bluuebunny/crossref_metadata_2025_split huggingface dataset:
Functions of Two Types of NADH Oxidases in Energy Metabolism and Oxidative Stress of
Streptococcus mutans
Authors: Higuchi Masako , Yamamoto Yuji , Poole Leslie B. , Shimada Mamoru , Sato Yutaka , Takahashi Nobuhiro , Kamio Yoshiyuki 
Published: December 2019

ABSTRACT

We have previously identified two distinct NADH oxidases corresponding to H
2
O
2
-forming oxidase (Nox-1) and H
2
O-forming oxidase (Nox-2) induced in
Streptococcus mutans
. Sequence analyses indicated a strong similarity between Nox-1 and AhpF, the flavoprotein component of
Salmonella typhimurium
alkyl hydroperoxide reductase; an open reading frame upstream of
nox-1
also showed homology to AhpC, the direct peroxide-reducing component of
S. typhimurium
alkyl hydroperoxide reductase. To determine their physiological functions in
S. mutans
, we constructed knockout mutants of Nox-1, Nox-2, and/or the AhpC homologue; we verified that Nox-2 plays an important role in energy metabolism through the regeneration of NAD
+
but Nox-1 contributes negligibly. The Nox-2 mutant exhibited greatly reduced aerobic growth on mannitol, whereas there was no significant effect of aerobiosis on the growth on mannitol of the other strains or growth on glucose of any of the strains. Although the Nox-2 mutants grew well on glucose aerobically, the end products of glucose fermentation by the Nox-2 mutant were substantially shifted to higher ratios of lactic acid to acetic acid compared with wild-type cells. The resistance to cumene hydroperoxide of
Escherichia coli
TA4315 (
ahpCF
-defective mutant) transformed with pAN119 containing both
nox-1
and
ahpC
genes was not only restored but enhanced relative to that of
E. coli
K-12 (parent strain), indicating a clear function for Nox-1 as part of an alkyl hydroperoxide reductase system in vivo in combination with AhpC. Surprisingly, the Nox-1 and/or AhpC deficiency had no effect on the sensitivity of
S. mutans
to cumene hydroperoxide and H
2
O
2
, implying that the existence of some other antioxidant system(s) independent of Nox-1 in
S. mutans
compensates for the deficiency.
Regulation of
pelD
and
pelE
, Encoding Major Alkaline Pectate Lyases in
Erwinia chrysanthemi
: Involvement of the Main Transcriptional Factors
Authors: Rouanet Carine , Nomura Kinya , Tsuyumu Shinji , Nasser William 
Published: December 2019

ABSTRACT

The main virulence factors of the phytopathogenic bacterium
Erwinia chrysanthemi
are pectinases which attack pectin, the major constituent of the plant cell wall. Of these enzymes, the alkaline isoenzyme named PelD in strain 3937 and PelE in strain EC16 has been described as being particularly important, based on virulence studies of plants. Expression of the
pelD
and
pelE
genes is tightly modulated by various regulators, including the KdgR repressor and the cyclic AMP-cyclic AMP receptor protein (CRP) activator complex. The use of a
lacZ
reporter gene allowed us to quantify the repression of
E. chrysanthemi
3937
pelD
expression exerted by PecS, another repressor of pectinase synthesis. In vitro DNA-protein interaction experiments, centered on the
pelD
and
pelE
wild-type or
pelE
mutated promoter regions, allowed us to define precisely the sequences involved in the binding of these three regulators and of RNA polymerase (RNAP). These studies revealed an unusual binding of the KdgR repressor and suggested the presence of a UP (upstream) element in the
pelD
and
pelE
genes. Investigation of the simultaneous binding of CRP, KdgR, PecS, and the RNAP to the regulatory region of the
pelD
and
pelE
genes showed that (i) CRP and RNAP bind cooperatively, (ii) PecS partially inhibits binding of the CRP activator and of the CRP-RNAP complex, and (iii) KdgR stabilizes the binding of PecS and prevents transcriptional initiation by RNAP. Taken together, our data suggest that PecS attenuates
pelD
and
pelE
expression rather than acting as a true repressor like KdgR. Overall, control of the
pelD
and
pelE
genes of
E. chrysanthemi
appears to be both complex and novel.
The Propanediol Utilization (
pdu
) Operon of
Salmonella enterica
Serovar Typhimurium LT2 Includes Genes Necessary for Formation of Polyhedral Organelles Involved in Coenzyme B
12
-Dependent 1,2-Propanediol Degradation
Authors: Bobik Thomas A. , Havemann Gregory D. , Busch Robert J. , Williams Donna S. , Aldrich Henry C. 
Published: December 2019

ABSTRACT

The propanediol utilization (
pdu
) operon of
Salmonella enterica
serovar Typhimurium LT2 contains genes needed for the coenzyme B
12
-dependent catabolism of 1,2-propanediol. Here the completed DNA sequence of the
pdu
operon is presented. Analyses of previously unpublished
pdu
DNA sequence substantiated previous studies indicating that the
pdu
operon was acquired by horizontal gene transfer and allowed the identification of 16 hypothetical genes. This brings the total number of genes in the
pdu
operon to 21 and the total number of genes at the
pdu
locus to 23. Of these, six encode proteins of unknown function and are not closely related to sequences of known function found in GenBank. Two encode proteins involved in transport and regulation. Six probably encode enzymes needed for the pathway of 1,2-propanediol degradation. Two encode proteins related to those used for the reactivation of adenosylcobalamin (AdoCbl)-dependent diol dehydratase. Five encode proteins related to those involved in the formation of polyhedral organelles known as carboxysomes, and two encode proteins that appear distantly related to those involved in carboxysome formation. In addition, it is shown that
S. enterica
forms polyhedral bodies that are involved in the degradation of 1,2-propanediol. Polyhedra are formed during either aerobic or anaerobic growth on propanediol, but not during growth on other carbon sources. Genetic tests demonstrate that genes of the
pdu
operon are required for polyhedral body formation, and immunoelectron microscopy shows that AdoCbl-dependent diol dehydratase is associated with these polyhedra. This is the first evidence for a B
12
-dependent enzyme associated with a polyhedral body. It is proposed that the polyhedra consist of AdoCbl-dependent diol dehydratase (and perhaps other proteins) encased within a protein shell that is related to the shell of carboxysomes. The specific function of these unusual polyhedral bodies was not determined, but some possibilities are discussed.
Plasmid RK2 ParB Protein: Purification and Nuclease Properties
Authors: Johnson Erik P. , Mincer Tracy , Schwab Helmut , Burgin Alex B. , Helinski Donald R. 
Published: December 2019

ABSTRACT

The
parCBA
operon of the 3.2-kb stabilization region of plasmid RK2 encodes three cotranslated proteins. ParA mediates site-specific recombination to resolve plasmid multimers, ParB has been shown to be a nuclease, and the function of ParC is unknown. In this study ParB was overexpressed by cotranslation with ParC in
Escherichia coli
by using a plasmid construct that contained the
parC
and
parB
genes under the control of the T7 promoter. Purification was achieved by treatment of extracts with Polymin P, followed by ammonium sulfate precipitation and heparin and ion-exchange chromatography. Sizing-column analysis indicated that ParB exists as a monomer in solution. Analysis of the enzymatic properties of purified ParB indicated that the protein preferentially cleaves single-stranded DNA. ParB also nicks supercoiled plasmid DNA preferably at sites with potential single-stranded character, like AT-rich regions and sequences that can form cruciform structures. ParB also exhibits 5′→3′ exonuclease activity. This ParB activity on a 5′-end-labeled, double-stranded DNA substrate produces a 3′,5′-phosphorylated dinucleotide which is further cleaved to a 3′,5′-phosphorylated mononucleotide. The role of the ParB endonuclease and exonuclease activities in plasmid RK2 stabilization remains to be determined.
Use of Heme-Protein Complexes by the
Yersinia enterocolitica
HemR Receptor: Histidine Residues Are Essential for Receptor Function
Authors: Bracken Charles S. , Baer Michael T. , Abdur-Rashid Asiya , Helms Whitney , Stojiljkovic Igor 
Published: December 2019

ABSTRACT

The abilities of two bacterial active heme transporters, HmbR of
Neisseria meningitidis
and HemR of
Yersinia enterocolitica
, to use different heme sources were compared. While HmbR-expressing cells used only hemoglobin (Hb) and heme, HemR-expressing bacteria were able to grow on Hb, heme, myoglobin, hemopexin, catalase, human and bovine serum albumin-heme, and haptoglobin-hemoglobin complexes as sources of iron. Expression of functional HemR allowed
Escherichia coli
cells to respond to heme-containing peptides, microperoxidases MP-8, MP-9, and MP-11, suggesting the ability of HemR to transport heme covalently linked to other molecules. Comparison of HemR with other heme receptors identified several highly conserved histidine residues as well as two conserved amino acid motifs, the FRAP and NPNL boxes. A site-directed mutagenesis approach was used to investigate the roles of His128, His192, His352, and His461 residues in HemR function. The HemR receptor with histidine changed to lysine at position 128 (HemR
H128K
), HemR
H461L
, HemR
H461A
, and HemR
H128A,H461A
mutant receptors were unable to use Hb, human serum albumin-heme, and myoglobin as sources of porphyrin and iron. Utilization of free heme was also severely affected, with some residual heme uptake in cells expressing HemR
H128K
, HemR
H461A
, and HemR
H461L
. Conversely, the HemR
H192T
, HemR
H352A
, HemR
H352K
, and HemR
H192T,H352K
mutant receptors were fully functional. All mutant HemR proteins were expressed in the outer membrane at levels similar to that of the wild-type HemR receptor. Nonfunctional HemRs were able to bind heme- and Hb-agarose. A hypothetical model of the HemR function in which two conserved histidine residues, His128 and His461, participate in the transport of heme through the receptor pore is postulated.
Three Asparagine Synthetase Genes of
Bacillus subtilis
Authors: Yoshida Ken-Ichi , Fujita Yasutaro , Ehrlich S. Dusko 
Published: December 2019

ABSTRACT

Three asparagine synthetase genes,
asnB
,
asnH
, and
asnO
(
yisO
), were predicted from the sequence of the
Bacillus subtilis
genome. We show here that the three genes are expressed differentially during cell growth. In a rich sporulation medium, expression of
asnB
was detected only during exponential growth, that of
asnH
was drastically elevated at the transition between exponential growth and stationary phase, and that of
asnO
was seen only later in sporulation. In a minimal medium, both
asnB
and
asnH
were expressed constitutively during exponential growth and in stationary phase, while the expression of
asnO
was not detected in either phase. However, when the minimal medium was supplemented with asparagine, only the expression of
asnH
was partially repressed. Transcription analyses revealed that
asnB
was possibly cotranscribed with a downstream gene,
ytnA
, while the
asnH
gene was transcribed as the fourth gene of an operon comprising
yxbB
,
yxbA
,
yxnB
,
asnH
, and
yxaM
. The
asnO
gene is a monocistronic operon, the expression of which was dependent on one of the sporulation sigma factors, sigma-E. Each of the three genes, carried on a low-copy-number plasmid, complemented the asparagine deficiency of an
Escherichia coli
strain lacking asparagine synthetases, indicating that all encode an asparagine synthetase. In
B. subtilis
, deletion of
asnO
or
asnH
, singly or in combination, had essentially no effect on growth rates in media with or without asparagine. In contrast, deletion of
asnB
led to a slow-growth phenotype, even in the presence of asparagine. A strain lacking all three genes still grew without asparagine, albeit very slowly, implying that
B. subtilis
might have yet another asparagine synthetase, not recognized by sequence analysis. The strains lacking
asnO
failed to sporulate, indicating an involvement of this gene in sporulation.
Analysis of Mutations in the Pore-Forming Region Essential for Insecticidal Activity of a
Bacillus thuringiensis
δ-Endotoxin
Authors: Manoj Kumar A. S. , Aronson A. I. 
Published: December 2019

ABSTRACT

The
Bacillus thuringiensis
insecticidal δ-endotoxins have a three-domain structure, with the seven amphipathic helices which comprise domain I being essential for toxicity. To better define the function of these helices in membrane insertion and toxicity, either site-directed or random mutagenesis of two regions was performed. Thirty-nucleotide segments in the
B. thuringiensis cry1Ac1
gene, encoding parts of helix α4 and the loop connecting helices α4 and α5, were randomly mutagenized. This hydrophobic region of the toxin probably inserts into the membrane as a hairpin. Site-directed mutations were also created in specific surface residues of helix α3 in order to increase its hydrophobicity. Among 12 random mutations in helix α4, 5 resulted in the total loss of toxicity for
Manduca sexta
and
Heliothis virescens
, another caused a significant increase in toxicity, and one resulted in decreased toxicity. None of the nontoxic mutants was altered in toxin stability, binding of toxin to a membrane protein, or the ability of the toxin to aggregate in the membrane. Mutations in the loop connecting helices α4 and α5 did not affect toxicity, nor did mutations in α3, which should have enhanced the hydrophobic properties of this helix. In contrast to mutations in helix α5, those in helix α4 which inactivated the toxin did not affect its capacity to oligomerize in the membrane. Despite the formation of oligomers, there was no ion flow as measured by light scattering. Helix α5 is important for oligomerization and perhaps has other functions, whereas helix α4 must have a more direct role in establishing the properties of the channel.
FlbT Couples Flagellum Assembly to Gene Expression in
Caulobacter crescentus
Authors: Mangan Erin K. , Malakooti Jaleh , Caballero Anthony , Anderson Paul , Ely Bert , Gober James W. 
Published: December 2019

ABSTRACT

The biogenesis of the polar flagellum of
Caulobacter crescentus
is regulated by the cell cycle as well as by a
trans
-acting regulatory hierarchy that functions to couple flagellum assembly to gene expression. The assembly of early flagellar structures (MS ring, switch, and flagellum-specific secretory system) is required for the transcription of class III genes, which encode the remainder of the basal body and the external hook structure. Similarly, the assembly of class III gene-encoded structures is required for the expression of the class IV flagellins, which are incorporated into the flagellar filament. Here, we demonstrate that mutations in
flbT
, a flagellar gene of unknown function, can restore flagellin protein synthesis and the expression of
fljK
::
lacZ
(25-kDa flagellin) protein fusions in class III flagellar mutants. These results suggest that FlbT functions to negatively regulate flagellin expression in the absence of flagellum assembly. Deletion analysis shows that sequences within the 5′ untranslated region of the
fljK
transcript are sufficient for FlbT regulation. To determine the mechanism of FlbT-mediated regulation, we assayed the stability of
fljK
mRNA. The half-life (
t
1/2
) of
fljK
mRNA in wild-type cells was approximately 11 min and was reduced to less than 1.5 min in a
flgE
(hook) mutant. A
flgE flbT
double mutant exhibited an mRNA
t
1/2
of greater than 30 min. This suggests that the primary effect of FlbT regulation is an increased turnover of flagellin mRNA. The increased
t
1/2
of
fljK
mRNA in a
flbT
mutant has consequences for the temporal expression of
fljK
. In contrast to the case for wild-type cells,
fljK
::
lacZ
protein fusions in the mutant are expressed almost continuously throughout the
C. crescentus
cell cycle, suggesting that coupling of flagellin gene expression to assembly has a critical influence on regulating cell cycle expression.
Purification and Characterization of a Novel Naphthalene Dioxygenase from
Rhodococcus
sp. Strain NCIMB12038
Authors: Larkin Michael J. , Allen Christopher C. R. , Kulakov Leonid A. , Lipscomb David A. 
Published: December 2019

ABSTRACT

We report here the characterization of the catalytic component (ISP
NAR
) of a new naphthalene dioxygenase from
Rhodococcus
sp. strain NCIMB12038. The genes encoding the two subunits of ISP
NAR
are not homologous to their previously characterized counterparts in
Pseudomonas
. The deduced amino acid sequences have only 33 and 29% identity with the corresponding subunits in
Pseudomonas putida
NCIB 9816-4, for which the tertiary structure has been reported.
Assembly of XcpR in the Cytoplasmic Membrane Is Required for Extracellular Protein Secretion in
Pseudomonas aeruginosa
Authors: Ball Geneviève , Chapon-Hervé Virginie , Bleves Sophie , Michel Gérard , Bally Marc 
Published: December 2019

ABSTRACT

A broad range of extracellular proteins secreted by
Pseudomonas aeruginosa
use the type II or general secretory pathway (GSP) to reach the medium. This pathway requires the expression of at least 12
xcp
gene products. XcpR, a putative nucleotide-binding protein, is essential for the secretion process across the outer membrane even though the protein contains no hydrophobic sequence that could target or anchor it to the bacterial envelope. For a better understanding of the relationship between XcpR and the other Xcp proteins which are located in the envelope, we have studied its subcellular localization. In a wild-type
P. aeruginosa
strain, XcpR was found associated with the cytoplasmic membrane. This association depends on the presence of the XcpY protein, which also appears to be necessary for XcpR stability. Functional complementation of an
xcpY
mutant required the XcpY protein to be expressed at a low level. Higher expression precluded the complementing activity of XcpY, although membrane association of XcpR was restored. This behavior suggested that an excess of free XcpY might interfere with the secretion by formation of inactive XcpR-XcpY complexes which cannot properly interact with their natural partners in the secretion machinery. These data show that a precise stoichiometric ratio between several components may be crucial for the functioning of the GSP.
Discontinuous Occurrence of the
hsp70
(
dnaK
) Gene among
Archaea
and Sequence Features of HSP70 Suggest a Novel Outlook on Phylogenies Inferred from This Protein
Authors: Gribaldo Simonetta , Lumia Valentina , Creti Roberta , Conway de Macario Everly , Sanangelantoni Annamaria , Cammarano Piero 
Published: December 2019

ABSTRACT

Occurrence of the
hsp70
(
dnaK
) gene was investigated in various members of the domain
Archaea
comprising both euryarchaeotes and crenarchaeotes and in the hyperthermophilic bacteria
Aquifex pyrophilus
and
Thermotoga maritima
representing the deepest offshoots in phylogenetic trees of bacterial 16S rRNA sequences. The gene was not detected in 8 of 10 archaea examined but was found in
A. pyrophilus
and
T. maritima
, from which it was cloned and sequenced. Comparative analyses of the HSP70 amino acid sequences encoded in these genes, and others in the databases, showed that (i) in accordance with the vicinities seen in rRNA-based trees, the proteins from
A. pyrophilus
and
T. maritima
form a thermophilic cluster with that from the green nonsulfur bacterium
Thermomicrobium roseum
and are unrelated to their counterparts from gram-positive bacteria, proteobacteria/mitochondria, chlamydiae/spirochetes, deinococci, and cyanobacteria/chloroplasts; (ii) the
T. maritima
HSP70 clusters with the homologues from the archaea
Methanobacterium thermoautotrophicum
and
Thermoplasma acidophilum
, in contrast to the postulated unique kinship between archaea and gram-positive bacteria; and (iii) there are exceptions to the reported association between an insert in HSP70 and gram negativity, or vice versa, absence of insert and gram positivity. Notably, the HSP70 from
T. maritima
lacks the insert, although
T. maritima
is phylogenetically unrelated to the gram-positive bacteria. These results, along with the absence of
hsp70
(
dnaK
) in various archaea and its presence in others, suggest that (i) different taxa retained either one or the other of two
hsp70
(
dnaK
) versions (with or without insert), regardless of phylogenetic position; and (ii) archaea are aboriginally devoid of
hsp70
(
dnaK
), and those that have it must have received it from phylogenetically diverse bacteria via lateral gene transfer events that did not involve replacement of an endogenous
hsp70
(
dnaK
) gene.
A Glucan Synthase
FKS1
Homolog in
Cryptococcus neoformans
Is Single Copy and Encodes an Essential Function
Authors: Thompson John R. , Douglas Cameron M. , Li Weili , Jue Chong K. , Pramanik Barnali , Yuan Xiling , Rude Thomas H. , Toffaletti Dena L. , Perfect John R. , Kurtz Myra 
Published: December 2019

ABSTRACT

Cryptococcal meningitis is a fungal infection, caused by
Cryptococcus neoformans
, which is prevalent in immunocompromised patient populations. Treatment failures of this disease are emerging in the clinic, usually associated with long-term treatment with existing antifungal agents. The fungal cell wall is an attractive target for drug therapy because the syntheses of cell wall glucan and chitin are processes that are absent in mammalian cells. Echinocandins comprise a class of lipopeptide compounds known to inhibit 1,3-β-glucan synthesis, and at least two compounds belonging to this class are currently in clinical trials as therapy for life-threatening fungal infections. Studies of
Saccharomyces cerevisiae
and
Candida albicans
mutants identify the membrane-spanning subunit of glucan synthase, encoded by the
FKS
genes, as the molecular target of echinocandins. In vitro, the echinocandins show potent antifungal activity against
Candida
and
Aspergillus
species but are much less potent against
C. neoformans
. In order to examine why
C. neoformans
cells are less susceptible to echinocandin treatment, we have cloned a homolog of
S. cerevisiae FKS1
from
C. neoformans
. We have developed a generalized method to evaluate the essentiality of genes in
Cryptococcus
and applied it to the
FKS1
gene. The method relies on homologous integrative transformation with a plasmid that can integrate in two orientations, only one of which will disrupt the target gene function. The results of this analysis suggest that the
C. neoformans FKS1
gene is essential for viability. The
C. neoformans FKS1
sequence is closely related to the
FKS1
sequences from other fungal species and appears to be single copy in
C. neoformans
. Furthermore, amino acid residues known to be critical for echinocandin susceptibility in
Saccharomyces
are conserved in the
C. neoformans FKS1
sequence.
Transcription of Two Sigma 70 Homologue Genes,
sigA
and
sigB
, in Stationary-Phase
Mycobacterium tuberculosis
Authors: Hu Yanmin , Coates Anthony R. M. 
Published: December 2019

ABSTRACT

The
sigA
and
sigB
genes of
Mycobacterium tuberculosis
encode two sigma 70-like sigma factors of RNA polymerase. While transcription of the
sigA
gene is growth rate independent,
sigB
transcription is increased during entry into stationary phase. The
sigA
gene transcription is unresponsive to environmental stress but that of
sigB
is very responsive, more so in stationary-phase growth than in log-phase cultures. These data suggest that SigA is a primary sigma factor which, like ς
70
, controls the transcription of the housekeeping type of promoters. In contrast, SigB, although showing some overlap in function with SigA, is more like the alternative sigma factor, ς
S
, which controls the transcription of the gearbox type of promoters. Primer extension analysis identified the RNA start sites for both genes as 129 nucleotides upstream to the GTG start codon of
sigA
and 27 nucleotides from the ATG start codon of
sigB
. The −10 promoter of
sigA
but not that of
sigB
was similar to the ς
70
promoter. The half-life of the
sigA
transcript was very long, and this is likely to play an important part in its regulation. In contrast, the half-life of the
sigB
transcript was short, about 2 min. These results demonstrate that the
sigB
gene may control the regulons of stationary phase and general stress resistance, while
sigA
may be involved in the housekeeping regulons.