bluuebunny/crossref_metadata_2025_split huggingface dataset:
Ras Proteins Have Multiple Functions in Vegetative Cells of Dictyostelium
Authors: Bolourani Parvin , Spiegelman George , Weeks Gerald 
Published: September 2010

ABSTRACT

During the aggregation of
Dictyostelium
cells, signaling through RasG is more important in regulating cyclic AMP (cAMP) chemotaxis, whereas signaling through RasC is more important in regulating the cAMP relay. However, RasC is capable of substituting for RasG for chemotaxis, since
rasG
−
cells are only partially deficient in chemotaxis, whereas
rasC
−
/
rasG
−
cells are totally incapable of chemotaxis. In this study we have examined the possible functional overlap between RasG and RasC in vegetative cells by comparing the vegetative cell properties of
rasG
−
,
rasC
−
, and
rasC
−
/
rasG
−
cells. In addition, since RasD, a protein not normally found in vegetative cells, is expressed in vegetative
rasG
−
and
rasC
−
/
rasG
−
cells and appears to partially compensate for the absence of RasG, we have also examined the possible functional overlap between RasG and RasD by comparing the properties of
rasG
−
and
rasC
−
/
rasG
−
cells with those of the mutant cells expressing higher levels of RasD. The results of these two lines of investigation show that RasD is capable of totally substituting for RasG for cytokinesis and growth in suspension, whereas RasC is without effect. In contrast, for chemotaxis to folate, RasC is capable of partially substituting for RasG, but RasD is totally without effect. Finally, neither RasC nor RasD is able to substitute for the role that RasG plays in regulating actin distribution and random motility. These specificity studies therefore delineate three distinct and none-overlapping functions for RasG in vegetative cells.
Recognition of Yeast by Murine Macrophages Requires Mannan but Not Glucan
Authors: Keppler-Ross Sabine , Douglas Lois , Konopka James B. , Dean Neta 
Published: September 2010

ABSTRACT

The first barrier against infection by
Candida albicans
involves fungal recognition and destruction by phagocytic cells of the innate immune system. It is well established that interactions between different phagocyte receptors and components of the fungal cell wall trigger phagocytosis and subsequent immune responses, but the fungal ligands mediating the initial stage of recognition have not been identified. Here, we describe a novel assay for fungal recognition and uptake by macrophages which monitors this early recognition step independently of other downstream events of phagocytosis. To analyze infection in live macrophages, we validated the neutrality of a codon-optimized red fluorescent protein (yEmRFP) biomarker in
C. albicans
; growth, hyphal formation, and virulence in infected mice and macrophages were unaffected by yEmRFP production. This permitted a new approach for studying phagocytosis by carrying out competition assays between red and green fluorescent yeast cells to measure the efficiency of yeast uptake by murine macrophages as a function of dimorphism or cell wall defects. These competition experiments demonstrate that, given a choice, macrophages display strong preferences for phagocytosis based on genus, species, and morphology.
Candida glabrata
and
Saccharomyces cerevisiae
are taken up by J774 macrophage cells more rapidly than
C. albicans
, and
C. albicans
yeast cells are favored over hyphal cells. Significantly, these preferences are mannan dependent. Mutations that affect mannan, but not those that affect glucan or chitin, reduce the uptake of yeast challenged with wild-type competitors by both J774 and primary murine macrophages. These results suggest that mannose side chains or mannosylated proteins are the ligands recognized by murine macrophages prior to fungal uptake.
Extracellular Secretion of Overexpressed Glycosylphosphatidylinositol-Linked Cell Wall Protein Utr2/Crh2p as a Novel Protein Quality Control Mechanism in Saccharomyces cerevisiae
Authors: Miller Kelly A. , DiDone Louis , Krysan Damian J. 
Published: September 2010

ABSTRACT

Eukaryotic cells employ a variety of mechanisms to maintain protein quality control and homeostasis. Here we provide evidence that one such mechanism in
Saccharomyces cerevisiae
involves the regulated release of excess or misfolded proteins to the extracellular space. The overexpression of an epitope-tagged allele of the glycosylphosphatidylinositol (GPI)-linked cell wall protein Utr2/Crh2p (Utr2/Crh2-green fluorescent protein [GFP] or -hemagglutinin [HA]) causes endoplasmic reticulum (ER) stress and the secretion of Crh2-GFP/HA into the extracellular space. Secretion is dependent on two GPI-linked aspartyl proteases (Yps1p/2p) and components of the unfolded protein response (Ire1p and Hac1p) but is independent of ER-associated degradation (ERAD) components such as Hrd1p and Doa10p. Supporting the idea that this process represents a mechanism for protein quality control, the level of Crh2-HA is increased in strains lacking Bst1p, a protein required for the proteasomal degradation of GPI-linked proteins. Furthermore, secretion is dependent on Sec18p, indicating that it requires ER-to-Golgi trafficking, and accordingly, Crh2-HA accumulates in the ER in
ire1
Δ and
bst1
Δ mutants by cycloheximide chase experiments. Since a fraction of Utr2/Crh2-GFP properly localizes to the cell wall in an
ire1
Δ mutant, extracellular secretion appears to occur through a pathway that is distinct from the normal GPI protein-trafficking pathway. Taken together, these data support a model in which the unfolded protein response (UPR)/yapsin-mediated extracellular release of overexpressed Utr2/Crh2-HA or -GFP is an alternative pathway for the removal of excess or misfolded secretory proteins functioning in parallel with proteasome-mediated degradation in
S. cerevisiae
. This model provides an explanation for the deleterious effects of Yps1/2p on the industrial production of some recombinant proteins in
S. cerevisiae
.
SpdR, a Response Regulator Required for Stationary-Phase Induction of
Caulobacter crescentus cspD
Authors: da Silva Carolina A. P. T. , Balhesteros Heloise , Mazzon Ricardo R. , Marques Marilis V. 
Published: September 2010

ABSTRACT

The cold shock protein (CSP) family includes small polypeptides that are induced upon temperature downshift and stationary phase. The genome of the alphaproteobacterium
Caulobacter crescentus
encodes four CSPs, with two being induced by cold shock and two at the onset of stationary phase. In order to identify the environmental signals and cell factors that are involved in
cspD
expression at stationary phase, we have analyzed
cspD
transcription during growth under several nutrient conditions. The results showed that expression of
cspD
was affected by the medium composition and was inversely proportional to the growth rate. The maximum levels of expression were decreased in a
spoT
mutant, indicating that ppGpp may be involved in the signalization for carbon starvation induction of
cspD
. A Tn
5
mutant library was screened for mutants with reduced
cspD
expression, and 10 clones that showed at least a 50% reduction in expression were identified. Among these, a strain with a transposon insertion into a response regulator of a two-component system showed no induction of
cspD
at stationary phase. This protein (SpdR) was able to acquire a phosphate group from its cognate histidine kinase, and gel mobility shift assay and DNase I footprinting experiments showed that it binds to an inverted repeat sequence of the
cspD
regulatory region. A mutated SpdR with a substitution of the conserved aspartyl residue that is the probable phosphorylation site is unable to bind to the
cspD
regulatory region and to complement the
spdR
mutant phenotype.
Residue Histidine 669 Is Essential for the Catalytic Activity of
Bacillus anthracis
Lethal Factor
Authors: Cao Sha , Guo Aizhen , Wu Gaobing , Liu Ziduo , Chen Wei , Feng Chunfang , Zhang Cheng-Cai , Chen Huanchun 
Published: September 2010

ABSTRACT

The lethal factor (LF) of
Bacillus anthracis
is a Zn
2+
-dependent metalloprotease which plays an important role in anthrax virulence. This study was aimed at identifying the histidine residues that are essential to the catalytic activities of LF. The site-directed mutagenesis was employed to replace the 10 histidine residues in domains II, III, and IV of LF with alanine residues, respectively. The cytotoxicity of these mutants was tested, and the results revealed that the alanine substitution for His-669 completely abolished toxicity to the lethal toxin (LT)-sensitive RAW264.7 cells. The reason for the toxicity loss was further explored. The zinc content of this LF mutant was the same as that of the wild type. Also this LF mutant retained its protective antigan (PA)-binding activity. Finally, the catalytic cleavage activity of this mutant was demonstrated to be drastically reduced. Thus, we conclude that residue His-669 is crucial to the proteolytic activity of LF.
The Two-Component System BvrR/BvrS Regulates the Expression of the Type IV Secretion System VirB inBrucella abortus
Authors: Martínez-Núñez Carola , Altamirano-Silva Pamela , Alvarado-Guillén Francisco , Moreno Edgardo , Guzmán-Verri Caterina , Chaves-Olarte Esteban 
Published: September 2010

ABSTRACTThe pathogenesis ofBrucellais related to the ability to multiply intracellularly, an event controlled by the two-component system BvrR/BvrS (TCS BvrRS) and the type IV secretion machinery VirB (T4SS VirB). We have hypothesized that the TCS BvrRS transcriptionally regulates the T4SS VirB. To test this hypothesis, we have compared the levels of VirB proteins in the wild-type strainBrucella abortus2308 and mutant strains devoid of the sensor and regulator genes (bvrSandbvrRmutants, respectively). While thebvrRandbvrSmutants showed low levels of the VirB1, VirB5, VirB8, and VirB9 proteins, the same proteins were overexpressed in thebvrRmutant complemented with a plasmid carrying a functionalbvrRgene. Quantitation ofvirB5mRNA confirmed these data and indicated that the influence of the TCS BvrRS on the T4SS VirB occurs at the transcriptional level. The expression of the transcriptional activator VjbR also depended on the TCS BvrRS. In addition, we demonstrate a direct interaction between the promoter region of the VirB operon and the response regulator BvrR. Altogether these data demonstrate that the TCS BvrRS controls the expression of the T4SS VirB through direct and indirect mechanisms.
Role of Cross Talk in Regulating the Dynamic Expression of the FlagellarSalmonellaPathogenicity Island 1 and Type 1 Fimbrial Genes
Authors: Saini Supreet , Slauch James M. , Aldridge Phillip D. , Rao Christopher V. 
Published: September 2010

ABSTRACTSalmonella enterica, a common food-borne pathogen, differentially regulates the expression of multiple genes during the infection cycle. These genes encode systems related to motility, adhesion, invasion, and intestinal persistence. Key among them is a type three secretion system (T3SS) encoded withinSalmonellapathogenicity island 1 (SPI1). In addition to the SPI1 T3SS, other systems, including flagella and type 1 fimbriae, have been implicated inSalmonellapathogenesis. In this study, we investigated the dynamic expression of the flagellar, SPI1, and type 1 fimbrial genes. We demonstrate that these genes are expressed in a temporal hierarchy, beginning with the flagellar genes, followed by the SPI1 genes, and ending with the type 1 fimbrial genes. This hierarchy could mirror the roles of these three systems during the infection cycle. As multiple studies have shown that extensive regulatory cross talk exists between these three systems, we also tested how removing different regulatory links between them affects gene expression dynamics. These results indicate that cross talk is critical for regulating gene expression during transitional phases in the gene expression hierarchy. In addition, we identified a novel regulatory link between flagellar and type 1 fimbrial gene expression dynamics, where we found that the flagellar regulator, FliZ, represses type 1 fimbrial gene expression through the posttranscriptional regulation of FimZ. The significance of these results is that they provide the first systematic study of the effect of regulatory cross talk on the expression dynamics of flagellar, SPI1, and type 1 fimbrial genes.
Proteomic Analysis ofNeorickettsia sennetsuSurface-Exposed Proteins and Porin Activity of the Major Surface Protein P51
Authors: Gibson Kathryn , Kumagai Yumi , Rikihisa Yasuko 
Published: September 2010

ABSTRACTNeorickettsia sennetsuis an obligate intracellular bacterium of monocytes and macrophages and is the etiologic agent of human Sennetsu neorickettsiosis.Neorickettsiaproteins expressed in mammalian host cells, including the surface proteins ofNeorickettsiaspp., have not been defined. In this paper, we isolated surface-exposed proteins fromN. sennetsuby biotin surface labeling followed by streptavidin-affinity chromatography. Forty-two of the total of 936 (4.5%)N. sennetsuopen reading frames (ORFs) were detected by liquid chromatography-tandem mass spectrometry (LC/MS/MS), including six hypothetical proteins. Among the major proteins identified were the two major β-barrel proteins: the 51-kDa antigen (P51) andNeorickettsiasurface protein 3 (Nsp3). Immunofluorescence labeling not only confirmed surface exposure of these proteins but also showed rosary-like circumferential labeling with anti-P51 for the majority of bacteria and polar to diffuse punctate labeling with anti-Nsp3 for a minority of bacteria. We found that the isolated outer membrane ofN. sennetsuhad porin activity, as measured by a proteoliposome swelling assay. This activity allowed the diffusion ofl-glutamine, the monosaccharides arabinose and glucose, and the tetrasaccharide stachyose, which could be inhibited with anti-P51 antibody. We purified native P51 and Nsp3 under nondenaturing conditions. When reconstituted into proteoliposomes, purified P51, but not Nsp3, exhibited prominent porin activity. This the first proteomic study of aNeorickettsiasp. showing new sets of proteins evolved as major surface proteins forNeorickettsiaand the first identification of a porin for the genusNeorickettsia.
l
-Lysine Catabolism Is Controlled by
l
-Arginine and ArgR in
Pseudomonas aeruginosa
PAO1
Authors: Chou Han Ting , Hegazy Mohamed , Lu Chung-Dar 
Published: September 2010

ABSTRACT

In comparison to other pseudomonads,
Pseudomonas aeruginosa
grows poorly in
l
-lysine as a sole source of nutrient. In this study, the
ldcA
gene (
l
ysine
d
e

c

arboxylase
A
; PA1818), previously identified as a member of the ArgR regulon of
l
-arginine metabolism, was found essential for
l
-lysine catabolism in this organism. LdcA was purified to homogeneity from a recombinant strain of
Escherichia coli
, and the results of enzyme characterization revealed that this pyridoxal-5-phosphate-dependent decarboxylase takes
l
-lysine, but not
l
-arginine, as a substrate. At an optimal pH of 8.5, cooperative substrate activation by
l
-lysine was depicted from kinetics studies, with calculated

K
m

and
V
max
values of 0.73 mM and 2.2 μmole/mg/min, respectively. Contrarily, the
ldcA
promoter was induced by exogenous
l
-arginine but not by
l
-lysine in the wild-type strain PAO1, and the binding of ArgR to this promoter region was demonstrated by electromobility shift assays. This peculiar arginine control on lysine utilization was also noted from uptake experiments in which incorporation of radioactively labeled
l
-lysine was enhanced in cells grown in the presence of
l
-arginine but not
l
-lysine. Rapid growth on
l
-lysine was detected in a mutant devoid of the main arginine catabolic pathway and with a higher basal level of the intracellular
l
-arginine pool and hence elevated ArgR-responsive regulons, including
ldcA
. Growth on
l
-lysine as a nitrogen source can also be enhanced when the
aruH
gene encoding an arginine/lysine:pyruvate transaminase was expressed constitutively from plasmids; however, no growth of the
ldcA
mutant on
l
-lysine suggests a minor role of this transaminase in
l
-lysine catabolism. In summary, this study reveals a tight connection of lysine catabolism to the arginine regulatory network, and the lack of lysine-responsive control on lysine uptake and decarboxylation provides an explanation of
l
-lysine as a poor nutrient for
P. aeruginosa
.
Regulation of a Phage Endolysin by Disulfide Caging
Authors: Kuty Gabriel F. , Xu Min , Struck Douglas K. , Summer Elizabeth J. , Young Ry 
Published: September 2010

ABSTRACT

In contrast to canonical phage endolysins, which require holin-mediated disruption of the membrane to gain access to attack the cell wall, signal anchor release (SAR) endolysins are secreted by the host
sec
system, where they accumulate in an inactive form tethered to the membrane by their N-terminal SAR domains. SAR endolysins become activated by various mechanisms upon release from the membrane. In its inactive form, the prototype SAR endolysin, Lyz
P1
, of coliphage P1, has an active-site Cys covalently blocked by a disulfide bond; activation involves a disulfide bond isomerization driven by a thiol in the newly released SAR domain, unblocking the active-site Cys. Here, we report that Lyz
103
, the endolysin of
Erwinia
phage ERA103, is also a SAR endolysin. Although Lyz
103
does not have a catalytic Cys, genetic evidence suggests that it also is activated by a thiol-disulfide isomerization triggered by a thiol in the SAR domain. In this case, the inhibitory disulfide in nascent Lyz
103
is formed between cysteine residues flanking a catalytic glutamate, caging the active site. Thus, Lyz
P1
and Lyz
103
define subclasses of SAR endolysins that differ in the nature of their inhibitory disulfide, and Lyz
103
is the first enzyme found to be regulated by disulfide bond caging of its active site.
Analysis of the Intact Surface Layer ofCaulobacter crescentusby Cryo-Electron Tomography
Authors: Amat Fernando , Comolli Luis R. , Nomellini John F. , Moussavi Farshid , Downing Kenneth H. , Smit John , Horowitz Mark 
Published: September 2010

ABSTRACTThe surface layers (S layers) of those bacteria and archaea that elaborate these crystalline structures have been studied for 40 years. However, most structural analysis has been based on electron microscopy of negatively stained S-layer fragments separated from cells, which can introduce staining artifacts and allow rearrangement of structures prone to self-assemble. We present a quantitative analysis of the structure and organization of the S layer on intact growing cells of the Gram-negative bacteriumCaulobacter crescentususing cryo-electron tomography (CET) and statistical image processing. Instead of the expected long-range order, we observed different regions with hexagonally organized subunits exhibiting short-range order and a broad distribution of periodicities. Also, areas of stacked double layers were found, and these increased in extent when the S-layer protein (RsaA) expression level was elevated by addition of multiplersaAcopies. Finally, we combined high-resolution amino acid residue-specific Nanogold labeling and subtomogram averaging of CET volumes to improve our understanding of the correlation between the linear protein sequence and the structure at the 2-nm level of resolution that is presently available. The results support the view that the U-shaped RsaA monomer predicted from negative-stain tomography proceeds from the N terminus at one vertex, corresponding to the axis of 3-fold symmetry, to the C terminus at the opposite vertex, which forms the prominent 6-fold symmetry axis. Such information will help future efforts to analyze subunit interactions and guide selection of internal sites for display of heterologous protein segments.
Latency of the Lipid A Deacylase PagL Is Involved in Producing a Robust Permeation Barrier in the Outer Membrane of
Salmonella enterica
Authors: Kawasaki Kiyoshi , Manabe Takayuki 
Published: September 2010

ABSTRACT

Lipid A deacylase PagL, which detoxifies endotoxin, is latent in
Salmonella enterica
. This study determined the biological significance of this latency. PagL latency was beneficial for bacteria in producing a robust permeation barrier through lipid A modifications under host-mimetic conditions that induced the modification enzymes, including PagL.
Legionella pneumophilaStrain 130b Possesses a Unique Combination of Type IV Secretion Systems and Novel Dot/Icm Secretion System Effector Proteins
Authors: Schroeder Gunnar N. , Petty Nicola K. , Mousnier Aurélie , Harding Clare R. , Vogrin Adam J. , Wee Bryan , Fry Norman K. , Harrison Timothy G. , Newton Hayley J. , Thomson Nicholas R. , Beatson Scott A. , Dougan Gordon , Hartland Elizabeth L. , Frankel Gad 
Published: September 2010

ABSTRACTLegionella pneumophilais a ubiquitous inhabitant of environmental water reservoirs. The bacteria infect a wide variety of protozoa and, after accidental inhalation, human alveolar macrophages, which can lead to severe pneumonia. The capability to thrive in phagocytic hosts is dependent on the Dot/Icm type IV secretion system (T4SS), which translocates multiple effector proteins into the host cell. In this study, we determined the draft genome sequence ofL. pneumophilastrain 130b (Wadsworth). We found that the 130b genome encodes a unique set of T4SSs, namely, the Dot/Icm T4SS, a Trb-1-like T4SS, and two Lvh T4SS gene clusters. Sequence analysis substantiated that a core set of 107 Dot/Icm T4SS effectors was conserved among the sequencedL. pneumophilastrains Philadelphia-1, Lens, Paris, Corby, Alcoy, and 130b. We also identified new effector candidates and validated the translocation of 10 novel Dot/Icm T4SS effectors that are not present inL. pneumophilastrain Philadelphia-1. We examined the prevalence of the new effector genes among 87 environmental and clinicalL. pneumophilaisolates. Five of the new effectors were identified in 34 to 62% of the isolates, while less than 15% of the strains tested positive for the other five genes. Collectively, our data show that the core set of conserved Dot/Icm T4SS effector proteins is supplemented by a variable repertoire of accessory effectors that may partly account for differences in the virulences and prevalences of particularL. pneumophilastrains.
Genome Sequence of the Dioxin-Mineralizing Bacterium
Sphingomonas wittichii
RW1
Authors: Miller Todd R. , Delcher Arthur L. , Salzberg Steven L. , Saunders Elizabeth , Detter John C. , Halden Rolf U. 
Published: September 2010

ABSTRACT

Pollutants such as polychlorinated biphenyls and dioxins pose a serious threat to human and environmental health. Natural attenuation of these compounds by microorganisms provides one promising avenue for their removal from contaminated areas. Over the past 2 decades, studies of the bacterium
Sphingomonas wittichii
RW1 have provided a wealth of knowledge about how bacteria metabolize chlorinated aromatic hydrocarbons. Here we describe the finished genome sequence of
S. wittichii
RW1 and major findings from its annotation.