bluuebunny/crossref_metadata_2025 huggingface dataset:
Analysis of Mutations in the Pore-Forming Region Essential for Insecticidal Activity of a
Bacillus thuringiensis
δ-Endotoxin
Authors: Manoj Kumar A. S. , Aronson A. I.
Published: December 2019

The
Bacillus thuringiensis
insecticidal δ-endotoxins have a three-domain structure, with the seven amphipathic helices which comprise domain I being essential for toxicity. To better define the function of these helices in membrane insertion and toxicity, either site-directed or random mutagenesis of two regions was performed. Thirty-nucleotide segments in the
B. thuringiensis cry1Ac1
gene, encoding parts of helix α4 and the loop connecting helices α4 and α5, were randomly mutagenized. This hydrophobic region of the toxin probably inserts into the membrane as a hairpin. Site-directed mutations were also created in specific surface residues of helix α3 in order to increase its hydrophobicity. Among 12 random mutations in helix α4, 5 resulted in the total loss of toxicity for
Manduca sexta
and
Heliothis virescens
, another caused a significant increase in toxicity, and one resulted in decreased toxicity. None of the nontoxic mutants was altered in toxin stability, binding of toxin to a membrane protein, or the ability of the toxin to aggregate in the membrane. Mutations in the loop connecting helices α4 and α5 did not affect toxicity, nor did mutations in α3, which should have enhanced the hydrophobic properties of this helix. In contrast to mutations in helix α5, those in helix α4 which inactivated the toxin did not affect its capacity to oligomerize in the membrane. Despite the formation of oligomers, there was no ion flow as measured by light scattering. Helix α5 is important for oligomerization and perhaps has other functions, whereas helix α4 must have a more direct role in establishing the properties of the channel.
FlbT Couples Flagellum Assembly to Gene Expression in
Caulobacter crescentus
Authors: Mangan Erin K. , Malakooti Jaleh , Caballero Anthony , Anderson Paul , Ely Bert , Gober James W.
Published: December 2019

The biogenesis of the polar flagellum of
Caulobacter crescentus
is regulated by the cell cycle as well as by a
trans
-acting regulatory hierarchy that functions to couple flagellum assembly to gene expression. The assembly of early flagellar structures (MS ring, switch, and flagellum-specific secretory system) is required for the transcription of class III genes, which encode the remainder of the basal body and the external hook structure. Similarly, the assembly of class III gene-encoded structures is required for the expression of the class IV flagellins, which are incorporated into the flagellar filament. Here, we demonstrate that mutations in
flbT
, a flagellar gene of unknown function, can restore flagellin protein synthesis and the expression of
fljK
::
lacZ
(25-kDa flagellin) protein fusions in class III flagellar mutants. These results suggest that FlbT functions to negatively regulate flagellin expression in the absence of flagellum assembly. Deletion analysis shows that sequences within the 5′ untranslated region of the
fljK
transcript are sufficient for FlbT regulation. To determine the mechanism of FlbT-mediated regulation, we assayed the stability of
fljK
mRNA. The half-life (
t
1/2
) of
fljK
mRNA in wild-type cells was approximately 11 min and was reduced to less than 1.5 min in a
flgE
(hook) mutant. A
flgE flbT
double mutant exhibited an mRNA
t
1/2
of greater than 30 min. This suggests that the primary effect of FlbT regulation is an increased turnover of flagellin mRNA. The increased
t
1/2
of
fljK
mRNA in a
flbT
mutant has consequences for the temporal expression of
fljK
. In contrast to the case for wild-type cells,
fljK
::
lacZ
protein fusions in the mutant are expressed almost continuously throughout the
C. crescentus
cell cycle, suggesting that coupling of flagellin gene expression to assembly has a critical influence on regulating cell cycle expression.
Purification and Characterization of a Novel Naphthalene Dioxygenase from
Rhodococcus
sp. Strain NCIMB12038
Authors: Larkin Michael J. , Allen Christopher C. R. , Kulakov Leonid A. , Lipscomb David A.
Published: December 2019

We report here the characterization of the catalytic component (ISP
NAR
) of a new naphthalene dioxygenase from
Rhodococcus
sp. strain NCIMB12038. The genes encoding the two subunits of ISP
NAR
are not homologous to their previously characterized counterparts in
Pseudomonas
. The deduced amino acid sequences have only 33 and 29% identity with the corresponding subunits in
Pseudomonas putida
NCIB 9816-4, for which the tertiary structure has been reported.
Assembly of XcpR in the Cytoplasmic Membrane Is Required for Extracellular Protein Secretion in
Pseudomonas aeruginosa
Authors: Ball Geneviève , Chapon-Hervé Virginie , Bleves Sophie , Michel Gérard , Bally Marc
Published: December 2019

A broad range of extracellular proteins secreted by
Pseudomonas aeruginosa
use the type II or general secretory pathway (GSP) to reach the medium. This pathway requires the expression of at least 12
xcp
gene products. XcpR, a putative nucleotide-binding protein, is essential for the secretion process across the outer membrane even though the protein contains no hydrophobic sequence that could target or anchor it to the bacterial envelope. For a better understanding of the relationship between XcpR and the other Xcp proteins which are located in the envelope, we have studied its subcellular localization. In a wild-type
P. aeruginosa
strain, XcpR was found associated with the cytoplasmic membrane. This association depends on the presence of the XcpY protein, which also appears to be necessary for XcpR stability. Functional complementation of an
xcpY
mutant required the XcpY protein to be expressed at a low level. Higher expression precluded the complementing activity of XcpY, although membrane association of XcpR was restored. This behavior suggested that an excess of free XcpY might interfere with the secretion by formation of inactive XcpR-XcpY complexes which cannot properly interact with their natural partners in the secretion machinery. These data show that a precise stoichiometric ratio between several components may be crucial for the functioning of the GSP.
Discontinuous Occurrence of the
hsp70
(
dnaK
) Gene among
Archaea
and Sequence Features of HSP70 Suggest a Novel Outlook on Phylogenies Inferred from This Protein
Authors: Gribaldo Simonetta , Lumia Valentina , Creti Roberta , Conway de Macario Everly , Sanangelantoni Annamaria , Cammarano Piero
Published: December 2019

Occurrence of the
hsp70
(
dnaK
) gene was investigated in various members of the domain
Archaea
comprising both euryarchaeotes and crenarchaeotes and in the hyperthermophilic bacteria
Aquifex pyrophilus
and
Thermotoga maritima
representing the deepest offshoots in phylogenetic trees of bacterial 16S rRNA sequences. The gene was not detected in 8 of 10 archaea examined but was found in
A. pyrophilus
and
T. maritima
, from which it was cloned and sequenced. Comparative analyses of the HSP70 amino acid sequences encoded in these genes, and others in the databases, showed that (i) in accordance with the vicinities seen in rRNA-based trees, the proteins from
A. pyrophilus
and
T. maritima
form a thermophilic cluster with that from the green nonsulfur bacterium
Thermomicrobium roseum
and are unrelated to their counterparts from gram-positive bacteria, proteobacteria/mitochondria, chlamydiae/spirochetes, deinococci, and cyanobacteria/chloroplasts; (ii) the
T. maritima
HSP70 clusters with the homologues from the archaea
Methanobacterium thermoautotrophicum
and
Thermoplasma acidophilum
, in contrast to the postulated unique kinship between archaea and gram-positive bacteria; and (iii) there are exceptions to the reported association between an insert in HSP70 and gram negativity, or vice versa, absence of insert and gram positivity. Notably, the HSP70 from
T. maritima
lacks the insert, although
T. maritima
is phylogenetically unrelated to the gram-positive bacteria. These results, along with the absence of
hsp70
(
dnaK
) in various archaea and its presence in others, suggest that (i) different taxa retained either one or the other of two
hsp70
(
dnaK
) versions (with or without insert), regardless of phylogenetic position; and (ii) archaea are aboriginally devoid of
hsp70
(
dnaK
), and those that have it must have received it from phylogenetically diverse bacteria via lateral gene transfer events that did not involve replacement of an endogenous
hsp70
(
dnaK
) gene.
A Glucan Synthase
FKS1
Homolog in
Cryptococcus neoformans
Is Single Copy and Encodes an Essential Function
Authors: Thompson John R. , Douglas Cameron M. , Li Weili , Jue Chong K. , Pramanik Barnali , Yuan Xiling , Rude Thomas H. , Toffaletti Dena L. , Perfect John R. , Kurtz Myra
Published: December 2019

Cryptococcal meningitis is a fungal infection, caused by
Cryptococcus neoformans
, which is prevalent in immunocompromised patient populations. Treatment failures of this disease are emerging in the clinic, usually associated with long-term treatment with existing antifungal agents. The fungal cell wall is an attractive target for drug therapy because the syntheses of cell wall glucan and chitin are processes that are absent in mammalian cells. Echinocandins comprise a class of lipopeptide compounds known to inhibit 1,3-β-glucan synthesis, and at least two compounds belonging to this class are currently in clinical trials as therapy for life-threatening fungal infections. Studies of
Saccharomyces cerevisiae
and
Candida albicans
mutants identify the membrane-spanning subunit of glucan synthase, encoded by the
FKS
genes, as the molecular target of echinocandins. In vitro, the echinocandins show potent antifungal activity against
Candida
and
Aspergillus
species but are much less potent against
C. neoformans
. In order to examine why
C. neoformans
cells are less susceptible to echinocandin treatment, we have cloned a homolog of
S. cerevisiae FKS1
from
C. neoformans
. We have developed a generalized method to evaluate the essentiality of genes in
Cryptococcus
and applied it to the
FKS1
gene. The method relies on homologous integrative transformation with a plasmid that can integrate in two orientations, only one of which will disrupt the target gene function. The results of this analysis suggest that the
C. neoformans FKS1
gene is essential for viability. The
C. neoformans FKS1
sequence is closely related to the
FKS1
sequences from other fungal species and appears to be single copy in
C. neoformans
. Furthermore, amino acid residues known to be critical for echinocandin susceptibility in
Saccharomyces
are conserved in the
C. neoformans FKS1
sequence.
Transcription of Two Sigma 70 Homologue Genes,
sigA
and
sigB
, in Stationary-Phase
Mycobacterium tuberculosis
Authors: Hu Yanmin , Coates Anthony R. M.
Published: December 2019

The
sigA
and
sigB
genes of
Mycobacterium tuberculosis
encode two sigma 70-like sigma factors of RNA polymerase. While transcription of the
sigA
gene is growth rate independent,
sigB
transcription is increased during entry into stationary phase. The
sigA
gene transcription is unresponsive to environmental stress but that of
sigB
is very responsive, more so in stationary-phase growth than in log-phase cultures. These data suggest that SigA is a primary sigma factor which, like ς
70
, controls the transcription of the housekeeping type of promoters. In contrast, SigB, although showing some overlap in function with SigA, is more like the alternative sigma factor, ς
S
, which controls the transcription of the gearbox type of promoters. Primer extension analysis identified the RNA start sites for both genes as 129 nucleotides upstream to the GTG start codon of
sigA
and 27 nucleotides from the ATG start codon of
sigB
. The −10 promoter of
sigA
but not that of
sigB
was similar to the ς
70
promoter. The half-life of the
sigA
transcript was very long, and this is likely to play an important part in its regulation. In contrast, the half-life of the
sigB
transcript was short, about 2 min. These results demonstrate that the
sigB
gene may control the regulons of stationary phase and general stress resistance, while
sigA
may be involved in the housekeeping regulons.
CtaA ofStaphylococcus aureusIs Required for Starvation Survival, Recovery, and Cytochrome Biosynthesis
Authors: Clements Mark O. , Watson Sean P. , Poole Robert K. , Foster Simon J.
Published: December 2019

AStaphylococcus aureusmutant (SPW3) apparently unable to survive long-term starvation was shown to have a transposon insertion within a gene homologous toctaAofBacillus subtiliswhich encodes a heme A synthase. Analysis of the cytochrome profiles of SPW3 revealed the absence of heme A-containing cytochromes compared to the parental 8325-4 strain. SPW3 demonstrated a 100-fold reduction in the ability to survive starvation induced by glucose limitation, under aerated conditions, compared to 8325-4. Analysis of starved cultures revealed that greater than 90% of the cells which demonstrated metabolism (as shown by rhodamine 123 accumulation) were unable to recover and form colonies on agar. Analysis of the lag phase and initial growth kinetics of those cells which could recover also showed a defect. This recovery defect could be partially alleviated by the inclusion of catalase in the recovery medium, indicating the probable involvement of oxidative stress. SPW3 also exhibited reduced colony size similar to that of a small-colony variant, increased resistance to aminoglycoside antibiotics, and reduced hemolysin and toxic shock syndrome toxin 1 production, but no alteration in the ability to form lesions in a subcutaneous mouse infection model.
cis
- and
trans
-Acting Elements Involved in Regulation of
aniA
, the Gene Encoding the Major Anaerobically Induced Outer Membrane Protein in
Neisseria gonorrhoeae
Authors: Householder Tracey C. , Belli Wesley A. , Lissenden Sarah , Cole Jeffrey A. , Clark Virginia L.
Published: December 2019

AniA (formerly Pan1) is the major anaerobically induced outer membrane protein in
Neisseria gonorrhoeae
. AniA has been shown to be a major antigen in patients with gonococcal disease, and we have been studying its regulation in order to understand the gonococcal response to anaerobiosis and its potential role in virulence. This study presents a genetic analysis of
aniA
regulation. Through deletion analysis of the upstream region, we have determined the minimal promoter region necessary for
aniA
expression. This 130-bp region contains a sigma 70-type promoter and an FNR (fumarate and nitrate reductase regulator protein) binding site, both of which are absolutely required for anaerobic expression. Also located in the minimal promoter region are three T-rich direct repeats and several potential NarP binding sites. This 80-bp region is required for induction by nitrite. By site-directed mutagenesis of promoter sequences, we have determined that the transcription of
aniA
is initiated only from the sigma 70-type promoter. The gearbox promoter, previously believed to be the major promoter, does not appear to be active during anaerobiosis. The gonococcal FNR and NarP homologs are involved in the regulation of
aniA
, and we demonstrate that placing
aniA
under the control of the
tac
promoter compensates for the inability of a gonococcal
fnr
mutant to grow anaerobically.
The
ompB
Operon Partially Determines Differential Expression of OmpC in
Salmonella typhi
and
Escherichia coli
Authors: Martínez-Flores Irma , Cano Roxana , Bustamante Víctor H. , Calva Edmundo , Puente JoséLuis
Published: December 2019

Expression of the
Escherichia coli
OmpC and OmpF outer membrane proteins is regulated by the osmolarity of the culture media. In contrast, expression of OmpC in
Salmonella typhi
is not influenced by osmolarity, while OmpF is regulated as in
E. coli
. To better understand the lack of osmoregulation of OmpC expression in
S. typhi
, we compared the expression of the
ompC
gene in
S. typhi
and
E. coli
, using
ompC-lacZ
fusions and outer membrane protein (OMP) electrophoretic profiles.
S. typhi ompC
expression levels in
S. typhi
were similar at low and high osmolarity along the growth curve, whereas osmoregulation of
E. coli ompC
in
E. coli
was observed during the exponential phase. Both genes were highly expressed at high and low osmolarity when present in
S. typhi
, while expression of both was regulated by osmolarity in
E. coli
. Complementation experiments with either the
S. typhi
or
E. coli ompB
operon in an
S. typhi ΔompB
strain carrying the
ompC-lacZ
fusions showed that both
S. typhi
and
E. coli ompC
were not regulated by osmolarity when they were under the control of
S. typhi ompB
. Interestingly, in the same strain, both genes were osmoregulated under
E. coli ompB
. Surprisingly, in
E. coli ΔompB
, they were both osmoregulated under
S. typhi
or
E. coli ompB
. Thus, the lack of osmoregulation of OmpC expression in
S. typhi
is determined in part by the
ompB
operon, as well as by other unknown
trans
-acting elements present in
S. typhi
.
The LysR Homolog LrhA Promotes RpoS Degradation by Modulating Activity of the Response Regulator SprE
Authors: Gibson Katherine E. , Silhavy Thomas J.
Published: December 2019

Synthesis of the OmpF porin of
Escherichia coli
is regulated in response to environmental and growth phase signals. In order to identify constituents of the various regulatory pathways involved in modulating
ompF
transcriptional expression, transposon insertion mutagenesis was performed and mutations that increased
ompF′-lacZ
activity were identified as previously described. Mutations mapping to a previously identified gene of unknown function,
lrhA
, were obtained. We found that LrhA, a LysR homolog, functions as a regulatory component in the RpoS-dependent growth phase repression of
ompF
. In addition to altered growth phase regulation of
ompF
, these
lrhA
mutants have pleiotropic stationary-phase defects as a result of decreased RpoS levels. We provide evidence that LrhA promotes degradation of RpoS by functioning within a genetic pathway that includes the response regulator SprE and the ClpXP protease. LrhA functions upstream of the other components in the pathway and appears to modulate the activity of SprE.
Phosphorylation and Functional Properties of the IIA Domain of the Lactose Transport Protein of
Streptococcus thermophilus
Authors: Gunnewijk Marga G. W. , Postma Pieter W. , Poolman Bert
Published: December 2019

The lactose-H
+
symport protein (LacS) of
Streptococcus thermophilus
has a carboxyl-terminal regulatory domain (IIA
LacS
) that is homologous to a family of proteins and protein domains of the phosphoenolpyruvate:carbohydrate phosphotransferase system (PTS) in various organisms, of which IIA
Glc
of
Escherichia coli
is the best-characterized member. On the basis of these similarities, it was anticipated that IIA
LacS
would be able to perform one or more functions associated with IIA
Glc
, i.e., carry out phosphoryl transfer and/or affect other catabolic functions. The gene fragment encoding IIA
LacS
was overexpressed in
Escherichia coli
, and the protein was purified in two steps by metal affinity and anion-exchange chromatography. IIA
LacS
was unable to restore glucose uptake in a IIA
Glc
-deficient strain, which is consistent with a very low rate of phosphorylation of IIA
LacS
by phosphorylated HPr (HPr∼P) from
E. coli
. With HPr∼P from
S. thermophilus
, the rate was more than 10-fold higher, but the rate constants for the phosphorylation of IIA
LacS
(
k
1
= 4.3 × 10
2
M
−1
s
−1
) and dephosphorylation of IIA
LacS
∼P by HPr (
k
−1
= 1.1 × 10
3
M
−1
s
−1
) are still at least 4 orders of magnitude lower than for the phosphoryltransfer between IIA
Glc
and HPr from
E. coli
. This finding suggests that IIA
LacS
has evolved into a protein domain whose main function is not to transfer phosphoryl groups rapidly. On the basis of sequence alignment of IIA proteins with and without putative phosphoryl transfer functions and the known structure of IIA
Glc
, we constructed a double mutant [IIA
LacS
(I548E/G556D)] that was predicted to have increased phosphoryl transfer activity. Indeed, the phosphorylation rate of IIA
LacS
(I548E/G556D) by HPr∼P increased (
k
1
= 4.0 × 10
3
M
−1
s
−1
) and became nearly independent of the source of HPr∼P (
S. thermophilus
,
Bacillus subtilis
, or
E. coli
). The increased phosphoryl transfer rate of IIA
LacS
(I548E/G556D) was insufficient to complement IIA
Glc
in PTS-mediated glucose transport in
E. coli
. Both IIA
LacS
and IIA
LacS
(I548E/G556D) could replace IIA
Glc
, but in another function: they inhibited glycerol kinase (inducer exclusion) when present in the unphosphorylated form.
The Mithramycin Gene Cluster of
Streptomyces argillaceus
Contains a Positive Regulatory Gene and Two Repeated DNA Sequences That Are Located at Both Ends of the Cluster
Authors: Lombó Felipe , Braña Alfredo F. , Méndez Carmen , Salas José A.
Published: December 2019

Sequencing of a 4.3-kb DNA region from the chromosome of
Streptomyces argillaceus
, a mithramycin producer, revealed the presence of two open reading frames (ORFs). The first one (
orfA
) codes for a protein that resembles several transport proteins. The second one (
mtmR
) codes for a protein similar to positive regulators involved in antibiotic biosynthesis (DnrI, SnoA, ActII-orf4, CcaR, and RedD) belonging to the
Streptomyces
antibiotic regulatory protein (SARP) family. Both ORFs are separated by a 1.9-kb, apparently noncoding region. Replacement of the
mtmR
region by an antibiotic resistance cassette completely abolished mithramycin biosynthesis. Expression of
mtmR
in a high-copy-number vector in
S. argillaceus
caused a 16-fold increase in mithramycin production. The
mtmR
gene restored actinorhodin production in
Streptomyces coelicolor
JF1 mutant, in which the actinorhodin-specific activator ActII-orf4 is inactive, and also stimulated actinorhodin production by
Streptomyces lividans
TK21. A 241-bp region located 1.9 kb upstream of
mtmR
was found to be repeated approximately 50 kb downstream of
mtmR
at the other end of the mithramycin gene cluster. A model to explain a possible route for the acquisition of the mithramycin gene cluster by
S. argillaceus
is proposed.
Cyclic AMP Receptor Protein and TyrR Are Required for Acid pH and Anaerobic Induction of
hyaB
and
aniC
in
Salmonella typhimurium
Authors: Park Kyeong R. , Giard Jean-Christophe , Eom Juno H. , Bearson Shawn , Foster John W.
Published: December 2019

Two acid-inducible genes,
aniC
and
aciK
, that require anaerobiosis and tyrosine for expression were identified as
orf326a
encoding a potential amino acid/polyamine antiporter and
hyaB
encoding hydrogenase I, respectively. Cyclic AMP (cAMP) receptor protein, cAMP, and TyrR, regulator of aromatic amino acid metabolism, were strong positive regulators of both genes.
Genetic and Biochemical Characterization of a High-Affinity Betaine Uptake System (BusA) in
Lactococcus lactis
Reveals a New Functional Organization within Bacterial ABC Transporters
Authors: Obis David , Guillot Alain , Gripon Jean-Claude , Renault Pierre , Bolotin Alexander , Mistou Michel-Yves
Published: December 2019

The cytoplasmic accumulation of exogenous betaine stimulates the growth of
Lactococcus lactis
cultivated under hyperosmotic conditions. We report that
L. lactis
possesses a single betaine transport system that belongs to the ATP-binding cassette (ABC) superfamily of transporters. Through transposon mutagenesis, a mutant deficient in betaine transport was isolated. We identified two genes,
busAA
and
busAB
, grouped in an operon,
busA
(betaine uptake system). The transcription of
busA
is strongly regulated by the external osmolality of the medium. The
busAA
gene codes for the ATP-binding protein.
busAB
encodes a 573-residue polypeptide which presents two striking features: (i) a fusion between the regions encoding the transmembrane domain (TMD) and the substrate-binding domain (SBD) and (ii) a swapping of the SBD subdomains when compared to the
Bacillus subtilis
betaine-binding protein, OpuAC. BusA of
L. lactis
displays a high affinity towards betaine (

K
m

= 1.7 μM) and is an osmosensor whose activity is tightly regulated by external osmolality, leading the betaine uptake capacity of
L. lactis
to be under dual control at the biochemical and genetic levels. A protein presenting the characteristics predicted for BusAB was detected in the membrane fraction of
L. lactis
. The fusion between the TMD and the SBD is the first example of a new organization within prokaryotic ABC transporters.
Cloning and Sequence Analysis of Two
Pseudomonas
Flavoprotein Xenobiotic Reductases
Authors: Blehert David S. , Fox Brian G. , Chambliss Glenn H.
Published: December 2019

The genes encoding flavin mononucleotide-containing oxidoreductases, designated xenobiotic reductases, from
Pseudomonas putida
II-B and
P. fluorescens
I-C that removed nitrite from nitroglycerin (NG) by cleavage of the nitroester bond were cloned, sequenced, and characterized. The
P. putida
gene,
xenA
, encodes a 39,702-Da monomeric, NAD(P)H-dependent flavoprotein that removes either the terminal or central nitro groups from NG and that reduces 2-cyclohexen-1-one but did not readily reduce 2,4,6-trinitrotoluene (TNT). The
P. fluorescens
gene,
xenB
, encodes a 37,441-Da monomeric, NAD(P)H-dependent flavoprotein that exhibits fivefold regioselectivity for removal of the central nitro group from NG and that transforms TNT but did not readily react with 2-cyclohexen-1-one. Heterologous expression of
xenA
and
xenB
was demonstrated in
Escherichia coli
DH5α. The transcription initiation sites of both
xenA
and
xenB
were identified by primer extension analysis. BLAST analyses conducted with the
P. putida xenA
and the
P. fluorescens xenB
sequences demonstrated that these genes are similar to several other bacterial genes that encode broad-specificity flavoprotein reductases. The prokaryotic flavoprotein reductases described herein likely shared a common ancestor with old yellow enzyme of yeast, a broad-specificity enzyme which may serve a detoxification role in antioxidant defense systems.
rpoS
Function Is Essential for
bgl
Silencing Caused by C-Terminally Truncated H-NS in
Escherichia coli
Authors: Ohta Tomoko , Ueguchi Chiharu , Mizuno Takeshi
Published: December 2019

From evolutionary and physiological viewpoints, the
Escherichia coli bgl
operon is intriguing because its expression is silent (Bgl
−
phenotype), at least under several laboratory conditions. H-NS, a nucleoid protein, is known as a DNA-binding protein involved in
bgl
silencing. However, we previously found that
bgl
expression is still silent in a certain subset of
hns
mutations, each of which results in a defect in its DNA-binding ability. Based on this fact, we proposed a model in which a postulated DNA-binding protein(s) has an adapter function by interacting with both the
cis
-acting element of the
bgl
promoter and the mutated H-NS. To identify such a presumed adapter molecule, we attempted to isolate mutants exhibiting the Bgl
+
phenotype in the background of
hns60
, encoding the mutant H-NS protein lacking the DNA-binding domain by random insertion mutagenesis with the mini-Tn
10cam
transposon. These isolated mutations were mapped to five loci on the chromosome. Among these loci, three appeared to be
leuO
,
hns
, and
bglJ
, which were previously characterized, while the other two were novel. Genetic analysis revealed that the two insertions are within the
rpoS
gene and in front of the
lrhA
gene, respectively. The former encodes the stationary-phase-specific sigma factor, ς
S
, and the latter encodes a LysR-like DNA-binding protein. It was found that ς
S
is defective in both types of mutant cells. These results showed that the
rpoS
function is involved in the mechanism underlying
bgl
silencing, at least in the
hns60
background used in this study. We also examined whether the H-NS homolog StpA has such an adapter function, as was previously proposed. Our results did not support the idea that StpA has an adapter function in the genetic background used.
Campylobacter jejuni
Contains Two Fur Homologs: Characterization of Iron-Responsive Regulation of Peroxide Stress Defense Genes by the PerR Repressor
Authors: van Vliet Arnoud H. M. , Baillon Marie-Louise A. , Penn Charles W. , Ketley Julian M.
Published: December 2019

Expression of the peroxide stress genes alkyl hydroperoxide reductase (
ahpC
) and catalase (
katA
) of the microaerophile
Campylobacter jejuni
is repressed by iron. Whereas iron repression in gram-negative bacteria is usually carried out by the Fur protein, previous work showed that this is not the case in
C. jejuni
, as these genes are still iron repressed in a
C. jejuni fur
mutant. An open reading frame encoding a Fur homolog (designated PerR for “peroxide stress regulator”) was identified in the genome sequence of
C. jejuni
. The
perR
gene was disrupted by a kanamycin resistance cassette in
C. jejuni
wild-type and
fur
mutant strains. Subsequent characterization of the
C. jejuni perR
mutants showed derepressed expression of both AhpC and KatA at a much higher level than that obtained by iron limitation, suggesting that expression of these genes is controlled by other regulatory factors in addition to the iron level. Other iron-regulated proteins were not affected by the
perR
mutation. The
fur perR
double mutant showed derepressed expression of known iron-repressed genes. Further phenotypic analysis of the
perR
mutant,
fur
mutant, and the
fur perR
double mutant showed that the
perR
mutation made
C. jejuni
hyperresistant to peroxide stress caused by hydrogen peroxide and cumene hydroperoxide, a finding consistent with the high levels of KatA and AhpC expression, and showed that these enzymes were functional. Quantitative analysis of KatA expression showed that both the
perR
mutation and the
fur
mutation had profound effects on catalase activity, suggesting additional non-iron-dependent regulation of KatA and, by inference, AhpC. The PerR protein is a functional but nonhomologous substitution for the OxyR protein, which regulates peroxide stress genes in other gram-negative bacteria. Regulation of peroxide stress genes by a Fur homolog has recently been described for the gram-positive bacterium
Bacillus subtilis. C. jejuni
is the first gram-negative bacterium where non-OxyR regulation of peroxide stress genes has been described and characterized.
A Novel Role for
Escherichia coli
Endonuclease VIII in Prevention of Spontaneous G→T Transversions
Authors: Blaisdell Jeffrey O. , Hatahet Zafer , Wallace Susan S.
Published: December 2019

In the bacterium
Escherichia coli
, oxidized pyrimidines are removed by two DNA glycosylases, endonuclease III and endonuclease VIII (endo VIII), encoded by the
nth
and
nei
genes, respectively. Double mutants lacking both of these activities exhibit a high spontaneous mutation frequency, and here we show that all of the mutations observed in the double mutants were G:C→A:T transitions; no thymine mutations were found. These findings are in agreement with the preponderance of C→T transitions in the oxidative and spontaneous mutational databases. The major oxidized purine lesion in DNA, 7,8-dihydro-8-oxoguanine (8-oxoG), is processed by two DNA glycosylases, formamidopyrimidine DNA glycosylase (Fpg), which removes 8-oxoG opposite C, and MutY DNA glycosylase, which removes misincorporated A opposite 8-oxoG. The high spontaneous mutation frequency previously observed in
fpg mutY
double mutants was significantly enhanced by the addition of the
nei
mutation, suggesting an overlap in the substrate specificities between endo VIII and Fpg/MutY. When the mutational specificity was examined, all of the mutations observed were G:C→T:A transversions, indicating that in the absence of Fpg and MutY, endo VIII serves as a backup activity to remove 8-oxoG. This was confirmed by showing that, indeed, endo VIII can recognize 8-oxoG in vitro.
Mechanism of Repression of the
aroP
P2 Promoter by the TyrR Protein of
Escherichia coli
Authors: Yang Ji , Wang Peixiang , Pittard A. J.
Published: December 2019

Previously, we have shown that expression of the
Escherichia coli aroP
P2 promoter is partially repressed by the TyrR protein alone and strongly repressed by the TyrR protein in the presence of the coeffector tyrosine or phenylalanine (P. Wang, J. Yang, and A. J. Pittard, J. Bacteriol. 179:4206–4212, 1997). Here we present in vitro results showing that the TyrR protein and RNA polymerase can bind simultaneously to the
aroP
P2 promoter. In the presence of tyrosine, the TyrR protein inhibits open complex formation at the P2 promoter, whereas in the absence of any coeffector or in the presence of phenylalanine, the TyrR protein inhibits a step(s) following the formation of open complexes. We also present mutational evidence which implicates the N-terminal domain of the TyrR protein in the repression of P2 expression. The TyrR binding site of
aroP
, which includes one weak and one strong TyrR box, is located 5 bp downstream of the transcription start site of P2. Results from a mutational analysis show that the strong box (which is located more closely to the P2 promoter), but not the weak box, plays a critical role in P2 repression.
Identification and Characterization of the Single-Stranded DNA-Binding Protein of Bacteriophage P1
Authors: Lehnherr Hansjörg , Bendtsen Jannick D. , Preuss Fabian , Ilyina Tatiana V.
Published: December 2019

The genome of bacteriophage P1 harbors a gene coding for a 162-amino-acid protein which shows 66% amino acid sequence identity to the
Escherichia coli
single-stranded DNA-binding protein (SSB). The expression of the P1 gene is tightly regulated by P1 immunity proteins. It is completely repressed during lysogenic growth and only weakly expressed during lytic growth, as assayed by an
ssb
-P1/
lacZ
fusion construct. When cloned on an intermediate-copy-number plasmid, the P1 gene is able to suppress the temperature-sensitive defect of an
E. coli ssb
mutant, indicating that the two proteins are functionally interchangeable. Many bacteriophages and conjugative plasmids do not rely on the SSB protein provided by their host organism but code for their own SSB proteins. However, the close relationship between SSB-P1 and the SSB protein of the P1 host,
E. coli
, raises questions about the functional significance of the phage protein.
Expression of P
II
and Glutamine Synthetase Is Regulated by P
II
, the
ntrBC
Products, and Processing of the
glnBA
mRNA in
Rhodospirillum rubrum
Authors: Cheng Jiujun , Johansson Magnus , Nordlund Stefan
Published: December 2019

We have studied the transcription of the
glnB
and
glnA
genes in
Rhodospirillum rubrum
with firefly luciferase as a reporter enzyme. Under NH
4
+
and N
2
conditions,
glnBA
was cotranscribed from a weak and a strong promoter. In nitrogen-fixing cultures, activity of the latter was highly enhanced by NtrC, but transcription from both promoters occurred under both conditions. There is no promoter controlling transcription of
glnA
alone, supporting our proposal that the
glnA
mRNA is produced by processing.
Contribution of the Cell Wall Component Teichuronopeptide to pH Homeostasis and Alkaliphily in the Alkaliphile
Bacillus lentus
C-125
Authors: Aono Rikizo , Ito Masahiro , Machida Takayoshi
Published: December 2019

A teichuronopeptide (TUP) is one of major structural components of the cell wall of the facultative alkaliphilic strain
Bacillus lentus
C-125. A mutant defective in TUP synthesis grows slowly at alkaline pH. An upper limit of pH for growth of the mutant was 10.4, while that of the parental strain C-125 was 10.8. Gene
tupA
, directing synthesis of TUP, was cloned from C-125 chromosomal DNA. The primary translation product of this gene is likely a cytoplasmic protein (57.3 kDa) consisting of 489 amino acid residues. Introduction of the
tupA
gene into the TUP-defective mutant complemented the mutation responsible for the pleiotropic phenotypes of the mutant, leading to simultaneous disappearance of the defect in TUP synthesis, the diminished ability for cytoplasmic pH homeostasis, and the low tolerance for alkaline conditions. These results demonstrate that the acidic polymer TUP in the cell wall plays a role in pH homeostasis in this alkaliphile.
The
hetC
Gene Is a Direct Target of the NtcA Transcriptional Regulator in Cyanobacterial Heterocyst Development
Authors: Muro-Pastor Alicia M. , Valladares Ana , Flores Enrique , Herrero Antonia
Published: December 2019

The heterocyst is the site of nitrogen fixation in aerobically grown cultures of some filamentous cyanobacteria. Heterocyst development in
Anabaena
sp. strain PCC 7120 is dependent on the global nitrogen regulator NtcA and requires, among others, the products of the
hetR
and
hetC
genes. Expression of
hetC
, tested by RNA- DNA hybridization, was impaired in an
ntcA
mutant. A nitrogen-regulated, NtcA-dependent putative transcription start point was localized at nucleotide −571 with respect to the
hetC
translational start. Sequences upstream from this transcription start point exhibit the structure of the canonical cyanobacterial promoter activated by NtcA, and purified NtcA protein specifically bound to a DNA fragment containing this promoter. Activation of expression of
hetC
during heterocyst development appears thus to be directly operated by NtcA. NtcA-mediated activation of
hetR
expression was not impaired in a
hetC
mutant, indicating that HetC is not an NtcA-dependent element required for
hetR
induction.
Evidence of Horizontal Transfer of the
Eco
O109I Restriction-Modification Gene to
Escherichia coli
Chromosomal DNA
Authors: Kita Keiko , Tsuda Junko , Kato Toshinobu , Okamoto Kenji , Yanase Hideshi , Tanaka Masashi
Published: December 2019

A DNA fragment carrying the genes coding for
Eco
O109I endonuclease and
Eco
O109I methylase, which recognize the nucleotide sequence 5′-(A/G)GGNCC(C/T)-3′, was cloned from the chromosomal DNA of
Escherichia coli
H709c. The
Eco
O109I restriction-modification (R-M) system was found to be inserted between the
int
and
psu
genes from satellite bacteriophage P4, which were lysogenized in the chromosome at the P4 phage attachment site of the corresponding
leuX
gene observed in
E. coli
K-12 chromosomal DNA. The
sid
gene of the prophage was inactivated by insertion of one copy of IS
21
. These findings may shed light on the horizontal transfer and stable maintenance of the R-M system.
Expression of the
Escherichia coli
Ada Regulon in Stationary Phase: Evidence for
rpoS
-Dependent Negative Regulation of
alkA
Transcription
Authors: Landini Paolo , Busby Stephen J. W.
Published: December 2019

The
Escherichia coli
Ada protein activates ς
70
-dependent transcription at three different promoters (
ada
,
aidB
, and
alkA
) in response to alkylation damage of DNA. During stationary phase, however, the methylated form of Ada shuts off expression of
alkA
; this repression is specific for ς
S
-dependent transcription. Thus, at the
alkA
promoter, the Ada protein can act as both a positive and negative modulator of the adaptive response to alkylation damage, depending on the cell’s physiological state.
TheAgrobacterium tumefaciensChaperone-Like Protein, VirE1, Interacts with VirE2 at Domains Required for Single-Stranded DNA Binding and Cooperative Interaction
Authors: Sundberg Christopher D. , Ream Walt
Published: December 2019

Agrobacterium tumefacienstransfers single-stranded DNA (ssDNA) into plants. Efficient tumorigenesis requires VirE1-dependent export of ssDNA-binding (SSB) protein VirE2. VirE1 binds VirE2 domains involved in SSB and self-association, and VirE1 may facilitate VirE2 export by preventing VirE2 aggregation and the premature binding of VirE2 to ssDNA.
Partition of the Linear Plasmid N15: Interactions of N15 Partition Functions with the
sop
Locus of the F Plasmid
Authors: Ravin N. , Lane D.
Published: December 2019

A locus close to one end of the linear N15 prophage closely resembles the
sop
operon which governs partition of the F plasmid; the promoter region contains similar operator sites, and the two putative gene products have extensive amino acid identity with the SopA and -B proteins of F. Our aim was to ascertain whether the N15
sop
homologue functions in partition, to identify the centromere site, and to examine possible interchangeability of function with the F Sop system. When expressed at a moderate level, N15 SopA and -B proteins partly stabilize mini-F which lacks its own
sop
operon but retains the
sopC
centromere. The stabilization does not depend on increased copy number. Likewise, an N15 mutant with most of its
sop
operon deleted is partly stabilized by F Sop proteins and fully stabilized by its own. Four inverted repeat sequences similar to those of
sopC
were located in N15. They are distant from the
sop
operon and from each other. Two of these were shown to stabilize a mini-F
sop
deletion mutant when N15 Sop proteins were provided. Provision of the SopA homologue to plasmids with a
sopA
deletion resulted in further destabilization of the plasmid. The N15 Sop proteins exert effective, but incomplete, repression at the F
sop
promoter. We conclude that the N15
sop
locus determines stable inheritance of the prophage by using dispersed centromere sites. The SopB-centromere and SopA-operator interactions show partial functional overlap between N15 and F. SopA of each plasmid appears to interact with SopB of the other, but in a way that is detrimental to plasmid maintenance.
The Bacteriophage P1 HumD Protein Is a Functional Homolog of the Prokaryotic UmuD′-Like Proteins and Facilitates SOS Mutagenesis in
Escherichia coli
Authors: McLenigan Mary P. , Kulaeva Olga I. , Ennis Don G. , Levine Arthur S. , Woodgate Roger
Published: December 2019

The
Escherichia coli umuD
and
umuC
genes comprise an operon and encode proteins that are involved in the mutagenic bypass of normally replication-inhibiting DNA lesions. UmuD is, however, unable to function in this process until it undergoes a RecA-mediated cleavage reaction to generate UmuD′. Many homologs of
umuDC
have now been identified. Most are located on bacterial chromosomes or on broad-host-range R plasmids. One such putative homolog,
humD
(homolog of
umuD
) is, however, found on the bacteriophage P1 genome. Interestingly,
humD
differs from other
umuD
homologs in that it encodes a protein similar in size to the posttranslationally generated UmuD′ protein and not UmuD, nor is it in an operon with a cognate
umuC
partner. To determine if HumD is, in fact, a bona fide homolog of the prokaryotic UmuD′-like mutagenesis proteins, we have analyzed the ability of HumD to complement UmuD′ functions in vivo as well as examined HumD’s physical properties in vitro. When expressed from a high-copy-number plasmid, HumD restored cellular mutagenesis and increased UV survival to normally nonmutable
recA430 lexA
(Def) and UV-sensitive Δ
umuDC recA718 lexA
(Def) strains, respectively. Complementing activity was reduced when HumD was expressed from a low-copy-number plasmid, but this observation is explained by immunoanalysis which indicates that HumD is normally poorly expressed in vivo. In vitro analysis revealed that like UmuD′, HumD forms a stable dimer in solution and is able to interact with
E. coli
UmuC and RecA nucleoprotein filaments. We conclude, therefore, that bacteriophage P1 HumD is a functional homolog of the UmuD′-like proteins, and we speculate as to the reasons why P1 might require the activity of such a protein in vivo.
Preprotein Translocation by a Hybrid Translocase Composed of
Escherichia coli
and
Bacillus subtilis
Subunits
Authors: Swaving Jelto , van Wely Karel H. M. , Driessen Arnold J. M.
Published: December 2019

Bacterial protein translocation is mediated by translocase, a multisubunit membrane protein complex that consists of a peripheral ATPase SecA and a preprotein-conducting channel with SecY, SecE, and SecG as subunits. Like
Escherichia coli
SecG, the
Bacillus subtilis
homologue, YvaL, dramatically stimulated the ATP-dependent translocation of precursor PhoB (prePhoB) by the
B. subtilis
SecA-SecYE complex. To systematically determine the functional exchangeability of translocase subunits, all of the relevant combinations of the
E. coli
and
B. subtilis secY
,
secE
, and
secG
genes were expressed in
E. coli
. Hybrid SecYEG complexes were overexpressed at high levels. Since SecY could not be overproduced without SecE, these data indicate a stable interaction between the heterologous SecY and SecE subunits.
E. coli
SecA, but not
B. subtilis
SecA, supported efficient ATP-dependent translocation of the
E. coli
precursor OmpA (proOmpA) into inner membrane vesicles containing the hybrid SecYEG complexes, if
E. coli
SecY and either
E. coli
SecE or
E. coli
SecG were present. Translocation of
B. subtilis
prePhoB, on the other hand, showed a strict dependence on the translocase subunit composition and occurred efficiently only with the homologous translocase. In contrast to
E. coli
SecA,
B. subtilis
SecA binds the SecYEG complexes only with low affinity. These results suggest that each translocase subunit contributes in an exclusive manner to the specificity and functionality of the complex.
Two New Early Bacteriophage T4 Genes,
repEA
and
repEB
, That Are Important for DNA Replication Initiated from Origin E
Authors: Vaiskunaite Rita , Miller Andrew , Davenport Laura , Mosig Gisela
Published: December 2019

Two new, small, early bacteriophage T4 genes,
repEA
and
repEB
, located within the origin E (
oriE
) region of T4 DNA replication, affect functioning of this origin. An important and unusual property of the
oriE
region is that it is transcribed at early and late periods after infection, but in opposite directions (from complementary DNA strands). The early transcripts are mRNAs for RepEA and RepEB proteins, and they can serve as primers for leading-strand DNA synthesis. The late transcripts, which are genuine antisense RNAs for the early transcripts, direct synthesis of virion components. Because the T4 genome contains several origins, and because recombination can bypass a primase requirement for retrograde synthesis, neither defects in a single origin nor primase deficiencies are lethal in T4 (Mosig et al., FEMS Microbiol. Rev. 17:83–98, 1995). Therefore,
repEA
and
repEB
were expected and found to be important for T4 DNA replication only when activities of other origins were reduced. To investigate the in vivo roles of the two
repE
genes, we constructed nonsense mutations in each of them and combined them with the
motA
mutation
sip1
that greatly reduces initiation from other origins. As expected, T4 DNA synthesis and progeny production were severely reduced in the double mutants as compared with the single
motA
mutant, but early transcription of
oriE
was reduced neither in the
motA
nor in the
repE
mutants. Moreover, residual DNA replication and growth of the double mutants were different at different temperatures, suggesting different functions for
repEA
and
repEB
. We surmise that the different structures and protein requirements for functioning of the different origins enhance the flexibility of T4 to adapt to varied growth conditions, and we expect that different origins in other organisms with multiorigin chromosomes might differ in structure and function for similar reasons.
Inactivation of the Tricarboxylic Acid Cycle Aconitase Gene from
Streptomyces viridochromogenes
Tü494 Impairs Morphological and Physiological Differentiation
Authors: Schwartz D. , Kaspar S. , Kienzlen G. , Muschko K. , Wohlleben W.
Published: December 2019

The tricarboxylic acid (TCA) cycle aconitase gene
acnA
from
Streptomyces viridochromogenes
Tü494 was cloned and analyzed. AcnA catalyzes the isomerization of citrate to isocitrate in the TCA cycle, as indicated by the ability of
acnA
to complement the aconitase-deficient
Escherichia coli
mutant JRG3259. An
acnA
mutant was unable to develop aerial mycelium and to sporulate, resulting in a bald phenotype. Furthermore, the mutant did not produce the antibiotic phosphinothricin tripeptide, demonstrating that AcnA also affects physiological differentiation.
An Improved Transposon for the Halophilic Archaeon
Haloarcula hispanica
Authors: Woods Wayne G. , Ngui Katrina , Dyall-Smith Michael L.
Published: December 2019

An improved transposon (ThD
73
) for
Haloarcula hispanica
is described. Based on the halobacterial insertion sequence ISH
28
, it showed little target sequence specificity but was biased toward a lower G+C content. Twenty randomly selected ThD
73
mutants were analyzed, and the DNA flanking their insertions revealed several recognizable sequences, including two (unrelated) ISH elements.