bluuebunny/crossref_metadata_2025_split huggingface dataset:
Slt2 and Rim101 Contribute Independently to the Correct Assembly of the Chitin Ring at the Budding Yeast Neck in
Saccharomyces cerevisiae
Authors: Gomez Alberto , Perez Jaqueline , Reyes Abigail , Duran Angel , Roncero Cesar 
Published: July 2009

ABSTRACT

In
Saccharomyces cerevisiae
, the simultaneous absence of Slt2 and Rim101 prevents growth in nonosmotically stabilized media (F. Castrejon et al., Eukaryot. Cell 5:507-517, 2006). The double mutant
slt2
Δ
rim101
Δ displays altered chitin rings, together with a significant reduction in the overall levels of chitin. Cultures of this mutant lyse upon transfer to nonosmotically stabilized media, mostly through the bud, and such lysis is partially prevented by deletion of the chitinase gene (
CTS1
). Growth of the
slt2
Δ
rim101
Δ double mutant was restored by the overexpression of the
GFA1
or
CCT7
genes, which code for two biologically unrelated proteins. Further characterization of the mutant and its suppressors indicated that both Slt2 and Rim101 were independently required for the correct assembly of the septum machinery and that their concomitant absence reduced Chs3 accumulation at the neck, leading to lower levels of chitin.
GFA1
overexpression, as well as the addition of glucosamine to the growth medium, specifically suppressed the growth defects by activating chitin synthesis at the neck and restoring the normal assembly of the chitin ring. In contrast, overexpression of
CCT7
, a Cct chaperonin subunit, alleviated the defect in the septum machinery without affecting chitin synthesis. Both suppressors thus act by reducing neck fragility through different mechanisms and allow growth in nonstabilized media. This work reports new roles for Slt2 and Rim101 in septum formation in budding yeast and confirms the homeostatic role of the chitin ring in the maintenance of neck integrity during cell division.
Functional Dissection of the Catalytic Carboxyl-Terminal Domain of Origin Recognition Complex Subunit 1 (PfORC1) of the Human Malaria Parasite
Plasmodium falciparum
Authors: Gupta Ashish , Mehra Parul , Deshmukh Abhijeet , Dar Ashraf , Mitra Pallabi , Roy Nilanjan , Dhar Suman Kumar 
Published: July 2009

ABSTRACT

Origin recognition complex subunit 1 (ORC1) is essential for DNA replication in eukaryotes. The deadly human malaria parasite
Plasmodium falciparum
contains an ORC1/CDC6 homolog with several interesting domains at the catalytic carboxyl-terminal region that include a putative nucleoside triphosphate-binding and hydrolysis domain, a putative PCNA-interacting-protein (PIP) motif, and an extreme C-terminal region that shows poor homology with other ORC1 homologs. Due to the unavailability of a dependable inducible gene expression system, it is difficult to study the structure and function of essential genes in
Plasmodium
. Using a genetic yeast complementation system and biochemical experiments, here we show that the putative PIP domain in ORC1 that facilitates in vitro physical interaction with PCNA is functional in both yeast (
Saccharomyces cerevisiae
) and
Plasmodium
in vivo, confirming its essential biological role in eukaryotes. Furthermore, despite having less sequence homology, the extreme C-terminal region can be swapped between
S. cerevisiae
and
P. falciparum
and it binds to DNA directly, suggesting a conserved role of this region in DNA replication. These results not only provide us a useful system to study the function of the essential genes in
Plasmodium
, they help us to identify the previously undiscovered unique features of replication proteins in general.
Fate of Glycosylphosphatidylinositol (GPI)-Less Procyclin and Characterization of Sialylated Non-GPI-Anchored Surface Coat Molecules of Procyclic-Form
Trypanosoma brucei
Authors: Güther Maria Lucia Sampaio , Beattie Kenneth , Lamont Douglas J. , James John , Prescott Alan R. , Ferguson Michael A. J. 
Published: July 2009

ABSTRACT

A
Trypanosoma brucei
TbGPI12 null mutant that is unable to express cell surface procyclins and free glycosylphosphatidylinositols (GPI) revealed that these are not the only surface coat molecules of the procyclic life cycle stage. Here, we show that non-GPI-anchored procyclins are N-glycosylated, accumulate in the lysosome, and appear as proteolytic fragments in the medium. We also show, using lectin agglutination and galactose oxidase-NaB
3
H
4
labeling, that the cell surface of the TbGPI12 null parasites contains glycoconjugates that terminate in sialic acid linked to galactose. Following desialylation, a high-apparent-molecular-weight glycoconjugate fraction was purified by ricin affinity chromatography and gel filtration and shown to contain mannose, galactose,
N
-acetylglucosamine, and fucose. The latter has not been previously reported in
T. brucei
glycoproteins. A proteomic analysis of this fraction revealed a mixture of polytopic transmembrane proteins, including P-type ATPase and vacuolar proton-translocating pyrophosphatase. Immunolocalization studies showed that both could be labeled on the surfaces of wild-type and TbGPI12 null cells. Neither galactose oxidase-NaB
3
H
4
labeling of the non-GPI-anchored surface glycoconjugates nor immunogold labeling of the P-type ATPase was affected by the presence of procyclins in the wild-type cells, suggesting that the procyclins do not, by themselves, form a macromolecular barrier.
Characterization of Novel Alleles of the
Escherichia coli umuDC
Genes Identifies Additional Interaction Sites of UmuC with the Beta Clamp
Authors: Beuning Penny J. , Chan Sarah , Waters Lauren S. , Addepalli Haripriya , Ollivierre Jaylene N. , Walker Graham C. 
Published: July 2009

ABSTRACT

Translesion synthesis is a DNA damage tolerance mechanism by which damaged DNA in a cell can be replicated by specialized DNA polymerases without being repaired. The
Escherichia coli umuDC
gene products, UmuC and the cleaved form of UmuD, UmuD′, comprise a specialized, potentially mutagenic translesion DNA polymerase, polymerase V (UmuD′
2
C). The full-length UmuD protein, together with UmuC, plays a role in a primitive DNA damage checkpoint by decreasing the rate of DNA synthesis. It has been proposed that the checkpoint is manifested as a cold-sensitive phenotype that is observed when the
umuDC
gene products are overexpressed. Elevated levels of the beta processivity clamp along with elevated levels of the
umuDC
gene products, UmuD′C, exacerbate the cold-sensitive phenotype. We used this observation as the basis for genetic selection to identify two alleles of
umuD
′ and seven alleles of
umuC
that do not exacerbate the cold-sensitive phenotype when they are present in cells with elevated levels of the beta clamp. The variants were characterized to determine their abilities to confer the
umuD
′
C
-specific phenotype UV-induced mutagenesis. The
umuD
variants were assayed to determine their proficiencies in UmuD cleavage, and one variant (G129S) rendered UmuD noncleaveable. We found at least two UmuC residues, T243 and L389, that may further define the beta binding region on UmuC. We also identified UmuC S31, which is predicted to bind to the template nucleotide, as a residue that is important for UV-induced mutagenesis.
Molecular Basis of ChvE Function in Sugar Binding, Sugar Utilization, and Virulence in
Agrobacterium tumefaciens
Authors: He Fanglian , Nair Gauri R. , Soto Cinque S. , Chang Yehchung , Hsu Lillian , Ronzone Erik , DeGrado William F. , Binns Andrew N. 
Published: July 2009

ABSTRACT

ChvE is a chromosomally encoded protein in
Agrobacterium tumefaciens
that mediates a sugar-induced increase in virulence (
vir
) gene expression through the activities of the VirA/VirG two-component system and has also been suggested to be involved in sugar utilization. The ChvE protein has homology to several bacterial periplasmic sugar-binding proteins, such as the ribose-binding protein and the galactose/glucose-binding protein of
Escherichia coli
. In this study, we provide direct evidence that ChvE specifically binds the
vir
gene-inducing sugar
d
-glucose with high affinity. Furthermore, ChvE mutations resulting in altered
vir
gene expression phenotypes have been isolated and characterized. Three distinct categories of mutants have been identified. Strains expressing the first class are defective in both virulence and
d
-glucose utilization as a result of mutations to residues lining the sugar-binding cleft. Strains expressing a second class of mutants are not adversely affected in sugar binding but are defective in virulence, presumably due to impaired interactions with the sensor kinase VirA. A subset of this second class of mutants includes variants of ChvE that also result in defective sugar utilization. We propose that these mutations affect not only interactions with VirA but also interactions with a sugar transport system. Examination of a homology model of ChvE shows that the mutated residues associated with the latter two phenotypes lie in two overlapping solvent-exposed sites adjacent to the sugar-binding cleft where conformational changes associated with the binding of sugar might have a maximal effect on ChvE's interactions with its distinct protein partners.
Characterization and Comparative Analysis of the Genes EncodingHaemophilus parasuisOuter Membrane Proteins P2 and P5
Authors: Mullins Michael A. , Register Karen B. , Bayles Darrell O. , Loving Crystal L. , Nicholson Tracy L. , Brockmeier Susan L. , Dyer David W. , Phillips Gregory J. 
Published: July 2009

ABSTRACTHaemophilus parasuisis a swine pathogen of significant industry concern, but little is known about how the organism causes disease. A related human pathogen,Haemophilus influenzae, has been better studied, and many of its virulence factors have been identified. Two of these, outer membrane proteins P2 and P5, are known to have important virulence properties. The goals of this study were to identify, analyze, and compare the genetic relatedness of orthologous genes encoding P2 and P5 proteins in a diverse group of 35H. parasuisstrains. Genes encoding P2 and P5 proteins were detected in allH. parasuisstrains evaluated. The predicted amino acid sequences for both P2 and P5 proteins exhibit considerable heterogeneity, particularly in regions corresponding to predicted extracellular loops. Twenty-five variants of P2 and 17 variants of P5 were identified. The P2 proteins of seven strains were predicted to contain a highly conserved additional extracellular loop compared to the remaining strains and toH. influenzaeP2. Antigenic-site predictions coincided with predicted extracellular loop regions of both P2 and P5. Neighbor-joining trees constructed using P2 and P5 sequences predicted divergent evolutionary histories distinct from those predicted by a multilocus sequence typing phylogeny based on partial sequencing of seven housekeeping genes. Real-time reverse transcription-PCR indicated that both genes are expressed in all of the strains.
Feedback Control of DnaA-Mediated Replication Initiation by Replisome-Associated HdaA Protein inCaulobacter
Authors: Collier Justine , Shapiro Lucy 
Published: July 2009

ABSTRACTChromosome replication inCaulobacter crescentusis tightly regulated to ensure that initiation occurs at the right time and only once during the cell cycle. The timing of replication initiation is controlled by both CtrA and DnaA. CtrA binds to and silences the origin. Upon the clearance of CtrA from the cell, the DnaA protein accumulates and allows loading of the replisome at the origin. Here, we identify an additional layer of replication initiation control that is mediated by the HdaA protein. InEscherichia coli, the Hda protein inactivates DnaA after replication initiation. We show that theCaulobacterHdaA homologue is necessary to restrict the initiation of DNA replication to only once per cell cycle and that it dynamically colocalizes with the replisome throughout the cell cycle. Moreover, the transcription ofhdaAis directly activated by DnaA, providing a robust feedback regulatory mechanism that adjusts the levels of HdaA to inactivate DnaA.
Isolation of a Variant of Subtilosin A with Hemolytic Activity
Authors: Huang Tai , Geng Hao , Miyyapuram Venugopal R. , Sit Clarissa S. , Vederas John C. , Nakano Michiko M. 
Published: July 2009

ABSTRACT

Bacillus subtilis
produces an anionic bacteriocin called subtilosin A that possesses antibacterial activity against certain gram-positive bacteria. In this study, we uncovered a hemolytic mutant of
B. subtilis
that produces an altered form of subtilosin A. The mutant bacteriocin, named subtilosin A1, has a replacement of threonine at position 6 with isoleucine. In addition to the hemolytic activity, subtilosin A1 was found to exhibit enhanced antimicrobial activity against specific bacterial strains. The
B. subtilis albB
mutant that does not produce a putative immunity peptide was more sensitive to both subtilosin A and subtilosin A1. A spontaneous suppressor mutation of
albB
that restored resistance to subtilosin A and subtilosin A1 was obtained. The
sbr
(
s
u
b
tilosin
r
esistance) mutation conferring the resistance is not linked to the
sboA-alb
locus. The
sbr
mutation does not increase the resistance of
B. subtilis
to other cell envelope-targeted antimicrobial agents, indicating that the mutation specifically confers the resistance to subtilosins. The findings suggest possible bioengineering approaches for obtaining anionic bacteriocins with enhanced and/or altered bactericidal activity. Furthermore, future identification of the subtilosin-resistant mutation could provide insights into the mechanism of subtilosin A activity.
Role of the Twin-Arginine Translocation Pathway in
Staphylococcus
Authors: Biswas Lalitha , Biswas Raja , Nerz Christiane , Ohlsen Knut , Schlag Martin , Schäfer Tina , Lamkemeyer Tobias , Ziebandt Anne-Kathrin , Hantke Klaus , Rosenstein Ralf , Götz Friedrich 
Published: July 2009

ABSTRACT

In
Staphylococcus
, the twin-arginine translocation (Tat) pathway is present only in some species and is composed of TatA and TatC. The
tatAC
operon is associated with the
fepABC
operon, which encodes homologs to an iron-binding lipoprotein, an iron-dependent peroxidase (FepB), and a high-affinity iron permease. The FepB protein has a typical twin-arginine (RR) signal peptide. The
tat
and
fep
operons constitute an entity that is not present in all staphylococcal species. Our analysis was focused on
Staphylococcus aureus
and
S. carnosus
strains. Tat deletion mutants (
ΔtatAC
) were unable to export active FepB, indicating that this enzyme is a Tat substrate. When the RR signal sequence from FepB was fused to prolipase and protein A, their export became Tat dependent. Since no other protein with a Tat signal could be detected, the
fepABC
-
tatAC
genes comprise not only a genetic but also a functional unit. We demonstrated that FepABC drives iron import, and in a mouse kidney abscess model, the bacterial loads of Δ
tatAC
and Δ
tat
-
fep
mutants were decreased. For the first time, we show that the Tat pathway in
S. aureus
is functional and serves to translocate the iron-dependent peroxidase FepB.
The
pgaABCD
Locus of
Acinetobacter baumannii
Encodes the Production of Poly-β-1-6-
N
-Acetylglucosamine, Which Is Critical for Biofilm Formation
Authors: Choi Alexis H. K. , Slamti Leyla , Avci Fikri Y. , Pier Gerald B. , Maira-Litrán Tomás 
Published: July 2009

ABSTRACT

We found that
Acinetobacter baumannii
contains a
pgaABCD
locus that encodes proteins that synthesize cell-associated poly-β-(1-6)-
N
-acetylglucosamine (PNAG). Both a mutant with an in-frame deletion of the
pga
locus (S1Δ
pga
) and a transcomplemented strain (S1Δ
pga
-c) of
A. baumannii
were constructed, and the PNAG production by these strains was compared using an immunoblot assay. Deleting the
pga
locus resulted in an
A. baumannii
strain without PNAG, and transcomplementation of the S1Δ
pga
strain with the
pgaABCD
genes fully restored the wild-type PNAG phenotype. Heterologous expression of the
A. baumannii pga
locus in
Escherichia coli
led to synthesis of significant amounts of PNAG, while no polysaccharide was detected in
E. coli
cells harboring an empty vector. Nuclear magnetic resonance analysis of the extracellular polysaccharide material isolated from
A. baumannii
confirmed that it was PNAG, but notably only 60% of the glucosamine amino groups were acetylated. PCR analysis indicated that all 30 clinical
A. baumannii
isolates examined had the
pga
genes, and immunoblot assays indicated that 14 of the 30 strains strongly produced PNAG, 14 of the strains moderately to weakly produced PNAG, and 2 strains appeared to not produce PNAG. Deletion of the
pga
locus led to loss of the strong biofilm phenotype, which was restored by complementation. Confocal laser scanning microscopy studies combined with COMSTAT analysis demonstrated that the biovolume, mean thickness, and maximum thickness of 16-h and 48-h-old biofilms formed by wild-type and
pga
-complemented
A. baumannii
strains were significantly greater than the biovolume, mean thickness, and maximum thickness of 16-h and 48-h-old biofilms formed by the S1Δ
pga
mutant strain. Biofilm-dependent production of PNAG could be an important virulence factor for this emerging pathogen that has few known virulence factors.
Participation of Regulator AscG of the β-Glucoside Utilization Operon in Regulation of the Propionate Catabolism Operon
Authors: Ishida Yuji , Kori Ayako , Ishihama Akira 
Published: July 2009

ABSTRACT

The
asc
operon of
Escherichia coli
is one of the cryptic genetic systems for β-
d
-galactoside utilization as a carbon source. The
ascFB
genes for β-
d
-galactoside transport and catabolism are repressed by the AscG regulator. After genomic SELEX screening, AscG was found to recognize and bind the consensus palindromic sequence TGAAACC-GGTTTCA. AscG binding was detected at two sites upstream of the
ascFB
promoter and at three sites upstream of the
prpBC
operon for propionate catabolism. In an
ascG
-disrupted mutant, transcription of
ascFB
was enhanced, in agreement with the repressor model of AscG. This repression was indicated to be due to interference of binding of cyclic AMP-CRP to the CRP box, which overlaps with the AscG-binding site 1, as well as binding of RNA polymerase to the promoter. Under conditions of steady-state
E. coli
growth in a rich medium, the intracellular level of AscG stayed constant at a level supposedly leading to tight repression of the
ascFB
operon. The level of
prpR
, encoding the activator of
prpBCDE
, was also increased in the absence of AscG, indicating the involvement of AscG in repression of
prpR
. Taken together, these data suggest a metabolic link through interplay between the
asc
and
prp
operons.
Downregulation of the
Escherichia coli guaB
Promoter by Upstream-Bound Cyclic AMP Receptor Protein
Authors: Husnain Seyyed I. , Busby Stephen J. W. , Thomas Mark S. 
Published: July 2009

ABSTRACT

The
Escherichia coli guaB
promoter (P

guaB

) is responsible for directing transcription of the
guaB
and
guaA
genes, which specify the biosynthesis of the nucleotide GMP. P

guaB

is subject to growth rate-dependent control (GRDC) and possesses an UP element that is required for this regulation. In addition, P

guaB

contains a discriminator, three binding sites for the nucleoid-associated protein FIS, and putative binding sites for the regulatory proteins DnaA, PurR, and cyclic AMP receptor protein (CRP). Here we show that the CRP-cyclic AMP (cAMP) complex binds to a site located over 100 bp upstream of the
guaB
transcription start site, where it serves to downregulate P

guaB

. The CRP-mediated repression of P

guaB

activity increases in media that support lower growth rates. Inactivation of the
crp
or
cyaA
gene or ablation/translocation of the CRP site relieves repression by CRP and results in a loss of GRDC of P

guaB

. Thus, GRDC of P

guaB

involves a progressive increase in CRP-mediated repression of the promoter as the growth rate decreases. Our results also suggest that the CRP-cAMP complex does not direct GRDC at P

guaB

and that at least one other regulatory factor is required for conferring GRDC on this promoter. However, PurR and DnaA are not required for this regulatory mechanism.
Potential Role of Group IIC-attCIntrons in Integron Cassette Formation
Authors: Léon Grégory , Roy Paul H. 
Published: July 2009

ABSTRACTIntegrons are natural expression vectors in which gene cassettes are integrated downstream of a promoter region by a site-specific recombinase. Gene cassettes usually consist of a single gene followed by a recombination site designatedattC. A major unanswered question is how a gene becomes associated with anattCsite. Here, we investigate the potential role of a specific lineage of group IIC introns, named group IIC-attC, in cassette formation. Group IIC-attCintrons preferentially targetattCwhile retaining the ability to target transcriptional terminators. We show using a PCR-based mobility assay withEscherichia colithat theS.ma.I2 intron from the genome of a clinical isolate ofSerratia marcescenscan target bothattCsite and putative terminator motifs of resistance genes. Quantitative results showed thatS.ma.I2 is more efficient in targeting variousattCsequences than three group IIC-attCintrons (54 to 64% sequence identity) from the genomes of environmental isolates. We also show that purified group IIC-attCintron-encoded reverse transcriptases have both RNA-dependent and DNA-dependent DNA polymerase activities in vitro. These data permit us to suggest a new model for gene cassette formation, in which a group IIC-attCintron targets separately a transcriptional terminator adjoining a gene and an isolatedattC, joins the gene and theattCby homologous recombination, and then splices and reverse transcribes a gene-attCRNA template, leading to the formation of a cassette.
Significant Bias against the ACA Triplet in the tmRNA Sequence of
Escherichia coli
K-12
Authors: Baik Sarah , Inoue Koichi , Ouyang Ming , Inouye Masayori 
Published: July 2009

ABSTRACT

The toxin MazF in
Escherichia coli
cleaves single-stranded RNAs specifically at ACA sequences. MazF overexpression virtually eliminates all cellular mRNAs to completely block protein synthesis. However, protein synthesis can continue on an mRNA that is devoid of ACA triplets. The finding that ribosomal RNAs remain intact in the face of complete translation arrest suggested a purpose for such preservation. We therefore examined the sequences of all transcribed RNAs to determine if there was any statistically significant bias against ACA. While ACA motifs are absent from tmRNA, 4.5S RNA, and seven of the eight 5S rRNAs, statistical analysis revealed that only for tmRNA was the absence nonrandom. The introduction of single-strand ACAs makes tmRNA highly susceptible to MazF cleavage. Furthermore, analysis of tmRNA sequences from 442 bacteria showed that the discrimination against ACA in tmRNAs was seen mostly in enterobacteria. We propose that the unusual bias against ACA in tmRNA may have coevolved with the acquisition of MazF.