRC
genes into the
E. coli
chromosome. Like the cells of other enterobacteria,
E. coli
cells acquire β-lactam resistance by
ampD
mutation. Here we show that starvation and stress responses provoke
ampD
β-lactam resistance mutagenesis. When starved on lactose medium, Lac
−
strains used in mutagenesis studies accumulate
ampD
β-lactam resistance mutations independent of Lac reversion. DNA double-strand break repair (DSBR) proteins and the SOS and RpoS stress responses are required for this mutagenesis, in agreement with the results obtained for
lac
reversion in these cells. Surprisingly, the stress-induced
ampD
mutations require DinB (DNA polymerase IV) and partially require error-prone DNA polymerase V, unlike
lac
mutagenesis, which requires only DinB. This assay demonstrates that real-world stressors, such as starvation, can induce clinically relevant resistance mutations. Finally, we used the
ampD
system to observe the true forward-mutation sequence spectrum of DSBR-associated stress-induced mutagenesis, for which previously only frameshift reversions were studied. We found that base substitutions outnumber frameshift mutations, as seen in other experimental systems showing stress-induced mutagenesis. The important evolutionary implication is that not only loss-of-function mutations but also change-of-function mutations can be generated by this mechanism.
NtrC-Dependent Regulatory Network for Nitrogen Assimilation in
Pseudomonas putida
Authors: Hervás Ana B. , Canosa Inés , Little Richard , Dixon Ray , Santero Eduardo 
Published: August 2009

ABSTRACT

Pseudomonas putida
KT2440 is a model strain for studying bacterial biodegradation processes. However, very little is known about nitrogen regulation in this strain. Here, we show that the nitrogen regulatory NtrC proteins from
P. putida
and
Escherichia coli
are functionally equivalent and that substitutions leading to partially active forms of enterobacterial NtrC provoke the same phenotypes in
P. putida
NtrC.
P. putida
has only a single P
II
-like protein, encoded by
glnK
, whose expression is nitrogen regulated. Two contiguous NtrC binding sites located upstream of the σ
N
-dependent
glnK
promoter have been identified by footprinting analysis. In vitro experiments with purified proteins demonstrated that
glnK
transcription was directly activated by NtrC and that open complex formation at this promoter required integration host factor. Transcription of genes orthologous to enterobacterial
codB
,
dppA
, and
ureD
genes, whose transcription is dependent on σ
70
and which are activated by Nac in
E. coli
, has also been analyzed for
P. putida
. Whereas
dppA
does not appear to be regulated by nitrogen via NtrC, the
codB
and
ureD
genes have σ
N
-dependent promoters and their nitrogen regulation was exerted directly by NtrC, thus avoiding the need for Nac, which is missing in this bacterial species. Based upon these results, we propose a simplified nitrogen regulatory network in
P. putida
(compared to that in enterobacteria), which involves an indirect-feedback autoregulation of
glnK
using NtrC as an intermediary.
mosR
, a Novel Transcriptional Regulator of Hypoxia and Virulence in
Mycobacterium tuberculosis
Authors: Abomoelak Bassam , Hoye Elizabeth A. , Chi Jing , Marcus Sarah A. , Laval Francoise , Bannantine John P. , Ward Sarah K. , Daffé Mamadou , Liu Hong Di , Talaat Adel M. 
Published: August 2009

ABSTRACT

Latent tuberculosis represents a high-risk burden for one-third of the world population. Previous analysis of murine tuberculosis identified a novel transcriptional regulator encoded by Rv0348 that could control the establishment of persistent tuberculosis. Disruption of the Rv0348 gene from the genome of the virulent H37Rv strain of
Mycobacterium tuberculosis
revealed a global impact on the transcriptional profiles of 163 genes, including induction of the mammalian cell entry (
mce1
) operon and the repression of a significant number of genes involved in hypoxia and starvation responses. Nonetheless, gel shift assays did not reveal direct binding between Rv0348 and a set of regulated promoters, suggesting an indirect regulatory role. However, when expressed in
Mycobacterium smegmatis
, the Rv0348 transcripts were significantly responsive to different levels of hypoxia and the encoded protein was shown to regulate genes involved in hypoxia [e.g., Rv3130c (
tgs1
)] and intracellular survival (e.g.,
mce1
), among other genes. Interestingly, the colonization level of the Δ
mosR
mutant strain was significantly lower than that of the wild-type strain of
M. tuberculosis
, suggesting its attenuation in the murine model of tuberculosis. Taken together, our analyses indicated that the Rv0348 gene encodes a novel transcriptional factor that regulates several operons involved in mycobacterial survival, especially during hypoxia; hence, we propose that Rv0348 be renamed
mosR
for
r
egulator of
m
ycobacterial
o
perons of
s
urvival.
Contributions of Zur-Controlled Ribosomal Proteins to Growth under Zinc Starvation Conditions
Authors: Gabriel Scott E. , Helmann John D. 
Published: August 2009

ABSTRACTMaintaining intracellular zinc levels is critical, because zinc serves as a cofactor for many required enzymes and is toxic in excess.Bacillus subtilisZur, a Fur family repressor, controls the zinc starvation response including two ribosomal proteins (r-proteins) paralogous to L31 and S14. Biochemical analyses suggest that Zur-controlled r-proteins (which lack the two CXXC metal-binding motifs) may functionally replace their cognate zinc-requiring proteins during zinc limitation. We demonstrate here that Zur regulates the expression of an additional r-protein paralog, RpmGC (L33c), and, using strains defective in zinc uptake, we investigate the physiological contributions of all three Zur-regulated r-proteins. In the 168 lineage,rpmGCis a pseudogene containing a frameshift mutation. Correction of this mutation allows expression of a functional L33c that can suppress the poor growth phenotype of anrpmGA rpmGB(encoding L33a, L33b) double mutant. Similarly, we provide physiological evidence in support of the “failsafe” model (Y. Natori et al., Mol. Microbiol. 63:294-307, 2007) in which the Zur-regulated S14 paralog YhzA allows continued ribosome synthesis when there is insufficient zinc to support S14 function. The L31 paralog YtiA can replace L31 and complement the growth defect of anrpmEmutant (Nanamiya et al., Mol. Microbiol. 52:273-283). We show that, under zinc starvation conditions, derepression of YtiA significantly increases the growth of cells in which preexisting ribosomes carry, as the sole L31 protein, RpmE (containing zinc), but not if they carry YtiA (which lacks zinc). These results support a direct and physiologically relevant role for YtiA in mobilizing zinc from ribosomes.
Involvement of NADH:Acceptor Oxidoreductase and Butyryl Coenzyme A Dehydrogenase in Reversed Electron Transport during Syntrophic Butyrate Oxidation by
Syntrophomonas wolfei
Authors: Müller Nicolai , Schleheck David , Schink Bernhard 
Published: August 2009

ABSTRACT

Methanogenic oxidation of butyrate to acetate requires a tight cooperation between the syntrophically fermenting
Syntrophomonas wolfei
and the methanogen
Methanospirillum hungatei
, and a reversed electron transport system in
S. wolfei
was postulated to shift electrons from butyryl coenzyme A (butyryl-CoA) oxidation to the redox potential of NADH for H
2
generation. The metabolic activity of butyrate-oxidizing
S. wolfei
cells was measured via production of formazan and acetate from butyrate, with 2,3,5-triphenyltetrazolium chloride as electron acceptor. This activity was inhibited by trifluoperazine (TPZ), an antitubercular agent known to inhibit NADH:menaquinone oxidoreductase. In cell extracts of
S. wolfei
, the oxidation of NADH could be measured with quinones, viologens, and tetrazolium dyes as electron acceptors, and also this activity was inhibited by TPZ. The TPZ-sensitive NADH:acceptor oxidoreductase activity appeared to be membrane associated but could be dissociated from the membrane as a soluble protein and was semipurified by anion-exchange chromatography. Recovered proteins were identified by peptide mass fingerprinting, which indicated the presence of an NADH:acceptor oxidoreductase as part of a three-component [FeFe] hydrogenase complex and a selenocysteine-containing formate dehydrogenase. Furthermore, purification of butyryl-CoA dehydrogenase (Bcd) activity and peptide mass fingerprinting revealed two Bcd proteins different from the Bcd subunit of the Bcd/electron-transfer flavoprotein complex (Bcd/EtfAB) predicted from the genome sequence of
S. wolfei
. The results suggest that syntrophic oxidation of butyrate in
S. wolfei
involves a membrane-associated TPZ-sensitive NADH:acceptor oxidoreductase as part of a hydrogenase complex similar to the recently discovered “bifurcating” hydrogenase in
Thermotoga maritima
and butyryl-CoA dehydrogenases that are different from Bcd of the Bcd/EtfAB complex.
Trichosporon mycotoxinivorans
, a Novel Respiratory Pathogen in Patients with Cystic Fibrosis
Authors: Hickey Patrick W. , Sutton Deanna A. , Fothergill Annette W. , Rinaldi Michael G. , Wickes Brian L. , Schmidt Howard J. , Walsh Thomas J. 
Published: August 2009

ABSTRACT

This report describes the molecular epidemiology, in vitro susceptibility, colonial and microscopic morphologies, and biochemical features of
Trichosporon mycotoxinivorans
, a newly recognized pathogen that appears to have a propensity for patients with cystic fibrosis. The index patient died with histologically documented
Trichosporon
pneumonia complicating cystic fibrosis. This is also the first report of disease caused by a
Trichosporon
species in a nontransplant patient with cystic fibrosis. As
T. mycotoxinivorans
has not previously been recognized as a respiratory pathogen, the significance of its recovery from sputum samples was not initially appreciated. Genetic analysis of archived clinical samples found three additional cases of
T. mycotoxinivorans
infection which had previously been identified as other members of the genus. An additional isolate of
T. mycotoxinivorans
was identified from a clinical sample on initial testing. Three of these four cases were also patients with cystic fibrosis. All isolates had MICs at 48 h of amphotericin B of ≥1 μg/ml and of echinocandins of ≥16 μg/ml, but they displayed various susceptibilities to the triazoles. In summary,
Trichosporon mycotoxinivorans
is a newly recognized human pathogen that is associated with cystic fibrosis.
Comparative Evaluation of the ExaVir Load Version 3 Reverse Transcriptase Assay for Measurement of Human Immunodeficiency Virus Type 1 Plasma Load
Authors: Labbett Wendy , Garcia-Diaz Ana , Fox Zoe , Clewley Gillian S. , Fernandez Thomas , Johnson Margaret , Geretti Anna Maria 
Published: August 2009

ABSTRACT

In resource-limited settings, the virological monitoring of antiretroviral therapy is limited by high cost and the lack of infrastructure. The Cavidi ExaVir Load assay employs a simple and inexpensive enzyme-linked immunosorbent assay format to measure human immunodeficiency virus (HIV) reverse transcriptase activity, which correlates with plasma RNA load. The version 3 assay has been described as having improved precision and sensitivity. There are limited data on its performance relative to those of current real-time assays. Our objective was to compare HIV type 1 (HIV-1) RNA load measurement in plasma by ExaVir Load version 3 (designated ExaVir), Abbott M2000sp/M2000rt RealTime HIV-1 assay (designated RealTime), and Roche COBAS Ampliprep/COBAS TaqMan HIV-1 version 1 assay (designated TaqMan). Plasma from 119 patients (34 with subtype B infection, 85 with non-subtype B infection [A-H, CRF01, CRF02, CRF06, CRF12, CRF14, and complex]; 48 subjects were treatment experienced, 71 were naive) and serial dilutions of the second international standard (IS) were tested. Assay relationship and agreement were determined by linear regression, correlation analysis, and the Bland-Altman method. The ExaVir assay quantified 77/83 (92.8%) samples with viral loads of >2.3 log
10
copies/ml by the molecular assays. Results were linearly associated and strongly correlated with RealTime and TaqMan measurements (
R
of 0.94 and 0.92, respectively) for both subtype B (
R
of 0.97 and 0.95, respectively) and non-subtype B (
R
of 0.93 and 0.91, respectively) samples. Mean differences were 0.28 and 0.18 log
10
copies/ml in favor of the two molecular assays; 7/119 (5.9%) and 5/119 (4.2%) samples were outside the 95% level of agreement. ExaVir underquantified the IS by a mean of 0.2 (range, 0.0 to 0.5) log
10
copies/ml. The ExaVir assay showed excellent concordance with real-time molecular assays, offering a suitable option for virological monitoring in settings with limited infrastructure.
Community-Associated Methicillin-Resistant
Staphylococcus aureus
Mediastinitis
Authors: Calvano Tatjana P. , Ferraro David M. , Prakash Vidhya , Mende Katrin , Hospenthal Duane R. 
Published: August 2009

ABSTRACT

Community-associated methicillin (meticillin)-resistant
Staphylococcus aureus
(CA-MRSA) continues to emerge as a cause of serious infections, chiefly of the skin and soft tissues. We present the first documented case of CA-MRSA mediastinitis in an adult. Blood and mediastinal isolates were characterized as CA-MRSA by pulsed-field gel electrophoresis and susceptibility testing.