bluuebunny/crossref_metadata_2025_split huggingface dataset:
Characterization of Insertions of IS
476
and Two Newly Identified Insertion Sequences, IS
1478
and IS
1479
, in
Xanthomonas campestris
pv. campestris
Authors: Chen Jiann-Hwa , Hsieh Yu-Ying , Hsiau Su-Lian , Lo Ta-Chun , Shau Chen-Chun 
Published: December 2019

ABSTRACT

Thirty-two plasmid insertion mutants were independently isolated from two strains of
Xanthomonas campestris
pv. campestris in Taiwan. Of the 32 mutants, 14 (44%), 8 (25%), and 4 (12%) mutants resulted from separate insertions of an IS
3
family member, IS
476
, and two new insertion sequences (IS), IS
1478
and IS
1479
. While IS
1478
does not have significant sequence homology with any IS elements in the EMBL/GenBank/DDBJ database, IS
1479
demonstrated 73% sequence homology with IS
1051
in
X. campestris
pv. dieffenbachiae, 62% homology with IS
52
in
Pseudomonas syringae
pv. glycinea, and 60% homology with IS
5
in
Escherichia coli
. Based on the predicted transposase sequences as well as the terminal nucleotide sequences, IS
1478
by itself constitutes a new subfamily of the widespread IS
5
family, whereas IS
1479
, along with IS
1051
, IS
52
, and IS
5
, belongs to the IS
5
subfamily of the IS
5
family. All but one of the IS
476
insertions had duplications of 4 bp at the target sites without sequence preference and were randomly distributed. An IS
476
insertion carried a duplication of 952 bp at the target site. A model for generating these long direct repeats is proposed. Insertions of IS
1478
and IS
1479
, on the other hand, were not random, and IS
1478
and IS
1479
each showed conservation of PyPuNTTA and PyTAPu sequences (Py is a pyrimidine, Pu is a purine, and N is any nucleotide) for duplications at the target sites. The results of Southern blot hybridization analysis indicated that multiple copies of IS
476
, IS
1478
, and IS
1479
are present in the genomes of all seven
X. campestris
pv. campestris strains tested and several
X. campestris
pathovars.
Rapid Purification and Properties of Betaine Aldehyde Dehydrogenase fromPseudomonas aeruginosa
Authors: Velasco-García Roberto , Mújica-Jiménez Carlos , Mendoza-Hernández Guillermo , Muñoz-Clares Rosario A. 
Published: December 2019

ABSTRACTBetaine aldehyde dehydrogenase (BADH) (EC1.2.1.8) catalyzes the last, irreversible step in the synthesis of the osmoprotectant glycine betaine from choline. InPseudomonas aeruginosathis reaction is also an obligatory step in the assimilation of carbon and nitrogen when bacteria are growing in choline or choline precursors. We present here a method for the rapid purification to homogeneity of this enzyme by the use of ion-exchange and affinity chromatographies on 2′,5′-ADP–Sepharose, which results in a high yield of pure enzyme with a specific activity at 30°C and pH 7.4 of 74.5 U/mg of protein. Analytical ultracentrifugation, gel filtration, chemical cross-linking, and sodium dodecyl sulfate-polyacrylamide gel electrophoresis suggest that BADH fromP. aeruginosais a homodimer with 61-kDa subunits. The amino acid composition and the N-terminal sequence of 21 amino acid residues showed significant similarity with those of the enzymes fromXanthomonas translucensandEscherichia coli. Neither BADH activity nor BADH protein was found in cell extracts from bacteria grown in the absence of choline. In contrast to other BADHs studied to date, thePseudomonasenzyme cannot use positively charged aldehydes other than betaine aldehyde as substrates. The oxidation reaction has an activation energy of 39.8 kJ mol−1. The pH dependence of the velocity indicated an optimum at pH 8.0 to 8.5 and the existence of two ionizable groups with macroscopic pK values of 7.0 ± 0.1 and 9.7 ± 0.1 involved in catalysis and/or binding of substrates. The enzyme is inactivated at 40°C, but activity is regained when the heated enzyme is cooled to 30°C or lower. At the optimum pH of 8.0, the enzyme is inactivated by dilution, but it is stable at pH 6.5 even at very low concentrations. Also,P. aeruginosaBADH activity is rapidly lost on removal of K+. In all cases studied, inactivation involves a biphasic process, which was dependent on the enzyme concentration only in the case of inactivation by dilution. NADP+considerably protected the enzyme against these inactivating conditions.
IS
1634
, a Novel Insertion Element Creating Long, Variable-Length Direct Repeats Which Is Specific for
Mycoplasma mycoides
subsp.
mycoides
Small-Colony Type
Authors: Vilei Edy M. , Nicolet Jacques , Frey Joachim 
Published: December 2019

ABSTRACT

A new insertion sequence, IS
1634
, has been identified in
Mycoplasma mycoides
subsp.
mycoides
small-colony type (SC). IS
1634
shows structural and functional similarities to IS
1549
of
Mycobacterium smegmatis
and with it seems to form a new class or family of insertion sequences. IS
1634
has a size of 1,872 bp, including two 13-bp terminal inverted repeats. It contains an open reading frame (ORF) encoding a product of 533 amino acids which shows similarity to the transposase of IS
1549
and to a lesser extent to the transposases of IS elements of the IS
4
family. IS
1634
is present at about 30 copies in the genome of all 22 different field strains of
M. mycoides
subsp.
mycoides
SC tested. Characteristic of IS
1634
are the long and variable-length direct repeats at the sites of insertion which were found to reach up to about 500 bp. IS
1634
is specific to
M. mycoides
subsp.
mycoides
SC and is not present in any of the other members of the
M. mycoides
cluster. Neither was it found in other closely related
Mycoplasma
species of ruminants.
Natural Competence for DNA Transformation by
Legionella pneumophila
and Its Association with Expression of Type IV Pili
Authors: Stone Barbara J. , Kwaik Yousef Abu 
Published: December 2019

ABSTRACT

We have recently described the expression of two pili of different lengths on the surface of
Legionella pneumophila
(B. J. Stone and Y. Abu Kwaik, Infect. Immun. 66:1768–1775, 1998). Production of long pili requires a functional
pilE
L
locus, encoding a type IV pilin protein. Since type IV pili in
Neisseria gonorrhoeae
are associated with competence for DNA transformation, we examined the competence of
L. pneumophila
for DNA transformation under conditions that allowed the expression of type IV pili. We show that
L. pneumophila
is naturally competent for DNA transformation by isogenic chromosomal DNA and by plasmid DNA containing
L. pneumophila
DNA. Many different
L. pneumophila
loci are able to transform
L. pneumophila
after addition of plasmid DNA, including
gspA
,
ppa
,
asd
, and
pilE
L
. The transformation frequency is reduced when competing DNA containing either
L. pneumophila
DNA or vector sequences is added to the bacteria, suggesting that uptake-specific sequences may not be involved in DNA uptake. Competence for DNA transformation correlates with expression of the type IV pili, and a
pilE
L
mutant defective in expression of type IV pili is not competent for DNA transformation. Complementation of the mutant for competence is restored by the reintroduction of a cosmid that restores production of type IV pili. Minimal competence is restored to the mutant by introduction of
pilE
L
alone. We conclude that competence for DNA transformation in
L. pneumophila
is associated with expression of the type IV pilus and results in recombination of
L. pneumophila
DNA into the chromosome. Since expression of type IV pili also facilitates attachment of
L. pneumophila
to mammalian cells and protozoa, we designated the type IV pili CAP (for competence- and adherence-associated pili).
Mutational Analysis of
Bacillus subtilis
Glutamine Phosphoribosylpyrophosphate Amidotransferase Propeptide Processing
Authors: Li Songtao , Smith Janet L. , Zalkin Howard 
Published: December 2019

ABSTRACT

Glutamine phosphoribosylpyrophosphate amidotransferase from
Bacillus subtilis
is a member of an N-terminal nucleophile hydrolase enzyme superfamily, several of which undergo autocatalytic propeptide processing to generate the mature active enzyme. A series of mutations was analyzed to determine whether amino acid residues required for catalysis are also used for propeptide processing. Propeptide cleavage was strongly inhibited by replacement of the cysteine nucleophile and two residues of an oxyanion hole that are required for glutaminase function. However, significant propeptide processing was retained in a deletion mutant with multiple defects in catalysis that was devoid of enzyme activity. Intermolecular processing of noncleaved mutant enzyme subunits by active wild-type enzyme subunits was not detected in hetero-oligomers obtained from a coexpression experiment. While direct in vitro evidence for autocatalytic propeptide cleavage was not obtained, the results indicate that some but not all of the amino acid residues that have a role in catalysis are also needed for propeptide processing.
Cloning and Expression of the
algL
Gene, Encoding the
Azotobacter chroococcum
Alginate Lyase: Purification and Characterization of the Enzyme
Authors: Peciña Ana , Pascual Alberto , Paneque Antonio 
Published: December 2019

ABSTRACT

The alginate lyase-encoding gene (
algL
) of
Azotobacter chroococcum
was localized to a 3.1-kb
Eco
RI DNA fragment that revealed an open reading frame of 1,116 bp. This open reading frame encodes a protein of 42.98 kDa, in agreement with the value previously reported by us for this protein. The deduced protein has a potential N-terminal signal peptide that is consistent with its proposed periplasmic location. The analysis of the deduced amino acid sequence indicated that the gene sequence has a high homology (90% identity) to the
Azotobacter vinelandii
gene sequence, which has very recently been deposited in the GenBank database, and that it has 64% identity to the
Pseudomonas aeruginosa
gene sequence but that it has rather low homology (15 to 22% identity) to the gene sequences encoding alginate lyase in other bacteria. The
A. chroococcum
AlgL protein was overproduced in
Escherichia coli
and purified to electrophoretic homogeneity in a two-step chromatography procedure on hydroxyapatite and phenyl-Sepharose. The kinetic and molecular parameters of the recombinant alginate lyase are similar to those found for the native enzyme.
Inhibition of Translation and Cell Growth by Minigene Expression
Authors: Tenson Tanel , Herrera Jesús Vega , Kloss Patricia , Guarneros Gabriel , Mankin Alexander S. 
Published: December 2019

ABSTRACT
A random five-codon gene library was used to isolate minigenes whose expression causes cell growth arrest. Eight different deleterious minigenes were isolated, five of which had in-frame stop codons; the predicted expressed peptides ranged in size from two to five amino acids. Mutational analysis demonstrated that translation of the inhibitory minigenes is essential for growth arrest. Pulse-labeling experiments showed that expression of at least some of the selected minigenes results in inhibition of cellular protein synthesis. Expression of the deleterious minigenes in cells deficient in peptidyl-tRNA hydrolase causes accumulation of families of peptidyl-tRNAs corresponding to the last minigene codon; the inhibitory action of minigene expression could be suppressed by overexpression of the tRNA corresponding to the last sense codon in the minigene. Experimental data are compatible with the model that the deleterious effect of minigene expression is mediated by depletion of corresponding pools of free tRNAs.
A Cold Shock-Induced Cyanobacterial RNA Helicase
Authors: Chamot Danuta , Magee Wendy C. , Yu Esther , Owttrim George W. 
Published: December 2019

ABSTRACT

The ability to modify RNA secondary structure is crucial for numerous cellular processes. We have characterized two RNA helicase genes,
crhB
and
crhC
, which are differentially expressed in the cyanobacterium
Anabaena
sp. strain PCC 7120.
crhC
transcription is limited specifically to cold shock conditions while
crhB
is expressed under a variety of conditions, including enhanced expression in the cold. This implies that both RNA helicases are involved in the cold acclimation process in cyanobacteria; however, they presumably perform different roles in this adaptation. Although both CrhB and CrhC belong to the DEAD box subfamily of RNA helicases, CrhC encodes a novel RNA helicase, as the highly conserved SAT motif is modified to FAT. This alteration may affect CrhC function and its association with specific RNA targets and/or accessory proteins, interactions required for cold acclimation. Primer extension and analysis of the 5′ untranslated region of
crhC
revealed the transcriptional start site, as well as a number of putative cold shock-responsive elements. The potential role(s) performed by RNA helicases in the acclimation of cyanobacteria to cold shock is discussed.
Physiological States of Individual
Salmonella typhimurium
Cells Monitored by In Situ Reverse Transcription-PCR
Authors: Holmstrøm Kim , Tolker-Nielsen Tim , Molin Søren 
Published: December 2019

ABSTRACT

The possibility of using levels of specific mRNAs in individual bacteria as indicators of single-cell physiology was investigated. Estimates of the numbers of
groEL
and
tsf
mRNAs per cell in
Salmonella typhimurium
cells in different physiological states were obtained by Northern analysis. The average number of
groEL
mRNAs per cell was estimated to be 22 in fast-growing cultures and 197 in heat-shocked cultures. The average number of
tsf
mRNAs per cell was estimated to be 37 in fast-growing cultures, 4 in slow-growing cultures, and 0 in nongrowing cultures. The potential of mRNA-targeted in situ reverse transcription (RT)-PCR to monitor quantitatively different levels of
groEL
and
tsf
mRNA in individual cells and thus monitor both specific gene induction and general growth activity was assessed. Neither
groEL
nor
tsf
mRNA was present in stationary-phase cells, but it was shown that stationary-phase cells contain other RNA species at high levels, which may provide a possibility for monitoring directly stationary-phase individual cells by the use of in situ RT-PCR. The outcome of the in situ RT-PCR analyses indicated that a population of fast-growing cells is heterogeneous with respect to
groEL
mRNA single-cell contents, suggesting a cell-cycle-controlled expression of
groEL
in
S. typhimurium
, whereas a fast-growing culture is homogeneous with respect to
tsf
mRNA single-cell contents, suggesting that the level of
tsf
mRNA is relatively constant during the cell cycle.
Functional Identification of the Product of the
Bacillus subtilis yvaL
Gene as a SecG Homologue
Authors: van Wely Karel H. M. , Swaving Jelto , Broekhuizen Cees P. , Rose Matthias , Quax Wim J. , Driessen Arnold J. M. 
Published: December 2019

ABSTRACT

Protein export in
Escherichia coli
is mediated by translocase, a multisubunit membrane protein complex with SecA as the peripheral subunit and the SecY, SecE, and SecG proteins as the integral membrane domain. In the gram-positive bacterium
Bacillus subtilis
, SecA, SecY, and SecE have been identified through genetic analysis. Sequence comparison of the
Bacillus
chromosome identified a potential homologue of SecG, termed YvaL. A chromosomal disruption of the
yvaL
gene results in mild cold sensitivity and causes a β-lactamase secretion defect. The cold sensitivity is exacerbated by overexpression of the secretory protein α-amylase, whereas growth and β-lactamase secretion are restored by coexpression of
yvaL
or the
E. coli secG
gene. These results indicate that the
yvaL
gene codes for a protein that is functionally homologous to SecG.
Role of ArgR in Activation of the
ast
Operon, Encoding Enzymes of the Arginine Succinyltransferase Pathway in
Salmonella typhimurium
Authors: Lu Chung-Dar , Abdelal Ahmed T. 
Published: December 2019

ABSTRACT

The
ast
operon, encoding enzymes of the arginine succinyltransferase (AST) pathway, was cloned from
Salmonella typhimurium
, and the nucleotide sequence for the upstream flanking region was determined. The control region contains several regulatory consensus sequences, including binding sites for NtrC, cyclic AMP receptor protein (CRP), and ArgR. The results of DNase I footprintings and gel retardation experiments confirm binding of these regulatory proteins to the identified sites. Exogenous arginine induced AST under nitrogen-limiting conditions, and this induction was abolished in an
argR
derivative. AST was also induced under carbon starvation conditions; this induction required functional CRP as well as functional ArgR. The combined data are consistent with the hypothesis that binding of one or more ArgR molecules to a region between the upstream binding sites for NtrC and CRP and two putative promoters plays a pivotal role in modulating expression of the
ast
operon in response to nitrogen or carbon limitation.
Mutational Analysis of the
phoD
Promoter in
Bacillus subtilis
: Implications for PhoP Binding and Promoter Activation of Pho Regulon Promoters
Authors: Eder Steve , Liu Wei , Hulett F. Marion 
Published: December 2019

ABSTRACT

The PhoP-PhoR two-component regulatory system controls the phosphate deficiency response in
B. subtilis
. A number of Pho regulon genes which require PhoP∼P for activation or repression have been identified. The studies reported here were initiated to understand the PhoP-DNA interaction necessary for Pho promoter regulation. The regulatory region of
phoD
was characterized in detail using oligo-directed mutagenesis, DNase I footprinting, and in vivo transcription assays. These data reveal basic principles of PhoP binding relevant to PhoP’s interaction with other Pho regulon promoters. Our results show that: (i) a dimer of PhoP∼P is able to bind two consensus repeats in a stable fashion; (ii) PhoP binding is highly cooperative within the core promoter region, which is located from −66 to −17 on the coding strand and contains four TT(A/T/C)ACA-like repeats; (iii) specific bases comprising the TT(A/T/C)ACA consensus are essential for transcriptional activation, but the specific base pairs of the intervening sequences separating the consensus repeats are not important for either PhoP binding or promoter activation; (iv) the spacing between two consensus repeats within a putative dimer binding site in the core region is important for both PhoP binding and promoter activation; (v) the exact spacing between two dimer binding sites within the core region is important for promoter activation but less so for PhoP binding affinity, as long as the repeats are on the same face of the helix; and (vi) the 5′ secondary binding region is important for coordinated PhoP binding to the core binding region, making it nearly essential for promoter activation.
Role of BkdR, a Transcriptional Activator of the SigL-Dependent Isoleucine and Valine Degradation Pathway in
Bacillus subtilis
Authors: Debarbouille Michel , Gardan Rozenn , Arnaud Maryvonne , Rapoport George 
Published: December 2019

ABSTRACT

A new gene,
bkdR
(formerly called
yqiR
), encoding a regulator with a central (catalytic) domain was found in
Bacillus subtilis
. This gene controls the utilization of isoleucine and valine as sole nitrogen sources. Seven genes, previously called
yqiS
,
yqiT
,
yqiU
,
yqiV
,
bfmBAA
,
bfmBAB
, and
bfmBB
and now referred to as
ptb
,
bcd
,
buk
,
lpd
,
bkdA1
,
bkdA2
, and
bkdB
, are located downstream from the
bkdR
gene in
B. subtilis
. The products of these genes are similar to phosphate butyryl coenzyme A transferase, leucine dehydrogenase, butyrate kinase, and four components of the branched-chain keto acid dehydrogenase complex: E3 (dihydrolipoamide dehydrogenase), E1α (dehydrogenase), E1β (decarboxylase), and E2 (dihydrolipoamide acyltransferase). Isoleucine and valine utilization was abolished in
bcd
and
bkdR
null mutants of
B. subtilis
. The seven genes appear to be organized as an operon,
bkd
, transcribed from a −12, −24 promoter. The expression of the
bkd
operon was induced by the presence of isoleucine or valine in the growth medium and depended upon the presence of the sigma factor SigL, a member of the sigma 54 family. Transcription of this operon was abolished in strains containing a null mutation in the regulatory gene
bkdR
. Deletion analysis showed that upstream activating sequences are involved in the expression of the
bkd
operon and are probably the target of
bkdR
. Transcription of the
bkd
operon is also negatively controlled by CodY, a global regulator of gene expression in response to nutritional conditions.
Analysis of the Role of
trans
-Translation in the Requirement of tmRNA for λ
imm
P22
Growth in
Escherichia coli
Authors: Withey Jeffrey , Friedman David 
Published: December 2019

ABSTRACT

The small, stable RNA molecule encoded by
ssrA
, known as tmRNA or 10Sa RNA, is required for the growth of certain hybrid λ
imm
P22
phages in
Escherichia coli
. tmRNA has been shown to tag partially synthesized proteins for degradation in vivo by attaching a short peptide sequence, encoded by tmRNA, to the carboxyl termini of these proteins. This tag sequence contains, at its C terminus, an amino acid sequence that is recognized by cellular proteases and leads to degradation of tagged proteins. A model describing this function of tmRNA, the
trans
-translation model (K. C. Keiler, P. R. Waller, and R. T. Sauer, Science 271:990–993, 1996), proposes that tmRNA acts first as a tRNA and then as a mRNA, resulting in release of the original mRNA template from the ribosome and translocation of the nascent peptide to tmRNA. Previous work from this laboratory suggested that tmRNA may also interact specifically with DNA-binding proteins, modulating their activity. However, more recent results indicate that interactions between tmRNA and DNA-binding proteins are likely nonspecific. In light of this new information, we examine the effects on λ
imm
P22
growth of mutations eliminating activities postulated to be important for two different steps in the
trans
-translation model, alanine charging of tmRNA and degradation of tagged proteins. This mutational analysis suggests that, while charging of tmRNA with alanine is essential for λ
imm
P22
growth in
E. coli
, degradation of proteins tagged by tmRNA is required only to achieve optimal levels of phage growth. Based on these results, we propose that
trans
-translation may have two roles, the primary role being the release of stalled ribosomes from their mRNA template and the secondary role being the tagging of truncated proteins for degradation.