bluuebunny/crossref_metadata_2025_split huggingface dataset:
Protein Kinase A Regulatory Subunit Isoforms Regulate Growth and Differentiation in Mucor circinelloides: Essential Role of PKAR4
Authors: Ocampo J. , McCormack B. , Navarro E. , Moreno S. , Garre V. , Rossi S. 
Published: May 2012

ABSTRACTThe protein kinase A (PKA) signaling pathway plays a role in regulating growth and differentiation in the dimorphic fungusMucor circinelloides. PKA holoenzyme is comprised of two catalytic (C) and two regulatory (R) subunits. InM. circinelloides, four genes encode the PKAR1, PKAR2, PKAR3, and PKAR4 isoforms of R subunits. We have constructed null mutants and demonstrate that each isoform has a different role in growth and differentiation. The most striking finding is thatpkaR4is an essential gene, because only heterokaryons were obtained in knockout experiments. Heterokaryons with low levels of wild-type nuclei showed an impediment in the emission of the germ tube, suggesting a pivotal role of this gene in germ tube emergence. The remaining null strains showed different alterations in germ tube emergence, sporulation, and volume of the mother cell. ThepkaR2null mutant showed an accelerated germ tube emission and was the only mutant that germinated under anaerobic conditions when glycine was used as a nitrogen source, suggesting thatpkaR2participates in germ tube emergence by repressing it. From the measurement of the mRNA and protein levels of each isoform in the wild-type and knockout strains, it can be concluded that the expression of each subunit has its own mechanism of differential regulation. The PKAR1 and PKAR2 isoforms are posttranslationally modified by ubiquitylation, suggesting another regulation point in the specificity of the signal transduction. The results indicate that each R isoform has a different role inM. circinelloidesphysiology, controlling the dimorphism and contributing to the specificity of cyclic AMP (cAMP)-PKA pathway.
The Heat-Induced Molecular Disaggregase Hsp104 of Candida albicans Plays a Role in Biofilm Formation and Pathogenicity in a Worm Infection Model
Authors: Fiori Alessandro , Kucharíková Soňa , Govaert Gilmer , Cammue Bruno P. A. , Thevissen Karin , Van Dijck Patrick 
Published: May 2012

ABSTRACT

The consequences of deprivation of the molecular chaperone Hsp104 in the fungal pathogen
Candida albicans
were investigated. Mutants lacking
HSP104
became hypersusceptible to lethally high temperatures, similarly to the corresponding mutants of
Saccharomyces cerevisiae
, whereas normal susceptibility was restored upon reintroduction of the gene. By use of a strain whose only copy of
HSP104
is an ectopic gene under the control of a tetracycline-regulated promoter, expression of Hsp104 prior to the administration of heat shock could be demonstrated to be sufficient to confer protection from the subsequent temperature increase. This result points to a key role for Hsp104 in orchestrating the cell response to elevated temperatures. Despite their not showing evident growth or morphological defects, biofilm formation by cells lacking
HSP104
proved to be defective in two established
in vitro
models that use polystyrene and polyurethane as the substrates. Biofilms formed by the wild-type and
HSP104
-reconstituted strains showed patterns of intertwined hyphae in the extracellular matrix. In contrast, biofilm formed by the
hsp104
Δ/
hsp104
Δ mutant showed structural defects and appeared patchy and loose. Decreased virulence of the
hsp104
Δ/
hsp104
Δ mutant was observed in the
Caenorhabditis elegans
infection model, in which high
in vivo
temperature does not play a role. In agreement with the view that stress responses in fungal pathogens may have evolved to provide niche-specific adaptation to environmental conditions, these results provide an indication of a temperature-independent role for Hsp104 in support of
Candida albicans
virulence, in addition to its key role in governing thermotolerance.
Characterization of a Vibrio fischeri Aminopeptidase and Evidence for Its Influence on an Early Stage of Squid Colonization
Authors: Fidopiastis Pat M. , Rader Bethany A. , Gerling David G. , Gutierrez Nestor A. , Watkins Katherine H. , Frey Michelle West , Nyholm Spencer V. , Whistler Cheryl A. 
Published: May 2012

ABSTRACT

Vibrio fischeri
cells are the sole colonists of a specialized light organ in the mantle cavity of the sepiolid squid
Euprymna scolopes
. The process begins when the bacteria aggregate in mucus secretions outside the light organ. The cells eventually leave the aggregate, enter the light organ, and encounter a rich supply of peptides. The need to dissociate from mucus and presumably utilize peptides led us to hypothesize that protease activity is integral to the colonization process. Protease activity associated with whole cells of
Vibrio fischeri
strain ES114 was identified as the product of a putative cell membrane-associated aminopeptidase (PepN). To characterize this activity, the aminopeptidase was cloned, overexpressed, and purified. Initial steady-state kinetic studies revealed that the aminopeptidase has broad activity, with a preference for basic and hydrophobic side chains and
k
cat
and

K
m

values that are lower and smaller, respectively, than those of
Escherichia coli
PepN. A
V. fischeri
mutant unable to produce PepN is significantly delayed in its ability to colonize squid within the first 12 h, but eventually it establishes a wild-type colonization level. Likewise, in competition with the wild type for colonization, the mutant is outcompeted at 12 h postinoculation but then competes evenly by 24 h. Also, the PepN-deficient strain fails to achieve wild-type levels of cells in aggregates, suggesting an explanation for the initial colonization delay. This study provides a foundation for more studies on PepN expression, localization, and role in the early stages of squid colonization.
Caldicellulosiruptor Core and Pangenomes Reveal Determinants for Noncellulosomal Thermophilic Deconstruction of Plant Biomass
Authors: Blumer-Schuette Sara E. , Giannone Richard J. , Zurawski Jeffrey V. , Ozdemir Inci , Ma Qin , Yin Yanbin , Xu Ying , Kataeva Irina , Poole Farris L. , Adams Michael W. W. , Hamilton-Brehm Scott D. , Elkins James G. , Larimer Frank W. , Land Miriam L. , Hauser Loren J. , Cottingham Robert W. , Hettich Robert L. , Kelly Robert M. 
Published: May 2012

ABSTRACT

Extremely thermophilic bacteria of the genus
Caldicellulosiruptor
utilize carbohydrate components of plant cell walls, including cellulose and hemicellulose, facilitated by a diverse set of glycoside hydrolases (GHs). From a biofuel perspective, this capability is crucial for deconstruction of plant biomass into fermentable sugars. While all species from the genus grow on xylan and acid-pretreated switchgrass, growth on crystalline cellulose is variable. The basis for this variability was examined using microbiological, genomic, and proteomic analyses of eight globally diverse
Caldicellulosiruptor
species. The open
Caldicellulosiruptor
pangenome (4,009 open reading frames [ORFs]) encodes 106 GHs, representing 43 GH families, but only 26 GHs from 17 families are included in the core (noncellulosic) genome (1,543 ORFs). Differentiating the strongly cellulolytic
Caldicellulosiruptor
species from the others is a specific genomic locus that encodes multidomain cellulases from GH families 9 and 48, which are associated with cellulose-binding modules. This locus also encodes a novel adhesin associated with type IV pili, which was identified in the exoproteome bound to crystalline cellulose. Taking into account the core genomes, pangenomes, and individual genomes, the ancestral
Caldicellulosiruptor
was likely cellulolytic and evolved, in some cases, into species that lost the ability to degrade crystalline cellulose while maintaining the capacity to hydrolyze amorphous cellulose and hemicellulose.
Cellular Characterization of the Primosome and Rep Helicase in Processing and Restoration of Replication following Arrest by UV-Induced DNA Damage in Escherichia coli
Authors: Courcelle Charmain T. , Landstrom Allison J. , Anderson Brittany , Courcelle Justin 
Published: May 2012

ABSTRACT

Following arrest by UV-induced DNA damage, replication is restored through a sequence of steps that involve partial resection of the nascent DNA by RecJ and RecQ, branch migration and processing of the fork DNA surrounding the lesion by RecA and RecF-O-R, and resumption of DNA synthesis once the blocking lesion has been repaired or bypassed.
In vitro
, the primosomal proteins (PriA, PriB, and PriC) and Rep are capable of initiating replication from synthetic DNA fork structures, and they have been proposed to catalyze these events when replication is disrupted by certain impediments
in vivo
. Here, we characterized the role that PriA, PriB, PriC, and Rep have in processing and restoring replication forks following arrest by UV-induced DNA damage. We show that the partial degradation and processing of the arrested replication fork occurs normally in both
rep
and primosome mutants. In each mutant, the nascent degradation ceases and DNA synthesis initially resumes in a timely manner, but the recovery then stalls in the absence of PriA, PriB, or Rep. The results demonstrate a role for the primosome and Rep helicase in overcoming replication forks arrested by UV-induced damage
in vivo
and suggest that these proteins are required for the stability and efficiency of the replisome when DNA synthesis resumes but not to initiate
de novo
replication downstream of the lesion.
Sinorhizobium meliloti ExoR Is the Target of Periplasmic Proteolysis
Authors: Lu Hai-Yang , Luo Li , Yang Meng-Hua , Cheng Hai-Ping 
Published: May 2012

ABSTRACT

Sinorhizobium meliloti
ExoR regulates the production of succinoglycan and flagella through the ExoS/ChvI two-component regulatory system. ExoR has been proposed to inhibit the ExoS sensor through direct interaction in the periplasm. To understand how ExoR suppression of ExoS is relieved, which is required for the expression of ExoS/ChvI-regulated symbiosis genes, we characterized wild-type ExoR and ExoR95 mutant proteins. In addition to the previously identified precursor and mature forms of ExoR (designated ExoR
p
and ExoR
m
, respectively), we detected a 20-kDa form of ExoR (designated ExoR
c20
) derived from the wild-type ExoR protein, but not from the ExoR95 mutant protein. ExoR
c20
was isolated directly from
S. meliloti
periplasm to identify its N-terminal amino acids and the site of the proteolysis, which is highly conserved among ExoR homologs. ExoR
c20
retains the C terminus of the wild-type ExoR. When expressed directly, ExoR
c20
did not complement the
exoR95
mutation, suggesting that ExoR
c20
does not function directly in the ExoR-ExoS/ChvI regulatory pathway and that ExoR
m
is the functional form of ExoR. A single-amino-acid change (ExoRL81A) at the site of ExoR periplasmic proteolysis resulted in the reduction of the amount of ExoR
m
and the loss of the regulatory function of the ExoR protein. These findings suggest that ExoR
m
is a target of periplasmic proteolysis and that the amount of ExoR
m
could be reduced through effective proteolysis to relieve its suppression of ExoS.
FeoB2 Functions in Magnetosome Formation and Oxidative Stress Protection in Magnetospirillum gryphiswaldense Strain MSR-1
Authors: Rong Chengbo , Zhang Chan , Zhang Yiting , Qi Lei , Yang Jing , Guan Guohua , Li Ying , Li Jilun 
Published: May 2012

ABSTRACT

Magnetotactic bacteria (MTB) synthesize unique organelles, the magnetosomes, which are intracellular nanometer-sized, membrane-enveloped magnetite. The biomineralization of magnetosomes involves the uptake of large amounts of iron. However, the iron metabolism of MTB is not well understood. The genome of the magnetotactic bacterium
Magnetospirillum gryphiswaldense
strain MSR-1 contains two ferrous iron transport genes,
feoB1
and
feoB2
. The FeoB1 protein was reported to be responsible mainly for the transport of ferrous iron and to play an accessory role in magnetosome formation. To determine the role of
feoB2
, we constructed an
feoB2
deletion mutant (MSR-1 Δ
feoB2
) and an
feoB1 feoB2
double deletion mutant (MSR-1 N
feoB
). The single
feoB2
mutation did not affect magnetite crystal biomineralization. MSR-1 N
feoB
had a significantly lower average magnetosome number per cell (∼65%) than MSR-1 Δ
feoB1
, indicating that FeoB2 plays a role in magnetosome formation when the
feoB1
gene is deleted. Our findings showed that FeoB1 has a greater ferrous iron transport ability than FeoB2 and revealed the differential roles of FeoB1 and FeoB2 in MSR-1 iron metabolism. Interestingly, compared to the wild type, the
feoB
mutants showed increased sensitivity to oxidative stress and lower activities of the enzymes superoxide dismutase and catalase, indicating that the FeoB proteins help protect bacterial cells from oxidative stress.
Expanded Roles for Multicargo and Class 1B Effector Chaperones in Type III Secretion
Authors: Thomas Nikhil A. , Ma Irene , Prasad Madhulika E. , Rafuse Cheryl 
Published: May 2012

ABSTRACT
Bacterial type III secretion systems (T3SS) are complex protein assemblies that mediate the secretion of protein substrates outside the cell. Type III secretion chaperones (T3SC) are always found associated with T3SS, and they serve in multiple roles to ensure that protein substrates are efficiently targeted for secretion. Bacterial pathogens with T3SS express T3SC proteins that bind effectors, a process important for effector protein delivery into eukaryotic cells during infection. In this minireview, we focus on multicargo and class 1B T3SC that associate with effectors within significant pathogens of animals and plants. As a primary role, multicargo and class 1B T3SC form homodimers and specifically bind different effectors within the cytoplasm, maintaining the effectors in a secretion-competent state. This role makes T3SC initial and central contributors to effector-mediated pathogenesis. Recent findings have greatly expanded our understanding of cellular events linked to multicargo T3SC function. New binding interactions with T3SS components have been reported in different systems, thereby implicating multicargo T3SC in critical roles beyond effector binding. Three notable interactions with the YscN, YscV, and YscQ family members are well represented in the literature. Similar T3SC interactions are reported in the putative related flagellar T3SS, suggesting that secretion mechanisms may be more similar than previously thought. The evidence implicates multicargo and class 1B T3SC in effector binding and stabilization, in addition to T3SS recruitment and docking events.
OmpA Is the Principal Nonspecific Slow Porin of Acinetobacter baumannii
Authors: Sugawara Etsuko , Nikaido Hiroshi 
Published: May 2012

ABSTRACTAcinetobacterspecies show high levels of intrinsic resistance to many antibiotics. The major protein species in the outer membrane ofAcinetobacter baumanniidoes not belong to the high-permeability trimeric porin family, which includesEscherichia coliOmpF/OmpC, and instead is a close homolog ofE. coliOmpA andPseudomonas aeruginosaOprF. We characterized the pore-forming function of this OmpA homolog, OmpAAb, by a reconstitution assay. OmpAAbproduced very low pore-forming activity, about 70-fold lower than that of OmpF and an activity similar to that ofE. coliOmpA andP. aeruginosaOprF. The pore size of the OmpAAbchannel was similar to that of OprF, i.e., about 2 nm in diameter. The low permeability of OmpAAbis not due to the inactivation of this protein during purification, because the permeability of the wholeA. baumanniiouter membrane was also very low. Furthermore, the outer membrane permeability to cephalothin and cephaloridine, measured in intact cells, was about 100-fold lower than that ofE. coliK-12. The permeability of cephalothin and cephaloridine inA. baumanniiwas decreased 2- to 3-fold when theompAAbgene was deleted. These results show that OmpAAbis the major nonspecific channel inA. baumannii. The low permeability of this porin, together with the presence of constitutive β-lactamases and multidrug efflux pumps, such as AdeABC and AdeIJK, appears to be essential for the high levels of intrinsic resistance to a number of antibiotics.
Genome Sequencing of a Genetically Tractable Pyrococcus furiosus Strain Reveals a Highly Dynamic Genome
Authors: Bridger Stephanie L. , Lancaster W. Andrew , Poole Farris L. , Schut Gerrit J. , Adams Michael W. W. 
Published: May 2012

ABSTRACT

The model archaeon
Pyrococcus furiosus
grows optimally near 100°C on carbohydrates and peptides. Its genome sequence (NCBI) was determined 12 years ago. A genetically tractable strain, COM1, was very recently reported, and here we describe its genome sequence. Of 1,909,827 bp in size, it is 1,571 bp longer (0.1%) than the reference NCBI sequence. The COM1 genome contains numerous chromosomal rearrangements, deletions, and single base changes. COM1 also has 45 full or partial insertion sequences (ISs) compared to 35 in the reference NCBI strain, and these have resulted in the direct deletion or insertional inactivation of 13 genes. Another seven genes were affected by chromosomal deletions and are predicted to be nonfunctional. In addition, the amino acid sequences of another 102 of the 2,134 predicted gene products are different in COM1. These changes potentially impact various cellular functions, including carbohydrate, peptide, and nucleotide metabolism; DNA repair; CRISPR-associated defense; transcriptional regulation; membrane transport; and growth at 72°C. For example, the IS-mediated inactivation of riboflavin synthase in COM1 resulted in a riboflavin requirement for growth. Nevertheless, COM1 grew on cellobiose, malto-oligosaccharides, and peptides in complex and minimal media at 98 and 72°C to the same extent as did both its parent strain and a new culture collection strain (DSMZ 3638). This was in spite of COM1 lacking several metabolic enzymes, including nonphosphorylating glyceraldehyde-3-phosphate dehydrogenase and beta-glucosidase. The
P. furiosus
genome is therefore of high plasticity, and the availability of the COM1 sequence will be critical for the future studies of this model hyperthermophile.
Bacterium-Generated Nitric Oxide Hijacks Host Tumor Necrosis Factor Alpha Signaling and Modulates the Host Cell Cycle
In Vitro
Authors: Mocca Brian , Wang Wei 
Published: May 2012

ABSTRACT

In mammalian cells, nitric oxide (NO·) is an important signal molecule with concentration-dependent and often controversial functions of promoting cell survival and inducing cell death. An inducible nitric oxide synthase (iNOS) in various mammalian cells produces higher levels of NO· from
l
-arginine upon infections to eliminate pathogens. In this study, we reveal novel pathogenic roles of NO· generated by bacteria in bacterium-host cell cocultures using
Moraxella catarrhalis
, a respiratory tract disease-causing bacterium, as a biological producer of NO·. We recently demonstrated that
M. catarrhalis
cells that express the nitrite reductase (AniA protein) can produce NO· by reducing nitrite. Our study suggests that, in the presence of pathophysiological levels of nitrite, this opportunistic pathogen hijacks host cell signaling and modulates host gene expression through its ability to produce NO· from nitrite. Bacterium-generated NO· significantly increases the secretion of tumor necrosis factor alpha (TNF-α) and modulates the expression of apoptotic proteins, therefore triggering host cell programmed death partially through TNF-α signaling. Furthermore, our study reveals that bacterium-generated NO· stalls host cell division and directly results in the death of dividing cells by reducing the levels of an essential regulator of cell division. This study provides unique insight into why NO· may exert more severe cytotoxic effects on fast growing cells, providing an important molecular basis for NO·-mediated pathogenesis in infections and possible therapeutic applications of NO·-releasing molecules in tumorigenesis. This study strongly suggests that bacterium-generated NO· can play important pathogenic roles during infections.
Role of the Fused Corrinoid/Methyl Transfer Protein CmtA during CO-Dependent Growth of Methanosarcina acetivorans
Authors: Vepachedu Venkata R. , Ferry James G. 
Published: May 2012

ABSTRACT

The genome of
Methanosarcina acetivorans
encodes three homologs, initially annotated as hypothetical fused corrinoid/methyl transfer proteins, which are highly elevated in CO-grown cells versus cells grown with alternate substrates. Based only on phenotypic analyses of deletion mutants, it was previously concluded that the homologs are strictly dimethylsulfide:coenzyme M (CoM) methyltransferases not involved in the metabolism of CO (E. Oelgeschlager and M. Rother, Mol. Microbiol. 72:1260 –1272, 2009). The homolog encoded by MA4383 (here designated CmtA) was reexamined via biochemical characterization of the protein overproduced in
Escherichia coli
. Purified CmtA reconstituted with methylcob(III)alamin contained a molar ratio of cobalt to protein of 1.0 ± 0.2. The UV-visible spectrum was typical of methylated corrinoid-containing proteins, with absorbance maxima at 370 and 420 nm and a band of broad absorbance between 450 and 600 nm with maxima at 525, 490, and 550 nm. CmtA reconstituted with aquocobalamin showed methyl-tetrahydromethanopterin:CoM (CH
3
-THMPT:HS-CoM) methyltransferase activity (0.31 μmol/min/mg) with apparent

K
m

values of 135 μM for CH
3
-THMPT and 277 μM for HS-CoM. The ratio of CH
3
-THMPT:HS-CoM methyltransferase activity in the soluble versus membrane cellular fractions was 15-fold greater in CO-grown versus methanol-grown cells. A mutant strain deleted for the CmtA gene showed lower growth rates and final yields when cultured with growth-limiting partial pressures of CO, demonstrating a role for CmtA during growth with this substrate. The results establish that CmtA is a soluble CH
3
-THSPT:HS-CoM methyltransferase postulated to supplement the membrane-bound CH
3
-THMPT:HS-CoM methyltransferase during CO-dependent growth of
M. acetivorans
. Thus, we propose that the name of the enzyme encoded by MA4384 be CmtA (for
c
ytoplasmic
m
ethyl
t
ransferase).
Enterococcus faecalis PrgJ, a VirB4-Like ATPase, Mediates pCF10 Conjugative Transfer through Substrate Binding
Authors: Li Feng , Alvarez-Martinez Cristina , Chen Yuqing , Choi Kyoung-Jae , Yeo Hye-Jeong , Christie Peter J. 
Published: May 2012

ABSTRACT

The
Enterococcus faecalis prg
and
pcf
genes of plasmid pCF10 encode a type IV secretion system (T4SS) required for conjugative transfer. PrgJ is a member of the VirB4 family of ATPases that are universally associated with T4SSs. Here, we report that purified PrgJ dimers displayed ATP binding and hydrolysis activities. A PrgJ nucleoside triphosphate (NTP) binding site mutation (K471E) slightly diminished ATP binding but abolished ATP hydrolysis
in vitro
and blocked pCF10 transfer
in vivo
. As shown with affinity pulldown assays, PrgJ and the K471E mutant protein interacted with the substrate receptor PcfC and with relaxase PcfG and accessory factor PcfF, which together form the relaxosome at the
oriT
sequence to initiate plasmid processing. The purified PrgJ and K471E proteins also bound single- and double-stranded DNA substrates without sequence specificity
in vitro
, and both PrgJ derivatives bound pCF10
in vivo
by a mechanism dependent on an intact
oriT
sequence and cosynthesis of PcfC, PcfF, and PcfG, as shown by a formaldehyde-cross-linking assay. Our findings support a model in which the PcfC receptor coordinates with the PrgJ ATPase to drive early steps of pCF10 processing/transfer: (i) PcfC first binds the pCF10 relaxosome through contacts with PcfF, PcfG, and DNA; (ii) PcfC delivers the plasmid substrate to PrgJ; and (iii) PrgJ catalyzes substrate transfer to the membrane translocase. Substrate engagement with a VirB4-like subunit has not been previously described; consequently, our studies point to a novel function for these signature T4SS ATPases in mediating early steps of type IV secretion.