bluuebunny/crossref_metadata_2025_split huggingface dataset:
Characterization of MarR Superrepressor Mutants
Authors: Alekshun Michael N. , Levy Stuart B. 
Published: December 2019

ABSTRACT

MarR negatively regulates expression of the multiple antibiotic resistance (
mar
) locus in
Escherichia coli
. Superrepressor mutants, generated in order to study regions of MarR required for function, exhibited altered inducer recognition properties in whole cells and increased DNA binding to
marO
in vitro. Mutations occurred in three areas of the relatively small MarR protein (144 amino acids). It is surmised that superrepression results from increased DNA binding activities of these mutant proteins.
Isolation and Characterization of Mutations in
Bacillus subtilis
That Allow Spore Germination in the Novel Germinant
d
-Alanine
Authors: Paidhungat Madan , Setlow Peter 
Published: December 2019

ABSTRACT

Bacillus subtilis
spores break their metabolic dormancy through a process called germination. Spore germination is triggered by specific molecules called germinants, which are thought to act by binding to and stimulating spore receptors. Three homologous operons,
gerA
,
gerB
, and
gerK
, were previously proposed to encode germinant receptors because inactivating mutations in those genes confer a germinant-specific defect in germination. To more definitely identify genes that encode germinant receptors, we isolated mutants whose spores germinated in the novel germinant
d
-alanine, because such mutants would likely contain gain-of-function mutations in genes that encoded preexisting germinant receptors. Three independent mutants were isolated, and in each case the mutant phenotype was shown to result from a single dominant mutation in the
gerB
operon. Two of the mutations altered the
gerBA
gene, whereas the third affected the
gerBB
gene. These results suggest that
gerBA
and
gerBB
encode components of the germinant receptor. Furthermore, genetic interactions between the wild-type
gerB
and the mutant
gerBA
and
gerBB
alleles suggested that the germinant receptor might be a complex containing GerBA, GerBB, and probably other proteins. Thus, we propose that the
gerB
operon encodes at least two components of a multicomponent germinant receptor.
Maltose Metabolism in the Hyperthermophilic Archaeon
Thermococcus litoralis
: Purification and Characterization of Key Enzymes
Authors: Xavier Karina B. , Peist Ralf , Kossmann Marina , Boos Winfried , Santos Helena 
Published: December 2019

ABSTRACT

Maltose metabolism was investigated in the hyperthermophilic archaeon
Thermococcus litoralis
. Maltose was degraded by the concerted action of 4-α-glucanotransferase and maltodextrin phosphorylase (MalP). The first enzyme produced glucose and a series of maltodextrins that could be acted upon by MalP when the chain length of glucose residues was equal or higher than four, to produce glucose-1-phosphate. Phosphoglucomutase activity was also detected in
T. litoralis
cell extracts. Glucose derived from the action of 4-α-glucanotransferase was subsequently metabolized via an Embden-Meyerhof pathway. The closely related organism
Pyrococcus furiosus
used a different metabolic strategy in which maltose was cleaved primarily by the action of an α-glucosidase, a
p
-nitrophenyl-α-
d
-glucopyranoside (PNPG)-hydrolyzing enzyme, producing glucose from maltose. A PNPG-hydrolyzing activity was also detected in
T. litoralis
, but maltose was not a substrate for this enzyme. The two key enzymes in the pathway for maltose catabolism in
T. litoralis
were purified to homogeneity and characterized; they were constitutively synthesized, although phosphorylase expression was twofold induced by maltodextrins or maltose. The gene encoding MalP was obtained by complementation in
Escherichia coli
and sequenced (calculated molecular mass, 96,622 Da). The enzyme purified from the organism had a specific activity for maltoheptaose, at the temperature for maximal activity (98°C), of 66 U/mg. A

K
m

of 0.46 mM was determined with heptaose as the substrate at 60°C. The deduced amino acid sequence had a high degree of identity with that of the putative enzyme from the hyperthermophilic archaeon
Pyrococcus horikoshii
OT3 (66%) and with sequences of the enzymes from the hyperthermophilic bacterium
Thermotoga maritima
(60%) and
Mycobacterium tuberculosis
(31%) but not with that of the enzyme from
E. coli
(13%). The consensus binding site for pyridoxal 5′-phosphate is conserved in the
T. litoralis
enzyme.
Growth Phase-Regulated Induction of
Salmonella
-Induced Macrophage Apoptosis Correlates with Transient Expression of SPI-1 Genes
Authors: Lundberg Urban , Vinatzer Ursula , Berdnik Daniela , von Gabain Alexander , Baccarini Manuela 
Published: December 2019

ABSTRACT

Invasive
Salmonella
has been reported to induce apoptosis in a fraction of infected macrophages within 2 to 14 h from the time of infection by a mechanism involving the type III secretion machinery encoded by the
Salmonella
pathogenicity island 1 (SPI-1). Here, we show that bacteria in the transition from logarithmic to stationary phase cause 90% of the macrophages to undergo phagocytosis-independent, caspase-mediated apoptosis within 30 to 60 min of infection. The ability of
Salmonella
to induce this rapid apoptosis was growth phase regulated and cell type restricted, with epithelial cells being resistant. Apoptosis induction was also abrogated by disruption of the
hilA
gene (encoding a regulator of SPI-1 genes) and by the expression of a constitutively active PhoPQ.
hilA
itself and a subset of SPI-1 genes were transiently expressed during aerobic growth in liquid medium. Interestingly, however,
hilA
was found to be required only for the expression of the
prgH
gene, while
sipB
,
invA
, and
invF
were expressed in a
hilA
-independent manner. The expression of SPI-1 genes and the secretion of invasion-associated proteins correlated temporally with the induction of apoptosis and are likely to represent its molecular basis. Thus, growth phase transition regulates the expression and secretion of virulence determinants and represents the most efficient environmental cue for apoptosis induction reported to date.
Regulation of Alginate Biosynthesis in
Pseudomonas syringae
pv. syringae
Authors: Fakhr Mohamed K. , Peñaloza-Vázquez Alejandro , Chakrabarty Ananda M. , Bender Carol L. 
Published: December 2019

ABSTRACT

Both
Pseudomonas aeruginosa
and the phytopathogen
P. syringae
produce the exopolysaccharide alginate. However, the environmental signals that trigger alginate gene expression in
P. syringae
are different from those in
P. aeruginosa
with copper being a major signal in
P. syringae
. In
P. aeruginosa
, the alternate sigma factor encoded by
algT
(ς
22
) and the response regulator AlgR1 are required for transcription of
algD
, a gene which encodes a key enzyme in the alginate biosynthetic pathway. In the present study, we cloned and characterized the gene encoding AlgR1 from
P. syringae
. The deduced amino acid sequence of AlgR1 from
P. syringae
showed 86% identity to its
P. aeruginosa
counterpart. Sequence analysis of the region flanking
algR1
in
P. syringae
revealed the presence of
argH
,
algZ
, and
hemC
in an arrangement virtually identical to that reported in
P. aeruginosa
. An
algR1
mutant,
P. syringae
FF5.32, was defective in alginate production but could be complemented when
algR1
was expressed in
trans
. The
algD
promoter region in
P. syringae
(
PsalgD
) was also characterized and shown to diverge significantly from the
algD
promoter in
P. aeruginosa
. Unlike
P. aeruginosa
,
algR1
was not required for the transcription of
algD
in
P. syringae
, and
PsalgD
lacked the consensus sequence recognized by AlgR1. However, both the
algD
and
algR1
upstream regions in
P. syringae
contained the consensus sequence recognized by ς
22
, suggesting that
algT
is required for transcription of both genes.
Regulation of
hmp
Gene Transcription in
Mycobacterium tuberculosis
: Effects of Oxygen Limitation and Nitrosative and Oxidative Stress
Authors: Hu Yanmin , Butcher Philip D. , Mangan Joseph A. , Rajandream Marie-Adele , Coates Anthony R. M. 
Published: December 2019

ABSTRACT

The
Mycobacterium tuberculosis hmp
gene encodes a protein which is homologous to flavohemoglobin in
Escherichia coli
. Northern blotting analysis demonstrated that
hmp
transcription increased when a microaerophilic culture became oxygen limited as it entered stationary phase at 20 days. There was a fivefold increase of the
hmp
transcripts during early stationary phase compared with the value which was observed in the exponential growth phase. This induction of
hmp
transcription was not due to changes in the mRNA stability since the half-life of
hmp
mRNA was very short in a 20-day microaerophilic culture. No induction of
hmp
mRNA was observed during entry into stationary phase when the culture was continuously aerated.
hmp
transcription was induced after a short exposure of a late-exponential-phase culture to anaerobic conditions. These data indicate that oxygen limitation is the trigger for
hmp
gene transcription. In addition, when a microaerophilic culture entered into the stationary phase at 20 days, transcription of
hmp
increased to a small extent after exposure to
S
-nitrosoglutathione (a nitric oxide [NO] releaser) and sodium nitroprusside (an NO
+
donor) and decreased after exposure to paraquat (a superoxide generator) and H
2
O
2
. In log phase (4 days) and late stationary phase (40 days), the transcription of
hmp
was unaffected by nitrosative and oxidative stress. Three primer extension products were observed. The −10 region is 100% identical to that of promoter T3 in mycobacteria and shows a strong similarity to the −10 sequence of
hmp
and
rpoS
promoters in
E. coli
. These observations of
hmp
mRNA induction in response to O
2
limitation and nitrosative stress suggest that the
hmp
gene of
M. tuberculosis
may have a role in protection of the organism from NO killing under microaerophilic conditions.
Carbon-13 Nuclear Magnetic Resonance Study of Metabolism of Propionate by
Escherichia coli
Authors: London Robert E. , Allen Devon L. , Gabel Scott A. , DeRose Eugene F. 
Published: December 2019

ABSTRACT

We have evaluated the use of [1,2-
13
C
2
]propionate for the analysis of propionic acid metabolism, based on the ability to distinguish between the methylcitrate and methylmalonate pathways. Studies using propionate-adapted
Escherichia coli
MG1655 cells were performed. Preservation of the
13
C-
13
C-
12
C carbon skeleton in labeled alanine and alanine-containing peptides involved in cell wall recycling is indicative of the direct formation of pyruvate from propionate via the methylcitrate cycle, the enzymes of which have recently been demonstrated in
E. coli
. Additionally, formation of
13
C-labeled formate from pyruvate by the action of pyruvate-formate lyase is also consistent with the labeling of pyruvate C-1. Carboxylation of the labeled pyruvate leads to formation of [1,2-
13
C
2
]oxaloacetate and to multiply labeled glutamate and succinate isotopomers, also consistent with the flux through the methylcitrate pathway, followed by the tricarboxylic acid (TCA) cycle. Additional labeling of TCA intermediates arises due to the formation of [1-
13
C]acetyl coenzyme A from the labeled pyruvate, formed via pyruvate-formate lyase. Labeling patterns in trehalose and glycine are also interpreted in terms of the above pathways. The information derived from the [1,2-
13
C
2
]propionate label is contrasted with information which can be derived from singly or triply labeled propionate and shown to be more useful for distinguishing the different propionate utilization pathways via nuclear magnetic resonance analysis.
The Glucuronic Acid Utilization Gene Cluster from
Bacillus stearothermophilus
T-6
Authors: Shulami Smadar , Gat Orit , Sonenshein Abraham L. , Shoham Yuval 
Published: December 2019

ABSTRACT

A λ-EMBL3 genomic library of
Bacillus stearothermophilus
T-6 was screened for hemicellulolytic activities, and five independent clones exhibiting β-xylosidase activity were isolated. The clones overlap each other and together represent a 23.5-kb chromosomal segment. The segment contains a cluster of xylan utilization genes, which are organized in at least three transcriptional units. These include the gene for the extracellular xylanase, xylanase T-6; part of an operon coding for an intracellular xylanase and a β-xylosidase; and a putative 15.5-kb-long transcriptional unit, consisting of 12 genes involved in the utilization of α-
d
-glucuronic acid (GlcUA). The first four genes in the potential GlcUA operon (
orf1
, -
2
, -
3
, and -
4
) code for a putative sugar transport system with characteristic components of the binding-protein-dependent transport systems. The most likely natural substrate for this transport system is aldotetraouronic acid [2-
O
-α-(4-
O
-methyl-α-
d
-glucuronosyl)-xylotriose] (MeGlcUAXyl
3
). The following two genes code for an intracellular α-glucuronidase (
aguA
) and a β-xylosidase (
xynB
). Five more genes (
kdgK
,
kdgA
,
uxaC
,
uxuA
, and
uxuB
) encode proteins that are homologous to enzymes involved in galacturonate and glucuronate catabolism. The gene cluster also includes a potential regulatory gene,
uxuR
, the product of which resembles repressors of the GntR family. The apparent transcriptional start point of the cluster was determined by primer extension analysis and is located 349 bp from the initial ATG codon. The potential operator site is a perfect 12-bp inverted repeat located downstream from the promoter between nucleotides +170 and +181. Gel retardation assays indicated that UxuR binds specifically to this sequence and that this binding is efficiently prevented in vitro by MeGlcUAXyl
3
, the most likely molecular inducer.
The Morphological Transition ofHelicobacter pyloriCells from Spiral to Coccoid Is Preceded by a Substantial Modification of the Cell Wall
Authors: Costa Katyssulla , Bacher Gerold , Allmaier Günter , Dominguez-Bello María Gloria , Engstrand Lars , Falk Per , de Pedro Miguel A. , García-del Portillo Francisco 
Published: December 2019

ABSTRACTThe peptidoglycan (murein) ofHelicobacter pylorihas been investigated by high-performance liquid chromatography and mass spectrometric techniques. Murein fromH. pyloricorresponded to the A1γ chemotype, but the muropeptide elution patterns were substantially different from the one forEscherichia coliin that the former produced high proportions of muropeptides with a pentapeptide side chain (about 60 mol%), with Gly residues as the C-terminal amino acid (5 to 10 mol%), and with (1→6)anhydro-N-acetylmuramic acid (13 to 18 mol%).H. pylorimurein also lacks murein-bound lipoprotein, trimeric muropeptides, and (l-d) cross-linked muropeptides. Cessation of growth and transition to coccoid shape triggered an increase inN-acetylglucosaminyl-N-acetylmuramyl–l-Ala–d-Glu (approximately 20 mol%), apparently at the expense of monomeric muropeptides with tri- and tetrapeptide side chains. Muropeptides with (1→6)anhydro-muramic acid and with Gly were also more abundant in resting cells.
Transcriptional Organization and In Vivo Role of the
Escherichia coli rsd
Gene, Encoding the Regulator of RNA Polymerase Sigma D
Authors: Jishage Miki , Ishihama Akira 
Published: December 2019

ABSTRACT

The regulator of sigma D (Rsd) was identified as an RNA polymerase ς
70
-associated protein in stationary-phase
Escherichia coli
with the inhibitory activity of ς
70
-dependent transcription in vitro (M. Jishage and A. Ishihama, Proc. Natl. Acad. Sci. USA 95:4953–4958, 1998). Primer extension analysis of
rsd
mRNA indicated the presence of two promoters, ς
S
-dependent P1 and ς
70
-dependent P2 with the gearbox sequence. To get insight into the in vivo role of Rsd, the expression of a reporter gene fused to either the ς
70
- or ς
S
-dependent promoter was analyzed in the absence of Rsd or the presence of overexpressed Rsd. In the
rsd
null mutant, the ς
70
- and ς
S
-dependent gene expression was increased or decreased, respectively. On the other hand, the ς
70
- or ς
S
-dependent transcription was reduced or enhanced, respectively, after overexpression of Rsd. The repression of the ς
S
-dependent transcription in the
rsd
mutant is overcome by increased production of the ς
S
subunit. Together these observations support the prediction that Rsd is involved in replacement of the RNA polymerase ς subunit from ς
70
to ς
S
during the transition from exponential growth to the stationary phase.
Transcriptional Regulation in the Hyperthermophilic Archaeon
Pyrococcus furiosus
: Coordinated Expression of Divergently Oriented Genes in Response to β-Linked Glucose Polymers
Authors: Voorhorst Wilfried G. B. , Gueguen Yannick , Geerling Ans C. M. , Schut Gerti , Dahlke Isabell , Thomm Michael , van der Oost John , de Vos Willem M. 
Published: December 2019

ABSTRACT

The genetic organization, expression, and regulation of the
celB
locus of the hyperthermophilic archaeon
Pyrococcus furiosus
were analyzed. This locus includes the
celB
gene, which codes for an intracellular β-glucosidase, and a divergently orientated gene cluster,
adhA-adhB-lamA
, which codes for two alcohol dehydrogenases and an extracellular β-1,3-endoglucanase that is transcribed as a polycistronic messenger (the
lamA
operon). During growth of
P. furiosus
on either the β-1,4-linked glucose dimer cellobiose or the β-1,3-linked glucose polymer laminarin, the activities of both β-glucosidase and endoglucanase were increased at least fivefold compared with levels during growth on maltose or pyruvate. Northern blot analysis revealed an enhanced transcription of both the
celB
gene and the
lamA
operon in the presence of these glucose-containing substrates. The in vivo and in vitro transcription initiation sites of both the
celB
gene and the
lamA
operon were identified 25 nucleotides downstream of conserved TATA box motifs. A number of repeating sequences have been recognized in the
celB-adhA
intergenic region, some of which might be part of a transcriptional regulator-binding site.
Analysis of Quorum-Sensing-Dependent Control of Rhizosphere-Expressed (
rhi
) Genes in
Rhizobium leguminosarum
bv. viciae
Authors: Rodelas Belen , Lithgow James K. , Wisniewski-Dye Florence , Hardman Andrea , Wilkinson Adam , Economou Anastassios , Williams Paul , Downie J. Allan 
Published: December 2019

ABSTRACT

The
rhi
genes of
Rhizobium leguminosarum
biovar viciae are expressed in the rhizosphere and play a role in the interaction with legumes, such as the pea. Previously (K. M. Gray, J. P. Pearson, J. A. Downie, B. E. A. Boboye, and E. P. Greenberg, J. Bacteriol. 178:372–376, 1996) the
rhiABC
operon had been shown to be regulated by RhiR and to be induced by added
N
-(3-hydroxy-7-
cis
-tetradecenoyl)-
l
-homoserine lactone (3OH,C
14:1
-HSL). Mutagenesis of a cosmid carrying the
rhiABC
and
rhiR
gene region identified a gene (
rhiI
) that affects the level of
rhiA
expression. Mutation of
rhiI
slightly increased the number of nodules formed on the pea. The
rhiI
gene is (like
rhiA
) regulated by
rhiR
in a cell density-dependent manner. RhiI is similar to LuxI and other proteins involved in the synthesis of
N
-acyl-homoserine lactones (AHLs). Chemical analyses of spent culture supernatants demonstrated that RhiI produces
N
-(hexanoyl)-
l
-homoserine lactone (C
6
-HSL) and
N
-(octanoyl)-
l
-homoserine lactone (C
8
-HSL). Both of these AHLs induced
rhiA-lacZ
and
rhiI-lacZ
expression on plasmids introduced into an
Agrobacterium
strain that produces no AHLs, showing that
rhiI
is positively regulated by autoinduction. However, in this system no induction of
rhiA
or
rhiI
with 3OH,C
14:1
-HSL was observed. Analysis of the spent culture supernatant of the wild-type
R. leguminosarum
bv. viciae revealed that at least seven different AHLs are made. Mutation of
rhiI
decreased the amounts of C
6
-HSL and C
8
-HSL but did not block their formation, and in this background the
rhiI
mutation did not significantly affect the expression levels of the
rhiI
gene or
rhiABC
genes or the accumulation of RhiA protein. These observations suggest that there are additional loci involved in AHL production in
R. leguminosarum
bv. viciae and that they affect
rhiI
and
rhiABC
expression. We postulate that the previously observed induction of
rhiA
by 3OH,C
14:1
-HSL may be due to an indirect effect caused by induction of other AHL production loci.
AGT1
, Encoding an α-Glucoside Transporter Involved in Uptake and Intracellular Accumulation of Trehalose in
Saccharomyces cerevisiae
Authors: Plourde-Owobi Lucile , Durner Sophie , Parrou Jean-Luc , Wieczorke Roman , Goma Gerard , François Jean 
Published: December 2019

ABSTRACT

The trehalose content in
Saccharomyces cerevisiae
can be significantly manipulated by including trehalose at an appropriate level in the growth medium. Its uptake is largely dependent on the expression of
AGT1
, which encodes an α-glucoside transporter. The trehalose found in a
tps1
mutant of trehalose synthase may therefore largely reflect its uptake from the enriched medium that was employed.
Genotype, Phenotype, and Protein Structure in a Regulator of Sporulation: Effects of Mutations in the
spoIIAA
Gene of
Bacillus subtilis
Authors: Barillà Daniela , Lucet Isabelle , Kuhlmann Anne , Yudkin Michael D. 
Published: December 2019

ABSTRACT

SpoIIAA, a phosphorylatable protein, is essential to the regulation of ς
F
, the first sporulation-specific transcription factor of
Bacillus subtilis
. The solution structure of SpoIIAA has recently been published. Here we examine four mutant SpoIIAA proteins and correlate their properties with the phenotypes of the corresponding
B. subtilis
mutant strains. Two of the mutations severely disrupted the structure of the protein, a third greatly diminished the rate of its phosphorylation and abolished dephosphorylation, and the fourth left phosphorylation unaffected but reduced the rate of dephosphorylation about 10-fold.
Amino Acid Residues in the ω-Minus Region Participate in Cellular Localization of Yeast Glycosylphosphatidylinositol-Attached Proteins
Authors: Hamada Kenji , Terashima Hiromichi , Arisawa Mikio , Yabuki Nami , Kitada Kunio 
Published: December 2019

ABSTRACT

The final destination of glycosylphosphatidylinositol (GPI)-attached proteins in
Saccharomyces cerevisiae
is the plasma membrane or the cell wall. Two kinds of signals have been proposed for their cellular localization: (i) the specific amino acid residues V, I, or L at the site 4 or 5 amino acids upstream of the GPI attachment site (the ω site) and Y or N at the site 2 amino acids upstream of the ω site for cell wall localization and (ii) dibasic residues in the region upstream of the ω site (the ω-minus region) for plasma membrane localization. The relationships between these amino acid residues and efficiencies of cell wall incorporation were examined by constructing fusion reporter proteins from open reading frames encoding putative GPI-attached proteins. The levels of incorporation were high in the constructs containing the specific amino acid residues and quite low in those containing two basic amino acid residues in the ω-minus region. With constructs that contained neither specific residues nor two basic residues, levels of incorporation were moderate. These correlations clearly suggest that GPI-attached proteins have two different signals which act positively or negatively in cell wall incorporation for their cellular localization.