bluuebunny/crossref_metadata_2025_split huggingface dataset:
Negative Regulation of
hrp
Genes in
Pseudomonas syringae
by HrpV
Authors: Preston Gail , Deng Wen-Ling , Huang Hsiou-Chen , Collmer Alan 
Published: December 2019

ABSTRACT

Mutations in the five
hrp
and
hrc
genes in the
hrpC
operon of the phytopathogen
Pseudomonas syringae
pv. syringae 61 have different effects on bacterial interactions with host and nonhost plants. The
hrcC
gene within the
hrpC
operon encodes an outer membrane component of the Hrp secretion system that is conserved in all type III protein secretion systems and is required for most pathogenic phenotypes and for secretion of the HrpZ harpin to the bacterial milieu. The other four genes (in order),
hrpF
,
hrpG
, (
hrcC
),
hrpT
, and
hrpV
, appear to be unique to the group I
hrp
clusters found in certain phytopathogens (e.g.,
P. syringae
and
Erwinia amylovora
) and are less well understood. We initiated an examination of their role in Hrp regulation and secretion by determining the effects of functionally nonpolar
nptII
cartridge insertions in each gene on the production and secretion of HrpZ, as determined by immunoblot analysis of cell fractions.
P. syringae
pv. syringae 61
hrpF
,
hrpG
, and
hrpT
mutants were unable to secrete HrpZ, whereas the
hrpV
mutant overproduced and secreted the protein. This suggested that HrpV is a negative regulator of HrpZ production. Further immunoblot assays showed that the
hrpV
mutant produced higher levels of proteins encoded by all three of the major
hrp
operons tested—HrcJ (
hrpZ
operon), HrcC (
hrpC
operon), and HrcQ
B
(
hrpU
operon)—and that constitutive expression of
hrpV
in
trans
abolished the production of each of these proteins. To determine the hierarchy of HrpV regulation in the
P. syringae
pv. syringae 61 positive regulatory cascade, which is composed of HrpRS (proteins homologous with ς
54
-dependent promoter-enhancer-binding proteins) and HrpL (alternate sigma factor), we tested the ability of constitutively expressed
hrpV
to repress the activation of HrcJ production that normally accompanies constitutive expression of
hrpL
or
hrpRS
. No repression was observed, indicating that HrpV acts upstream of HrpRS in the cascade. The effect of HrpV levels on transcription of the
hrpZ
operon was determined by monitoring the levels of β-glucuronidase produced by a
hrpA
′::
uidA
transcriptional fusion plasmid in different
P. syringae
pv. syringae 61 strains. The
hrpV
mutant produced higher levels of β-glucuronidase than the wild type, a
hrcU
(type III secretion) mutant produced the same level as the wild type, and the strain constitutively expressing
hrpV
in
trans
produced low levels equivalent to that of a
hrpS
mutant. These results suggest that HrpF, HrpG, and HrpT are all components of the type III protein secretion system whereas HrpV is a negative regulator of transcription of the Hrp regulon.
Bacillus subtilis
Cells Lacking Penicillin-Binding Protein 1 Require Increased Levels of Divalent Cations for Growth
Authors: Murray Thomas , Popham David L. , Setlow Peter 
Published: December 2019

ABSTRACT

Bacillus subtilis
strains lacking penicillin-binding protein 1 (PBP1), encoded by
ponA
, required greater amounts of Mg
2+
or Ca
2+
for vegetative growth or spore outgrowth than the wild-type strain and strains lacking other high-molecular-weight (HMW) PBPs. Growth of
ponA
cells in a medium low in Mg
2+
also resulted in greatly increased cell bending compared to wild-type cells or cells lacking other HMW PBPs. The addition of high levels of Mg
2+
to growth media eliminated these phenotypes of a
ponA
mutant. In contrast to the effects of divalent cations, NaCl did not restore
ponA
cell growth in a divalent-cation-deficient medium. Surprisingly, wild-type cells swelled and then lysed during both vegetative growth and spore outgrowth when 500 mM NaCl was included in a divalent-cation-deficient medium. Again, Mg
2+
addition was sufficient to allow normal vegetative growth and spore outgrowth of both wild-type and
ponA
cells in a medium with 500 mM NaCl. These studies demonstrate that (i) while HMW PBPs possess largely redundant functions in rich medium, when divalent cations are limiting, PBP1 is required for cell growth and spore outgrowth; and (ii) high levels of NaCl induce cell lysis in media deficient in divalent cations during both vegetative growth and spore outgrowth.
Cloning and Identification of Conjugative Transfer Origins in the
Rhizobium meliloti
Genome
Authors: Herrera-Cervera Jose A. , Sanjuan-Pinilla Julio M. , Olivares Jose , Sanjuan Juan 
Published: December 2019

ABSTRACT

A simple approach was used to identify
Rhizobium meliloti
DNA regions with the ability to convert a nontransmissible vector into a mobilizable plasmid, i.e., to contain origins of conjugative transfer (
oriT
,
mob
). RecA-defective
R. meliloti
merodiploid populations, where each individual contained a hybrid cosmid from an
R. meliloti
GR4 gene library, were used as donors en masse in conjugation with another
R. meliloti
recipient strain, selecting transconjugants for vector-encoded antibiotic resistance. Restriction analysis of cosmids isolated from individual transconjugants resulted in the identification of 11 nonoverlapping DNA regions containing potential
oriT
s. Individual hybrid cosmids were confirmed to be mobilized from the original
recA
donors at frequencies ranging from 10
−2
to 10
−5
per recipient cell. DNA hybridization experiments showed that seven
mob
DNA regions correspond to plasmid replicons: four on symbiotic megaplasmid 1 (pSym1), one on pSym2, and another two on each of the two cryptic plasmids harbored by
R. meliloti
GR4. Another three
mob
clones could not be located to any plasmid and were therefore preliminarily assigned to the chromosome. With this strategy, we were able to characterize the
oriT
of the conjugative plasmid pRmeGR4a, which confirmed the reliability of the approach to select for
oriT
s. Moreover, transfer of the 11
mob
cosmids from
R. meliloti
into
Escherichia coli
occurred at frequencies as high as 10
−1
, demonstrating the
R. meliloti
gene transfer capacity is not limited to the family
Rhizobiaceae
. Our results show that the
R. meliloti
genome contains multiple
oriT
s that allow efficient DNA mobilization to rhizobia as well as to phylogenetically distant gram-negative bacteria.
SdeK Is Required for Early Fruiting Body Development in
Myxococcus xanthus
Authors: Garza Anthony G. , Pollack Jeffrey S. , Harris Baruch Z. , Lee Albert , Keseler Ingrid M. , Licking Ellen F. , Singer Mitchell 
Published: December 2019

ABSTRACT

Myxococcus xanthus
cells carrying the Ω4408 Tn
5lac
insertion at the
sde
locus show defects in fruiting body development and sporulation. Our analysis of
sde
expression patterns showed that this locus is induced early in the developmental program (0 to 2 h) and that expression increases approximately fivefold after 12 h of development. Further studies showed that expression of
sde
is induced as growing cells enter stationary phase, suggesting that activation of the
sde
locus is not limited to the developmental process. Because the peak levels of
sde
expression in both an
sde
+
and an
sde
mutant background were similar, we conclude that the
sde
locus is not autoregulated. Characterization of the
sde
locus by DNA sequence analysis indicated that the Ω4408 insertion occurred within the
sdeK
gene. Primer extension analyses localized the 5′ end of
sde
transcript to a guanine nucleotide 307 bp upstream of the proposed start for the SdeK coding sequence. The DNA sequence in the −12 and −24 regions upstream of the
sde
transcriptional start site shows similarity to the ς
54
family of promoters. The results of complementation studies suggest that the defects in development and sporulation caused by the Ω4408 insertion are due to an inactivation of
sdeK
. The predicted amino acid sequence of SdeK was found to have similarity to the sequences of the histidine protein kinases of two-component regulatory systems. Based on our results, we propose that SdeK may be part of a signal transduction pathway required for the activation and propagation of the early developmental program.
Functional Dissection of the Molybdate-Responsive Transcription Regulator, ModE, from
Escherichia coli
Authors: McNicholas Paul M. , Mazzotta Mandy M. , Rech Sabine A. , Gunsalus Robert P. 
Published: December 2019

ABSTRACT

The product of the
Escherichia coli modE
gene, ModE, is a member of a unique class of molybdate-responsive DNA binding proteins. Here we investigated the roles of the N- and C-terminal domains of ModE in mediating DNA binding and protein dimerization, respectively. Compared to the full-length protein, the N-terminal half of ModE has a greatly diminished capacity to bind the
modA
promoter in vitro and to repress expression from a
modA-lacZ
operon fusion in vivo. Fusing a protein dimerization domain, encoded by the C terminus of λ CI repressor protein, to the truncated ModE protein generated a ModE-CI fusion protein that not only displayed a greatly increased in vivo repressor activity but could also substitute for ModE at the
moaA
and
dmsA
promoters. In the reciprocal experiment, we restored repressor activity to a truncated CI protein by addition of the C-terminal domain of ModE, which is comprised of two MopI-like subdomains. By an in vivo competition assay, we also demonstrated that the CI-ModE chimeric protein retained the ability to interact with wild-type ModE. Finally, specific deletions within the ModE portion of the CI-ModE protein chimera abolished both in vivo repression and the ability to interact with wild-type ModE. Together, these data demonstrate that the N-terminal domain of ModE is sufficient to mediate DNA binding, although efficient binding requires that ModE form a dimer, a function that is supplied by the C-terminal MopI-like subdomains.
Analysis of the Exochelin Locus in
Mycobacterium smegmatis
: Biosynthesis Genes Have Homology with Genes of the Peptide Synthetase Family
Authors: Yu Shengwei , Fiss Ellen , Jacobs William R. 
Published: December 2019

ABSTRACT

Mycobacteria secrete the siderophore exochelin when grown under iron-limiting conditions. In order to understand iron uptake mechanisms in mycobacteria, we have taken a genetic approach to identify those genes involved in exochelin biosynthesis and transport in
Mycobacterium smegmatis
. Of the 6,000 chemically mutagenized clones of
M. smegmatis
mc
2
155 screened on agar plates containing chrome azural S, 19 mutants that had lost the ability to produce or secrete exochelin were identified. Thirteen of these mutants were complemented by a single
M. smegmatis
cosmid. Sequence analysis of this cosmid revealed nine open reading frames, three of which are homologous to genes encoding transporter proteins, which are likely involved in exochelin transport. Complementation and Tn
10
mutagenesis analysis identified two new genes,
fxbB
and
fxbC
, which are required for exochelin biosynthesis. The
fxbB
and
fxbC
genes encode large proteins of 257 and 497 kDa, respectively, which are highly homologous to peptide synthetases, indicating that exochelin biosynthesis occurs by a nonribosomal mechanism.
Mutations in
toxR
and
toxS
That Separate Transcriptional Activation from DNA Binding at the Cholera Toxin Gene Promoter
Authors: Pfau James D. , Taylor Ronald K. 
Published: December 2019

ABSTRACT

ToxR and ToxS are integral membrane proteins that activate the transcription of virulence genes in
Vibrio cholerae
. ToxR can be separated into three different domains: an N-terminal cytoplasmic DNA binding domain, a central transmembrane domain, and a C-terminal periplasmic domain. ToxS is thought to enhance ToxR-mediated transcriptional activation through a periplasmic interaction. By P22 challenge phage selection for DNA binding, in combination with a screen for cholera toxin gene transcription, 12
toxR
and
toxS
positive control mutants producing variant ToxR proteins from the
toxRS
operon that bind to the cholera toxin promoter but that fail to activate transcription were isolated. One mutation in
toxR
specifies an E82K change in the predicted helix-loop-helix DNA binding domain and destroys ToxR-mediated activation. Seven
toxR
mutations included frameshifts and stop codons introduced into the periplasmic domain, and six of these mutations appeared to produce proteolytically processed shorter forms of ToxR, suggesting that even short periplasmic deletions alter the folding of ToxR in the periplasm. Deletion of
toxS
did not alter the steady-state level of ToxR, and ToxR was found to be capable of binding to DNA in the absence of ToxS even though it did not activate transcription. However, the ToxS L33S variant rendered ToxR susceptible to proteolysis, suggesting that the natural function of ToxS is to complex with ToxR. Therefore, certain alterations that map to the ToxR cytoplasmic DNA binding domain, to the periplasmic domain, or to ToxS separate DNA binding activity from activator function. These data support a model where proper assembly or stability of the periplasmic domain of ToxR is enhanced by ToxS. This chaperone-like activity of ToxS may be required for the formation of the transcriptional activation complex but not the ToxR-DNA complex.
Mutational Inactivation of a Gene Homologous to
Escherichia coli ptsP
Affects Poly-β-Hydroxybutyrate Accumulation and Nitrogen Fixation in
Azotobacter vinelandii
Authors: Segura Daniel , Espín Guadalupe 
Published: December 2019

ABSTRACT

Strain DS988, an
Azotobacter vinelandii
mutant with a reduced capacity to accumulate poly-β-hydroxybutyrate, was isolated after mini-Tn
5
mutagenesis of the UW136 strain. Cloning and nucleotide sequencing of the affected locus revealed a gene homologous to
Escherichia coli ptsP
which encodes enzyme I
Ntr
, a homologue of enzyme I of the phosphoenol pyruvate-sugar phosphotransferase system with an N-terminal domain similar to the N-terminal domain of some NifA proteins. Strain DS988 was unable to grow diazotrophically with 10 mM glucose as a carbon source. Diazotrophic growth on alternative carbon sources such as gluconate was only slightly affected. Glucose uptake, as well as glucose kinase and glucose-6-phosphate-dehydrogenase activities that lead to the synthesis of gluconate-6-phosphate, were not affected by the
ptsP
mutation. The inability of DS988 to grow diazotrophically in 10 mM glucose was overcome by supplying ammonium or other sources of fixed nitrogen. Acetylene reduction activity but not transcription of the nitrogenase structural gene
nifH
was shown to be impaired in strain DS988 when it was incubated in 10 mM glucose. The diazotrophic growth defect of DS988 was restored either by increasing the glucose concentration to above 20 mM or by lowering the oxygen concentration. These data suggest that a mutation in
ptsP
leads to a failure in poly-β-hydroxybutyrate metabolism and in the respiratory protection of nitrogenase under carbon-limiting conditions.
A Natural Large Chromosomal Inversion in
Lactococcus lactis
Is Mediated by Homologous Recombination between Two Insertion Sequences
Authors: Daveran-Mingot Marie-Line , Campo Nathalie , Ritzenthaler Paul , Le Bourgeois Pascal 
Published: December 2019

ABSTRACT

Comparative analysis of chromosomal macrorestriction polymorphism of the two closely related
Lactococcus lactis
subsp.
cremoris
strains MG1363 and NCDO763 revealed the presence of a large inversion covering half of the genome. To determine what kind of genetic element could be implicated in this rearrangement, the two inversion junctions of MG1363 and NCDO763 chromosomes were cloned and characterized. Nucleotide sequence analysis showed the presence of one copy of the lactococcal IS
905
element in each junction. Each copy of this element contained the same nucleotide mutation that inactivates the putative transposase. Comparison of the sequences surrounding the insertion sequence demonstrated that the large inversion arose from a single-step homologous recombination event between the two defective copies of the IS
905
element. The large inversion presumably conferred no selective disadvantage on strain NCDO763 because this rearrangement did not alter the
oriC-terC
symmetry of the chromosome and the local genetic environment.
The
ggpS
Gene from
Synechocystis
sp. Strain PCC 6803 Encoding Glucosyl-Glycerol-Phosphate Synthase Is Involved in Osmolyte Synthesis
Authors: Marin Kay , Zuther Ellen , Kerstan Thomas , Kunert Anja , Hagemann Martin 
Published: December 2019

ABSTRACT

A salt-sensitive mutant of
Synechocystis
sp. strain PCC 6803 defective in the synthesis of the compatible solute glucosylglycerol (GG) was used to search for the gene encoding GG-phosphate synthase (GGPS), the key enzyme in GG synthesis. Cloning and sequencing of the mutated region and the corresponding wild-type region revealed that a deletion of about 13 kb occurred in the genome of mutant 11. This deletion affected at least 10 open reading frames, among them regions coding for proteins showing similarities to trehalose (
otsA
homolog)- and glycerol-3-phosphate-synthesizing enzymes. After construction and characterization of mutants defective in these genes, it became obvious that an
otsA
homolog (
sll
1566) (T. Kaneko et al., DNA Res. 3:109–136, 1996) encodes GGPS, since only the mutant affected in
sll
1566 showed salt sensitivity combined with a complete absence of GG accumulation. Furthermore, the overexpression of
sll
1566 in
Escherichia coli
led to the appearance of GGPS activity in the heterologous host. The overexpressed protein did not show the salt dependence that is characteristic for the GGPS in crude protein extracts of
Synechocystis
.
Oxidative Stress Response and Characterization of theoxyR-ahpCandfurA-katGLoci inMycobacterium marinum
Authors: Pagán-Ramos E. , Song J. , McFalone M. , Mudd M. H. , Deretic V. 
Published: December 2019

ABSTRACTOxidative stress response in pathogenic mycobacteria is believed to be of significance for host-pathogen interactions at various stages of infection. It also plays a role in determining the intrinsic susceptibility to isoniazid in mycobacterial species. In this work, we characterized theoxyR-ahpCandfurA-katGloci in the nontuberculous pathogenMycobacterium marinum. In contrast toMycobacterium smegmatisand likeMycobacterium tuberculosisandMycobacterium leprae,M. marinumwas shown to possess a closely linked and divergently oriented equivalents of the regulator of peroxide stress responseoxyRand its subordinate geneahpC, encoding a homolog of alkyl hydroperoxide reductase. Purified mycobacterial OxyR was found to bind to theoxyR-ahpCpromoter region fromM. marinumand additional mycobacterial species. Mobility shift DNA binding analyses using OxyR binding sites from several mycobacteria and a panel of in vitro-generated mutants validated the proposed consensus mycobacterial recognition sequence.M. marinumAhpC levels detected by immunoblotting, were increased upon treatment with H2O2, in keeping with the presence of a functional OxyR and its binding site within the promoter region ofahpC. In contrast, OxyR did not bind to the sequences upstream of thekatGstructural gene, andkatGexpression did not follow the pattern seen withahpC. Instead, a new open reading frame encoding a homolog of the ferric uptake regulator Fur was identified immediately upstream ofkatGinM. marinum. ThefurA-katGlinkage and arrangement are ubiquitous in mycobacteria, suggesting the presence of additional regulators of oxidative stress response and potentially explaining the observed differences inahpCandkatGexpression. Collectively, these findings broaden our understanding of oxidative stress response in mycobacteria. They also suggest thatM. marinumwill be useful as a model system for studying the role of oxidative stress response in mycobacterial physiology, intracellular survival, and other host-pathogen interactions associated with mycobacterial diseases.
Spore Photoproduct Lyase from
Bacillus subtilis
Spores Is a Novel Iron-Sulfur DNA Repair Enzyme Which Shares Features with Proteins such as Class III Anaerobic Ribonucleotide Reductases and Pyruvate-Formate Lyases
Authors: Rebeil Roberto , Sun Yubo , Chooback Lilian , Pedraza-Reyes Mario , Kinsland Cynthia , Begley Tadhg P. , Nicholson Wayne L. 
Published: December 2019

ABSTRACT

The major photoproduct in UV-irradiated spore DNA is the unique thymine dimer 5-thyminyl-5,6-dihydrothymine, commonly referred to as spore photoproduct (SP). An important determinant of the high UV resistance of
Bacillus subtilis
spores is the accurate in situ reversal of SP during spore germination by the DNA repair enzyme SP lyase. To study the molecular aspects of SP lyase-mediated SP repair, the cloned
B. subtilis splB
gene was engineered to encode SP lyase with a molecular tag of six histidine residues at its amino terminus. The engineered six-His-tagged SP lyase expressed from the
amyE
locus restored UV resistance to spores of a UV-sensitive mutant
B. subtilis
strain carrying a deletion-insertion mutation which removed the entire
splAB
operon at its natural locus and was shown to repair SP in vivo during spore germination. The engineered SP lyase was purified both from dormant
B. subtilis
spores and from an
Escherichia coli
overexpression system by nickel-nitrilotriacetic acid (NTA) agarose affinity chromatography and was shown by Western blotting, UV-visible spectroscopy, and iron and acid-labile sulfide analysis to be a 41-kDa iron-sulfur (Fe-S) protein, consistent with its amino acid sequence homology to the 4Fe-4S clusters in anaerobic ribonucleotide reductases and pyruvate-formate lyases. SP lyase was capable of reversing SP from purified SP-containing DNA in an in vitro reaction either when present in a cell-free extract prepared from dormant spores or after purification on nickel-NTA agarose. SP lyase activity was dependent upon reducing conditions and addition of
S
-adenosylmethionine as a cofactor.
Two Functionally Distinct Regions Upstream of the

cbb
I

Operon of
Rhodobacter sphaeroides
Regulate Gene Expression
Authors: Dubbs James M. , Tabita F. Robert 
Published: December 2019

ABSTRACT

A number of

cbbF
I

::
lacZ
translational fusion plasmids containing various lengths of sequence 5′ to the form I (

cbb
I

) Calvin-Benson-Bassham cycle operon (

cbbF
I
cbbP
I
cbbA
I
cbbL
I
cbbS
I

) of
Rhodobacter sphaeroides
were constructed. Expression of β-galactosidase was monitored under a variety of growth conditions. It was found that 103 bp of sequence upstream of the

cbbF
I

transcription start was sufficient to confer low levels of regulated

cbb
I

promoter expression; this activity was dependent on the presence of an intact
cbbR
gene. Additionally,
R. sphaeroides
CbbR was shown to bind to the region between 9 and 100 bp 5′ to the

cbbF
I

transcription start. Inclusion of an additional upstream sequence, from 280 to 636 bp 5′ to

cbbF
I

, resulted in a significant increase in regulated

cbb
I

promoter expression under all growth conditions tested. A 50-bp region responsible for the majority of this increase occurs between 280 and 330 bp 5′ to

cbbF
I

. The additional 306 bp of upstream sequence from 330 to 636 bp also appears to play a positive regulatory role. A 4-bp deletion 281 to 284 bp 5′ to

cbbF
I

significantly reduced

cbb
I

expression while the proper regulatory pattern was retained. These studies provide evidence for the presence of two functionally distinct regions of the

cbb
I

promoter, with the distal domain providing significant regulated promoter activity that adheres to the normal pattern of expression.
Transcriptional Analysis and Mutation of a
dnaA
-Like Gene in
Synechocystis
sp. Strain PCC 6803
Authors: Richter Stefan , Hagemann Martin , Messer Walter 
Published: December 2019

ABSTRACT

Transcription of the
dnaA
gene of the cyanobacterium
Synechocystis
sp. strain PCC 6803 is light dependent and yields a monocistronic mRNA, as determined by Northern analysis. Surprisingly, mutants with inactivated
dnaA
were viable. In batch cultures under standard conditions, the mutants grew like the wild type and did not show an aberrant phenotype. We conclude that, unlike the situation in other bacteria,
dnaA
of
Synechocystis
sp. cannot have an essential function, such as initiation of DNA replication.