bluuebunny/crossref_metadata_2025_split huggingface dataset:
ς
K
Can Negatively Regulate
sigE
Expression by Two Different Mechanisms during Sporulation of
Bacillus subtilis
Authors: Zhang Bin , Struffi Paolo , Kroos Lee 
Published: December 2019

ABSTRACT

Temporal and spatial gene regulation during
Bacillus subtilis
sporulation involves the activation and inactivation of multiple sigma subunits of RNA polymerase in a cascade. In the mother cell compartment of sporulating cells, expression of the
sigE
gene, encoding the earlier-acting sigma factor, ς
E
, is negatively regulated by the later-acting sigma factor, ς
K
. Here, it is shown that the negative feedback loop does not require SinR, an inhibitor of
sigE
transcription. Production of ς
K
about 1 h earlier than normal does affect Spo0A, which when phosphorylated is an activator of
sigE
transcription. A mutation in the
spo0A
gene, which bypasses the phosphorelay leading to the phosphorylation of Spo0A, diminished the negative effect of early ς
K
production on
sigE
expression early in sporulation. Also, early production of ς
K
reduced expression of other Spo0A-dependent genes but not expression of the Spo0A-independent
ald
gene. In contrast, both
sigE
and
ald
were overexpressed late in development of cells that fail to make ς
K
. The
ald
promoter, like the
sigE
promoter, is believed to be recognized by ς
A
RNA polymerase, suggesting that ς
K
may inhibit ς
A
activity late in sporulation. To exert this negative effect, ς
K
must be transcriptionally active. A mutant form of ς
K
that associates with core RNA polymerase, but does not direct transcription of a ς
K
-dependent gene, failed to negatively regulate expression of
sigE
or
ald
late in development. On the other hand, the negative effect of early ς
K
production on
sigE
expression early in sporulation did not require transcriptional activity of ς
K
RNA polymerase. These results demonstrate that ς
K
can negatively regulate
sigE
expression by two different mechanisms, one observed when ς
K
is produced earlier than normal, which does not require ς
K
to be transcriptionally active and affects Spo0A, and the other observed when ς
K
is produced at the normal time, which requires ς
K
RNA polymerase transcriptional activity. The latter mechanism facilitates the switch from ς
E
to ς
K
in the cascade controlling mother cell gene expression.
Isolation and Properties of
Enterococcus hirae
Mutants Defective in the Potassium/Proton Antiport System
Authors: Kakinuma Yoshimi , Igarashi Kazuei 
Published: December 2019

ABSTRACT

A K
+
/H
+
antiporter regulates cytoplasmic pH in
Enterococcus hirae
growing at alkaline pH. Mutants defective in this antiport activity were alkaline pH sensitive. One mutant, Pop1, lacked both K
+
/methylamine exchange at pH 9.5 and concomitant acidification of cytoplasmic pH. Pop1 grew well at pHs below 8 but did not at pHs above 9, conditions under which cytoplasmic pH was not fully acidified.
Overexpression ofMethanococcus voltaeFlagellin Subunits inEscherichia coliandPseudomonas aeruginosa: a Source of Archaeal Preflagellin
Authors: Bayley Douglas P. , Jarrell Ken F. 
Published: December 2019

ABSTRACTMethanococcus voltaeis a flagellated member of theArchaea. Four highly similar flagellin genes have previously been cloned and sequenced, and the presence of leader peptides has been demonstrated. While the flagellins ofM. voltaeare predicted from their gene sequences to be approximately 22 to 25 kDa, sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) analysis of purified flagella revealed flagellin subunits with apparent molecular masses of 31 and 33 kDa. Here we describe the expression of aM. voltaeflagellin in the bacteriaEscherichia coliandPseudomonas aeruginosa. Both of these systems successfully generated a specific expression product with an apparently uncleaved leader peptide migrating at approximately 26.5 kDa. This source of preflagellin was used to detect the presence of preflagellin peptidase activity in the membranes ofM. voltae. In addition to the native flagellin, a hybrid flagellin gene containing the sequence encoding theM. voltaeFlaB2 mature protein fused to theP. aeruginosapilin (PilA) leader peptide was constructed and transformed into both wild-typeP. aeruginosaand a prepilin peptidase (pilD) mutant ofP. aeruginosa. Based on migration in SDS-PAGE, the leader peptide appeared to be cleaved in the wild-type cells. However, the archaeal flagellin could not be detected by immunoblotting when expressed in thepilDmutant, indicating a role of the peptidase in the ultimate stability of the fusion product. When the +5 position of the mature flagellin portion of the pilin-flagellin fusion was changed from glycine to glutamic acid (as in theP. aeruginosapilin) and expressed in both wild-type andpilDmutantP. aeruginosa, the product detected by immunoblotting migrated slightly more slowly in thepilDmutant, indicating that the fusion was likely processed by the prepilin peptidase present in the wild type. Potential assembly of the cleaved fusion product by the type IV pilin assembly system in aP. aeruginosaPilA-deficient strain was tested, but no filaments were noted on the cell surface by electron microscopy.
Eikenella corrodens
Phase Variation Involves a Posttranslational Event in Pilus Formation
Authors: Villar Maria T. , Helber Jennifer T. , Hood Becky , Schaefer Michael R. , Hirschberg Rona L. 
Published: December 2019

ABSTRACT

The human pathogen
Eikenella corrodens
synthesizes type IV pili and exhibits a phase variation involving the irreversible transition from piliated to nonpiliated variants. On solid medium, piliated variants form small (S-phase), corroding colonies whereas nonpiliated variants form large (L-phase), noncorroding colonies. We are studying the molecular basis of this phase variation in the clinical isolate
E. corrodens
VA1. A genomic fragment encoding the major type IV pilin was cloned from the S-phase variant of strain VA1. Sequence analysis of the fragment revealed four tandemly arranged potential open reading frames (ORFs), designated
pilA1
,
pilA2
,
pilB
, and
hagA
. Both
pilA1
and
pilA2
predict a type IV pilin. The protein predicted by
pilB
shares sequence identity with the
Dichelobacter nodosus
FimB fimbrial assembly protein. The protein predicted by
hagA
resembles a hemagglutinin. The region containing these four ORFs was designated the
pilA
locus. DNA hybridization and sequence analysis showed that the
pilA
locus of an L-phase variant of strain VA1 was identical to that of the S-phase variant. An abundant
pilA1
transcript initiating upstream of
pilA1
and terminating at a predicted hairpin structure between
pilA1
and
pilA2
was detected by several assays, as was a less abundant read-through transcript encompassing
pilA1
,
pilA2
, and
pilB
. Transcription from the
pilA
locus was nearly indistinguishable between S- and L-phase variants. Electron microscopy and immunochemical analysis showed that S-phase variants synthesize, export, and assemble pilin into pili. In contrast, L-phase variants synthesize pilin but do not export and assemble it into pili. These data suggest that a posttranslational event, possibly involving an alteration in pilin export and assembly, is responsible for phase variation in
E. corrodens
.
Heterologous Expression of Correctly Assembled Methylamine Dehydrogenase in
Rhodobacter sphaeroides
Authors: Graichen M. Elizabeth , Jones Limei H. , Sharma Bethel V. , van Spanning Rob J. M. , Hosler Jonathan P. , Davidson Victor L. 
Published: December 2019

ABSTRACT

The biosynthesis of methylamine dehydrogenase (MADH) from
Paracoccus denitrificans
requires four genes in addition to those that encode the two structural protein subunits,
mauB
and
mauA
. The accessory gene products appear to be required for proper export of the protein to the periplasm, synthesis of the tryptophan tryptophylquinone (TTQ) prosthetic group, and formation of several structural disulfide bonds. To accomplish the heterologous expression of correctly assembled MADH, eight genes from the methylamine utilization gene cluster of
P. denitrificans
,
mauFBEDACJG
, were placed under the regulatory control of the
coxII
promoter of
Rhodobacter sphaeroides
and introduced into
R. sphaeroides
by using a broad-host-range vector. The heterologous expression of MADH was constitutive with respect to carbon source, whereas the native
mau
promoter allows induction only when cells are grown in the presence of methylamine as a sole carbon source and is repressed by other carbon sources. The recombinant MADH was localized exclusively in the periplasm, and its physical, spectroscopic, kinetic and redox properties were indistinguishable from those of the enzyme isolated from
P. denitrificans
. These results indicate that
mauM
and
mauN
are not required for MADH or TTQ biosynthesis and that
mauFBEDACJG
are sufficient for TTQ biosynthesis, since
R. sphaeroides
cannot synthesize TTQ. A similar construct introduced into
Escherichia coli
did not produce detectable MADH activity or accumulation of the
mauB
and
mauA
gene products but did lead to synthesizes of amicyanin, the
mauC
gene product. This finding suggests that active recombinant MADH is not expressed in
E. coli
because one of the accessory gene products is not functionally expressed. This study illustrates the potential utility of
R. sphaeroides
and the
coxII
promoter for heterologous expression of complex enzymes such as MADH which cannot be expressed in
E. coli
. These results also provide the foundation for future studies on the molecular mechanisms of MADH and TTQ biosynthesis, as well as a system for performing site-directed mutagenesis of the MADH gene and other
mau
genes.
Site-Specific Recombination of Bacteriophage P22 Does Not Require Integration Host Factor
Authors: Cho Eun Hee , Nam Chan-Eun , Alcaraz Renato , Gardner Jeffrey F. 
Published: December 2019

ABSTRACT

Site-specific recombination by phages λ and P22 is carried out by multiprotein-DNA complexes. Integration host factor (IHF) facilitates λ site-specific recombination by inducing DNA bends necessary to form an active recombinogenic complex. Mutants lacking IHF are over 1,000-fold less proficient in supporting λ site-specific recombination than wild-type cells. Although the
attP
region of P22 contains strong IHF binding sites, in vivo measurements of integration and excision frequencies showed that infecting P22 phages can perform site-specific recombination to its maximum efficiency in the absence of IHF. In addition, a plasmid integration assay showed that integrative recombination occurs equally well in wild-type and
ihfA
mutant cells. P22 integrative recombination is also efficient in
Escherichia coli
in the absence of functional IHF. These results suggest that nucleoprotein structures proficient for recombination can form in the absence of IHF or that another factor(s) can substitute for IHF in the formation of complexes.
Genetic and Molecular Analysis of
cglB
, a Gene Essential for Single-Cell Gliding in
Myxococcus xanthus
Authors: Rodriguez Ana M. , Spormann Alfred M. 
Published: December 2019

ABSTRACT

Gliding movements of individual isolated
Myxococcus xanthus
cells depend on the genes of the A-motility system (
agl
and
cgl
genes). Mutants carrying defects in those genes are unable to translocate as isolated cells on solid surfaces. The motility defect of
cgl
mutants can be transiently restored to wild type by extracellular complementation upon mixing mutant cells with wild-type or other motility mutant cells. To develop a molecular understanding of the function of a Cgl protein in gliding motility, we cloned the
cglB
wild-type allele by genetic complementation of the mutant phenotype. The nucleotide sequence of a 2.85-kb fragment was determined and shown to encode two complete open reading frames. The CglB protein was determined to be a 416-amino-acid putative lipoprotein with an unusually high cysteine content. The CglB antigen localized to the membrane fraction. The swarming and gliding defects of a constructed Δ
cglB
mutant were fully restored upon complementation with the
cglB
wild-type allele. Experiments with a
cglB
allele encoding a CglB protein with a polyhistidine tag at the C terminus showed that this allele also promoted wild-type levels of swarming and single-cell gliding, but was unable to stimulate Δ
cglB
cells to move. Possible functions of CglB as a mechanical component or as a signal protein in single cell gliding are discussed.
Lysis and Lysis Inhibition in Bacteriophage T4:
rV
Mutations Reside in the Holin
t
Gene
Authors: Dressman Holly Kloos , Drake John W. 
Published: December 2019

ABSTRACT

Upon infecting populations of susceptible host cells, T-even bacteriophages maximize their yield by switching from lysis at about 25 to 35 min at 37°C after infection by a single phage particle to long-delayed lysis (lysis inhibition) under conditions of sequential infection occurring when free phages outnumber host cells. The timing of lysis depends upon gene
t
and upon one or more rapid-lysis (
r
) genes whose inactivation prevents lysis inhibition.
t
encodes a holin that mediates the movement of the T4 endolysin though the inner cell membrane to its target, the cell wall. The rI protein has been proposed to sense superinfection. Of the five reasonably well characterized
r
genes, only two,
rI
and
rV
, are clearly obligatory for lysis inhibition. We show here that
rV
mutations are alleles of
t
that probably render the t protein unable to respond to the lysis inhibition signal. The
tr
alleles cluster in the 5′ third of
t
and produce a strong r phenotype, whereas conditional-lethal
t
alleles produce the classical t phenotype (inability to lyse) and other
t
alleles produce additional, still poorly understood phenotypes.
tr
mutations are dominant to
t
+
, a result that suggests specific ways to probe T4 holin function.
Water Transport across Yeast Vacuolar and Plasma Membrane-Targeted Secretory Vesicles Occurs by Passive Diffusion
Authors: Coury Larry A. , Hiller Mark , Mathai John C. , Jones Elizabeth W. , Zeidel Mark L. , Brodsky Jeffrey L. 
Published: December 2019

ABSTRACT

To determine whether solute transport across yeast membranes was facilitated, we measured the water and solute permeations of vacuole-derived and late secretory vesicles in
Saccharomyces cerevisiae
; all permeations were consistent with passive diffusive flow. We also overexpressed Fps1p, the putative glycerol facilitator in
S. cerevisiae
, in secretory vesicles but observed no effect on water, glycerol, formamide, or urea permeations. However, spheroplasts prepared from the strain overexpressing Fps1p showed enhanced glycerol uptake, suggesting that Fps1p becomes active only upon insertion in the plasma membrane.
Autogenous Regulation of the
Bacillus anthracis pag
Operon
Authors: Hoffmaster Alex R. , Koehler Theresa M. 
Published: December 2019

ABSTRACT

Protective antigen (PA) is an important component of the edema and lethal toxins produced by
Bacillus anthracis
. PA is essential for binding the toxins to the target cell receptor and for facilitating translocation of the enzymatic toxin components, edema factor and lethal factor, across the target cell membrane. The structural gene for PA,
pagA
(previously known as
pag
), is located on the 182-kb virulence plasmid pXO1 at a locus distinct from the edema factor and lethal factor genes. Here we show that a 300-bp gene located downstream of
pagA
is cotranscribed with
pagA
and represses expression of the operon. We have designated this gene
pagR
(for protective antigen repressor). Two
pagA
mRNA transcripts were detected in cells producing PA: a short, 2.7-kb transcript corresponding to the
pagA
gene, and a longer, 4.2-kb transcript representing a bicistronic message derived from
pagA
and
pagR
. The 3′ end of the short transcript mapped adjacent to an inverted repeat sequence, suggesting that the sequence can act as a transcription terminator. Attenuation of termination at this site results in transcription of
pagR
. A
pagR
mutant exhibited increased steady-state levels of
pagA
mRNA, indicating that
pagR
negatively controls expression of the operon. Autogenous control of the operon may involve
atxA
, a
trans
-acting positive regulator of
pagA
. The steady-state level of
atxA
mRNA was also increased in the
pagR
mutant. The mutant phenotype was complemented by addition of
pagR
in
trans
on a multicopy plasmid.
Outer Membrane Changes in a Toluene-Sensitive Mutant of Toluene-Tolerant
Pseudomonas putida
IH-2000
Authors: Kobayashi Hideki , Takami Hideto , Hirayama Hisako , Kobata Kuniko , Usami Ron , Horikoshi Koki 
Published: December 2019

ABSTRACT

We isolated a toluene-sensitive mutant, named mutant No. 32, which showed unchanged antibiotic resistance levels, from toluene-tolerant
Pseudomonas putida
IH-2000 by transposon mutagenesis with Tn
5
. The gene disrupted by insertion of Tn
5
was identified as
cyoC
, which is one of the subunits of cytochrome
o
. The membrane protein, phospholipid, and lipopolysaccharide (LPS) of IH-2000 and that of mutant No. 32 were examined and compared. Some of the outer membrane proteins showed a decrease in mutant No. 32. The fatty acid components of LPS were found to be dodecanoic acid, 2-hydroxydodecanoic acid, 3-hydroxydodecanoic acid, and 3-hydroxydecanoic acid in both IH-2000 and No. 32; however, the relative proportions of these components differed in the two strains. Furthermore, cell surface hydrophobicity was increased in No. 32. These data suggest that mutation of
cyoC
caused the decrease in outer membrane proteins and the changing fatty acid composition of LPS. These changes in the outer membrane would cause an increase in cell surface hydrophobicity, and mutant No. 32 is considered to be sensitive to toluene.
Escherichia coli
Open Reading Frame 696 Is
idi
, a Nonessential Gene Encoding Isopentenyl Diphosphate Isomerase
Authors: Hahn Frederick M. , Hurlburt Anthony P. , Poulter C. Dale 
Published: December 2019

ABSTRACT

Isopentenyl diphosphate isomerase catalyzes the interconversion of isopentenyl diphosphate (IPP) and dimethylallyl diphosphate (DMAPP). In eukaryotes, archaebacteria, and some bacteria, IPP is synthesized from acetyl coenzyme A by the mevalonate pathway. The subsequent isomerization of IPP to DMAPP activates the five-carbon isoprene unit for subsequent prenyl transfer reactions. In
Escherichia coli
, the isoprene unit is synthesized from pyruvate and glyceraldehyde-3-phosphate by the recently discovered nonmevalonate pathway. An open reading frame (ORF696) encoding a putative IPP isomerase was identified in the
E. coli
chromosome at 65.3 min. ORF696 was cloned into an expression vector; the 20.5 kDa recombinant protein was purified in three steps, and its identity as an IPP isomerase was established biochemically. The gene for IPP isomerase,
idi
, is not clustered with other known genes for enzymes in the isoprenoid pathway.
E. coli
FH12 was constructed by disruption of the chromosomal
idi
gene with the aminoglycoside 3′-phosphotransferase gene and complemented by the wild-type
idi
gene on plasmid pFMH33 with a temperature-sensitive origin of replication. FH12/pFMH33 was able to grow at the restrictive temperature of 44°C and FH12 lacking the plasmid grew on minimal medium, thereby establishing that
idi
is a nonessential gene. Although the
V
max
of the bacterial protein was 20-fold lower than that of its yeast counterpart, the catalytic efficiencies of the two enzymes were similar through a counterbalance in

K
m

s. The
E. coli
protein requires Mg
2+
or Mn
2+
for activity. The enzyme contains conserved cysteine and glutamate active-site residues found in other IPP isomerases.
areABC
Genes Determine the Catabolism of Aryl Esters in
Acinetobacter
sp. Strain ADP1
Authors: Jones Rheinallt M. , Collier Lauren S. , Neidle Ellen L. , Williams Peter A. 
Published: December 2019

ABSTRACT

Acinetobacter
sp. strain ADP1 is able to grow on a range of esters of aromatic alcohols, converting them to the corresponding aromatic carboxylic acids by the sequential action of three inducible enzymes: an
areA
-encoded esterase, an
areB
-encoded benzyl alcohol dehydrogenase, and an
areC
-encoded benzaldehyde dehydrogenase. The
are
genes, adjacent to each other on the chromosome and transcribed in the order
areCBA
, were located 3.5 kbp upstream of
benK. benK
, encoding a permease implicated in benzoate uptake, is at one end of the
ben-cat
supraoperonic cluster for benzoate catabolism by the β-ketoadipate pathway. Two open reading frames which may encode a transcriptional regulator,
areR
, and a porin,
benP
, separate
benK
from
areC
. Each
are
gene was individually expressed to high specific activity in
Escherichia coli
. The relative activities against different substrates of the cloned enzymes were, within experimental error, identical to that of wild-type
Acinetobacter
sp. strain ADP1 grown on either benzyl acetate, benzyl alcohol, or 4-hydroxybenzyl alcohol as the carbon source. The substrate preferences of all three enzymes were broad, encompassing a range of substituted aromatic compounds and in the case of the AreA esterase, different carboxylic acids. The
areA
,
areB
, and
areC
genes were individually disrupted on the chromosome by insertion of a kanamycin resistance cassette, and the rates at which the resultant strains utilized substrates of the aryl ester catabolic pathway were severely reduced as determined by growth competitions between the mutant and wild-type strains.
A
Zymomonas mobilis
Mutant with Delayed Growth on High Glucose Concentrations
Authors: Douka Eugenia , Koukkou Anna Irini , Vartholomatos Georgios , Frillingos Stathis , Papamichael Emmanuel M. , Drainas Constantin 
Published: December 2019

ABSTRACT

Exponentially growing cells of
Zymomonas mobilis
normally exhibit a lag period of up to 3 h when transferred from 0.11 M (2%) to 0.55 M (10%) glucose liquid medium. A mutant of
Z. mobilis
(CU1Rif2), fortuitously isolated, showed more than a 20-h lag period when grown under the same conditions, whereas on 0.55 M glucose solid medium, it failed to grow. The growth of CU1Rif2 on elevated concentrations of other fermentable (0.55 M sucrose or fructose) or nonfermentable (0.11 M glucose plus 0.44 M maltose or xylose) sugars appeared to be normal. Surprisingly, CU1Rif2 cells grew without any delay on 0.55 M glucose on which wild-type cells had been incubated for 3 h and removed at the beginning of their exponential phase. This apparent preconditioning was not observed with medium obtained from wild-type cells grown on 0.11 M glucose and supplemented to 0.55 M after removal of the wild-type cells. Undelayed growth of CU1Rif2 on 0.55 M glucose previously conditioned by the wild type was impaired by heating or protease treatment. It is suggested that in
Z. mobilis
, a diffusible proteinaceous heat-labile factor, transitionally not present in 0.55 M glucose CU1Rif2 cultures, triggers growth on 0.55 M glucose. Biochemical analysis of glucose uptake and glycolytic enzymes implied that glucose assimilation was not directly involved in the phenomenon. By use of a wild-type
Z. mobilis
genomic library, a 4.5-kb DNA fragment which complemented in low copy number the glucose-defective phenotype as well as glucokinase and glucose uptake of CU1Rif2 was isolated. This fragment carries a gene cluster consisting of four putative coding regions, encoding 167, 167, 145, and 220 amino acids with typical
Z. mobilis
codon usage, −35 and −10 promoter elements, and individual Shine-Dalgarno consensus sites. However, strong homologies were not detected in a BLAST2 (EMBL-Heidelberg) computer search with known protein sequences.