bluuebunny/crossref_metadata_2025_split huggingface dataset:
Analysis of Genes Involved in Biosynthesis of Coronafacic Acid, the Polyketide Component of the Phytotoxin Coronatine
Authors: Rangaswamy Vidhya , Mitchell Robin , Ullrich Matthias , Bender Carol 
Published: December 2019

ABSTRACT

Coronafacic acid (CFA) is the polyketide component of coronatine (COR), a phytotoxin produced by the plant-pathogenic bacterium
Pseudomonas syringae
. The genes involved in CFA biosynthesis are encoded by a single transcript which encompasses 19 kb of the COR gene cluster. In the present study, the nucleotide sequence was determined for a 4-kb region located at the 3′ end of the CFA biosynthetic gene cluster. Three open reading frames were identified and designated
cfa8
,
cfa9
, and
tnp1
; the predicted translation products of these genes showed relatedness to oxidoreductases, thioesterases, and transposases, respectively. The translational products of
cfa8
and
cfa9
were overproduced in
Escherichia coli
BL21; however,
tnp1
was not translated in these experiments. Mutagenesis and complementation analysis indicated that
cfa8
is required for the production of CFA and COR. Analysis of a
cfa9
mutant indicated that this gene is dispensable for CFA and COR production but may increase the release of enzyme-bound products from the COR pathway;
tnp1
, however, had no obvious function in CFA or COR biosynthesis. A genetic strategy was used to produce CFA in a
P. syringae
strain which lacks the COR gene cluster; this approach will be useful in future studies designed to investigate biosynthetic products of the CFA gene cluster.
The V Antigen of
Yersinia pestis
Regulates Yop Vectorial Targeting as Well as Yop Secretion through Effects on YopB and LcrG
Authors: Nilles Matthew L. , Fields Kenneth A. , Straley Susan C. 
Published: December 2019

ABSTRACT

Yersinia pestis
expresses a set of secreted proteins called Yops and the bifunctional LcrV, which has both regulatory and antihost functions. Yops and LcrV expression and the activity of the type III mechanism for their secretion are coordinately regulated by environmental signals such as Ca
2+
concentration and eukaryotic cell contact. In vitro, Yops and LcrV are secreted into the culture medium in the absence of Ca
2+
as part of the low-Ca
2+
response (LCR). The LCR is induced in a tissue culture model by contact with eukaryotic cells that results in Yop translocation into cells and subsequent cytotoxicity. The secretion mechanism is believed to indirectly regulate expression of
lcrV
and
yop
operons by controlling the intracellular concentration of a secreted negative regulator. LcrG, a secretion-regulatory protein, is thought to block secretion of Yops and LcrV, possibly at the inner face of the inner membrane. A recent model proposes that when the LCR is induced, the increased expression of LcrV yields an excess of LcrV relative to LcrG, and this is sufficient for LcrV to bind LcrG and unblock secretion. To test this LcrG titration model, LcrG and LcrV were expressed alone or together in a newly constructed
lcrG
deletion strain, a Δ
lcrG2
mutant, of
Y. pestis
that produces low levels of LcrV and constitutively expresses and secretes Yops. Overexpression of LcrG in this mutant background was able to block secretion and depress expression of Yops in the presence of Ca
2+
and to dramatically decrease Yop expression and secretion in growth medium lacking Ca
2+
. Overexpression of both LcrG and LcrV in the Δ
lcrG2
strain restored wild-type levels of Yop expression and Ca
2+
control of Yop secretion. Surprisingly, when HeLa cells were infected with the Δ
lcrG2
strain, no cytotoxicity was apparent and translocation of Yops was abolished. This correlated with an altered distribution of YopB as measured by accessibility to trypsin. These effects were not due to the absence of LcrG, because they were alleviated by restoration of LcrV expression and secretion alone. LcrV itself was found to enter HeLa cells in a nonpolarized manner. These studies supported the LcrG titration model of LcrV’s regulatory effect at the level of Yop secretion and revealed a further role of LcrV in the deployment of YopB, which in turn is essential for the vectorial translocation of Yops into eukaryotic cells.
Evolution of Cyanobacteria by Exchange of Genetic Material among Phyletically Related Strains
Authors: Rudi Knut , Skulberg Olav M. , Jakobsen Kjetill S. 
Published: December 2019

ABSTRACT

The cyanobacterial radiation consists of several lineages of phyletically (morphologically and genetically) related organisms. Several of these organisms show a striking resemblance to fossil counterparts. To investigate the molecular mechanisms responsible for stabilizing or homogenizing cyanobacterial characters, we compared the evolutionary rates and phylogenetic origins of the small-subunit rRNA-encoding DNA (16S rDNA), the conserved gene
rbcL
(encoding
d
-ribulose 1,5-bisphosphate carboxylase-oxygenase large subunit), and the less conserved gene
rbcX
. This survey includes four categories of phyletically related organisms: 16 strains of
Microcystis
, 6 strains of
Tychonema
, 10 strains of
Planktothrix
, and 12 strains of
Nostoc
. Both
rbcL
and
rbcX
can be regarded as neutrally evolving genes, with 95 to 100% and 50 to 80% synonymous nucleotide substitutions, respectively. There is generally low sequence divergence within the
Microcystis
,
Tychonema
, and
Planktothrix
categories both for
rbcLX
and 16S rDNA. The
Nostoc
category, on the other hand, consists of three genetically clustered lineages for these loci. The 16S rDNA and
rbcLX
phylogenies are not congruent for strains within the clustered groups. Furthermore, analysis of the phyletic structure for
rbcLX
indicates recombinational events between the informative sites within this locus. Thus, our results are best explained by a model involving both intergenic and intragenic recombinations. This evolutionary model explains the DNA sequence clustering for the modern species as a result of sequence homogenization (concerted evolution) caused by exchange of genetic material for neutrally evolving genes. The morphological clustering, on the other hand, is explained by structural and functional stability of these characters. We also suggest that exchange of genetic material for neutrally evolving genes may explain the apparent stability of cyanobacterial morphological characters, perhaps over billions of years.
Secretion of Elastinolytic Enzymes and Their Propeptides by
Pseudomonas aeruginosa
Authors: Braun Peter , de Groot Arjan , Bitter Wilbert , Tommassen Jan 
Published: December 2019

ABSTRACT

Elastase of
Pseudomonas aeruginosa
is synthesized as a preproenzyme. The signal sequence is cleaved off during transport across the inner membrane and, in the periplasm, proelastase is further processed. We demonstrate that the propeptide and the mature elastase are both secreted but that the propeptide is degraded extracellularly. In addition, reduction of the extracellular proteolytic activity led to the accumulation of unprocessed forms of LasA and LasD in the extracellular medium, which shows that these enzymes are secreted in association with their propeptides. Furthermore, a hitherto undefined protein with homology to a
Streptomyces griseus
aminopeptidase accumulated under these conditions.
Identification of the Repressor-Encoding Gene of the
Lactobacillus
Bacteriophage A2
Authors: Ladero Victor , García Pilar , Bascarán Victoria , Herrero Mónica , Alvarez Miguel A. , Suárez Juan E. 
Published: December 2019

ABSTRACT

The repressor gene of the
Lactobacillus
phage A2 has the following properties: it (i) encodes a 224-residue polypeptide with DNA binding and RecA cleavage motifs, (ii) is expressed in lysogenic cultures, and (iii) confers superinfection immunity on the host. Adjacent, but divergently transcribed, lies another open reading frame whose product resembles the λ Cro protein. In the 161-bp intergenic segment, putative promoters and operators have been detected.
Aberrant Cell Division and Random FtsZ Ring Positioning in
Escherichia coli cpxA
* Mutants
Authors: Pogliano Joe , Dong Jian Ming , De Wulf Peter , Furlong Dierdre , Boyd Dana , Losick Richard , Pogliano Kit , Lin E. C. C. 
Published: December 2019

ABSTRACT

In
Escherichia coli
, certain mutations in the
cpxA
gene (encoding a sensor kinase of a two-component signal transduction system) randomize the location of FtsZ ring assembly and dramatically affect cell division. However, deletion of the
cpxRA
operon, encoding the sensor kinase and its cognate regulator CpxR, has no effect on division site biogenesis. It appears that certain mutant sensor kinases (CpxA*) either exhibit hyperactivity on CpxR or extend their signalling activity to one or more noncognate response regulators involved in cell division.
Proposed Signal Transduction Role for Conserved CheY Residue Thr87, a Member of the Response Regulator Active-Site Quintet
Authors: Appleby Jeryl L. , Bourret Robert B. 
Published: December 2019

ABSTRACT

CheY serves as a structural prototype for the response regulator proteins of two-component regulatory systems. Functional roles have previously been defined for four of the five highly conserved residues that form the response regulator active site, the exception being the hydroxy amino acid which corresponds to Thr87 in CheY. To investigate the contribution of Thr87 to signaling, we characterized, genetically and biochemically, several
cheY
mutants with amino acid substitutions at this position. The hydroxyl group appears to be necessary for effective chemotaxis, as a Thr→Ser substitution was the only one of six tested which retained a Che
+
swarm phenotype. Although nonchemotactic,
cheY
mutants with amino acid substitutions T87A and T87C could generate clockwise flagellar rotation either in the absence of CheZ, a protein that stimulates dephosphorylation of CheY, or when paired with a second site-activating mutation, Asp13→Lys, demonstrating that a hydroxy amino acid at position 87 is not essential for activation of the flagellar switch. All purified mutant proteins examined phosphorylated efficiently from the CheA kinase in vitro but were impaired in autodephosphorylation. Thus, the mutant CheY proteins are phosphorylated to a greater degree than wild-type CheY yet support less clockwise flagellar rotation. The data imply that Thr87 is important for generating and/or stabilizing the phosphorylation-induced conformational change in CheY. Furthermore, the various position 87 substitutions differentially affected several properties of the mutant proteins. The chemotaxis and autodephosphorylation defects were tightly linked, suggesting common structural elements, whereas the effects on self-catalyzed and CheZ-mediated dephosphorylation of CheY were uncorrelated, suggesting different structural requirements for the two dephosphorylation reactions.
Identification of a Gene Product Induced by Hard-Surface Contact of
Colletotrichum gloeosporioides
Conidia as a Ubiquitin-Conjugating Enzyme by Yeast Complementation
Authors: Liu Zhi-Mei , Kolattukudy Pappachan E. 
Published: December 2019

ABSTRACT

The germinating conidia of many phytopathogenic fungi on hosts must differentiate into an infection structure called the appressorium in order to penetrate their hosts. Chemical signals, such as the host’s surface wax or fruit ripening hormone, ethylene, trigger germination and appressorium formation of the avocado pathogen
Colletotrichum gloeosporioides
only after the conidia are in contact with a hard surface. What role this contact plays is unknown. Here, we describe isolation of genes expressed during the early stage of hard-surface treatment by a differential-display method and report characterization of one of these cloned genes,
chip1
(
Colletotrichum
hard-surface induced protein 1 gene), which encodes a ubiquitin-conjugating enzyme. RNA blots clearly showed that it is induced by hard-surface contact and that ethylene treatment enhanced this induction. The predicted open reading frame (
ubc1
Cg
) would encode a 16.2-kDa ubiquitin-conjugating enzyme, which shows 82% identity to the
Saccharomyces cerevisiae
UBC4-UBC5 E2 enzyme, comprising a major part of total ubiquitin-conjugating activity in stressed yeast cells. UBC1
Cg
can complement the proteolysis deficiency of the
S. cerevisiae ubc4 ubc5
mutant, indicating that ubiquitin-dependent protein degradation is involved in conidial germination and appressorial differentiation.
Circadian Rhythm of Nitrogenase Gene Expression in the Diazotrophic Filamentous Nonheterocystous Cyanobacterium
Trichodesmium
sp. Strain IMS 101
Authors: Chen Yi-Bu , Dominic Benny , Mellon Mark T. , Zehr Jonathan P. 
Published: December 2019

ABSTRACT

Recent studies suggested that the daily cycle of nitrogen fixation activity in the marine filamentous nonheterocystous cyanobacterium
Trichodesmium
sp. is controlled by a circadian rhythm. In this study, we evaluated the rhythm of nitrogen fixation in
Trichodesmium
sp. strain IMS 101 by using the three criteria for an endogenous rhythm. Nitrogenase transcript abundance oscillated with a period of approximately 24 h, and the cycle was maintained even under constant light conditions. The cyclic pattern of transcript abundance was maintained when the culture was grown at 24 and 28.5°C, although the period was slightly longer (26 h) at the higher temperature. The cycle of gene expression could be entrained with light-dark cues. Results of inhibitor experiments indicated that transcript abundance was regulated primarily by transcription initiation, rather than by degradation. The circadian rhythm, the first conclusively demonstrated endogenous rhythm in a filamentous cyanobacterium, was also reflected in nitrogenase MoFe protein abundance and patterns of Fe protein posttranslational modification-demodification.
Differential Regulation of
Rhizobium etli rpoN2
Gene Expression during Symbiosis and Free-Living Growth
Authors: Michiels Jan , Moris Martine , Dombrecht Bruno , Verreth Christel , Vanderleyden Jos 
Published: December 2019

ABSTRACT

The
Rhizobium etli rpoN1
gene, encoding the alternative sigma factor ς
54
(RpoN), was recently characterized and shown to be involved in the assimilation of several nitrogen and carbon sources during free-living aerobic growth (J. Michiels, T. Van Soom, I. D’hooghe, B. Dombrecht, T. Benhassine, P. de Wilde, and J. Vanderleyden, J. Bacteriol. 180:1729–1740, 1998). We identified a second
rpoN
gene copy in
R. etli
,
rpoN2
, encoding a 54.0-kDa protein which displays 59% amino acid identity with the
R. etli
RpoN1 protein. The
rpoN2
gene is cotranscribed with a short open reading frame,
orf180
, which codes for a protein with a size of 20.1 kDa that is homologous to several prokaryotic and eukaryotic proteins of similar size. In contrast to the
R. etli rpoN1
mutant strain, inactivation of the
rpoN2
gene did not produce any phenotypic defects during free-living growth. However, symbiotic nitrogen fixation was reduced by approximately 90% in the
rpoN2
mutant, whereas wild-type levels of nitrogen fixation were observed in the
rpoN1
mutant strain. Nitrogen fixation was completely abolished in the
rpoN1 rpoN2
double mutant. Expression of
rpoN1
was negatively autoregulated during aerobic growth and was reduced during microaerobiosis and symbiosis. In contrast,
rpoN2-gusA
and
orf180-gusA
fusions were not expressed aerobically but were strongly induced at low oxygen tensions or in bacteroids. Expression of
rpoN2
and
orf180
was abolished in
R. etli rpoN1 rpoN2
and
nifA
mutants under all conditions tested. Under free-living microaerobic conditions, transcription of
rpoN2
and
orf180
required the RpoN1 protein. In symbiosis, expression of
rpoN2
and
orf180
occurred independently of the
rpoN1
gene, suggesting the existence of an alternative symbiosis-specific mechanism of transcription activation.
The Central, Surface-Exposed Region of the Flagellar Hook Protein FlgE of
Campylobacter jejuni
Shows Hypervariability among Strains
Authors: Lüneberg Edeltraud , Glenn-Calvo Eduardo , Hartmann Maike , Bär Werner , Frosch Matthias 
Published: December 2019

ABSTRACT

In a previous study, we observed that monoclonal antibodies raised against the hook protein FlgE of
Campylobacter jejuni
LIO 36, isolate 5226, bound exclusively to this strain. The aim of this study was to elucidate the molecular basis for these binding specificities. The hook protein-encoding gene
flgE
of
C. jejuni
was cloned in
Escherichia coli
and sequenced. The
flgE
genes of four additional
C. jejuni
strains were amplified by PCR and also sequenced. Comparison of the deduced amino acid sequences revealed a high degree of variability in the central parts of the FlgE proteins among the strains, including variable and hypervariable domains. These findings may indicate a selective pressure of
C. jejuni
hosts, forcing the bacteria to generate variations in surface-exposed antigenic determinants.
Photosynthetic Electron Transport Involved in PxcA-Dependent Proton Extrusion in
Synechocystis
sp. Strain PCC6803: Effect of
pxcA
Inactivation on CO
2
, HCO
3
−
, and NO
3
−
Uptake
Authors: Sonoda Masatoshi , Katoh Hirokazu , Vermaas Wim , Schmetterer George , Ogawa Teruo 
Published: December 2019

ABSTRACT

The product of
pxcA
(formerly known as
cotA
) is involved in light-induced Na
+
-dependent proton extrusion. In the presence of 2,5-dimethyl-
p
-benzoquinone, net proton extrusion by
Synechocystis
sp. strain PCC6803 ceased after 1 min of illumination and a postillumination influx of protons was observed, suggesting that the PxcA-dependent, light-dependent proton extrusion equilibrates with a light-independent influx of protons. A photosystem I (PS I) deletion mutant extruded a large number of protons in the light. Thus, PS II-dependent electron transfer and proton translocation are major factors in light-driven proton extrusion, presumably mediated by ATP synthesis. Inhibition of CO
2
fixation by glyceraldehyde in a cytochrome
c
oxidase (COX) deletion mutant strongly inhibited the proton extrusion. Leakage of PS II-generated electrons to oxygen via COX appears to be required for proton extrusion when CO
2
fixation is inhibited. At pH 8.0, NO
3
−
uptake activity was very low in the
pxcA
mutant at low [Na
+
] (∼100 μM). At pH 6.5, the
pxcA
strain did not take up CO
2
or NO
3
−
at low [Na
+
] and showed very low CO
2
uptake activity even at 15 mM Na
+
. A possible role of PxcA-dependent proton exchange in charge and pH homeostasis during uptake of CO
2
, HCO
3
−
, and NO
3
−
is discussed.
Transcriptional Analysis of Different Promoters in the
sar
Locus in
Staphylococcus aureus
Authors: Manna Adhar C. , Bayer Manfred G. , Cheung Ambrose L. 
Published: December 2019

ABSTRACT

The expression of extracellular virulence determinants in
Staphylococcus aureus
is controlled by a 510-nucleotide RNA molecule (RNAIII) which is a part of the
agr
system. The
agr
operon, which encodes a multicomponent signal transduction system, is partially under the influence of an unlinked regulatory locus called
sar
. The
sar
locus is composed of three overlapping transcripts, designated
sarA
(0.56 kb),
sarC
(0.8 kb), and
sarB
(1.2 kb), originating from the P1, P3, and P2 promoters, respectively. In this study, we analyzed the differential expression of these promoters by using transcriptional fusion with the
xylE
reporter gene to study the activation of the
sar
locus. The data confirm the existence of three independent promoters with different promoter activities. Maximal promoter activity was observed with the combined fusion of P
2
-P
3
-P
1
promoters. Expression studies with a
sigB
mutant revealed that the P3 promoter is SigB dependent. Analysis of these transcriptional fusions in a
sarA
mutant and in complemented strains with each of the
sar
transcriptional units revealed that the
sar
locus is autoregulatory, with SarA acting as a positive regulator. From various transcriptional fusion studies of the upstream region of the P1 promoter, we have localized a 34-bp sequence which seems to play a role in down-modulating P1 transcription. Using heparin-Sepharose and DNA-specific columns, we partially purified a 12-kDa protein, possibly a repressor, which binds to the promoter regions upstream of P2 and P1 and which also binds to the 34-bp sequence. These data indicated that the regulation of the
sar
locus is complex and may involve the
sar
gene product(s) and other regulatory protein(s).
YscO of
Yersinia pestis
Is a Mobile Core Component of the Yop Secretion System
Authors: Payne Patricia L. , Straley Susan C. 
Published: December 2019

ABSTRACT

The
Yersinia pestis
low-Ca
2+
response stimulon is responsible for the temperature- and Ca
2+
-regulated expression and secretion of plasmid pCD1-encoded antihost proteins (V antigen and Yops). We have previously shown that
lcrD
,
yscC
,
yscD
,
yscG
, and
yscR
encode proteins that are essential for high-level expression and secretion of V antigen and Yops at 37°C in the absence of Ca
2+
. In this study, we characterized
yscO
of the Yop secretion (
ysc
) operon that contains
yscN
through
yscU
by determining the localization of its gene product and the phenotype of an in-frame deletion. The
yscO
mutant grew and expressed the same levels of Yops as the parent at 37°C in the presence of Ca
2+
. In the absence of Ca
2+
, the mutant grew independently of Ca
2+
, expressed only basal levels of V antigen and Yops, and failed to secrete these. These defects could be partially complemented by providing
yscO
in
trans
in the
yscO
mutant. Overexpression of YopM and V antigen in the mutant failed to restore the export of either protein, showing that the mutation had a direct effect on secretion. These results indicated that the
yscO
gene product is required for high-level expression and secretion of V antigen and Yops. YscO was found by immunoblot analysis in the soluble and membrane fractions of bacteria growing at 37°C irrespective of the presence of Ca
2+
and in the culture medium in the absence of Ca
2+
. YscO is the only mobile protein identified so far in the
Yersinia
species that is required for secretion of V antigen and Yops.
The Conjugal Intermediate of Plasmid RSF1010 Inhibits
Agrobacterium tumefaciens
Virulence and VirB-Dependent Export of VirE2
Authors: Stahl Lisa E. , Jacobs Amy , Binns Andrew N. 
Published: December 2019

ABSTRACT

Agrobacterium tumefaciens
causes crown gall disease by transferring oncogenic, single-stranded DNA (T strand), covalently attached to the VirD2 protein, across the bacterial envelope into plant cells where its expression results in tumor formation. The single-stranded DNA binding protein VirE2 is also transferred into the plant cell, though the location at which VirE2 interacts with the T strand is still under investigation. The movement of the transferred DNA and VirE2 from
A. tumefaciens
to the plant cell depends on the membrane-localized VirB and VirD4 proteins. Further, the movement of the IncQ broad-host-range plasmid RSF1010 between
Agrobacterium
strains or from
Agrobacterium
to plants also requires the
virB
-encoded transfer system. Our earlier studies showed that the presence of the RSF1010 plasmid in wild-type strains of
Agrobacterium
inhibits both their virulence and their capacity to transport VirE2, as assayed by coinfection with
virE
mutants. Here we demonstrate that the capacity to form a conjugal intermediate of RSF1010 is necessary for this inhibition, suggesting that the transferred form of the plasmid competes with the VirD2-T strand and/or VirE2 for a common export site.