bluuebunny/crossref_metadata_2025_split huggingface dataset:
Functional Analysis of a Rickettsial OmpA Homology Domain of
Shigella flexneri
IcsA
Authors: Charles Macarthur , Magdalena Juana , Theriot Julie A. , Goldberg Marcia B. 
Published: December 2019

ABSTRACT

Shigella flexneri
is a gram-negative bacterium that causes diarrhea and dysentery by invasion and spread through the colonic epithelium. Bacteria spread by assembling actin and other cytoskeletal proteins of the host into “actin tails” at the bacterial pole; actin tail assembly provides the force required to move bacteria through the cell cytoplasm and into adjacent cells. The 120-kDa
S. flexneri
outer membrane protein IcsA is essential for actin assembly. IcsA is anchored in the outer membrane by a carboxy-terminal domain (the β domain), such that the amino-terminal 706 amino acid residues (the α domain) are exposed on the exterior of the bacillus. The α domain is therefore likely to contain the domains that are important to interactions with host factors. We identify and characterize a domain of IcsA within the α domain that bears significant sequence similarity to two repeated domains of rickettsial OmpA, which has been implicated in rickettsial actin tail formation. Strains of
S. flexneri
and
Escherichia coli
that carry derivatives of IcsA containing deletions within this domain display loss of actin recruitment and increased accessibility to IcsA-specific antibody on the surface of intracytoplasmic bacteria. However, site-directed mutagenesis of charged residues within this domain results in actin assembly that is indistinguishable from that of the wild type, and in vitro competition of a polypeptide of this domain fused to glutathione
S
-transferase did not alter the motility of the wild-type construct. Taken together, our data suggest that the rickettsial homology domain of IcsA is required for the proper conformation of IcsA and that its disruption leads to loss of interactions of other IcsA domains within the amino terminus with host cytoskeletal proteins.
Cra-Dependent Transcriptional Activation of the
icd
Gene of
Escherichia coli
Authors: Prost Jean-François , Nègre Didier , Oudot Christelle , Murakami Katsuhiko , Ishihama Akira , Cozzone Alain J. , Cortay Jean-Claude 
Published: December 2019

ABSTRACT

The
icd
gene of
Escherichia coli
, encoding isocitrate dehydrogenase, was shown to be expressed from two different promoters: the previously identified
icd
P1 and a newly detected second promoter,
icd
P2, whose expression is positively regulated by the catabolite repressor-activator protein Cra, formerly called FruR. In each case, we determined the mRNA start site by primer extension analysis of in vivo transcripts and examined the interaction of the
icd
control region with either RNA polymerase or Cra. We observed that (i) the Cra factor binds to and activates transcription from a site centered at position −76.5 within the
icd
P2 promoter region and (ii) three particular mutations in the C-terminal end of the α subunit of RNA polymerase (L262A, R265A, and N268A) considerably diminish transcription initiating from the
icd
P2 promoter, as shown by in vitro experiments performed in the presence of mutant RNA polymerases carrying Ala substitutions.
Effect of
wzx
(
rfbX
) Mutations on A-Band and B-Band Lipopolysaccharide Biosynthesis in
Pseudomonas aeruginosa
O5
Authors: Burrows Lori L. , Lam Joseph S. 
Published: December 2019

ABSTRACT

The
wbp
cluster of
Pseudomonas aeruginosa
O5 encodes a number of proteins involved in biosynthesis of the heteropolymeric and Wzy-dependent B-band O antigen, including Wzy, the O-antigen polymerase, and Wzz, the regulator of O-antigen chain length. A gene (formerly
wbpF
), contiguous with
wzy
in the
wbp
cluster, is predicted to encode a highly hydrophobic protein with multiple membrane-spanning domains. This secondary structure is consistent with that of Wzx (RfbX), the putative O-antigen unit translocase or “flippase.” Insertion of a Gm
r
cassette at two separate sites within the putative
wzx
gene led in both cases to the loss of B-band lipopolysaccharide (LPS) O-antigen production. To our knowledge, this is the first report of the successful generation of chromosomal
wzx
gene replacement mutations. Surprisingly, inactivation of
wzx
also led to a marked delay in production of the ATP-binding cassette–transporter-dependent,
d
-rhamnose homopolymer, A-band LPS. This effect on A-band LPS synthesis was alleviated by supplying multiple copies of WbpL in
trans
. WbpL, a WecA (Rfe) homologue, was shown recently to be essential for the initiation of both A-band and B-band LPS synthesis in
P. aeruginosa
O5 (H. L. Rocchetta, L. L. Burrows, J. C. Pacan, and J. S. Lam, Mol. Microbiol. 28:1103–1119, 1998). These results suggest that the delay in A-band LPS production may arise from insufficient access to WbpL when the completed B-band O unit is not successfully translocated to the periplasm. Without adequate WbpL, A-band LPS synthesis is delayed. A subset of
wzx
mutants appeared to have accumulated second-site mutations which either restored the normal expression of A-band LPS or abolished A-band expression completely. Complementation studies showed that all of the additional mutations affecting LPS synthesis that were characterized in this study were located within the B-band LPS genes.
Cell Density-Dependent Starvation Survival of
Rhizobium leguminosarum
bv. phaseoli: Identification of the Role of an N- Acyl Homoserine Lactone in Adaptation to Stationary-Phase Survival
Authors: Thorne Stephen H. , Williams Huw D. 
Published: December 2019

ABSTRACT

The cell density dependence of stationary-phase survival of
Rhizobium leguminosarum
has been investigated. Following starvation by exhaustion of carbon or nitrogen, but not of phosphorus, the survival of cultures was dependent on the cell density at entry into stationary phase. High-density cultures survived with little or no loss of viability over a 20-day period in stationary phase. In contrast, low-density cultures lost viability rapidly but consisted of a heterogeneous population, a small fraction of which successfully adapted and eventually formed a stable, surviving population. The threshold density above which the cultures survived successfully in stationary phase was dependent on the growth conditions and the strain used. We took advantage of the fact that
R. leguminosarum
survives poorly following starvation by resuspension in carbon-free medium to demonstrate that cell density-dependent survival was mediated by a component accumulating in the growth medium. The effects of this medium component on survival in resuspension assays could be mimicked by an
N
-acyl homoserine lactone,
N
-(3
R
-hydroxy-7-
cis
-tetradecanoyl)-
l
-homoserine lactone, previously demonstrated to have a role in controlling cell density-dependent phenomena in
R. leguminosarum
. The Sym plasmids pRP2JI and pRL1JI were found to be essential for the production of the extracellular factor, which could also be made in
Escherichia coli
carrying the cosmid clone pIJ1086 containing a specific region of pRL1JI.
The Signal Transduction Protein GlnK Is Required for NifL-Dependent Nitrogen Control of
nif
Gene Expression in
Klebsiella pneumoniae
Authors: Jack Rachael , De Zamaroczy Miklos , Merrick Mike 
Published: December 2019

ABSTRACT

In
Klebsiella pneumoniae
, transcription of the nitrogen fixation (
nif
) genes is regulated in response to molecular oxygen or availability of fixed nitrogen by the coordinated activities of the
nifA
and
nifL
gene products. NifA is a
nif
-specific transcriptional activator, the activity of which is inhibited by interaction with NifL. Nitrogen control of NifL occurs at two levels: transcription of the
nifLA
operon is regulated by the global
ntr
system, and the inhibitory activity of NifL is controlled in response to fixed nitrogen by an unknown factor.
K. pneumoniae
synthesizes two P
II
-like signal transduction proteins, GlnB, which we have previously shown not to be involved in the response of NifL to fixed nitrogen, and the recently identified protein GlnK. We have now cloned the
K. pneumoniae glnK
gene, studied its expression, and shown that a null mutation in
glnK
prevents NifL from responding to the absence of fixed nitrogen, i.e., from relieving the inhibition of NifA activity. Hence, GlnK appears to be involved, directly or indirectly, in NifL-dependent regulation of
nif
gene expression in
K. pneumoniae
. Comparison of the GlnB and GlnK amino acid sequences from six species of proteobacteria identifies five residues (residues 3, 5, 52, 54, and 64) which serve to distinguish the GlnB and GlnK proteins.
An Unusual Oxygen-Sensitive, Iron- and Zinc-Containing Alcohol Dehydrogenase from the Hyperthermophilic Archaeon
Pyrococcus furiosus
Authors: Ma Kesen , Adams Michael W. W. 
Published: December 2019

ABSTRACT

Pyrococcus furiosus
is a hyperthermophilic archaeon that grows optimally at 100°C by the fermentation of peptides and carbohydrates to produce acetate, CO
2
, and H
2
, together with minor amounts of ethanol. The organism also generates H
2
S in the presence of elemental sulfur (S
0
). Cell extracts contained NADP-dependent alcohol dehydrogenase activity (0.2 to 0.5 U/mg) with ethanol as the substrate, the specific activity of which was comparable in cells grown with and without S
0
. The enzyme was purified by multistep column chromatography. It has a subunit molecular weight of 48,000 ± 1,000, appears to be a homohexamer, and contains iron (∼1.0 g-atom/subunit) and zinc (∼1.0 g-atom/subunit) as determined by chemical analysis and plasma emission spectroscopy. Neither other metals nor acid-labile sulfur was detected. Analysis using electron paramagnetic resonance spectroscopy indicated that the iron was present as low-spin Fe(II). The enzyme is oxygen sensitive and has a half-life in air of about 1 h at 23°C. It is stable under anaerobic conditions even at high temperature, with half-lives at 85 and 95°C of 160 and 7 h, respectively. The optimum pH for ethanol oxidation was between 9.4 and 10.2 (at 80°C), and the apparent

K
m

s (at 80°C) for ethanol, acetaldehyde, NADP, and NAD were 29.4, 0.17, 0.071, and 20 mM, respectively.
P. furiosus
alcohol dehydrogenase utilizes a range of alcohols and aldehydes, including ethanol, 2-phenylethanol, tryptophol, 1,3-propanediol, acetaldehyde, phenylacetaldehyde, and methyl glyoxal. Kinetic analyses indicated a marked preference for catalyzing aldehyde reduction with NADPH as the electron donor. Accordingly, the proposed physiological role of this unusual alcohol dehydrogenase is in the production of alcohols. This reaction simultaneously disposes of excess reducing equivalents and removes toxic aldehydes, both of which are products of fermentation.
Purification and Molecular Characterization of the Tungsten-Containing Formaldehyde Ferredoxin Oxidoreductase from the Hyperthermophilic Archaeon
Pyrococcus furiosus
: the Third of a Putative Five-Member Tungstoenzyme Family
Authors: Roy Roopali , Mukund Swarnalatha , Schut Gerrit J. , Dunn Dianne M. , Weiss Robert , Adams Michael W. W. 
Published: December 2019

ABSTRACT

Pyrococcus furiosus
is a hyperthermophilic archaeon which grows optimally near 100°C by fermenting peptides and sugars to produce organic acids, CO
2
, and H
2
. Its growth requires tungsten, and two different tungsten-containing enzymes, aldehyde ferredoxin oxidoreductase (AOR) and glyceraldehyde-3-phosphate ferredoxin oxidoreductase (GAPOR), have been previously purified from
P. furiosus
. These two enzymes are thought to function in the metabolism of peptides and carbohydrates, respectively. A third type of tungsten-containing enzyme, formaldehyde ferredoxin oxidoreductase (FOR), has now been characterized. FOR is a homotetramer with a mass of 280 kDa and contains approximately 1 W atom, 4 Fe atoms, and 1 Ca atom per subunit, together with a pterin cofactor. The low recovery of FOR activity during purification was attributed to loss of sulfide, since the purified enzyme was activated up to fivefold by treatment with sulfide (HS
−
) under reducing conditions. FOR uses
P. furiosus
ferredoxin as an electron acceptor (

K
m

= 100 μM) and oxidizes a range of aldehydes. Formaldehyde (

K
m

= 15 mM for the sulfide-activated enzyme) was used in routine assays, but the physiological substrate is thought to be an aliphatic C
5
semi- or dialdehyde, e.g., glutaric dialdehyde (

K
m

= 1 mM). Based on its amino-terminal sequence, the gene encoding FOR (
for
) was identified in the genomic database, together with those encoding AOR and GAPOR. The amino acid sequence of FOR corresponded to a mass of 68.7 kDa and is highly similar to those of the subunits of AOR (61% similarity and 40% identity) and GAPOR (50% similarity and 23% identity). The three genes are not linked on the
P. furiosus
chromosome. Two additional (and nonlinked) genes (termed
wor4
and
wor5
) that encode putative tungstoenzymes with 57% (WOR4) and 56% (WOR5) sequence similarity to FOR were also identified. Based on sequence motif similarities with FOR, both WOR4 and WOR5 are also proposed to contain a tungstobispterin site and one [4Fe-4S] cluster per subunit.
Active Efflux and Diffusion Are Involved in Transport of
Pseudomonas aeruginosa
Cell-to-Cell Signals
Authors: Pearson James P. , Van Delden Christian , Iglewski Barbara H. 
Published: December 2019

ABSTRACT

Many gram-negative bacteria communicate by
N
-acyl homoserine lactone signals called autoinducers (AIs). In
Pseudomonas aeruginosa
, cell-to-cell signaling controls expression of extracellular virulence factors, the type II secretion apparatus, a stationary-phase sigma factor (ς
s
), and biofilm differentiation. The fact that a similar signal,
N
-(3-oxohexanoyl) homoserine lactone, freely diffuses through
Vibrio fischeri
and
Escherichia coli
cells has led to the assumption that all AIs are freely diffusible. In this work, transport of the two
P. aeruginosa
AIs,
N
-(3-oxododecanoyl) homoserine lactone (3OC
12
-HSL) (formerly called PAI-1) and
N
-butyryl homoserine lactone (C
4
-HSL) (formerly called PAI-2), was studied by using tritium-labeled signals. When [
3
H]C
4
-HSL was added to cell suspensions of
P. aeruginosa
, the cellular concentration reached a steady state in less than 30 s and was nearly equal to the external concentration, as expected for a freely diffusible compound. In contrast, [
3
H]3OC
12
-HSL required about 5 min to reach a steady state, and the cellular concentration was 3 times higher than the external level. Addition of inhibitors of the cytoplasmic membrane proton gradient, such as azide, led to a strong increase in cellular accumulation of [
3
H]3OC
12
-HSL, suggesting the involvement of active efflux. A defined mutant lacking the
mexA-mexB-oprM
-encoded active-efflux pump accumulated [
3
H]3OC
12
-HSL to levels similar to those in the azide-treated wild-type cells. Efflux experiments confirmed these observations. Our results show that in contrast to the case for C
4
-HSL,
P. aeruginosa
cells are not freely permeable to 3OC
12
-HSL. Instead, the
mexA-mexB-oprM
-encoded efflux pump is involved in active efflux of 3OC
12
-HSL. Apparently the length and/or degree of substitution of the
N
-acyl side chain determines whether an AI is freely diffusible or is subject to active efflux by
P. aeruginosa
.
Inhibition of Homologous Recombination by the Plasmid MucA′B Complex
Authors: Venderbure Claude , Chastanet Arnaud , Boudsocq François , Sommer Suzanne , Bailone Adriana 
Published: December 2019

ABSTRACT

By its functional interaction with a RecA polymer, the mutagenic UmuD′C complex possesses an antirecombination activity. We show here that MucA′B, a functional homolog of the UmuD′C complex, inhibits homologous recombination as well. In F
−
recipients expressing MucA′B from a P

tac

promoter, Hfr × F
−
recombination decreased with increasing MucA′B concentrations down to 50-fold. In damage-induced pKM101-containing cells expressing MucA′B from the native promoter, recombination between a UV-damaged F
lac
plasmid and homologous chromosomal DNA decreased 10-fold. Overexpression of MucA′B together with UmuD′C resulted in a synergistic inhibition of recombination. RecA[UmuR] proteins, which are resistant to UmuD′C inhibition of recombination, are inhibited by MucA′B while promoting MucA′B-promoted mutagenesis efficiently. The data suggest that MucA′B and UmuD′C contact a RecA polymer at distinct sites. The MucA′B complex was more active than UmuD′C in promoting UV mutagenesis, yet it did not inhibit recombination more than UmuD′C does. The enhanced mutagenic potential of MucA′B may result from its inherent superior capacity to assist DNA polymerase in
trans
-lesion synthesis. In the course of this work, we found that the natural plasmid pKM101 expresses around 45,000 MucA and 13,000 MucB molecules per
lexA
(Def) cell devoid of LexA. These molecular Muc concentrations are far above those of the chromosomally encoded Umu counterparts. Plasmid pKM101 belongs to a family of broad-host-range conjugative plasmids. The elevated levels of the Muc proteins might be required for successful installation of pKM101-like plasmids into a variety of host cells.
Conversion of 3-Chlorocatechol by Various Catechol 2,3-Dioxygenases and Sequence Analysis of the Chlorocatechol Dioxygenase Region of
Pseudomonas putida
GJ31
Authors: Mars Astrid E. , Kingma Jaap , Kaschabek Stefan R. , Reineke Walter , Janssen Dick B. 
Published: December 2019

ABSTRACT

Pseudomonas putida
GJ31 contains an unusual catechol 2,3-dioxygenase that converts 3-chlorocatechol and 3-methylcatechol, which enables the organism to use both chloroaromatics and methylaromatics for growth. A 3.1-kb region of genomic DNA of strain GJ31 containing the gene for this chlorocatechol 2,3-dioxygenase (
cbzE
) was cloned and sequenced. The
cbzE
gene appeared to be plasmid localized and was found in a region that also harbors genes encoding a transposase, a ferredoxin that was homologous to XylT, an open reading frame with similarity to a protein of a
meta
-cleavage pathway with unknown function, and a 2-hydroxymuconic semialdehyde dehydrogenase. CbzE was most similar to catechol 2,3-dioxygenases of the 2.C subfamily of type 1 extradiol dioxygenases (L. D. Eltis and J. T. Bolin, J. Bacteriol. 178:5930–5937, 1996). The substrate range and turnover capacity with 3-chlorocatechol were determined for CbzE and four related catechol 2,3-dioxygenases. The results showed that CbzE was the only enzyme that could productively convert 3-chlorocatechol. Besides, CbzE was less susceptible to inactivation by methylated catechols. Hybrid enzymes that were made of CzbE and the catechol 2,3-dioxygenase of
P. putida
UCC2 (TdnC) showed that the resistance of CbzE to suicide inactivation and its substrate specificity were mainly determined by the C-terminal region of the protein.
The Lethal Effect of a Benzamide Derivative, 3-Methoxybenzamide, Can Be Suppressed by Mutations within a Cell Division Gene,
ftsZ
, in
Bacillus subtilis
Authors: Ohashi Yoshiaki , Chijiiwa Yoshie , Suzuki Koichiro , Takahashi Kouki , Nanamiya Hideaki , Sato Tsutomu , Hosoya Yoshiko , Ochi Kozo , Kawamura Fujio 
Published: December 2019

ABSTRACT

3-Methoxybenzamide (3-MBA), which is known to be an inhibitor of ADP-ribosyltransferase, inhibits cell division in
Bacillus subtilis
, leading to filamentation and eventually lysis of cells. Our genetic analysis of 3-MBA-resistant mutants indicated that the primary target of the drug is the cell division system involving FtsZ function during both vegetative growth and sporulation.
Components of the
Salmonella
Flagellar Export Apparatus and Classification of Export Substrates
Authors: Minamino Tohru , Macnab Robert M. 
Published: December 2019

ABSTRACT

Until now, identification of components of the flagellar protein export apparatus has been indirect. We have now identified these components directly by establishing whether mutants defective in putative export components could translocate export substrates across the cytoplasmic membrane into the periplasmic space. Hook-type proteins could be exported to the periplasm of rod mutants, indicating that rod protein export does not have to precede hook-type protein export and therefore that both types of proteins belong to a single export class, the rod/hook-type class, which is distinct from the filament-type class. Hook-capping protein (FlgD) and hook protein (FlgE) required FlhA, FlhB, FliH, FliI, FliO, FliP, FliQ, and FliR for their export to the periplasm. In the case of flagellin as an export substrate, because of the phenomenon of hook-to-filament switching of export specificity, it was necessary to use temperature-sensitive mutants and establish whether flagellin could be exported to the cell exterior following a shift from the permissive to the restrictive temperature. Again, FlhA, FlhB, FliH, FliI, and FliO were required for its export. No suitable temperature-sensitive
fliQ
or
fliR
mutants were available. FliP appeared not to be required for flagellin export, but we suspect that the temperature-sensitive FliP protein continued to function at the restrictive temperature if incorporated at the permissive temperature. Thus, we conclude that these eight proteins are general components of the flagellar export pathway. FliJ was necessary for export of hook-type proteins (FlgD and FlgE); we were unable to test whether FliJ is needed for export of filament-type proteins. We suspect that FliJ may be a cytoplasmic chaperone for the hook-type proteins and possibly also for FliE and the rod proteins. FlgJ was not required for the export of the hook-type proteins; again, because of lack of a suitable temperature-sensitive mutant, we were unable to test whether it was required for export of filament-type proteins. Finally, it was established that there is an interaction between the processes of outer ring assembly and of penetration of the outer membrane by the rod and nascent hook, the latter process being of course necessary for passage of export substrates into the external medium. During the brief transition stage from completion of rod assembly and initiation of hook assembly, the L ring and perhaps the capping protein FlgD can be regarded as bona fide export components, with the L ring being in a formal sense the equivalent of the outer membrane secretin structure of type III virulence factor export systems.
An Lrp-Like Protein of the Hyperthermophilic Archaeon
Sulfolobus solfataricus
Which Binds to Its Own Promoter
Authors: Napoli Alessandra , van der Oost John , Sensen Christoph W. , Charlebois Robert L. , Rossi Mosé , Ciaramella Maria 
Published: December 2019

ABSTRACT

Regulation of gene expression in the domain
Archaea
, and specifically hyperthermophiles, has been poorly investigated so far. Biochemical experiments and genome sequencing have shown that, despite the prokaryotic cell and genome organization, basal transcriptional elements of members of the domain
Archaea
(i.e., TATA box-like sequences, RNA polymerase, and transcription factors TBP, TFIIB, and TFIIS) are of the eukaryotic type. However, open reading frames potentially coding for bacterium-type transcription regulation factors have been recognized in different archaeal strains. This finding raises the question of how bacterial and eukaryotic elements interact in regulating gene expression in
Archaea
. We have identified a gene coding for a bacterium-type transcription factor in the hyperthermophilic archaeon
Sulfolobus solfataricus
. The protein, named Lrs14, contains a potential helix-turn-helix motif and is related to the Lrp-AsnC family of regulators of gene expression in the class
Bacteria
. We show that Lrs14, expressed in
Escherichia coli
, is a highly thermostable DNA-binding protein. Bandshift and DNase I footprint analyses show that Lrs14 specifically binds to multiple sequences in its own promoter and that the region of binding overlaps the TATA box, suggesting that, like the
E. coli
Lrp, Lrs14 is autoregulated. We also show that the
lrs14
transcript is accumulated in the late growth stages of
S. solfataricus
.