bluuebunny/crossref_metadata_2025_split huggingface dataset:
Visualization of AqpZ-Mediated Water Permeability in
Escherichia coli
by Cryoelectron Microscopy
Authors: Delamarche Christian , Thomas Daniel , Rolland Jean-Paul , Froger Alexandrine , Gouranton Jean , Svelto Maria , Agre Peter , Calamita Giuseppe 
Published: December 2019

ABSTRACT

Transport of water across the plasma membrane is a fundamental process occurring in all living organisms. In bacteria, osmotic movement of water across the cytoplasmic membrane is needed to maintain cellular turgor; however, the molecular mechanisms of this process are poorly defined. Involvement of aquaporin water channels in bacterial water permeability was suggested by the recent discovery of the aquaporin gene,
aqpZ
, in
Escherichia coli
. By employing cryoelectron microscopy to compare
E. coli
cells containing (AqpZ
+
) and lacking (AqpZ
−
) aquaporin, we show that the AqpZ water channel rapidly mediates large water fluxes in response to sudden changes in extracellular osmolarity. These findings (i) demonstrate for the first time functional expression of a prokaryotic water channel, (ii) evidence the bidirectional water channel feature of AqpZ, (iii) document a role for AqpZ in bacterial osmoregulation, and (iv) define a suitable model for studying the physiology of prokaryotic water transport.
The Unique Chaperone Operon of
Thermotoga maritima
: Cloning and Initial Characterization of a Functional Hsp70 and Small Heat Shock Protein
Authors: Michelini Edward T. , Flynn Gregory C. 
Published: December 2019

ABSTRACT

The hyperthermophilic eubacterium
Thermotoga maritima
possesses an operon encoding an Hsp70 molecular chaperone protein and a protein with meaningful homology to the small heat shock protein family of chaperones. This represents the first demonstrated co-operon organization for these two important classes of molecular chaperones. We have cloned and initially characterized these proteins as functional chaperones in vitro: the Hsp70 is capable of ATP hydrolysis and substrate binding, and the small heat shock protein can suppress protein aggregation and stably bind a refolding-competent substrate. In addition, the primary sequence of the Hsp70 is used to infer the phylogenetic relationships of
T. maritima
, one of the deepest-branching eubacteria known.
A Mycobacterial Extracytoplasmic Sigma Factor Involved in Survival following Heat Shock and Oxidative Stress
Authors: Fernandes Norvin D. , Wu Qi-long , Kong Dequan , Puyang Xiaoling , Garg Sumeet , Husson Robert N. 
Published: December 2019

ABSTRACT

Extracytoplasmic function (ECF) sigma factors are a heterogeneous group of alternative sigma factors that regulate gene expression in response to a variety of conditions, including stress. We previously characterized a mycobacterial ECF sigma factor, SigE, that contributes to survival following several distinct stresses. A gene encoding a closely related sigma factor,
sigH
, was cloned from
Mycobacterium tuberculosis
and
Mycobacterium smegmatis
. A single copy of this gene is present in these and other fast- and slow-growing mycobacteria, including
M. fortuitum
and
M. avium
. While the
M. tuberculosis
and
M. smegmatis sigH
genes encode highly similar proteins, there are multiple differences in adjacent genes. The single in vivo transcriptional start site identified in
M. smegmatis
and one of two identified in
M. bovis
BCG were found to have −35 promoter sequences that match the ECF-dependent −35 promoter consensus. Expression from these promoters was strongly induced by 50°C heat shock. In comparison to the wild type, an
M. smegmatis sigH
mutant was found to be more susceptible to cumene hydroperoxide stress but to be similar in logarithmic growth, stationary-phase survival, and survival following several other stresses. Survival of an
M. smegmatis sigH sigE
double mutant was found to be markedly decreased following 53°C heat shock and following exposure to cumene hydroperoxide. Expression of the second gene in the
sigH
operon is required for complementation of the
sigH
stress phenotypes. SigH is an alternative sigma factor that plays a role in the mycobacterial stress response.
The Chemotactic Response of
Vibrio anguillarum
to Fish Intestinal Mucus Is Mediated by a Combination of Multiple Mucus Components
Authors: O’Toole Ronan , Lundberg Susanne , Fredriksson Sten-Åke , Jansson Anita , Nilsson Bo , Wolf-Watz Hans 
Published: December 2019

ABSTRACT

Chemotactic motility has previously been shown to be essential for the virulence of
Vibrio anguillarum
in waterborne infections of fish. To investigate the mechanisms by which chemotaxis may function during infection, mucus was isolated from the intestinal and skin epithelial surfaces of rainbow trout. Chemotaxis assays revealed that
V. anguillarum
swims towards both types of mucus, with a higher chemotactic response being observed for intestinal mucus. Work was performed to examine the basis, in terms of mucus composition, of this chemotactic response. Intestinal mucus was analyzed by using chromatographic and mass spectrometric techniques, and the compounds identified were tested in a chemotaxis assay to determine the attractants present. A number of mucus-associated components, in particular, amino acids and carbohydrates, acted as chemoattractants for
V. anguillarum
. Importantly, only upon combination of these attractants into a single mixture were levels of chemotactic activity similar to those of intestinal mucus generated. A comparative analysis of skin mucus revealed its free amino acid and carbohydrate content to be considerably lower than that of the more chemotactically active intestinal mucus. To study whether host specificity exists in relation to vibrio chemotaxis towards mucus, comparisons with a human
Vibrio
pathogen were made. A
cheR
mutant of a
Vibrio cholerae
El Tor strain was constructed, and it was found that
V. cholerae
and
V. anguillarum
exhibit a chemotactic response to mucus from several animal sources in addition to that from the human jejunum and fish epithelium, respectively.
A Region of ς
K
Involved in Promoter Activation by GerE in
Bacillus subtilis
Authors: Wade Kathryn H. , Schyns Ghislain , Opdyke Jason A. , Moran Charles P. 
Published: December 2019

ABSTRACT

During endospore formation in
Bacillus subtilis
, the DNA binding protein GerE stimulates transcription from several promoters that are used by RNA polymerase containing ς
K
. GerE binds to a site on one of these promoters,
cotX
, that overlaps its −35 region. We tested the model that GerE interacts with ς
K
at the
cotX
promoter by seeking amino acid substitutions in ς
K
that interfered with GerE-dependent activation of the
cotX
promoter but which did not affect utilization of the ς
K
-dependent, GerE-independent promoter
gerE
. We identified two amino acid substitutions in ς
K
, E216K and H225Y, that decrease
cotX
promoter utilization but do not affect
gerE
promoter activity. Alanine substitutions at these positions had similar effects. We also examined the effects of the E216A and H225Y substitutions in ς
K
on transcription in vitro. We found that these substitutions specifically reduced utilization of the
cotX
promoter. These and other results suggest that the amino acid residues at positions 216 and 225 are required for GerE-dependent
cotX
promoter activity, that the histidine at position 225 of ς
K
may interact with GerE at the
cotX
promoter, and that this interaction may facilitate the initial binding of ς
K
RNA polymerase to the
cotX
promoter. We also found that the alanine substitutions at positions 216 and 225 of ς
K
had no effect on utilization of the GerE-dependent promoter
cotD
, which contains GerE binding sites that do not overlap with its −35 region.
Structural Characterization of the Lactoferrin Receptor from
Neisseria meningitidis
Authors: Prinz Thorsten , Meyer Markus , Pettersson Annika , Tommassen Jan 
Published: December 2019

ABSTRACT
The meningococcal lactoferrin receptor is composed of the integral outer membrane protein LbpA and the peripheral lipoprotein LbpB. Homooligomeric complexes of LbpA and heterooligomers consisting of LbpA and LbpB were identified. Furthermore, five cell surface-exposed loops of LbpA were identified, which partially confirms a previously proposed topology model.
Growth Phase-Dependent Regulation of Vsr Endonuclease May Contribute to 5-Methylcytosine Mutational Hot Spots in
Escherichia coli
Authors: Macintyre Georgina , Pitsikas Photini , Cupples Claire G. 
Published: December 2019

ABSTRACT

Using rabbit polyclonal antibodies, we have shown that the Dcm cytosine methylase of
Escherichia coli
is maintained at a constant level during cell growth, while Vsr endonuclease levels are growth phase dependent. Decreased production of Vsr relative to Dcm during the log phase may contribute substantially to the mutability of 5-methylcytosine.
Role of TolR N-Terminal, Central, and C-Terminal Domains in Dimerization and Interaction with TolA and TolQ
Authors: Journet Laure , Rigal Alain , Lazdunski Claude , Bénédetti Hélène 
Published: December 2019

ABSTRACT

The Tol-PAL system of
Escherichia coli
is a multiprotein system involved in maintaining the cell envelope integrity and is necessary for the import of some colicins and phage DNA into the bacterium. It is organized into two complexes, one near the outer membrane between TolB and PAL and one in the cytoplasmic membrane between TolA, TolQ, and TolR. In the cytoplasmic membrane, all of the Tol proteins have been shown to interact with each other. Cross-linking experiments have shown that the TolA transmembrane domain interacts with TolQ and TolR. Suppressor mutant analyses have localized the TolQ-TolA interaction to the first transmembrane domain of TolQ and have shown that the third transmembrane domain of TolQ interacts with the transmembrane domain of TolR. To get insights on the composition of the cytoplasmic membrane complex and its possible contacts with the outer membrane complex, we focused our attention on TolR. Cross-linking and immunoprecipitation experiments allowed the identification of Tol proteins interacting with TolR. The interactions of TolR with TolA and TolQ were confirmed, TolR was shown to dimerize, and the resulting dimer was shown to interact with TolQ. Deletion mutants of TolR were constructed, and they allowed us to determine the TolR domains involved in each interaction. The TolR transmembrane domain was shown to be involved in the TolA-TolR and TolQ-TolR interactions, while TolR central and C-terminal domains appeared to be involved in TolR dimerization. The role of the TolR C-terminal domain in the TolA-TolR interaction and its association with the membranes was also demonstrated. Furthermore, phenotypic studies clearly showed that the three TolR domains (N terminal, central, and C terminal) and the level of TolR production are important for colicin A import and for the maintenance of cell envelope integrity.
Characterization of an Atypical Superoxide Dismutase from
Sinorhizobium meliloti
Authors: Santos Renata , Bocquet Stephane , Puppo Alain , Touati Danièle 
Published: December 2019

ABSTRACT

Sinorhizobium meliloti
Rm5000 is an aerobic bacterium that can live free in the soil or in symbiosis with the roots of leguminous plants. A single detectable superoxide dismutase (SOD) was found in free-living growth conditions. The corresponding gene was isolated from a genomic library by using a
sod
fragment amplified by PCR from degenerate primers as a probe. The
sodA
gene was located in the chromosome. It is transcribed monocistronically and encodes a 200-amino-acid protein with a theoretical
M
r
of 22,430 and pI of 5.8.
S. meliloti
SOD complemented a deficient
E. coli
mutant, restoring aerobic growth of a
sodA sodB recA
strain, when the gene was expressed from the synthetic
tac
promoter but not from its own promoter. Amino acid sequence alignment showed great similarity with Fe-containing SODs (FeSODs), but the enzyme was not inactivated by H
2
O
2
. The native enzyme was purified and found to be a dimeric protein, with a specific activity of 4,000 U/mg. Despite its Fe-type sequence, atomic absorption spectroscopy showed manganese to be the cofactor (0.75 mol of manganese and 0.24 mol of iron per mol of monomer). The apoenzyme was prepared from crude extracts of
S. meliloti
. Activity was restored by dialysis against either MnCl
2
or Fe(NH
4
)
2
(SO
4
)
2
, demonstrating the cambialistic nature of the
S. meliloti
SOD. The recovered activity with manganese was sevenfold higher than with iron. Both reconstituted enzymes were resistant to H
2
O
2
. Sequence comparison with 70 FeSODs and MnSODs indicates that
S. meliloti
SOD contains several atypical residues at specific sites that might account for the activation by manganese and resistance to H
2
O
2
of this unusual Fe-type SOD.
The Structure of Multiple Polypeptide Domains Determines the Signal Recognition Particle Targeting Requirement of
Escherichia coli
Inner Membrane Proteins
Authors: Newitt John A. , Ulbrandt Nancy D. , Bernstein Harris D. 
Published: December 2019

ABSTRACT

The signal recognition particle (SRP) targeting pathway is required for the efficient insertion of many polytopic inner membrane proteins (IMPs) into the
Escherichia coli
inner membrane, but in the absence of SRP protein export proceeds normally. To define the properties of IMPs that impose SRP dependence, we analyzed the targeting requirements of bitopic IMPs that are structurally intermediate between exported proteins and polytopic IMPs. We found that disruption of the SRP pathway inhibited the insertion of only a subset of bitopic IMPs. Studies on a model bitopic AcrB-alkaline phosphatase fusion protein (AcrB 265-AP) showed that the SRP requirement for efficient insertion correlated with the presence of a large periplasmic domain (P1). As previously reported, perturbation of the SRP pathway also affected the insertion of a polytopic AcrB-AP fusion. Even exhaustive SRP depletion, however, failed to block the insertion of any AcrB derivative by more than 50%. Taken together, these data suggest that many proteins that are normally targeted by SRP can utilize alternative targeting pathways and that the structure of both hydrophilic and membrane-spanning domains determines the degree to which the biogenesis of a protein is SRP dependent.
Cloning, Sequencing, and Characterization of the
cgmB
Gene of
Sinorhizobium meliloti
Involved in Cyclic β-Glucan Biosynthesis
Authors: Wang Ping , Ingram-Smith Cheryl , Hadley Jill A. , Miller Karen J. 
Published: December 2019

ABSTRACT

Periplasmic cyclic β-glucans of
Rhizobium
species provide important functions during plant infection and hypo-osmotic adaptation. In
Sinorhizobium meliloti
(also known as
Rhizobium meliloti
), these molecules are highly modified with phosphoglycerol and succinyl substituents. We have previously identified an
S. meliloti
Tn
5
insertion mutant, S9, which is specifically impaired in its ability to transfer phosphoglycerol substituents to the cyclic β-glucan backbone (M. W. Breedveld, J. A. Hadley, and K. J. Miller, J. Bacteriol. 177:6346–6351, 1995). In the present study, we have cloned, sequenced, and characterized this mutation at the molecular level. By using the Tn
5
flanking sequences (amplified by inverse PCR) as a probe, an
S. meliloti
genomic library was screened, and two overlapping cosmid clones which functionally complement S9 were isolated. A 3.1-kb
Hin
dIII-
Eco
RI fragment found in both cosmids was shown to fully complement mutant S9. Furthermore, when a plasmid containing this 3.1-kb fragment was used to transform
Rhizobium leguminosarum
bv. trifolii TA-1JH, a strain which normally synthesizes only neutral cyclic β-glucans, anionic glucans containing phosphoglycerol substituents were produced, consistent with the functional expression of an
S. meliloti
phosphoglycerol transferase gene. Sequence analysis revealed the presence of two major, overlapping open reading frames within the 3.1-kb fragment. Primer extension analysis revealed that one of these open reading frames, ORF1, was transcribed and its transcription was osmotically regulated. This novel locus of
S. meliloti
is designated the
cgm
(cyclic glucan modification) locus, and the product encoded by ORF1 is referred to as CgmB.
OxyR and SoxRS Regulation of
fur
Authors: Zheng Ming , Doan Bernard , Schneider Thomas D. , Storz Gisela 
Published: December 2019

ABSTRACT

The cytotoxic effects of reactive oxygen species are largely mediated by iron. Hydrogen peroxide reacts with iron to form the extremely reactive and damaging hydroxyl radical via the Fenton reaction. Superoxide anion accelerates this reaction because the dismutation of superoxide leads to increased levels of hydrogen peroxide and because superoxide elevates the intracellular concentration of iron by attacking iron-sulfur proteins. We found that regulators of the
Escherichia coli
responses to oxidative stress, OxyR and SoxRS, activate the expression of Fur, the global repressor of ferric ion uptake. A transcript encoding Fur was induced by hydrogen peroxide in a wild-type strain but not in a Δ
oxyR
strain, and DNase I footprinting assays showed that OxyR binds to the
fur
promoter. In cells treated with the superoxide-generating compound paraquat, we observed the induction of a longer transcript encompassing both
fur
and its immediate upstream gene
fldA
, which encodes a flavodoxin. This polycistronic mRNA is induced by paraquat in a wild-type strain but not in a Δ
soxRS
strain, and SoxS was shown to bind to the
fldA
promoter. These results demonstrate that iron metabolism is coordinately regulated with the oxidative stress defenses.
Influence of a Putative ECF Sigma Factor on Expression of the Major Outer Membrane Protein, OprF, in Pseudomonas aeruginosa and Pseudomonas fluorescens
Authors: Brinkman Fiona S. L. , Schoofs Geert , Hancock Robert E. W. , De Mot René 
Published: December 2019

ABSTRACTThe gene encoding OprF, a major outer membrane protein inPseudomonasspecies (formerly known as type 1 pseudomonads), was thought to be constitutively transcribed from a single sigma 70 promoter immediately upstream of the gene. We now report the identification of a novel putative ECF (extracytoplasmic function) sigma factor gene,sigX, located immediately upstream ofoprFin bothPseudomonas aeruginosaPAO1 andPseudomonas fluorescensOE 28.3 and show that disruption of this gene significantly reduces OprF expression. InP. aeruginosa, Northern analysis demonstrated that this reduction was a result of an effect on transcription of monocistronicoprFcombined with a polar effect due to termination of a transcript containingsigXandoprF. Comparison ofsigX-disrupted and wild-type cell transcripts by primer extension indicated that monocistronic transcription ofoprFoccurs from two overlapping promoters, one that is SigX-dependent and resembles ECF sigma factor promoters in its minus-35 region and another promoter that is independent of SigX and is analogous to the sigma 70-type promoter previously reported. Complementation of theP. aeruginosa sigX-disrupted mutant with plasmid-encoded OprF did not resolve the phenotypes associated with this mutant, which included a markedly reduced logarithmic-phase growth rate in rich medium (compared to that in minimal medium), further reduction of the growth rate in a low-osmolarity environment, secretion of an unidentified pigment, and increased sensitivity to the antibiotic imipenem. This indicates that SigX is involved in the regulation of other genes inP. aeruginosa. Disruption of thesigXgene inP. fluorescensalso had an effect on the logarithmic-phase growth rate in rich medium. A conservedsigXgene was also identified in aPseudomonas syringaeisolate and sixP. aeruginosaclinical isolates. Collectively, these data indicate that an ECF sigma factor plays a role in the regulation and expression of OprF and also affects other genes.
Toxicity of Copper, Cobalt, and Nickel Salts Is Dependent on Histidine Metabolism in the YeastSaccharomyces cerevisiae
Authors: Pearce David A. , Sherman Fred 
Published: December 2019

ABSTRACTThe pH-dependent inhibition of 22 metal salts have been systematically investigated for the yeastSaccharomyces cerevisiae. We have established that the inhibition of growth by Cu, Co, or Ni salts is markedly enhanced by histidine auxotrophy and by increasing the pH of the medium. Each of thehis1-his7mutant strains were unable to grow in the presence of elevated levels of Cu, Co, or Ni at nearly neutral pHs, in contrast to His+strains, which grew under these conditions. The Cu, Co, or Ni inhibition was reversed by the addition of histidine to the medium. Deletion of the high-affinity histidine permease Hip1p in His−strains resulted in even greater sensitivity to Cu, Co, and Ni and the requirement of an even higher level of histidine to reverse the inhibition. These results suggest that intracellular histidine, most likely in the vacuole, diminishes the pH-dependent toxicity of Cu, Co, and Ni. Furthermore, the toxicity of many salts is exacerbated in strains with a defective vacuolar H+-ATPase, which abolishes the ability of yeast to maintain an acidic vacuole, a compartment known to sequester metal compounds. We suggest that the accumulation of histidine in the vacuole is a normal process used to detoxify Cu, Co, and Ni.
Hemoglobinase Activity of the Lysine Gingipain Protease (Kgp) of Porphyromonas gingivalis W83
Authors: Lewis Janina P. , Dawson Janet A. , Hannis James C. , Muddiman David , Macrina Francis L. 
Published: December 2019

ABSTRACT

Porphyromonas gingivalis, an important periodontal disease pathogen, forms black-pigmented colonies on blood agar. Pigmentation is believed to result from accumulation of iron protoporphyrin IX (FePPIX) derived from erythrocytic hemoglobin. The Lys-X (Lys-gingipain) and Arg-X (Arg-gingipain) cysteine proteases ofP. gingivalis bind and degrade erythrocytes. We have observed that mutations abolishing activity of the Lys-X-specific cysteine protease, Kgp, resulted in loss of black pigmentation ofP. gingivalis W83. Because the hemagglutinating and hemolytic potentials of mutant strains were reduced but not eliminated, we hypothesized that this protease played a role in acquisition of FePPIX from hemoglobin. In contrast to Arg-gingipain, Lys-gingipain was not inhibited by hemin, suggesting that this protease played a role near the cell surface where high concentrations of hemin confer the black pigmentation. Human hemoglobin contains 11 Lys residues in the α chain and 10 Lys residues in the β chain. In contrast, there are only three Arg residues in each of the α and β chains. These observations are consistent with human hemoglobin being a preferred substrate for Lys-gingipain but not Arg-gingipain. The ability of the Lys-gingipain to cleave human hemoglobin at Lys residues was confirmed by electrospray ionization Fourier transform ion cyclotron resonance mass spectrometry of hemoglobin fragments resulting from digestion with the purified protease. We were able to detect several of the predicted hemoglobin fragments rendered by digestion with purified Lys-gingipain. Thus, we postulate that the Lys-gingipain of P. gingivalisis a hemoglobinase which plays a role in heme and iron uptake by effecting the accumulation of FePPIX on the bacterial cell surface.