bluuebunny/crossref_metadata_2025_split huggingface dataset:
The pur7 Gene from the Puromycin Biosynthetic pur Cluster of Streptomyces alboniger Encodes a Nudix Hydrolase
Authors: Espinosa J. C. , Tercero J. A. , Rubio M. A. , Jiménez A. 
Published: December 2019

ABSTRACTPur7 is the product of a gene from the puromycin biosyntheticpurcluster ofStreptomyces alboniger. It was expressed inEscherichia colias a recombinant protein fused to a His tag and then was highly purified through a Ni2+column. It showed a 3′-amino-3′-dATP pyrophosphohydrolase (nudix) activity which produced 3′-amino-3′-dAMP and pyrophosphate. This is consistent with the presence of a nudix box in its amino acid sequence. As observed with other nudix hydrolases, Pur7 has an alkaline pH optimum and a requirement for Mg2+. Among a large variety of other nucleotides tested, only 3′-amino-3′-dTTP was a Pur7 substrate, although at lower reaction rates than 3′-amino-3′-dATP. These findings suggest that Pur7 has a high specificity for the 3′ amino group at the ribofuranoside moiety of these two substrates. TheKmandVmaxvalues for these dATP and dTTP derivatives were 120 μM and 17 μM/min and 3.45 mM and 12.5 μM/min, respectively. Since it is well known that 3′-amino-3′-dATP is a strong inhibitor of DNA-dependent RNA polymerase, whereas 3′-amino-3′-dAMP is not, Pur7 appears to be similar to other nudix enzymes in terms of being a housecleaning agent that permits puromycin biosynthesis to proceed through nontoxic intermediates. Finally, the identification of this activity has allowed a revision of the previously proposed puromycin biosynthetic pathway.
InvF Is Required for Expression of Genes Encoding Proteins Secreted by the SPI1 Type III Secretion Apparatus inSalmonella typhimurium
Authors: Heran Darwin K. , Miller Virginia L. 
Published: December 2019

ABSTRACT
The expression of genes encoding proteins secreted by the SPI1 (Salmonella pathogenicity island) type III secretion apparatus is known to require the transcriptional activators SirA and HilA. However, neither SirA nor HilA is believed to directly activate the promoters of these genes. invF, the first gene of theinv-spa gene cluster, is predicted to encode an AraC-type transcriptional activator and is required for invasion into cultured epithelial cells. However, the genes which are regulated by InvF have not been identified. In this work, an in-frame deletion ininvF was constructed and tested for the expression of Φ(sigD-lacZYA),sipC::Tn5lacZY, and a plasmid-encoded Φ(sicA-lacZYA). SigD (Salmonella invasion gene) is a secreted protein required for the efficient invasion ofSalmonella typhimurium into cultured eucaryotic cells.sicA (Salmonella invasion chaperone) is the first gene of a putative operon encoding the Sip/Ssp (Salmonella invasion/Salmonella secreted proteins) invasion proteins secreted by the SPI1 type III export apparatus. invF was required for the expression of thesigD, sicA, and sipC fusions. This is the first demonstration that there is a functional promoter in the intergenic sequence between spaS and sicA. In addition, several proteins were either absent from or found in reduced amounts in the culture supernatants of the invF mutant. Therefore, invF is required for the optimal expression of several genes encoding SPI1-secreted proteins. Genetic evidence is also presented suggesting there is HilA-dependent readthrough transcription from the invF promoter at least through sipC.
Regulation of the lic Operon ofBacillus subtilis and Characterization of Potential Phosphorylation Sites of the LicR Regulator Protein by Site-Directed Mutagenesis
Authors: Tobisch Steffen , Stülke Jörg , Hecker Michael 
Published: December 2019

ABSTRACTThelicoperon ofBacillus subtilisis required for the transport and degradation of oligomeric β-glucosides, which are produced by extracellular enzymes on substrates such as lichenan or barley glucan. Thelicoperon is transcribed from a ςA-dependent promoter and is inducible by lichenan, lichenan hydrolysate, and cellobiose. Induction of the operon requires a DNA sequence with dyad symmetry located immediately upstream of thelicBCAHpromoter. Expression of thelicoperon is positively controlled by the LicR regulator protein, which contains two potential helix-turn-helix motifs, two phosphoenolpyruvate:carbohydrate phosphotransferase system (PTS) regulation domains (PRDs), and a domain similar to PTS enzyme IIA (EIIA). The activity of LicR is stimulated by modification (probably phosphorylation) of both PRD-I and PRD-II by the general PTS components and is negatively regulated by modification (probably phosphorylation) of its EIIA domain by the specific EIILicin the absence of oligomeric β-glucosides. This was shown by the analysis oflicRmutants affected in potential phosphorylation sites. Moreover, thelicoperon is subject to carbon catabolite repression (CCR). CCR takes place via a CcpA-dependent mechanism and a CcpA-independent mechanism in which the general PTS enzyme HPr is involved.
Purification and Ligand Binding of EmrR, a Regulator of a Multidrug Transporter
Authors: Brooun Alexei , Tomashek John J. , Lewis Kim 
Published: December 2019

ABSTRACTEmrR, the repressor of theemrRABoperon ofEscherichia coli, was purified to 95% homogeneity. EmrR was found to bind putative ligands of the EmrAB pump—2,4-dinitrophenol, carbonyl cyanidem-chlorophenylhydrazone, and carbonyl cyanidep-(trifluoro-methoxy)phenylhydrazone—with affinities in the micromolar range. Equilibrium dialysis experiments suggested one bound ligand per monomer of the dimeric EmrR.
16S rRNA Is Bound to Era of
Streptococcus pneumoniae
Authors: Meier Timothy I. , Peery Robert B. , Jaskunas S. Richard , Zhao Genshi 
Published: December 2019

ABSTRACT

Era is an essential membrane-associated GTPase that is present in bacteria and mycoplasmas. Era appears to play an important role in the regulation of the bacterial cell cycle. In this study, we expressed the native and glutathione
S
-transferase (GST) fusion forms of
Streptococcus pneumoniae
Era in
Escherichia coli
and purified both proteins to homogeneity. We showed that RNA was copurified with the GST-Era protein of
S. pneumoniae
during affinity purification and remained associated with the protein after removal of the GST tag by thrombin cleavage. The thrombin-treated and untreated GST-Era proteins could bind and hydrolyze GTP and exhibited similar kinetic properties (dissociation constant [

k
D

],

K
m

, and
V
max
). However, the native Era protein purified by using different chromatographic columns had a much lower GTPase activity than did GST-Era, although it had a similar

k
D

. In addition, RNA was not associated with the protein. Purified GST-Era protein was shown to be present as high (600-kDa)- and low (120-kDa)-molecular-mass forms. The high-molecular-mass form of GST-Era was associated with RNA and exhibited a very high GTPase activity. Approximately 40% of purified GST-Era protein was associated with RNA, and removal of the RNA resulted in a significant reduction in GTPase activity. The RNA associated with GST-Era was shown to be predominantly 16S rRNA. The native Era protein isolated directly from
S. pneumoniae
was also present as a high-molecular-mass species (600 kDa) complexed with RNA. Together, our results suggest that 16S rRNA is associated with Era and might stimulate its GTPase activity.
The 17-Gene Ethanolamine (
eut
) Operon of
Salmonella typhimurium
Encodes Five Homologues of Carboxysome Shell Proteins
Authors: Kofoid Eric , Rappleye Chad , Stojiljkovic Igor , Roth John 
Published: December 2019

ABSTRACT

The
eut
operon of
Salmonella typhimurium
encodes proteins involved in the cobalamin-dependent degradation of ethanolamine. Previous genetic analysis revealed six
eut
genes that are needed for aerobic use of ethanolamine; one (
eutR
), encodes a positive regulator which mediates induction of the operon by vitamin B
12
plus ethanolamine. The DNA sequence of the
eut
operon included 17 genes, suggesting a more complex pathway than that revealed genetically. We have correlated an open reading frame in the sequence with each of the previously identified genes. Nonpolar insertion and deletion mutations made with the Tn
10
-derived transposable element T-POP showed that at least 10 of the 11 previously undetected
eut
genes have no Eut phenotype under the conditions tested. Of the dispensable
eut
genes, five encode apparent homologues of proteins that serve (in other organisms) as shell proteins of the carboxysome. This bacterial organelle, found in photosynthetic and sulfur-oxidizing bacteria, may contribute to CO
2
fixation by concentrating CO
2
and excluding oxygen. The presence of these homologues in the
eut
operon of
Salmonella
suggests that CO
2
fixation may be a feature of ethanolamine catabolism in
Salmonella
.
Comparative Genetics of Capsular Polysaccharide Biosynthesis in
Streptococcus pneumoniae
Types Belonging to Serogroup 19
Authors: Morona Judy K. , Morona Renato , Paton James C. 
Published: December 2019

ABSTRACT

The genetic basis for the structural diversity of capsule polysaccharide (CPS) in
Streptococcus pneumoniae
serogroup 19 (consisting of types 19F, 19A, 19B, and 19C) has been determined for the first time. In this study, the genetic basis for the 19A and 19C serotypes is described, and the structures of all four serogroup 19
cps
loci and their flanking sequences are compared. Transformation studies show that the structural difference between the 19A and 19F CPSs is likely to be a consequence of differences between their respective polysaccharide polymerase genes (
cps19aI
and
cps19fI
). The CPS of type 19C differs from that of type 19B by the addition of glucose. We have identified a single gene difference between the two
cps
loci (
cps19cS
), which is likely to encode a glucosyl transferase. The arrangement of the genes within the
cps19
loci is highly conserved, with 13 genes (
cps19A
to -
H
and
cps19K
to -
O
) common to all four serogroup 19 members. These
cps
genes encode functions required for the synthesis of the shared trisaccharide component of the group 19 CPS repeat unit structures. Furthermore, the genetic differences between the group 19
cps
loci identified are consistent with the CPS structures of the individual serotypes. Functions have been assigned to nearly all of the
cps19
gene products, based on either gene complementation or similarity to other proteins with known functions, and putative biosynthetic pathways for production of all four group 19 CPSs have been proposed.
An Engineered Cytochrome
b
6
c
1
Complex with a Split Cytochrome
b
Is Able To Support Photosynthetic Growth of
Rhodobacter capsulatus
Authors: Saribas A. Sami , Mandaci Sevnur , Daldal Fevzi 
Published: December 2019

ABSTRACT

The ubihydroquinone-cytochrome
c
oxidoreductase (or the cytochrome
bc
1
complex) from
Rhodobacter capsulatus
is composed of the Fe-S protein, cytochrome
b
, and cytochrome
c
1
subunits encoded by
petA
(
fbcF
),
petB
(
fbcB
), and
petC
(
fbcC
) genes organized as an operon. In the work reported here,
petB
(
fbcB
) was split genetically into two cistrons,
petB6
and
petBIV
, which encoded two polypeptides corresponding to the four amino-terminal and four carboxyl-terminal transmembrane helices of cytochrome
b
, respectively. These polypeptides resembled the cytochrome
b
6
and su IV subunits of chloroplast cytochrome
b
6
f
complexes, and together with the unmodified subunits of the cytochrome
bc
1
complex, they formed a novel enzyme, named cytochrome
b
6
c
1
complex. This membrane-bound multisubunit complex was functional, and despite its smaller amount, it was able to support the photosynthetic growth of
R. capsulatus
. Upon further mutagenesis, a mutant overproducing it, due to a C-to-T transition at the second base of the second codon of
petBIV
, was obtained. Biochemical analyses, including electron paramagnetic spectroscopy, with this mutant revealed that the properties of the cytochrome
b
6
c
1
complex were similar to those of the cytochrome
bc
1
complex. In particular, it was highly sensitive to inhibitors of the cytochrome
bc
1
complex, including antimycin A, and the redox properties of its
b
- and
c
-type heme prosthetic groups were unchanged. However, the optical absorption spectrum of its cytochrome
b
L
heme was modified in a way reminiscent of that of a cytochrome
b
6
f
complex. Based on the work described here and that with
Rhodobacter sphaeroides
(R. Kuras, M. Guergova-Kuras, and A. R. Crofts, Biochemistry 37:16280–16288, 1998), it appears that neither the inhibitor resistance nor the redox potential differences observed between the bacterial (or mitochondrial) cytochrome
bc
1
complexes and the chloroplast cytochrome
b
6
f
complexes are direct consequences of splitting cytochrome
b
into two separate polypeptides. The overall findings also illustrate the possible evolutionary relationships among various cytochrome
bc
oxidoreductases.
Comparison of the
d
-Glutamate-Adding Enzymes from Selected Gram-Positive and Gram-Negative Bacteria
Authors: Walsh Ann W. , Falk Paul J. , Thanassi Jane , Discotto Linda , Pucci Michael J. , Ho Hsu-Tso 
Published: December 2019

ABSTRACT

The biochemical properties of the
d
-glutamate-adding enzymes (MurD) from
Escherichia coli
,
Haemophilus influenzae
,
Enterococcus faecalis
, and
Staphylococcus aureus
were investigated to detect any differences in the activity of this enzyme between gram-positive and gram-negative bacteria. The genes (
murD
) that encode these enzymes were cloned into pMAL-c2 fusion vector and overexpressed as maltose-binding protein–MurD fusion proteins. Each fusion protein was purified to homogeneity by affinity to amylose resin. Proteolytic treatments of the fusion proteins with factor Xa regenerated the individual MurD proteins. It was found that these fusion proteins retain
d
-glutamate-adding activity and have

K
m

and
V
max
values similar to those of the regenerated MurDs, except for the
H. influenzae
enzyme. Substrate inhibition by UDP-
N
-acetylmuramyl-
l
-alanine, the acceptor substrate, was observed at concentrations greater than 15 and 30 μM for E. coli and
H. influenzae
MurD, respectively. Such substrate inhibition was not observed with the
E. faecalis
and
S. aureus
enzymes, up to a substrate concentration of 1 to 2 mM. In addition, the two MurDs of gram-negative origin were shown to require monocations such as NH
4
+
and/or K
+
, but not Na
+
, for optimal activity, while anions such as Cl
−
and SO
4
2−
had no effect on the enzyme activities. The activities of the two MurDs of gram-positive origin, on the other hand, were not affected by any of the ions tested. All four enzymes required Mg
2+
for the ligase activity and exhibited optimal activities around pH 8. These differences observed between the gram-positive and gram-negative MurDs indicated that the two gram-negative bacteria may apply a more stringent regulation of cell wall biosynthesis at the early stage of peptidoglycan biosynthesis pathway than do the two gram-positive bacteria. Therefore, the MurD-catalyzed reaction may constitute a fine-tuning step necessary for the gram-negative bacteria to optimally maintain its relatively thin yet essential cell wall structure during all stages of growth.
The Frequency of Conjugative Transposition of Tn
916
Is Not Determined by the Frequency of Excision
Authors: Marra Diana , Pethel Beth , Churchward Gordon G. , Scott June R. 
Published: December 2019

ABSTRACT

Excision and formation of a covalently closed circular transposon molecule are required for conjugative transposition of Tn
916
but are not the only factors that limit the frequency of conjugative transposition from one host to another. We found that in gram-positive bacteria, an increase in the frequency of excision and circularization of Tn
916
caused by expression of integrase (Int) and excisionase (Xis) from a xylose-inducible promoter does not lead to an increase in the frequency of conjugative transposition. We also found that the concentration of Int and Xis in the recipient cell does not limit the frequency of conjugative transposition and that increased excision does not result in increased expression of transfer functions required to mobilize a plasmid containing the Tn
916
origin of transfer. We conclude that in gram-positive hosts in which the Tn
916
functions Int and Xis are overexpressed, the frequency of conjugative transposition is limited by the availability of transfer functions.
ZmaR, a Novel and Widespread Antibiotic Resistance Determinant That Acetylates Zwittermicin A
Authors: Stohl Elizabeth A. , Brady Sean F. , Clardy Jon , Handelsman Jo 
Published: December 2019

ABSTRACT

ZmaR is a resistance determinant of unusual abundance in the environment and confers on gram-positive and gram-negative bacteria resistance to zwittermicin A, a novel broad-spectrum antibiotic produced by species of
Bacillus
. The ZmaR protein has no sequence similarity to proteins of known function; thus, the purpose of the present study was to determine the function of ZmaR in vitro. Cell extracts of
E. coli
containing
zmaR
inactivated zwittermicin A by covalent modification. Chemical analysis of inactivated zwittermicin A by
1
H NMR,
13
C NMR, and high- and low-resolution mass spectrometry demonstrated that the inactivated zwittermicin A was acetylated. Purified ZmaR protein inactivated zwittermicin A, and biochemical assays for acetyltransferase activity with [
14
C]acetyl coenzyme A demonstrated that ZmaR catalyzes the acetylation of zwittermicin A with acetyl coenzyme A as a donor group, suggesting that ZmaR may constitute a new class of acetyltransferases. Our results allow us to assign a biochemical function to a resistance protein that has no sequence similarity to proteins of known function, contributing fundamental knowledge to the fields of antibiotic resistance and protein function.
Molecular Characterization of the PhoP-PhoQ Two-Component System in
Escherichia coli
K-12: Identification of Extracellular Mg
2+
-Responsive Promoters
Authors: Kato Akinori , Tanabe Hiroyuki , Utsumi Ryutaro 
Published: December 2019

ABSTRACT

We identified Mg
2+
-responsive promoters of the
phoPQ
,
mgtA
, and
mgrB
genes of
Escherichia coli
K-12 by S1 nuclease analysis. Expression of these genes was induced by magnesium limitation and depended on PhoP and PhoQ. The transcription start sites were also determined, which allowed us to find a (T/G)GTTTA direct repeat in their corresponding promoter regions.
A Hyperactive NAD(P)H:Rubredoxin Oxidoreductase from the Hyperthermophilic Archaeon
Pyrococcus furiosus
Authors: Ma Kesen , Adams Michael W. W. 
Published: December 2019

ABSTRACT

NAD(P)H:rubredoxin oxidoreductase (NROR) has been purified from the hyperthermophilic archaeon
Pyrococcus furiosus
. The enzyme is exceedingly active in catalyzing the NADPH-dependent reduction of rubredoxin, a small (5.3-kDa) iron-containing redox protein that had previously been purified from this organism. The apparent
V
max
at 80°C is 20,000 μmol/min/mg, which corresponds to a
k
cat
/

K
m

value of 300,000 mM
−1
s
−1
. The apparent

K
m

values measured at 80°C and pH 8.0 for rubredoxin, NADPH, and NADH were 50, 5, and 34 μM, respectively. The enzyme did not reduce
P. furiosus
ferredoxin. NROR is a monomer with a molecular mass of 45 kDa and contains one flavin adenine dinucleotide molecule per mole but lacks metals and inorganic sulfide. The possible physiological role of this hyperactive enzyme is discussed.
Inactivation and Regulation of the Aerobic C
4
-Dicarboxylate Transport (
dctA
) Gene of
Escherichia coli
Authors: Davies Suzanne J. , Golby Paul , Omrani Davood , Broad Susan A. , Harrington Vikki L. , Guest John R. , Kelly David J. , Andrews Simon C. 
Published: December 2019

ABSTRACT

The gene (
dctA
) encoding the aerobic C
4
-dicarboxylate transporter (DctA) of
Escherichia coli
was previously mapped to the 79-min region of the linkage map. The nucleotide sequence of this region reveals two candidates for the
dctA
gene:
f428
at 79.3 min and the
o157a-o424-o328
(or
orfQMP
) operon at 79.9 min. The
f428
gene encodes a homologue of the
Sinorhizobium meliloti
and
Rhizobium leguminosarum
H
+
/C
4
-dicarboxylate symporter, DctA, whereas the
orfQMP
operon encodes homologues of the aerobic periplasmic-binding protein- dependent C
4
-dicarboxylate transport system (DctQ, DctM, and DctP) of
Rhodobacter capsulatus
. To determine which, if either, of these loci specify the
E. coli
DctA system, the chromosomal
f428
and
orfM
genes were inactivated by inserting Sp
r
or Ap
r
cassettes, respectively. The resulting
f428
mutant was unable to grow aerobically with fumarate or malate as the sole carbon source and grew poorly with succinate. Furthermore, fumarate uptake was abolished in the
f428
mutant and succinate transport was ∼10-fold lower than that of the wild type. The growth and fumarate transport deficiencies of the
f428
mutant were complemented by transformation with an
f428
-containing plasmid. No growth defect was found for the
orfM
mutant. In combination, the above findings confirm that
f428
corresponds to the
dctA
gene and indicate that the
orfQMP
products play no role in C
4
-dicarboxylate transport. Regulation studies with a
dctA-lacZ
(
f428-lacZ
) transcriptional fusion showed that
dctA
is subject to cyclic AMP receptor protein (CRP)-dependent catabolite repression and ArcA-mediated anaerobic repression and is weakly induced by the DcuS-DcuR system in response to C
4
-dicarboxylates and citrate. Interestingly, in a
dctA
mutant, expression of
dctA
is constitutive with respect to C
4
-dicarboxylate induction, suggesting that DctA regulates its own synthesis. Northern blot analysis revealed a single, monocistronic
dctA
transcript and confirmed that
dctA
is subject to regulation by catabolite repression and CRP. Reverse transcriptase-mediated primer extension indicated a single transcriptional start site centered 81 bp downstream of a strongly predicted CRP-binding site.
A Nonessential Signal Peptidase II (Lsp) of
Myxococcus xanthus
Might Be Involved in Biosynthesis of the Polyketide Antibiotic TA
Authors: Paitan Yossi , Orr Elisha , Ron Eliora Z. , Rosenberg Eugene 
Published: December 2019

ABSTRACT

Myxococcus xanthus
is a gram-negative soil bacterium that produces the polyketide antibiotic TA. In this study, we describe the analysis of an
M. xanthus
gene which encodes a homologue of the prolipoprotein signal peptidase II (SPase II;
lsp
). Overexpression of the
M. xanthus
SPase II in
Escherichia coli
confers high levels of globomycin resistance, confirming its function as an SPase II. The
M. xanthus
gene encoding the
lsp
homologue is nonessential for growth, as determined by specific gene disruption. It has been mapped to the antibiotic TA gene cluster, and the disrupted mutants do not produce the antibiotic, indicating a probable involvement in TA production. These results suggest the existence of more than one SPase II protein in
M. xanthus
, where one is a system-specific SPase II (for TA biosynthesis).