bluuebunny/crossref_metadata_2025_split huggingface dataset:
Anaerobic Growth, a Property Horizontally Transferred by an Hfr-Like Mechanism among Extreme Thermophiles
Authors: Ramírez-Arcos Sandra , Fernández-Herrero Luis A. , Marín Irma , Berenguer José 
Published: December 2019

ABSTRACTDespite the fact that the extreme thermophilic bacteria belonging to the genusThermusare classified as strict aerobes, we have shown thatThermus thermophilusHB8 (ATCC 27634) can grow anaerobically when nitrate is present in the growth medium. This strain-specific property is encoded by a respiratory nitrate reductase gene cluster (nar) whose expression is induced by anoxia and nitrate (S. Ramı́rez-Arcos, L. A. Fernández-Herrero, and J. Berenguer, Biochim. Biophys. Acta, 1396:215–1997). We show here that thisnaroperon can be transferred by conjugation to an aerobicThermusstrain, enabling it to grow under anaerobic conditions. We show that this transfer takes place through a DNase-insensitive mechanism which, as for the Hfr (high frequency of recombination) derivatives ofEscherichia coli, can also mobilize other chromosomal markers in a time-dependent way. Three lines of evidence are presented to support a genetic linkage betweennarand a conjugative plasmid integrated into the chromosome. First, thenaroperon is absent from a plasmid-free derivative and from a closely related strain. Second, we have identified an origin for autonomous replication (oriV) overlapping the last gene of thenarcluster. Finally, the mating time required for the transfer of thenaroperon is in good agreement with the time expected if the transfer origin (oriT) were located nearby and downstream ofnar.
The ms
2
io
6
A37 Modification of tRNA in
Salmonella typhimurium
Regulates Growth on Citric Acid Cycle Intermediates
Authors: Persson Britt C. , Ólafsson Ólafur , Lundgren Hans K. , Hederstedt Lars , Björk Glenn R. 
Published: December 2019

ABSTRACT

The modified nucleoside 2-methylthio-
N
-6-isopentenyl adenosine (ms
2
i
6
A) is present in position 37 (adjacent to and 3′ of the anticodon) of tRNAs that read codons beginning with U except tRNA
I,V
Ser
in
Escherichia coli
. In
Salmonella typhimurium
, 2-methylthio-
N
-6-(
cis
-hydroxy)isopentenyl adenosine (ms
2
io
6
A; also referred to as 2-methylthio
cis
-ribozeatin) is found in tRNA, most likely in the species that have ms
2
i
6
A in
E. coli
. Mutants (
miaE
) of
S. typhimurium
in which ms
2
i
6
A hydroxylation is blocked are unable to grow aerobically on the dicarboxylic acids of the citric acid cycle. Such mutants have normal uptake of dicarboxylic acids and functional enzymes of the citric acid cycle and the aerobic respiratory chain. The ability of
S. typhimurium
to grow on succinate, fumarate, and malate is dependent on the state of modification in position 37 of those tRNAs normally having ms
2
io
6
A37 and is not due to a second cellular function of tRNA (ms
2
io
6
A37)hydroxylase, the
miaE
gene product. We suggest that
S. typhimurium
senses the hydroxylation status of the isopentenyl group of the tRNA and will grow on succinate, fumarate, or malate only if the isopentenyl group is hydroxylated.
Streptomyces griseus
Protease B: Secretion Correlates with the Length of the Propeptide
Authors: Baardsnes J. , Sidhu S. , MacLeod A. , Elliott J. , Morden D. , Watson J. , Borgford T. 
Published: December 2019

ABSTRACT

Streptomyces griseus
protease B, a member of the chymotrypsin superfamily, is encoded by a gene that expresses a pre-pro-mature protein. During secretion the precursor protein is processed into a mature, fully folded protease. In this study, we constructed a family of genes which encode deletions at the amino-terminal end of the propeptide. The secretion of active protease B was seen to decrease in an exponential manner according to the length of the deletion. The results underscore the intimate relationship between folding and secretion in bacterial protease expression. They further suggest that the propeptide segment of the zymogen stabilizes the folding of the mature enzyme through many small binding interactions over the entire surface of the peptide rather than through a few specific contacts.
Characterization of the
glnK-amtB
Operon of
Azotobacter vinelandii
Authors: Meletzus Dietmar , Rudnick Paul , Doetsch Natalie , Green Andrew , Kennedy Christina 
Published: December 2019

ABSTRACT

To determine whether in
Azotobacter vinelandii
the P
II
protein influences the regulation of
nif
gene expression in response to fluxes in the ammonium supply, the gene encoding P
II
was isolated and characterized. Its deduced translation product was highly similar to P
II
proteins from other organisms, with the greatest degree of relatedness being exhibited to the
Escherichia coli glnK
gene product. A gene designated
amtB
was found downstream of and was cotranscribed with
glnK
as in
E. coli
. The AmtB protein is similar to functionally characterized ammonium transport proteins from a few other eukaryotes and one other prokaryote.
glnK
and
amtB
comprise an operon. Attempts to isolate a stable
glnK
mutant strain were unsuccessful, suggesting that
glnK
, like
glnA
, is an essential gene in
A. vinelandii
.
amtB
mutants were isolated, and although growth on limiting amounts of ammonium was similar in the mutant and wild-type strains, the mutants were unable to transport [
14
C]methylammonium.
Efflux Pumps Involved in Toluene Tolerance in
Pseudomonas putida
DOT-T1E
Authors: Ramos Juan L. , Duque Estrella , Godoy Patricia , Segura Ana 
Published: December 2019

ABSTRACT

The basic mechanisms underlying solvent tolerance in
Pseudomonas putida
DOT-T1E are efflux pumps that remove the solvent from bacterial cell membranes. The solvent-tolerant
P. putida
DOT-T1E grows in the presence of high concentrations (e.g., 1% [vol/vol]) of toluene and octanol. Growth of
P. putida
DOT-T1E cells in LB in the presence of toluene supplied via the gas phase has a clear effect on cell survival: the sudden addition of 0.3% (vol/vol) toluene to
P. putida
DOT-T1E pregrown with toluene in the gas phase resulted in survival of almost 100% of the initial cell number, whereas only 0.01% of cells pregrown in the absence of toluene tolerated exposure to this aromatic hydrocarbon. One class of toluene-sensitive octanol-tolerant mutant was isolated after Tn
5
-′
phoA
mutagenesis of wild-type
P. putida
DOT-T1E cells. The mutant, called
P. putida
DOT-T1E-18, was extremely sensitive to 0.3% (vol/vol) toluene added when cells were pregrown in the absence of toluene, whereas pregrowth on toluene supplied via the gas phase resulted in survival of about 0.0001% of the initial number. Solvent exclusion was tested with 1,2,4-[
14
C]trichlorobenzene. The levels of radiochemical accumulated in wild-type cells grown in the absence and in the presence of toluene were not significantly different. In contrast, the mutant was unable to remove 1,2,4-[
14
C]trichlorobenzene from the cell membranes when grown on Luria-Bertani (LB) medium but was able to remove the aromatic compound when pregrown on LB medium with toluene supplied via the gas phase. The amount of
14
C-labeled substrate in whole cells increased in competition assays in which toluene and xylenes were the unlabeled competitors, whereas this was not the case when benzene was the competitor. This finding suggests that the exclusion system works specifically with certain aromatic substrates. The mutation in
P. putida
DOT-T1E-18 was cloned, and the knockedout gene was sequenced and found to be homologous to the drug exclusion gene
mexB
, which belongs to the efflux pump family of the resistant nodulator division type.
Replication Terminator Protein-Based Replication Fork-Arrest Systems in Various
Bacillus
Species
Authors: Griffiths A. A. , Andersen P. A. , Wake R. G. 
Published: December 2019

ABSTRACT

The replication terminator protein (RTP) of
Bacillus subtilis
interacts with its cognate DNA terminators to cause replication fork arrest, thereby ensuring that the forks approaching one another at the conclusion of a round of replication meet within a restricted terminus region. A similar situation exists in
Escherichia coli
, but it appears that the fork-arrest systems in these two organisms have evolved independently of one another. In the present work, RTP homologs in four species closely related to
B. subtilis
(
B. atrophaeus
,
B. amyloliquefaciens
,
B. mojavensis
, and
B. vallismortis
) have been identified and characterized. An RTP homolog could not be identified in another closely related species,
B. licheniformis
. The nucleotide and amino acid changes from
B. subtilis
among the four homologs are consistent with the recently established phylogenetic tree for these species. The GC contents of the
rtp
genes raise the possibility that these organisms arose within this branch of the tree by horizontal transfer into a common ancestor after their divergence from
B. licheniformis
. Only 5 amino acid residue positions were changed among the four homologs, despite an up to 17.2% change in the nucleotide sequence, a finding that highlights the importance of the precise folded structure to the functioning of RTP. The absence of any significant change in the proposed DNA-binding region of RTP emphasizes the importance of its high affinity for the DNA terminator in its functioning. By coincidence, the single change (E30K) found in the
B. mojavensis
RTP corresponds exactly to that purposefully introduced by others into
B. subtilis
RTP to implicate a crucial role for E30 in the fork-arrest mechanism. The natural occurrence of this variant is difficult to reconcile with such an implication, and it was shown directly that RTP.E30K functions normally in fork arrest in
B. subtilis
in vivo. Additional DNA terminators were identified in the new RTP homolog-containing strains, allowing the definition of a
Bacillus
terminator consensus and identification of two more terminators in the
B. subtilis
168 genome sequence to bring the total to nine.
Membrane-Bound Lytic Endotransglycosylase in
Escherichia coli
Authors: Kraft Angelika R. , Templin Markus F. , Höltje Joachim-Volker 
Published: December 2019

ABSTRACT

The gene for a novel endotype membrane-bound lytic transglycosylase,
emtA
, was mapped at 26.7 min of the
E. coli
chromosome. EmtA is a lipoprotein with an apparent molecular mass of 22 kDa. Overexpression of the
emtA
gene did not result in bacteriolysis in vivo, but the enzyme was shown to hydrolyze glycan strands isolated from murein by amidase treatment. The formation of tetra- and hexasaccharides, but no disaccharides, reflects the endospecificity of the enzyme. The products are characterized by the presence of 1,6-anhydromuramic acid, indicating a lytic transglycosylase reaction mechanism. EmtA may function as a formatting enzyme that trims the nascent murein strands produced by the murein synthesis machinery into proper sizes, or it may be involved in the formation of tightly controlled minor holes in the murein sacculus to facilitate the export of bulky compounds across the murein barrier.
Mutants of
Citrobacter freundii
That Transport and Utilize Melibiose
Authors: Okazaki Noriko , Xu Xing Jue , Shimamoto Toshi , Kuroda Masayuki , Wilson Thomas H. , Tsuchiya Tomofusa 
Published: December 2019

ABSTRACT

We have isolated mutants of
Citrobacter freundii
that can grow on melibiose. Inducible α-galactosidase activity and melibiose transport activity were detected in the mutant cells but not in the wild-type cells. We detected a DNA region which hybridized with
melB
(the gene for the melibiose transporter) DNA of
Escherichia coli
in the chromosomal DNA of wild-type
C. freundii
. Protons, but not sodium ions, were found to be the coupling cations for melibiose (and methyl-β-
d
-thiogalactoside) transport in the mutant cells.
KtrAB, a New Type of Bacterial K
+
-Uptake System from
Vibrio alginolyticus
Authors: Nakamura Tatsunosuke , Yuda Rieko , Unemoto Tsutomu , Bakker Evert P. 
Published: December 2019

ABSTRACT

Vibrio alginolyticus
contained two adjacent genes,
ktrA
and
ktrB
, which encode a new type of bacterial K
+
-uptake system. KtrA and KtrB are peripheral and integral membrane proteins, respectively. Six of the nine sequenced bacterial genomes contain homologs to both
ktrA
and
ktrB
, suggesting that KtrAB is widespread.
Identification of
cpxR
as a Positive Regulator Essential for Expression of the
Shigella sonnei virF
Gene
Authors: Nakayama Shu-ichi , Watanabe Haruo 
Published: December 2019

ABSTRACT

virF
is the master regulator which activates the virulence determinant genes of
Shigella
spp. such as
ipaBCD
and
virG
. We previously reported that expression of
virF
itself is regulated in a pH-dependent manner and that
cpxA
, a sensor of a two-component regulatory system, is involved in this regulation (S. Nakayama and H. Watanabe, J. Bacteriol. 177:5062–5069, 1995). Disruption of
cpxR
, which has been thought to be the cognate response regulator of
cpxA
(J. Dong, S. Iuchi, H.-S. Kwan, Z. Lue, and E. C. C. Lin, Gene 136:227–230, 1993), abolished
virF
expression almost completely. Purified CpxR bound directly to the upstream region of
virF
. Binding capacity was enhanced when CpxR was phosphorylated by coincubation with acetyl phosphate in vitro. Furthermore, we observed that phosphorylated CpxR could activate
virF
transcription in vitro. These results clearly indicated that CpxR was an essential activator for
virF
expression and strongly suggested that the binding of phosphorylated CpxR to the target site upstream of the
virF
gene induced a direct activation of
virF
transcription.
Regulation of Expression of
GLT1
, the Gene Encoding Glutamate Synthase in
Saccharomyces cerevisiae
Authors: Valenzuela Lourdes , Ballario Paola , Aranda Cristina , Filetici Patrizia , González Alicia 
Published: December 2019

ABSTRACT

Saccharomyces cerevisiae
glutamate synthase (GOGAT) is an oligomeric enzyme composed of three 199-kDa identical subunits encoded by
GLT1
. In this work, we analyzed
GLT1
transcriptional regulation.
GLT1-lacZ
fusions were prepared and
GLT1
expression was determined in a
GDH1
wild-type strain and in a
gdh1
mutant derivative grown in the presence of various nitrogen sources. Null mutants impaired in
GCN4
,
GLN3
,
GAT1/NIL1
, or
UGA43/DAL80
were transformed with a
GLT1-lacZ
fusion to determine whether the above-mentioned transcriptional factors had a role in
GLT1
expression. A collection of increasingly larger 5′ deletion derivatives of the
GLT1
promoter was constructed to identify DNA sequences that could be involved in
GLT1
transcriptional regulation. The effect of the lack of
GCN4
,
GLN3
, or
GAT1/NIL1
was also tested in the pertinent 5′ deletion derivatives. Our results indicate that (i)
GLT1
expression is negatively modulated by glutamate-mediated repression and positively regulated by Gln3p- and Gcn4p-dependent transcriptional activation; (ii) two
cis
-acting elements, a CGGN
15
CCG palindrome and an imperfect poly(dA-dT), are present and could play a role in
GLT1
transcriptional activation; and (iii)
GLT1
expression is moderately regulated by
GCN4
under amino acid deprivation. Our results suggest that in a wild-type strain grown on ammonium, GOGAT constitutes an ancillary pathway for glutamate biosynthesis.
Expression of a Gene for a Porin-Like Protein of the OmpA Family fromMycobacterium tuberculosisH37Rv
Authors: Senaratne Ryan H. , Mobasheri Hamid , Papavinasasundaram K. G. , Jenner Peter , Lea Edward J. A. , Draper Philip 
Published: December 2019

ABSTRACTAn open reading frame in the genomic database ofMycobacterium tuberculosisH37Rv was identified as having homology with an outer membrane protein. We found that the gene specified a protein belonging to the OmpA family, which includes some porins of gram-negative organisms. The gene was amplified by PCR and cloned intoEscherichia coli. Overexpression of the gene was toxic to the host, but limited amounts could be purified from cells before growth ceased. A truncated gene devoid of the code for a presumed signal sequence was well expressed, but the protein had no pore-forming activity in the liposome swelling assay. However, the intact protein, OmpATb, behaved as a porin of low specific activity, with a pore diameter of 1.4 to 1.8 nm, and was also active in planar lipid bilayers, showing a single-channel conductance of 700 pS. The protein had a molecular mass of about 38 kDa in sodium dodecyl sulfate-polyacrylamide gel electrophoresis. A polyclonal rabbit antiserum raised to the truncated protein recognized a protein of similar molecular mass in detergent extracts of brokenM. tuberculosiscells. Reverse transcription-PCR confirmed that the gene for OmpATb was expressed inM. tuberculosiscells growing in culture. Comparison of the purified protein with that in the detergent-extracted preparation using liposomes and planar lipid bilayers showed that the two materials had similar pore-forming properties. OmpATb is different from either of the mycobacterial porins described so far. This is the first report of a porin-like molecule fromM. tuberculosis; the porin is likely to be important in controlling the access of hydrophilic molecules to the bacterial cell.
The
d
-Xylose-Binding Protein, XylF, from
Thermoanaerobacter ethanolicus
39E: Cloning, Molecular Analysis, and Expression of the Structural Gene
Authors: Erbeznik Milutin , Strobel Herbert J. , Dawson Karl A. , Jones Chris R. 
Published: December 2019

ABSTRACT

Immediately downstream from the
Thermoanaerobacter ethanolicus xylAB
operon, comprising genes that encode
d
-xylose isomerase and
d
-xylulose kinase, lies a 1,101-bp open reading frame that exhibits 61% amino acid sequence identity to the
Escherichia coli
d
-xylose binding periplasmic receptor, XylF, a component of the high-affinity binding-protein-dependent
d
-xylose transport. The 25-residue N-terminal fragment of the deduced
T. ethanolicus
XylF has typical features of bacterial leader peptides. The C-terminal portion of this leader sequence matches the cleavage consensus for lipoproteins and is followed by a 22-residue putative linker sequence rich in serine, threonine, and asparagine. The putative mature 341-amino-acid-residue XylF (calculated molecular mass of 37,069 Da) appears to be a lipoprotein attached to the cell membrane via a lipid anchor covalently linked to the N-terminal cysteine, as demonstrated by metabolic labelling of the recombinant XylF with [
14
C]palmitate. The induced
E. coli
avidly bound
d
-[
14
C]xylose, yielding additional evidence that
T. ethanolicus
XylF is the
d
-xylose-binding protein. On the basis of sequence comparison of XylFs to other monosaccharide-binding proteins, we propose that the sequence signature of binding proteins specific for hexoses and pentoses be refined as (KDQ)(LIVFAG)
3
IX
3
(DN)(SGP)X
3
(GS)X(LIVA)
2
X
2
A. Transcription of the monocistronic 1.3-kb
xylF
mRNA is inducible by xylose and unaffected by glucose. Primer extension analysis indicated that
xylF
transcription initiates from two +1 sites, both situated within the
xylAB
operon. Unlike in similar transport systems in other bacteria, the genes specifying the membrane components (e.g., ATP-binding protein and permease) of the high-affinity
d
-xylose uptake system are not located in the vicinity of
xylF
in
T. ethanolicus
. This is the first report of a gene encoding a xylose-binding protein in a gram-positive or thermophilic bacterium.
A Region in the
Bacillus subtilis
Transcription Factor Spo0A That Is Important for
spoIIG
Promoter Activation
Authors: Buckner Cindy M. , Schyns Ghislain , Moran Charles P. 
Published: December 2019

ABSTRACT

Spo0A is a DNA binding protein in
Bacillus subtilis
required for the activation of
spoIIG
and other promoters at the onset of endospore formation. Activation of some of these promoters may involve interaction of Spo0A and the ς
A
subunit of RNA polymerase. Previous studies identified two single-amino-acid substitutions in ς
A
, K356E and H359R, that specifically impaired Spo0A-dependent transcription in vivo. Here we report the identification of an amino acid substitution in Spo0A (S231F) that suppressed the sporulation deficiency due to the H359R substitution in ς
A
. We also found that the S231F substitution partially restored use of the
spoIIG
promoter by the ς
A
H359R RNA polymerase in vitro. Alanine substitutions in the 231 region of Spo0A revealed an additional amino acid residue important for
spoIIG
promoter activation, I229. This amino acid substitution in Spo0A did not affect repression of
abrB
transcription, indicating that the alanine-substituted Spo0A was not defective in DNA binding. Moreover, the alanine-substituted Spo0A protein activated the
spoIIA
promoter; therefore, this region of Spo0A is probably not required for Spo0A-dependent, ς
H
-directed transcription. These and other results suggest that the region of Spo0A near position 229 is involved in ς
A
-dependent promoter activation.
Molecular Characterization and Postsplicing Fate of Three Introns within the Single rRNA Operon of the Hyperthermophilic Archaeon
Aeropyrum pernix
K1
Authors: Nomura Norimichi , Sako Yoshihiko , Uchida Aritsune 
Published: December 2019

ABSTRACT

The single rRNA operon (
arnS-arnL
) of the hyperthermophilic archaeon
Aeropyrum pernix
K1 was sequenced. The DNA sequence data and detailed RNA analyses disclosed an unusual feature: the presence of three introns at hitherto undescribed insertion positions within the rRNA genes. The 699-nucleotide (nt) intron Iα was located at position 908 (
Escherichia coli
numbering [H. F. Noller, Annu. Rev. Biochem. 53:119–162, 1984]) of the 16S rRNA, while the 202-nt intron Iβ and 575-nt intron Iγ were located at positions 1085 and 1927 (
E. coli
numbering), respectively, of the 23S rRNA. They were located within highly conserved sites which have been implicated as crucial for rRNA function in
E. coli
. All three introns were remarkably AT rich (41.5 to 43.1 mol% G+C) compared with the mature rRNAs (67.7 and 69.2 mol% G+C for 16S and 23S rRNAs, respectively). No obvious primary sequence similarities were detected among them. After splicing from rRNA transcripts in vivo, a large quantity of intronic RNAs were stably retained in the linear monomeric form, whereas a trace of topoisomeric RNA molecules also appeared, as characterized by their behavior in two-dimensional gel electrophoresis. Secondary structural models of the Iα-, Iβ-, and Iγ-containing rRNA precursors agree with the bulge-helix-bulge motif. Two of the introns, Iα and Iγ, contained open reading frames whose protein translation exhibited no overall similarity with proteins reported so far. However, both share a LAGLI-DADG motif characteristic of homing endonucleases.
Evidence of Two Oxidative Reaction Steps Initiating Anaerobic Degradation of Resorcinol (1,3-Dihydroxybenzene) by the Denitrifying Bacterium
Azoarcus anaerobius
Authors: Philipp Bodo , Schink Bernhard 
Published: December 2019

ABSTRACT

The denitrifying bacterium
Azoarcus anaerobius
LuFRes1 grows anaerobically with resorcinol (1,3-dihydroxybenzene) as the sole source of carbon and energy. The anaerobic degradation of this compound was investigated in cell extracts. Resorcinol reductase, the key enzyme for resorcinol catabolism in fermenting bacteria, was not present in this organism. Instead, resorcinol was hydroxylated to hydroxyhydroquinone (HHQ; 1,2,4-trihydroxybenzene) with nitrate or K
3
Fe(CN)
6
as the electron acceptor. HHQ was further oxidized with nitrate to 2-hydroxy-1,4-benzoquinone as identified by high-pressure liquid chromatography, UV/visible light spectroscopy, and mass spectroscopy. Average specific activities were 60 mU mg of protein
−1
for resorcinol hydroxylation and 150 mU mg of protein
−1
for HHQ dehydrogenation. Both activities were found nearly exclusively in the membrane fraction and were only barely detectable in extracts of cells grown with benzoate, indicating that both reactions were specific for resorcinol degradation. These findings suggest a new strategy of anaerobic degradation of aromatic compounds involving oxidative steps for destabilization of the aromatic ring, different from the reductive dearomatization mechanisms described so far.