bluuebunny/crossref_metadata_2025 huggingface dataset:
Inactivation and Regulation of the Aerobic C
4
-Dicarboxylate Transport (
dctA
) Gene of
Escherichia coli
Authors: Davies Suzanne J. , Golby Paul , Omrani Davood , Broad Susan A. , Harrington Vikki L. , Guest John R. , Kelly David J. , Andrews Simon C.
Published: December 2019

The gene (
dctA
) encoding the aerobic C
4
-dicarboxylate transporter (DctA) of
Escherichia coli
was previously mapped to the 79-min region of the linkage map. The nucleotide sequence of this region reveals two candidates for the
dctA
gene:
f428
at 79.3 min and the
o157a-o424-o328
(or
orfQMP
) operon at 79.9 min. The
f428
gene encodes a homologue of the
Sinorhizobium meliloti
and
Rhizobium leguminosarum
H
+
/C
4
-dicarboxylate symporter, DctA, whereas the
orfQMP
operon encodes homologues of the aerobic periplasmic-binding protein- dependent C
4
-dicarboxylate transport system (DctQ, DctM, and DctP) of
Rhodobacter capsulatus
. To determine which, if either, of these loci specify the
E. coli
DctA system, the chromosomal
f428
and
orfM
genes were inactivated by inserting Sp
r
or Ap
r
cassettes, respectively. The resulting
f428
mutant was unable to grow aerobically with fumarate or malate as the sole carbon source and grew poorly with succinate. Furthermore, fumarate uptake was abolished in the
f428
mutant and succinate transport was ∼10-fold lower than that of the wild type. The growth and fumarate transport deficiencies of the
f428
mutant were complemented by transformation with an
f428
-containing plasmid. No growth defect was found for the
orfM
mutant. In combination, the above findings confirm that
f428
corresponds to the
dctA
gene and indicate that the
orfQMP
products play no role in C
4
-dicarboxylate transport. Regulation studies with a
dctA-lacZ
(
f428-lacZ
) transcriptional fusion showed that
dctA
is subject to cyclic AMP receptor protein (CRP)-dependent catabolite repression and ArcA-mediated anaerobic repression and is weakly induced by the DcuS-DcuR system in response to C
4
-dicarboxylates and citrate. Interestingly, in a
dctA
mutant, expression of
dctA
is constitutive with respect to C
4
-dicarboxylate induction, suggesting that DctA regulates its own synthesis. Northern blot analysis revealed a single, monocistronic
dctA
transcript and confirmed that
dctA
is subject to regulation by catabolite repression and CRP. Reverse transcriptase-mediated primer extension indicated a single transcriptional start site centered 81 bp downstream of a strongly predicted CRP-binding site.
A Nonessential Signal Peptidase II (Lsp) of
Myxococcus xanthus
Might Be Involved in Biosynthesis of the Polyketide Antibiotic TA
Authors: Paitan Yossi , Orr Elisha , Ron Eliora Z. , Rosenberg Eugene
Published: December 2019

Myxococcus xanthus
is a gram-negative soil bacterium that produces the polyketide antibiotic TA. In this study, we describe the analysis of an
M. xanthus
gene which encodes a homologue of the prolipoprotein signal peptidase II (SPase II;
lsp
). Overexpression of the
M. xanthus
SPase II in
Escherichia coli
confers high levels of globomycin resistance, confirming its function as an SPase II. The
M. xanthus
gene encoding the
lsp
homologue is nonessential for growth, as determined by specific gene disruption. It has been mapped to the antibiotic TA gene cluster, and the disrupted mutants do not produce the antibiotic, indicating a probable involvement in TA production. These results suggest the existence of more than one SPase II protein in
M. xanthus
, where one is a system-specific SPase II (for TA biosynthesis).
Bacterial 2,4-Dioxygenases: New Members of the α/β Hydrolase-Fold Superfamily of Enzymes Functionally Related to Serine Hydrolases
Authors: Fischer Frank , Künne Stefan , Fetzner Susanne
Published: December 2019

1
H
-3-hydroxy-4-oxoquinoline 2,4-dioxygenase (Qdo) from
Pseudomonas putida
33/1 and 1
H
-3-hydroxy-4-oxoquinaldine 2,4-dioxygenase (Hod) from
Arthrobacter ilicis
Rü61a catalyze an N-heterocyclic-ring cleavage reaction, generating
N
-formylanthranilate and
N
-acetylanthranilate, respectively, and carbon monoxide. Amino acid sequence comparisons between Qdo, Hod, and a number of proteins belonging to the α/β hydrolase-fold superfamily of enzymes and analysis of the similarity between the predicted secondary structures of the 2,4-dioxygenases and the known secondary structure of haloalkane dehalogenase from
Xanthobacter autotrophicus
GJ10 strongly suggested that Qdo and Hod are structurally related to the α/β hydrolase-fold enzymes. The residues S95 and H244 of Qdo were found to be arranged like the catalytic nucleophilic residue and the catalytic histidine, respectively, of the α/β hydrolase-fold enzymes. Investigation of the potential functional significance of these and other residues of Qdo through site-directed mutagenesis supported the hypothesis that Qdo is structurally as well as functionally related to serine hydrolases, with S95 being a possible catalytic nucleophile and H244 being a possible catalytic base. A hypothetical reaction mechanism for Qdo-catalyzed 2,4-dioxygenolysis, involving formation of an ester bond between the catalytic serine residue and the carbonyl carbon of the substrate and subsequent dioxygenolysis of the covalently bound anionic intermediate, is discussed.
A 5′ RNA Stem-Loop Participates in the Transcription Attenuation Mechanism That Controls Expression of the
Bacillus subtilis trpEDCFBA
Operon
Authors: Sudershana Subita , Du Hansen , Mahalanabis Madhumita , Babitzke Paul
Published: December 2019

The
trp
RNA-binding attenuation protein (TRAP) regulates expression of the
Bacillus subtilis trpEDCFBA
operon by transcription attenuation. Tryptophan-activated TRAP binds to the nascent
trp
leader transcript by interacting with 11 (G/U)AG repeats. TRAP binding prevents formation of an antiterminator structure, thereby promoting formation of an overlapping terminator, and hence transcription is terminated before RNA polymerase can reach the
trp
structural genes. In addition to the antiterminator and terminator, a stem-loop structure is predicted to form at the 5′ end of the
trp
leader transcript. Deletion of this structure resulted in a dramatic increase in expression of a
trpE′-′lacZ
translational fusion and a reduced ability to regulate expression in response to tryptophan. By introducing a series of point mutations in the 5′ stem-loop, we found that both the sequence and the structure of the hairpin are important for its regulatory function and that compensatory changes that restored base pairing partially restored wild-type-like expression levels. Our results indicate that the 5′ stem-loop functions primarily through the TRAP-dependent regulatory pathway. Gel shift results demonstrate that the 5′ stem-loop increases the affinity of TRAP for
trp
leader RNA four- to fivefold, suggesting that the 5′ structure interacts with TRAP. In vitro transcription results indicate that this 5′ structure functions in the attenuation mechanism, since deletion of the stem-loop caused an increase in transcription readthrough. An oligonucleotide complementary to a segment of the 5′ stem-loop was used to demonstrate that formation of the 5′ structure is required for proper attenuation control of this operon.
Distribution of Tetrahydromethanopterin-Dependent Enzymes in Methylotrophic Bacteria and Phylogeny of Methenyl Tetrahydromethanopterin Cyclohydrolases
Authors: Vorholt Julia A. , Chistoserdova Ludmila , Stolyar Sergei M. , Thauer Rudolf K. , Lidstrom Mary E.
Published: December 2019

The methylotrophic proteobacterium
Methylobacterium extorquens
AM1 possesses tetrahydromethanopterin (H
4
MPT)-dependent enzymes, which are otherwise specific to methanogenic and sulfate-reducing archaea and which have been suggested to be involved in formaldehyde oxidation to CO
2
in
M. extorquens
AM1. The distribution of H
4
MPT-dependent enzyme activities in cell extracts of methylotrophic bacteria from 13 different genera are reported. H
4
MPT-dependent activities were detected in all of the methylotrophic and methanotrophic proteobacteria tested that assimilate formaldehyde by the serine or ribulose monophosphate pathway. H
4
MPT-dependent activities were also found in autotrophic
Xanthobacter
strains. However, no H
4
MPT-dependent enzyme activities could be detected in other autotrophic α-proteobacteria or in gram-positive methylotrophic bacteria. Genes encoding methenyl H
4
MPT cyclohydrolase (
mch
genes) were cloned and sequenced from several proteobacteria. Bacterial and archaeal Mch sequences have roughly 35% amino acid identity and form distinct groups in phylogenetic analysis.
Pathways of Assimilative Sulfur Metabolism in
Pseudomonas putida
Authors: Vermeij Paul , Kertesz Michael A.
Published: December 2019

Cysteine and methionine biosynthesis was studied in
Pseudomonas putida
S-313 and
Pseudomonas aeruginosa
PAO1. Both these organisms used direct sulfhydrylation of
O
-succinylhomoserine for the synthesis of methionine but also contained substantial levels of
O
-acetylserine sulfhydrylase (cysteine synthase) activity. The enzymes of the transsulfuration pathway (cystathionine γ-synthase and cystathionine β-lyase) were expressed at low levels in both pseudomonads but were strongly upregulated during growth with cysteine as the sole sulfur source. In
P. aeruginosa
, the reverse transsulfuration pathway between homocysteine and cysteine, with cystathionine as the intermediate, allows
P. aeruginosa
to grow rapidly with methionine as the sole sulfur source.
P. putida
S-313 also grew well with methionine as the sulfur source, but no cystathionine γ-lyase, the key enzyme of the reverse transsulfuration pathway, was found in this species. In the absence of the reverse transsulfuration pathway,
P. putida
desulfurized methionine by the conversion of methionine to methanethiol, catalyzed by methionine γ-lyase, which was upregulated under these conditions. A transposon mutant of
P. putida
that was defective in the alkanesulfonatase locus (
ssuD
) was unable to grow with either methanesulfonate or methionine as the sulfur source. We therefore propose that in
P. putida
methionine is converted to methanethiol and then oxidized to methanesulfonate. The sulfonate is then desulfonated by alkanesulfonatase to release sulfite for reassimilation into cysteine.
The C-Terminal Domain of the
Bordetella pertussis
Autotransporter BrkA Forms a Pore in Lipid Bilayer Membranes
Authors: Shannon Jennifer L. , Fernandez Rachel C.
Published: December 2019

BrkA is a 103-kDa outer membrane protein of
Bordetella pertussis
that mediates resistance to antibody-dependent killing by complement. It is proteolytically processed into a 73-kDa N-terminal domain and a 30-kDa C-terminal domain as determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. BrkA is also a member of the autotransporter family of proteins. Translocation of the N-terminal domain of the protein across the outer membrane is hypothesized to occur through a pore formed by the C-terminal domain. To test this hypothesis, we performed black lipid bilayer experiments with purified recombinant protein. The BrkA C-terminal protein showed an average single-channel conductance of 3.0 nS in 1 M KCl. This result strongly suggests that the C-terminal autotransporter domain of BrkA is indeed capable of forming a pore.
Cloning and Characterization of a Sulfonate/α-Ketoglutarate Dioxygenase from
Saccharomyces cerevisiae
Authors: Hogan Deborah A. , Auchtung Thomas A. , Hausinger Robert P.
Published: December 2019

The
Saccharomyces cerevisiae
open reading frame YLL057c is predicted to encode a gene product with 31.5% amino acid sequence identity to
Escherichia coli
taurine/α-ketoglutarate dioxygenase and 27% identity to
Ralstonia eutropha
TfdA, a herbicide-degrading enzyme. Purified recombinant yeast protein is shown to be an Fe(II)-dependent sulfonate/α-ketoglutarate dioxygenase. Although taurine is a poor substrate, a variety of other sulfonates are utilized, with the best natural substrates being isethionate and taurocholate. Disruption of the gene encoding this enzyme negatively affects the use of isethionate and taurine as sulfur sources by
S. cerevisiae
, providing strong evidence that YLL057c plays a role in sulfonate catabolism.
Partitioning, Movement, and Positioning of Nucleoids in
Mycoplasma capricolum
Authors: Seto Shintaro , Miyata Makoto
Published: December 2019

The nucleoids in
Mycoplasma capricolum
cells were visualized by phase-combined fluorescence microscopy of DAPI (4′,6-diamidino-2-phenylindole)-stained cells. Most growing cells in a rich medium had one or two nucleoids in a cell, and no anucleate cells were found. The nucleoids were positioned in the center in mononucleoid cells and at one-quarter and three-quarters of the cell length in binucleoid cells. These formations may have the purpose of ensuring delivery of replicated DNA to daughter cells. Internucleoid distances in binucleoid cells correlated with the cell lengths, and the relationship of DNA content to cell length showed that cell length depended on DNA content in binucleoid cells but not in mononucleoid cells. These observations suggest that cell elongation takes place in combination with nucleoid movement. Lipid synthesis was inhibited by transfer of cells to a medium lacking supplementation for lipid synthesis. The transferred cells immediately stopped dividing and elongated while regular spaces were maintained between the nucleoids for 1 h. After 1 h, the cells changed their shapes from rod-like to round, but the proportion of multinucleoid cells increased. Inhibition of protein synthesis by chloramphenicol induced nucleoid condensation and abnormal positioning, although partitioning was not inhibited. These results suggest that nucleoid partitioning does not require lipid or protein synthesis, while regular positioning requires both. When DNA replication was inhibited, the cells formed branches, and the nucleoids were positioned at the branching points. A model for the reproduction process of
M. capricolum
, including nucleoid migration and cell division, is discussed.
Mutational Analysis of the RecJ Exonuclease of
Escherichia coli
: Identification of Phosphoesterase Motifs
Authors: Sutera Vincent A. , Han Eugene S. , Rajman Luis A. , Lovett Susan T.
Published: December 2019

The
recJ
gene, identified in
Escherichia coli
, encodes a Mg
+2
-dependent 5′-to-3′ exonuclease with high specificity for single-strand DNA. Genetic and biochemical experiments implicate RecJ exonuclease in homologous recombination, base excision, and methyl-directed mismatch repair. Genes encoding proteins with strong similarities to RecJ have been found in every eubacterial genome sequenced to date, with the exception of
Mycoplasma
and
Mycobacterium tuberculosis
. Multiple genes encoding proteins similar to RecJ are found in some eubacteria, including
Bacillus
and
Helicobacter
, and in the archaea. Among this divergent set of sequences, seven conserved motifs emerge. We demonstrate here that amino acids within six of these motifs are essential for both the biochemical and genetic functions of
E. coli
RecJ. These motifs may define interactions with Mg
2+
ions or substrate DNA. A large family of proteins more distantly related to RecJ is present in archaea, eubacteria, and eukaryotes, including a hypothetical protein in the MgPa adhesin operon of
Mycoplasma
, a domain of putative polyA polymerases in
Synechocystis
and
Aquifex
, PRUNE of
Drosophila
, and an exopolyphosphatase (PPX1) of
Saccharomyces cereviseae
. Because these six RecJ motifs are shared between exonucleases and exopolyphosphatases, they may constitute an ancient phosphoesterase domain now found in all kingdoms of life.
Analysis of F Factor TraD Membrane Topology by Use of Gene Fusions and Trypsin-Sensitive Insertions
Authors: Lee Martin H. , Kosuk Nick , Bailey Jeannie , Traxler Beth , Manoil Colin
Published: December 2019

This report describes a procedure for characterizing membrane protein topology which combines the analysis of reporter protein hybrids and trypsin-sensitive 31-amino-acid insertions generated by using transposons ISphoA/in and ISlacZ/in. Studies of the F factor TraD protein imply that the protein takes on a structure with two membrane-spanning sequences and amino and carboxyl termini facing the cytoplasm. It was possible to assign the subcellular location of one region for which the behavior of fused reporter proteins was ambiguous, based on the trypsin cleavage behavior of a 31-residue insertion.
Insertion Mutations in
pilE
Differentially Alter Gonococcal Pilin Antigenic Variation
Authors: Howell-Adams Becky , Seifert H. Steven
Published: December 2019

Pilus antigenic variation in
Neisseria gonorrhoeae
occurs by the high-frequency, unidirectional transfer of DNA sequences from one of several silent pilin loci (
pilS
) into the expressed pilin gene (
pilE
), resulting in a change in the primary pilin protein sequence. Previously, we investigated the effects of large or small heterologous insertions in conserved and variable portions of a
pilS
copy on antigenic variation. We observed differential effects on pilin recombination by the various insertions, and the severity of the defect correlated with the disruption or displacement of a conserved pilin DNA sequence called
cys2
. In this study, we show that disruption or displacement of the
pilE cys2
sequence by the same insertions or a deletion also affects pilin recombination. However, in contrast to the insertions in
pilS
, the analogous insertions in
pilE
impaired, but did not block, recombination of the flanking pilin sequences. These results, the change in the spectrum of donor silent copies used during variation, and our previous results with
pilS
mutations show that the donor
pilS
and recipient
pilE
play different roles in antigenic variation. We conclude that when high-frequency recombination mechanisms are blocked, alternative mechanisms are operative.
4,4′-Diapophytoene Desaturase: Catalytic Properties of an Enzyme from the C
30
Carotenoid Pathway of
Staphylococcus aureus
Authors: Raisig Axel , Sandmann Gerhard
Published: December 2019

Staphylococcus aureus
synthesizes C
30
carotenoids. Their formation involves the introduction of three double bonds, which is catalyzed by a single enzyme. This enzyme, 4,4′-diapophytoene desaturase from
S. aureus
, was overexpressed in
Escherichia coli
and purified in one step by affinity chromatography, and then the protein was characterized with respect to substrate specificity, cofactor requirement, and oligomerization.
Spore Peptidoglycan Structure in a
cwlD dacB
Double Mutant of
Bacillus subtilis
Authors: Popham David L. , Meador-Parton Jennifer , Costello Catherine E. , Setlow Peter
Published: December 2019

Bacillus subtilis cwlD
and
dacB
mutants produce spore peptidoglycan (PG) with increased cross-linking but with little change in spore core hydration compared to the wild type. A
cwlD dacB
double mutant produced spores with a two- to fourfold greater increase in PG cross-linking and novel muropeptides containing glycine residues but no significant changes in spore resistance or core hydration.
Characterization of the Type 8 Capsular Gene Cluster of
Streptococcus pneumoniae
Authors: Muñoz Rosario , Mollerach Marta , López Rubens , García Ernesto
Published: December 2019

The complete nucleotide sequence of the capsular gene cluster (
cap8
) responsible for the biosynthesis of the capsular polysaccharide of
Streptococcus pneumoniae
type 8 has been determined. The
cap8
gene cluster, located between the genes
dexB and aliA
, is composed of 12 open reading frames. A 14.7-kb DNA fragment embracing the
cap8
genes was sufficient to transform an unencapsulated type 3
S. pneumoniae
strain to a strain with the type 8 capsule. A possible scenario for the evolution of pneumococcal types 2 and 8 is outlined.
Function of Trehalose and Glycogen in Cell Cycle Progression and Cell Viability in
Saccharomyces cerevisiae
Authors: Silljé H. H. W. , Paalman J. W. G. , ter Schure E. G. , Olsthoorn S. Q. B. , Verkleij A. J. , Boonstra J. , Verrips C. T.
Published: December 2019

Trehalose and glycogen accumulate in
Saccharomyces cerevisiae
when growth conditions deteriorate. It has been suggested that aside from functioning as storage factors and stress protectants, these carbohydrates may be required for cell cycle progression at low growth rates under carbon limitation. By using a mutant unable to synthesize trehalose and glycogen, we have investigated this requirement of trehalose and glycogen under carbon-limited conditions in continuous cultures. Trehalose and glycogen levels increased with decreasing growth rates in the wild-type strain, whereas no trehalose or glycogen was detected in the mutant. However, the mutant was still able to grow and divide at low growth rates with doubling times similar to those for the wild-type strain, indicating that trehalose and glycogen are not essential for cell cycle progression. Nevertheless, upon a slight increase of extracellular carbohydrates, the wild-type strain degraded its reserve carbohydrates and was able to enter a cell division cycle faster than the mutant. In addition, wild-type cells survived much longer than the mutant cells when extracellular carbon was exhausted. Thus, trehalose and glycogen have a dual role under these conditions, serving as storage factors during carbon starvation and providing quickly a higher carbon and ATP flux when conditions improve. Interestingly, the CO
2
production rate and hence the ATP flux were higher in the mutant than in the wild-type strain at low growth rates. The possibility that the mutant strain requires this steady higher glycolytic flux at low growth rates for passage through Start is discussed.
Heat-Induced Synthesis of ς
32
in
Escherichia coli
: Structural and Functional Dissection of
rpoH
mRNA Secondary Structure
Authors: Morita Miyo , Kanemori Masaaki , Yanagi Hideki , Yura Takashi
Published: December 2019

The heat shock response in
Escherichia coli
depends primarily on the increased synthesis and stabilization of otherwise scarce and unstable ς
32
(
rpoH
gene product), which is required for the transcription of heat shock genes. The heat-induced synthesis of ς
32
occurs at the level of translation, and genetic evidence has suggested the involvement of a secondary structure at the 5′ portion (nucleotides −19 to +247) of
rpoH
mRNA in regulation. We now present evidence for the mRNA secondary structure model by means of structure probing of RNA with chemical and enzymatic probes. A similar analysis of several mutant RNAs with a mutation predicted to alter a base pairing or with two compensatory mutations revealed altered secondary structures consistent with the expression and heat inducibility of the corresponding fusion constructs observed in vivo. These findings led us to assess the possible roles of each of the stem-loop structures by analyzing an additional set of deletions and base substitutions. The results indicated not only the primary importance of base pairings between the translation initiation region of ca. 20 nucleotides (the AUG initiation codon plus the “downstream box”) and the internal region of
rpoH
mRNA but also the requirement of appropriate stability of mRNA secondary structures for characteristic thermoregulation, i.e., repression at a low temperature and induction upon a temperature upshift.
BglF, the
Escherichia coli
β-Glucoside Permease and Sensor of the
bgl
System: Domain Requirements of the Different Catalytic Activities
Authors: Chen Qing , Amster-Choder Orna
Published: December 2019

The
Escherichia coli
BglF protein, an enzyme II of the phosphoenolpyruvate-dependent carbohydrate phosphotransferase system, has several enzymatic activities. In the absence of β-glucosides, it phosphorylates BglG, a positive regulator of
bgl
operon transcription, thus inactivating BglG. In the presence of β-glucosides, it activates BglG by dephosphorylating it and, at the same time, transports β-glucosides into the cell and phosphorylates them. BglF is composed of two hydrophilic domains, IIA
bgl
and IIB
bgl
, and a membrane-bound domain, IIC
bgl
, which are covalently linked in the order IIBCA
bgl
. Cys-24 in the IIB
bgl
domain is essential for all the phosphorylation and dephosphorylation activities of BglF. We have investigated the domain requirement of the different functions carried out by BglF. To this end, we cloned the individual BglF domains, as well as the domain pairs IIBC
bgl
and IICA
bgl
, and tested which domains and which combinations are required for the catalysis of the different functions, both in vitro and in vivo. We show here that the IIB and IIC domains, linked to each other (IIBC
bgl
), are required for the sugar-driven reactions, i.e., sugar phosphotransfer and BglG activation by dephosphorylation. In contrast, phosphorylated IIB
bgl
alone can catalyze BglG inactivation by phosphorylation. Thus, the sugar-induced and noninduced functions have different structural requirements. Our results suggest that catalysis of the sugar-induced functions depends on specific interactions between IIB
bgl
and IIC
bgl
which occur upon the interaction of BglF with the sugar.
A Polypeptide with Similarity to Phycocyanin α-Subunit Phycocyanobilin Lyase Involved in Degradation of Phycobilisomes
Authors: Dolganov Nadia , Grossman Arthur R.
Published: December 2019

To optimize the utilization of photosynthate and avoid damage that can result from the absorption of excess excitation energy, photosynthetic organisms must rapidly modify the synthesis and activities of components of the photosynthetic apparatus in response to environmental cues. During nutrient-limited growth, cyanobacteria degrade their light-harvesting complex, the phycobilisome, and dramatically reduce the rate of photosynthetic electron transport. In this report, we describe the isolation and characterization of a cyanobacterial mutant that does not degrade its phycobilisomes during either sulfur or nitrogen limitation and exhibits an increased ratio of phycocyanin to chlorophyll during nutrient-replete growth. The mutant phenotype was complemented by a gene encoding a polypeptide with similarities to polypeptides that catalyze covalent bond formation between linear tetrapyrrole chromophores and subunits of apophycobiliproteins. The complementing gene, designated
nblB
, is expressed at approximately the same level in cells grown in nutrient-replete medium and medium devoid of either sulfur or nitrogen. These results suggest that the NblB polypeptide may be a constitutive part of the machinery that coordinates phycobilisome degradation with environmental conditions.
Dissecting the VanRS Signal Transduction Pathway with Specific Inhibitors
Authors: Ulijasz Andrew T. , Weisblum Bernard
Published: December 2019

The VanRS two-component signal transduction pathway from
Enterococcus faecium
was reconstituted in vitro from partially purified components and shown to be inhibited by the halophenyl isothiazolone LY-266,400, inhibitor A, a compound shown previously to reduce expression of the AlgR1-AlgR2 two-component signal transduction pathway in
Pseudomonas aeruginosa
(S. Roychoudhury, N. A. Zielinski, A. J. Ninfa, N. E. Allen, L. N. Jungheim, T. I. Nicas, and A. M. Chakrabarty, Proc. Natl. Acad. Sci. USA 90:965–969, 1993). Inhibitor A attenuates phosphoryl transfer from VanS∼P to VanR by its action on the ability of VanR to accept. We observed an apparent stimulatory effect of inhibitor A on VanS autophosphorylation which is attributable to the accumulation of VanS∼P as an intermediate unable to transfer P
i
to the inhibited VanR. Thus, inhibitor A acts on the second of two sequential steps which lead to transcriptional activation of the VanHAXYZ gene cluster and the resultant expression of vancomycin resistance.
Targeted Mutagenesis by Duplication Insertion in the Radioresistant Bacterium
Deinococcus radiodurans
: Radiation Sensitivities of Catalase (
katA
) and Superoxide Dismutase (
sodA
) Mutants
Authors: Markillie Lye Meng , Varnum Susan M. , Hradecky Preston , Wong Kwong-Kwok
Published: December 2019

Deinococcus radiodurans
R1 is extremely resistant to both oxidative stress and ionizing radiation. A simple and general targeted mutagenesis method was developed to generate catalase (
katA
) and superoxide dismutase (
sodA
) mutants. Both mutants were shown to be more sensitive to ionizing radiation than the wild type.
Subunit II ofBacillus subtilisCytochromecOxidase Is a Lipoprotein
Authors: Bengtsson Jenny , Tjalsma Harold , Rivolta Carlo , Hederstedt Lars
Published: December 2019

The sequence of the N-terminal end of the deducedctaCgene product ofBacillusspecies has the features of a bacterial lipoprotein. CtaC is the subunit II of cytochromecaa3, which is a cytochromecoxidase. UsingBacillus subtilismutants blocked in lipoprotein synthesis, we show that CtaC is a lipoprotein and that synthesis of the membrane-bound protein and covalent binding of heme to the cytochromecdomain is not dependent on processing at the N-terminal part of the protein. Mutants blocked in prolipoprotein diacylglyceryl transferase (Lgt) or signal peptidase type II (Lsp) are, however, deficient in cytochromecaa3enzyme activity. Removal of the signal peptide from the CtaC polypeptide, but not lipid modification, is seemingly required for formation of functional enzyme.
Analysis of Protein Synthesis Rates after Initiation of Chromosome Replication in
Escherichia coli
Authors: Bechtloff Dorothée , Grünenfelder Björn , Åkerlund Thomas , Nordström Kurt
Published: December 2019

The aim of this study was to investigate whether the synthesis rates of some proteins change after the initiation of replication in
Escherichia coli
. An
intR1
strain, in which chromosome replication is under the control of an R1 replicon integrated into an inactivated
oriC
, was used to synchronize chromosome replication, and the rates of protein synthesis were analyzed by two-dimensional polyacrylamide gel electrophoresis of pulse-labeled proteins. Computerized image analysis was used to search for proteins whose expression levels changed at least threefold after initiation of a single round of chromosome replication, which revealed 7 out of about 1,000 detected proteins. The various synthesis rates of three of these proteins turned out to be caused by unbalanced growth and the synthesis of one protein was suppressed in the
intR1
strain. The rates of synthesis of the remaining three could be correlated only to the synchronous initiation of replication. These three proteins were analyzed by peptide mass mapping and appeared to be the products of the
dps
,
gapA
, and
pyrI
genes. Thus, the expression of the vast majority of proteins is not influenced by the state of chromosome replication, and a possible role of the replication-associated expression changes of the three identified proteins in the cell cycle is not clear.
Functional Analysis of Glycosyltransferase Genes from
Lactococcus lactis
and Other Gram-Positive Cocci: Complementation, Expression, and Diversity
Authors: van Kranenburg Richard , Vos Harmjan R. , van Swam Iris I. , Kleerebezem Michiel , de Vos Willem M.
Published: December 2019

Sixteen exopolysaccharide (EPS)-producing
Lactococcus lactis
strains were analyzed for the chemical compositions of their EPSs and the locations, sequences, and organization of the
eps
genes involved in EPS biosynthesis. This allowed the grouping of these strains into three major groups, representatives of which were studied in detail. Previously, we have characterized the
eps
gene cluster of strain NIZO B40 (group I) and determined the function of three of its glycosyltransferase (GTF) genes. Fragments of the
eps
gene clusters of strains NIZO B35 (group II) and NIZO B891 (group III) were cloned, and these encoded the NIZO B35 priming galactosyltransferase, the NIZO B891 priming glucosyltransferase, and the NIZO B891 galactosyltransferase involved in the second step of repeating-unit synthesis. The NIZO B40 priming glucosyltransferase gene
epsD
was replaced with an erythromycin resistance gene, and this resulted in loss of EPS production. This
epsD
deletion was complemented with priming GTF genes from gram-positive organisms with known function and substrate specificity. Although no EPS production was found with priming galactosyltransferase genes from
L. lactis
or
Streptococcus thermophilus
, complementation with priming glucosyltransferase genes involved in
L. lactis
EPS and
Streptococcus pneumoniae
capsule biosynthesis could completely restore or even increase EPS production in
L. lactis
.
Sequence and Organization of pXO1, the Large
Bacillus anthracis
Plasmid Harboring the Anthrax Toxin Genes
Authors: Okinaka R. T. , Cloud K. , Hampton O. , Hoffmaster A. R. , Hill K. K. , Keim P. , Koehler T. M. , Lamke G. , Kumano S. , Mahillon J. , Manter D. , Martinez Y. , Ricke D. , Svensson ⋕ R. , Jackson P. J.
Published: December 2019

The
Bacillus anthracis
Sterne plasmid pXO1 was sequenced by random, “shotgun” cloning. A circular sequence of 181,654 bp was generated. One hundred forty-three open reading frames (ORFs) were predicted using GeneMark and GeneMark.hmm, comprising only 61% (110,817 bp) of the pXO1 DNA sequence. The overall guanine-plus-cytosine content of the plasmid is 32.5%. The most recognizable feature of the plasmid is a “pathogenicity island,” defined by a 44.8-kb region that is bordered by inverted IS
1627
elements at each end. This region contains the three toxin genes (
cya
,
lef
, and
pagA
), regulatory elements controlling the toxin genes, three germination response genes, and 19 additional ORFs. Nearly 70% of the ORFs on pXO1 do not have significant similarity to sequences available in open databases. Absent from the pXO1 sequence are homologs to genes that are typically required to drive theta replication and to maintain stability of large plasmids in
Bacillus
spp. Among the ORFs with a high degree of similarity to known sequences are a collection of putative transposases, resolvases, and integrases, suggesting an evolution involving lateral movement of DNA among species. Among the remaining ORFs, there are three sequences that may encode enzymes responsible for the synthesis of a polysaccharide capsule usually associated with serotype-specific virulent streptococci.
Localization of Periplasmic Redox Proteins of
Alcaligenes faecalis
by a Modified General Method for Fractionating Gram-Negative Bacteria
Authors: Zhu Zhenyu , Sun Dapeng , Davidson Victor L.
Published: December 2019

A lysozyme-osmotic shock method is described for fractionation of
Alcaligenes faecalis
which uses glucose to adjust osmotic strength and multiple osmotic shocks. During phenylethylamine-dependent growth, aromatic amine dehydrogenase, azurin, and a single cytochrome
c
were localized in the periplasm. Their induction patterns are different from those for the related quinoprotein methylamine dehydrogenase and its associated redox proteins.
Purification, Characterization, and Gene Analysis of a Chitosanase (ChoA) from
Matsuebacter chitosanotabidus
3001
Authors: Park Jae Kweon , Shimono Kumiko , Ochiai Nobuhisa , Shigeru Kazutaka , Kurita Masako , Ohta Yukari , Tanaka Katsunori , Matsuda Hideyuki , Kawamukai Makoto
Published: December 2019

The extracellular chitosanase (34,000
M
r
) produced by a novel gram-negative bacterium
Matsuebacter chitosanotabidus
3001 was purified. The optimal pH of this chitosanase was 4.0, and the optimal temperature was between 30 and 40°C. The purified chitosanase was most active on 90% deacetylated colloidal chitosan and glycol chitosan, both of which were hydrolyzed in an endosplitting manner, but this did not hydrolyze chitin, cellulose, or their derivatives. Among potential inhibitors, the purified chitosanase was only inhibited by Ag
+
. Internal amino acid sequences of the purified chitosanase were obtained. A PCR fragment corresponding to one of these amino acid sequences was then used to screen a genomic library for the entire
choA
gene encoding chitosanase. Sequencing of the
choA
gene revealed an open reading frame encoding a 391-amino-acid protein. The N-terminal amino acid sequence had an excretion signal, but the sequence did not show any significant homology to other proteins, including known chitosanases. The 80-amino-acid excretion signal of ChoA fused to green fluorescent protein was functional in
Escherichia coli
. Taken together, these results suggest that we have identified a novel, previously unreported chitosanase.
Exponential-Phase Glycogen Recycling Is Essential for Growth of
Mycobacterium smegmatis
Authors: Belanger Aimee E. , Hatfull Graham F.
Published: December 2019

Bacterial glycogen is a polyglucose storage compound that is thought to prolong viability during stationary phase. However, a specific role for glycogen has not been determined. We have characterized SMEG53, a temperature-sensitive mutant of
Mycobacterium smegmatis
that contains a mutation in
glgE
, encoding a putative glucanase. This mutation causes exponentially growing SMEG53 cells to stop growing at 42°C in response to high levels of glycogen accumulation. The mutation in
glgE
is also associated with an altered growth rate and colony morphology at permissive temperatures; the severity of these phenotypes correlates with the amount of glycogen accumulated by the mutant. Suppression of the temperature-sensitive phenotype, via a decrease in glycogen accumulation, is mediated by growth in certain media or multicopy expression of
garA
. The function of GarA is unknown, but the presence of a forkhead-associated domain suggests that this protein is a member of a serine-threonine kinase signal transduction pathway. Our results suggest that in
M. smegmatis
glycogen is continuously synthesized and then degraded by GlgE throughout exponential growth. In turn, this constant recycling of glycogen controls the downstream availability of carbon and energy. Thus, in addition to its conventional storage role, glycogen may also serve as a carbon capacitor for glycolysis during the exponential growth of
M. smegmatis
.
Genetic and Structural Analyses of Cytoplasmic Filaments of Wild-Type
Treponema phagedenis
and a Flagellar Filament-Deficient Mutant
Authors: Izard Jacques , Samsonoff William A. , Kinoshita Mary Beth , Limberger Ronald J.
Published: December 2019

Unique cytoplasmic filaments are found in the treponeme genus of spirochete bacteria. Their function is unknown, but their location underneath the periplasmic flagellar filaments (PFF) suggests a role in motility and/or cell structure. To better understand these unique structures, the gene coding for the cytoplasmic filaments,
cfpA
, was identified in various treponemal species.
Treponema phagedenis cfpA
was 2,037 nucleotides long, and the encoded polypeptide showed 78 to 100% amino acid sequence identity with the partial sequence of CfpA from
T. denticola
,
T. vincentii
, and
T. pallidum
subsp.
pertenue
. Wild-type
T. phagedenis
and a PFF-deficient isolate were analyzed by electron microscopy to assess the structural relationship of the cytoplasmic filaments and the PFF. The number of cytoplasmic filaments per cell of
T. phagedenis
(mean, 5.7) was compared with the number of PFF at each end of the cell (mean, 4.7); the results suggest that there is no direct one-to-one correlation at the cell end. Moreover, a structural link between these structures could not be demonstrated. The cytoplasmic filaments were also analyzed by electron microscopy at different stages of cell growth; this analysis revealed that they are cleaved before or during septum formation and before the nascent formation of PFF. A PFF-deficient mutant of
T. phagedenis
possessed cytoplasmic filaments similar to those of the wild type, suggesting that intact PFF are not required for their assembly and regulation. The extensive conservation of CfpA among pathogenic spirochetes suggests an important function, and structural analysis suggests that it is unlikely that the cytoplasmic filaments and the flagellar apparatus are physically linked.
Imbalanced Base Excision Repair Increases Spontaneous Mutation and Alkylation Sensitivity in
Escherichia coli
Authors: Posnick Lauren M. , Samson Leona D.
Published: December 2019

Inappropriate expression of 3-methyladenine (3MeA) DNA glycosylases has been shown to have harmful effects on microbial and mammalian cells. To understand the underlying reasons for this phenomenon, we have determined how DNA glycosylase activity and substrate specificity modulate glycosylase effects in
Escherichia coli
. We compared the effects of two 3MeA DNA glycosylases with very different substrate ranges, namely, the
Saccharomyces cerevisiae
Mag1 and the
E. coli
Tag glycosylases. Both glycosylases increased spontaneous mutation, decreased cell viability, and sensitized
E. coli
to killing by the alkylating agent methyl methanesulfonate. However, Tag had much less harmful effects than Mag1. The difference between the two enzymes’ effects may be accounted for by the fact that Tag almost exclusively excises 3MeA lesions, whereas Mag1 excises a broad range of alkylated and other purines. We infer that the DNA lesions responsible for changes in spontaneous mutation, viability, and alkylation sensitivity are abasic sites and secondary lesions resulting from processing abasic sites via the base excision repair pathway.
The CpxRA Signal Transduction System of
Escherichia coli
: Growth-Related Autoactivation and Control of Unanticipated Target Operons
Authors: De Wulf Peter , Kwon Ohsuk , Lin E. C. C.
Published: December 2019

In
Escherichia coli
, the CpxRA two-component signal transduction system senses and responds to aggregated and misfolded proteins in the bacterial envelope. We show that CpxR-P (the phosphorylated form of the cognate response regulator) activates
cpxRA
expression in conjunction with RpoS, suggesting an involvement of the Cpx system in stationary-phase survival. Engagement of the CpxRA system in functions beyond protein management is indicated by several putative targets identified after a genomic screening for the CpxR-P recognition consensus sequence. Direct negative control of the newly identified targets
motABcheAW
(specifying motility and chemotaxis) and
tsr
(encoding the serine chemoreceptor) by CpxR-P was shown by electrophoretic mobility shift analysis and Northern hybridization. The results suggest that the CpxRA system plays a core role in an extensive stress response network in which the coordination of protein turnover and energy conservation may be the unifying element.
The
Vibrio cholerae
O139 Calcutta Bacteriophage CTXφ Is Infectious and Encodes a Novel Repressor
Authors: Davis Brigid M. , Kimsey Harvey H. , Chang William , Waldor Matthew K.
Published: December 2019

CTXφ is a lysogenic, filamentous bacteriophage. Its genome includes the genes encoding cholera toxin (
ctxAB
), one of the principal virulence factors of
Vibrio cholerae
; consequently, nonpathogenic strains of
V. cholerae
can be converted into toxigenic strains by CTXφ infection. O139 Calcutta strains of
V. cholerae
, which were linked to cholera outbreaks in Calcutta, India, in 1996, are novel pathogenic strains that carry two distinct CTX prophages integrated in tandem: CTX
ET
, the prophage previously characterized within El Tor strains, and a new CTX Calcutta prophage (CTX
calc
). We found that the CTX
calc
prophage gives rise to infectious virions; thus, CTX
ET
φ is no longer the only known vector for transmission of
ctxAB
. The most functionally significant differences between the nucleotide sequences of CTX
calc
φ and CTX
ET
φ are located within the phages’ repressor genes (
rstR
calc
and
rstR
ET
, respectively) and their RstR operators. RstR
calc
is a novel, allele-specific repressor that regulates replication of CTX
calc
φ by inhibiting the activity of the
rstA
calc
promoter. RstR
calc
has no inhibitory effect upon the classical and El Tor
rstA
promoters, which are instead regulated by their cognate RstRs. Consequently, production of RstR
calc
renders a CTX
calc
lysogen immune to superinfection by CTX
calc
φ but susceptible (heteroimmune) to infection by CTX
ET
φ. Analysis of the prophage arrays generated by sequentially integrated CTX phages revealed that pathogenic
V. cholerae
O139 Calcutta probably arose via infection of an O139 CTX
ET
φ lysogen by CTX
calc
φ.
Mutations in the Regulatory Gene
hrpG
of
Xanthomonas campestris
pv. vesicatoria Result in Constitutive Expression of All
hrp
Genes
Authors: Wengelnik Kai , Rossier Ombeline , Bonas Ulla
Published: December 2019

hrpG
is a key regulatory gene for transcriptional activation of pathogenicity genes (
hrp
) of
Xanthomonas campestris
pv. vesicatoria. We identified three mutations in
hrpG
which render
hrp
gene expression constitutive in normally suppressing medium. The mutations in
hrpG
result in novel amino acid substitutions compared to mutations in related proteins, such as OmpR. In addition, mutated
hrpG
enhances the timing and intensity of plant reactions in infection assays.
The BldD Protein from
Streptomyces coelicolor
Is a DNA-Binding Protein
Authors: Elliot Marie A. , Leskiw Brenda K.
Published: December 2019

Gel mobility shift assays with His-tagged BldD isolated from
Escherichia coli
have illustrated that BldD is capable of specifically recognizing its own promoter region. DNase I and hydroxyl radical footprinting assays have served to delimit the BldD binding site, revealing that BldD recognizes and binds to a site just upstream from, and overlapping with, the −10 region of the promoter. How BldD binds to its promoter and the effect this binding has on the expression of BldD are discussed.
The IntI1 Integron Integrase Preferentially Binds Single-Stranded DNA of the
attC
Site
Authors: Francia M. Victoria , Zabala Juan C. , de la Cruz Fernando , García Lobo Juan M.
Published: December 2019

IntI1 integrase is a member of the prokaryotic DNA integrase superfamily. It is responsible for mobility of antibiotic resistance cassettes found in integrons. IntI1 protein, as well as IntI1-COOH, a truncated form containing its carboxy-terminal domain, has been purified. Electrophoretic mobility shift assays were carried out to study the ability of IntI1 to bind the integrase primary target sites
attI
and
aadA1 attC
. When using double-stranded DNA as a substrate, we observed IntI1 binding to
attI
but not to
attC
. IntI1-COOH did not bind either
attI
or
attC
, indicating that the N-terminal domain of IntI1 was required for binding to double-stranded
attI
. On the other hand, when we used single-stranded (ss) DNA substrates, IntI1 bound strongly and specifically to ss
attC
DNA. Binding was strand specific, since only the bottom DNA strand was bound. Protein IntI1-COOH bound ss
attC
as well as did the complete integrase, indicating that the ability of the protein to bind ss
aadA1 attC
was contained in the region between amino acids 109 and 337 of IntI1. Binding to ss
attI
DNA by the integrase, but not by IntI1-COOH, was also observed and was specific for the
attI
bottom strand, indicating similar capabilities of IntI1 for binding
attI
DNA in either double-stranded or ss conformation. Footprinting analysis showed that IntI1 protected at least 40 bases of
aadA1 attC
against DNase I attack. The protected sequence contained two of the four previously proposed IntI1 DNA binding sites, including the crossover site. Preferential ssDNA binding can be a significant activity of IntI1 integrase, which suggests the utilization of extruded cruciforms in the reaction mechanisms leading to cassette excision and integration.