bluuebunny/crossref_metadata_2025_split huggingface dataset:
Involvement of Superoxide Dismutase in Spore Coat Assembly in
Bacillus subtilis
Authors: Henriques Adriano O. , Melsen Lawrence R. , Moran Charles P. 
Published: December 2019

ABSTRACT

Endospores of
Bacillus subtilis
are enclosed in a proteinaceous coat which can be differentiated into a thick, striated outer layer and a thinner, lamellar inner layer. We found that the N-terminal sequence of a 25-kDa protein present in a preparation of spore coat proteins matched that of the Mn-dependent superoxide dismutase (SOD) encoded by the
sodA
locus.
sodA
is transcribed throughout the growth and sporulation of a wild-type strain and is responsible for the SOD activity detected in total cell extracts prepared from
B. subtilis
. Disruption of the
sodA
locus produced a mutant that lacked any detectable SOD activity during vegetative growth and sporulation. The
sodA
mutant was not impaired in the ability to form heat- or lysozyme-resistant spores. However, examination of the coat layers of
sodA
mutant spores revealed increased extractability of the tyrosine-rich outer coat protein CotG. We showed that this condition was not accompanied by augmented transcription of the
cotG
gene in sporulating cells of the
sodA
mutant. We conclude that SodA is required for the assembly of CotG into the insoluble matrix of the spore and suggest that CotG is covalently cross-linked into the insoluble matrix by an oxidative reaction dependent on SodA. Ultrastructural analysis revealed that the inner coat formed by a
sodA
mutant was incomplete. Moreover, the outer coat lacked the characteristic striated appearance of wild-type spores, a pattern that was accentuated in a
cotG
mutant. These observations suggest that the SodA-dependent formation of the insoluble matrix containing CotG is largely responsible for the striated appearance of this coat layer.
Nucleotide Sequence and Spatiotemporal Expression of the
Vibrio cholerae vieSAB
Genes during Infection
Authors: Lee Sang Ho , Angelichio Michael J. , Mekalanos John J. , Camilli Andrew 
Published: December 2019

ABSTRACT

The
iviVII
gene of
Vibrio cholerae
was previously identified by a screen for genes induced during intestinal infection. In the present study, nucleotide sequence analysis revealed that
iviVII
is a 1,659-bp open reading frame, herein designated
vieB
, that is predicted to be last in a tricistronic operon (
vieSAB
). The deduced amino acid sequence of VieS exhibited similarity to the sensor kinase component, and those of VieA and VieB were similar to the response regulator components, respectively, of the two-component signal transduction family. Analysis of transcriptional fusions to a site-specific DNA recombinase reporter,
tnpR
, revealed that
vieS
and
vieA
are transcribed during in vitro growth in a
vieAB
-independent and
vieA
-dependent manner, respectively. In contrast, transcription of
vieB
occurred exclusively during infection and was not dependent upon VieB. We conclude that the
vieSAB
genes are differentially regulated, at least during laboratory growth. Use of a
V. cholerae
strain harboring a
vieB
::
tnpR
transcriptional fusion allowed the kinetics and location of
vieB
expression within the intestine to be determined. We found that
vieB
transcription is induced shortly after infection of the proximal and mid-small intestine.
2-Ketocyclohexanecarboxyl Coenzyme A Hydrolase, the Ring Cleavage Enzyme Required for Anaerobic Benzoate Degradation by
Rhodopseudomonas palustris
Authors: Pelletier Dale A. , Harwood Caroline S. 
Published: December 2019

ABSTRACT

2-Ketocyclohexanecarboxyl coenzyme A (2-ketochc-CoA) hydrolase has been proposed to catalyze an unusual hydrolytic ring cleavage reaction as the last unique step in the pathway of anaerobic benzoate degradation by bacteria. This enzyme was purified from the phototrophic bacterium
Rhodopseudomonas palustris
by sequential Q-Sepharose, phenyl-Sepharose, gel filtration, and hydroxyapatite chromatography. The sequence of the 25 N-terminal amino acids of the purified hydrolase was identical to the deduced amino acid sequence of the
badI
gene, which is located in a cluster of genes involved in anaerobic degradation of aromatic acids. The deduced amino acid sequence of
badI
indicates that 2-ketochc-CoA hydrolase is a member of the crotonase superfamily of proteins. Purified BadI had a molecular mass of 35 kDa as determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and a native molecular mass of 134 kDa as determined by gel filtration. This indicates that the native form of the enzyme is a homotetramer. The purified enzyme was insensitive to oxygen and catalyzed the hydration of 2-ketochc-CoA to yield pimelyl-CoA with a specific activity of 9.7 μmol min
−1
mg of protein
−1
. Immunoblot analysis using polyclonal antiserum raised against the purified hydrolase showed that the synthesis of BadI is induced by growth on benzoate and other proposed benzoate pathway intermediates but not by growth on pimelate or succinate. An
R. palustris
mutant, carrying a chromosomal disruption of
badI
, did not grow with benzoate and other proposed benzoate pathway intermediates but had wild-type doubling times on pimelate and succinate. These data demonstrate that BadI, the 2-ketochc-CoA hydrolase, is essential for anaerobic benzoate metabolism by
R. palustris.
Substrate Specificities of Hybrid Naphthalene and 2,4-Dinitrotoluene Dioxygenase Enzyme Systems
Authors: Parales Rebecca E. , Emig Matthew D. , Lynch Nancy A. , Gibson David T. 
Published: December 2019

ABSTRACTBacterial three-component dioxygenase systems consist of reductase and ferredoxin components which transfer electrons from NAD(P)H to a terminal oxygenase. In most cases, the oxygenase consists of two different subunits (α and β). To assess the contributions of the α and β subunits of the oxygenase to substrate specificity, hybrid dioxygenase enzymes were formed by coexpressing genes from two compatible plasmids inEscherichia coli. The activities of hybrid naphthalene and 2,4-dinitrotoluene dioxygenases containing four different β subunits were tested with four substrates (indole, naphthalene, 2,4-dinitrotoluene, and 2-nitrotoluene). In the active hybrids, replacement of small subunits affected the rate of product formation but had no effect on the substrate range, regiospecificity, or enantiomeric purity of oxidation products with the substrates tested. These studies indicate that the small subunit of the oxygenase is essential for activity but does not play a major role in determining the specificity of these enzymes.
Transcriptional Repression Mediated by LysR-Type Regulator CatR Bound at Multiple Binding Sites
Authors: Chugani Sudha A. , Parsek Matthew R. , Chakrabarty A. M. 
Published: December 2019

ABSTRACTThecatBCAoperon ofPseudomonas putidaencodes enzymes involved in the catabolism of benzoate. Transcription of this operon requires the LysR-type transcriptional regulator CatR and an inducer molecule,cis,cis-muconate. Previous gel shift assays and DNase I footprinting have demonstrated that CatR occupies two adjacent sites proximal to thecatBCApromoter in the presence of the inducer. We report the presence of an additional binding site for CatR downstream of thecatBCApromoter within thecatBstructural gene. This site, called the internal binding site (IBS), extends from +162 to +193 with respect to thecatBtranscriptional start site and lies within thecatBopen reading frame. Gel shift analysis and DNase I footprinting determined that CatR binds to this site with low affinity. CatR binds cooperatively with higher affinity to the IBS in the presence of the two upstream binding sites. Parallel in vivo and in vitro studies were conducted to determine the role of the internal binding site. We measured β-galactosidase activity ofcatB-lacZtranscriptional fusions in vivo. Our results suggest a probablecis-acting repressor function for the internal binding site. Site-directed mutagenesis of the IBS verified this finding. The location of the IBS within thecatBstructural gene, the cooperativity observed in footprinting studies, and phasing studies suggest that the IBS likely participates in the interaction of CatR with the upstream binding sites by looping out the intervening DNA.
A Low pH-Inducible, PhoPQ-Dependent Acid Tolerance Response Protects
Salmonella typhimurium
against Inorganic Acid Stress
Authors: Bearson Bradley L. , Wilson Lee , Foster John W. 
Published: December 2019

ABSTRACT

The acid tolerance response enables
Salmonella typhimurium
to survive exposures to potentially lethal acidic environments. The acid stress imposed in a typical assay for acid tolerance (log-phase cells in minimal glucose medium) was shown to comprise both inorganic (i.e., low pH) and organic acid components. A gene previously determined to affect acid tolerance,
atbR
, was identified as
pgi
, the gene encoding phosphoglucoisomerase. Mutations in
pgi
were shown to increase acid tolerance by preventing the synthesis of organic acids. Protocols designed to separate the stresses of inorganic from organic acids revealed that the regulators ς
38
(RpoS), Fur, and Ada have major effects on tolerance to organic acid stress but only minor effects on inorganic acid stress. In contrast, the two-component regulatory system PhoP (identified as acid shock protein ASP29) and PhoQ proved to be important for tolerance to organic acid stress but had little effect against organic acid stress. PhoP mutants also failed to induce four ASPs, confirming a role for this regulator in acid tolerance. Acid shock induction of PhoP appears to occur at the transcriptional level and requires the PhoPQ system. Furthermore, induction by acid occurs even in the presence of high concentrations of magnesium, the ion known to be sensed by PhoQ. These results suggest that PhoQ can sense both Mg
2+
and pH. Since
phoP
mutants are avirulent, the low pH activation of this system has important implications concerning the pathogenesis of
S. typhimurium
. The involvement of four regulators, two of which are implicated in virulence, underscores the complexity of the acid tolerance stress response and further suggests that features of acid tolerance and virulence are interwoven.
A Novel 2-Aminomuconate Deaminase in the Nitrobenzene Degradation Pathway of
Pseudomonas pseudoalcaligenes
JS45
Authors: He Zhongqi , Spain Jim C. 
Published: December 2019

ABSTRACT

2-Aminomuconate, an intermediate in the metabolism of tryptophan in mammals, is also an intermediate in the biodegradation of nitrobenzene by
Pseudomonas pseudoalcaligenes
JS45. Strain JS45 hydrolyzes 2-aminomuconate to 4-oxalocrotonic acid, with the release of ammonia, which serves as the nitrogen source for growth of the microorganism. As an initial step in studying the novel deamination mechanism, we report here the purification and some properties of 2-aminomuconate deaminase. The purified enzyme migrates as a single band with a molecular mass of 16.6 kDa in 15% polyacrylamide gel electrophoresis under denaturing conditions. The estimated molecular mass of the native enzyme was 100 kDa by gel filtration and 4 to 20% gradient nondenaturing polyacrylamide gel electrophoresis, suggesting that the enzyme consists of six identical subunits. The enzyme was stable at room temperature and exhibited optimal activity at pH 6.6. The

K
m

for 2-aminomuconate was approximately 67 μM, and the
V
max
was 125 μmol · min
−1
· mg
−1
. The N-terminal amino acid sequence of the enzyme did not show any significant similarity to any sequence in the databases. The purified enzyme converted 2-aminomuconate directly to 4-oxalocrotonate, rather than 2-hydroxymuconate, which suggests that the deamination was carried out via an imine intermediate.
A Gene Cluster Encoding Steps in Conversion of Naphthalene to Gentisate in
Pseudomonas
sp. Strain U2
Authors: Fuenmayor Sergio L. , Wild Mark , Boyes Alastair L. , Williams Peter A. 
Published: December 2019

ABSTRACT

Pseudomonas
sp. strain U2 was isolated from oil-contaminated soil in Venezuela by selective enrichment on naphthalene as the sole carbon source. The genes for naphthalene dioxygenase were cloned from the plasmid DNA of strain U2 on an 8.3-kb
Bam
HI fragment. The genes for the naphthalene dioxygenase genes
nagAa
(for ferredoxin reductase),
nagAb
(for ferredoxin), and
nagAc
and
nagAd
(for the large and small subunits of dioxygenase, respectively) were located by Southern hybridizations and by nucleotide sequencing. The genes for
nagB
(for naphthalene
cis
-dihydrodiol dehydrogenase) and
nagF
(for salicylaldehyde dehydrogenase) were inferred from subclones by their biochemical activities. Between
nagAa
and
nagAb
were two open reading frames, homologs of which have also been identified in similar locations in two nitrotoluene-using strains (J. V. Parales, A. Kumar, R. E. Parales, and D. T. Gibson, Gene 181:57–61, 1996; W.-C. Suen, B. Haigler, and J. C. Spain, J. Bacteriol. 178:4926–4934, 1996) and a naphthalene-using strain (G. J. Zylstra, E. Kim, and A. K. Goyal, Genet. Eng. 19:257–269, 1997). Recombinant
Escherichia coli
strains with plasmids carrying this region were able to convert salicylate to gentisate, which was identified by a combination of gas chromatography-mass spectrometry and nuclear magnetic resonance. The first open reading frame, designated
nagG
, encodes a protein with characteristics of a Rieske-type iron-sulfur center homologous to the large subunits of dihydroxylating dioxygenases, and the second open reading frame, designated
nagH
, encodes a protein with limited homology to the small subunits of the same dioxygenases. Cloned together in
E. coli
,
nagG
,
nagH
, and
nagAb
, were able to convert salicylate (2-hydroxybenzoate) into gentisate (2,5-dihydroxybenzoate) and therefore encode a salicylate 5-hydroxylase activity. Single-gene knockouts of
nagG
,
nagH
, and
nagAb
demonstrated their functional roles in the formation of gentisate. It is proposed that NagG and NagH are structural subunits of salicylate 5-hydroxylase linked to an electron transport chain consisting of NagAb and NagAa, although
E. coli
appears to be able to partially substitute for the latter. This constitutes a novel mechanism for monohydroxylation of the aromatic ring. Salicylate hydroxylase and catechol 2,3-dioxygenase in strain U2 could not be detected either by enzyme assay or by Southern hybridization. However growth on both naphthalene and salicylate caused induction of gentisate 1,2-dioxygenase, confirming this route for salicylate catabolism in strain U2. Sequence comparisons suggest that the novel gene order
nagAa-nagG-nagH-nagAb-nagAc-nagAd-nagB-nagF
represents the archetype for naphthalene strains which use the gentisate pathway rather than the
meta
cleavage pathway of catechol.
Purification and Biochemical Characterization of the Lambda Holin
Authors: Smith David L. , Struck Douglas K. , Scholtz J. Martin , Young Ry 
Published: December 2019

ABSTRACT

Holins are small phage-encoded cytoplasmic membrane proteins, remarkable for their ability to make membranes permeable in a temporally regulated manner. The purification of S105, the λ holin, and one of the two products of gene
S
is described. Because the wild-type S105 holin could be only partially purified from membrane extracts by ion-exchange chromatography, an oligohistidine tag was added internally to the S105 sequence for use in immobilized metal affinity chromatography. An acceptable site for the tag was found between residues 94 and 95 in the highly charged C-terminal domain of S. This allele, designated
S105H94
, had normal lysis timing under physiological expression conditions. The S105H94 protein was overproduced, purified, and characterized by circular dichroism spectroscopy, which revealed approximately 40% alpha-helix conformation, consistent with the presence of two transmembrane helices. The purified protein was then used to achieve release of fluorescent dye loaded in liposomes in vitro, whereas protein from an isogenic construct carrying an
S
mutation known to abolish hole formation was inactive in this assay. These results suggest that S is a bitopic membrane protein capable of forming aqueous holes in bilayers.
Regulation of a New Cell Wall Hydrolase Gene,
cwlF
, Which Affects Cell Separation in
Bacillus subtilis
Authors: Ishikawa Shu , Hara Yoshiko , Ohnishi Ryo , Sekiguchi Junichi 
Published: December 2019

ABSTRACT

Bacillus subtilis
produces a 35-kDa cell wall hydrolase, CwlF, during vegetative growth. The CwlF protein was extracted from
B. subtilis cwlB sigD
mutant cells and separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. N-terminal amino acid sequencing revealed that its sequence is completely identical to that of the internal region of the
papQ
gene product. Disruption of the
papQ
gene in the
B. subtilis
chromosome led to the complete loss of CwlF, indicating that
papQ
is identical to
cwlF
. CwlF exhibits high sequence similarity to the p60 proteins of
Listeria
species, NlpC proteins of
Escherichia coli
and
Haemophilus influenzae
, and Enp2 protein of
Bacillus sphaericus
. The β-galactosidase activity of the
cwlF-lacZ
transcriptional fusion and Northern blot analysis of the
cwlF
gene indicated that the gene is expressed as a monocistronic operon during the exponential growth phase, and primer extension analysis suggested that the
cwlF
gene is transcribed mainly by EςA RNA polymerase and weakly by EςH RNA polymerase. While the cells of the
cwlF
-deficient mutant were about twice as long as those of the wild-type strain, the
cwlF sigD
double mutant cells exhibited extraordinary microfiber formation, in contrast to the filamentation of the
sigD
mutant. The CwlF production was not affected by the pleiotropic mutations
flaD1
and
degU32
(Hy), which endow cells with the ability of extensive filamentation.
Small Abundant DNA Binding Proteins from the Thermoacidophilic Archaeon
Sulfolobus shibatae
Constrain Negative DNA Supercoils
Authors: Mai Viet Q. , Chen Xulin , Hong Ray , Huang Li 
Published: December 2019

ABSTRACT

Major DNA binding proteins, designated Ssh7, were purified from the thermoacidophilic archaeon
Sulfolobus shibatae
. The Ssh7 proteins have an apparent molecular mass of 6.5 kDa and are similar to the 7-kDa DNA binding proteins from
Sulfolobus acidocaldarius
and
Sulfolobus solfataricus
in N-terminal amino acid sequence. The proteins constitute about 4.8% of the cellular protein. Upon binding to DNA, the Ssh7 proteins constrain negative supercoils. At the tested Ssh7/DNA mass ratios (0 to 1.65), one negative supercoil was taken up by approximately 20 Ssh7 molecules. Our results, together with the observation that the viral DNA isolated from
S. shibatae
is relaxed, suggest that regions of free DNA in the
S. shibatae
genome, if present, are highly positively supercoiled.
Identification and Enzymatic Characterization of the Maltose-Inducible α-Glucosidase MalL (Sucrase-Isomaltase-Maltase) of
Bacillus subtilis
Authors: Schönert Stefan , Buder Thomas , Dahl Michael K. 
Published: December 2019

ABSTRACT

A gene coding for a putative α-glucosidase has been identified in the open reading frame
yvdL
(now termed
malL
), which was sequenced as part of the
Bacillus subtilis
genome project. The enzyme was overproduced in
Escherichia coli
and purified. Further analyses indicate that MalL is a specific oligo-1,4-1,6-α-glucosidase (sucrase-maltase-isomaltase). MalL expression in
B. subtilis
requires maltose induction and is subject to carbon catabolite repression by glucose and fructose. Insertional mutagenesis of
malL
resulted in a complete inactivation of the maltose-inducible α-glucosidase activity in crude protein extracts and a Mal
−
phenotype.
Roles of Low-Molecular-Weight Penicillin-Binding Proteins in
Bacillus subtilis
Spore Peptidoglycan Synthesis and Spore Properties
Authors: Popham David L. , Gilmore Meghan E. , Setlow Peter 
Published: December 2019

ABSTRACT

The peptidoglycan cortex of endospores of
Bacillus
species is required for maintenance of spore dehydration and dormancy, and the structure of the cortex may also allow it to function in attainment of spore core dehydration. A significant difference between spore and growing cell peptidoglycan structure is the low degree of peptide cross-linking in cortical peptidoglycan; regulation of the degree of this cross-linking is exerted by
d
,
d
-carboxypeptidases. We report here the construction of mutant
B. subtilis
strains lacking all combinations of two and three of the four apparent
d
,
d
-carboxypeptidases encoded within the genome and the analysis of spore phenotypic properties and peptidoglycan structure for these strains. The data indicate that while the
dacA
and
dacC
products have no significant role in spore peptidoglycan formation, the
dacB
and
dacF
products both function in regulating the degree of cross-linking of spore peptidoglycan. The spore peptidoglycan of a
dacB dacF
double mutant was very highly cross-linked, and this structural modification resulted in a failure to achieve normal spore core dehydration and a decrease in spore heat resistance. A model for the specific roles of DacB and DacF in spore peptidoglycan synthesis is proposed.
The
Azotobacter vinelandii
Response Regulator AlgR Is Essential for Cyst Formation
Authors: Núñez Cinthia , Moreno Soledad , Soberón-Chávez Gloria , Espín Guadalupe 
Published: December 2019

ABSTRACT

Azotobacter vinelandii
produces the exopolysaccharide alginate, which is essential for the encystment process. In
Pseudomonas aeruginosa
, as well as in
A. vinelandii
, the ς
E
factor encoded by
algU
is required for transcription of
algD
, which encodes a key enzyme of the alginate biosynthetic pathway. The
P. aeruginosa
response regulator AlgR activates transcription of
algD
.
fimS
, located upstream
algR
, is proposed to encode the AlgR cognate sensor kinase. We have cloned and characterized the
A. vinelandii algR
gene; the deduced amino acid sequence of the protein encoded by this gene shows 79% identity with its
P. aeruginosa
homolog. Sequence analysis around the
algR
gene revealed the absence of a
fimS
homolog. Inactivation of
A. vinelandii algR
diminished alginate production by 50%, but did not affect
algD
transcription, and completely impaired the capacity to form mature cysts. Electron microscopy of the cyst structures formed by the
algR
mutant revealed that the encystment process is blocked at the step of exine formation. The transcriptional regulation of the
A. vinelandii algR
gene and the role of AlgR in alginate production differ significantly from those of its
P. aeruginosa
counterparts. These differences could be due to the fact that in
A. vinelandii
, alginate plays a role in encystment, a function not found in
P. aeruginosa
.
Protein Mobility in the Cytoplasm of
Escherichia coli
Authors: Elowitz Michael B. , Surette Michael G. , Wolf Pierre-Etienne , Stock Jeffry B. , Leibler Stanislas 
Published: December 2019

ABSTRACT

The rate of protein diffusion in bacterial cytoplasm may constrain a variety of cellular functions and limit the rates of many biochemical reactions in vivo. In this paper, we report noninvasive measurements of the apparent diffusion coefficient of green fluorescent protein (GFP) in the cytoplasm of
Escherichia coli
. These measurements were made in two ways: by photobleaching of GFP fluorescence and by photoactivation of a red-emitting fluorescent state of GFP (M. B. Elowitz, M. G. Surette, P. E. Wolf, J. Stock, and S. Leibler, Curr. Biol. 7:809–812, 1997). The apparent diffusion coefficient,

D
a

, of GFP in
E. coli
DH5α was found to be 7.7 ± 2.5 μm
2
/s. A 72-kDa fusion protein composed of GFP and a cytoplasmically localized maltose binding protein domain moves more slowly, with

D
a

of 2.5 ± 0.6 μm
2
/s. In addition, GFP mobility can depend strongly on at least two factors: first,

D
a

is reduced to 3.6 ± 0.7 μm
2
/s at high levels of GFP expression; second, the addition to GFP of a small tag consisting of six histidine residues reduces

D
a

to 4.0 ± 2.0 μm
2
/s. Thus, a single effective cytoplasmic viscosity cannot explain all values of

D
a

reported here. These measurements have implications for the understanding of intracellular biochemical networks.