bluuebunny/crossref_metadata_2025_split huggingface dataset:
The Amino-Terminal 100 Residues of the Nitrogen Assimilation Control Protein (NAC) Encode All Known Properties of NAC from
Klebsiella aerogenes
and
Escherichia coli
Authors: Muse Wilson B. , Bender Robert A. 
Published: December 2019

ABSTRACT

The nitrogen assimilation control protein (NAC) from
Klebsiella aerogenes
or
Escherichia coli
(NAC
K
or NAC
E
, respectively) is a transcriptional regulator that is both necessary and sufficient to activate transcription of the histidine utilization (
hut
) operon and to repress transcription of the glutamate dehydrogenase (
gdh
) operon in
K. aerogenes
. Truncated NAC polypeptides, generated by the introduction of stop codons within the
nac
open reading frame, were tested for the ability to activate
hut
and repress
gdh
in vivo. Most of the NAC
K
and NAC
E
fragments with 100 or more amino acids (wild-type NAC
K
and NAC
E
both have 305 amino acids) were functional in activating
hut
and repressing
gdh
expression in vivo. Full-length NAC
K
and NAC
E
were isolated as chimeric proteins with the maltose-binding protein (MBP). NAC
K
and NAC
E
released from such chimeras were able to activate
hut
transcription in a purified system in vitro, as were NAC
K
129 and NAC
E
100 (a NAC
K
fragment of 129 amino acids and a NAC
E
fragment of 100 amino acids) released from comparable chimeras. A set of NAC
E
and NAC
K
fragments carrying nickel-binding histidine tags (his
6
) at their C termini were also generated. All such constructs derived from NAC
E
were insoluble, as was NAC
E
itself. Of the his
6
-tagged constructs derived from NAC
K
, NAC
K
100 was inactive, but NAC
K
120 was active. Several NAC fragments were tested for dimerization. NAC
K
120-his
6
and NAC
K
100-his
6
were dimers in solution. MBP-NAC
K
and MBP-NAC
K
129 were monomers in solution but dimerized when the MBP was released by cleavage with factor Xa. MBP-NAC
E
was readily cleaved by factor Xa, but the resulting NAC
E
was also degraded by the protease. However, MBP-NAC
E
-his6 was completely resistant to cleavage by factor Xa, suggesting an interaction between the C and N termini of this protein.
EmrE, a Small
Escherichia coli
Multidrug Transporter, Protects
Saccharomyces cerevisiae
from Toxins by Sequestration in the Vacuole
Authors: Yelin Rodrigo , Rotem Dvir , Schuldiner Shimon 
Published: December 2019

ABSTRACT

In this report we describe the functional expression of EmrE, a 110-amino-acid multidrug transporter from
Escherichia coli
, in the yeast
Saccharomyces cerevisiae
. To allow for phenotypic complementation, a mutant strain sensitive to a series of cationic lipophilic drugs was first identified. A hemagglutinin epitope-tagged version of EmrE (HA-EmrE) conferring resistance to a wide variety of drugs, including acriflavine, ethidium, methyl viologen, and the neurotoxin 1-methyl-4-phenylpyridinium (MPP
+
), was functionally expressed in this strain. HA-EmrE is expressed in yeast at relatively high levels (0.5 mg/liter), is soluble in a mixture of organic solvents, and can be functionally reconstituted in proteoliposomes. In bacterial cells, EmrE removes toxic compounds by active transport through the plasma membrane, lowering their cytosolic concentration. However, yeast cells expressing HA-EmrE take up
14
C-methyl viologen as well as control cells do. Thus, we investigated the basis of the enhanced resistance to the above compounds. Using Cu
2+
ions or methylamine, we could selectively permeabilize the plasma membrane or deplete the proton electrochemical gradients across the vacuolar membrane, respectively. Incubation of yeast cells with copper ions caused an increase in
14
C-methyl viologen uptake. In contrast, treatment with methylamine markedly diminished the extent of uptake. Conversely, the effect of Cu
2+
and methylamine on a plasma membrane uptake system, proline, was essentially the opposite: while inhibited by the addition of Cu
2+
, it remained unaffected when cells were treated with methylamine. To examine the intracellular distribution of HA-EmrE, a functional chimera between HA-EmrE and the green fluorescent protein (HA-EmrE-GFP) was prepared. The pattern of HA-EmrE-GFP fluorescence distribution was virtually identical to that of the vacuolar marker FM 4-64, indicating that the transporter is found mainly in this organelle. Therefore, HA-EmrE protects yeast cells by lowering the cytoplasmic concentrations through removal of the toxin to the vacuole. This novel way of detoxification has been previously suggested to function in organisms in which a large vacuolar compartment exists. This report represents the first molecular description of such a mechanism.
Physiological Characterization of
Pseudomonas aeruginosa
during Exotoxin A Synthesis: Glutamate, Iron Limitation, and Aconitase Activity
Authors: Somerville Greg , Mikoryak Carole Ann , Reitzer Larry 
Published: December 2019

ABSTRACT

Glutamate enhances the yield of exotoxin A (ETA), which is induced by iron limitation, from
Pseudomonas aeruginosa
. We tested the possibility that glutamate affects growth during iron restriction. We confirmed that iron limitation caused early entry into stationary phase but had no effect on the exponential growth rate. We showed that glutamate, as well as citrate and isocitrate, partially overcame this growth limitation. Glutamate had no effect on
toxA
(ETA-encoding) transcription, which implies that glutamate primarily increases the number of toxin-producing cells. In contrast, citrate and isocitrate diminished
toxA
transcription. Since glutamate, citrate, and isocitrate stimulated growth, we suspected a block in the citric acid cycle. Iron limitation reduced the activity of the iron-containing aconitase 12-fold but had no effect on isocitrate dehydrogenase activity, which was assayed as a control. There is a reciprocal relationship between aconitase activity and ETA synthesis, and this correlation does not appear to be coincidental because aconitase-specific effectors affect ETA synthesis. We tested whether a metabolic block is sufficient to induce ETA synthesis, but an aconitase-specific inhibitor diminished ETA production, which argues against this possibility. Finally, we present preliminary evidence that iron limitation may reversibly and posttranslationally inactivate aconitase in vivo. In summary, the environmental factors that stimulate ETA synthesis are related: glutamate bypasses an iron limitation-dependent metabolic block that causes entry into stationary phase. We speculate that one or more of the aconitases in
P. aeruginosa
may contribute to the control of virulence factor synthesis.
Genetic and Transcriptional Analyses of the
Vibrio cholerae
Mannose-Sensitive Hemagglutinin Type 4 Pilus Gene Locus
Authors: Marsh Jane W. , Taylor Ronald K. 
Published: December 2019

ABSTRACT

The mannose-sensitive hemagglutinin (MSHA) of the
Vibrio cholerae
O1 El Tor biotype is a member of the family of type 4 pili. Type 4 pili are found on the surface of a variety of gram-negative bacteria and have demonstrated importance as host colonization factors, bacteriophage receptors, and mediators of DNA transfer. The gene locus required for the assembly and secretion of the MSHA pilus has been localized to a 16.7-kb region of the
V. cholerae
chromosome. Sixteen genes required for hemagglutination, including five that encode prepilin or prepilin-like proteins, have been identified. Examination of MSHA-specific cDNAs has localized two promoters that drive expression of these genes. This evidence indicates that the MSHA gene locus is transcriptionally organized into two operons, one encoding the secretory components and the other encoding the structural subunits, an arrangement unique among previously characterized type 4 pilus loci. The genes flanking the MSHA locus encode proteins that show homology to YhdA and MreB of
Escherichia coli
. In
E. coli
, the
yhdA
and
mreB
genes are adjacent to each other on the chromosome. The finding that the MSHA locus lies between these two
E. coli
homologs and that it is flanked by a 7-bp direct repeat suggests that the MSHA locus may have been acquired as a mobile genetic element.
Identification of a
Streptococcus pneumoniae
Gene Locus Encoding Proteins of an ABC Phosphate Transporter and a Two-Component Regulatory System
Authors: Novak Rodger , Cauwels Anje , Charpentier Emmanuelle , Tuomanen Elaine 
Published: December 2019

ABSTRACT

The
Escherichia coli
Pst system belongs to the family of ABC transporters. It is part of a phosphate (PHO) regulon which is regulated by extracellular phosphate. Under conditions of phosphate limitation, the response regulator PhoB is phosphorylated by the histidine kinase PhoR and binds to promoters that share a consensus PHO box. Under conditions of phosphate excess, PhoR, Pst, and PhoU downregulate the PHO regulon. Screening of a library of pneumococcal mutants with defects in exported proteins revealed a putative two-component regulatory system, PnpR-PnpS, and a downstream ABC transporter, similar to the Pst system in
E. coli
including a gene encoding a PhoU protein. Similar to
E. coli
, mutagenesis of the ATP-binding cassette gene,
pstB
, resulted in decreased uptake of phosphate. The effects of the loss of the pneumococcal Pst system extended to decreased transformation and lysis. Withdrawal of phosphate led to transformation deficiency in the parent strain R6x but not to penicillin tolerance, suggesting that reduced bacterial death was independent of phosphate. None of these phenotypes was observed in the pneumococcal loss-of-function mutant
phoU
. By using a
lacZ
reporter construct, it was demonstrated that expression of the two-component regulatory system PnpR-PnpS was not influenced by different concentrations of phosphate. These results suggest a more complex role of the Pst system in pneumococcal physiology than in that of
E. coli.
Analysis of
rpoS
mRNA in
Salmonella dublin
: Identification of Multiple Transcripts with Growth-Phase-Dependent Variation in Transcript Stability
Authors: Paesold G. , Krause M. 
Published: December 2019

ABSTRACT

In
Salmonella dublin
,
rpoS
encodes an alternative sigma factor of the RNA polymerase that activates a variety of stationary-phase-induced genes, including some virulence-associated genes. In this work, we studied the regulation and transcriptional organization of
rpoS
during growth. We found two transcripts, 2.3 and 1.6 kb in length, that represent the complete
rpoS
sequence. The 2.3-kb transcript is a polycistronic message that also includes the upstream
nlpD
gene. It is driven by a weak promoter with increasing activity when cells enter early stationary growth. The 1.6-kb message includes 566 bp upstream of the
rpoS
start codon. It is transcribed from a strong ς
70
RNA polymerase-dependent promoter which is independent of growth. The decay of this transcript decreases substantially in early stationary growth, resulting in a significant net increase in
rpoS
mRNA levels. These levels are approximately 10-fold higher than the levels of the 2.3-kb mRNA, indicating that the 1.6-kb message is mainly responsible for RpoS upregulation. In addition to the 2.3- and 1.6-kb transcripts, two smaller 1.0- and 0.4-kb RNA species are produced from the
nlpD-rpoS
locus. They do not allow translation of full-length RpoS; hence their significance for
rpoS
regulation remains unclear. We conclude that of four transcripts arising from the
nlpD-rpoS
locus, only one plays a significant role in
rpoS
expression in
S. dublin
. Its upregulation when cells enter stationary growth is due primarily to an increase in transcript stability.
Functional Determinants of the
Escherichia coli fis
Promoter: Roles of −35, −10, and Transcription Initiation Regions in the Response to Stringent Control and Growth Phase-Dependent Regulation
Authors: Walker Kimberly A. , Atkins Carey L. , Osuna Robert 
Published: December 2019

ABSTRACT

Escherichia coli
Fis is a small DNA binding and bending protein that has been implicated in a variety of biological processes. A minimal promoter sequence consisting of 43 bp is sufficient to generate its characteristic growth phase-dependent expression pattern and is also subject to negative regulation by stringent control. However, information about the precise identification of nucleotides contributing to basal promoter activity and its regulation has been scant. In this work, 72 independent mutations were generated in the
fis
promoter (
fis
P) region from −108 to +78 using both random and site-directed PCR mutagenesis. β-Galactosidase activities from mutant promoters fused to the (
trp-lac
)
W200
fusion on a plasmid were used to conclusively identify the sequences TTTCAT and TAATAT as the −35 and −10 regions, respectively, which are optimally separated by 17 bp. We found that four consecutive substitutions within the GC-rich sequence just upstream of +1 and mutations in the −35 region, but not in the −10 region, significantly reduced the response to stringent control. Analysis of the effects of mutations on growth phase-dependent regulation showed that replacing the predominant transcription initiation nucleotide +1C with a preferred nucleotide (A or G) profoundly altered expression such that high levels of
fis
P mRNA were detected during late logarithmic and early stationary phases. A less dramatic effect was seen with improvements in the −10 and −35 consensus sequences. These results suggest that the acute growth phase-dependent regulation pattern observed with this promoter requires an inefficient transcription initiation process that is achieved with promoter sequences deviating from the −10 and −35 consensus sequences and, more importantly, a dependence upon the availability of the least favored transcription initiation nucleotide, CTP.
Molecular Cloning, Sequencing, Purification, and Characterization of
Pseudomonas aeruginosa
Ribosome Recycling Factor
Authors: Ohnishi Makoto , Janosi Laszlo , Shuda Masahiro , Matsumoto Hideki , Hayashi Tetsuya , Terawaki Yoshiro , Kaji Akira 
Published: December 2019

ABSTRACT

Ribosome recycling factor (RRF) is required for release of 70S ribosomes from mRNA on reaching the termination codon for the next cycle of protein synthesis. The RRF-encoding gene (
frr
) of
Pseudomonas aeruginosa
PAO1 was functionally cloned by using a temperature-sensitive
frr
mutant of
Escherichia coli
and sequenced. The
P. aeruginosa frr
was mapped at 30 to 32 min of the
P. aeruginosa
chromosome. The deduced amino acid sequence of RRF showed a 64% identity to that of
E. coli
RRF. In an assay including
E. coli
polysome and elongation factor G, purified recombinant RRF of
P. aeruginosa
released monosomes from polysomes. This is the first case in which an RRF homologue was found to be active in heterogeneous ribosome recycling machinery. The genes for ribosomal protein S2 (
rpsB
), elongation factor Ts (
tsf
), and UMP kinase (
pyrH
) are located upstream of
frr
. The arrangement of the genes,
rpsB-tsf-pyrH-frr
, resembles those reported for
E. coli
and
Bacillus subtilis
. Even in the cyanobacterium genome, the arrangement
pyrH-frr
is conserved. Although RRF homologues are found in eukaryotic cells, phylogenetic analysis suggests that they were originally present within the members of the phylogenetic tree of prokaryotic RRF. This finding suggests that the ribosome recycling step catalyzed by RRF is specific for prokaryotic cells and that eukaryotic RRF is required for protein synthesis in organelles, which are believed to be phylogenetically originated from prokaryotes.
Auxins Upregulate Expression of the Indole-3-Pyruvate Decarboxylase Gene in
Azospirillum brasilense
Authors: Vande Broek Ann , Lambrecht Mark , Eggermont Kristel , Vanderleyden Jos 
Published: December 2019

ABSTRACT

Transcription of the
Azospirillum brasilense ipdC
gene, encoding an indole-3-pyruvate decarboxylase involved in the biosynthesis of indole-3-acetic acid (IAA), is induced by IAA as determined by
ipdC-gusA
expression studies and Northern analysis. Besides IAA, exogenously added synthetic auxins such as 1-naphthaleneacetic acid, 2,4-dichlorophenoxypropionic acid, and
p
-chlorophenoxyacetic acid were also found to upregulate
ipdC
expression. No upregulation was observed with tryptophan, acetic acid, or propionic acid or with the IAA conjugates IAA ethyl ester and IAA-
l
-phenylalanine, indicating structural specificity is required for
ipdC
induction. This is the first report describing the induction of a bacterial gene by auxin.
Bacteriophage T4 gp2 Interferes with Cell Viability and with Bacteriophage Lambda Red Recombination
Authors: Appasani Krishnarao , Thaler David S. , Goldberg Edward B. 
Published: December 2019

ABSTRACT
The T4 head protein, gp2, promotes head-tail joining during phage morphogenesis and is also incorporated into the phage head. It protects the injected DNA from degradation by exonuclease V during the subsequent infection. In this study, we show that recombinant gp2, a very basic protein, rapidly kills the cells in which it is expressed. To further illustrate the protectiveness of gp2 for DNA termini, we compare the effect of gp2 expression on Red-mediated and Int-mediated recombination. Red-mediated recombination is nonspecific and requires the transient formation of double-stranded DNA termini. Int-mediated recombination, on the other hand, is site specific and does not require chromosomal termini. Red-mediated recombination is inhibited to a much greater extent than is Int-mediated recombination. We conclude from the results of these physiological and genetic experiments that T4 gp2 expression, like Mu Gam expression, kills bacteria by binding to double-stranded DNA termini, the most likely mode for its protection of entering phage DNA from exonuclease V.
The Virulence Plasmid of
Salmonella typhimurium
Is Self-Transmissible
Authors: Ahmer Brian M. M. , Tran Mimi , Heffron Fred 
Published: December 2019

ABSTRACT

Most isolates of
Salmonella enterica
serovar Typhimurium contain a 90-kb virulence plasmid. This plasmid is reported to be mobilizable but nonconjugative. However, we have determined that the virulence plasmid of strains LT2, 14028, and SR-11 is indeed self-transmissible. The plasmid of strain SL1344 is not. Optimal conjugation frequency requires filter matings on M9 minimal glucose plates with a recipient strain lacking the virulence plasmid. These conditions result in a frequency of 2.9 × 10
−4
transconjugants/donor. Matings on Luria-Bertani plates, liquid matings, or matings with a recipient strain carrying the virulence plasmid reduce the efficiency by up to 400-fold. Homologs of the F plasmid conjugation genes are physically located on the virulence plasmid and are required for the conjugative phenotype.
3-Hydroxylaminophenol Mutase from
Ralstonia eutropha
JMP134 Catalyzes a Bamberger Rearrangement
Authors: Schenzle Andreas , Lenke Hiltrud , Spain Jim C. , Knackmuss Hans-Joachim 
Published: December 2019

ABSTRACT

3-Hydroxylaminophenol mutase from
Ralstonia eutropha
JMP134 is involved in the degradative pathway of 3-nitrophenol, in which it catalyzes the conversion of 3-hydroxylaminophenol to aminohydroquinone. To show that the reaction was really catalyzed by a single enzyme without the release of intermediates, the corresponding protein was purified to apparent homogeneity from an extract of cells grown on 3-nitrophenol as the nitrogen source and succinate as the carbon and energy source. 3-Hydroxylaminophenol mutase appears to be a relatively hydrophobic but soluble and colorless protein consisting of a single 62-kDa polypeptide. The pI was determined to be at pH 4.5. In a database search, the NH
2
-terminal amino acid sequence of the undigested protein and of two internal sequences of 3-hydroxylaminophenol mutase were found to be most similar to those of glutamine synthetases from different species. Hydroxylaminobenzene, 4-hydroxylaminotoluene, and 2-chloro-5-hydroxylaminophenol, but not 4-hydroxylaminobenzoate, can also serve as substrates for the enzyme. The enzyme requires no oxygen or added cofactors for its reaction, which suggests an enzymatic mechanism analogous to the acid-catalyzed Bamberger rearrangement.
Mechanism of Citrate Metabolism in
Lactococcus lactis
: Resistance against Lactate Toxicity at Low pH
Authors: Magni Christian , de Mendoza Diego , Konings Wil N. , Lolkema Juke S. 
Published: December 2019

ABSTRACT

Measurement of the flux through the citrate fermentation pathway in resting cells of
Lactococcus lactis
CRL264 grown in a pH-controlled fermentor at different pH values showed that the pathway was constitutively expressed, but its activity was significantly enhanced at low pH. The flux through the citrate-degrading pathway correlated with the magnitude of the membrane potential and pH gradient that were generated when citrate was added to the cells. The citrate degradation rate and proton motive force were significantly higher when glucose was metabolized at the same time, a phenomenon that could be mimicked by the addition of lactate, the end product of glucose metabolism. The results clearly demonstrate that citrate metabolism in
L. lactis
is a secondary proton motive force-generating pathway. Although the proton motive force generated by citrate in cells grown at low pH was of the same magnitude as that generated by glucose fermentation, citrate metabolism did not affect the growth rate of
L. lactis
in rich media. However, inhibition of growth by lactate was relieved when citrate also was present in the growth medium. Citrate did not relieve the inhibition by other weak acids, suggesting a specific role of the citrate transporter CitP in the relief of inhibition. The mechanism of citrate metabolism presented here provides an explanation for the resistance to lactate toxicity. It is suggested that the citrate metabolic pathway is induced under the acidic conditions of the late exponential growth phase to make the cells (more) resistant to the inhibitory effects of the fermentation product, lactate, that accumulates under these conditions.
Peptidoglycan-Hydrolyzing Activity of the FlgJ Protein, Essential for Flagellar Rod Formation in
Salmonella typhimurium
Authors: Nambu Takayuki , Minamino Tohru , Macnab Robert M. , Kutsukake Kazuhiro 
Published: December 2019

ABSTRACT

Because the rod structure of the flagellar basal body crosses the inner membrane, the periplasmic space, and the outer membrane, its formation must involve hydrolysis of the peptidoglycan layer. So far, more than 10 genes have been shown to be required for rod formation in
Salmonella typhimurium
. Some of them encode the component proteins of the rod structure, and most of the remaining genes are believed to encode proteins involved in the export process of the component proteins. Although FlgJ has also been known to be involved in rod formation, its exact role has not been understood. Recently, it was suggested that the C-terminal half of the FlgJ protein has homology to the active center of some muramidase enzymes from gram-positive bacteria. In this study, we showed that the purified FlgJ protein from
S. typhimurium
has a peptidoglycan-hydrolyzing activity and that this activity is localized in its C-terminal half. Through oligonucleotide-directed mutagenesis, we constructed
flgJ
mutants with amino acid substitutions in the putative active center of the muramidase. The resulting mutants produced FlgJ proteins with reduced enzymatic activity and showed poor motility. These results indicate that the muramidase activity of FlgJ is essential for flagellar formation. Immunoblotting analysis with the fractionated cell extracts revealed that FlgJ is exported to the periplasmic space, where the peptidoglycan layer is localized. On the basis of these results, we conclude that FlgJ is the flagellum-specific muramidase which hydrolyzes the peptidoglycan layer to assemble the rod structure in the periplasmic space.