bluuebunny/crossref_metadata_2025 huggingface dataset:
Intracellular Immunization of Prokaryotic Cells against a Bacteriotoxin
Authors: Chames Patrick , Fieschi Jacques , Baty Daniel , Duché Denis
Published: December 2019

Intracellularly expressed antibodies have been designed to bind and inactivate target molecules inside eukaryotic cells. Here we report that an antibody fragment can be used to probe the periplasmic localization of the colicin A N-terminal domain. Colicins form voltage-gated ion channels in the inner membrane of
Escherichia coli
. To reach their target, they bind to a receptor located on the outer membrane and then are translocated through the envelope. The N-terminal domain of colicins is involved in the translocation step and therefore is thought to interact with proteins of the translocation system. To compete with this system, a single-chain variable fragment (scFv) directed against the N-terminal domain of the colicin A was synthesized and exported into the periplasmic space of
E. coli
. The periplasmic scFv inhibited the lethal activity of colicin A and had no effect on the lethal activity of other colicins. Moreover, the scFv was able to specifically inactivate hybrid colicins possessing the colicin A N-terminal domain without affecting their receptor binding. Hence, the periplasmic scFv prevents the translocation of colicin A and probably its interaction with import machinery. This indicates that the N-terminal domain of the toxin is accessible in the periplasm. Moreover, we show that production of antibody fragments to interfere with a biological function can be applied to prokaryotic systems.
Transcriptional and Translational Regulation of Photosystem I and II Genes in Light-Dark- and Continuous-Light-Grown Cultures of the Unicellular Cyanobacterium
Cyanothece
sp. Strain ATCC 51142
Authors: Colón-López Milagros S. , Sherman Louis A.
Published: December 2019

Cyanothece
sp. strain ATCC 51142, a unicellular, diazotrophic cyanobacterium, demonstrated extensive metabolic periodicities of photosynthesis, respiration, and nitrogen fixation when grown under N
2
-fixing conditions. This report describes the relationship of the biosynthesis of photosynthesis genes to changes in the oligomerization state of the photosystems. Transcripts of the
psbA
gene family, encoding the photosystem II (PSII) reaction center protein D1, accumulated primarily during the light period, and net transcription reached a peak between 2 to 6 h in the light in light-dark (LD) growth and between 4 to 10 h in the subjective light when grown under continuous light (LL). The relative amount of the D1 protein (form 1 versus form 2) appeared to change during this diurnal cycle, along with changes in the PSII monomer/dimer ratio. D1 form 1 accumulated at approximately equal levels throughout the 24-h cycle, whereas D1 form 2 accumulated at significantly higher levels at approximately 8 to 10 h in the light or subjective light. The
psbD
gene, encoding the reaction center protein D2, also demonstrated differences between the two copies of this gene, with one copy transcribed more heavily around 6 to 8 h in the light. Accumulation of the PSI reaction center proteins PsaA and PsaB was maximal in the dark or subjective-dark periods, a period during which PSI was primarily in the trimeric form. We conclude that photosystem organization changes during the diurnal cycle to favor either noncyclic electron flow, which leads to O
2
evolution and CO
2
fixation, or cyclic electron flow, which favors ATP synthesis.
Genetic Determinants of Immunity and Integration of Temperate
Myxococcus xanthus
Phage Mx8
Authors: Salmi Daniel , Magrini Vincent , Hartzell Patricia L. , Youderian Philip
Published: December 2019

An 8.1-kb fragment of the temperate
Myxococcus xanthus
phage Mx8 genome, when cloned into a plasmid vector, permits site-specific integration of the plasmid and confers superinfection immunity. Sequence analysis of a 9.5-kb region of Mx8 DNA containing this fragment reveals 19 densely packed open reading frames, four of which have predicted products with known or suspected activities. The Mx8
imm
gene, required for superinfection immunity, has a sequence similar to that of
Arabidopsis thaliana
G-box-binding factor 1. Mx8 makes a DNA adenine methylase, Mox, and integrase, Int, related to other methylases and integrases. The
int
gene has two alternate translation initiation codons within the extensively overlapping
uoi
(upstream of int) gene. Comparison of the predicted product of the
uoi
gene with
Salmonella
phage P22 and
Streptomyces
plasmid Xis proteins shows that temperate phage excisionases may use variations of a helix-turn-helix motif to recognize specific DNA sequences.
Deletion of
algK
in Mucoid
Pseudomonas aeruginosa
Blocks Alginate Polymer Formation and Results in Uronic Acid Secretion
Authors: Jain Sumita , Ohman Dennis E.
Published: December 2019

Chronic pulmonary infection with
Pseudomonas aeruginosa
is a common and serious problem in patients with cystic fibrosis (CF). The
P. aeruginosa
isolates from these patients typically have a mucoid colony morphology due to overproduction of the exopolysaccharide alginate, which contributes to the persistence of the organisms in the CF lung. Most of the alginate biosynthetic genes are clustered in the
algD
operon, located at 34 min on the chromosome. Alginate biosynthesis begins with the formation of an activated monomer, GDP-mannuronate, which is known to occur via the products of the
algA
,
algC
, and
algD
genes. Polymannuronate forms in the periplasm, but the gene products involved in mannuronate translocation across the inner membrane and its polymerization are not known. One locus of the operon which remained uncharacterized was a new gene called
algK
between
alg44
and
algE
. We sequenced
algK
from the mucoid CF isolate FRD1 and expressed it in
Escherichia coli
, which revealed a polypeptide of the predicted size (52 kDa). The sequence of AlgK showed an apparent signal peptide characteristic of a lipoprotein. AlgK-PhoA fusion proteins were constructed and shown to be active, indicating that AlgK has a periplasmic subcellular localization. To test the phenotype of an AlgK
−
mutant, the
algK
coding sequence was replaced with a nonpolar gentamicin resistance cassette to avoid polar effects on genes downstream of
algK
that are essential for polymer formation. The
algK
Δ mutant was nonmucoid, demonstrating that AlgK was required for alginate production. Also, AlgK
−
mutants demonstrated a small-colony phenotype on L agar, suggesting that the loss of AlgK also caused a growth defect. The mutant phenotypes were complemented by a plasmid expressing
algK
in
trans
. When the
algK
Δ mutation was placed in an
algJ
::Tn
501
background, where
algA
was not expressed due to polar transposon effects, the growth defect was not observed. AlgK
−
mutants appeared to accumulate a toxic extracellular product, and we hypothesized that this could be an unpolymerized alginate precursor. High levels of low-molecular-weight uronic acid were produced by the AlgK
−
mutant. When AlgK
−
culture supernatants were subjected to dialysis, high levels of uronic acids diffused out of the dialysis sac, and no uronic acids were detectable after extensive dialysis. In contrast, the mucoid wild-type strain produced only polymerized uronic acids (i.e., alginate), whereas the
algKΔ algJ
::Tn
501
mutant produced no uronic acids. Thus, the alginate pathway in an AlgK
−
mutant was blocked after transport but at a step before polymerization, suggesting that AlgK plays an important role in the polymerization of mannuronate to alginate.
Archaeal Binding Protein-Dependent ABC Transporter: Molecular and Biochemical Analysis of the Trehalose/Maltose Transport System of the Hyperthermophilic Archaeon
Thermococcus litoralis
Authors: Horlacher Reinhold , Xavier Karina B. , Santos Helena , DiRuggiero Jocelyne , Kossmann Marina , Boos Winfried
Published: December 2019

We report the cloning and sequencing of a gene cluster encoding a maltose/trehalose transport system of the hyperthermophilic archaeon
Thermococcus litoralis
that is homologous to the
malEFG
cluster encoding the
Escherichia coli
maltose transport system. The deduced amino acid sequence of the
malE
product, the trehalose/maltose-binding protein (TMBP), shows at its N terminus a signal sequence typical for bacterial secreted proteins containing a glyceride lipid modification at the N-terminal cysteine. The
T. litoralis malE
gene was expressed in
E. coli
under control of an inducible promoter with and without its natural signal sequence. In addition, in one construct the endogenous signal sequence was replaced by the
E. coli
MalE signal sequence. The secreted, soluble recombinant protein was analyzed for its binding activity towards trehalose and maltose. The protein bound both sugars at 85°C with a

K
d

of 0.16 μM. Antibodies raised against the recombinant soluble TMBP recognized the detergent-soluble TMBP isolated from
T. litoralis
membranes as well as the products from all other DNA constructs expressed in
E. coli
. Transmembrane segments 1 and 2 as well as the N-terminal portion of the large periplasmic loop of the
E. coli
MalF protein are missing in the
T. litoralis
MalF. MalG is homologous throughout the entire sequence, including the six transmembrane segments. The conserved EAA loop is present in both proteins. The strong homology found between the components of this archaeal transport system and the bacterial systems is evidence for the evolutionary conservation of the binding protein-dependent ABC transport systems in these two phylogenetic branches.
Transfer of Tn
5385
, a Composite, Multiresistance Chromosomal Element from
Enterococcus faecalis
Authors: Rice Louis B. , Carias Lenore L.
Published: December 2019

Tn
5385
is a ca. 65-kb element integrated into the chromosomes of clinical
Enterococcus faecalis
strains CH19 and CH116. It confers resistance to erythromycin, gentamicin, mercuric chloride, streptomycin, tetracycline-minocycline, and penicillin via β-lactamase production. Tn
5385
is a composite structure containing regions previously found in staphylococcal and enterococcal plasmids. Several transposons and transposon-like elements within Tn
5385
have been identified, including conjugative transposon Tn
5381
, composite transposon Tn
5384
, and elements indistinguishable from staphylococcal transposons Tn
4001
and Tn
552
. The divergent regions of Tn
5385
are linked by a series of insertion sequence (IS) elements (IS
256
, IS
257
, and IS
1216
) of staphylococcal and enterococcal origin. The ends of Tn
5385
consist of directly repeated copies of enterococcal IS
1216
. Within the chromosomes of strains CH19 and CH116, Tn
5385
has interrupted an open reading frame with substantial homology to previously described alkyl hydrogen peroxide reductase genes. Segments of this open reading frame in both CH19 and CH116 have been deleted, but the amount of deleted DNA differs for the two insertions. Transfer of Tn
5385
from both donors into
E. faecalis
recipients occurs at a low frequency. Two types of transconjugants have been identified. In one type, the target alkyl hydrogen peroxide reductase open reading frame has been deleted, and sequences flanking Tn
5385
in the respective donors are carried over to the transconjugants. These data suggest that the mechanism of Tn
5385
insertion into the recipient chromosome in these transconjugants was recombination across flanking regions in the donors and homologous sequences in the recipients. The second type of transconjugant appears to have resulted from excision of Tn
5385
from the CH19 chromosome by recombination across the terminal IS
1216
elements and insertion into the recipient chromosome by recombination across Tn
5381
(within Tn
5385
) and a previously transferred Tn
5381
copy in the recipient chromosome. These data confirm that Tn
5385
is a composite structure with genetic material from diverse genera and suggest that it is a functional transposon. They also suggest that chromosomal recombination is a mechanism of genetic exchange in enterococci.
Expression of Invasin and Motility Are Coordinately Regulated in
Yersinia enterocolitica
Authors: Badger Julie L. , Miller Virginia L.
Published: December 2019

The
Yersinia enterocolitica inv
gene encodes the primary invasion factor invasin, which has been previously shown to be critical in the initial stages of infection. The expression of
inv
is influenced by growth phase and temperature and is maximal during late exponential-early stationary phase at 23°C. In addition, motility of
Y. enterocolitica
is regulated by temperature.
Y. enterocolitica
cells are motile when grown at lower temperatures (30°C or below), while bacteria grown at 37°C are nonmotile. This study was initiated to determine the molecular basis for the temperature regulation of
inv
expression. Two mutants were isolated that both showed a significant decrease in invasin expression but are hypermotile when grown at 23°C. The first mutant (JB1A8v) was a result of a random mTn
5
Km insertion into the
uvrC
gene. The
uvrC
mutant JB1A8v demonstrated a significant decrease in
inv
and an increase in
fleB
(encodes flagellin) expression. These results suggest that expression of
inv
and flagellin genes is coordinated at the level of transcription. The second regulatory mutant, JB16v, was a result of a targeted insertion into a locus similar to
sspA
which in
E. coli
encodes a stationary-phase regulator. The
E. coli sspA
gene was cloned and assayed for complementation in both of the regulatory mutants. It was determined that
E. coli sspA
restored invasin expression in both the
uvrC
mutant and the
sspA
mutant. In addition, the complementing clone decreased flagellin levels in these mutants.
CpxP, a Stress-Combative Member of the Cpx Regulon
Authors: Danese Paul N. , Silhavy Thomas J.
Published: December 2019

The CpxA/R two-component signal transduction system of
Escherichia coli
can combat a variety of extracytoplasmic protein-mediated toxicities. The Cpx system performs this function, in part, by increasing the synthesis of the periplasmic protease, DegP. However, other factors are also employed by the Cpx system for this stress-combative function. In an effort to identify these remaining factors, we screened a collection of random
lacZ
operon fusions for those fusions whose transcription is regulated by CpxA/R. Through this approach, we have identified a new locus,
cpxP
, whose transcription is stimulated by activation of the Cpx pathway.
cpxP
specifies a periplasmic protein that can combat the lethal phenotype associated with the synthesis of a toxic envelope protein. In addition, we show that
cpxP
transcription is strongly induced by alkaline pH in a CpxA-dependent manner and that
cpxP
and
cpx
mutant strains display hypersensitivity to growth in alkaline conditions.
Identification of AlcR, an AraC-Type Regulator of Alcaligin Siderophore Synthesis in
Bordetella bronchiseptica
and
Bordetella pertussis
Authors: Pradel Elizabeth , Guiso Nicole , Locht Camille
Published: December 2019

A Fur titration assay was used to isolate DNA fragments bearing putative Fur binding sites (FBS) from a partial
Bordetella bronchiseptica
genomic DNA library. A recombinant plasmid bearing a 3.5-kb DNA insert was further studied. Successive deletions in the cloned fragment enabled us to map a putative FBS at about 2 kb from one end. Sequence analysis revealed the presence of an FBS upstream from a new gene encoding an AraC-type transcriptional regulator. The deduced protein displays similarity to PchR, an activator of pyochelin siderophore and ferripyochelin receptor synthesis in
Pseudomonas aeruginosa
. Homologous genes in
Bordetella pertussis
and
Bordetella parapertussis
were PCR amplified, and sequence comparisons indicated a very high conservation in the three species. The
B. pertussis
and
B. bronchiseptica
chromosomal genes were inactivated by allelic exchange. Under low-iron growth conditions, the mutants did not secrete the alcaligin siderophore and lacked AlcC, an alcaligin biosynthetic enzyme. Alcaligin production was restored after transformation with a plasmid bearing the wild-type gene. On the basis of its role in regulation of alcaligin biosynthesis, the new gene was designated
alcR
. Additional sequence determination showed that
alcR
is located about 2 kb downstream from the
alcABC
operon and is transcribed in the same orientation. Two tightly linked open reading frames,
alcD
and
alcE
, were identified between
alcC
and
alcR
. AlcE is a putative iron-sulfur protein; AlcD shows no homology with the proteins in the database. The production of major virulence factors and colonization in the mouse respiratory infection model are AlcR independent.
Molecular Characterization of a Phage-Inducible Middle Promoter and Its Transcriptional Activator from the Lactococcal Bacteriophage φ31
Authors: Walker Shirley A. , Klaenhammer Todd R.
Published: December 2019

An inducible middle promoter from the lactococcal bacteriophage φ31 was isolated previously by shotgun cloning an 888-bp fragment (P
15A10
) upstream of the β-galactosidase (β-Gal) gene (
lacZ.st
) from
Streptococcus thermophilus
(D. J. O’Sullivan, S. A. Walker, S. G. West, and T. R. Klaenhammer, Bio/Technology 14:82–87, 1996). The promoter showed low levels of constitutive β-Gal activity which could be induced two- to threefold over baseline levels after phage infection. During this study, the fragment was subcloned and characterized to identify a smaller, tightly regulated promoter fragment which allowed no β-Gal activity until after phage infection. This fragment, defined within nucleotides 566 to 888 (P
566–888
; also called fragment 566–888), contained tandem, phage-inducible transcription start sites at nucleotides 703 and 744 (703/744 start sites). Consensus −10 regions were present upstream of both start sites, but no consensus −35 regions were identified for either start site. A transcriptional activator, encoded by an open reading frame (ORF2) upstream of the 703/744 start sites, was identified for P
566–888
. ORF2 activated P
566–888
when provided in
trans
in
Escherichia coli
. In addition, when combined with pTRK391 (P
15A10
::
lacZ.st
) in
Lactococcus lactis
NCK203, an antisense ORF2 construct was able to retard induction of the phage-inducible promoter as measured by β-Gal activity levels. Finally, gel shift assays showed that ORF2 was able to bind to promoter fragment 566–888. Deletion analysis of the region upstream from the tandem promoters identified a possible binding site for transcriptional activation of the phage promoters. The DNA-binding ability of ORF2 was eliminated upon deletion of part of this region, which lies centered approximately 35 bp upstream of start site 703. Deletion analysis and mutagenesis studies also elucidated a critical region downstream of the 703/744 start sites, where mutagenesis resulted in a two- to threefold increase in β-Gal activity. With these improvements, the level of expression achieved by an explosive-expression strategy was elevated from 3,000 to 11,000 β-Gal units within 120 min after induction.
Identification of the Fucose Synthetase Gene in the Colanic Acid Gene Cluster of
Escherichia coli
K-12
Authors: Andrianopoulos Kanella , Wang Lei , Reeves Peter R.
Published: December 2019

GDP–
l
-fucose, the substrate for fucosyltransferases for addition of fucose to polysaccharides or glycoproteins in both procaryotes and eucaryotes, is made from GDP–
d
-mannose.
l
-Fucose is a component of bacterial surface antigens, including the extracellular polysaccharide colanic acid produced by most
Escherichia coli
strains. We previously sequenced the
E. coli
colanic acid gene cluster and identified one of the GDP–
l
-fucose biosynthetic pathway genes,
gmd
. We report here the identification of the gene (
fcl
), located downstream of
gmd
, encoding the fucose synthetase.
Msn2p and Msn4p Control a Large Number of Genes Induced at the Diauxic Transition Which Are Repressed by Cyclic AMP in
Saccharomyces cerevisiae
Authors: Boy-Marcotte Emmanuelle , Perrot Michel , Bussereau Françoise , Boucherie Hélian , Jacquet Michel
Published: December 2019

The multicopy suppressors of the
snf1
defect, Msn2p and Msn4p transcription factors (Msn2/4p), activate genes through the stress-responsive
cis
element (CCCCT) in response to various stresses. This
cis
element is also the target for repression by the cyclic AMP (cAMP)-signaling pathway. We analyzed the two-dimensional gel electrophoresis pattern of protein synthesis of the
msn2 msn4
double mutant and compared it with that of the wild-type strain during exponential growth phase and at the diauxic transition. Thirty-nine gene products (including those of
ALD3
,
GDH3
,
GLK1
,
GPP2
,
HSP104
,
HXK1
,
PGM2
,
SOD2
,
SSA3
,
SSA4
,
TKL2
,
TPS1
, and
YBR149W
) are dependent upon Msn2/4p for their induction at the diauxic transition. The expression of all these genes is repressed by cAMP. Thirty other genes identified during this study are still inducible in the mutant. A subset of these genes were found to be superinduced at the diauxic transition, and others were subject to cAMP repression (including
ACH1
,
ADH2
,
ALD6
,
ATP2
,
GPD1
,
ICL1
, and
KGD2
). We conclude from this analysis that Msn2/4p control a large number of genes induced at the diauxic transition but that other, as-yet-uncharacterized regulators, also contribute to this response. In addition, we show here that cAMP repression applies to both Msn2/4p-dependent and -independent control of gene expression at the diauxic shift. Furthermore, the fact that all the Msn2/4p gene targets are subject to cAMP repression suggests that these regulators could be targets for the cAMP-signaling pathway.
Characterization of a Protocatechuate Catabolic Gene Cluster from
Rhodococcus opacus
1CP: Evidence for a Merged Enzyme with 4-Carboxymuconolactone-Decarboxylating and 3-Oxoadipate Enol-Lactone-Hydrolyzing Activity
Authors: Eulberg Dirk , Lakner Silvia , Golovleva Ludmila A. , Schlömann Michael
Published: December 2019

The catechol and protocatechuate branches of the 3-oxoadipate pathway, which are important for the bacterial degradation of aromatic compounds, converge at the common intermediate 3-oxoadipate enol-lactone. A 3-oxoadipate enol-lactone-hydrolyzing enzyme, purified from benzoate-grown cells of
Rhodococcus opacus
(
erythropolis
) 1CP, was found to have a larger molecular mass under denaturing conditions than the corresponding enzymes previously purified from γ-proteobacteria. Sequencing of the N terminus and of tryptic peptides allowed cloning of the gene coding for the 3-oxoadipate enol-lactone hydrolase by using PCR with degenerate primers. Sequencing showed that the gene belongs to a protocatechuate catabolic gene cluster. Most interestingly, the hydrolase gene, usually termed
pcaD
, was fused to a second gene, usually termed
pcaC
, which encodes the enzyme catalyzing the preceding reaction, i.e., 4-carboxymuconolactone decarboxylase. The two enzymatic activities could not be separated chromatographically. At least six genes of protocatechuate catabolism appear to be transcribed in the same direction and in the following order:
pcaH
and
pcaG
, coding for the subunits of protocatechuate 3,4-dioxygenase, as shown by N-terminal sequencing of the subunits of the purified protein; a gene termed
pcaB
due to the homology of its gene product to 3-carboxy-
cis
,
cis
-muconate cycloisomerases;
pcaL
, the fused gene coding for PcaD and PcaC activities;
pcaR
, presumably coding for a regulator of the IclR-family; and a gene designated
pcaF
because its product resembles 3-oxoadipyl coenzyme A (3-oxoadipyl-CoA) thiolases. The presumed
pcaI
, coding for a subunit of succinyl-CoA:3-oxoadipate CoA-transferase, was found to be transcribed divergently from
pcaH.
Multiple Regions on the
Escherichia coli
Heat Shock Transcription Factor ς
32
Determine Core RNA Polymerase Binding Specificity
Authors: Joo Daniel M. , Nolte Audrey , Calendar Richard , Zhou Yan Ning , Jin Ding Jun
Published: December 2019

We have analyzed the core RNA polymerase (RNAP) binding activity of the purified products of nine defective alleles of the
rpoH
gene, which encodes ς
32
in
Escherichia coli
. All mutations studied here lie outside of the putative core RNAP binding regions 2.1 and 2.2. Based on the estimated

K
s

s for the mutant sigma and core RNAP interaction determined by in vitro transcription and by glycerol gradient sedimentation, we have divided the mutants into three classes. The class III mutants showed greatly decreased affinity for core RNAP, whereas the class II mutants’ effect on core RNAP interaction was only clearly seen in the presence of ς
70
competitor. The class I mutant behaved nearly identically to the wild type in core RNAP binding. Two point mutations in class III altered residues that were distant from one another. One was found in conserved region 4.2, and the other was in a region conserved only among heat shock sigma factors. These data suggest that there is more than one core RNAP binding region in ς
32
and that differences in contact sites probably exist among sigma factors.
Identification of Essential Glutamates in the Acetate Kinase from
Methanosarcina thermophila
Authors: Singh-Wissmann Kavita , Ingram-Smith Cheryl , Miles Rebecca D. , Ferry James G.
Published: December 2019

Acetate kinase catalyzes the reversible phosphorylation of acetate (CH
3
COO
−
+ ATP⇄CH
3
CO
2
PO
3
2−
+ ADP). A mechanism which involves a covalent phosphoryl-enzyme intermediate has been proposed, and chemical modification studies of the enzyme from
Escherichia coli
indicate an unspecified glutamate residue is phosphorylated (J. A. Todhunter and D. L. Purich, Biochem. Biophys. Res. Commun. 60:273–280, 1974). Alignment of the amino acid sequences for the acetate kinases from
E. coli
(
Bacteria
domain),
Methanosarcina thermophila
(
Archaea
domain), and four other phylogenetically divergent microbes revealed high identity which included five glutamates. These glutamates were replaced in the
M. thermophila
enzyme to determine if any are essential for catalysis. The histidine-tagged altered enzymes were produced in
E. coli
and purified to electrophoretic homogeneity by metal affinity chromatography. Replacements of E384 resulted in either undetectable or extremely low kinase activity, suggesting E384 is essential for catalysis which supports the proposed mechanism. Replacement of E385 influenced the

K
m

values for acetate and ATP with only moderate decreases in
k
cat
, which suggests that this residue is involved in substrate binding but not catalysis. The unaltered acetate kinase was not inactivated by
N
-ethylmaleimide; however, replacement of E385 with cysteine conferred sensitivity to
N
-ethylmaleimide which was prevented by preincubation with acetate, acetyl phosphate, ATP, or ADP, suggesting that E385 is located near the active site. Replacement of E97 decreased the

K
m

value for acetate but not ATP, suggesting this residue is involved in binding acetate. Replacement of either E32 or E334 had no significant effects on the kinetic constants, which indicates that neither residue is essential for catalysis or significantly influences the binding of acetate or ATP.
Chromosomal Regions Specific to Pathogenic Isolates of
Escherichia coli
Have a Phylogenetically Clustered Distribution
Authors: Boyd E. Fidelma , Hartl Daniel L.
Published: December 2019

We studied the ancestry of virulence-associated genes in
Escherichia coli
by examining chromosomal regions specific to pathogenic isolates. The four virulence determinants examined were the alpha-hemolysin (
hly
) loci
hlyI
and
hlyII
, the type II capsule gene cluster
kps
, and the P (
pap
) and S (
sfa
) fimbria gene clusters. All four loci were shown previously to be associated with pathogenicity islands of uropathogenic
E. coli
isolates. The
hly
,
kps
,
sfa
, and
pap
regions each have an unexpected clustered distribution among the
E. coli
collection of reference (ECOR) strains, but all these regions were absent from a collection of diarrheagenic
E. coli
isolates. Strains in the ECOR subgroup B2 typically had a combination of at least three of the four loci, and all strains in subgroup D had a copy of the
kps
and
pap
clusters. In contrast, only four strains in subgroup A had either
hly
,
kps
,
sfa
, or
pap
, and no subgroup A strains had all four together. Strains of subgroup B1 were devoid of all four virulence regions, with the exception of one isolate that had a copy of the
sfa
gene cluster. This phylogenetic distribution of strain-specific sequences corresponds to the ECOR groups with the largest genome size, namely, B2 and D. We propose that the pathogenicity islands are ancestral to subgroups B2 and D and were acquired after speciation, with subsequent horizontal transfer into some group A, B1, and E lineages. These results suggest that the
hly
,
kps
,
sfa
, and
pap
pathogenicity determinants may play a role in the evolution of enteric bacteria quite apart from, and perhaps with precedence over, their ability to cause disease.
The
nac
(Nitrogen Assimilation Control) Gene from
Escherichia coli
Authors: Muse Wilson B. , Bender Robert A.
Published: December 2019

The nitrogen assimilation control gene,
nac
, was detected in
Escherichia coli
but not in
Salmonella typhimurium
by Southern blotting, using a probe from the
Klebsiella aerogenes nac
(

nac
K

) gene. The
E. coli nac
gene (

nac
E

) was isolated from a cosmid clone by complementation of a
nac
mutation in

K. aerogenes. nac
E

was fully functional in this complementation assay. DNA sequence analysis showed considerable divergence between

nac
E

and

nac
K

, with a predicted amino acid sequence identity of only 79% and most of the divergence in the C-terminal half of the protein sequence. The total predicted size of NAC
E
is 305 amino acids, the same as for NAC
K
. A null mutation,
nac-28
, was generated by reverse genetics. Mutants bearing
nac-28
have a variety of phenotypes related to nitrogen metabolism, including slower growth on cytosine, faster growth on arginine, and suppression of the failure of an Ntr-constitutive mutant to grow with serine as sole nitrogen source. In addition to a loss of nitrogen regulation of histidase formation,
nac-28
mutants also showed a loss of a weak repression of glutamate dehydrogenase formation. This repression was unexpected because it is balanced by a NAC-independent activation of glutamate dehydrogenase formation during nitrogen-limited growth. Attempts to purify NAC
E
by using methods established for NAC
K
failed, and NAC
E
appears to be degraded with a half-life at 30°C as short as 15 min during inhibition of protein synthesis.
MppA, a Periplasmic Binding Protein Essential for Import of the Bacterial Cell Wall Peptide
l
-Alanyl-γ-
d
-Glutamyl-
meso
-Diaminopimelate
Authors: Park James T. , Raychaudhuri Debabrata , Li Hongshan , Normark Staffan , Mengin-Lecreulx Dominique
Published: December 2019

Mutants of a diaminopimelic acid (Dap)-requiring strain of
Escherichia coli
were isolated which failed to grow on media in which Dap was replaced by the cell wall murein tripeptide,
l
-alanyl-γ-
d
-glutamyl-
meso
-diaminopimelate. In one such mutant, which is oligopeptide permease (Opp) positive, we have identified a new gene product, designated MppA (murein peptide permease A), that is about 46% identical to OppA, the periplasmic binding protein for Opp. A plasmid carrying the wild-type
mppA
gene allows the mutant to grow on tripeptide. Two other mutants that failed to grow on tripeptide were resistant to triornithine toxicity, indicating a defect in the
opp
operon. An
E. coli
strain whose entire
opp
operon was deleted but which carried the
mppA
locus was unable to grow on murein tripeptide unless it was provided with
oppBCDF
genes in
trans
. Our data suggest a model whereby the periplasmic MppA binds the murein tripeptide, which is then transported into the cytoplasm via membrane-bound and cytoplasmic OppBCDF. In assessing the affinity of MppA for non-cell wall peptides, we have found that proline auxotrophy can be satisfied with the peptide Pro-Phe-Lys, which utilizes either MppA or OppA in conjunction with OppBCDF for its uptake. Thus, MppA, OppA, and perhaps the third OppA paralog revealed by the
E. coli
genome sequence may each bind a particular family of peptides but interact with common membrane-associated components for transport of their bound ligands into the cell. As to the physiological function of MppA, the possibility that it may be involved in signal transduction pathway(s) is discussed.
fbfB
, a Gene Encoding a Putative Galactose Oxidase, Is Involved in
Stigmatella aurantiaca
Fruiting Body Formation
Authors: Silakowski Barbara , Ehret Heidi , Schairer Hans Ulrich
Published: December 2019

Stigmatella aurantiaca
is a gram-negative bacterium which forms, under conditions of starvation in a multicellular process, characteristic three-dimensional structures: the fruiting bodies. For studying this complex process, mutants impaired in fruiting body formation have been induced by transposon insertion with a Tn
5
-derived transposon. The gene affected (
fbfB
) in one of the mutants (AP182) was studied further. Inactivation of
fbfB
results in mutants which form only clumps during starvation instead of wild-type fruiting bodies. This mutant phenotype can be partially rescued, if cells of mutants impaired in
fbfB
function are mixed with those of some independent mutants defective in fruiting before starvation. The
fbfB
gene is expressed about 14 h after induction of fruiting body formation as determined by measuring β-galactosidase activity in a merodiploid strain harboring the wild-type gene and an
fbfB-Δtrp-lacZ
fusion gene or by Northern (RNA) analysis with the
Rhodobacter capsulatus pufBA
fragment fused to
fbfB
as an indicator. The predicted polypeptide FbfB has a molecular mass of 57.8 kDa and shows a significant homology to the galactose oxidase (GaoA) of the fungus
Dactylium dendroides
. Galactose oxidase catalyzes the oxidation of galactose and primary alcohols to the corresponding aldehydes.
Molecular Characterization of the α-Glucosidase Gene (malA) from the Hyperthermophilic ArchaeonSulfolobus solfataricus
Authors: Rolfsmeier Michael , Haseltine Cynthia , Bini Elisabetta , Clark Amy , Blum Paul
Published: December 2019

Acidic hot springs are colonized by a diversity of hyperthermophilic organisms requiring extremes of temperature and pH for growth. To clarify how carbohydrates are consumed in such locations, the structural gene (malA) encoding the major soluble α-glucosidase (maltase) and flanking sequences fromSulfolobus solfataricuswere cloned and characterized. This is the first report of an α-glucosidase gene from the archaeal domain.malAis 2,083 bp and encodes a protein of 693 amino acids with a calculated mass of 80.5 kDa. It is flanked on the 5′ side by an unusual 1-kb intergenic region. Northern blot analysis of themalAregion identified transcripts formalAand an upstream open reading frame located 5′ to the 1-kb intergenic region. ThemalAtranscription start site was located by primer extension analysis to a guanine residue 8 bp 5′ of themalAstart codon. Gel mobility shift analysis of themalApromoter region suggests that sequences 3′ to position −33, including a consensus archaeal TATA box, play an essential role inmalAexpression.malAhomologs were detected by Southern blot analysis in otherS. solfataricusstrains and inSulfolobus shibatae, while no homologs were evident inSulfolobus acidocaldarius, lending further support to the proposed revision of the genusSulfolobus. Phylogenetic analyses indicate that the closestS. solfataricusα-glucosidase homologs are of mammalian origin. Characterization of the recombinant enzyme purified fromEscherichia colirevealed differences from the natural enzyme in thermostability and electrophoretic behavior. Glycogen is a substrate for the recombinant enzyme. Unlike maltose hydrolysis, glycogen hydrolysis is optimal at the intracellular pH of the organism. These results indicate a unique role for theS. solfataricusα-glucosidase in carbohydrate metabolism.
Opposite Patterns of Expression of Two
Aspergillus nidulans
Xylanase Genes with Respect to Ambient pH
Authors: Maccabe A. P. , Orejas M. , Pérez-González J. A. , Ramón D.
Published: December 2019

The
Aspergillus nidulans
xylanase genes
xlnA
and
xlnB
are subject to regulation by ambient pH via the zinc finger transcription factor PacC. In the presence of
d
-xylose,
xlnA
is expressed under conditions of alkaline ambient pH while
xlnB
is expressed at acidic ambient pH. These data have been confirmed for acidity- and alkalinity-mimicking
A. nidulans
mutants.
Contribution of the Disulfide Bond of the A Subunit to the Action of
Escherichia coli
Heat-Labile Enterotoxin
Authors: Okamoto Keinosuke , Nomura Tomohiko , Fujii Yoshio , Yamanaka Hiroyasu
Published: December 2019

Escherichia coli
heat-labile enterotoxin (LT) consists of an A subunit and five B subunits. These subunits oligomerize into an assembled holotoxin within the periplasm. Structural analysis of LT has revealed that the A subunit interacts with the B subunit through its carboxy terminus. This indicates that the carboxy-terminal portion of the protein is required for assembly of holotoxin in the periplasm. However, it is not known whether other regions of the A subunit contribute to the assembly. The A subunit constituting the holotoxin contains a disulfide bond between Cys-187 and Cys-199. It has been observed in many proteins that the intramolecular disulfide bond is deeply involved in the function and tertiary structure of the protein. We speculated that the disulfide bond of the A subunit contributes to the assembly in the periplasm, although the bond is not a structural element of the carboxy-terminal portion of the A subunit. We replaced these cysteine residues of the A subunit by oligonucleotide-directed site-specific mutagenesis and analyzed the LTs produced by cells containing the mutant LT genes. The amount of the mutant holotoxin produced was small compared with that of the wild-type strain, indicating that the disulfide bond of the A subunit contributes to the structure which functions as the site of nucleation in the assembly. A reconstitution experiment in vitro supported the notion. Subsequently, we found that the mutant A subunit constituting holotoxin is easily degraded by trypsin and that in cells incubated with mutant LTs, the lag until the intracellular cyclic AMP begins to accumulate is longer than in cells incubated with native LTs. These results might be useful for the analysis of the interaction of LT with target cells at the molecular level.
Physiological and Genetic Analyses Leading to Identification of a Biochemical Role for the
moeA
(Molybdate Metabolism) Gene Product in
Escherichia coli
Authors: Hasona Adnan , Ray Ramesh M. , Shanmugam K. T.
Published: December 2019

A unique class of chlorate-resistant mutants of
Escherichia coli
which produced formate hydrogenlyase and nitrate reductase activities only when grown in medium with limiting amounts of sulfur compounds was isolated. These mutants failed to produce the two molybdoenzyme activities when cultured in rich medium or glucose-minimal medium. The mutations in these mutants were localized in the
moeA
gene. Mutant strains with polar mutations in
moeA
which are also
moeB
did not produce active molybdoenzymes in any of the media tested.
moeA
mutants with a second mutation in either
cysDNCJI
or
cysH
gene lost the ability to produce active molybdoenzyme even when grown in medium limiting in sulfur compounds. The CysDNCJIH proteins along with CysG catalyze the conversion of sulfate to sulfide. Addition of sulfide to the growth medium of
moeA cys
double mutants suppressed the MoeA
−
phenotype. These results suggest that in the absence of MoeA protein, the sulfide produced by the sulfate activation/reduction pathway combines with molybdate in the production of activated molybdenum. Since hydrogen sulfide is known to interact with molybdate in the production of thiomolybdate, it is possible that the MoeA-catalyzed activated molybdenum is a form of thiomolybdenum species which is used in the synthesis of molybdenum cofactor from Mo-free molybdopterin.
Biochemical Characterization of the 20S Proteasome from the Methanoarchaeon
Methanosarcina thermophila
Authors: Maupin-Furlow Julie A. , Aldrich Henry C. , Ferry James G.
Published: December 2019

The 20S proteasome from the methanoarchaeon
Methanosarcina thermophila
was produced in
Escherichia coli
and characterized. The biochemical properties revealed novel features of the archaeal 20S proteasome. A fully active 20S proteasome could be assembled in vitro with purified native α ring structures and β prosubunits independently produced in
Escherichia coli
, which demonstrated that accessory proteins are not essential for processing of the β prosubunits or assembly of the 20S proteasome. A protein complex with a molecular mass intermediate to those of the α
7
ring and the 20S proteasome was detected, suggesting that the 20S proteasome is assembled from precursor complexes. The heterologously produced
M. thermophila
20S proteasome predominately catalyzed cleavage of peptide bonds carboxyl to the acidic residue Glu (postglutamyl activity) and the hydrophobic residues Phe and Tyr (chymotrypsinlike activity) in short chromogenic and fluorogenic peptides. Low-level hydrolyzing activities were also detected carboxyl to the acidic residue Asp and the basic residue Arg (trypsinlike activity). Sodium dodecyl sulfate and divalent or monovalent ions stimulated chymotrypsinlike activity and inhibited postglutamyl activity, whereas ATP stimulated postglutamyl activity but had little effect on the chymotrypsinlike activity. The results suggest that the 20S proteasome is a flexible protein which adjusts to binding of substrates. The 20S proteasome also hydrolyzed large proteins. Replacement of the nucleophilic Thr
1
residue with an Ala in the β subunit abolished all activities, which suggests that only one active site is responsible for the multisubstrate activity. Replacement of β subunit active-site Lys
33
with Arg reduced all activities, which further supports the existence of one catalytic site; however, this result also suggests a role for Lys
33
in polarization of the Thr
1
N, which serves to strip a proton from the active-site Thr
1
Oγ nucleophile. Replacement of Asp
51
with Asn had no significant effect on trypsinlike activity, enhanced postglutamyl and trypsinlike activities, and only partially reduced lysozyme-hydrolyzing activity, which suggested that this residue is not essential for multisubstrate activity.
Lambda Xis Degradation In Vivo by Lon and FtsH
Authors: Leffers Gerald G. , Gottesman Susan
Published: December 2019

Lambda Xis, which is required for site-specific excision of phage lambda from the bacterial chromosome, has a much shorter functional half-life than Int, which is required for both integration and excision (R. A. Weisberg and M. E. Gottesman, p. 489–500,
in
A. D. Hershey, ed.,
The Bacteriophage Lambda
, 1971). We found that Xis is degraded in vivo by two ATP-dependent proteases, Lon and FtsH (HflB). Xis was stabilized two- to threefold more than in the wild type in a
lon
mutant and as much as sixfold more in a
lon ftsH
double mutant at the nonpermissive temperature for the
ftsH
mutation. Integration of lambda into the bacterial chromosome was delayed in the
lon ftsH
background, suggesting that accumulation of Xis in vivo interferes with integration. Overexpression of Xis in wild-type cells from a multicopy plasmid inhibited integration of lambda and promoted curing of established lysogens, confirming that accumulation of Xis interferes with the ability of Int to establish and maintain an integrated prophage.
Identification and Characterization of a Novel Competence Gene,
comC
, Required for DNA Binding and Uptake in
Acinetobacter
sp. Strain BD413
Authors: Link Caroline , Eickernjäger Sandra , Porstendörfer Dirk , Averhoff Beate
Published: December 2019

A gene (
comC
) essential for natural transformation was identified in
Acinetobacter
sp. strain BD413. ComC has a typical leader sequence and is similar to different type IV pilus assembly factors. A
comC
mutant (T308) is not able to bind or take up DNA but exhibits a piliation phenotype indistinguishable from the transformation wild type as revealed by electron microscopy.
Characterization of the
cvaA
and
cvi
Promoters of the Colicin V Export System: Iron-Dependent Transcription of
cvaA
Is Modulated by Downstream Sequences
Authors: Boyer Anne E. , Tai Phang C.
Published: December 2019

Secretion of the
Escherichia coli
toxin colicin V was previously determined to be iron regulated via the Fur (ferric uptake regulator) protein, based on studies in
fur
mutants. The iron dependence of transcription and expression of
cvaA
, which encodes a transporter accessory protein, and
cvi
, encoding the colicin V immunity protein, was assessed under conditions of iron excess or depletion. Immunoblots showed that production of both Cvi and CvaA is iron dependent. The iron-dependent transcriptional start for
cvaA
identified by primer extension and S1 nuclease analysis, P1, lies 320 bp upstream of the translational start and is associated with a newly identified Fur binding site. β-Galactosidase activity in transcriptional
lacZ
fusions with the P1 promoter alone is higher than with downstream sequences present and is induced 10-fold by iron depletion. Including immediate downstream regions with P1 enhances activity from P1 even more but reduces the induction by iron depletion fivefold. Including subsequent downstream sequences, however, down-modulates overall transcription from P1 almost fourfold. Deletion of a long stem-loop structure in this region alleviates the down-modulation by increasing transcription, indicating that the sequences or structure of this element may contribute to this down-regulation. Characterization of the
cvi
promoter by primer extension showed that it resides where predicted, about 50 bp upstream of
cvi
associated with a previously identified Fur binding site. The
cvi
promoter is also inducible by iron depletion. The modulating sequences from
cvaA
were placed downstream of the
cvi
promoter to test their effects in transcriptional fusions of the
cvi
promoter to
lacZ
. The fusion results showed that these sequences also modulate transcription of the
cvi
promoter in a manner similar to that of the
cvaA
promoter. The potential for up- and down-regulation within the long untranslated region downstream of the
cvaA
promoter suggests a novel mechanism that fine-tunes expression of the colicin V secretion genes.
Characterization of thebvgRLocus ofBordetella pertussis
Authors: Merkel Tod J. , Barros Cassia , Stibitz Scott
Published: December 2019

Bordetella pertussis, the causative agent of whooping cough, produces a wide array of factors that are associated with its ability to cause disease. The expression and regulation of these virulence factors is dependent upon thebvglocus (originally designated thevirlocus), which encodes two proteins: BvgA, a 23-kDa cytoplasmic protein, and BvgS, a 135-kDa transmembrane protein. It is proposed that BvgS responds to environmental signals and interacts with BvgA, a transcriptional regulator which upon modification by BvgS binds to specific promoters and activates transcription. An additional class of genes is repressed by thebvglocus. Expression of this class, thebvg-repressed genes (vrgs [forvir-repressed genes]), is reduced under conditions in which expression of the aforementionedbvg-activated virulence factors is maximal; this repression is dependent upon the presence of an intactbvgASlocus. We have previously identified a locus required for regulation of all of the knownbvg-repressed genes inB. pertussis. This locus, designatedbvgR, maps to a location immediately downstream ofbvgAS. We have undertaken deletion and complementation studies, as well as sequence analysis, in order to identify thebvgRopen reading frame and identify thecis-acting sequences required for regulated expression ofbvgR. Studies utilizing transcriptional fusions ofbvgRto the gene encoding alkaline phosphatase have demonstrated thatbvgRis activated at the level of transcription and that this activation is dependent upon an intactbvgASlocus.