bluuebunny/crossref_metadata_2025_split huggingface dataset:
Control of
Herbaspirillum seropedicae
NifA Activity by Ammonium Ions and Oxygen
Authors: Souza E. M. , Pedrosa F. O. , Drummond M. , Rigo L. U. , Yates M. G. 
Published: December 2019

ABSTRACT

The activity of a truncated form of
Herbaspirillum seropedicae
NifA in different genetic backgrounds showed that its regulatory domain is involved in nitrogen control but not in O
2
sensitivity or Fe dependence. The model for nitrogen control involving PII could thus apply to the proteobacteria at large. NifA may have a role in controlling ADP-ribosylation of nitrogenase in
Azospirillum brasilense
.
Regulation of the Transposase of Tn
4652
by the Transposon-Encoded Protein TnpC
Authors: Hõrak Rita , Kivisaar Maia 
Published: December 2019

ABSTRACT

Transposition is a DNA reorganization reaction potentially deleterious for the host. The frequency of transposition is limited by the amount of transposase. Therefore, strict regulation of a transposase is required to keep control over the destructive multiplication of the mobile element. We have shown previously that the expression of the transposase (
tnpA
) of the
Pseudomonas putida
PaW85 transposon Tn
4652
is positively affected by integration host factor. Here, we present evidence that the amount of the transposase of Tn
4652
in
P. putida
cells is controlled by the transposon-encoded protein (TnpC). Sequence analysis of the 120-amino-acid-long TnpC, coded just downstream of the
tnpA
gene, showed that it has remarkable similarity to the putative polypeptide encoded by the mercury resistance transposon Tn
5041
. As determined by quantitative Western blot analysis, the abundance of TnpA was reduced up to 10-fold in the intact
tnpC
background. In vivo experiments using transcriptional and translational fusions of the
tnpA
gene and the reporter gene
gusA
indicated that TnpC operates in the regulation of the transposase of Tn
4652
at the post-transcriptional level.
MinDE-Dependent Pole-to-Pole Oscillation of Division Inhibitor MinC in
Escherichia coli
Authors: Raskin David M. , de Boer Piet A. J. 
Published: December 2019

ABSTRACT

By inhibiting FtsZ ring formation near the cell ends, the MinC protein plays a critical role in proper positioning of the division apparatus in
Escherichia coli
. MinC activity requires that of MinD, and the MinE peptide provides topological specificity by suppressing MinC-MinD-mediated division inhibition specifically at the middle of the cell. We recently presented evidence that MinE not only accumulates in an FtsZ-independent ring structure at the cell’s middle but also imposes a unique dynamic localization pattern upon MinD in which the latter accumulates alternately in either one of the cell halves in what appears to be a rapidly oscillating membrane association-dissociation cycle. Here we show that functional green fluorescent protein-MinC displays a very similar oscillatory behavior which is dependent on both MinD and MinE and independent of FtsZ. The results support a model in which MinD recruits MinC to its site of action and in which FtsZ ring assembly at each of the cell ends is blocked in an intermittent and alternate fashion.
Functional Genomics: Expression Analysis of
Escherichia coli
Growing on Minimal and Rich Media
Authors: Tao Han , Bausch Christoph , Richmond Craig , Blattner Frederick R. , Conway Tyrrell 
Published: December 2019

ABSTRACT

DNA arrays of the entire set of
Escherichia coli
genes were used to measure the genomic expression patterns of cells growing in late logarithmic phase on minimal glucose medium and on Luria broth containing glucose. Ratios of the transcript levels for all 4,290
E. coli
protein-encoding genes (cds) were obtained, and analysis of the expression ratio data indicated that the physiological state of the cells under the two growth conditions could be ascertained. The cells in the rich medium grew faster, and expression of the majority of the translation apparatus genes was significantly elevated under this growth condition, consistent with known patterns of growth rate-dependent regulation and increased rate of protein synthesis in rapidly growing cells. The cells grown on minimal medium showed significantly elevated expression of many genes involved in biosynthesis of building blocks, most notably the amino acid biosynthetic pathways. Nearly half of the known RpoS-dependent genes were expressed at significantly higher levels in minimal medium than in rich medium, and
rpoS
expression was similarly elevated. The role of RpoS regulation in these logarithmic phase cells was suggested by the functions of the RpoS dependent genes that were induced. The hallmark features of
E. coli
cells growing on glucose minimal medium appeared to be the formation and excretion of acetate, metabolism of the acetate, and protection of the cells from acid stress. A hypothesis invoking RpoS and UspA (universal stress protein, also significantly elevated in minimal glucose medium) as playing a role in coordinating these various aspects and consequences of glucose and acetate metabolism was generated. This experiment demonstrates that genomic expression assays can be applied in a meaningful way to the study of whole-bacterial-cell physiology for the generation of hypotheses and as a guide for more detailed studies of particular genes of interest.
Identification and Characterization of Major Lipid Particle Proteins of the Yeast
Saccharomyces cerevisiae
Authors: Athenstaedt Karin , Zweytick Dagmar , Jandrositz Anita , Kohlwein Sepp Dieter , Daum Günther 
Published: December 2019

ABSTRACT

Lipid particles of the yeast
Saccharomyces cerevisiae
were isolated at high purity, and their proteins were separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Major lipid particle proteins were identified by mass spectrometric analysis, and the corresponding open reading frames (ORFs) were deduced. In silicio analysis revealed that all lipid particle proteins contain several hydrophobic domains but none or only few (hypothetical) transmembrane spanning regions. All lipid particle proteins identified by function so far, such as Erg1p, Erg6p, and Erg7p (ergosterol biosynthesis) and Faa1p, Faa4p, and Fat1p (fatty acid metabolism), are involved in lipid metabolism. Based on sequence homology, another group of three lipid particle proteins may be involved in lipid degradation. To examine whether lipid particle proteins of unknown function are also involved in lipid synthesis, mutants with deletions of the respective ORFs were constructed and subjected to systematic lipid analysis. Deletion of YDL193w resulted in a lethal phenotype which could not be suppressed by supplementation with ergosterol or fatty acids. Other deletion mutants were viable under standard conditions. Strains with YBR177c, YMR313c, and YKL140w deleted exhibited phospholipid and/or neutral lipid patterns that were different from the wild-type strain and thus may be further candidate ORFs involved in yeast lipid metabolism.
Substitution, Insertion, Deletion, Suppression, and Altered Substrate Specificity in Functional Protocatechuate 3,4-Dioxygenases
Authors: D’Argenio David A. , Vetting Mathew W. , Ohlendorf Douglas H. , Ornston L. Nicholas 
Published: December 2019

ABSTRACT

Protocatechuate 3,4-dioxygenase is a member of a family of bacterial enzymes that cleave the aromatic rings of their substrates between two adjacent hydroxyl groups, a key reaction in microbial metabolism of varied environmental chemicals. In an appropriate genetic background, it is possible to select for
Acinetobacter
strains containing spontaneous mutations blocking expression of
pcaH
or -
G
, genes encoding the α and β subunits of protocatechuate 3,4-dioxygenase. The crystal structure of the
Acinetobacter
oxygenase has been determined, and this knowledge affords us the opportunity to understand how mutations alter function in the enzyme. An earlier investigation had shown that a large fraction of spontaneous mutations inactivating
Acinetobacter
protocatechuate oxygenase are either insertions or large deletions. Therefore, the prior procedure of mutant selection was modified to isolate
Acinetobacter
strains in which mutations within
pcaH
or -
G
cause a heat-sensitive phenotype. These mutations affected residues distributed throughout the linear amino acid sequences of PcaH and PcaG and impaired the dioxygenase to various degrees. Four of 16 mutants had insertions or deletions in the enzyme ranging in size from 1 to 10 amino acid residues, highlighting areas of the protein where large structural changes can be tolerated. To further understand how protein structure influences function, we isolated strains in which the phenotypes of three different deletion mutations in
pcaH
or -
G
were suppressed either by a spontaneous mutation or by a PCR-generated random mutation introduced into the
Acinetobacter
chromosome by natural transformation. The latter procedure was also used to identify a single amino acid substitution in PcaG that conferred activity towards catechol sufficient for growth with benzoate in a strain in which catechol 1,2-dioxygenase was inactivated.
Horizontal Transfer of Genetic Material among
Saccharomyces
Yeasts
Authors: Marinoni Gaelle , Manuel Martine , Petersen Randi Føns , Hvidtfeldt Jeanne , Sulo Pavol , Piškur Jure 
Published: December 2019

ABSTRACT

The genus
Saccharomyces
consists of several species divided into the sensu stricto and the sensu lato groups. The genomes of these species differ in the number and organization of nuclear chromosomes and in the size and organization of mitochondrial DNA (mtDNA). In the present experiments we examined whether these yeasts can exchange DNA and thereby create novel combinations of genetic material. Several putative haploid, heterothallic yeast strains were isolated from different
Saccharomyces
species. All of these strains secreted an a- or α-like pheromone recognized by
S. cerevisiae
tester strains. When interspecific crosses were performed by mass mating between these strains, hybrid zygotes were often detected. In general, the less related the two parental species were, the fewer hybrids they gave. For some crosses, viable hybrids could be obtained by selection on minimal medium and their nuclear chromosomes and mtDNA were examined. Often the frequency of viable hybrids was very low. Sometimes putative hybrids could not be propagated at all. In the case of sensu stricto yeasts, stable viable hybrids were obtained. These contained both parental sets of chromosomes but mtDNA from only one parent. In the case of sensu lato hybrids, during genetic stabilization one set of the parental chromosomes was partially or completely lost and the stable mtDNA originated from the same parent as the majority of the nuclear chromosomes. Apparently, the interspecific hybrid genome was genetically more or less stable when the genetic material originated from phylogenetically relatively closely related parents; both sets of nuclear genetic material could be transmitted and preserved in the progeny. In the case of more distantly related parents, only one parental set, and perhaps some fragments of the other one, could be found in genetically stabilized hybrid lines. The results obtained indicate that
Saccharomyces
yeasts have a potential to exchange genetic material. If
Saccharomyces
isolates could mate freely in nature, horizontal transfer of genetic material could have occurred during the evolution of modern yeast species.
Divergent Regulation of the Evolutionarily Closely Related Promoters of the
Saccharomyces cerevisiae STA2
and
MUC1
Genes
Authors: Gagiano Marco , Van Dyk Dewald , Bauer Florian F. , Lambrechts Marius G. , Pretorius Isak S. 
Published: December 2019

ABSTRACT

The 5′ upstream regions of the
Saccharomyces cerevisiae
glucoamylase-encoding genes
STA1
to -
3
and of the
MUC1
(or
FLO11
) gene, which is critical for pseudohyphal development, invasive growth, and flocculation, are almost identical, and the genes are coregulated to a large extent. Besides representing the largest yeast promoters identified to date, these regions are of particular interest from both a functional and an evolutionary point of view. Transcription of the genes indeed seems to be dependent on numerous transcription factors which integrate the information of a complex network of signaling pathways, while the very limited sequence differences between them should allow the study of promoter evolution on a molecular level. To investigate the transcriptional regulation, we compared the transcription levels conferred by the
STA2
and
MUC1
promoters under various growth conditions. Our data show that transcription of both genes responded similarly to most environmental signals but also indicated significant divergence in some aspects. We identified distinct areas within the promoters that show specific responses to the activating effect of Flo8p, Msn1p (or Mss10p, Fup1p, or Phd2p), and Mss11p as well as to carbon catabolite repression. We also identified the
STA10
repressive effect as the absence of Flo8p, a transcriptional activator of flocculation genes in
S. cerevisiae.
Genetic Analysis of
nif
Regulatory Genes by Utilizing the Yeast Two-Hybrid System Detected Formation of a NifL-NifA Complex That Is Implicated in Regulated Expression of
nif
Genes
Authors: Lei Shi , Pulakat Lakshmidevi , Gavini Narasaiah 
Published: December 2019

ABSTRACT

In diazotrophic organisms, nitrogenase synthesis and activity are tightly regulated. Two genes,
nifL
and
nifA
, are implicated as playing a major role in this regulation. NifA is a transcriptional activator, and its activity is inhibited by NifL in response to availability of excess fixed nitrogen and high O
2
tension. It was postulated that NifL binds to NifA to inhibit NifA-mediated transcriptional activation of
nif
genes. Mutational analysis combined with transcriptional activation studies clearly is in agreement with the proposal that NifL interacts with NifA. However, several attempts to identify NifA-NifL interactions by using methods such as coimmunoprecipitations and chemical cross-linking experiments failed to detect direct interactions between these proteins. Here we have taken a genetic approach, the use of a yeast two-hybrid protein-protein interaction assay system, to investigate NifL interaction with NifA. A DNA fragment corresponding to the kinase-like domain of
nifL
was PCR amplified and was used to generate translation fusions with the DNA binding domain and the DNA activation domain of the yeast transcriptional activator GAL4 in yeast two-hybrid vectors. Similarly, a DNA fragment corresponding to the catalytic domain of
nifA
was PCR amplified and used to generate translation fusions with the DNA-binding domain and the DNA-activation domain of GAL4 in yeast two-hybrid vectors. After introducing appropriate plasmid combinations in yeast cells, the existance of direct interaction between NifA and NifL was analyzed with the MATCHMAKER yeast two-hybrid system by testing for the expression of
lacZ
and
his3
genes. These analyses showed that the kinase-like domain of NifL directly interacts with the catalytic domain of NifA.
Porins in the Cell Wall of
Mycobacterium tuberculosis
Authors: Kartmann Bettina , Stengler Steffen , Niederweis Michael 
Published: December 2019

ABSTRACT

Lipid bilayer experiments indicated that the cell wall of
Mycobacterium tuberculosis
contains at least two different porins: (i) a cation-selective, heat-sensitive 0.7-nS channel which has a short-lived open state and is probably composed of 15-kDa subunits and (ii) a 3-nS, >60-kDa channel with a long-lived open state, resembling porins from fast-growing mycobacteria.
Gene Disruption Studies of Penicillin-Binding Proteins 1a, 1b, and 2a in
Streptococcus pneumoniae
Authors: Hoskins JoAnn , Matsushima Patti , Mullen Deborah L. , Tang Joseph , Zhao Genshi , Meier Timothy I. , Nicas Thalia I. , Jaskunas S. Richard 
Published: December 2019

ABSTRACT

The effects of inactivation of the genes encoding penicillin-binding protein 1a (PBP1a), PBP1b, and PBP2a in
Streptococcus pneumoniae
were examined. Insertional mutants did not exhibit detectable changes in growth rate or morphology, although a
pbp1a pbp1b
double-disruption mutant grew more slowly than its parent did. Attempts to generate a
pbp1a pbp2a
double-disruption mutant failed. The
pbp2a
mutants, but not the other mutants, were more sensitive to moenomycin, a transglycosylase inhibitor. These observations suggest that individually the
pbp1a
,
pbp1b
, and
pbp2a
genes are dispensable but that either
pbp1a
or
pbp2a
is required for growth in vitro. These results also suggest that PBP2a is a functional transglycosylase in
S. pneumoniae
.
Occurrence of Free
d
-Amino Acids and Aspartate Racemases in Hyperthermophilic Archaea
Authors: Matsumoto Megumi , Homma Hiroshi , Long Zhiqun , Imai Kazuhiro , Iida Toshii , Maruyama Tadashi , Aikawa Yuko , Endo Isao , Yohda Masafumi 
Published: December 2019

ABSTRACT

The occurrence of free
d
-amino acids and aspartate racemases in several hyperthermophilic archaea was investigated. Aspartic acid in all the hyperthermophilic archaea was highly racemized. The ratio of
d
-aspartic acid to total aspartic acid was in the range of 43.0 to 49.1%. The crude extracts of the hyperthermophiles exhibited aspartate racemase activity at 70°C, and aspartate racemase homologous genes in them were identified by PCR.
d
-Enantiomers of other amino acids (alanine, leucine, phenylalanine, and lysine) in
Thermococcus
strains were also detected. Some of them might be by-products of aspartate racemase. It is proven that
d
-amino acids are produced in some hyperthermophilic archaea, although their function is unknown.
The Modality of Enterobacterial Common Antigen Polysaccharide Chain Lengths Is Regulated by
o349
of the
wec
Gene Cluster of
Escherichia coli
K-12
Authors: Barr Kathleen , Klena John , Rick Paul D. 
Published: December 2019

ABSTRACT

The assembly of the phosphoglyceride-linked form of enterobacterial common antigen (ECA
PG
) occurs by a mechanism that involves modulation of polysaccharide chain length. However, the genetic determinant of this modulation has not been identified. Site-directed mutagenesis of
o349
of the
Escherichia coli
K-12
wec
gene cluster revealed that this locus encodes a Wzz protein that specifically modulates the chain length of ECA
PG
polysaccharides, and we have designated this locus
wzz
ECA
. The Wzz
ECA
-mediated modulation of ECA
PG
polysaccharide chains is the first demonstrated example of Wzz regulation involving a polysaccharide that is not linked to the core-lipid A structure of lipopolysaccharide.
On the Origin of Branches in
Escherichia coli
Authors: Gullbrand Björn , Åkerlund Thomas , Nordström Kurt 
Published: December 2019

ABSTRACT

Some
Escherichia coli
strains with impaired cell division form branched cells at high frequencies during certain growth conditions. Here, we show that neither FtsI nor FtsZ activity is required for the development of branches. Buds did not form at specific positions along the cell surface during high-branching conditions. Antibiotics affecting cell wall synthesis had a positive effect on branch formation in the case of ampicillin, cephalexin, and penicillin G, whereas mecillinam and
d
-cycloserine had no substantial effect. Altering the cell morphology by nutritional shifts showed that changes in morphology preceded branching, indicating that the cell’s physiological state rather than specific medium components induced branching. Finally, there was no increased probability for bud formation in the daughters of a cell with a bud or branch, showing that bud formation is a random event. We suggest that branch formation is caused by abnormalities in cell wall elongation rather than by aberrant cell division events.
Characterization of a Hank’s Type Serine/Threonine Kinase and Serine/Threonine Phosphoprotein Phosphatase in
Pseudomonas aeruginosa
Authors: Mukhopadhyay Subhendu , Kapatral Vinayak , Xu Wenbin , Chakrabarty A. M. 
Published: December 2019

ABSTRACT

Pseudomonas aeruginosa
is an opportunistic pathogen that causes infections in eye, urinary tract, burn, and immunocompromised patients. We have cloned and characterized a serine/threonine (Ser/Thr) kinase and its cognate phosphoprotein phosphatase. By using oligonucleotides from the conserved regions of Ser/Thr kinases of mycobacteria, an 800-bp probe was used to screen
P. aeruginosa
PAO1 genomic library. A 20-kb cosmid clone was isolated, from which a 4.5-kb DNA with two open reading frames (ORFs) were subcloned. ORF1 was shown to encode Ser/Thr phosphatase (Stp1), which belongs to the PP2C family of phosphatases. Overlapping with the
stp1
ORF, an ORF encoding Hank’s type Ser/Thr kinase was identified. Both ORFs were cloned in pGEX-4T1 and expressed in
Escherichia coli
. The overexpressed proteins were purified by glutathione-Sepharose 4B affinity chromatography and were biochemically characterized. The Stk1 kinase is 39 kDa and undergoes autophosphorylation and can phosphorylate eukaryotic histone H1. A site-directed Stk1 (K86A) mutant was shown to be incapable of autophosphorylation. A two-dimensional phosphoamino acid analysis of Stk1 revealed strong phosphorylation at a threonine residue and weak phosphorylation at a serine residue. The Stp1 phosphatase is 27 kDa and is an Mn
2+
-, but not a Ca
2+
- or a Mg
2+
-, dependent Ser/Thr phosphatase. Its activity is inhibited by EDTA and NaF, but not by okadaic acid, and is similar to that of PP2C phosphatase.
Transcriptional Activation of the Chlorocatechol Degradative Genes of
Ralstonia eutropha
NH9
Authors: Ogawa Naoto , McFall Sally M. , Klem Thomas J. , Miyashita Kiyotaka , Chakrabarty A. M. 
Published: December 2019

ABSTRACT

Ralstonia eutropha
(formerly
Alcaligenes eutrophus
) NH9 degrades 3-chlorobenzoate via the modified
ortho
-cleavage pathway. A ca. 5.7-kb six-gene cluster is responsible for chlorocatechol degradation: the
cbnABCD
operon encoding the degradative enzymes (including
orfX
of unknown function) and the divergently transcribed
cbnR
gene encoding the LysR-type transcriptional regulator of the
cbn
operon. The
cbnRAB orfXCD
gene cluster is nearly identical to the chlorocatechol genes (
tcbRCD orfXEF
) of the 1,2,4-trichlorobenzene-degrading bacterium
Pseudomonas
sp. strain P51. Transcriptional fusion studies demonstrated that
cbnR
regulates the expression of
cbnABCD
positively in the presence of either 3-chlorobenzoate or benzoate, which are catabolized via 3-chlorocatechol and catechol, respectively. In vitro transcription assays confirmed that 2-chloro-
cis
,
cis
-muconate (2-CM) and
cis
,
cis
-muconate (CCM), intermediate products from 3-chlorocatechol and catechol, respectively, were inducers of this operon. This inducer-recognizing specificity is different from those of the homologous catechol (
catBCA
) and chlorocatechol (
clcABD
) operons of
Pseudomonas putida
, in which only the intermediates of the regulated pathway, CCM for
catBCA
and 2-CM for
clcABD
, act as significant inducers. Specific binding of CbnR protein to the
cbnA
promoter region was demonstrated by gel shift and DNase I footprinting analysis. In the absence of inducer, a region of ca. 60 bp from position −20 to position −80 upstream of the
cbnA
transcriptional start point was protected from DNase I cleavage by CbnR, with a region of hypersensitivity to DNase I cleavage clustered at position −50. Circular permutation gel shift assays demonstrated that CbnR bent the
cbnA
promoter region to an angle of 78° and that this angle was relaxed to 54° upon the addition of inducer. While a similar relaxation of bending angles upon the addition of inducer molecules observed with the
catBCA
and
clcABD
promoters may indicate a conserved transcriptional activation mechanism of
ortho
-cleavage pathway genes, CbnR is unique in having a different specificity of inducer recognition and the extended footprint as opposed to the restricted footprint of CatR without CCM.