bluuebunny/crossref_metadata_2025_split huggingface dataset:
Molecular Analyses of a Three-Subunit Euryarchaeal Clamp Loader Complex from
Methanosarcina acetivorans
Authors: Chen Yi-Hsing , Lin Yuyen , Yoshinaga Aya , Chhotani Benazir , Lorenzini Jenna L. , Crofts Alexander A. , Mei Shou , Mackie Roderick I. , Ishino Yoshizumi , Cann Isaac K. O. 
Published: August 2009

ABSTRACT

Chromosomal DNA replication is dependent on processive DNA synthesis. Across the three domains of life and in certain viruses, a toroidal sliding clamp confers processivity to replicative DNA polymerases by encircling the DNA and engaging the polymerase in protein/protein interactions. Sliding clamps are ring-shaped; therefore, they have cognate clamp loaders that open and load them onto DNA. Here we use biochemical and mutational analyses to study the structure/function of the
Methanosarcina acetivorans
clamp loader or replication factor C (RFC) homolog.
M. acetivorans
RFC (RFC

Ma

), which represents an intermediate between the common archaeal RFC and the eukaryotic RFC, comprises two different small subunits (RFCS1 and RFCS2) and a large subunit (RFCL). Size exclusion chromatography suggested that RFCS1 exists in oligomeric states depending on protein concentration, while RFCS2 exists as a monomer. Protein complexes of RFCS1/RFCS2 formed in solution; however, they failed to stimulate DNA synthesis by a cognate DNA polymerase in the presence of its clamp. Determination of the subunit composition and previous mutational analysis allowed the prediction of the spatial distribution of subunits in this new member of the clamp loader family. Three RFCS1 subunits are flanked by an RFCS2 and an RFCL. The spatial distribution is, therefore, reminiscent of the minimal
Escherichia coli
clamp loader that exists in space as three γ-subunits (motor) flanked by the δ′ (stator) and the δ (wrench) subunits. Mutational analysis, however, suggested that the similarity between the two clamp loaders does not translate into the complete conservation of the functions of individual subunits within the RFC

Ma

complex.
Mycobacterium tuberculosis
Is Able To Accumulate and Utilize Cholesterol
Authors: Brzostek Anna , Pawelczyk Jakub , Rumijowska-Galewicz Anna , Dziadek Bozena , Dziadek Jaroslaw 
Published: August 2009

ABSTRACT

It is expected that the obligatory human pathogen
Mycobacterium tuberculosis
must adapt metabolically to the various nutrients available during its cycle of infection, persistence, and reactivation. Cholesterol, which is an important part of the mammalian cytoplasmic membrane, is a potential energy source. Here, we show that
M. tuberculosis
grown in medium containing a carbon source other than cholesterol is able to accumulate cholesterol in the free-lipid zone of its cell wall. This cholesterol accumulation decreases the permeability of the cell wall for the primary antituberculosis drug, rifampin, and partially masks the mycobacterial surface antigens. Furthermore,
M. tuberculosis
was able to grow on mineral medium supplemented with cholesterol as the sole carbon source. Targeted disruption of the Rv3537 (
kstD
) gene inhibited growth due to inactivation of the cholesterol degradation pathway, as evidenced by accumulation of the intermediate, 9-hydroxy-4-androstene-3,17-dione. Our findings that
M. tuberculosis
is able to accumulate cholesterol in the presence of alternative nutrients and use it when cholesterol is the sole carbon source in vitro may facilitate future studies into the pathophysiology of this important deadly pathogen.
Investigation of the Three-Dimensional Architecture of the Collagen Adhesin EmaA ofAggregatibacter actinomycetemcomitansby Electron Tomography
Authors: Yu Chunxiao , Mintz Keith P. , Ruiz Teresa 
Published: August 2009

ABSTRACTThe periodontal pathogenAggregatibacter actinomycetemcomitansdisplays on the bacterial surface a nonfimbrial adhesin, EmaA, which is required for collagen binding. In this study, electron tomography was used to characterize the three-dimensional (3D) architecture of this adhesin. The antenna-like surface appendages, corresponding to EmaA, were found to be composed of an ellipsoidal domain capping a rod-like domain that adopts either a straight or a bent conformation at various positions along the length. The most common flexible point along the length of the EmaA appendage was localized 29.4 nm away from the distal end. One-fifth of the appendages were straight and the remaining showed angles distributed between 140° and 170° at this location. Deletion analysis mapped this bend to amino acids 611 to 640 of the protein sequence. The 3D structure of the collagen binding domain of EmaA was generated by alignment and averaging of 9 subvolumes of the adhesin extracted from tomograms. The structure contains three subdomains: a globular structure with a diameter of ∼5 nm and a cylindrical domain (∼4.4 nm by 5.8 nm) separated by a linker region with a diameter of ∼3 nm, followed by a cylindrical domain (∼4.6 nm by 6.6 nm). This is the first 3D structure of a trimeric autotransporter protein ofA. actinomycetemcomitans.
A Novel Glucosyltransferase Involved in O-Antigen Modification of
Shigella flexneri
Serotype 1c
Authors: Stagg Robert M. , Tang Swee-Seong , Carlin Nils I. A. , Talukder Kaisar A. , Cam Phung D. , Verma Naresh K. 
Published: August 2009

ABSTRACT

The O antigen of serotype 1c differs from the unmodified O antigen of serotype Y by the addition of a disaccharide (two glucosyl groups) to the tetrasaccharide repeating unit. It was shown here that addition of the first glucosyl group is mediated by the previously characterized
gtrI
cluster, which is found within a cryptic prophage at the
proA
locus in the bacterial chromosome. Transposon mutagenesis was performed to disrupt the gene responsible for addition of the second glucosyl group, causing reversion to serotype 1a. Colony immunoblotting was used to identify the desired revertants, and subsequent sequencing, cloning, and functional expression successfully identified the gene encoding serotype 1c-specific O-antigen modification. This gene (designated
gtrIC
) was present as part of a three-gene cluster, similar to other
S. flexneri
glucosyltransferase genes. Relative to the other
S. flexneri gtr
clusters, the
gtrIC
cluster is more distantly related and appears to have arrived in
S. flexneri
from outside the species. Analysis of surrounding sequence suggests that the
gtrIC
cluster arrived via a novel bacteriophage that was subsequently rendered nonfunctional by a series of insertion events.
Crystal Structure of theSalmonella entericaSerovar Typhimurium Virulence Factor SrfJ, a Glycoside Hydrolase Family Enzyme
Authors: Kim Yeon-Gil , Kim Jin-Hong , Kim Kyung-Jin 
Published: August 2009

ABSTRACTTo cause infection,Salmonella entericaserovar Typhimurium uses type III secretion systems, which are encoded on twoSalmonellapathogenicity islands, SPI-1 and SPI-2, the latter of which is thought to play a crucial role in bacterial proliferation inSalmonella-containing vacuoles (SCVs) after invading cells.S.Typhimurium SrfJ, located outside SPI-2, is also known to be involved inSalmonellapathogenicity and has high amino acid sequence homology with human lysosomal glucosylceramidase (GlcCerase). We present the first crystal structure of SrfJ at a resolution of 1.8 Å. The overall fold of SrfJ shares high structure similarities with that of human GlcCerase, comprising two distinctive domains: a (β/α)8-barrel catalytic domain and a β-sandwich domain. As in human GlcCerase, the pocket-shaped active site of SrfJ is located on the C-terminal side of the barrel, and two conserved glutamic acid residues are used for the enzyme catalysis. Moreover, a glycerol-bound form of SrfJ reveals that the glucose ring moiety of the substrate might similarly bind to the enzyme as to human GlcCerase, suggesting that SrfJ might function as a glycoside hydrolase. Although some structural differences are observed between SrfJ and human GlcCerase in the substrate entrance of the active site, we speculate that, based on the high structural similarities to human GlcCerase in the overall fold and the active-site environment, SrfJ might have a GlcCerase activity and use the activity to enhanceSalmonellavirulence by modifying SCV membrane lipids.
Facilitated Oligomerization of Mycobacterial GroEL: Evidence for Phosphorylation-Mediated Oligomerization
Authors: Kumar C. M. Santosh , Khare Garima , Srikanth C. V. , Tyagi Anil K. , Sardesai Abhijit A. , Mande Shekhar C. 
Published: August 2009

ABSTRACT

The distinctive feature of the GroES-GroEL chaperonin system in mediating protein folding lies in its ability to exist in a tetradecameric state, form a central cavity, and encapsulate the substrate via the GroES lid. However, recombinant GroELs of
Mycobacterium tuberculosis
are unable to act as effective molecular chaperones when expressed in
Escherichia coli
. We demonstrate here that the inability of
M. tuberculosis
GroEL1 to act as a functional chaperone in
E. coli
can be alleviated by facilitated oligomerization. The results of directed evolution involving random DNA shuffling of the genes encoding
M. tuberculosis
GroEL homologues followed by selection for functional entities suggested that the loss of chaperoning ability of the recombinant mycobacterial GroEL1 and GroEL2 in
E. coli
might be due to their inability to form canonical tetradecamers. This was confirmed by the results of domain-swapping experiments that generated
M. tuberculosis-E. coli
chimeras bearing mutually exchanged equatorial domains, which revealed that
E. coli
GroEL loses its chaperonin activity due to alteration of its oligomerization capabilities and vice versa for
M. tuberculosis
GroEL1. Furthermore, studying the oligomerization status of native GroEL1 from cell lysates of
M. tuberculosis
revealed that it exists in multiple oligomeric forms, including single-ring and double-ring variants. Immunochemical and mass spectrometric studies of the native
M. tuberculosis
GroEL1 revealed that the tetradecameric form is phosphorylated on serine-393, while the heptameric form is not, indicating that the switch between the single- and double-ring variants is mediated by phosphorylation.
Different Minimal Signal Peptide Lengths Recognized by the Archaeal Prepilin-Like Peptidases FlaK and PibD
Authors: Ng Sandy Y. M. , VanDyke David J. , Chaban Bonnie , Wu John , Nosaka Yoshika , Aizawa Shin-Ichi , Jarrell Ken F. 
Published: August 2009

ABSTRACT

In
Archaea
, the preflagellin peptidase (a type IV prepilin-like peptidase designated FlaK in
Methanococcus voltae
and
Methanococcus maripaludis
) is the enzyme that cleaves the N-terminal signal peptide from preflagellins. In methanogens and several other archaeal species, the typical flagellin signal peptide length is 11 to 12 amino acids, while in other archaea preflagellins possess extremely short signal peptides. A systematic approach to address the signal peptide length requirement for preflagellin processing is presented in this study.
M. voltae
preflagellin FlaB2 proteins with signal peptides 3 to 12 amino acids in length were generated and used as a substrate in an in vitro assay utilizing
M. voltae
membranes as an enzyme source. Processing by FlaK was observed in FlaB2 proteins containing signal peptides shortened to 5 amino acids; signal peptides 4 or 3 amino acids in length were unprocessed. In the case of
Sulfolobus solfataricus
, where the preflagellin peptidase PibD has broader substrate specificity, some predicted substrates have predicted signal peptides as short as 3 amino acids. Interestingly, the shorter signal peptides of the various mutant FlaB2 proteins not processed by FlaK were processed by PibD, suggesting that some archaeal preflagellin peptidases are likely adapted toward cleaving shorter signal peptides. The functional complementation of signal peptidase activity by FlaK and PibD in an
M. maripaludis
Δ
flaK
mutant indicated that processing of preflagellins was detected by complementation with either FlaK or PibD, yet only FlaK-complemented cells were flagellated. This suggested that a block in an assembly step subsequent to signal peptide removal occurred in the PibD complementation.
Structure of Chlorosomes from the Green Filamentous Bacterium
Chloroflexus aurantiacus
Authors: Pšenčík Jakub , Collins Aaron M. , Liljeroos Lassi , Torkkeli Mika , Laurinmäki Pasi , Ansink Hermanus M. , Ikonen Teemu P. , Serimaa Ritva E. , Blankenship Robert E. , Tuma Roman , Butcher Sarah J. 
Published: August 2009

ABSTRACT

The green filamentous bacterium
Chloroflexus aurantiacus
employs chlorosomes as photosynthetic antennae. Chlorosomes contain bacteriochlorophyll aggregates and are attached to the inner side of a plasma membrane via a protein baseplate. The structure of chlorosomes from
C. aurantiacus
was investigated by using a combination of cryo-electron microscopy and X-ray diffraction and compared with that of
Chlorobi
species. Cryo-electron tomography revealed thin chlorosomes for which a distinct crystalline baseplate lattice was visualized in high-resolution projections. The baseplate is present only on one side of the chlorosome, and the lattice dimensions suggest that a dimer of the CsmA protein is the building block. The bacteriochlorophyll aggregates inside the chlorosome are arranged in lamellae, but the spacing is much greater than that in
Chlorobi
species. A comparison of chlorosomes from different species suggested that the lamellar spacing is proportional to the chain length of the esterifying alcohols.
C. aurantiacus
chlorosomes accumulate larger quantities of carotenoids under high-light conditions, presumably to provide photoprotection. The wider lamellae allow accommodation of the additional carotenoids and lead to increased disorder within the lamellae.
An Extracytoplasmic Function Sigma Factor Controls β-Lactamase Gene Expression inBacillus anthracisand OtherBacillus cereusGroup Species
Authors: Ross Cana L. , Thomason Kerrie S. , Koehler Theresa M. 
Published: August 2009

ABSTRACTThe susceptibility of mostBacillus anthracisstrains to β-lactam antibiotics is intriguing considering that the closely related speciesBacillus cereusandBacillus thuringiensistypically produce β-lactamases and theB. anthracisgenome harbors two β-lactamase genes,bla1andbla2. We show that β-lactamase activity associated withB. anthracisis affected by two genes,sigP(BA2502) andrsiP(BA2503), predicted to encode an extracytoplasmic function sigma factor and an anti-sigma factor, respectively. Deletion of thesigP-rsiPlocus abolished β-lactamase activity in a naturally occurring penicillin-resistant strain and had no effect on β-lactamase activity in a prototypical penicillin-susceptible strain. Complementation withsigPandrsiPfrom the penicillin-resistant strain, but not withsigPandrsiPfrom the penicillin-susceptible strain, conferred constitutive β-lactamase activity in both mutants. These results are attributed to a nucleotide deletion near the 5′ end ofrsiPin the penicillin-resistant strain that is predicted to result in a nonfunctional protein.B. cereusandB. thuringiensis sigPandrsiPhomologues are required for inducible penicillin resistance in these species. Expression of theB. cereusorB. thuringiensis sigPandrsiPgenes in aB. anthracis sigP-rsiP-null mutant confers inducible production of β-lactamase activity, suggesting that whileB. anthraciscontains the genes necessary for sensing β-lactam antibiotics, theB. anthracis sigPandrsiPgene products are not sufficient forblainduction.
One of the Two Genes Encoding Nucleoid-Associated HU Proteins inStreptomyces coelicolorIs Developmentally Regulated and Specifically Involved in Spore Maturation
Authors: Salerno Paola , Larsson Jessica , Bucca Giselda , Laing Emma , Smith Colin P. , Flärdh Klas 
Published: August 2009

ABSTRACTStreptomycesgenomes encode two homologs of the nucleoid-associated HU proteins. One of them, here designated HupA, is of a conventional type similar toE. coliHUα and HUβ, while the other, HupS, is a two-domain protein. In addition to the N-terminal part that is similar to that of HU proteins, it has a C-terminal domain that is similar to the alanine- and lysine-rich C termini of eukaryotic linker histones. Such two-domain HU proteins are found only amongActinobacteria. In this phylum some organisms have only a single HU protein of the type with a C-terminal histone H1-like domain (e.g., Hlp inMycobacterium smegmatis), while others have only a single conventional HU. Yet others, including the streptomycetes, produce both types of HU proteins. We show here that the two HU genes inStreptomyces coelicolorare differentially regulated and thathupSis specifically expressed during sporulation, whilehupAis expressed in vegetative hyphae. The developmental upregulation ofhupSoccurred in sporogenic aerial hyphal compartments and was dependent on the developmental regulatorswhiA,whiG, andwhiI. HupS was found to be nucleoid associated in spores, and ahupSdeletion mutant had an average nucleoid size in spores larger than that in the parent strain. The mutant spores were also defective in heat resistance and spore pigmentation, although they possessed apparently normal spore walls and displayed no increased sensitivity to detergents. Overall, the results show that HupS is specifically involved in sporulation and may affect nucleoid architecture and protection in spores ofS. coelicolor.
Cell Wall Hydrolases Affect Germination, Vegetative Growth, and Sporulation in
Streptomyces coelicolor
Authors: Haiser Henry J. , Yousef Mary R. , Elliot Marie A. 
Published: August 2009

ABSTRACT

Peptidoglycan is a major cell wall constituent of gram-positive bacteria. It is a dynamic macromolecule that is actively remodeled to enable cell growth and differentiation through a tightly choreographed interplay of hydrolytic and biosynthetic enzyme activities. The filamentous bacterium
Streptomyces coelicolor
has a complex life cycle that likely requires considerable cell wall remodeling to enable both extension of vegetative hyphae and formation of differentiated cell types. In silico analysis of the
S. coelicolor
genome enabled identification of 56 candidate cell wall hydrolase genes. We found that seven of these genes shared a highly conserved 5′ untranslated region and were expressed during both vegetative growth and sporulation; four of these genes were selected for more extensive biochemical and biological characterization. The proteins encoded by these genes, termed RpfA, SwlA, SwlB, and SwlC, were confirmed to be hydrolytic enzymes, as they could efficiently cleave
S. coelicolor
cell walls. Phenotypic analyses revealed that these enzymes are important throughout development; deletion of each hydrolase gene resulted in a mutant strain that was heat sensitive, defective in spore formation, and either altered in vegetative growth or delayed in spore germination. Our results indicate that these enzymes play key roles at multiple stages in the growth and development of
S. coelicolor
, highlighting both the lack of redundancy in hydrolase activity and the importance of cell wall remodeling in the
S. coelicolor
life cycle.
The ArsR Repressor Mediates Arsenite-Dependent Regulation of Arsenate Respiration and Detoxification Operons of
Shewanella
sp. Strain ANA-3
Authors: Murphy Julie N. , Saltikov Chad W. 
Published: August 2009

ABSTRACT

Microbial arsenate reduction affects the fate and transport of arsenic in the environment. Arsenate respiratory (
arr
) and detoxifying (
ars
) reduction pathways in
Shewanella
sp. strain ANA-3 are induced by arsenite and under anaerobic conditions. Here it is shown that an ArsR family protein, called ArsR2, regulates the arsenate respiratory reduction pathway in response to elevated arsenite under anaerobic conditions. Strains lacking
arsR2
grew faster in the presence of high levels of arsenite (3 mM). Moreover, expression of
arrA
and
arsC
(arsenate reductase-encoding genes) in the Δ
arsR2
mutant of ANA-3 were increased in cells grown under anaerobic conditions and in the absence of arsenic. Mutations in putative arsenic binding amino acid residues in ArsR2 (substitutions of Cys-30 and Cys-32 with Ser) resulted in ANA-3 strains that exhibited anaerobic growth deficiencies with high levels of arsenite and arsenate. DNA binding studies with purified ArsR2 showed that ArsR2 binding to the
arr
promoter region was impaired by trivalent arsenicals such as arsenite and phenylarsine oxide. However, ArsR2 binding occurred in the presence of arsenate. A second known regulator of the
arr
operon, cyclic AMP (cAMP)-cAMP receptor protein (CRP), could bind simultaneously with ArsR2 within the
arr
promoter region. It is concluded that ArsR2 is most likely the major arsenite-dependent regulator of
arr
and
ars
operons in
Shewanella
sp. strain ANA-3. However, anaerobic growth on arsenate will require coregulation with global regulators such as cAMP-CRP.
Dam Methylation Controls O-Antigen Chain Length in
Salmonella enterica
Serovar Enteritidis by Regulating the Expression of Wzz Protein
Authors: Sarnacki Sebastián H. , Marolda Cristina L. , Noto Llana Mariángeles , Giacomodonato Mónica N. , Valvano Miguel A. , Cerquetti María Cristina 
Published: August 2009

ABSTRACT

We reported previously that a
Salmonella enterica
serovar Enteritidis
dam
mutant expressing a truncated Dam protein does not agglutinate in the presence of specific antibodies against O9 polysaccharide. Here we investigate the participation of Dam in lipopolysaccharide (LPS) synthesis in
Salmonella
. The LPS O-antigen profiles of a
dam
null mutant (SEΔ
dam
) and the
Salmonella
serovar Enteritidis parental strain were examined by using electrophoresis and silver staining. Compared to the parental strain, SEΔ
dam
produced LPS with shorter O-antigen polysaccharide chains. Since Wzz is responsible for the chain length distribution of the O antigen, we investigated whether Dam methylation is involved in regulating
wzz
expression. Densitometry analysis showed that the amount of Wzz produced by SEΔ
dam
is threefold lower than the amount of Wzz produced by the parental strain. Concomitantly, the activity of the
wzz
promoter in SEΔ
dam
was reduced nearly 50% in logarithmic phase and 25% in stationary phase. These results were further confirmed by reverse transcription-PCR showing that
wzz
gene expression was threefold lower in the
dam
mutant than in the parental strain. Our results demonstrate that
wzz
gene expression is downregulated in a
dam
mutant, indicating that Dam methylation activates expression of this gene. This work indicates that
wzz
is a new target regulated by Dam methylation and demonstrates that DNA methylation not only affects the production of bacterial surface proteins but also the production of surface polysaccharides.