bluuebunny/crossref_metadata_2025_split huggingface dataset:
Catabolite Regulation of the
pta
Gene as Part of Carbon Flow Pathways in
Bacillus subtilis
Authors: Presecan-Siedel Elena , Galinier Anne , Longin Robert , Deutscher Josef , Danchin Antoine , Glaser Philippe , Martin-Verstraete Isabelle 
Published: December 2019

ABSTRACT

In
Bacillus subtilis
, the products of the
pta
and
ackA
genes, phosphotransacetylase and acetate kinase, play a crucial role in the production of acetate, one of the most abundant by-products of carbon metabolism in this gram-positive bacterium. Although these two enzymes are part of the same pathway, only mutants with inactivated
ackA
did not grow in the presence of glucose. Inactivation of
pta
had only a weak inhibitory effect on growth. In contrast to
pta
and
ackA
in
Escherichia coli
, the corresponding
B. subtilis
genes are not cotranscribed. Expression of the
pta
gene was increased in the presence of glucose, as has been reported for
ackA
. The effects of the predicted
cis
-acting catabolite response element (CRE) located upstream from the promoter and of the
trans
-acting proteins CcpA, HPr, Crh, and HPr kinase on the catabolite regulation of
pta
were investigated. As for
ackA
, glucose activation was abolished in
ccpA
and
hprK
mutants and in the
ptsH1 crh
double mutant. Footprinting experiments demonstrated an interaction between CcpA and the
pta
CRE sequence, which is almost identical to the proposed CRE consensus sequence. This interaction occurs only in the presence of Ser-46-phosphorylated HPr (HPrSer-P) or Ser-46-phosphorylated Crh (CrhSer-P) and fructose-1,6-bisphosphate (FBP). In addition to CcpA, carbon catabolite activation of the
pta
gene therefore requires at least two other cofactors, FBP and either HPr or Crh, phosphorylated at Ser-46 by the ATP-dependent Hpr kinase.
Characterization of a Periplasmic ATP-Binding Cassette Iron Import System of
Brachyspira
(
Serpulina
)
hyodysenteriae
Authors: Dugourd Dominique , Martin Christine , Rioux Clément R. , Jacques Mario , Harel Josée 
Published: December 2019

ABSTRACT

The nucleotide sequence of the pathogenic spirochete
Brachyspira hyodysenteriae bit
(for “
Brachyspira
iron transport”) genomic region has been determined. The
bit
region is likely to encode an iron ATP-binding cassette transport system with some homology to those encountered in gram-negative bacteria. Six open reading frames oriented in the same direction and physically linked have been identified. This system possesses a protein containing ATP-binding motifs (BitD), two hydrophobic cytoplasmic membrane permeases (BitE and BitF), and at least three lipoproteins (BitA, BitB, and BitC) with homology to iron periplasmic binding proteins. These periplasmic binding proteins exhibit lipoprotein features. They are labeled by [
3
H]palmitate when tested in recombinant
Escherichia coli
, and their signal peptides are typical for substrates of the type II secretory peptidase. The FURTA system and Congo red assay indicate that BitB and BitC are involved in iron binding. The Bit system is detected only in
B. hyodysenteriae
and is absent from
B. innocens
and
B. pilosicoli
.
The Respiratory System and Diazotrophic Activity of
Acetobacter diazotrophicus
PAL5
Authors: Flores-Encarnación M. , Contreras-Zentella M. , Soto-Urzua L. , Aguilar G. R. , Baca B. E. , Escamilla J. E. 
Published: December 2019

ABSTRACT

The characteristics of the respiratory system of
Acetobacter diazotrophicus
PAL5 were investigated. Increasing aeration (from 0.5 to 4.0 liters of air min
−1
liter of medium
−1
) had a strong positive effect on growth and on the diazotrophic activity of cultures. Cells obtained from well-aerated and diazotrophically active cultures possessed a highly active, membrane-bound electron transport system with dehydrogenases for NADH, glucose, and acetaldehyde as the main electron donors. Ethanol, succinate, and gluconate were also oxidized but to only a minor extent. Terminal cytochrome
c
oxidase-type activity was poor as measured by reduced
N
,
N
,
N
,
N
′-tetramethyl-
p
-phenylenediamine, but quinol oxidase-type activity, as measured by 2,3,5,6-tetrachloro-1,4-benzenediol, was high. Spectral and high-pressure liquid chromatography analysis of membranes revealed the presence of cytochrome
ba
as a putative oxidase in cells obtained from diazotrophically active cultures. Cells were also rich in
c
-type cytochromes; four bands of high molecular mass (i.e., 67, 56, 52, and 45 kDa) were revealed by a peroxidase activity stain in sodium dodecyl sulfate-polyacrylamide gel electrophoresis. KCN inhibition curves of respiratory oxidase activities were biphasic, with a highly resistant component. Treatment of membranes with 0.2% Triton X-100 solubilized
c
-type cytochromes and resulted in a preparation that was significantly more sensitive to cyanide. Repression of diazotrophic activity in well-aerated cultures by 40 mM (NH
4
)
2
SO
4
caused a significant decrease of the respiratory activities. It is noteworthy that the levels of glucose dehydrogenase and putative oxidase
ba
decreased 6.8- and 10-fold, respectively. In these cells, a
bd
-type cytochrome seems to be the major terminal oxidase. Thus, it would seem that glucose dehydrogenase and cytochrome
ba
are key components of the respiratory system of
A. diazotrophicus
during aerobic diazotrophy.
Control of Synthesis and Secretion of the
Bacillus subtilis
Protein YqxM
Authors: Stöver Axel G. , Driks Adam 
Published: December 2019

ABSTRACT

yqxM
is a
Bacillus subtilis
gene of unknown function residing in an operon with
sipW
, which encodes a signal peptidase, and
tasA
, which encodes an antibiotic protein secreted in a
sipW
-dependent manner. YqxM was undetectable during growth in a variety of rich media, including Luria-Bertani (LB) medium, or in minimal media or under heat shock or ethanol stress conditions but was synthesized and secreted during growth in LB medium supplemented with 1.2 M NaCl. Consistent with the possible involvement of
sipW
in YqxM secretion, inactivation of
sipW
prevented YqxM secretion. YqxM was produced and secreted in a
sipW
-dependent manner during growth in LB medium when the sequences upstream of
yqxM
were replaced with those of the inducible P

spac

promoter. Coexpression of
yqxM
and
sipW
in
Escherichia coli
resulted in a decrease in the apparent molecular mass of YqxM, consistent with the removal of a signal peptide. These experiments suggest that YqxM production is induced by a high concentration of salt and that YqxM is secreted under the control of SipW. We hypothesize that during most conditions of growth, YqxM is present at very low levels or is not synthesized at all and that this low level or absence is due, at least in part, to posttranscriptional repression.
PHR1
and
PHR2
of
Candida albicans
Encode Putative Glycosidases Required for Proper Cross-Linking of β-1,3- and β-1,6-Glucans
Authors: Fonzi William A. 
Published: December 2019

ABSTRACT

PHR1
and
PHR2
encode putative glycosylphosphatidylinositol-anchored cell surface proteins of the opportunistic fungal pathogen
Candida albicans
. These proteins are functionally related, and their expression is modulated in relation to the pH of the ambient environment in vitro and in vivo. Deletion of either gene results in a pH-conditional defect in cell morphology and virulence. Multiple sequence alignments demonstrated a distant relationship between the Phr proteins and β-galactosidases. Based on this alignment, site-directed mutagenesis of the putative active-site residues of Phr1p and Phr2p was conducted and two conserved glutamate residues were shown to be essential for activity. By taking advantage of the pH-conditional expression of the genes, a temporal analysis of cell wall changes was performed following a shift of the mutants from permissive to nonpermissive pH. The mutations did not grossly affect the amount of polysaccharides in the wall but did alter their distribution. The most immediate alteration to occur was a fivefold increase in the rate of cross-linking between β-1,6-glycosylated mannoproteins and chitin. This increase was followed shortly thereafter by a decline in β-1,3-glucan-associated β-1,6-glucans and, within several generations, a fivefold increase in the chitin content of the walls. The increased accumulation of chitin-linked glucans was not due to a block in subsequent processing as determined by pulse-chase analysis. Rather, the results suggest that the glucans are diverted to chitin linkage due to the inability of the mutants to establish cross-links between β-1,6- and β-1,3-glucans. Based on these and previously published results, it is suggested that the Phr proteins process β-1,3-glucans and make available acceptor sites for the attachment of β-1,6-glucans.
Mercury Resistance in
Bacillus cereus
RC607: Transcriptional Organization and Two New Open Reading Frames
Authors: Gupta Amit , Phung Le T. , Chakravarty Leena , Silver Simon 
Published: December 2019

ABSTRACT

The chromosomal mercury resistance determinant of
Bacillus cereus
RC607 confers resistance to inorganic mercury and to organomercurials. The order of genes in the completed mercury resistance determinant is operator-promoter 1 (O/P1)
merR1 merT
open reading frame 3 (ORF3) ORF4
merA
O/P2
merR2 merB2 merB1
. The previously undetermined 1-kb DNA sequence between the
merA
and
merB1
genes includes two significant ORFs, whose predicted protein products are homologous with MerR (the transcriptional regulator) and MerB (the organomercurial lyase enzyme). Two transcriptional start sites (promoters), O/P1 at the beginning of the determinant and O/P2 immediately upstream of the sixth ORF, the newly identified
merR2
, were mapped by reverse transcriptase (RT) primer extension. A long 6.3-kb mRNA traversing all eight ORFs was shown by RT-PCR. Growth sensitivity measurements in liquid media and cellular mercury volatization assays characterized inducibility and differences in functional activity in
B. cereus
RC607 and after cloning of the
mer
determinant into plasmids in
Escherichia coli
.
Transcriptional Analysis of the Rubrerythrin and Superoxide Dismutase Genes of
Clostridium perfringens
Authors: Geissmann Thomas A. , Teuber Michael , Meile Leo 
Published: December 2019

ABSTRACT

We cloned and sequenced a 2.7-kb fragment of chromosomal DNA from
Clostridium perfringens
containing the superoxide dismutase-encoding gene,
sod
. Previously, rubrerythrin from
C. perfringens
had been isolated and its gene (
rbr
) had been cloned (Y. Lehmann, L. Meile, and M. Teuber, J. Bacteriol. 178:7152–7158, 1996). Northern blot experiments revealed a length of approximately 800 bases for each transcript of
rbr
and
sod
of
C. perfringens
. Thus,
rbr
and
sod
each represent a monocistronic operon. Their transcription start points were located by primer extension analyses.
sod
transcription was shown to depend on the growth phase, and it reached a maximum during the transition from log phase to stationary phase. Neither
sod
nor
rbr
transcription was influenced by oxidative stress.
Bacillus subtilis yckG
and
yckF
Encode Two Key Enzymes of the Ribulose Monophosphate Pathway Used by Methylotrophs, and
yckH
Is Required for Their Expression
Authors: Yasueda Hisashi , Kawahara Yoshio , Sugimoto Shin-ichi 
Published: December 2019

ABSTRACT

The ribulose monophosphate (RuMP) pathway is one of the metabolic pathways for the synthesis of compounds containing carbon-carbon bonds from one-carbon units and is found in many methane- and methanol-utilizing bacteria, which are known as methylotrophs. The characteristic enzymes of this pathway are 3-hexulose-6-phosphate synthase (HPS) and 6-phospho-3-hexuloisomerase (PHI), neither of which was thought to exist outside methylotrophs. However, the presumed
yckG
gene product (YckG) of
Bacillus subtilis
shows a primary structure similar to that of methylotroph HPS (F. Kunst et al., Nature 390:249–256, 1997). We have also investigated the sequence similarity between the
yckF
gene product (YckF) and methylotroph PHI (Y. Sakai, R. Mitsui, Y. Katayama, H. Yanase, and N. Kato, FEMS Microbiol. Lett. 176:125–130, 1999) and found that the
yckG
and
yckF
genes of
B. subtilis
express enzymatic activities of HPS and PHI, respectively. Both of these activities were concomitantly induced in
B. subtilis
by formaldehyde, with induction showing dependence on the
yckH
gene, but were not induced by methanol, formate, or methylamine. Disruption of either gene caused moderate sensitivity to formaldehyde, suggesting that these enzymes may act as a detoxification system for formaldehyde in
B. subtilis
. In conclusion, we found an active
yckG
(for HPS)-
yckF
(for PHI) gene structure (now named
hxlA-hxlB
) in a nonmethylotroph,
B. subtilis
, which inherently preserves the RuMP pathway.
Renaturation of RecombinantTreponema pallidumRare Outer Membrane Protein 1 into a Trimeric, Hydrophobic, and Porin-Active Conformation
Authors: Zhang Hongwei H. , Blanco David R. , Exner Maurice M. , Shang Ellen S. , Champion Cheryl I. , Phillips Martin L. , Miller James N. , Lovett Michael A. 
Published: December 2019

ABSTRACTWe have previously observed that while nativeTreponema pallidumrare outer membrane protein 1 (Tromp1) is hydrophobic and has porin activity, recombinant forms of Tromp1 do not possess these properties. In this study we show that these properties are determined by conformation and can be replicated by proper renaturation of recombinant Tromp1. Native Tromp1, but not the 47-kDa lipoprotein, extracted from whole organisms by using Triton X-114, was found to lose hydrophobicity after treatment in 8 M urea, indicating that Tromp1’s hydrophobicity is conformation dependent. Native Tromp1 was purified from 0.1% Triton X-100 extracts of whole organisms by fast-performance liquid chromatography (FPLC) and shown to have porin activity in planar lipid bilayers. Cross-linking studies of purified native Tromp1 with an 11 Å cross-linking agent showed oligomeric forms consistent with dimers and trimers. For renaturation studies of recombinant Tromp1 (rTromp1), a 31,109-Da signal-less construct was expressed inEscherichia coliand purified by FPLC. FPLC-purified rTromp1 was denatured in 8 M urea and then renatured in the presence of 0.5% Zwittergent 3,14 during dialysis to remove the urea. Renatured rTromp1 was passed through a Sephacryl S-300 gel exclusion column previously calibrated with known molecular weight standards. While all nonrenatured rTromp1 eluted from the column at approximately the position of the carbonic anhydrase protein standard (29 kDa), all renatured rTromp1 eluted at the position of the phosphorylasebprotein standard (97 kDa), suggesting a trimeric conformation. Trimerization was confirmed by using an 11 Å cross-linking agent which showed both dimers and trimers similar to that of native Tromp1. Triton X-114 phase separations showed that all of renatured rTromp1, but none of nonrenatured rTromp1, phase separated exclusively into the hydrophobic detergent phase, similar to native Tromp1. Circular dichroism of nonrenatured and renatured rTromp1 showed a marked loss in alpha-helical secondary structure of renatured rTromp1 compared to the nonrenatured form. Finally, renatured rTromp1, but not the nonrenatured form, showed porin activity in planar liquid bilayers. These results demonstrate that proper folding of rTromp1 results in a trimeric, hydrophobic, and porin-active conformation similar to that of the native protein.
Interference with Murein Turnover Has No Effect on Growth but Reduces β-Lactamase Induction in
Escherichia coli
Authors: Kraft Angelika R. , Prabhu Julia , Ursinus Astrid , Höltje Joachim-Volker 
Published: December 2019

ABSTRACT

Physiological studies of a mutant of
Escherichia coli
lacking the three lytic transglycosylases Slt70, MltA, and MltB revealed that interference with murein turnover can prevent AmpC β-lactamase induction. The triple mutant, although growing normally, shows a dramatically reduced rate of murein turnover. Despite the reduction in the formation of low-molecular-weight murein turnover products, neither the rate of murein synthesis nor the amount of murein per cell was increased. This might be explained by assuming that during growth in the absence of the major lytic transglycosylases native murein strands are excised by the action of endopeptidases and directly reused without further breakdown to muropeptides. The reduced rate of murein turnover could be correlated with lowered cefoxitin-induced expression of β-lactamase, present on a plasmid carrying the
ampC
and
ampR
genes from
Enterobacter cloacae
. Overproduction of MltB stimulated β-lactamase induction, whereas specific inhibition of Slt70 by bulgecin repressed
ampC
expression. Thus, specific inhibitors of lytic transglycosylases can increase the potency of penicillins and cephalosporins against bacteria inducing AmpC-like β-lactamases.
Genetic Dissection of the Outer Membrane Secretin PulD: Are There Distinct Domains for Multimerization and Secretion Specificity?
Authors: Guilvout Ingrid , Hardie Kim R. , Sauvonnet Nathalie , Pugsley Anthony P. 
Published: December 2019

ABSTRACT

Linker and deletion mutagenesis and gene fusions were used to probe the possible domain structure of the dodecameric outer membrane secretin PulD from the pullulanase secretion pathway of
Klebsiella oxytoca
. Insertions of 24 amino acids close to or within strongly predicted and highly conserved amphipathic β strands in the C-terminal half of the polypeptide (the β domain) abolished sodium dodecyl sulfate (SDS)-resistant multimer formation that is characteristic of this protein, whereas insertions elsewhere generally had less dramatic effects on multimer formation. However, the β domain alone did not form SDS-resistant multimers unless part of the N-terminal region of the protein (the N domain) was produced in
trans
. All of the insertions except one, close to the C terminus of the protein, abolished function. The N domain alone was highly unstable and did not form SDS-resistant multimers even when the β domain was present in
trans
. We conclude that the β domain is a major determinant of multimer stability and that the N domain contributes to multimer formation. The entire or part of the N domain of PulD could be replaced by the corresponding region of the OutD secretin from the pectate lyase secretion pathway of
Erwinia chrysanthemi
without abolishing pullulanase secretion. This suggests that the N domain of PulD is not involved in substrate recognition, contrary to the role proposed for the N domain of OutD, which binds specifically to pectate lyase secreted by
E. chrysanthemi
(V. E. Shevchik, J. Robert-Badouy, and G. Condemine, EMBO J. 16:3007–3016, 1997).
Identification of the
miaB
Gene, Involved in Methylthiolation of Isopentenylated A37 Derivatives in the tRNA of
Salmonella typhimurium
and
Escherichia coli
Authors: Esberg Birgitta , Leung Hon-Chiu Eastwood , Tsui Ho-Ching Tiffany , Björk Glenn R. , Winkler Malcolm E. 
Published: December 2019

ABSTRACT

The tRNA of the
miaB2508
::Tn
10d
Cm mutant of
Salmonella typhimurium
is deficient in the methylthio group of the modified nucleoside
N
6
-(4-hydroxyisopentenyl)-2-methylthioadenosine (ms
2
io
6
A37). By sequencing, we found that the Tn
10d
Cm of this strain had been inserted into the
f474
(
yleA
) open reading frame, which is located close to the
nag
locus in both
S. typhimurium
and
Escherichia coli
. By complementation of the
miaB2508
::Tn
10d
Cm mutation with a minimal subcloned
f474
fragment, we showed that
f474
could be identified as the
miaB
gene, which is transcribed in the counterclockwise direction on the bacterial chromosome. Transcriptional studies revealed two promoters upstream of
miaB
in
E. coli
and
S. typhimurium
. A Rho-independent terminator was identified downstream of the
miaB
gene, at which the majority (96%) of the
miaB
transcripts terminate in
E. coli
, showing that the
miaB
gene is part of a monocistronic operon. A highly conserved motif with three cysteine residues was present in MiaB. This motif resembles iron-binding sites in other proteins. Only a weak similarity to an AdoMet-binding site was found, favoring the idea that the MiaB protein is involved in the thiolation step and not in the methylating reaction of ms
2
i(o)
6
A37 formation.
RNA Polymerase α and ς
70
Subunits Participate in Transcription of the
Escherichia coli uhpT
Promoter
Authors: Olekhnovich Igor N. , Kadner Robert J. 
Published: December 2019

ABSTRACT

Fundamental questions in bacterial gene regulation concern how multiple regulatory proteins interact with the transcription apparatus at a single promoter and what are the roles of protein contacts with RNA polymerase and changes in DNA conformation. Transcription of the
Escherichia coli uhpT
gene, encoding the inducible sugar phosphate transporter, is dependent on the response regulator UhpA and is stimulated by the cyclic AMP receptor protein (CAP). UhpA binds to multiple sites in the
uhpT
promoter between positions −80 and −32 upstream of the transcription start site, and CAP binds to a single site centered at position −103.5. The role in
uhpT
transcription of portions of RNA polymerase Eς
70
holoenzyme which affect regulation at other promoters was examined by using series of alanine substitutions throughout the C-terminal domains of RpoA (residues 255 to 329) and of RpoD (residues 570 to 613). Alanine substitutions that affected in vivo expression of a
uhpT-lacZ
transcriptional fusion were tested for their effect on in vitro transcription activity by using reconstituted holoenzymes. Consistent with the binding of UhpA near the −35 region, residues K593 and K599 in the C-terminal region of RpoD were necessary for efficient
uhpT
expression in response to UhpA alone. Their requirement was overcome when CAP was also present. In addition, residues R265, G296, and S299 in the DNA-binding surface of the C-terminal domain of RpoA (αCTD) were important for
uhpT
transcription even in the presence of CAP. Substitutions at several other positions had effects in cells but not during in vitro transcription with saturating levels of the transcription factors. Two DNase-hypersensitive sites near the upstream end of the UhpA-binding region were seen in the presence of all three transcription factors. Their appearance required functional αCTD but not the presence of upstream DNA. These results suggest that both transcription activators depend on or interact with different subunits of RNA polymerase, although their role in formation of proper DNA geometry may also be crucial.