bluuebunny/crossref_metadata_2025_split huggingface dataset:
Regulatory Conservation and Divergence of ς
32
Homologs from Gram-Negative Bacteria:
Serratia marcescens
,
Proteus mirabilis
,
Pseudomonas aeruginosa
, and
Agrobacterium tumefaciens
Authors: Nakahigashi Kenji , Yanagi Hideki , Yura Takashi 
Published: December 2019

ABSTRACT

The heat shock response in
Escherichia coli
is mediated primarily by the
rpoH
gene, encoding ς
32
, which is specifically required for transcription of heat shock genes. A number of ς
32
homologs have recently been cloned from gram-negative bacteria that belong to the gamma or alpha subdivisions of the proteobacteria. We report here some of the regulatory features of several such homologs (RpoH) expressed in
E. coli
as well as in respective cognate bacteria. When expressed in an
E. coli
Δ
rpoH
strain lacking its own ς
32
, these homologs activated the transcription of heat shock genes (
groE
and
dnaK
) from the start sites normally used in
E. coli
. The level of RpoH in
Serratia marcescens
and
Pseudomonas aeruginosa
cells was very low at 30°C but was elevated markedly upon a shift to 42°C, as found previously with
E. coli
. The increased RpoH levels upon heat shock resulted from both increased synthesis and stabilization of the normally unstable RpoH protein. In contrast, the RpoH level in
Proteus mirabilis
was relatively high at 30°C and increased less markedly upon heat shock, mostly by increased synthesis; this ς
32
homolog was already stable at 30°C, and little further stabilization occurred upon the shift to 42°C. The increased synthesis of RpoH homologs in all these gamma proteobacteria was observed even in the presence of rifampin, suggesting that the induction occurred at the level of translation. Thus, the basic regulatory strategy of the heat shock response by enhancing the RpoH level is well conserved in the gamma proteobacteria, but some divergence in the actual mechanisms used occurred during evolution.
Mutations Affecting the α Subunit of
Bordetella pertussis
RNA Polymerase Suppress Growth Inhibition Conferred by Short C-Terminal Deletions of the Response Regulator BvgA
Authors: Stibitz Scott 
Published: December 2019

ABSTRACT

The effects of short deletions of the C terminus of the BvgA response regulator protein of the BvgAS two-component system were examined in
Bordetella pertussis
. When present as a single copy in the chromosome, deletions removing as few as two amino acids conferred a completely Bvg
−
phenotype. When provided in
trans
, on the broad-host-range plasmid pRK290, under the control of the native
bvgAS
promoter, deletions of two or three amino acids conferred a profound growth inhibition which was dependent on the integrity and activity of the wild-type chromosomal
bvgAS
locus. It is proposed that this phenotype was the result of an inappropriate interaction of the mutant BvgA protein with the RNA polymerase enzyme, specifically the α subunit. Mutant strains in which this growth inhibition was relieved were isolated and characterized. Although most of the suppressor mutations affected either the mutant plasmid copy or the wild-type chromosomal
bvg
locus, three mutations which affected the α subunit of
B. pertussis
RNA polymerase were also isolated. Two of these resulted in increased levels of the α subunit, and one caused a substitution of glycine for the aspartic acid residue at position 171, in the N-terminal domain. All three mutations also resulted in a differential phenotype in that expression of
fha
was essentially normal, but expression of
ptx
was greatly reduced.
TheSaccharomyces cerevisiae YCC5(YCL025c) Gene Encodes an Amino Acid Permease, Agp1, Which Transports Asparagine and Glutamine
Authors: Schreve James L. , Sin Jennifer K. , Garrett Jinnie M. 
Published: December 2019

ABSTRACTThe yeastYCC5gene encodes a putative amino acid permease and is homologous toGNP1(encoding a high-affinity glutamine permease). Using strains with disruptions in the genes for multiple permeases, we demonstrated that Ycc5 (which we have renamed Agp1) is involved in the transport of asparagine and glutamine, performed a kinetic analysis of this activity, and showed thatAGP1expression is subject to nitrogen repression.
Identification and Analysis of “Extended −10” Promoters from Mycobacteria
Authors: Bashyam Murali D. , Tyagi Anil K. 
Published: December 2019

ABSTRACT

Earlier studies from our laboratory on randomly isolated transcriptional signals of mycobacteria had revealed that the −10 region of mycobacterial promoters and the corresponding binding domain in the major sigma factor are highly similar to their
Escherichia coli
counterparts. In contrast, the sequences in −35 regions of mycobacterial promoters and the corresponding binding domain in the major sigma factor are vastly different from their
E. coli
counterparts (M. D. Bashyam, D. Kaushal, S. K. Dasgupta, and A. K. Tyagi, J. Bacteriol. 178:4847–4853, 1996). We have now analyzed the role of the TGN motif present immediately upstream of the −10 region of mycobacterial promoters. Sequence analysis and site-specific mutagenesis of a
Mycobacterium tuberculosis
promoter and a
Mycobacterium smegmatis
promoter reveal that the TGN motif is an important determinant of transcriptional strength in mycobacteria. We show that mutation in the TGN motif can drastically reduce the transcriptional strength of a mycobacterial promoter. The influence of the TGN motif on transcriptional strength is also modulated by the sequences in the −35 region. Comparative assessment of these extended −10 promoters in mycobacteria and
E. coli
suggests that functioning of the TGN motif in promoters of these two species is similar.
Coordinated Repression In Vitro of the Divergent
fepA-fes
Promoters of
Escherichia coli
by the Iron Uptake Regulation (Fur) Protein
Authors: Escolar Lucía , Pérez-Martín José , de Lorenzo Víctor 
Published: December 2019

ABSTRACT

The mechanism involved in transcriptional repression of the
fepA-fes
divergent promoters of
Escherichia coli
by the Fur (ferric uptake regulation) protein has been examined in vitro. This DNA region includes a suboptimal and single Fur-binding site with two divergent and overlapped −35/−10 hexamers. Comparison of transcription patterns generated with runoff experiments in either the presence or the absence of heparin showed that access of the RNA polymerase to the principal −35/−10 hexamers was fully prevented by Fur-Mn
2+
bound to its target site within the divergent promoter region. Neither RNA polymerase bound to the
fes
and
fepA
promoters could be displaced by Fur-Mn
2+
, nor could the bound repressor be outcompeted by an excess of the enzyme. However, the repressor blocked reinitiation as soon as the polymerase moved away from the
fes
promoter during transcription. The spatial distribution of regulatory elements within the DNA region allowed the simultaneous binding of the RNA polymerase to the
fes
and
fepA
promoters and their coordinate regulation regardless of their different transcriptional activities. Comparisons with other iron-regulated systems support a general mechanism for Fur-controlled promoters that implies a direct competition between the polymerase and the regulator for overlapping target sites in the DNA.
Mutations That Extend the Specificity of the Endonuclease Activity of λ Terminase
Authors: Arens Jean Sippy , Hang Qi , Hwang Young , Tuma Bill , Max Sara , Feiss Mike 
Published: December 2019

ABSTRACT

Terminase, an enzyme encoded by the
Nu1
and
A
genes of bacteriophage lambda, is crucial for packaging concatemeric DNA into virions.
cosN
, a 22-bp segment, is the site on the virus chromosome where terminase introduces staggered nicks to cut the concatemer to generate unit-length virion chromosomes. Although
cosN
is rotationally symmetric, mutations in
cosN
have asymmetric effects. The
cosN
G
2
C mutation (a G-to-C change at position 2) in the left half of
cosN
reduces the phage yield 10-fold, whereas the symmetric mutation
cosN
C
11
G, in the right half of
cosN
, does not affect the burst size. The reduction in phage yield caused by
cosN
G
2
C is correlated with a defect in
cos
cleavage. Three suppressors of the
cosN
G
2
C mutation,

A-E
515
G

,

A-N
509
K

, and

A-R
504
C

, have been isolated that restore the yield of λ
cosN
G
2
C to the wild-type level. The suppressors are missense mutations that alter amino acids located near an ATPase domain of gpA. λ

A-E
515
G

,

A-N
509
K

, and

A-R
504
C

phages, which are
cosN
+
, also had wild-type burst sizes. In vitro
cos
cleavage experiments on
cosN
G
2
C C
11
G DNA showed that the rate of cleavage for

A-E
515
G

terminase is three- to fourfold higher than for wild-type terminase. The

A-E
515
G

mutation changes residue 515 of gpA from glutamic acid to glycine. Uncharged polar and hydrophobic residues at position 515 suppressed the growth defect of λ
cosN
G
2
C C
11
G. In contrast, basic (K, R) and acidic (E, D) residues at position 515 failed to suppress the growth defect of λ
cosN
G
2
C C
11
G. In a λ
cosN
+
background, all amino acids tested at position 515 were functional. These results suggest that

A-E
515
G

plays an indirect role in extending the specificity of the endonuclease activity of λ terminase.
Lactobacillus casei
64H Contains a Phosphoenolpyruvate-Dependent Phosphotransferase System for Uptake of Galactose, as Confirmed by Analysis of
ptsH
and Different
gal
Mutants
Authors: Bettenbrock Katja , Siebers Ulrike , Ehrenreich Petra , Alpert Carl-Alfred 
Published: December 2019

ABSTRACT

Galactose metabolism in
Lactobacillus casei
64H was analyzed by genetic and biochemical methods. Mutants with defects in
ptsH
,
galK
, or the tagatose 6-phosphate pathway were isolated either by positive selection using 2-deoxyglucose or 2-deoxygalactose or by an enrichment procedure with streptozotocin.
ptsH
mutations abolish growth on lactose, cellobiose,
N
-acetylglucosamine, mannose, fructose, mannitol, glucitol, and ribitol, while growth on galactose continues at a reduced rate. Growth on galactose is also reduced, but not abolished, in
galK
mutants. A mutation in
galK
in combination with a mutation in the tagatose 6-phosphate pathway results in sensitivity to galactose and lactose, while a
galK
mutation in combination with a mutation in
ptsH
completely abolishes galactose metabolism. Transport assays, in vitro phosphorylation assays, and thin-layer chromatography of intermediates of galactose metabolism also indicate the functioning of a permease/Leloir pathway and a phosphoenolpyruvate-dependent phosphotransferase system (PTS)/tagatose 6-phosphate pathway. The galactose-PTS is induced by growth on either galactose or lactose, but the induction kinetics for the two substrates are different.
Regulation of the
sol
Locus Genes for Butanol and Acetone Formation in
Clostridium acetobutylicum
ATCC 824 by a Putative Transcriptional Repressor
Authors: Nair Ramesh V. , Green Edward M. , Watson David E. , Bennett George N. , Papoutsakis Eleftherios T. 
Published: December 2019

ABSTRACT

A gene (
orf1
, now designated
solR
) previously identified upstream of the aldehyde/alcohol dehydrogenase gene
aad
(R. V. Nair, G. N. Bennett, and E. T. Papoutsakis, J. Bacteriol. 176:871–885, 1994) was found to encode a repressor of the
sol
locus (
aad
,
ctfA
,
ctfB
and
adc
) genes for butanol and acetone formation in
Clostridium acetobutylicum
ATCC 824. Primer extension analysis identified a transcriptional start site 35 bp upstream of the
solR
start codon. Amino acid comparisons of SolR identified a potential helix-turn-helix DNA-binding motif in the C-terminal half towards the center of the protein, suggesting a regulatory role. Overexpression of SolR in strain ATCC 824(pCO1) resulted in a solvent-negative phenotype owing to its deleterious effect on the transcription of the
sol
locus genes. Inactivation of
solR
in
C. acetobutylicum
via homologous recombination yielded mutants B and H (ATCC 824
solR
::pO1X) which exhibited deregulated solvent production characterized by increased flux towards butanol and acetone formation, earlier induction of
aad
, lower overall acid production, markedly improved yields of solvents on glucose, a prolonged solvent production phase, and increased biomass accumulation compared to those of the wild-type strain.
Exopolysaccharide Biosynthesis in
Lactococcus lactis
NIZO B40: Functional Analysis of the Glycosyltransferase Genes Involved in Synthesis of the Polysaccharide Backbone
Authors: van Kranenburg Richard , van Swam Iris I. , Marugg Joey D. , Kleerebezem Michiel , de Vos Willem M. 
Published: December 2019

ABSTRACT

We used homologous and heterologous expression of the glycosyltransferase genes of the
Lactococcus lactis
NIZO B40
eps
gene cluster to determine the activity and substrate specificities of the encoded enzymes and established the order of assembly of the trisaccharide backbone of the exopolysaccharide repeating unit. EpsD links glucose-1-phosphate from UDP-glucose to a lipid carrier, EpsE and EpsF link glucose from UDP-glucose to lipid-linked glucose, and EpsG links galactose from UDP-galactose to lipid-linked cellobiose. Furthermore, EpsJ appeared to be involved in EPS biosynthesis as a galactosyl phosphotransferase or an enzyme which releases the backbone oligosaccharide from the lipid carrier.
Genetic and Biochemical Analyses of the
tec
Operon Suggest a Route for Evolution of Chlorobenzene Degradation Genes
Authors: Beil Stefan , Timmis Kenneth N. , Pieper Dietmar H. 
Published: December 2019

ABSTRACT

The TecA broad-spectrum chlorobenzene dioxygenase of
Burkholderia
sp. strain PS12 catalyzes the first step in the mineralization of 1,2,4,5-tetrachlorobenzene. The catabolic genes were localized on a small plasmid that belongs to the IncPβ incompatibility group. PCR analysis of the genetic environment of the
tec
genes indicated high similarity to the transposon-organized catabolic
tcb
chlorobenzene degradation genes of
Pseudomonas
sp. strain P51. Sequence analysis of the regions flanking the
tecA
genes revealed an upstream open reading frame (ORF) with high similarity to the
todF
2-hydroxy-6-oxo-2,4-heptadienoate hydrolase gene of
Pseudomonas putida
F1 and a discontinuous downstream ORF showing high similarity to the
todE
catechol 2,3-dioxygenase gene of strain F1. Both homologues in strain P51 exist only as deletion remnants. We suggest that different genetic events thus led to inactivation of the perturbing
meta
-cleavage enzymes in strains P51 and PS12 during the evolution of efficient chlorobenzene degradation pathways. Biochemical characterization of TodF-like protein TlpF and a genetically refunctionalized TodE-like protein, TlpE, produced in
Escherichia coli
provided data consistent with the proposed relationships.
Evidence for a Chemiosmotic Model of Dehalorespiration in
Desulfomonile tiedjei
DCB-1
Authors: Louie Tai Man , Mohn William W. 
Published: December 2019

ABSTRACT

Desulfomonile tiedjei
DCB-1, a sulfate-reducing bacterium, conserves energy for growth from reductive dehalogenation of 3-chlorobenzoate by an uncharacterized chemiosmotic process. Respiratory electron transport components were examined in
D. tiedjei
cells grown under conditions for reductive dehalogenation, pyruvate fermentation, and sulfate reduction. Reductive dehalogenation was inhibited by the respiratory quinone inhibitor 2-heptyl-4-hydroxyquinoline
N
-oxide, suggesting that a respiratory quinoid is a component of the electron transport chain coupled to reductive dehalogenation. Moreover, reductive dehalogenation activity was dependent on 1,4-naphthoquinone, a possible precursor for a respiratory quinoid. However, no ubiquinone or menaquinone could be extracted from
D. tiedjei
. Rather, a UV-absorbing quinoid which is different from common respiratory quinones in chemical structure according to mass spectrometric and UV absorption spectroscopic analyses was extracted. ATP sulfurylase, adenosine phosphosulfate reductase, and desulfoviridin sulfite reductase, enzymes involved in sulfate reduction, were constitutively expressed in the cytoplasm of
D. tiedjei
cells grown under all three metabolic conditions. A periplasmic hydrogenase was detected in cells grown under reductive-dehalogenating and pyruvate-fermenting conditions. A membrane-bound, periplasm-oriented formate dehydrogenase was detected only in cells grown with formate as electron donor, while a cytoplasmic formate dehydrogenase was detected in cells grown under reductive-dehalogenating and pyruvate-fermenting conditions. Results from dehalogenation assays with
D. tiedjei
whole-cell suspensions and cell extracts suggest that the membrane-bound reductive dehalogenase is cytoplasm oriented. The data clearly demonstrate an enzyme topology in
D. tiedjei
which produces protons directly in the periplasm, generating a proton motive force by a scalar mechanism.
Characterization of Biosynthetic Enzymes for Ectoine as a Compatible Solute in a Moderately Halophilic Eubacterium,
Halomonas elongata
Authors: Ono Hisayo , Sawada Kazuhisa , Khunajakr Nonpanga , Tao Tao , Yamamoto Mihoko , Hiramoto Masayuki , Shinmyo Atsuhiko , Takano Mitsuo , Murooka Yoshikatsu 
Published: December 2019

ABSTRACT

1,4,5,6-Tetrahydro-2-methyl-4-pyrimidinecarboxylic acid (ectoine) is an excellent osmoprotectant. The biosynthetic pathway of ectoine from aspartic β-semialdehyde (ASA), in
Halomonas elongata
, was elucidated by purification and characterization of each enzyme involved. 2,4-Diaminobutyrate (DABA) aminotransferase catalyzed reversively the first step of the pathway, conversion of ASA to DABA by transamination with
l
-glutamate. This enzyme required pyridoxal 5′-phosphate and potassium ions for its activity and stability. The gel filtration estimated an apparent molecular mass of 260 kDa, whereas molecular mass measured by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) was 44 kDa. This enzyme exhibited an optimum pH of 8.6 and an optimum temperature of 25°C and had

K
m

s of 9.1 mM for
l
-glutamate and 4.5 mM for
dl
-ASA. DABA acetyltransferase catalyzed acetylation of DABA to γ-
N
-acetyl-α,γ-diaminobutyric acid (ADABA) with acetyl coenzyme A and exhibited an optimum pH of 8.2 and an optimum temperature of 20°C in the presence of 0.4 M NaCl. The molecular mass was 45 kDa by gel filtration. Ectoine synthase catalyzed circularization of ADABA to ectoine and exhibited an optimum pH of 8.5 to 9.0 and an optimum temperature of 15°C in the presence of 0.5 M NaCl. This enzyme had an apparent molecular mass of 19 kDa by SDS-PAGE and a

K
m

of 8.4 mM in the presence of 0.77 M NaCl. DABA acetyltransferase and ectoine synthase were stabilized in the presence of NaCl (>2 M) and DABA (100 mM) at temperatures below 30°C.
Overproduction of SecA Suppresses the Export Defect Caused by a Mutation in the Gene Encoding the
Escherichia coli
Export Chaperone SecB
Authors: Cook Heather A. , Kumamoto Carol A. 
Published: December 2019

ABSTRACT

SecB is a cytosolic protein required for rapid and efficient export of particular periplasmic and outer membrane proteins in
Escherichia coli
. SecB promotes export by stabilizing newly synthesized precursor proteins in a nonnative conformation and by targeting the precursors to the inner membrane. Biochemical studies suggest that SecB facilitates precursor targeting by binding to the SecA protein, a component of the membrane-embedded translocation apparatus. To gain more insight into the functional interaction of SecB and SecA, in vivo, mutations in the
secA
locus that compensate for the export defect caused by the
secB
missense mutation
secBL75Q
were isolated. Two suppressors were isolated, both of which led to the overproduction of wild-type SecA protein. In vivo studies demonstrated that the SecBL75Q mutant protein releases precursor proteins at a lower rate than does wild-type SecB. Increasing the level of SecA protein in the cell was found to reverse this slow-release defect, indicating that overproduction of SecA stimulates the turnover of SecBL75Q-precursor complexes. These findings lend additional support to the proposed pathway for precursor targeting in which SecB promotes targeting to the translocation apparatus by binding to the SecA protein.
Identification and Transcriptional Control of the Genes Encoding the
Caulobacter crescentus
ClpXP Protease
Authors: Østerås Magne , Stotz Agathe , Nuoffer Stefanie Schmid , Jenal Urs 
Published: December 2019

ABSTRACT

The region of the
Caulobacter crescentus
chromosome harboring the genes for the ClpXP protease was isolated and characterized. Comparison of the deduced amino acid sequences of the
C. crescentus
ClpP and ClpX proteins with those of their homologues from several gram-positive and gram-negative bacteria revealed stronger conservation for the ATPase regulatory subunit (ClpX) than for the peptidase subunit (ClpP). The
C. crescentus clpX
gene was shown by complementation analysis to be functional in
Escherichia coli
. However,
clpX
from
E. coli
was not able to substitute for the essential nature of the
clpX
gene in
C. crescentus
. The
clpP
and
clpX
genes are separated on the
C. crescentus
chromosome by an open reading frame pointing in the opposite direction from the
clp
genes, and transcription of
clpP
and
clpX
was found to be uncoupled.
clpP
is transcribed as a monocistronic unit with a promoter (P
P1
) located immediately upstream of the 5′ end of the gene and a terminator structure following its 3′ end. P
P1
is under heat shock control and is induced upon entry of the cells into the stationary phase. At least three promoters for
clpX
(P
X1
, P
X2
, and P
X3
) were mapped in the
clpP-clpX
intergenic region. In contrast to P
P1
, the
clpX
promoters were found to be downregulated after heat shock but were also subject to growth phase control. In addition, the
clpP
and
clpX
promoters showed different activity patterns during the cell cycle. Together, these results demonstrate that the genes coding for the peptidase and the regulatory subunits of the ClpXP protease are under independent transcriptional control in
C. crescentus
. Determination of the numbers of ClpP and ClpX molecules per cell suggested that ClpX is the limiting component compared with ClpP.
Role of the Mitogen-Activated Protein Kinase Hog1p in Morphogenesis and Virulence of
Candida albicans
Authors: Alonso-Monge R. , Navarro-García F. , Molero G. , Diez-Orejas R. , Gustin M. , Pla J. , Sánchez M. , Nombela C. 
Published: December 2019

ABSTRACT

The relevance of the mitogen-activated protein (MAP) kinase Hog1p in
Candida albicans
was addressed through the characterization of
C. albicans
strains without a functional
HOG1
gene. Analysis of the phenotype of
hog1
mutants under osmostressing conditions revealed that this mutant displays a set of morphological alterations as the result of a failure to complete the final stages of cytokinesis, with parallel defects in the budding pattern. Even under permissive conditions,
hog1
mutants displayed a different susceptibility to some compounds such as nikkomycin Z or Congo red, which interfere with cell wall functionality. In addition, the
hog1
mutant displayed a colony morphology different from that of the wild-type strain on some media which promote morphological transitions in
C. albicans
. We show that
C. albicans hog1
mutants are derepressed in the serum-induced hyphal formation and, consistently with this behavior, that
HOG1
overexpression in
Saccharomyces cerevisiae
represses the pseudodimorphic transition. Most interestingly, deletion of
HOG1
resulted in a drastic increase in the mean survival time of systemically infected mice, supporting a role for this MAP kinase pathway in virulence of pathogenic fungi. This finding has potential implications in antifungal therapy.