bluuebunny/crossref_metadata_2025_split huggingface dataset:
Specific Contacts between Residues in the DNA-Binding Domain of the TyrR Protein and Bases in the Operator of the
tyrP
Gene of
Escherichia coli
Authors: Hwang J. S. , Yang J. , Pittard A. J. 
Published: December 2019

ABSTRACT

In the presence of tyrosine, the TyrR protein of
Escherichia coli
represses the expression of the
tyrP
gene by binding to the double TyrR boxes which overlap the promoter. Previously, we have carried out methylation, uracil, and ethylation interference experiments and have identified both guanine and thymine bases and phosphates within the TyrR box sequences that are contacted by the TyrR protein (J. S. Hwang, J. Yang, and A. J. Pittard, J. Bacteriol. 179:1051–1058, 1997). In this study, we have used missing contact probing to test the involvement of all of the bases within the
tyrP
operator in the binding of TyrR. Our results indicate that nearly all the bases within the palindromic arms of the strong and weak boxes are important for the binding of the TyrR protein. Two alanine-substituted mutant TyrR proteins, HA494 and TA495, were purified, and their binding affinities for the
tyrP
operator were measured by a gel shift assay. HA494 was shown to be completely defective in binding to the
tyrP
operator in vitro, while, in comparison with wild-Type TyrR, TA495 had only a small reduction in DNA binding. Missing contact probing was performed by using the purified TA495 protein, and the results suggest that T495 makes specific contacts with adenine and thymine bases at the ±5 positions in the TyrR boxes.
The ArgR Regulatory Protein, a Helper to the Anaerobic Regulator ANR during Transcriptional Activation of the
arcD
Promoter in
Pseudomonas aeruginosa
Authors: Lu Chung-Dar , Winteler Harald , Abdelal Ahmed , Haas Dieter 
Published: December 2019

ABSTRACT

Pseudomonas aeruginosa
, when deprived of oxygen, generates ATP from arginine catabolism by enzymes of the arginine deiminase pathway, encoded by the
arcDABC
operon. Under conditions of low oxygen tension, the transcriptional activator ANR binds to a site centered 41.5 bp upstream of the
arcD
transcriptional start. ANR-mediated anaerobic induction was enhanced two- to threefold by extracellular arginine. This arginine effect depended, in
trans
, on the transcriptional regulator ArgR and, in
cis
, on an ArgR binding site centered at −73.5 bp in the
arcD
promoter. Binding of purified ArgR protein to this site was demonstrated by electrophoretic mobility shift assays and DNase I footprinting. This ArgR recognition site contained a sequence, 5′-TGACGC-3′, which deviated in only 1 base from the common sequence motif 5′-TGTCGC-3′ found in other ArgR binding sites of
P. aeruginosa
. Furthermore, an alignment of all known ArgR binding sites confirmed that they consist of two directly repeated half-sites. In the absence of ANR, arginine did not induce the
arc
operon, suggesting that ArgR alone does not activate the
arcD
promoter. According to a model proposed, ArgR makes physical contact with ANR and thereby facilitates initiation of
arc
transcription.
Isolation of
Helicobacter pylori
Genes That Modulate Urease Activity
Authors: McGee David J. , May Carrie A. , Garner Rachel M. , Himpsl Janette M. , Mobley Harry L. T. 
Published: December 2019

ABSTRACT

Helicobacter pylori
urease, a nickel-requiring metalloenzyme, hydrolyzes urea to NH
3
and CO
2
. We sought to identify
H. pylori
genes that modulate urease activity by constructing pHP8080, a plasmid which encodes both
H. pylori
urease and the NixA nickel transporter.
Escherichia coli
SE5000 and DH5α transformed with pHP8080 resulted in a high-level urease producer and a low-level urease producer, respectively. An
H. pylori
DNA library was cotransformed into SE5000 (pHP8080) and DH5α (pHP8080) and was screened for cotransformants expressing either lowered or heightened urease activity, respectively. Among the clones carrying urease-enhancing factors, 21 of 23 contained
hp0548
, a gene that potentially encodes a DNA helicase found within the
cag
pathogenicity island, and
hp0511
, a gene that potentially encodes a lipoprotein. Each of these genes, when subcloned, conferred a urease-enhancing activity in
E. coli
(pHP8080) compared with the vector control. Among clones carrying urease-decreasing factors, 11 of 13 clones contained the
flbA
(also known as
flhA
) flagellar biosynthesis/regulatory gene (
hp1041
), an
lcrD
homolog. The LcrD protein family is involved in type III secretion and flagellar secretion in pathogenic bacteria. Almost no urease activity was detected in
E. coli
(pHP8080) containing the subcloned
flbA
gene. Furthermore, there was significantly reduced synthesis of the urease structural subunits in
E. coli
(pHP8080) containing the
flbA
gene, as determined by Western blot analysis with UreA and UreB antiserum. Thus, flagellar biosynthesis and urease activity may be linked in
H. pylori
. These results suggest that
H. pylori
genes may modulate urease activity.
Accessory DNA in the Genomes of Representatives of the
Escherichia coli
Reference Collection
Authors: Hurtado Ana , Rodríguez-Valera Francisco 
Published: December 2019

ABSTRACT

Different strains of the
Escherichia coli
reference collection (ECOR) differ widely in chromosomal size. To analyze the nature of the differential gene pool carried by different strains, we have followed an approach in which random amplified polymorphic DNA (RAPD) was used to generate several PCR fragments. Those present in some but not all the strains were screened by hybridization to assess their distribution throughout the ECOR collection. Thirteen fragments with various degrees of occurrence were sequenced. Three of them corresponded to RAPD markers of widespread distribution. Of these, two were housekeeping genes shown by hybridization to be present in all the
E. coli
strains and in
Salmonella enterica
LT2; the third fragment contained a paralogous copy of
dnaK
with widespread, but not global, distribution. The other 10 RAPD markers were found in only a few strains. However, hybridization results demonstrated that four of them were actually present in a large selection of the ECOR collection (between 42 and 97% of the strains); three of these fragments contained open reading frames associated with phages or plasmids known in
E. coli
K-12. The remaining six fragments were present in only between one and four strains; of these, four fragments showed no similarity to any sequence in the databases, and the other two had low but significant similarity to a protein involved in the
Klebsiella
capsule synthesis and to RNA helicases of archaeal genomes, respectively. Their percent GC, dinucleotide content, and codon adaptation index suggested an exogenous origin by horizontal transfer. These results can be interpreted as reflecting the presence of a large pool of strain-specific genes, whose origin could be outside the species boundaries.
Azorhizobium caulinodans
P
II
and GlnK Proteins Control Nitrogen Fixation and Ammonia Assimilation
Authors: Michel-Reydellet Nathalie , Kaminski P. Alexandre 
Published: December 2019

ABSTRACT

We herein report that
Azorhizobium caulinodans
P
II
and GlnK are not necessary for glutamine synthetase (GS) adenylylation whereas both proteins are required for complete GS deadenylylation. The disruption of both
glnB
and
glnK
resulted in a high level of GS adenylylation under the condition of nitrogen fixation, leading to ammonium excretion in the free-living state. P
II
and GlnK also controlled
nif
gene expression because NifA activated
nifH
transcription and nitrogenase activity was derepressed in
glnB glnK
double mutants, but not in wild-type bacteria, grown in the presence of ammonia.
Sequence Analysis of the Four Plasmid Genes Required To Produce the Circular Peptide Antibiotic Microcin J25
Authors: Solbiati José O. , Ciaccio Mirella , Farías Ricardo N. , González-Pastor José E. , Moreno Felipe , Salomón Raúl A. 
Published: December 2019

ABSTRACT

A 4.8-kb plasmid region carrying the four genes
mcjABCD
necessary for production of and immunity to the cyclic peptide antibiotic microcin J25 (MccJ25) has been sequenced.
mcjA
encodes the primary structure of MccJ25 as a precursor endowed with an N-terminal extension of 37 amino acids. The products of
mcjB
and
mcjC
are thought to be involved in microcin maturation, which implies cleavage of McjA and head-tail linkage of the 21-residue pro-MccJ25. The predicted McjD polypeptide, which is highly similar to several ABC exporters, was found to be required for MccJ25 secretion, thus explaining its ability to confer immunity to MccJ25.
The Global Nitrogen Regulator NtcA Regulates Transcription of the Signal Transducer P
II
(GlnB) and Influences Its Phosphorylation Level in Response to Nitrogen and Carbon Supplies in the Cyanobacterium
Synechococcus
sp. Strain PCC 7942
Authors: Lee Hyun-Mi , Vázquez-Bermúdez María Félix , de Marsac Nicole Tandeau 
Published: December 2019

ABSTRACT

The P
II
protein is encoded by a unique
glnB
gene in
Synechococcus
sp. strain PCC 7942. Its expression has been analyzed in the wild type and in NtcA-null mutant cells grown under different conditions of nitrogen and carbon supply. RNA-DNA hybridization experiments revealed the presence of one transcript species 680 nucleotides long, whatever the nutrient conditions tested. A second transcript species, 620 nucleotides long, absent in the NtcA null mutant, was observed in wild-type cells that were nitrogen starved for 2 h under both high and low CO
2
and in the presence of nitrate under a high CO
2
concentration. Primer extension analysis indicated that the two transcript species are generated from two tandem promoters, a ς
70
Escherichia coli
-type promoter and an NtcA-dependent promoter, located 120 and 53 nucleotides, respectively, from the
glnB
initiation codon. The NtcA-dependent promoter is up-regulated under the conditions mentioned above, while the ς
70
E. coli
-type promoter displays constitutive levels of transcripts in the NtcA null mutant and slightly different levels in the wild-type cells, depending on the nitrogen and carbon supplies. In general, a good correlation between the amounts of the two transcript species and that of the P
II
protein was observed, as revealed by immunodetection with specific antibodies. The phosphorylation level of P
II
in the wild type is inversely correlated with nitrogen availability and directly correlated with higher CO
2
concentration. This regulation is correspondingly less stringent in the NtcA null mutant cells. In contrast, the dephosphorylation of P
II
is NtcA independent.
Membrane-Bound Division Proteins DivIB and DivIC of
Bacillus subtilis
Function Solely through Their External Domains in both Vegetative and Sporulation Division
Authors: Katis Vittorio L. , Wake R. Gerry 
Published: December 2019

ABSTRACT

The
Bacillus subtilis
membrane-bound division proteins, DivIB and DivIC, each contain a single transmembrane segment flanked by a short cytoplasmic N-terminal domain and a larger external C-terminal domain. Both proteins become localized at the division site prior to septation. Mutagenesis of both
divIB
and
divIC
was performed whereby the sequences encoding the cytoplasmic domains were replaced by the corresponding sequence of the other gene. Finally, the cytoplasmic-plus-transmembrane-encoding domain of each protein was replaced by a totally foreign sequence not involved in division, that encodes the N-terminal-plus-transmembrane domains of the
Escherichia coli
TolR protein.
B. subtilis
strains expressing the
divIB
and
divIC
hybrids, in the absence of the wild-type gene, were viable when grown under conditions in which the wild-type genes were found previously to be essential. Furthermore, these strains were able to sporulate to near normal levels. Thus, the cytoplasmic and transmembrane segments of DivIB and DivIC do not appear to have any specific functions other than to anchor these proteins correctly in the membrane. The implications of these findings are discussed.
An Operon That Confers UV Resistance by Evoking the SOS Mutagenic Response in Streptococcal Conjugative Transposon Tn
5252
Authors: Munoz-Najar Ursula , Vijayakumar Moses N. 
Published: December 2019

ABSTRACT

Streptococcus pneumoniae
Rx1 is capable of repairing lesions caused by DNA-damaging agents in an error-free manner but lacks a UV-inducible error-prone repair system due to the absence of chromosomally encoded UmuDC-like proteins. We have identified an operon-like structure 8 kb from the left end of the pneumococcal conjugative transposon Tn
5252
that confers SOS function in the host cells. DNA sequence analysis of this region revealed the presence of four open reading frames (ORFs). The deduced amino acid sequence of one of them, ORF13, which is capable of encoding a protein of 49.7 kDa, showed significant homology to UmuC, MucB, and other proteins involved in the SOS response. The carboxy-terminal region of another, ORF14, which is predicted to encode a 26-kDa polypeptide, shared similarity with UmuD- and MucA-like proteins that carry the amino acid residues recognized by the activated RecA* protein for proteolytic cleavage. The presence of plasmids carrying subcloned DNA from this region was found to restore UV-inducible mutagenic repair of chromosomal DNA in
Escherichia coli
cells defective in error-prone repair as well as in pneumococcus and
Enterococcus faecalis
UV202. Mutations within ORF13 abolished UV-induced mutagenesis but did not affect the conjugal transposition of the element.
The
Yersinia enterocolitica
Motility Master Regulatory Operon,
flhDC
, Is Required for Flagellin Production, Swimming Motility, and Swarming Motility
Authors: Young Glenn M. , Smith Michael J. , Minnich Scott A. , Miller Virginia L. 
Published: December 2019

ABSTRACT

The ability to move over and colonize surface substrata has been linked to the formation of biofilms and to the virulence of some bacterial pathogens. Results from this study show that the gastrointestinal pathogen
Yersinia enterocolitica
can migrate over and colonize surfaces by swarming motility, a form of cooperative multicellular behavior. Immunoblot analysis and electron microscopy indicated that swarming motility is dependent on the same flagellum organelle that is required for swimming motility, which occurs in fluid environments. Furthermore, motility genes such as
flgEF
,
flgMN
,
flhBA
, and
fliA
, known to be required for the production of flagella, are essential for swarming motility. To begin to investigate how environmental signals are processed and integrated by
Y. enterocolitica
to stimulate the production of flagella and regulate these two forms of cell migration, the motility master regulatory operon,
flhDC
, was cloned. Mutations within
flhDC
completely abolished swimming motility, swarming motility, and flagellin production. DNA sequence analysis revealed that this locus is similar to motility master regulatory operons of other gram-negative bacteria. Genetic complementation and functional analysis of
flhDC
indicated that it is required for the production of flagella. When
flhDC
was expressed from an inducible p
tac
promoter, flagellin production was shown to be dependent on levels of
flhDC
expression. Phenotypically, induction of the p
tac-flhDC
fusion also corresponded to increased levels of both swimming and swarming motility.
DNA Polymerase II (
polB
) Is Involved in a New DNA Repair Pathway for DNA Interstrand Cross-Links in
Escherichia coli
Authors: Berardini Mark , Foster Patricia L. , Loechler Edward L. 
Published: December 2019

ABSTRACT

DNA-DNA interstrand cross-links are the cytotoxic lesions for many chemotherapeutic agents. A plasmid with a single nitrogen mustard (HN2) interstrand cross-link (inter-HN2-pTZSV28) was constructed and transformed into
Escherichia coli
, and its replication efficiency (RE = [number of transformants from inter-HN2-pTZSV28]/[number of transformants from control]) was determined to be ∼0.6. Previous work showed that RE was high because the cross-link was repaired by a pathway involving nucleotide excision repair (NER) but not recombination. (In fact, recombination was precluded because the cells do not receive lesion-free homologous DNA.) Herein, DNA polymerase II is shown to be in this new pathway, since the replication efficiency (RE) is higher in a
polB
+
(∼0.6) than in a Δ
polB
(∼0.1) strain. Complementation with a
polB
+
-containing plasmid restores RE to wild-type levels, which corroborates this conclusion. In separate experiments,
E. coli
was treated with HN2, and the relative sensitivity to killing was found to be as follows: wild type <
polB
<
recA
<
polB recA
∼
uvrA
. Because cells deficient in either recombination (
recA
) or DNA polymerase II (
polB
) are hypersensitive to nitrogen mustard killing,
E. coli
appears to have two pathways for cross-link repair: an NER/recombination pathway (which is possible when the cross-links are formed in cells where recombination can occur because there are multiple copies of the genome) and an NER/DNA polymerase II pathway. Furthermore, these results show that some cross-links are uniquely repaired by each pathway. This represents one of the first clearly defined pathway in which DNA polymerase II plays a role in
E. coli
. It remains to be determined why this new pathway prefers DNA polymerase II and why there are two pathways to repair cross-links.
In Vitro Transcriptional Studies of the
bkd
Operon of
Pseudomonas putida
:
l
-Branched-Chain Amino Acids and
d
-Leucine Are the Inducers
Authors: Madhusudhan Kunapuli T. , Luo Jinhe , Sokatch John R. 
Published: December 2019

ABSTRACT

BkdR is the transcriptional activator of the
bkd
operon, which encodes the four proteins of the branched-chain keto acid dehydrogenase multienzyme complex of
Pseudomonas putida
. In this study, hydroxyl radical footprinting revealed that BkdR bound to only one face of DNA over the same region identified in DNase I protection assays. Deletions of even a few bases in the 5′ region of the BkdR-binding site greatly reduced transcription, confirming that the entire protected region is necessary for transcription. In vitro transcription of the
bkd
operon was obtained by using a vector containing the
bkdR-bkdA1
intergenic region plus the putative ρ-independent terminator of the
bkd
operon. Substrate DNA, BkdR, and any of the
l
-branched-chain amino acids or
d
-leucine was required for transcription. Branched-chain keto acids,
d
-valine, and
d
-isoleucine did not promote transcription. Therefore, the
l
-branched-chain amino acids and
d
-leucine are the inducers of the
bkd
operon. The concentration of
l
-valine required for half-maximal transcription was 2.8 mM, which is similar to that needed to cause half-maximal proteolysis due to a conformational change in BkdR. A model for transcriptional activation of the
bkd
operon by BkdR during enzyme induction which incorporates these results is presented.
Ribosomal −1 Frameshifting during Decoding ofBacillus subtilis cddOccurs at the Sequence CGA AAG
Authors: Mejlhede Nina , Atkins John F. , Neuhard Jan 
Published: December 2019

ABSTRACTDuring translation of theBacillus subtilis cddgene, encoding cytidine deaminase (CDA), a ribosomal −1 frameshift occurs near the stop codon, resulting in a CDA subunit extended by 13 amino acids. The frequency of the frameshift is approximately 16%, and it occurs both when thecddgene is expressed from a multicopy plasmid inEscherichia coliand when it is expressed from the chromosomal copy inB. subtilis. As a result, heterotetrameric forms of the enzyme are formed in vivo along with the dominant homotetrameric species. The different forms have approximately the same specific activity. Thecddgene was cloned in pUC19 such that thelacZ′ gene of the vector followed thecddgene in the −1 reading frame immediately after thecddstop codon. By using site-directed mutagenesis of thecdd-lacZ′ fusion, it was shown that frameshifting occurred at the sequence CGA AAG, 9 bp upstream of the in-framecddstop codon, and that it was stimulated by a Shine-Dalgarno-like sequence located 14 bp upstream of the shift site. The possible function of this frameshift in gene expression is discussed.
Synthesis of the ς
D
Protein Is Not Sufficient To Trigger Expression of Motility Functions in
Bacillus subtilis
Authors: Yang Dong-Hui , von Kalckreuth Johannes , Allmansberger Rudolf 
Published: December 2019

ABSTRACT

The gene encoding ς
D
,
sigD
, is transcribed from two promoter regions, the
fla/che
promoter region in front of the
fla/che
operon and P

sigD

directly in front of
sigD
. If ς
D
is translated from transcripts originating from P

sigD

, the cell is unable to express motility functions but synthesizes autolysins. Therefore, one function of the additional promoter is to allow the cell to express autolysins without expressing motility functions as well.
Evaluation of the Indirect Hemagglutination Assay for Diagnosis of Acute Leptospirosis
Authors: Levett Paul N. , Whittington Carol U. 
Published: December 2019

ABSTRACT
Serology plays an important role in the diagnosis of leptospirosis. Few laboratories have the resources and expertise to perform the microscopic agglutination test. There is a need for rapid and simple serological tests which facilitate the early diagnosis of leptospirosis, while antibiotic therapy may be most effective. A commercially available indirect hemagglutination assay (IHA; MRL Diagnostics, Cypress, Calif.) was evaluated with multiple serum specimens from 107 patients being investigated for leptospirosis. By using a combination of enzyme-linked immunosorbent assay (ELISA) methods for immunoglobulin M (IgM) and IgG antibodies and the microscopic agglutination test, 54 patients were found to have leptospirosis and 53 were found not to have leptospirosis. The sensitivity of IHA for the detection of acute leptospirosis was 100%, the specificity was 94%, the positive predictive value was 95%, and the negative predictive value was 100%. IHA was negative when 13 antinuclear antibody-positive sera, 24 serum specimens from patients with syphilis, and 16 serum specimens false positive by the Venereal Disease Research Laboratory test were tested. IHA was shown to detect both IgM and IgG classes of antibodies in human sera. Serum specimens from 27 dogs investigated for leptospirosis were studied: 3 samples gave nonspecific hemagglutination, but for all remaining samples, the results of IHA and an IgM ELISA were concordant. Performance of IHA was simple, and IHA requires no specialized equipment. It represents a useful assay for laboratories which require a leptospiral diagnostic capability but lack the expertise to perform specialist investigations.