bluuebunny/crossref_metadata_2025_split huggingface dataset:
Regulation of
carAB
Expression in
Escherichia coli
Occurs in Part through UTP-Sensitive Reiterative Transcription
Authors: Han Xiaosi , Turnbough Charles L. 
Published: December 2019

ABSTRACT

In
Escherichia coli
, expression of the
carAB
operon is subject to cumulative repression, which occurs by ArgR-mediated repression at a downstream promoter, P2, and by pyrimidine-mediated regulation at an upstream promoter, P1. In this study, we show that pyrimidine-mediated regulation occurs in part through a mechanism involving UTP-sensitive reiterative transcription (i.e., repetitive addition of U residues to the 3′ end of a nascent transcript due to transcript-template slippage). In this case, reiterative transcription occurs at the end of a run of three T · A base pairs in the initially transcribed region of the
carAB
P1 promoter. The sequence of this region is 5′-GTTTGC (nontemplate strand). In the proposed regulatory mechanism, increased intracellular levels of UTP promote reiterative transcription, which results in the synthesis of transcripts with the sequence GUUUU

n

(where
n
= 1 to >30). These transcripts are not extended downstream to include structural gene sequences. In contrast, lower levels of UTP enhance normal template-directed addition of a G residue at position 5 of the nascent transcript. This addition precludes reiterative transcription and permits normal transcript elongation capable of producing translatable
carAB
transcripts. Thus,
carAB
expression, which is necessary for pyrimidine nucleotide (and arginine) biosynthesis, increases in proportion to the cellular need for UTP. The proposed mechanism appears to function independently of a second pyrimidine-mediated control mechanism that involves the regulatory proteins CarP and integration host factor.
Curli Fibers Are Highly Conserved between
Salmonella typhimurium
and
Escherichia coli
with Respect to Operon Structure and Regulation
Authors: Römling Ute , Bian Zhao , Hammar Mårten , Sierralta Walter D. , Normark Staffan 
Published: December 2019

ABSTRACT

Mouse-virulent
Salmonella typhimurium
strains SR-11 and ATCC 14028-1s express curli fibers, thin aggregative fibers, at ambient temperature on plates as judged by Western blot analysis and electron microscopy. Concomitantly with curli expression, cells develop a rough and dry colony morphology and bind the dye Congo red (called the rdar morphotype). Cloning and characterization of the two divergently transcribed operons required for curli biogenesis,
csgBA(C)
and
csgDEFG
, from
S. typhimurium
SR-11 revealed the same gene order and flanking genes as in
Escherichia coli
. The divergence of the curli region between
S. typhimurium
and
E. coli
at the nucleotide level is above average (22.4%). However, a high level of conservation at the protein level, which ranged from 86% amino acid homology for the fiber subunit CsgA to 99% homology for the lipoprotein CsgG, implies functional constraints on the gene products. Consequently,
S. typhimurium
genes on low-copy-number plasmids were able to complement respective
E. coli
mutants, although not always to wild-type levels.
rpoS
and
ompR
are required for transcriptional activation of (at least) the
csgD
promoter. The high degree of conservation at the protein level and the identical regulation patterns in
E. coli
and
S. typhimurium
suggest similar roles of curli fibers in the same ecological niche in the two species.
Relationships among the O-Antigen Gene Clusters of
Salmonella enterica
Groups B, D1, D2, and D3
Authors: Curd Heather , Liu Dan , Reeves Peter R. 
Published: December 2019

ABSTRACT

The O antigen is an important cell wall antigen of gram-negative bacteria, and the genes responsible for its biosynthesis are located in a gene cluster. We have cloned and sequenced the DNA segment unique to the O-antigen gene cluster of
Salmonella enterica
group D3. This segment includes a novel O-antigen polymerase gene (
wzy
D3
). The polymerase gives α(1→6) linkages but has no detectable sequence similarity to that of group D2, which confers the same linkage. We find the remnant of a D3-like
wzy
gene in the O-antigen gene clusters of groups D1 and B and suggest that this is the original
wzy
gene of these O-antigen gene clusters.
Perturbation of Anion Balance during Inhibition of Growth ofEscherichia coliby Weak Acids
Authors: Roe Andrew J. , McLaggan Debra , Davidson Ian , O’Byrne Conor , Booth Ian R. 
Published: December 2019

ABSTRACTDuring inhibition of cell growth by weak acids, there is substantial accumulation of the weak acid anions in the cytoplasm. This study was undertaken to determine the impact of anion accumulation on cellular pools. At pH 6, growth in the presence of 8 mM acetate led to an internal pool of greater than 240 mM acetate anion and resulted in reduced levels of glutamate in the cell, but there were no significant changes in K+and Na+levels. At low osmolarity, the change in the glutamate pool compensated for only a small fraction of the accumulated acetate anion. However, at high osmolarity, glutamate compensated for over half of the accumulated acetate. Recovery of the normal cytoplasmic pH after the removal of acetate was dependent on the synthesis of glutamate.
Role of
rpoS
in Stress Survival and Virulence of
Vibrio cholerae
Authors: Yildiz Fitnat H. , Schoolnik Gary K. 
Published: December 2019

ABSTRACT

Vibrio cholerae
is known to persist in aquatic environments under nutrient-limiting conditions. To analyze the possible involvement of the alternative sigma factor encoded by
rpoS
, which is shown to be important for survival during nutrient deprivation in several other bacterial species, a
V. cholerae rpoS
homolog was cloned by functional complementation of an
Escherichia coli
mutant by using a wild-type genomic library. Sequence analysis of the complementing clone revealed an 1.008-bp open reading frame which is predicted to encode a 336-amino-acid protein with 71 to 63% overall identity to other reported
rpoS
gene products. To determine the functional role of
rpoS
in
V. cholerae
, we inactivated
rpoS
by homologous recombination.
V. cholerae
strains lacking
rpoS
are impaired in the ability to survive diverse environmental stresses, including exposure to hydrogen peroxide, hyperosmolarity, and carbon starvation. These results suggest that
rpoS
may be required for the persistence of
V. cholerae
in aquatic habitats. In addition, the
rpoS
mutation led to reduced production or secretion of hemagglutinin/protease. However,
rpoS
is not critical for in vivo survival, as determined by an infant mouse intestinal competition assay.
The
apbE
Gene Encodes a Lipoprotein Involved in Thiamine Synthesis in
Salmonella typhimurium
Authors: Beck Brian J. , Downs Diana M. 
Published: December 2019

ABSTRACT

Thiamine pyrophosphate is an essential cofactor that is synthesized de novo in
Salmonella typhimurium
. The biochemical steps and gene products involved in the conversion of aminoimidazole ribotide (AIR), a purine intermediate, to the 4-amino-5-hydroxymethyl-2-methyl pyrimidine (HMP) moiety of thiamine have yet to be elucidated. We have isolated mutations in a new locus (
Escherichia coli
open reading frame designation
yojK
) at 49 min on the
S. typhimurium
chromosome. Two significant phenotypes associated with lesions in this locus (
apbE
) were identified. First,
apbE purF
double mutants require thiamine, specifically the HMP moiety. Second, in the presence of adenine,
apbE
single mutants require thiamine, specifically both the HMP and the thiazole moieties. Together, the phenotypes associated with
apbE
mutants suggest that flux through the purine pathway has a role in regulating synthesis of the thiazole moiety of thiamine and are consistent with ApbE being involved in the conversion of AIR to HMP. The product of the
apbE
gene was found to be a 36-kDa membrane-associated lipoprotein, making it the second membrane protein implicated in thiamine synthesis.
Coordination of Initiation of Nuclear Division and Initiation of Cell Division in
Schizosaccharomyces pombe
: Genetic Interactions of Mutations
Authors: Grallert A. , Grallert B. , Ribar B. , Sipiczki M. 
Published: December 2019

ABSTRACT

sep1
+

encodes a
Schizosaccharomyces pombe
homolog of the HNF-3/forkhead family of the tissue-specific and developmental gene regulators identified in higher eukaryotes. Its mutant allele
sep1-1
causes a defect in cytokinesis and confers a mycelial morphology. Here we report on genetic interactions of
sep1-1
with the M-phase initiation mutations

wee1
−

,
cdc2-1w
, and
cdc25-22
. The double mutants

sep1-1 wee1
−

and
sep1-1 cdc2-1w
form dikaryon cells at high frequency, which is due to nuclear division in the absence of cell division. The dikaryosis is reversible and suppressible by
cdc25-22
. We propose that the genes

wee1
+

,

cdc2
+

,

cdc25
+

, and

sep1
+

form a regulatory link between the initiation of mitosis and the initiation of cell division.
A Major Outer Membrane Protein of
Rahnella aquatilis
Functions as a Porin and Root Adhesin
Authors: Achouak Wafa , Pages Jean-Marie , De Mot Rene , Molle Gerard , Heulin Thierry 
Published: December 2019

ABSTRACT

A 38-kDa major outer membrane protein (OMP) was isolated from the nitrogen-fixing enterobacterium
Rahnella aquatilis
CF3. This protein exists as a stable trimer in the presence of 2% sodium dodecyl sulfate at temperatures below 60°C. Single channel experiments showed that this major OMP of
R. aquatilis
CF3 is able to form pores in the planar lipid membrane. Two oligonucleotides encoding the N-terminal portion of the 38-kDa OMP and C-terminal portion of OmpC were used to amplify the 38-kDa gene by PCR. The deduced amino acid sequence showed a strong homology with
Escherichia coli
,
Klebsiella pneumoniae
,
Salmonella typhi
, and
Serratia marcescens
OmpC sequences, except loops L6 and L7, which are postulated to be cell surface exposed. On the basis of the OmpF-PhoE three-dimensional structure, it seems likely that this 38-kDa organizes three 16-strand β-barrel subunits. The relationship between the structure and the double functionality of this protein as porin and as a root adhesin is discussed.
Regulation of Riboflavin Biosynthesis in
Bacillus subtilis
Is Affected by the Activity of the Flavokinase/Flavin Adenine Dinucleotide Synthetase Encoded by
ribC
Authors: Mack Matthias , van Loon Adolphus P. G. M. , Hohmann Hans-Peter 
Published: December 2019

ABSTRACT

This work shows that the
ribC
wild-type gene product has both flavokinase and flavin adenine dinucleotide synthetase (FAD-synthetase) activities. RibC plays an essential role in the flavin metabolism of
Bacillus subtilis
, as growth of a
ribC
deletion mutant strain was dependent on exogenous supply of FMN and the presence of a heterologous FAD-synthetase gene in its chromosome. Upon cultivation with growth-limiting amounts of FMN, this
ribC
deletion mutant strain overproduced riboflavin, while with elevated amounts of FMN in the culture medium, no riboflavin overproduction was observed. In a
B. subtilis ribC820
mutant strain, the corresponding
ribC820
gene product has reduced flavokinase/FAD-synthetase activity. In this strain, riboflavin overproduction was also repressed by exogenous FMN but not by riboflavin. Thus, flavin nucleotides, but not riboflavin, have an effector function for regulation of riboflavin biosynthesis in
B. subtilis
, and RibC seemingly is not directly involved in the riboflavin regulatory system. The mutation
ribC820
leads to deregulation of riboflavin biosynthesis in
B. subtilis
, most likely by preventing the accumulation of the effector molecule FMN or FAD.
Isolation and Characterization of
Rhodobacter capsulatus
Mutants Affected in Cytochrome
cbb
3
Oxidase Activity
Authors: Koch Hans-Georg , Hwang Olivia , Daldal Fevzi 
Published: December 2019

ABSTRACT

The facultative phototrophic bacterium
Rhodobacter capsulatus
contains only one form of cytochrome (cyt)
c
oxidase, which has recently been identified as a

cbb
3

-type cyt
c
oxidase. This is unlike other related species, such as
Rhodobacter sphaeroides
and
Paracoccus denitrificans
, which contain an additional mitochondrial-like
aa
3
-type cyt
c
oxidase. An extensive search for mutants affected in cyt
c
oxidase activity in
R. capsulatus
led to the isolation of at least five classes of mutants. Plasmids complementing them to a wild-type phenotype were obtained for all but one of these classes from a chromosomal DNA library. The first class of mutants contained mutations within the structural genes (
ccoNOQP
) of the cyt
cbb
3
oxidase. Sequence analysis of these mutants and of the plasmids complementing them revealed that
ccoNOQP
in
R. capsulatus
is not flanked by the oxygen response regulator
fnr
, which is located upstream of these genes in other species. Genetic and biochemical characterizations of mutants belonging to this group indicated that the subunits CcoN, CcoO, and CcoP are required for the presence of an active cyt
cbb
3
oxidase, and unlike in
Bradyrhizobium japonicum
, no active CcoN-CcoO subcomplex was found in
R. capsulatus
. In addition, mutagenesis experiments indicated that the highly conserved open reading frame 277 located adjacent to
ccoNOQP
is required neither for cyt
cbb
3
oxidase activity or assembly nor for respiratory or photosynthetic energy transduction in
R. capsulatus
. The remaining cyt
c
oxidase-minus mutants mapped outside of
ccoNOQP
and formed four additional groups. In one of these groups, a fully assembled but inactive cyt
cbb
3
oxidase was found, while another group had only extremely small amounts of it. The next group was characterized by a pleiotropic effect on all membrane-bound
c
-type cytochromes, and the remaining mutants not complemented by the plasmids complementing the first four groups formed at least one additional group affecting the biogenesis of the cyt
cbb
3
oxidase of
R. capsulatus.
IS
1549
from
Mycobacterium smegmatis
Forms Long Direct Repeats upon Insertion
Authors: Plikaytis Bonnie B. , Crawford Jack T. , Shinnick Thomas M. 
Published: December 2019

ABSTRACT

A new insertion element, IS
1549
, was identified serendipitously from
Mycobacterium smegmatis
LR222 during experiments using a vector designed to detect the excision of IS
6110
from between the promoter region and open reading frame (ORF) of an aminoglycoside phosphotransferase gene. Six of the kanamycin-resistant isolates had a previously unidentified insertion element upstream of the ORF of the
aph
gene. The 1,634-bp sequence contained a single ORF of 504 amino acids with 85% G+C content in the third codon position. The putative protein sequence showed a distant relationship to the transposase of IS
231
, which is a member of the IS
4
family of insertion elements. IS
1549
contains 11-bp terminal inverted repeats and is characterized by the formation of unusually long and variable-length (71- to 246-bp) direct repeats of the target DNA during transposition. Southern blot analysis revealed that five copies of IS
1549
are present in LR222, but not all
M. smegmatis
strains carry this element. Only strains with a 65-kDa antigen gene with a PCR-restriction fragment length polymorphism type identical to that of
M. smegmatis
607 contain IS
1549
. None of 13 other species of
Mycobacterium
tested by PCR with two sets of primers specific for IS
1549
were positive for the expected amplified product.
Production of 1-Kestose in Transgenic Yeast Expressing a Fructosyltransferase from
Aspergillus foetidus
Authors: Rehm Jochen , Willmitzer Lothar , Heyer Arnd G. 
Published: December 2019

ABSTRACT

Sucrose-inducible secretory sucrose:sucrose 1-fructosyltransferase (1-SST) from
Aspergillus foetidus
has been purified and subjected to N-terminal amino acid sequence determination. The enzyme is extensively glycosylated, and the active form is probably represented by a dimer of identical subunits with an apparent molecular mass of 180 kDa as judged from mobility in seminative acrylamide gels. The enzyme catalyzes fructosyl transfer from sucrose to sucrose producing glucose and 1-kestose. Oligosaccharides with a higher degree of polymerization are not obtained with sucrose as the substrate. The cDNA encoding the
A. foetidus
1-SST has been cloned and sequenced. Sequence homology was found to be highest to levanases, but no hydrolytic activity was observed when levan was incubated with the enzyme. Expression of the cloned gene in an invertase-deficient mutant of
Saccharomyces cerevisiae
resulted in 1-kestose production, with 6-kestose and neokestose being side products of the reaction. Products were well distinguishable from those formed by yeast transformants expressing a cytosolic invertase.
Cloning and Sequencing of a 2,5-Dichlorohydroquinone Reductive Dehalogenase Gene Whose Product Is Involved in Degradation of γ-Hexachlorocyclohexane by
Sphingomonas paucimobilis
Authors: Miyauchi Keisuke , Suh Seug-Kyo , Nagata Yuji , Takagi Masamichi 
Published: December 2019

ABSTRACT

Sphingomonas
(formerly
Pseudomonas
)
paucimobilis
UT26 utilizes γ-hexachlorocyclohexane (γ-HCH), a halogenated organic insecticide, as a sole carbon and energy source. In a previous study, we showed that γ-HCH is degraded to 2,5-dichlorohydroquinone (2,5-DCHQ) (Y. Nagata, R. Ohtomo, K. Miyauchi, M. Fukuda, K. Yano, and M. Takagi, J. Bacteriol. 176:3117–3125, 1994). In the present study, we cloned and characterized a gene, designated
linD
, directly involved in the degradation of 2,5-DCHQ. The
linD
gene encodes a peptide of 343 amino acids and has a low level of similarity to proteins which belong to the glutathione
S
-transferase family. When LinD was overproduced in
Escherichia coli
, a 40-kDa protein was found after sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Northern blot analysis revealed that expression of the
linD
gene was induced by 2,5-DCHQ in
S. paucimobilis
UT26. Thin-layer chromatography and gas chromatography-mass spectrometry analyses with the LinD-overexpressing
E. coli
cells revealed that LinD converts 2,5-DCHQ rapidly to chlorohydroquinone (CHQ) and also converts CHQ slowly to hydroquinone. LinD activity in crude cell extracts was increased 3.7-fold by the addition of glutathione. All three of the Tn
5
-induced mutants of UT26, which lack 2,5-DCHQ dehalogenase activity, had rearrangements or a deletion in the
linD
region. These results indicate that LinD is a glutathione-dependent reductive dehalogenase involved in the degradation of γ-HCH by
S. paucimobilis
UT26.
Identification of a Sequence Motif That Confers SecB Dependence on a SecB-Independent Secretory Protein In Vivo
Authors: Kim Jinoh , Kendall Debra A. 
Published: December 2019

ABSTRACT

SecB is a cytosolic chaperone which facilitates the transport of a subset of proteins, including membrane proteins such as PhoE and LamB and some periplasmic proteins such as maltose-binding protein, in
Escherichia coli
. However, not all proteins require SecB for transport, and proteins such as ribose-binding protein are exported efficiently even in SecB-null strains. The characteristics which confer SecB dependence on some proteins but not others have not been defined. To determine the sequence characteristics that are responsible for the SecB requirement, we have inserted a systematic series of short, polymeric sequences into the SecB-independent protein alkaline phosphatase (PhoA). The extent to which these simple sequences convert alkaline phosphatase into a SecB-requiring protein was evaluated in vivo. Using this approach we have examined the roles of the polarity and charge of the sequence, as well as its location within the mature region, in conferring SecB dependence. We find that an insert with as few as 10 residues, of which 3 are basic, confers SecB dependence and that the mutant protein is efficiently exported in the presence of SecB. Remarkably, the basic motifs caused the protein to be translocated in a strict membrane potential-dependent fashion, indicating that the membrane potential is not a barrier to, but rather a requirement for, translocation of the motif. The alkaline phosphatase mutants most sensitive to the loss of SecB are those most sensitive to inhibition of SecA via azide treatment, consistent with the necessity for formation of a preprotein-SecB-SecA complex. Furthermore, the impact of the basic motif depends on location within the mature protein and parallels the accessibility of the location to the secretion apparatus.
Import and Metabolism of Glutathione by
Streptococcus mutans
Authors: Sherrill Christopher , Fahey Robert C. 
Published: December 2019

ABSTRACT

Glutathione (γ-GluCysGly, GSH) is not found in most gram-positive bacteria, but some appear to synthesize it and others, including
Streptococcus mutans
ATCC 33402, import it from their growth medium. Import of oxidized glutathione (GSSG) by
S. mutans
33402 in 7H9 medium was shown to require glucose and to occur with an apparent

K
m

of 18 ± 5 μM. GSSG, GSH,
S
-methylglutathione, and homocysteine-glutathione mixed disulfide (hCySSG) were imported at comparable rates (measured by depletion of substrate in the medium), as was the disulfide of γ-GluCys. In contrast, the disulfide of CysGly was not taken up at a measurable rate, indicating that the γ-Glu residue is important for efficient transport. During incubation with GSSG, little GSSG was detected in cells but GSH and γ-GluCys accumulated during the first 30 min and then declined. No significant intracellular accumulation of Cys or sulfide was found. Transient intracellular accumulation of
d
/
l
-homocysteine, as well as GSH and γ-GluCys, was observed during import of hCySSG. Although substantial levels of GSH were found in cells when
S. mutans
was grown on media containing glutathione, such GSH accumulation had no effect on the growth rate. However, the presence of cellular GSH did protect against growth inhibition by the thiol-oxidizing agent diamide. Import of glutathione by
S. mutans
ATCC 25175, which like strain 33402 does not synthesize glutathione, occurred at a rate comparable to that of strain 33402, but three species which appear to synthesize glutathione (
S. agalactiae
ATCC 12927,
S. pyogenes
ATCC 8668, and
Enterococcus faecalis
ATCC 29212) imported glutathione at negligible or markedly lower rates.
Activation of
Escherichia coli
rRNA Transcription by FIS during a Growth Cycle
Authors: Appleman J. Alex , Ross Wilma , Salomon Julia , Gourse Richard L. 
Published: December 2019

ABSTRACT

rRNA transcription in
Escherichia coli
is activated by the FIS protein, which binds upstream of
rrnp
1
promoters and interacts directly with RNA polymerase. Analysis of the contribution of FIS to
rrn
transcription under changing physiological conditions is complicated by several factors: the wide variation in cellular FIS concentrations with growth conditions, the contributions of several other regulatory systems to rRNA synthesis, and the pleiotropy of
fis
mutations. In this report, we show by in vivo footprinting and Western blot analysis that occupancy of the
rrnBp
1
FIS sites correlates with cellular levels of FIS. We find, using two methods of measurement (pulse induction of a FIS-activated hybrid promoter and primer extension from an unstable transcript made from
rrnBp
1
), that the extent of transcription activation by FIS parallels the degree of FIS site occupancy and therefore cellular FIS levels. FIS activates transcription throughout exponential growth at low culture density, but
rrnp
1
transcription increases independently of FIS immediately following upshift, before FIS accumulates. These results support the model that FIS is one of a set of overlapping signals that together contribute to transcription from
rrnp
1
promoters during steady-state growth.