bluuebunny/crossref_metadata_2025_split huggingface dataset:
cis
-Acting Sequences Required for NtcB-Dependent, Nitrite-Responsive Positive Regulation of the Nitrate Assimilation Operon in the Cyanobacterium
Synechococcus
sp. Strain PCC 7942
Authors: Maeda Shin-Ichi , Kawaguchi Yuriko , Ohe Taka-Aki , Omata Tatsuo 
Published: December 2019

ABSTRACT

There are three binding sites for NtcA (
nir
I,
nir
II, and
nir
III), the global nitrogen regulator of cyanobacteria, in the DNA region between the two divergently transcribed operons (
nirA
and
nirB
operons) involved in nitrate assimilation in
Synechococcus
sp. strain PCC 7942. Using the
luxAB
reporter system, we showed that
nir
I and
nir
III, which are located 23 bp upstream from the −10 promoter element of
nirA
and
nirB
, respectively, are required for induction by nitrogen depletion of the
nirA
and
nirB
operons, respectively. The induction of
nirA
operon transcription was a prerequisite for the nitrite-responsive positive regulation of the transcription by NtcB, a LysR-type protein. The NtcA-binding site
nir
II, located in the middle of the
nirA-nirB
intergenic region, and a potential binding site for a LysR-type protein (TGCAN
5
TGCA; designated L1), located between
nir
I and
nir
II, were required for the nitrite-responsive, NtcB-dependent enhancement of
nirA
operon transcription. Although the requirement for the L1 site was consistent with the involvement of the LysR family protein NtcB in transcriptional regulation, NtcB did not bind to the
nirA
regulatory region in vitro in the presence of nitrite and NtcA, suggesting the involvement of some additional factor(s) in the regulation. An L1-like inverted repeat with the consensus sequence TGCN
7
GCA was conserved in the
nirA
promoter region of cyanobacteria, being centered at position −23 with respect to the NtcA-binding site corresponding to
nir
I, which suggested the common occurrence of nitrite-responsive regulation of the nitrate assimilation operon among cyanobacteria.
The Tip of the Hydrophobic Hairpin of Colicin U Is Dispensable for Colicin U Activity but Is Important for Interaction with the Immunity Protein
Authors: Pilsl Holger , Smajs David , Braun Volkmar 
Published: December 2019

ABSTRACT

The hydrophobic C terminus of pore-forming colicins associates with and inserts into the cytoplasmic membrane and is the target of the respective immunity protein. The hydrophobic region of colicin U of
Shigella boydii
was mutated to identify determinants responsible for recognition of colicin U by the colicin U immunity protein. Deletion of the tip of the hydrophobic hairpin of colicin U resulted in a fully active colicin that was no longer inactivated by the colicin U immunity protein. Replacement of eight amino acids at the tip of the colicin U hairpin by the corresponding amino acids of the related colicin B resulted in colicin U(575–582ColB), which was inactivated by the colicin U immunity protein to 10% of the level of inactivation of the wild-type colicin U. The colicin B immunity protein inactivated colicin U(575–582ColB) to the same degree. These results indicate that the tip of the hydrophobic hairpin of colicin U and of colicin B mainly determines the interaction with the corresponding immunity proteins and is not required for colicin activity. Comparison of these results with published data suggests that interhelical loops and not membrane helices of pore-forming colicins mainly interact with the cognate immunity proteins and that the loops are located in different regions of the A-type and E1-type colicins. The colicin U immunity protein forms four transmembrane segments in the cytoplasmic membrane, and the N and C termini face the cytoplasm.
The Cyanobacterium
Synechocystis
sp. Strain PCC 6803 Expresses a DNA Methyltransferase Specific for the Recognition Sequence of the Restriction Endonuclease
Pvu
I
Authors: Scharnagl Matthias , Richter Stefan , Hagemann Martin 
Published: December 2019

ABSTRACT

By use of restriction endonucleases, the DNA of the cyanobacterium
Synechocystis
sp. strain PCC 6803 was analyzed for DNA-specific methylation. Three different recognition sites of methyltransferases, a
dam
-like site including N
6
-methyladenosine and two other sites with methylcytosine, were identified, whereas no activities of restriction endonucleases could be detected in this strain.
slr
0214, a
Synechocystis
gene encoding a putative methyltransferase that shows significant similarities to C
5
-methylcytosine-synthesizing enzymes, was amplified by PCR and cloned for further characterization. Mutations in
slr
0214 were generated by the insertion of an
aph
II gene cassette. Analyses of chromosomal DNAs of such mutants demonstrated that the methylation pattern was changed. The recognition sequence of the methyltransferase was identified as 5′-CGATCG-3′, corresponding to the recognition sequence of
Pvu
I. The specific methyltransferase activity was significantly reduced in protein extracts obtained from mutant cells. Mutation of
slr
0214 also led to changed growth characteristics of the cells compared to wild-type cells. These alterations led to the conclusion that the methyltransferase Slr0214 might play a regulatory role in
Synechocystis
. The Slr0214 protein was also overexpressed in
Escherichia coli
, and the purified protein demonstrated methyltransferase activity and specificity for
Pvu
I recognition sequences in vitro. We propose the designation
Syn
MI (
Synechocystis
methyltransferase I) for the
slr
0214-encoded enzyme.
A Mutation in a Purported Regulatory Gene Affects Control of Sterol Uptake in
Saccharomyces cerevisiae
Authors: Crowley James H. , Leak Frank W. , Shianna Kevin V. , Tove Shirley , Parks Leo W. 
Published: December 2019

ABSTRACT

Aerobically growing wild-type strains of
Saccharomyces cerevisiae
are unable to take exogenously supplied sterols from media. This aerobic sterol exclusion is vitiated under anaerobic conditions, in heme-deficient strains, and under some conditions of impaired sterol synthesis. Mutants which can take up sterols aerobically in heme-competent cells have been selected. One of these mutations, designated
upc2-1
, gives a pleiotropic phenotype in characteristics as diverse as aerobic accumulation of sterols, total lipid storage, sensitivity to metabolic inhibitors, response to altered sterol structures, and cation requirements. During experiments designed to ascertain the effects of various cations on yeast with sterol alterations, it was observed that
upc2-1
was hypersensitive to Ca
2+
. Using resistance to Ca
2+
as a screening vehicle, we cloned
UPC2
and showed that it is YDR213W, an open reading frame on chromosome IV. This belongs to a fungal regulatory family containing the Zn(II)2Cys6 binuclear cluster DNA binding domain. The single guanine-to-adenine transition in
upc2-1
gives a predicted amino acid change from glycine to aspartic acid. The regulatory defect explains the semidominance and pleiotropic effects of
upc2-1.
Fnr, NarP, and NarL Regulation of
Escherichia coli
K-12
napF
(Periplasmic Nitrate Reductase) Operon Transcription In Vitro
Authors: Darwin Andrew J. , Ziegelhoffer Eva C. , Kiley Patricia J. , Stewart Valley 
Published: December 2019

ABSTRACT

The expression of several
Escherichia coli
operons is activated by the Fnr protein during anaerobic growth and is further controlled in response to nitrate and nitrite by the homologous response regulators, NarL and NarP. Among these operons, the
napF
operon, encoding a periplasmic nitrate reductase, has unique features with respect to its Fnr-, NarL-, and NarP-dependent regulation. First, the Fnr-binding site is unusually located compared to the control regions of most other Fnr-activated operons, suggesting different Fnr-RNA polymerase contacts during transcriptional activation. Second, nitrate and nitrite activation is solely dependent on NarP but is antagonized by the NarL protein. In this study, we used DNase I footprint analysis to confirm our previous assignment of the unusual location of the Fnr-binding site in the
napF
control region. In addition, the in vivo effects of Fnr-positive control mutations on
napF
operon expression indicate that the
napF
promoter is atypical with respect to Fnr-mediated activation. The transcriptional regulation of
napF
was successfully reproduced in vitro by using a supercoiled plasmid template and purified Fnr, NarL, and NarP proteins. These in vitro transcription experiments demonstrate that, in the presence of Fnr, the NarP protein causes efficient transcription activation whereas the NarL protein does not. This suggests that Fnr and NarP may act synergistically to activate
napF
operon expression. As observed in vivo, this activation by Fnr and NarP is antagonized by the addition of NarL in vitro.
DNA Bending by AraC: a Negative Mutant
Authors: Saviola Beatrice , Seabold Robert R. , Schleif Robert F. 
Published: December 2019

ABSTRACT

We sought a mutation in the DNA binding domain of the arabinose operon regulatory protein, AraC, of
Escherichia coli
that allows the protein to bind DNA normally but not activate transcription. The mutation was isolated by mutagenizing a plasmid overproducing a chimeric leucine zipper-AraC DNA binding domain and screening for proteins that were
trans
dominant negative with regard to wild-type AraC protein. The mutant with the lowest transcription activation of the
araBAD
promoter was studied further. It proved to alter a residue that had previously been demonstrated to contact DNA. Because the overproduced mutant protein still bound DNA in vivo, it is deficient in transcription activation for some reason other than absence of DNA binding. Using the phase-sensitive DNA bending assay, we found that wild-type AraC bends DNA about 90° whereas the mutant bends DNA by a smaller amount.
Arginine Catabolism and the Arginine Succinyltransferase Pathway in
Escherichia coli
Authors: Schneider Barbara L. , Kiupakis Alexandros K. , Reitzer Lawrence J. 
Published: December 2019

ABSTRACT

Arginine catabolism produces ammonia without transferring nitrogen to another compound, yet the only known pathway of arginine catabolism in
Escherichia coli
(through arginine decarboxylase) does not produce ammonia. Our aims were to find the ammonia-producing pathway of arginine catabolism in
E. coli
and to examine its function. We showed that the only previously described pathway of arginine catabolism, which does not produce ammonia, accounted for only 3% of the arginine consumed. A search for another arginine catabolic pathway led to discovery of the ammonia-producing arginine succinyltransferase (AST) pathway in
E. coli
. Nitrogen limitation induced this pathway in both
E. coli
and
Klebsiella aerogenes
, but the mechanisms of activation clearly differed in these two organisms. We identified the
E. coli
gene for succinylornithine aminotransferase, the third enzyme of the AST pathway, which appears to be the first of an
astCADBE
operon. Its disruption prevented arginine catabolism, impaired ornithine utilization, and affected the synthesis of all the enzymes of the AST pathway. Disruption of
astB
eliminated succinylarginine dihydrolase activity and prevented arginine utilization but did not impair ornithine catabolism. Overproduction of AST enzymes resulted in faster growth with arginine and aspartate. We conclude that the AST pathway is necessary for aerobic arginine catabolism in
E. coli
and that at least one enzyme of this pathway contributes to ornithine catabolism.
Pas, a Novel Protein Required for Protein Secretion and Attaching and Effacing Activities of Enterohemorrhagic
Escherichia coli
Authors: Kresse Andreas U. , Schulze Kai , Deibel Christina , Ebel Frank , Rohde Manfred , Chakraborty Trinad , Guzmán Carlos A. 
Published: December 2019

ABSTRACT

Enterohemorrhagic
Escherichia coli
(EHEC) exhibits a pattern of localized adherence to host cells, with the formation of microcolonies, and induces a specific histopathological phenotype collectively known as the attaching and effacing lesion. The genes encoding the products responsible for this phenotype are located on a 35-kb pathogenicity island designated the locus of enterocyte effacement, which is also shared by enteropathogenic
E. coli
. We have identified an open reading frame (ORF) which is located upstream of the
espA
,
espB
, and
espD
genes on the complementary strand and which exhibits high homology to the genes
spiB
from
Salmonella
,
yscD
from
Yersinia
, and
pscD
from
Pseudomonas
. Localization studies showed that the encoded product is present in the cytoplasmic and inner membrane fractions of EHEC. The construction and characterization of a recombinant clone containing an in-frame deletion of this ORF demonstrated that the encoded product is a putative member of a type III system required for protein secretion. Disruption of this ORF, designated
pas
(protein associated with secretion), abolished the secretion of Esp proteins. The mutant adhered only poorly and lost its capacities to trigger attaching and effacing activity and to invade HeLa cells. These results demonstrate that Pas is a virulence-associated factor that plays an essential role in EHEC pathogenesis.
Denitrification by Actinomycetes and Purification of Dissimilatory Nitrite Reductase and Azurin from
Streptomyces thioluteus
Authors: Shoun Hirofumi , Kano Mitsuyoshi , Baba Ikuko , Takaya Naoki , Matsuo Masaru 
Published: December 2019

ABSTRACT

Many actinomycete strains are able to convert nitrate or nitrite to nitrous oxide (N
2
O). As a representative of actinomycete denitrification systems, the system of
Streptomyces thioluteus
was investigated in detail.
S. thioluteus
attained distinct cell growth upon anaerobic incubation with nitrate or nitrite with concomitant and stoichiometric conversion of nitrate or nitrite to N
2
O, suggesting that the denitrification acts as anaerobic respiration. Furthermore, a copper-containing, dissimilatory nitrite reductase (CuNir) and its physiological electron donor, azurin, were isolated. This is the first report to show that denitrification generally occurs among actinomycetes.
Genetic Linkage and Cotransfer of a Novel,
vanB
-Containing Transposon (Tn
5382
) and a Low-Affinity Penicillin-Binding Protein 5 Gene in a Clinical Vancomycin-Resistant
Enterococcus faecium
Isolate
Authors: Carias Lenore L. , Rudin Susan D. , Donskey Curtis J. , Rice Louis B. 
Published: December 2019

ABSTRACT

Mechanisms for the intercellular transfer of VanB-type vancomycin resistance determinants and for the almost universal association of these determinants with those for high-level ampicillin resistance remain poorly defined. We report the discovery of Tn
5382
, a ca. 27-kb putative transposon encoding VanB-type glycopeptide resistance in
Enterococcus faecium
. Open reading frames internal to the right end of Tn
5382
and downstream of the
vanX
B
dipeptidase gene exhibit significant homology to genes encoding the excisase and integrase of conjugative transposon Tn
916
. The ends of Tn
5382
are also homologous to the ends of Tn
916
, especially in regions bound by the integrase enzyme. PCR amplification experiments indicate that Tn
5382
excises to form a circular intermediate in
E. faecium
. Integration of Tn
5382
in the chromosome of
E. faecium
C68 has occurred 113 bp downstream of the stop codon for the
pbp5
gene, which encodes high-level ampicillin resistance in this clinical isolate. Transfer of vancomycin, ampicillin, and tetracycline resistance from C68 to an
E. faecium
recipient strain occurs at low frequency in vitro and is associated with acquisition of a 130- to 160-kb segment of DNA that contains Tn
5382
, the
pbp5
gene, and its putative repressor gene,
psr
. The interenterococcal transfer of this large chromosomal element appears to be the primary mechanism for
vanB
operon spread in northeast Ohio. These results expand the known family of Tn
916
-related transposons, suggest a mechanism for
vanB
operon entry into and dissemination among enterococci, and provide an explanation for the nearly universal association of vancomycin and high-level ampicillin resistance in clinical
E. faecium
strains.
The
Pseudomonas aeruginosa rhlG
Gene Encodes an NADPH-Dependent β-Ketoacyl Reductase Which Is Specifically Involved in Rhamnolipid Synthesis
Authors: Campos-García Jesús , Caro Alma Delia , Nájera Rebeca , Miller-Maier Raina M. , Al-Tahhan Ragheb A. , Soberón-Chávez Gloria 
Published: December 2019

ABSTRACT

A
Pseudomonas aeruginosa
gene homologous to the
fabG
gene, which encodes the NADPH-dependent β-ketoacyl-acyl carrier protein (ACP) reductase required for fatty acid synthesis, was identified. The insertional mutation of this
fabG
homolog (herein called
rhlG
) produced no apparent effect on the growth rate and total lipid content of
P. aeruginosa
cells, but the production of rhamnolipids was completely abrogated. These results suggest that the synthetic pathway for the fatty acid moiety of rhamnolipids is separate from the general fatty acid synthetic pathway, starting with a specific ketoacyl reduction step catalyzed by the RhlG protein. In addition, the synthesis of poly-β-hydroxyalkanoate (PHA) is delayed in this mutant, suggesting that RhlG participates in PHA synthesis, although it is not the only reductase involved in this pathway. Traits regulated by the quorum-sensing response, other than rhamnolipid production, including production of proteases, pyocyanine, and the autoinducer butanoyl-homoserine lactone (PAI-2), were not affected by the
rhlG
mutation. We conclude that the
P. aeruginosa rhlG
gene encodes an NADPH-dependent β-ketoacyl reductase absolutely required for the synthesis of the β-hydroxy acid moiety of rhamnolipids and that it has a minor role in PHA production. Expression of
rhlG
mRNA under different culture conditions is consistent with this conclusion.
Promoter Substitution and Deletion Analysis of Upstream Region Required for
rpoS
Translational Regulation
Authors: Cunning Christofer , Brown Larissa , Elliott Thomas 
Published: December 2019

ABSTRACT

The RpoS sigma factor of enteric bacteria is required for the increased expression of a number of genes that are induced during nutrient limitation and growth into stationary phase and in response to high osmolarity. RpoS is also a virulence factor for several pathogenic species, including
Salmonella typhimurium
. The activity of RpoS is regulated at both the level of synthesis and protein turnover. Here we investigate the posttranscriptional control of RpoS synthesis by using
rpoS-lac
protein and operon fusions. Substitution of the native
rpoS
promoters with the
tac
or
lac
UV5 promoters allowed essentially normal regulation after growth into stationary phase in rich medium or after osmotic challenge. Regulation of these fusions required the function of
hfq
, encoding the RNA-binding protein host factor I (HF-I). Short deletions from the 5′ end of the
rpoS
transcript did not affect regulation very much; however, a larger deletion mutation that still retains 220 bp upstream of the
rpoS
ATG codon, including a proposed antisense element inhibitory for
rpoS
translation, was no longer regulated by HF-I. Several models for regulation of
rpoS
expression by HF-I are discussed.
Intraspecific Variation of Unusual Phospholipids from
Corynebacterium
spp. Containing a Novel Fatty Acid
Authors: Niepel Tanja , Meyer Holger , Wray Victor , Abraham Wolf-Rainer 
Published: December 2019

ABSTRACT

The novel fatty acid
trans
-9-methyl-10-octadecenoic acid was isolated from the coryneform bacterial strain LMG 3820 (previously misidentified as
Arthrobacter globiformis
) and identified by spectroscopic methods and chemical derivatization. This fatty acid is attached to the unusual lipid acyl phosphatidylglycerol. Five different species of this lipid type were identified; their structures were elucidated by tandem mass spectrometry and are reported here for the first time. Additionally, we identified three different cardiolipins, two bearing the novel fatty acid. The characteristic 10-methyl-octadecanoic acid was present only in phosphatidylinositol. Because of the unusual fatty acid pattern of strain LMG 3820, the 16S rDNA sequence was determined and showed regions of identity to sequences of
Corynebacterium variabilis
DSM 20132
T
and DSM 20536. All three strains possessed the novel fatty acid, identifying
trans
-9-methyl-10-octadecenoic acid as a potential biomarker characteristic for this taxon. Surprisingly, the fatty acid and relative abundances of phospholipids of
Corynebacterium
sp. strain LMG 3820 were similar to those of the type strain but different from those of
Corynebacterium variabilis
DSM 20536, although all three strains possessed identical 16S rDNA sequences and strains DSM 20132
T
and DSM 20536 have 90.5% DNA-DNA homology. This is one of the rare cases wherein different organisms with identical 16S rDNA sequences have been observed to present recognizably different fatty acid and lipid compositions. Since methylation of a fatty acid considerably lowers the transition temperature of the corresponding lipid resulting in a more flexible cell membrane, the intraspecific variation in the lipid composition, coinciding with the morphological and Gram stain reaction variability of this species, probably offers an advantage for this species to inhabit different environmental niches.
Novel
Escherichia coli umuD
′ Mutants: Structure-Function Insights into SOS Mutagenesis
Authors: McLenigan Mary , Peat Thomas S. , Frank Ekaterina G. , McDonald John P. , Gonzalez Martín , Levine Arthur S. , Hendrickson Wayne A. , Woodgate Roger 
Published: December 2019

ABSTRACT

Although it has been 10 years since the discovery that the
Escherichia coli
UmuD protein undergoes a RecA-mediated cleavage reaction to generate mutagenically active UmuD′, the function of UmuD′ has yet to be determined. In an attempt to elucidate the role of UmuD′ in SOS mutagenesis, we have utilized a colorimetric papillation assay to screen for mutants of a hydroxylamine-treated, low-copy-number
umuD
′ plasmid that are unable to promote SOS-dependent spontaneous mutagenesis. Using such an approach, we have identified 14 independent
umuD
′ mutants. Analysis of these mutants revealed that two resulted from promoter changes which reduced the expression of wild-type UmuD′, three were nonsense mutations that resulted in a truncated UmuD′ protein, and the remaining nine were missense alterations. In addition to the hydroxylamine-generated mutants, we have subcloned the mutations found in three chromosomal
umuD1
,
umuD44
, and
umuD77
alleles into
umuD
′. All 17
umuD
′ mutants resulted in lower levels of SOS-dependent spontaneous mutagenesis but varied in the extent to which they promoted methyl methanesulfonate-induced mutagenesis. We have attempted to correlate these phenotypes with the potential effect of each mutation on the recently described structure of UmuD′.