bluuebunny/crossref_metadata_2025_split huggingface dataset:
Growth Phase and Temperature Influence Promoter Activity, Transcript Abundance, and Protein Stability during Biosynthesis of thePseudomonas syringaePhytotoxin Coronatine
Authors: Budde Ina P. , Rohde Bettina H. , Bender Carol L. , Ullrich Matthias S. 
Published: December 2019

ABSTRACTThe plant-pathogenic bacteriumPseudomonas syringaepv. glycinea PG4180.N9 synthesizes high levels of the polyketide phytotoxin coronatine (COR) at 18°C, whereas no detectable toxin is produced at 28°C. Previously, we reported that the temperature-sensitive activation of three promoters within the COR biosynthetic gene cluster might explain thermoregulation of COR biosynthesis. The present study was aimed at furthering our understanding of the transcriptional as well as the posttranslational effects of temperature on expression ofcmaB, which encodes an enzyme involved in COR biosynthesis. Transcriptional fusions using a promoterless glucuronidase gene and Northern blot analyses were used to monitor promoter activities and transcript abundance for thecmaABToperon during bacterial growth at 18 and 28°C. Promoter activity and transcription rates were maximal when cells were incubated at 18°C and sampled at mid-logarithmic phase. Transcription declined moderately during the transition to stationary phase but remained higher at 18°C than at 28°C. Western blot analysis indicated that CmaB accumulated in the late stationary phase ofP. syringaecultures grown at 18°C but not in cultures incubated at 28°C. Temperature shift experiments indicated that CmaB stability was more pronounced at 18°C than at 28°C. Although temperature has a strong impact on transcription of COR biosynthetic genes, we propose that thermoregulation of protein stability might also control COR synthesis.
External Loops at the C Terminus of
Erwinia chrysanthemi
Pectate Lyase C Are Required for Species-Specific Secretion through the Out Type II Pathway
Authors: Lindeberg Magdalen , Boyd Carol M. , Keen Noel T. , Collmer Alan 
Published: December 2019

ABSTRACT

The type II secretion system (main terminal branch of the general secretion pathway) is used by diverse gram-negative bacteria to secrete extracellular proteins. Proteins secreted by this pathway are synthesized with an N-terminal signal peptide which is removed upon translocation across the inner membrane, but the signals which target the mature proteins for secretion across the outer membrane are unknown. The plant pathogens
Erwinia chrysanthemi
and
Erwinia carotovora
secrete several isozymes of pectate lyase (Pel) by the
out
-encoded type II pathway. However, these two bacteria cannot secrete Pels encoded by heterologously expressed
pel
genes from the other species, suggesting the existence of species-specific secretion signals within these proteins. The functional cluster of
E. chrysanthemi out
genes carried on cosmid pCPP2006 enables
Escherichia coli
to secrete
E. chrysanthemi
, but not
E. carotovora
, Pels. We exploited the high sequence similarity between
E. chrysanthemi
PelC and
E. carotovora
Pel1 to construct 15 hybrid proteins in which different regions of PelC were replaced with homologous sequences from Pel1. The differential secretion of these hybrid proteins by
E. coli
(pCPP2006) revealed M118 to D175 and V215 to C329 as regions required for species-specific secretion of PelC. We propose that the primary targeting signal is contained within the external loops formed by G274 to C329 but is dependent on residues in M118 to D170 and V215 to G274 for proper positioning.
Characterization of
Rhizobium leguminosarum
Exopolysaccharide Glycanases That Are Secreted via a Type I Exporter and Have a Novel Heptapeptide Repeat Motif
Authors: Finnie Christine , Zorreguieta Angeles , Hartley Nigel M. , Downie J. Allan 
Published: December 2019

ABSTRACT

The
prsDE
genes encode a type I protein secretion system required for the secretion of the nodulation protein NodO and at least three other proteins from
Rhizobium leguminosarum
bv. viciae. At least one of these proteins was predicted to be a glycanase involved in processing of bacterial exopolysaccharide (EPS). Two strongly homologous genes (
plyA
and
plyB
) were identified as encoding secreted proteins with polysaccharide degradation activity. Both PlyA and PlyB degrade EPS and carboxymethyl cellulose (CMC), and these extracellular activities are absent in a
prsD
(protein secretion) mutant. The
plyA
gene is upstream of
prsD
but appears to be expressed at a very low level (if at all) in cultured bacteria. A
plyB
::Tn
5
mutant has a very large reduction in degradation of EPS and CMC. Cultures of
plyB
mutants contained an increased ratio of EPS repeat units to reducing ends, indicating that the EPS was present in a longer-chain form, and this correlated with a significant increase in culture viscosity. Thus, PlyB may play a role in processing of EPS. Analysis of the symbiotic properties of a
plyA plyB
double mutant revealed that these genes are not required for symbiotic nitrogen fixation and that nodulation was not significantly affected. PlyA and PlyB are similar to bacterial and fungal polysaccharide lyases; they contain 10 copies of what we propose as a novel heptapeptide repeat motif that may constitute a fold similar to that found in the family of extracellular pectate lyases. PlyA and PlyB lack the Ca
2+
-binding RTX nonapeptide repeat motifs usually found in proteins secreted via type I systems. We propose that PlyA and PlyB are members of a new family of proteins secreted via type I secretion systems and that they are involved in processing of EPS.
Identification and Function of the
pdxY
Gene, Which Encodes a Novel Pyridoxal Kinase Involved in the Salvage Pathway of Pyridoxal 5′-Phosphate Biosynthesis in
Escherichia coli
K-12
Authors: Yang Yong , Tsui Ho-Ching Tiffany , Man Tsz-Kwong , Winkler Malcolm E. 
Published: December 2019

ABSTRACT

pdxK
encodes a pyridoxine (PN)/pyridoxal (PL)/pyridoxamine (PM) kinase thought to function in the salvage pathway of pyridoxal 5′-phosphate (PLP) coenzyme biosynthesis. The observation that
pdxK
null mutants still contain PL kinase activity led to the hypothesis that
Escherichia coli
K-12 contains at least one other B
6
-vitamer kinase. Here we support this hypothesis by identifying the
pdxY
gene (formally, open reading frame
f287b
) at 36.92 min, which encodes a novel PL kinase. PdxY was first identified by its homology to PdxK in searches of the complete
E. coli
genome. Minimal clones of
pdxY
+
overexpressed PL kinase specific activity about 10-fold. We inserted an omega cassette into
pdxY
and crossed the resulting
pdxY
::ΩKan
r
mutation into the bacterial chromosome of a
pdxB
mutant, in which de novo PLP biosynthesis is blocked. We then determined the growth characteristics and PL and PN kinase specific activities in extracts of
pdxK
and
pdxY
single and double mutants. Significantly, the requirement of the
pdxB pdxK pdxY
triple mutant for PLP was not satisfied by PL and PN, and the triple mutant had negligible PL and PN kinase specific activities. Our combined results suggest that the PL kinase PdxY and the PN/PL/PM kinase PdxK are the only physiologically important B
6
vitamer kinases in
E. coli
and that their function is confined to the PLP salvage pathway. Last, we show that
pdxY
is located downstream from
pdxH
(encoding PNP/PMP oxidase) and essential
tyrS
(encoding aminoacyl-tRNA
Tyr
synthetase) in a multifunctional operon.
pdxY
is completely cotranscribed with
tyrS
, but about 92% of
tyrS
transcripts terminate at a putative Rho-factor-dependent attenuator located in the
tyrS-pdxY
intercistronic region.
Efficiency of T4 Gene
60
Translational Bypassing
Authors: Maldonado Rafael , Herr Alan J. 
Published: December 2019

ABSTRACT

Ribosomes translating bacteriophage T4 gene
60
mRNA bypass 50 noncoding nucleotides from a takeoff site at codon 46 to a landing site just upstream of codon 47. A key signal for efficient bypassing is contained within the nascent peptide synthesized prior to takeoff. Here we show that this signal is insensitive to the addition of coding information at its N terminus. In addition, analysis of amino-terminal fusions, which allow detection of all major products synthesized from the gene
60
mRNA, show that 50% of ribosomes bypass the coding gap while the rest either terminate at a UAG stop codon immediately following codon 46 or fail to resume coding. Bypassing efficiency estimates significantly lower than 50% were obtained with enzymatic reporter systems that relied on comparing test constructs to constructs with a precise excision of the gap (gap deletion). Further analysis showed that these estimates are distorted by differences between test and gap deletion functional mRNA levels. An internal translation initiation site at Met12 of gene
60
(which eliminates part of the essential nascent peptide) also distorts these estimates. Together, these results support an efficiency estimate of ∼50%, less than previously reported. This estimate suggests that bypassing efficiency is determined by the competition between reading signals and release factors and gives new insight into the kinetics of bypassing signal action.
Thioredoxin Is an Essential Protein Induced by Multiple Stresses in
Bacillus subtilis
Authors: Scharf Christian , Riethdorf Sabine , Ernst Henrik , Engelmann Susanne , Völker Uwe , Hecker Michael 
Published: December 2019

ABSTRACT

Thioredoxin, a small, ubiquitous protein which participates in redox reactions through the reversible oxidation of its active center dithiol to a disulfide, is an essential protein in
Bacillus subtilis
. A variety of stresses, including heat or salt stress or ethanol treatment, strongly enhanced the synthesis of thioredoxin in
B. subtilis
. The stress induction of the monocistronic
trxA
gene encoding thioredoxin occurs at two promoters. The general stress sigma factor, ς
B
, was required for the initiation of transcription at the upstream site, S
B
, and the promoter preceding the downstream start site, S
A
, was presumably recognized by the vegetative sigma factor, ς
A
. In contrast to the heat-inducible, ς
A
-dependent promoters preceding the chaperone-encoding operons
groESL
and
dnaK
, no CIRCE (for controlling inverted repeat of chaperone expression) was present in the vicinity of the start site, S
A
. The induction patterns of the promoters differed, with the upstream promoter displaying the typical stress induction of ς
B
-dependent promoters. Transcription initiating at S
A
, but not at S
B
, was also induced after treatment with hydrogen peroxide or puromycin. Such a double control of stress induction at two different promoters seems to be typical of a subgroup of class III heat shock genes of
B. subtilis
, like
clpC
, and it either allows the cells to raise the level of the antioxidant thioredoxin after oxidative stress or allows stressed cells to accumulate thioredoxin. These increased levels of thioredoxin might help stressed
B. subtilis
cells to maintain the native and reduced state of cellular proteins.
Proton Gradient-Driven Nickel Uptake by Vacuolar Membrane Vesicles of
Saccharomyces cerevisiae
Authors: Nishimura Ken , Igarashi Kazuei , Kakinuma Yoshimi 
Published: December 2019

ABSTRACT

A vacuolar H
+
-ATPase-negative mutant of
Saccharomyces cerevisiae
was highly sensitive to nickel ion. Accumulation of nickel ion in the cells of this mutant of less than 60% of the value for the parent strain arrested growth, suggesting a role for this ATPase in sequestering nickel ion into vacuoles. An artificially imposed pH gradient (interior acid) induced transient nickel ion uptake by vacuolar membrane vesicles, which was inhibited by collapse of the pH difference but not of the membrane potential. Nickel ion transport into vacuoles in a pH gradient-dependent manner is thus important for its detoxification in yeast.
Identification of the Ω4400 Regulatory Region, a Developmental Promoter of
Myxococcus xanthus
Authors: Brandner Janine P. , Kroos Lee 
Published: December 2019

ABSTRACT

Ω4400 is the site of a Tn
5 lac
insertion in the
Myxococcus xanthus
genome that fuses
lacZ
expression to a developmentally regulated promoter. Cell-cell interactions that occur during development, including C signaling, are required for normal expression of Tn
5 lac
Ω4400. The DNA upstream of the Ω4400 insertion has been cloned, the promoter has been localized, and a partial open reading frame has been identified. From the deduced amino acid sequence of the partial open reading frame, the gene disrupted by Tn
5 lac
Ω4400 may encode a protein with an ATP- or GTP-binding site. Expression of the gene begins 6 to 12 h after starvation initiates development, as measured by β-galactosidase production in cells containing Tn
5 lac
Ω4400. The putative transcriptional start site was mapped, and deletion analysis has shown that DNA downstream of −101 bp is sufficient for C-signal-dependent, developmental activation of this promoter. A deletion to −76 bp eliminated promoter activity, suggesting the involvement of an upstream activator protein. The promoter may be transcribed by RNA polymerase containing a novel sigma factor, since a mutation in the
M. xanthus sigB
or
sigC
gene did not affect Tn
5 lac
Ω4400 expression and the DNA sequence upstream of the transcriptional start site did not match the sequence of any
M. xanthus
promoter transcribed by a known form of RNA polymerase. However, the Ω4400 promoter does contain the sequence 5′-CATCCCT-3′ centered at −49 and the C-signal-dependent Ω4403 promoter also contains this sequence at the same position. Moreover, the two promoters match at five of six positions in the −10 regions, suggesting that these promoters may share one or more transcription factors. These results begin to define the
cis
-acting regulatory elements important for cell-cell interaction-dependent gene expression during the development of a multicellular prokaryote.
Isolation and Characterization of Methanophenazine and Function of Phenazines in Membrane-Bound Electron Transport ofMethanosarcina mazeiGö1
Authors: Abken Hans-Jörg , Tietze Mario , Brodersen Jens , Bäumer Sebastian , Beifuss Uwe , Deppenmeier Uwe 
Published: December 2019

ABSTRACTA hydrophobic, redox-active component with a molecular mass of 538 Da was isolated from lyophilized membranes ofMethanosarcina mazeiGö1 by extraction with isooctane. After purification on a high-performance liquid chromatography column, the chemical structure was analyzed by mass spectroscopy and nuclear magnetic resonance studies. The component was called methanophenazine and represents a 2-hydroxyphenazine derivative which is connected via an ether bridge to a polyisoprenoid side chain. Since methanophenazine was almost insoluble in aqueous buffers, water-soluble phenazine derivatives were tested for their ability to interact with membrane-bound enzymes involved in electron transport and energy conservation. The purified F420H2dehydrogenase fromM. mazeiGö1 showed highest activity with 2-hydroxyphenazine and 2-bromophenazine as electron acceptors when F420H2was added. Phenazine-1-carboxylic acid and phenazine proved to be less effective. TheKmvalues for 2-hydroxyphenazine and phenazine were 35 and 250 μM, respectively. 2-Hydroxyphenazine was also reduced by molecular hydrogen catalyzed by an F420-nonreactive hydrogenase which is present in washed membrane preparations. Furthermore, the membrane-bound heterodisulfide reductase was able to use reduced 2-hydroxyphenazine as an electron donor for the reduction of CoB-S-S-CoM. Considering all these results, it is reasonable to assume that methanophenazine plays an important role in vivo in membrane-bound electron transport ofM. mazeiGö1.
Availability of O
2
as a Substrate in the Cytoplasm of Bacteria under Aerobic and Microaerobic Conditions
Authors: Arras Tanja , Schirawski Jan , Unden Gottfried 
Published: December 2019

ABSTRACT

The growth rates of
Pseudomonas putida
KT2442 and mt-2 on benzoate, 4-hydroxybenzoate, or 4-methylbenzoate showed an exponential decrease with decreasing oxygen tensions (partial O
2
tension [pO
2
] values). The oxygen tensions resulting in half-maximal growth rates were in the range of 7 to 8 mbar of O
2
(corresponding to 7 to 8 μM O
2
) (1 bar = 10
5
Pa) for aromatic compounds, compared to 1 to 2 mbar for nonaromatic compounds like glucose or succinate. The decrease in the growth rates coincided with excretion of catechol or protocatechuate, suggesting that the activity of the corresponding oxygenases became limiting. The experiments directly establish that under aerobic and microaerobic conditions (about 10 mbar of O
2
), the diffusion of O
2
into the cytoplasm occurs at high rates sufficient for catabolic processes. This is in agreement with calculated O
2
diffusion rates. Below 10 mbar of O
2
, oxygen became limiting for the oxygenases, probably due to their high

K
m

values, but the diffusion of O
2
into the cytoplasm presumably should be sufficiently rapid to maintain ambient oxygen concentrations at oxygen tensions as low as 1 mbar of O
2
. The consequences of this finding for the availability of O
2
as a substrate or as a regulatory signal in the cytoplasm of bacterial cells are discussed.
Interaction of Native and Mutant MecI Repressors with Sequences That Regulate
mecA
, the Gene Encoding Penicillin Binding Protein 2a in Methicillin-Resistant Staphylococci
Authors: Sharma Vijay K. , Hackbarth Corinne J. , Dickinson Tanja M. , Archer Gordon L. 
Published: December 2019

ABSTRACT

Methicillin resistance in staphylococci is mediated by PBP2a, a penicillin binding protein with low affinity for β-lactam antibiotics. The gene encoding PBP2a,
mecA
, is transcriptionally regulated in some clinical isolates by
mecR1
and
mecI
, genes divergently transcribed from
mecA
that encode a signal transducer and repressor, respectively. The biochemical basis of MecI-mediated
mecA
transcriptional repression was investigated by using purified MecI. In DNase I protection studies, MecI protected a 30-bp palindrome encompassing the predicted
mecA
−10 and the
mecR1
−35 promoter sequences. The larger palindrome contained 15 bp of dyad symmetry within which was a smaller 6-bp palindrome. Electrophoretic mobility shift assays established a requirement for the entire 15-bp half-site for initial repressor binding. Fragments containing the 30-bp palindrome and the entire
mecA-mecR1
intergenic region were retarded in gels as multiple discrete bands varying in molecular size, characteristic of cooperative DNA binding. Glutaraldehyde cross-linking confirmed oligomerization of repressor in solution. A naturally occurring MecI mutant (MecI*; D39G) repressed
mecA
transcription sixfold less well than the wild type in vivo. Although MecI* protected the same target sequences and exhibited similar gel shift patterns to MecI, 5- to 10-fold more protein was required. MecI* exhibited defective oligomerization in solution, suggesting that the MecI amino terminus is important in protein-protein interactions and that protein oligomerization is necessary for optimum repression.
Developmental Regulation of Transcription of
whiE
, a Locus Specifying the Polyketide Spore Pigment in
Streptomyces coelicolor
A3(2)
Authors: Kelemen Gabriella H. , Brian Paul , Flärdh Klas , Chamberlin Leony , Chater Keith F. , Buttner Mark J. 
Published: December 2019

ABSTRACT

whiE
is a complex locus that specifies the polyketide spore pigment in
Streptomyces coelicolor
A3(2). Two divergently oriented promoters,
whiEP1
and
whiEP2
, were identified in the
whiE
gene cluster, and their activities were analyzed during colony development in wild-type and sporulation-deficient strains. Both promoters were developmentally regulated;
whiEP1
and
whiEP2
transcripts were detected transiently at approximately the time when sporulation septa were observed in the aerial hyphae, and transcription from both promoters depended on each of the six known “early”
whi
genes required for sporulation septum formation (
whiA
, -
B
, -
G
, -
H
, -
I
, and -
J
). Mutation of the late sporulation-specific sigma factor gene,
sigF
, had no effect on the activity of
whiEP1
but blocked transcription from
whiEP2
. However, ς
F
-containing holoenzyme was not sufficient to direct transcription of
whiEP2
in vitro. The
whiEP2
promoter controls expression of
whiE
ORFVIII, encoding a putative flavin adenine dinucleotide-dependent hydroxylase that catalyzes a late tailoring step in the spore pigment biosynthetic pathway. Disruption of
whiE
ORFVIII causes a change in spore color, from grey to greenish (T.-W. Yu and D. A. Hopwood, Microbiology 141:2779–2791, 1995). Consistent with these observations, construction of a
sigF
null mutant of
S. coelicolor
M145 caused the same change in spore color, showing that disruption of
sigF
in
S. coelicolor
changes the nature of the spore pigment rather than preventing its synthesis altogether.
Kinetics of
nirS
Expression (Cytochrome
cd
1
Nitrite Reductase) in
Pseudomonas stutzeri
during the Transition from Aerobic Respiration to Denitrification: Evidence for a Denitrification-Specific Nitrate- and Nitrite-Responsive Regulatory System
Authors: Härtig Elisabeth , Zumft Walter G. 
Published: December 2019

ABSTRACT

After shifting an oxygen-respiring culture of
Pseudomonas stutzeri
to nitrate or nitrite respiration, we directly monitored the expression of the
nirS
gene by mRNA analysis.
nirS
encodes the 62-kDa subunit of the homodimeric cytochrome
cd
1
nitrite reductase involved in denitrification. Information was sought about the requirements for gene activation, potential regulators of such activation, and signal transduction pathways triggered by the alternative respiratory substrates. We found that
nirS
, together with
nirT
and
nirB
(which encode tetra- and diheme cytochromes, respectively), is part of a 3.4-kb operon. In addition, we found a 2-kb monocistronic transcript. The half-life of each of these messages was approximately 13 min in denitrifying cells with a doubling time of around 2.5 h. When the culture was subjected to a low oxygen tension, we observed a transient expression of
nirS
lasting for about 30 min. The continued transcription of the
nirS
operon required the presence of nitrate or nitrite. This anaerobically manifested
N
-oxide response was maintained in nitrate sensor (NarX) and response regulator (NarL) knockout strains. Similar mRNA stability and transition kinetics were observed for the
norCB
operon, encoding the NO reductase complex, and the
nosZ
gene, encoding nitrous oxide reductase. Our results suggest that a nitrate- and nitrite-responsive regulatory circuit independent of NarXL is necessary for the activation of denitrification genes.