bluuebunny/crossref_metadata_2025_split huggingface dataset:
Novel Functions of (p)ppGpp and Cyclic di-GMP in Mycobacterial Physiology Revealed by Phenotype Microarray Analysis of Wild-Type and Isogenic Strains of Mycobacterium smegmatis
Authors: Gupta Kuldeepkumar Ramnaresh , Kasetty Sanjay , Chatterji Dipankar 
Published: January 2015

ABSTRACT

The bacterial second messengers (p)ppGpp and bis-(3′-5′)-cyclic dimeric GMP (c-di-GMP) regulate important functions, such as transcription, virulence, biofilm formation, and quorum sensing. In mycobacteria, they regulate long-term survival during starvation, pathogenicity, and dormancy. Recently, a
Pseudomonas aeruginosa
strain lacking (p)ppGpp was shown to be sensitive to multiple classes of antibiotics and defective in biofilm formation. We were interested to find out whether
Mycobacterium smegmatis
strains lacking the gene for either (p)ppGpp synthesis (Δ
rel
Msm
) or c-di-GMP synthesis (Δ
dcpA
) would display similar phenotypes. We used phenotype microarray technology to compare the growth of the wild-type and the knockout strains in the presence of several antibiotics. Surprisingly, the Δ
rel
Msm
and Δ
dcpA
strains showed enhanced survival in the presence of many antibiotics, but they were defective in biofilm formation. These strains also displayed altered surface properties, like impaired sliding motility, rough colony morphology, and increased aggregation in liquid cultures. Biofilm formation and surface properties are associated with the presence of glycopeptidolipids (GPLs) in the cell walls of
M. smegmatis
. Thin-layer chromatography analysis of various cell wall fractions revealed that the levels of GPLs and polar lipids were reduced in the knockout strains. As a result, the cell walls of the knockout strains were significantly more hydrophobic than those of the wild type and the complemented strains. We hypothesize that reduced levels of GPLs and polar lipids may contribute to the antibiotic resistance shown by the knockout strains. Altogether, our data suggest that (p)ppGpp and c-di-GMP may be involved in the metabolism of glycopeptidolipids and polar lipids in
M. smegmatis
.
Evolutionary Expansion of the Amidohydrolase Superfamily in Bacteria in Response to the Synthetic Compounds Molinate and Diuron
Authors: Sugrue Elena , Fraser Nicholas J. , Hopkins Davis H. , Carr Paul D. , Khurana Jeevan L. , Oakeshott John G. , Scott Colin , Jackson Colin J. 
Published: January 2015

ABSTRACT
The amidohydrolase superfamily has remarkable functional diversity, with considerable structural and functional annotation of known sequences. In microbes, the recent evolution of several members of this family to catalyze the breakdown of environmental xenobiotics is not well understood. An evolutionary transition from binuclear to mononuclear metal ion coordination at the active sites of these enzymes could produce large functional changes such as those observed in nature, but there are few clear examples available to support this hypothesis. To investigate the role of binuclear-mononuclear active-site transitions in the evolution of new function in this superfamily, we have characterized two recently evolved enzymes that catalyze the hydrolysis of the synthetic herbicides molinate (MolA) and phenylurea (PuhB). In this work, the crystal structures, mutagenesis, metal ion analysis, and enzyme kinetics of both MolA and PuhB establish that these enzymes utilize a mononuclear active site. However, bioinformatics and structural comparisons reveal that the closest putative ancestor of these enzymes had a binuclear active site, indicating that a binuclear-mononuclear transition has occurred. These proteins may represent examples of evolution modifying the characteristics of existing catalysts to satisfy new requirements, specifically, metal ion rearrangement leading to large leaps in activity that would not otherwise be possible.
Multigene Editing in the Escherichia coli Genome via the CRISPR-Cas9 System
Authors: Jiang Yu , Chen Biao , Duan Chunlan , Sun Bingbing , Yang Junjie , Yang Sheng 
Published: January 2015

ABSTRACT

An efficient genome-scale editing tool is required for construction of industrially useful microbes. We describe a targeted, continual multigene editing strategy that was applied to the
Escherichia coli
genome by using the
Streptococcus pyogenes
type II CRISPR-Cas9 system to realize a variety of precise genome modifications, including gene deletion and insertion, with a highest efficiency of 100%, which was able to achieve simultaneous multigene editing of up to three targets. The system also demonstrated successful targeted chromosomal deletions in
Tatumella citrea
, another species of the
Enterobacteriaceae
, with highest efficiency of 100%.
The Putative Mevalonate Diphosphate Decarboxylase from Picrophilus torridus Is in Reality a Mevalonate-3-Kinase with High Potential for Bioproduction of Isobutene
Authors: Rossoni Luca , Hall Stephen J. , Eastham Graham , Licence Peter , Stephens Gill 
Published: January 2015

ABSTRACT

Mevalonate diphosphate decarboxylase (MVD) is an ATP-dependent enzyme that catalyzes the phosphorylation/decarboxylation of (
R
)-mevalonate-5-diphosphate to isopentenyl pyrophosphate in the mevalonate (MVA) pathway. MVD is a key enzyme in engineered metabolic pathways for bioproduction of isobutene, since it catalyzes the conversion of 3-hydroxyisovalerate (3-HIV) to isobutene, an important platform chemical. The putative homologue from
Picrophilus torridus
has been identified as a highly efficient variant in a number of patents, but its detailed characterization has not been reported. In this study, we have successfully purified and characterized the putative MVD from
P. torridus.
We discovered that it is not a decarboxylase
per se
but an ATP-dependent enzyme, mevalonate-3-kinase (M3K), which catalyzes the phosphorylation of MVA to mevalonate-3-phosphate. The enzyme's potential in isobutene formation is due to the conversion of 3-HIV to an unstable 3-phosphate intermediate that undergoes consequent spontaneous decarboxylation to form isobutene. Isobutene production rates were as high as 507 pmol min
−1
g cells
−1
using
Escherichia coli
cells expressing the enzyme and 2,880 pmol min
−1
mg protein
−1
with the purified histidine-tagged enzyme, significantly higher than reported previously. M3K is a key enzyme of the novel MVA pathway discovered very recently in
Thermoplasma acidophilum
. We suggest that
P. torridus
metabolizes MVA by the same pathway.
Type III Secretion System and Virulence Markers Highlight Similarities and Differences between Human- and Plant-Associated Pseudomonads Related to Pseudomonas fluorescens and P. putida
Authors: Mazurier Sylvie , Merieau Annabelle , Bergeau Dorian , Decoin Victorien , Sperandio Daniel , Crépin Alexandre , Barbey Corinne , Jeannot Katy , Vicré-Gibouin Maïté , Plésiat Patrick , Lemanceau Philippe , Latour Xavier 
Published: January 2015

ABSTRACTPseudomonas fluorescensis commonly considered a saprophytic rhizobacterium devoid of pathogenic potential. Nevertheless, the recurrent isolation of strains from clinical human cases could indicate the emergence of novel strains originating from the rhizosphere reservoir, which could be particularly resistant to the immune system and clinical treatment. The importance of type three secretion systems (T3SSs) in the relatedPseudomonas aeruginosanosocomial species and the occurrence of this secretion system in plant-associatedP. fluorescensraise the question of whether clinical isolates may also harbor T3SSs. In this study, isolates associated with clinical infections and identified in hospitals as belonging toP. fluorescenswere compared with fluorescent pseudomonads harboring T3SSs isolated from plants. Bacterial isolates were tested for (i) their genetic relationships based on their 16S rRNA phylogeny, (ii) the presence of T3SS genes by PCR, and (iii) their infectious potential on animals and plants under environmental or physiological temperature conditions. Two groups of bacteria were delineated among the clinical isolates. The first group encompassed thermotolerant (41°C) isolates from patients suffering from blood infections; these isolates were finally found to not belong toP. fluorescensbut were closely related and harbored highly conserved T3SS genes belonging to the Ysc-T3SS family, like the T3SSs fromP. aeruginosa. The second group encompassed isolates from patients suffering from cystic fibrosis; these isolates belonged toP. fluorescensand harbored T3SS genes belonging to the Hrp1-T3SS family found commonly in plant-associatedP. fluorescens.
The Flagellar Regulator TviA Reduces Pyroptosis by Salmonella enterica Serovar Typhi
Authors: Winter Sebastian E. , Winter Maria G. , Atluri Vidya , Poon Victor , Romão Everton L. , Tsolis Renée M. , Bäumler Andreas J. 
Published: February 2015

ABSTRACT

To discern virulent from innocuous microbes, the innate immune system senses events associated with bacterial access to immunoprivileged sites such as the host cell cytosol. One such pathway is triggered by the cytosolic delivery of flagellin, the major subunit of the flagellum, by bacterial secretion systems. This leads to inflammasome activation and subsequent proinflammatory cell death (pyroptosis) of the infected phagocyte. In this study, we demonstrate that the causative agent of typhoid fever,
Salmonella enterica
serovar Typhi, can partially subvert this critical innate immune recognition event. The transcriptional regulator TviA, which is absent from
Salmonella
serovars associated with human gastroenteritis, repressed the expression of flagellin during infection of human macrophage-like (THP-1) cells. This mechanism allowed
S
. Typhi to dampen inflammasome activation, leading to reduced interleukin-1β (IL-1β) secretion and diminished cell death. Likewise, the introduction of the
tviA
gene in nontyphoidal
Salmonella enterica
serovar Typhimurium reduced flagellin-induced pyroptosis. These data suggest that gene regulation of virulence factors enables
S
. Typhi to evade innate immune recognition by concealing a pathogen-induced process from being sensed by the inflammasome.
A Novel Pyrroloquinoline Quinone-Dependent 2-Keto-
d
-Glucose Dehydrogenase from Pseudomonas aureofaciens
Authors: Umezawa Kiwamu , Takeda Kouta , Ishida Takuya , Sunagawa Naoki , Makabe Akiko , Isobe Kazuo , Koba Keisuke , Ohno Hiroyuki , Samejima Masahiro , Nakamura Nobuhumi , Igarashi Kiyohiko , Yoshida Makoto 
Published: February 2015

ABSTRACT

A gene encoding an enzyme similar to a pyrroloquinoline quinone (PQQ)-dependent sugar dehydrogenase from filamentous fungi, which belongs to new auxiliary activities (AA) family 12 in the CAZy database, was cloned from
Pseudomonas aureofaciens
. The deduced amino acid sequence of the cloned enzyme showed only low homology to previously characterized PQQ-dependent enzymes, and multiple-sequence alignment analysis showed that the enzyme lacks one of the three conserved arginine residues that function as PQQ-binding residues in known PQQ-dependent enzymes. The recombinant enzyme was heterologously expressed in an
Escherichia coli
expression system for further characterization. The UV-visible (UV-Vis) absorption spectrum of the oxidized form of the holoenzyme, prepared by incubating the apoenzyme with PQQ and CaCl
2
, revealed a broad peak at approximately 350 nm, indicating that the enzyme binds PQQ. With the addition of 2-keto-
d
-glucose (2KG) to the holoenzyme solution, a sharp peak appeared at 331 nm, attributed to the reduction of PQQ bound to the enzyme, whereas no effect was observed upon 2KG addition to authentic PQQ. Enzymatic assay showed that the recombinant enzyme specifically reacted with 2KG in the presence of an appropriate electron acceptor, such as 2,6-dichlorophenol indophenol, when PQQ and CaCl
2
were added.
1
H nuclear magnetic resonance (
1
H-NMR) analysis of reaction products revealed 2-keto-
d
-gluconic acid (2KGA) as the main product, clearly indicating that the recombinant enzyme oxidizes the C-1 position of 2KG. Therefore, the enzyme was identified as a PQQ-dependent 2KG dehydrogenase (
Pa
2KGDH). Considering the high substrate specificity, the physiological function of
Pa
2KGDH may be for production of 2KGA.
SepD/SepL-Dependent Secretion Signals of the Type III Secretion System Translocator Proteins in Enteropathogenic Escherichia coli
Authors: Deng Wanyin , Yu Hong B. , Li Yuling , Finlay B. Brett 
Published: February 2015

ABSTRACTThe type III protein secretion system (T3SS) encoded by the locus of enterocyte effacement (LEE) is essential for the pathogenesis of attaching/effacing bacterial pathogens, including enteropathogenicEscherichia coli(EPEC), enterohemorrhagicE. coli(EHEC), andCitrobacter rodentium. These pathogens use the T3SS to sequentially secrete three categories of proteins: the T3SS needle and inner rod protein components; the EspA, EspB, and EspD translocators; and many LEE- and non-LEE-encoded effectors. SepD and SepL are essential for translocator secretion, and mutations in either lead to hypersecretion of effectors. However, how SepD and SepL control translocator secretion and secretion hierarchy between translocators and effectors is poorly understood. In this report, we show that the secreted T3SS components, the translocators, and both LEE- and non-LEE-encoded effectors all carry N-terminal type III secretion and translocation signals. These signals all behave like those of the effectors and are sufficient for mediating type III secretion and translocation by wild-type EPEC and hypersecretion by thesepDandsepLmutants. Our results extended previous observations and suggest that the secretion hierarchy of the different substrates is determined by a signal other than the N-terminal secretion signal. We identified a domain located immediately downstream of the N-terminal secretion signal in the translocator EspB that is required for SepD/SepL-dependent secretion. We further demonstrated that this EspB domain confers SepD/SepL- and CesAB-dependent secretion on the secretion signal of effector EspZ. Our results thus suggest that SepD and SepL control and regulate secretion hierarchy between translocators and effectors by recognizing translocator-specific export signals.IMPORTANCEMany bacterial pathogens use a syringe-like protein secretion apparatus, termed the type III protein secretion system (T3SS), to secrete and inject numerous proteins directly into the host cells to cause disease. The secreted proteins perform different functions at various stages during infection and are classified into three substrate categories (T3SS components, translocators, and effectors). They all contain secretion signals at their N termini, but how their secretion hierarchy is determined is poorly understood. Here, we show that the N-terminal secretion signals from different substrate categories all behave the same and do not confer substrate specificity. We further characterize the secretion signals of the translocators and identify a translocator-specific signal, demonstrating that substrate-specific secretion signals are required in regulating T3SS substrate hierarchy.
Characterization of Novel Acyl Coenzyme A Dehydrogenases Involved in Bacterial Steroid Degradation
Authors: Ruprecht Amanda , Maddox Jaymie , Stirling Alexander J. , Visaggio Nicole , Seah Stephen Y. K. 
Published: February 2015

ABSTRACT

The acyl coenzyme A (acyl-CoA) dehydrogenases (ACADs) FadE34 and CasC, encoded by the cholesterol and cholate gene clusters of
Mycobacterium tuberculosis
and
Rhodococcus jostii
RHA1, respectively, were successfully purified. Both enzymes differ from previously characterized ACADs in that they contain two fused acyl-CoA dehydrogenase domains in a single polypeptide. Site-specific mutagenesis showed that only the C-terminal ACAD domain contains the catalytic glutamate base required for enzyme activity, while the N-terminal ACAD domain contains an arginine required for ionic interactions with the pyrophosphate of the flavin adenine dinucleotide (FAD) cofactor. Therefore, the two ACAD domains must associate to form a single active site. FadE34 and CasC were not active toward the 3-carbon side chain steroid metabolite 3-oxo-23,24-bisnorchol-4-en-22-oyl-CoA (4BNC-CoA) but were active toward steroid CoA esters containing 5-carbon side chains. CasC has similar specificity constants for cholyl-CoA, deoxycholyl-CoA, and 3β-hydroxy-5-cholen-24-oyl-CoA, while FadE34 has a preference for the last compound, which has a ring structure similar to that of cholesterol metabolites. Knockout of the
casC
gene in
R. jostii
RHA1 resulted in a reduced growth on cholate as a sole carbon source and accumulation of a 5-carbon side chain cholate metabolite. FadE34 and CasC represent unique members of ACADs with primary structures and substrate specificities that are distinct from those of previously characterized ACADs.

IMPORTANCE
We report here the identification and characterization of acyl-CoA dehydrogenases (ACADs) involved in the metabolism of 5-carbon side chains of cholesterol and cholate. The two homologous enzymes FadE34 and CasC, from
M. tuberculosis
and
Rhodococcus jostii
RHA1, respectively, contain two ACAD domains per polypeptide, and we show that these two domains interact to form a single active site. FadE34 and CasC are therefore representatives of a new class of ACADs with unique primary and quaternary structures. The bacterial steroid degradation pathway is important for the removal of steroid waste in the environment and for survival of the pathogen
M. tuberculosis
within host macrophages. FadE34 is a potential target for development of new antibiotics against tuberculosis.
Stability of the Osmoregulated Promoter-DerivedproPmRNA Is Posttranscriptionally Regulated by RNase III in Escherichia coli
Authors: Lim Boram , Lee Kangseok 
Published: February 2015

ABSTRACTThe enzymatic activity ofEscherichia coliendo-RNase III determines the stability of a subgroup of mRNA species, includingbdm,betT, andproU, whose protein products are associated with the cellular response to osmotic stress. Here, we report that the stability ofproPmRNA, which encodes a transporter of osmoprotectants, is controlled by RNase III in response to osmotic stress. We observed that steady-state levels ofproPmRNA and ProP protein are inversely correlated with cellular RNase III activity and, in turn, affect the proline uptake capacity of the cell.In vitroandin vivoanalyses ofproPmRNA revealed RNase III cleavage sites in a stem-loop within the 5′ untranslated region present only inproPmRNA species synthesized from the osmoregulated P1 promoter. Introduction of nucleotide substitutions in the cleavage site identified inhibited the ribonucleolytic activity of RNase III onproPmRNA, increasing the steady-state levels and half-life of the mRNA. In addition, decreased RNase III activity coincided with a significant increase in both the half-life and abundance ofproPmRNA under hyperosmotic stress conditions. Analysis of the RNA bound to RNase III viain vivocross-linking and immunoprecipitation indicated that this phenomenon is related to the decreased RNA binding capacity of RNase III. Our findings suggest the existence of an RNase III-mediated osmoregulatory network that rapidly balances the expression levels of factors associated with the cellular response to osmotic stress inE. coli.IMPORTANCEOur results demonstrate that RNase III activity onproPmRNA degradation is downregulated inEscherichia colicells under osmotic stress. In addition, we show that the downregulation of RNase III activity is associated with decreased RNA binding capacity of RNase III under hyperosmotic conditions. In particular, our findings demonstrate a link between osmotic stress and RNase III activity, underscoring the growing importance of posttranscriptional regulation in modulating rapid physiological adjustment to environmental changes.
The
STM4195
Gene Product (PanS) Transports Coenzyme A Precursors in Salmonella enterica
Authors: Ernst Dustin C. , Downs Diana M. 
Published: February 2015

ABSTRACT

Coenzyme A (CoA) is a ubiquitous coenzyme involved in fundamental metabolic processes. CoA is synthesized from pantothenic acid by a pathway that is largely conserved among bacteria and eukaryotes and consists of five enzymatic steps. While higher organisms, including humans, must scavenge pantothenate from the environment, most bacteria and plants are capable of
de novo
pantothenate biosynthesis. In
Salmonella enterica
, precursors to pantothenate can be salvaged, but subsequent intermediates are not transported due to their phosphorylated state, and thus the pathway from pantothenate to CoA is considered essential. Genetic analyses identified the
STM4195
gene product of
Salmonella enterica
serovar Typhimurium as a transporter of pantothenate precursors, ketopantoate and pantoate and, to a lesser extent, pantothenate. Further results indicated that STM4195 transports a product of CoA degradation that serves as a precursor to CoA and enters the biosynthetic pathway between PanC and CoaBC (
dfp
). The relevant CoA derivative is distinguishable from pantothenate, pantetheine, and pantethine and has spectral properties indicating the adenine moiety of CoA is intact. Taken together, the results presented here provide evidence of a transport mechanism for the uptake of ketopantoate, pantoate, and pantothenate and demonstrate a role for STM4195 in the salvage of a CoA derivative of unknown structure. The
STM4195
gene is renamed
panS
to reflect participation in
pan
tothenate
s
alvage that was uncovered herein.

IMPORTANCE
This manuscript describes a transporter for two pantothenate precursors in addition to the existence and transport of a salvageable coenzyme A (CoA) derivative. Specifically, these studies defined a function for an STM protein in
S. enterica
that was distinct from the annotated role and led to its designation as PanS (
pan
tothenate
s
alvage). The presence of a salvageable CoA derivative and a transporter for it suggests the possibility that this compound is present in the environment and may serve a role in CoA synthesis for some organisms. As such, this work raises important question about CoA salvage that can be pursued with future studies in bacteria and other organisms.
Role of Branched-Chain Amino Acid Transport in Bacillus subtilis CodY Activity
Authors: Belitsky Boris R. 
Published: February 2015

ABSTRACT

CodY is a branched-chain amino acid-responsive transcriptional regulator that controls the expression of several dozen transcription units in
Bacillus subtilis
. The presence of isoleucine, valine, and leucine in the growth medium is essential for achieving high activity of CodY and for efficient regulation of the target genes. We identified three permeases—BcaP, BraB, and BrnQ—that are responsible for the bulk of isoleucine and valine uptake and are also involved in leucine uptake. At least one more permease is capable of efficient leucine uptake, as well as low-affinity transport of isoleucine and valine. The lack of the first three permeases strongly reduced activity of CodY in an amino acid-containing growth medium. BcaP appears to be the most efficient isoleucine and valine permease responsible for their utilization as nitrogen sources. The previously described strong CodY-mediated repression of BcaP provides a mechanism for fine-tuning CodY activity by reducing the availability of amino acids and for delaying the utilization of isoleucine and valine as nitrogen and carbon sources under conditions of nutrient excess.

IMPORTANCE
Bacillus subtilis
CodY is a global transcriptional regulator that is activated by branched-chain amino acids (BCAA). Since the level of BCAA achieved by intracellular synthesis is insufficient to fully activate CodY, transport of BCAA from the environment is critical for CodY activation, but the permeases needed for such activation have not been previously identified. This study identifies three such permeases, reports their amino acid transport specificity, and reveals their impact on CodY activation.