bluuebunny/crossref_metadata_2025_split huggingface dataset:
Identification of the Protease and the Turnover Signal Responsible for Cell Cycle-Dependent Degradation of the
Caulobacter
FliF Motor Protein
Authors: Grünenfelder Björn , Tawfilis Sherif , Gehrig Stefanie , Østerås Magne , Eglin Daniel , Jenal Urs 
Published: July 2004

ABSTRACT

Flagellar ejection is tightly coupled to the cell cycle in
Caulobacter crescentus
. The MS ring protein FliF, which anchors the flagellar structure in the inner membrane, is degraded coincident with flagellar release. Previous work showed that removal of 26 amino acids from the C terminus of FliF prevents degradation of the protein and interferes with flagellar assembly. To understand FliF degradation in more detail, we identified the protease responsible for FliF degradation and performed a high-resolution mutational analysis of the C-terminal degradation signal of FliF. Cell cycle-dependent turnover of FliF requires an intact
clpA
gene, suggesting that the ClpAP protease is required for removal of the MS ring protein. Deletion analysis of the entire C-terminal cytoplasmic portion of FliF C confirmed that the degradation signal was contained in the last 26 amino acids that were identified previously. However, only deletions longer than 20 amino acids led to a stable FliF protein, while shorter deletions dispersed over the entire 26 amino acids critical for turnover had little effect on stability. This indicated that the nature of the degradation signal is not based on a distinct primary amino acid sequence. The addition of charged amino acids to the C-terminal end abolished cell cycle-dependent FliF degradation, implying that a hydrophobic tail feature is important for the degradation of FliF. Consistent with this, ClpA-dependent degradation was restored when a short stretch of hydrophobic amino acids was added to the C terminus of stable FliF mutant forms.
Circadian Rhythms in the Thermophilic Cyanobacterium
Thermosynechococcus elongatus
: Compensation of Period Length over a Wide Temperature Range
Authors: Onai Kiyoshi , Morishita Megumi , Itoh Shino , Okamoto Kazuhisa , Ishiura Masahiro 
Published: July 2004

ABSTRACT

Proteins derived from the thermophilic cyanobacterium
Thermosynechococcus elongatus
BP-1, which performs plant-type oxygenic photosynthesis, are suitable for biochemical, biophysical, and X-ray crystallographic studies. We developed an automated bioluminescence real-time monitoring system for the circadian clock in the thermophilic cyanobacterium
T. elongatus
BP-1 that uses a bacterial luciferase gene set (Xl
luxAB
) derived from
Xenorhabdus luminescens
as a bioluminescence reporter gene. A promoter region of the
psbA1
gene of
T. elongatus
was fused to the Xl
luxAB
gene set and inserted into a specific targeting site in the genome of
T. elongatus
. The bioluminescence from the cells of the
psbA1
-reporting strain was measured by an automated monitoring apparatus with photomultiplier tubes. The strain exhibited the circadian rhythms of bioluminescence with a 25-h period length for at least 10 days in constant light and temperature. The rhythms were reset by light-dark cycle, and their period length was almost constant over a wide range of temperatures (30 to 60°C). Theses results indicate that
T. elongatus
has the circadian clock that is widely temperature compensated.
6S RNA Function Enhances Long-Term Cell Survival
Authors: Trotochaud Amy E. , Wassarman Karen M. 
Published: July 2004

ABSTRACT

6S RNA was identified in
Escherichia coli
>30 years ago, but the physiological role of this RNA has remained elusive. Here, we demonstrate that 6S RNA-deficient cells are at a disadvantage for survival in stationary phase, a time when 6S RNA regulates transcription. Growth defects were most apparent as a decrease in the competitive fitness of cells lacking 6S RNA. To decipher the molecular mechanisms underlying the growth defects, we have expanded studies of 6S RNA effects on transcription. 6S RNA inhibition of σ
70
-dependent transcription was not ubiquitous, in spite of the fact that the vast majority of σ
70
-RNA polymerase is bound by 6S RNA during stationary phase. The σ
70
-dependent promoters inhibited by 6S RNA contain an extended −10 promoter element, suggesting that this feature may define a class of 6S RNA-regulated genes. We also discovered a secondary effect of 6S RNA in the activation of σ
S
-dependent transcription at several promoters. We conclude that 6S RNA regulation of both σ
70
and σ
S
activities contributes to increased cell persistence during nutrient deprivation.
The Global Regulator Genes from Biocontrol Strain
Serratia plymuthica
IC1270: Cloning, Sequencing, and Functional Studies
Authors: Ovadis Marianna , Liu Xiaoguang , Gavriel Sagi , Ismailov Zafar , Chet Ilan , Chernin Leonid 
Published: July 2004

ABSTRACT

The biocontrol activity of various fluorescent pseudomonads towards plant-pathogenic fungi is dependent upon the GacA/GacS-type two-component system of global regulators and the RpoS transcription sigma factor. In particular, these components are required for the production of antifungal antibiotics and exoenzymes. To investigate the effects of these global regulators on the expression of biocontrol factors by plant-associated bacteria other than
Pseudomonas
spp.,
gacA/gacS
and
rpoS
homologues were cloned from biocontrol strain IC1270 of
Serratia plymuthica
, which produces a set of antifungal compounds, including chitinolytic enzymes and the antibiotic pyrrolnitrin. The nucleotide and deduced protein sequence alignments of the cloned
gacA/gacS-
like genes
—
tentatively designated
grrA
(global response regulation activator) and
grrS
(global response regulation sensor) and of the cloned
rpoS
gene revealed 64 to 93% identity with matching genes and proteins of the enteric bacteria
Escherichia coli
,
Pectobacterium carotovora
subsp.
carotovora
, and
Serratia marcescens. grrA
,
grrS
, and
rpoS
gene replacement mutants of strain IC1270 were deficient in the production of pyrrolnitrin, an exoprotease, and
N
-acylhomoserine lactone quorum-sensing signal molecules. However, neither mutant appeared to differ from the parental strain in the production of siderophores, and only
grrA
and
grrS
mutants were deficient in the production of a 58-kDa endochitinase, representing the involvement of other sigma factors in the regulation of strain IC1270's chitinolytic activity. Compared to the parental strain, the
grrA
,
grrS
, and
rpoS
mutants were markedly less capable of suppressing
Rhizoctonia solani
and
Pythium aphanidermatum
under greenhouse conditions, indicating the dependence of strain IC1270's biocontrol property on the GrrA/GrrS and RpoS global regulators.
Intraspecific Phylogeny and Lineage Group Identification Based on theprfAVirulence Gene Cluster ofListeria monocytogenes†
Authors: Ward Todd J. , Gorski Lisa , Borucki Monica K. , Mandrell Robert E. , Hutchins Jan , Pupedis Kitty 
Published: July 2004

ABSTRACTListeria monocytogenesis a serious food-borne pathogen that can cause invasive disease in humans and other animals and has been the leading cause of food recalls due to microbiological concerns in recent years. In order to test hypotheses regardingL.monocytogeneslineage composition, evolution, ecology, and taxonomy, a robust intraspecific phylogeny was developed based onprfAvirulence gene cluster sequences from 113L.monocytogenesisolates. The results of the multigene phylogenetic analyses confirm thatL. monocytogenescomprises at least three evolutionary lineages, demonstrate that lineages most frequently (lineage 1) and least frequently (lineage 3) associated with human listeriosis are sister-groups, and reveal for the first time that the human epidemic associated serotype 4b is prevalent among strains from lineage 1 and lineage 3. In addition, a PCR-based test for lineage identification was developed and used in a survey of food products demonstrating that the low frequency of association between lineage 3 isolates and human listeriosis cases likely reflects rarity of exposure and not reduced virulence for humans as has been previously suggested. However, prevalence data do suggest lineage 3 isolates may be better adapted to the animal production environment than the food-processing environment. Finally, analyses of haplotype diversity indicate that lineage 1 has experienced a purge of genetic variation that was not observed in the other lineages, suggesting that the threeL. monocytogeneslineages may represent distinct species within the framework of the cohesion species concept.
Replication of
Enterococcus faecalis
Pheromone-Responding Plasmid pAD1: Location of the Minimal Replicon and
oriV
Site and RepA Involvement in Initiation of Replication
Authors: Francia Maria Victoria , Fujimoto Shuhei , Tille Patricia , Weaver Keith E. , Clewell Don B. 
Published: July 2004

ABSTRACT

The hemolysin-determining plasmid pAD1 is a member of a widely disseminated family of highly conjugative elements commonly present in clinical isolates of
Enterococcus faecalis
. The determinants
repA
,
repB
, and
repC
, as well as adjacent iteron sequences, are believed to play important roles in pAD1 replication and maintenance. The
repA
gene encodes an initiator protein, whereas
repB
and
repC
encode proteins related to stability and copy number. The present study focuses specifically on
repA
and identifies a replication origin (
oriV
) within a central region of the
repA
determinant. A small segment of
repA
carrying
oriV
was able to support replication in
cis
of a plasmid vector otherwise unable to replicate, if an intact RepA was supplied in
trans
. We demonstrate that under conditions in which RepA is expressed from an artificial promoter, a segment of DNA carrying only
repA
is sufficient for stable replication in
E. faecalis
. We also show that RepA binds specifically to
oriV
DNA at several sites containing inverted repeat sequences (i.e., IR-1) and nonspecifically to single-stranded DNA, and related genetic analyses confirm that these sequences play an important role in replication. Finally, we reveal a relationship between the internal structure of RepA and its ability to recognize
oriV
. An in-frame deletion within
repA
resulting in loss of 105 nucleotides, including at least part of
oriV
, did not eliminate the ability of the altered RepA protein to initiate replication using an intact origin provided in
trans
. The relationship of RepA to other known initiator proteins is also discussed.
Induction of a Novel Class of Diacylglycerol Acyltransferases and Triacylglycerol Accumulation in
Mycobacterium tuberculosis
as It Goes into a Dormancy-Like State in Culture
Authors: Daniel Jaiyanth , Deb Chirajyoti , Dubey Vinod S. , Sirakova Tatiana D. , Abomoelak Bassam , Morbidoni Hector R. , Kolattukudy Pappachan E. 
Published: July 2004

ABSTRACT

Mycobacterium tuberculosis
enters the host by inhalation of an infectious aerosol and replicates in the alveolar macrophages until the host's immune defense causes bacteriostasis, which leads the pathogen to go into nonreplicative drug-resistant dormancy. The dormant pathogen can survive for decades till the host's immune system is weakened and active tuberculosis develops. Even though fatty acids are thought to be the major energy source required for the persistence phase, the source of fatty acids used is not known. We postulate that the pathogen uses triacylglycerol (TG) as a storage form of fatty acids. Little is known about the biosynthesis of TG in
M. tuberculosis
. We show that 15 mycobacterial genes that we identified as putative triacylglycerol synthase (
tgs
) when expressed in
Escherichia coli
showed TGS activity, and we report some basic catalytic characteristics of the most active enzymes. We show that several
tgs
genes are induced when the pathogen goes into the nonreplicative drug-resistant state caused by slow withdrawal of O
2
and also by NO treatment, which is known to induce dormancy-associated genes. The gene (Rv3130c) that shows the highest TGS activity when expressed in
E. coli
shows the highest induction by hypoxia and NO treatment. Biochemical evidence shows that TG synthesis and accumulation occur under both conditions. We conclude that TG may be a form of energy storage for use during long-term dormancy. Therefore, TG synthesis may be an appropriate target for novel antilatency drugs that can prevent the organism from surviving dormancy and thus assist in the control of tuberculosis.
A Type II Protein Secretory Pathway Required for Levansucrase Secretion by
Gluconacetobacter diazotrophicus
Authors: Arrieta Juan G. , Sotolongo Mailin , Menéndez Carmen , Alfonso Dubiel , Trujillo Luis E. , Soto Melvis , Ramírez Ricardo , Hernández Lázaro 
Published: July 2004

ABSTRACT

The endophytic diazotroph
Gluconacetobacter diazotrophicus
secretes a constitutively expressed levansucrase (LsdA, EC 2.4.1.10) to utilize plant sucrose. LsdA, unlike other extracellular levansucrases from gram-negative bacteria, is transported to the periplasm by a signal-peptide-dependent pathway. We identified an unusually organized gene cluster encoding at least the components LsdG, -O, -E, -F, -H, -I, -J, -L, -M, -N, and -D of a type II secretory system required for LsdA translocation across the outer membrane. Another open reading frame, designated
lsdX
, is located between the operon promoter and
lsdG
, but it was not identified in BLASTX searches of the DDBJ/EMBL/GenBank databases. The
lsdX
,
-G
, and -
O
genes were isolated from a cosmid library of strain SRT4 by complementation of an ethyl methanesulfonate mutant unable to transport LsdA across the outer membrane. The downstream genes
lsdE
,
-F
,
-H
,
-I
,
-J
, -
L
,
-M
,
-N
, and -
D
were isolated through chromosomal walking. The high G+C content (64 to 74%) and the codon usage of the genes identified are consistent with the G+C content and codon usage of the standard
G. diazotrophicus
structural gene. Sequence analysis of the gene cluster indicated that a polycistronic transcript is synthesized. Targeted disruption of
lsdG
,
lsdO
, or
lsdF
blocked LsdA secretion, and the bacterium failed to grow on sucrose. Replacement of Cys
162
by Gly at the C terminus of the pseudopilin LsdG abolished the protein functionality, suggesting that there is a relationship with type IV pilins. Restriction fragment length polymorphism analysis revealed conservation of the type II secretion operon downstream of the levansucrase-levanase (
lsdA
-
lsdB
) locus in 14
G. diazotrophicus
strains representing 11 genotypes recovered from four different host plants in diverse geographical regions. To our knowledge, this is the first report of a type II pathway for protein secretion in the
Acetobacteraceae
.
Comparative Whole-Genome Hybridization Reveals Genomic Islands inBrucellaSpecies
Authors: Rajashekara Gireesh , Glasner Jeremy D. , Glover David A. , Splitter Gary A. 
Published: July 2004

ABSTRACTBrucellaspecies are responsible for brucellosis, a worldwide zoonotic disease causing abortion in domestic animals and Malta fever in humans. Based on host preference, the genus is divided into six species.Brucella abortus,B. melitensis, andB. suisare pathogenic to humans, whereasB. ovisandB. neotomaeare nonpathogenic to humans andB. canishuman infections are rare. Limited genome diversity exists amongBrucellaspecies. Comparison ofBrucellaspecies whole genomes is, therefore, likely to identify factors responsible for differences in host preference and virulence restriction. To facilitate such studies, we used the complete genome sequence ofB. melitensis16M, the species highly pathogenic to humans, to construct a genomic microarray. Hybridization of labeled genomic DNA fromBrucellaspecies to this microarray revealed a total of 217 open reading frames (ORFs) altered in fiveBrucellaspecies analyzed. These ORFs are often found in clusters (islands) in the 16M genome. Examination of the genomic context of these islands suggests that many are horizontally acquired. Deletions of genetic content identified inBrucellaspecies are conserved in multiple strains of the same species, and genomic islands missing in a given species are often restricted to that particular species. These findings suggest that, whereas the loss or gain of genetic material may be related to the host range and virulence restriction of certainBrucellaspecies for humans, independent mechanisms involving gene inactivation or altered expression of virulence determinants may also contribute to these differences.
Three-Dimensional Electron Microscopic Imaging of Membrane Invaginations in
Escherichia coli
Overproducing the Chemotaxis Receptor Tsr
Authors: Lefman Jonathan , Zhang Peijun , Hirai Teruhisa , Weis Robert M. , Juliani Jemma , Bliss Donald , Kessel Martin , Bos Erik , Peters Peter J. , Subramaniam Sriram 
Published: July 2004

ABSTRACT

Electron tomography is a powerful method for determining the three-dimensional structures of large macromolecular assemblies, such as cells, organelles, and multiprotein complexes, when crystallographic averaging methods are not applicable. Here we used electron tomographic imaging to determine the molecular architecture of
Escherichia coli
cells engineered to overproduce the bacterial chemotaxis receptor Tsr. Tomograms constructed from fixed, cryosectioned cells revealed that overproduction of Tsr led to formation of an extended internal membrane network composed of stacks and extended tubular structures. We present an interpretation of the tomogram in terms of the packing arrangement of Tsr using constraints derived from previous X-ray and electron-crystallographic studies of receptor clusters. Our results imply that the interaction between the cytoplasmic ends of Tsr is likely to stabilize the presence of the membrane networks in cells overproducing Tsr. We propose that membrane invaginations that are potentially capable of supporting axial interactions between receptor clusters in apposing membranes could also be present in wild-type
E. coli
and that such receptor aggregates could play an important role in signal transduction during bacterial chemotaxis.
The Homogentisate Pathway: a Central Catabolic Pathway Involved in the Degradation of
l
-Phenylalanine,
l
-Tyrosine, and 3-Hydroxyphenylacetate in
Pseudomonas putida
Authors: Arias-Barrau Elsa , Olivera Elías R. , Luengo José M. , Fernández Cristina , Galán Beatriz , García José L. , Díaz Eduardo , Miñambres Baltasar 
Published: July 2004

ABSTRACT

Pseudomonas putida
metabolizes Phe and Tyr through a peripheral pathway involving hydroxylation of Phe to Tyr (PhhAB), conversion of Tyr into 4-hydroxyphenylpyruvate (TyrB), and formation of homogentisate (Hpd) as the central intermediate. Homogentisate is then catabolized by a central catabolic pathway that involves three enzymes, homogentisate dioxygenase (HmgA), fumarylacetoacetate hydrolase (HmgB), and maleylacetoacetate isomerase (HmgC), finally yielding fumarate and acetoacetate. Whereas the
phh
,
tyr
, and
hpd
genes are not linked in the
P. putida
genome, the
hmgABC
genes appear to form a single transcriptional unit. Gel retardation assays and
lacZ
translational fusion experiments have shown that
hmgR
encodes a specific repressor that controls the inducible expression of the divergently transcribed
hmgABC
catabolic genes, and homogentisate is the inducer molecule. Footprinting analysis revealed that HmgR protects a region in the
Phmg
promoter that spans a 17-bp palindromic motif and an external direct repetition from position −16 to position 29 with respect to the transcription start site. The HmgR protein is thus the first IclR-type regulator that acts as a repressor of an aromatic catabolic pathway. We engineered a broad-host-range mobilizable catabolic cassette harboring the
hmgABC
,
hpd
, and
tyrB
genes that allows heterologous bacteria to use Tyr as a unique carbon and energy source. Remarkably, we show here that the catabolism of 3-hydroxyphenylacetate in
P. putida
U funnels also into the homogentisate central pathway, revealing that the
hmg
cluster is a key catabolic trait for biodegradation of a small number of aromatic compounds.
Secretion and Function ofSalmonellaSPI-2 Effector SseF Require Its Chaperone, SscB
Authors: Dai Shipan , Zhou Daoguo 
Published: July 2004

ABSTRACTSalmonellastrains utilize a type III secretion system for their successful survival and replications inside host cells. SseF is one of the several effector proteins that are required for conferring this survival ability by altering the trafficking of theSalmonella-containing vacuoles. These effector proteins often require appropriate chaperones to maintain their stabilities inside the bacteria. These chaperones are also known to assist the subsequent secretion and translocation of their substrates. We report here that SscB acts as the chaperone for SseF, an effector for theSalmonellapathogenicity island 2 (SPI-2). We found that thesscBgene is required for the formation ofSalmonellasp.-induced continuous filaments in epithelial cells. EfficientSalmonellareplication in macrophages requires SscB function. Intracellular and secretion levels of SseF are greatly reduced in ansscBmutant strain compared to the wild-type strain. A protein stability assay demonstrated that the half-life of SseF is significantly shortened in the absence of SscB. Transcriptional analysis of thesseFgene showed that the effect of SscB on the SseF level is not at the transcriptional level. A coprecipitation experiment indicated that SscB interacts with SseF. In summary, our results indicate that SscB is a chaperone for SPI-2 effector SseF to facilitate its secretion and function inside the host cells.
A Movable Surface: Formation of
Yersinia
sp. Biofilms on Motile
Caenorhabditis elegans
Authors: Tan Li , Darby Creg 
Published: July 2004

ABSTRACT

Bubonic plague is transmitted by fleas whose feeding is blocked by a mass of
Yersinia pestis
in the digestive tract.
Y. pestis
and the closely related
Y. pseudotuberculosis
also block the feeding of
Caenorhabditis elegans
by forming a biofilm on the nematode head.
C. elegans
mutants with severe motility defects acquire almost no biofilm, indicating that normal animals accumulate the biofilm matrix as they move through a
Yersinia
lawn. Using the lectin wheat germ agglutinin as a probe, we show that the matrix on
C. elegans
contains carbohydrate produced by
Yersinia
. The carbohydrate is present in bacterial lawns prior to addition of nematodes, indicating that biofilm formation does not involve signaling between the two organisms. Furthermore, biofilm accumulation depends on continuous
C. elegans
exposure to a lawn of
Yersinia
bacteria.