bluuebunny/crossref_metadata_2025_split huggingface dataset:
Characterization of the
ssnA
Gene, Which Is Involved in the Decline of Cell Viability at the Beginning of Stationary Phase in
Escherichia coli
Authors: Yamada Mamoru , Talukder Ali Azam , Nitta Takeshi 
Published: December 2019

ABSTRACT

When grown in rich medium,
Escherichia coli
exhibits a drastic reduction of the number of viable cells at the beginning of stationary phase. The decline of cell viability was retarded by disruption of the
ssnA
gene, which was identified as a gene subject to RpoS-dependent negative regulation. Moreover,
ssnA
expression was induced at the time of decline of cell viability at early stationary phase. The viability decline was augmented in the
rpoS
background, and this augmentation was suppressed by
ssnA
mutation. Cloning of the
ssnA
gene in a multicopy plasmid, pBR322, caused small colony formation and slow growth in liquid medium. Cells harboring the
ssnA
clone showed aberrant morphology that included enlarged and filamentous shapes. The gene product was identified as a 44-kDa soluble protein, but its function could not be deduced by homology searching. From these results, we conclude that
ssnA
is expressed in response to a phase-specific signal(s) and that its expression level is controlled by RpoS, by a mechanism which may contribute to determination of cell number in the stationary phase.
Nucleoid-Independent Identification of Cell Division Sites in
Escherichia coli
Authors: Cook William R. , Rothfield Lawrence I. 
Published: December 2019

ABSTRACT

The mechanism used by
Escherichia coli
to determine the correct site for cell division is unknown. In this report, we have attempted to distinguish between a model in which septal position is determined by the position of the nucleoids and a model in which septal position is predetermined by a mechanism that does not involve nucleoid position. To do this, filaments with extended nucleoid-free regions adjacent to the cell poles were produced by simultaneous inactivation of cell division and DNA replication. The positions of septa that formed within the nucleoid-free zones after division was allowed to resume were then analyzed. The results showed that septa were formed at a uniform distance from cell poles when division was restored, with no relation to the distance from the nearest nucleoid. In some cells, septa were formed directly over nucleoids. These results are inconsistent with models that invoke nucleoid positioning as the mechanism for determining the site of division site formation.
Site-Directed Mutagenesis of Loop L3 of Sucrose Porin ScrY Leads to Changes in Substrate Selectivity
Authors: Ulmke Christine , Kreth Jens , Lengeler Joseph W. , Welte Wolfram , Schmid Kurt 
Published: December 2019

ABSTRACT
The difference in substrate selectivity of the maltodextrin (LamB) and sucrose (ScrY) porins is attributed mainly to differences in loop L3, which is supposed to constrict the lumen of the pores. We show that even a single mutation (D201Y) in loop L3 leads to a narrowing of the substrate range of ScrY to that resembling LamB. In addition, we removed the putative N-terminal coiled-coil structure of ScrY and studied the effect of this deletion on sucrose transport.
Sugar- and Nitrogen-Dependent Regulation of an
Amanita muscaria
Phenylalanine Ammonium Lyase Gene
Authors: Nehls U. , Ecke M. , Hampp R. 
Published: December 2019

ABSTRACT
The cDNA of a key enzyme of secondary metabolism, phenylalanine ammonium lyase, was identified for an ectomycorrhizal fungus by differential screening of a mycorrhizal library. The gene was highly expressed in hyphae grown at low external monosaccharide concentrations, but its expression was 30-fold reduced at elevated concentrations. Gene repression was regulated by hexokinase.
A Missense Mutation Accounts for the Defect in the Glycerol-3-Phosphate Acyltransferase Expressed in the
plsB26
Mutant
Authors: Heath Richard J. , Rock Charles O. 
Published: December 2019

ABSTRACT

The
sn
-glycerol-3-phosphate acyltransferase (
plsB
) catalyzes the first step in membrane phospholipid formation. A conditional
Escherichia coli
mutant (
plsB26
) has a single missense mutation (G1045A) predicting the expression of an acyltransferase with an Ala349Thr substitution. The PlsB26 protein had a significantly reduced glycerol-3-phosphate acyltransferase specific activity coupled with an elevated

K
m

for glycerol-3-phosphate.
Expression of the
Methanobacterium thermoautotrophicum hpt
Gene, Encoding Hypoxanthine (Guanine) Phosphoribosyltransferase, in
Escherichia coli
Authors: Sauer Jørgen , Nygaard Per 
Published: December 2019

ABSTRACT

The
hpt
gene from the archaeon
Methanobacterium thermoautotrophicum
, encoding hypoxanthine (guanine) phosphoribosyltransferase, was cloned by functional complementation into
Escherichia coli
. The
hpt
-encoded amino acid sequence is most similar to adenine phosphoribosyltransferases, but the encoded enzyme has activity only with hypoxanthine and guanine. The synthesis of the recombinant enzyme is apparently limited by the presence of the rare arginine codons AGA and AGG and the rare isoleucine AUA codon on the
hpt
gene. The recombinant enzyme was purified to apparent homogeneity.
Chromosome Methylation and Measurement of Faithful, Once and Only Once per Cell Cycle Chromosome Replication in
Caulobacter crescentus
Authors: Marczynski Gregory T. 
Published: December 2019

ABSTRACT

Caulobacter crescentus
exhibits cell-type-specific control of chromosome replication and DNA methylation. Asymmetric cell division yields a replicating stalked cell and a nonreplicating swarmer cell. The motile swarmer cell must differentiate into a sessile stalked cell in order to replicate and execute asymmetric cell division. This program of cell division implies that chromosome replication initiates in the stalked cell only once per cell cycle. DNA methylation is restricted to the predivisional cell stage, and since DNA synthesis produces an unmethylated nascent strand, late DNA methylation also implies that DNA near the replication origin remains hemimethylated longer than DNA located further away. In this report, both assumptions are tested with an engineered Tn
5
-based transposon, Tn
5
Ω-MP. This allows a sensitive Southern blot assay that measures fully methylated, hemimethylated, and unmethylated DNA duplexes. Tn
5
Ω-MP was placed at 11 sites around the chromosome and it was clearly demonstrated that Tn
5
Ω-MP DNA near the replication origin remained hemimethylated longer than DNA located further away. One Tn
5
Ω-MP placed near the replication origin revealed small but detectable amounts of unmethylated duplex DNA in replicating stalked cells. Extra DNA synthesis produces a second unmethylated nascent strand. Therefore, measurement of unmethylated DNA is a critical test of the “once and only once per cell cycle” rule of chromosome replication in
C. crescentus
. Fewer than 1 in 1,000 stalked cells prematurely initiate a second round of chromosome replication. The implications for very precise negative control of chromosome replication are discussed with respect to the bacterial cell cycle.
acs1
of
Haemophilus influenzae
Type a Capsulation Locus Region II Encodes a Bifunctional Ribulose 5-Phosphate Reductase– CDP-Ribitol Pyrophosphorylase
Authors: Follens Anja , Veiga-da-Cunha Maria , Merckx Rita , van Schaftingen Emile , van Eldere Johan 
Published: December 2019

ABSTRACT

The serotype-specific, 5.9-kb region II of the
Haemophilus influenzae
type a capsulation locus was sequenced and found to contain four open reading frames termed
acs1
to
acs4
. Acs1 was 96% identical to
H. influenzae
type b Orf1, previously shown to have CDP-ribitol pyrophosphorylase activity (J. Van Eldere, L. Brophy, B. Loynds, P. Celis, I. Hancock, S. Carman, J. S. Kroll, and E. R. Moxon, Mol. Microbiol. 15:107–118, 1995). Low but significant homology to other pyrophosphorylases was only detected in the N-terminal part of Acs1, whereas the C-terminal part was homologous to several short-chain dehydrogenases/reductases, suggesting that Acs1 might be a bifunctional enzyme. To test this hypothesis,
acs1
was cloned in an expression vector and overexpressed in
Escherichia coli
. Cells expressing this protein displayed both ribitol 5-phosphate dehydrogenase and CDP-ribitol pyrophosphorylase activities, whereas these activities were not detectable in control cells. Acs1 was purified to near homogeneity and found to copurify with ribitol 5-phosphate dehydrogenase and CDP-ribitol pyrophosphorylase activities. These had superimposable elution profiles from DEAE-Sepharose and Blue-Sepharose columns. The dehydrogenase activity was specific for ribulose 5-phosphate and NADPH in one direction and for ribitol 5-phosphate and NADP
+
in the other direction and was markedly stimulated by CTP. The pyrophosphorylase showed activity with CTP and ribitol 5-phosphate or arabitol 5-phosphate. We conclude that
acs1
encodes a bifunctional enzyme that converts ribulose 5-phosphate into ribitol 5-phosphate and further into CDP-ribitol, which is the activated precursor form for incorporation of ribitol 5-phosphate into the
H. influenzae
type a capsular polysaccharide.
Role of Acid pH and Deficient Efflux of Pyrazinoic Acid in Unique Susceptibility of
Mycobacterium tuberculosis
to Pyrazinamide
Authors: Zhang Ying , Scorpio Angelo , Nikaido Hiroshi , Sun Zhonghe 
Published: December 2019

ABSTRACT

Pyrazinamide (PZA) is an important antituberculosis drug. Unlike most antibacterial agents, PZA, despite its remarkable in vivo activity, has no activity against
Mycobacterium tuberculosis
in vitro except at an acidic pH.
M. tuberculosis
is uniquely susceptible to PZA, but other mycobacteria as well as nonmycobacteria are intrinsically resistant. The role of acidic pH in PZA action and the basis for the unique PZA susceptibility of
M. tuberculosis
are unknown. We found that in
M. tuberculosis
, acidic pH enhanced the intracellular accumulation of pyrazinoic acid (POA), the active derivative of PZA, after conversion of PZA by pyrazinamidase. In contrast, at neutral or alkaline pH, POA was mainly found outside
M. tuberculosis
cells. PZA-resistant
M. tuberculosis
complex organisms did not convert PZA into POA. Unlike
M. tuberculosis
, intrinsically PZA-resistant
M. smegmatis
converted PZA into POA, but it did not accumulate POA even at an acidic pH, due to a very active POA efflux mechanism. We propose that a deficient POA efflux mechanism underlies the unique susceptibility of
M. tuberculosis
to PZA and that the natural PZA resistance of
M. smegmatis
is due to a highly active efflux pump. These findings may have implications with regard to the design of new antimycobacterial drugs.
Degradation of 1,2-Dibromoethane by
Mycobacterium
sp. Strain GP1
Authors: Poelarends Gerrit J. , van Hylckama Vlieg Johan E. T. , Marchesi Julian R. , Freitas Dos Santos Luisa M. , Janssen Dick B. 
Published: December 2019

ABSTRACT

The newly isolated bacterial strain GP1 can utilize 1,2-dibromoethane as the sole carbon and energy source. On the basis of 16S rRNA gene sequence analysis, the organism was identified as a member of the subgroup which contains the fast-growing mycobacteria. The first step in 1,2-dibromoethane metabolism is catalyzed by a hydrolytic haloalkane dehalogenase. The resulting 2-bromoethanol is rapidly converted to ethylene oxide by a haloalcohol dehalogenase, in this way preventing the accumulation of 2-bromoethanol and 2-bromoacetaldehyde as toxic intermediates. Ethylene oxide can serve as a growth substrate for strain GP1, but the pathway(s) by which it is further metabolized is still unclear. Strain GP1 can also utilize 1-chloropropane, 1-bromopropane, 2-bromoethanol, and 2-chloroethanol as growth substrates. 2-Chloroethanol and 2-bromoethanol are metabolized via ethylene oxide, which for both haloalcohols is a novel way to remove the halide without going through the corresponding acetaldehyde intermediate. The haloalkane dehalogenase gene was cloned and sequenced. The dehalogenase (DhaA
f
) encoded by this gene is identical to the haloalkane dehalogenase (DhaA) of
Rhodococcus rhodochrous
NCIMB 13064, except for three amino acid substitutions and a 14-amino-acid extension at the C terminus. Alignments of the complete dehalogenase gene region of strain GP1 with DNA sequences in different databases showed that a large part of a
dhaA
gene region, which is also present in
R. rhodochrous
NCIMB 13064, was fused to a fragment of a haloalcohol dehalogenase gene that was identical to the last 42 nucleotides of the
hheB
gene found in
Corynebacterium
sp. strain N-1074.
Disruption and Analysis of the
clpB
,
clpC
, and
clpE
Genes in
Lactococcus lactis
: ClpE, a New Clp Family in Gram-Positive Bacteria
Authors: Ingmer Hanne , Vogensen Finn K. , Hammer Karin , Kilstrup Mogens 
Published: December 2019

ABSTRACT

In the genome of the gram-positive bacterium
Lactococcus lactis
MG1363, we have identified three genes (
clpC
,
clpE
, and
clpB
) which encode Clp proteins containing two conserved ATP binding domains. The proteins encoded by two of the genes belong to the previously described ClpB and ClpC families. The
clpE
gene, however, encodes a member of a new Clp protein family that is characterized by a short N-terminal domain including a putative zinc binding domain (-CX
2
CX
22
CX
2
C-). Expression of the 83-kDa ClpE protein as well as of the two proteins encoded by
clpB
was strongly induced by heat shock and, while
clpC
mRNA synthesis was moderately induced by heat, we were unable to identify the ClpC protein. When we analyzed mutants with disruptions in
clpB
,
clpC
, or
clpE
, we found that although the genes are part of the
L. lactis
heat shock stimulon, the mutants responded like wild-type cells to heat and salt treatments. However, when exposed to puromycin, a tRNA analogue that results in the synthesis of truncated, randomly folded proteins,
clpE
mutant cells formed smaller colonies than wild-type cells and
clpB
and
clpC
mutant cells. Thus, our data suggest that ClpE, along with ClpP, which recently was shown to participate in the degradation of randomly folded proteins in
L. lactis
, could be necessary for degrading proteins generated by certain types of stress.
Iron Reductase for Magnetite Synthesis in the Magnetotactic Bacterium
Magnetospirillum magnetotacticum
Authors: Noguchi Yasushi , Fujiwara Taketomo , Yoshimatsu Katsuhiko , Fukumori Yoshihiro 
Published: December 2019

ABSTRACT

Ferric iron reductase was purified from magnetotactic bacterium
Magnetospirillum
(formerly
Aquaspirillum
)
magnetotacticum
(ATCC 31632) to an electrophoretically homogeneous state. The enzyme was loosely bound on the cytoplasmic face of the cytoplasmic membrane and was found more frequently in magnetic cells than in nonmagnetic cells. The molecular mass of the purified enzyme was calculated upon sodium dodecyl sulfate-polyacrylamide gel electrophoresis to be about 36 kDa, almost the same as that calibrated by gel filtration analysis. The enzyme required NADH and flavin mononucleotide (FMN) as optimal electron donor and cofactor, respectively, and the activity was strongly inhibited by Zn
2+
acting as a partial mixed-type inhibitor. The

K
m

values for NADH and FMN were 4.3 and 0.035 μM, respectively, and the

K
i

values for Zn
2+
were 19.2 and 23.9 μM for NADH and FMN, respectively. When the bacterium was grown in the presence of ZnSO
4
, the magnetosome number in the cells and the ferric iron reductase activity declined in parallel with an increase in the ZnSO
4
concentration of the medium, suggesting that the ferric iron reductase purified in the present study may participate in magnetite synthesis.
A Periplasmic
d
-Alanyl-
d
-Alanine Dipeptidase in the Gram-Negative Bacterium
Salmonella enterica
Authors: Hilbert Friederike , del Portillo Francisco García , Groisman Eduardo A. 
Published: December 2019

ABSTRACT

The VanX protein is a
d
-alanyl-
d
-alanine (
d
-Ala-
d
-Ala) dipeptidase essential for resistance to the glycopeptide antibiotic vancomycin. While this enzymatic activity has been typically associated with vancomycin- and teicoplainin-resistant enterococci, we now report the identification of a
d
-Ala-
d
-Ala dipeptidase in the gram-negative species
Salmonella enterica
. The
Salmonella
enzyme is only 36% identical to VanX but exhibits a similar substrate specificity: it hydrolyzes
d
-Ala-
d
-Ala,
dl
-Ala-
dl
-Phe, and
d
-Ala-Gly but not the tripeptides
d
-Ala-
d
-Ala-
d
-Ala and
dl
-Ala-
dl
-Lys-Gly or the dipeptides
l
-Ala-
l
-Ala,
N
-acetyl-
d
-Ala-
d
-Ala, and
l
-Leu-Pro. The
Salmonella
dipeptidase gene, designated
pcgL
, appears to have been acquired by horizontal gene transfer because
pcgL
-hybridizing sequences were not detected in related bacterial species and the G+C content of the
pcgL
-containing region (41%) is much lower than the overall G+C content of the
Salmonella
chromosome (52%). In contrast to wild-type
Salmonella
, a
pcgL
mutant was unable to use
d
-Ala-
d
-Ala as a sole carbon source. The
pcgL
gene conferred
d
-Ala-
d
-Ala dipeptidase activity upon
Escherichia coli
K-12 but did not allow growth on
d
-Ala-
d
-Ala. The PcgL protein localizes to the periplasmic space of
Salmonella
, suggesting that this dipeptidase participates in peptidoglycan metabolism.
Genes Coding for Phosphotransacetylase and Acetate Kinase in
Sinorhizobium meliloti
Are in an Operon That Is Inducible by Phosphate Stress and Controlled by PhoB
Authors: Summers Michael L. , Denton Michael C. , McDermott Timothy R. 
Published: December 2019

ABSTRACT

Recent work in this laboratory has shown that the gene coding for acetate kinase (
ackA
) in
Sinorhizobium meliloti
is up-regulated in response to phosphate limitation. Characterization of the region surrounding
ackA
revealed that it is adjacent to
pta
, which codes for phosphotransacetylase, and that these two genes are part of an operon composed of at least two additional genes in the following order: an open reading frame (
orfA
),
pta
,
ackA
, and the partial sequence of a gene with an inferred peptide that has a high degree of homology to enoyl-ACP reductase (
fabI
). Experiments combining enzyme assays, a chromosomal
lacZ
::
ackA
transcriptional fusion, complementation analysis with cosmid subclones, and the creation of mutations in
pta
and
ackA
all indicated that the
orfA-pta-ackA-fabI
genes are cotranscribed in response to phosphate starvation. Primer extension was used to map the position of the phosphate starvation-inducible transcriptional start sites upstream of
orfA
. The start sites were found to be preceded by a sequence having similarity to PHO boxes from other phosphate-regulated genes in
S. meliloti
and to the consensus PHO box in
Escherichia coli
. Introduction of a
phoB
mutation in the wild-type strain eliminated elevated levels of acetate kinase and phosphotransacetylase activities in response to phosphate limitation and also eliminated the phosphate stress-induced up-regulation of the
ackA
::
lacZ
fusion. Mutations in either
ackA
alone or both
pta
and
ackA
did not affect the nodulation or nitrogen fixation phenotype of
S. meliloti
.
Genetic Organization of the
Escherichia coli
K10 Capsule Gene Cluster: Identification and Characterization of Two Conserved Regions in Group III Capsule Gene Clusters Encoding Polysaccharide Transport Functions
Authors: Clarke Bradley R. , Pearce Rowan , Roberts Ian S. 
Published: December 2019

ABSTRACT

Analysis of the
Escherichia coli
K10 capsule gene cluster identified two regions, regions 1 and 3, conserved between different group III capsule gene clusters. Region 1 encodes homologues of KpsD, KpsM, KpsT, and KpsE proteins, and region 3 encodes homologues of the KpsC and KpsS proteins. An
rfaH
mutation abolished K10 capsule production, suggesting that expression of the K10 capsule was regulated by RfaH in a manner analogous to group II capsule gene clusters. An IS
3
element and a φR73-like prophage, both of which may have played a role in the acquisition of group III capsule gene clusters, were detected flanking the K10 capsule genes.
Cellular Locations of
Pseudomonas syringae
pv. syringae HrcC and HrcJ Proteins, Required for Harpin Secretion via the Type III Pathway
Authors: Deng Wen-Ling , Huang Hsiou-Chen 
Published: December 2019

ABSTRACT

The complete
hrp-hrc-hrmA
cluster of
Pseudomonas syringae
pv. syringae 61 encodes 28 polypeptides. A saprophytic bacterium carrying this cluster is capable of secreting HrpZ—a harpin encoded by
hrpZ
—in an
hrp
-dependent manner, which suggests that this cluster contains sufficient components to assemble functional type III secretion machinery. Sequence data show that HrcJ and HrcC are putative outer membrane proteins, and nonpolar mutagenesis demonstrates they are all required for HrpZ secretion. In this study, we investigated the cellular localization of the HrcC and HrcJ proteins by Triton solubilization, sucrose-gradient isopycnic centrifugation, and immunogold labeling of the bacterial cell surface. Our results indicate that HrcC is indeed an outer membrane protein and that HrcJ is located between both membranes. Their membrane localization suggests that they might be involved in the formation of a supramolecular structure for protein secretion.
The Blue Copper-Containing Nitrite Reductase fromAlcaligenes xylosoxidans: Cloning of thenirAGene and Characterization of the Recombinant Enzyme
Authors: Prudêncio Miguel , Eady Robert R. , Sawers Gary 
Published: December 2019

ABSTRACTThenirAgene encoding the blue dissimilatory nitrite reductase fromAlcaligenes xylosoxidanshas been cloned and sequenced. To our knowledge, this is the first report of the characterization of a gene encoding a blue copper-containing nitrite reductase. The deduced amino acid sequence exhibits a high degree of similarity to other copper-containing nitrite reductases from various bacterial sources. The full-length protein included a 24-amino-acid leader peptide. ThenirAgene was overexpressed inEscherichia coliand was shown to be exported to the periplasm. Purification was achieved in a single step, and analysis of the recombinant Nir enzyme revealed that cleavage of the signal peptide occurred at a position identical to that for the native enzyme isolated fromA. xylosoxidans. The recombinant Nir isolated directly was blue and trimeric and, on the basis of electron paramagnetic resonance spectroscopy and metal analysis, possessed only type 1 copper centers. This type 2-depleted enzyme preparation also had a low nitrite reductase enzyme activity. Incubation of the periplasmic fraction with copper sulfate prior to purification resulted in the isolation of an enzyme with a full complement of type 1 and type 2 copper centers and a high specific activity. The kinetic properties of the recombinant enzyme were indistinguishable from those of the native nitrite reductase isolated fromA. xylosoxidans. This rapid isolation procedure will greatly facilitate genetic and biochemical characterization of both wild-type and mutant derivatives of this protein.