bluuebunny/crossref_metadata_2025_split huggingface dataset:
Sequence and Function of LuxU: a Two-Component Phosphorelay Protein That Regulates Quorum Sensing in
Vibrio harveyi
Authors: Freeman Jeremy A. , Bassler Bonnie L. 
Published: December 2019

ABSTRACT

Vibrio harveyi
regulates the expression of bioluminescence (
lux
) in response to cell density, a phenomenon known as quorum sensing. In
V. harveyi
, two independent quorum-sensing systems exist, and each produces, detects, and responds to a specific cell density-dependent autoinducer signal. The autoinducers are recognized by two-component hybrid sensor kinases called LuxN and LuxQ, and sensory information from both systems is transduced by a phosphorelay mechanism to the response regulator protein LuxO. Genetic evidence suggests that LuxO-phosphate negatively regulates the expression of luminescence at low cell density in the absence of autoinducers. At high cell density, interaction of the sensors with their cognate autoinducers results in dephosphorylation and inactivation of the LuxO repressor. In the present report, we show that LuxN and LuxQ channel sensory information to LuxO via a newly identified phosphorelay protein that we have named LuxU. LuxU shows sequence similarity to other described phosphorelay proteins, including BvgS, ArcB, and Ypd1. A critical His residue (His 58) of LuxU is required for phosphorelay function.
Expression and Study of Recombinant ExoM, a β1-4 Glucosyltransferase Involved in Succinoglycan Biosynthesis in
Sinorhizobium meliloti
Authors: Lellouch Annemarie C. , Geremia Roberto A. 
Published: December 2019

ABSTRACT

Here we report on the overexpression and in vitro characterization of a recombinant form of ExoM, a putative β1-4 glucosyltransferase involved in the assembly of the octasaccharide repeating subunit of succinoglycan from
Sinorhizobium meliloti
. The open reading frame
exoM
was isolated by PCR and subcloned into the expression vector pET29b, allowing inducible expression under the control of the T7 promoter.
Escherichia coli
BL21(DE3)/pLysS containing
exoM
expressed a novel 38-kDa protein corresponding to ExoM in N-terminal fusion with the S-tag peptide. Cell fractionation studies showed that the protein is expressed in
E. coli
as a membrane-bound protein in agreement with the presence of a predicted C-terminal transmembrane region.
E. coli
membrane preparations containing ExoM were shown to be capable of transferring glucose from UDP-glucose to glycolipid extracts from an
S. meliloti
mutant strain which accumulates the ExoM substrate (Glcβ1-4Glcβ1-3Gal-pyrophosphate-polyprenol). Thin-layer chromatography of the glycosidic portion of the ExoM product showed that the oligosaccharide formed comigrates with an authentic standard. The oligosaccharide produced by the recombinant ExoM, but not the starting substrate, was sensitive to cleavage with a specific cellobiohydrolase, consistent with the formation of a β1-4 glucosidic linkage. No evidence for the transfer of multiple glucose residues to the glycolipid substrate was observed. It was also found that ExoM does not transfer glucose to an acceptor substrate that has been hydrolyzed from the polyprenol anchor. Furthermore, neither glucose, cellobiose, nor the trisaccharide Glcβ1-4Glcβ1-3Glc inhibited the transferase activity, suggesting that some feature of the lipid anchor is necessary for activity.
The
Enterococcus faecalis pyr
Operon Is Regulated by Autogenous Transcriptional Attenuation at a Single Site in the 5′ Leader
Authors: Ghim Sa-Youl , Kim Charles C. , Bonner Eric R. , D’Elia John N. , Grabner Gail K. , Switzer Robert L. 
Published: December 2019

ABSTRACT

The 5′ end of the
Enterococcus faecalis pyr
operon specifies, in order, the promoter, a 5′ untranslated leader, the
pyrR
gene encoding the regulatory protein for the operon, a 39-nucleotide (nt) intercistronic region, the
pyrP
gene encoding a uracil permease, a 13-nt intercistronic region, and the
pyrB
gene encoding aspartate transcarbamylase. The 5′ leader RNA is capable of forming stem-loop structures involved in attenuation control of the operon. No attenuation regions, such as those found in the
Bacillus subtilis pyr
operon, are present in the
pyrR-pyrP
or
pyrP-pyrB
intercistronic regions. Several lines of evidence demonstrate that the
E. faecalis pyr
operon is repressed by uracil via transcriptional attenuation at the single 5′ leader termination site and that attenuation is mediated by the PyrR protein.
Cell Surface Galactosylation Is Essential for Nonsexual Flocculation in
Schizosaccharomyces pombe
Authors: Tanaka Naotaka , Awai Atsuro , Bhuiyan M. Shah Alam , Fujita Kiyotaka , Fukui Hiroshi , Takegawa Kaoru 
Published: December 2019

ABSTRACT

We have isolated fission yeast mutants that constitutively flocculate upon growth in liquid media. One of these mutants, the
gsf1
mutant, was found to cause dominant, nonsexual, and calcium-dependent aggregation of cells into flocs. Its flocculation was inhibited by the addition of galactose but was not affected by the addition of mannose or glucose, unlike
Saccharomyces cerevisiae FLO
mutants. The
gsf1
mutant coflocculated with
Schizosaccharomyces pombe
wild-type cells, while no coflocculation was found with galactose-deficient (
gms1
Δ) cells. Moreover, flocculation of the
gsf1
mutant was also inhibited by addition of cell wall galactomannan from wild-type cells but not from
gms1
Δ cells. These results suggested that galactose residues in the cell wall glycoproteins may be receptors of
gsf1
-mediated flocculation, and therefore cell surface galactosylation is required for nonsexual flocculation in
S. pombe
.
Regulated Exopolysaccharide Production in
Myxococcus xanthus
Authors: Kim Sang-Hoon , Ramaswamy Srinivas , Downard John 
Published: December 2019

ABSTRACT

Myxococcus xanthus
fibrils are cell surface-associated structures composed of roughly equal amounts of polysaccharide and protein. The level of
M. xanthus
polysaccharide production under different conditions in the wild type and in several mutants known to have alterations in fibril production was investigated. Wild-type exopolysaccharide increased significantly as cells entered the stationary phase of growth or upon addition of Ca
2+
to growing cells, and the polysaccharide-induced cells exhibited an enhanced capacity for cell-cell agglutination. The activity of the key gluconeogenic pathway enzyme phosphoenolpyruvate carboxykinase (Pck) also increased under these conditions. Most fibril-deficient mutants failed to produce polysaccharide in a stationary-phase- or Ca
2+
-dependent fashion. However, regulation of Pck activity was generally unimpaired in these mutant strains. In an
stk
mutant, which overproduces fibrils, polysaccharide production and Pck activity were constitutively high under the conditions tested. Polysaccharide production increased in most fibril-deficient strains when an
stk
mutant allele was present, indicating that these fibril-deficient mutants retained the basic cellular components required for fibril polysaccharide production. In contrast to other divalent cations tested, Sr
2+
effectively replaced Ca
2+
in stimulating polysaccharide production, and either Ca
2+
or Sr
2+
was required for fruiting-body formation by wild-type cells. By using transmission electron microscopy of freeze-substituted log-phase wild-type cells, fibril material was observed as a cell surface-associated layer of uniform thickness composed of filaments with an ordered structure.
The
Escherichia coli
Ada Protein Can Interact with Two Distinct Determinants in the ς
70
Subunit of RNA Polymerase According to Promoter Architecture: Identification of the Target of Ada Activation at the
alkA
Promoter
Authors: Landini Paolo , Busby Stephen J. W. 
Published: December 2019

ABSTRACT

The methylated form of the Ada protein (
me
Ada) activates transcription from the
Escherichia coli ada
,
aidB
, and
alkA
promoters with different mechanisms. In this study we identify amino acid substitutions in region 4 of the RNA polymerase subunit ς
70
that affect Ada-activated transcription at
alkA
. Substitution to alanine of residues K593, K597, and R603 in ς
70
region 4 results in decreased Ada-dependent binding of RNA polymerase to the
alkA
promoter in vitro and impairs
alkA
transcription both in vivo and in vitro, suggesting that these residues define a determinant for
me
Ada-ς
70
interaction. In a previous study (P. Landini, J. A. Bown, M. R. Volkert, and S. J. W. Busby, J. Biol. Chem. 273:13307–13312, 1998), we showed that a set of negatively charged amino acids in ς
70
region 4 is involved in
me
Ada-ς
70
interaction at the
ada
and
aidB
promoters. However, the alanine substitutions of positively charged residues K593, K597, and R603 do not affect
me
Ada-dependent transcription at
ada
and
aidB
. Unlike the ς
70
amino acids involved in the interaction with
me
Ada at the
ada
and
aidB
promoters, K593, K597, and R603 are not conserved in ς
S
, an alternative ς subunit of RNA polymerase mainly expressed during the stationary phase of growth. While
me
Ada is able to promote transcription by the ς
S
form of RNA polymerase (Eς
S
) at
ada
and
aidB
, it fails to do so at
alkA
. We propose that
me
Ada can activate transcription at different promoters by contacting distinct determinants in ς
70
region 4 in a manner dependent on the location of the Ada binding site.
Roles of
Pseudomonas aeruginosa las
and
rhl
Quorum-Sensing Systems in Control of Twitching Motility
Authors: Glessner Alex , Smith Roger S. , Iglewski Barbara H. , Robinson Jayne B. 
Published: December 2019

ABSTRACT

Pseudomonas aeruginosa
is a ubiquitous environmental bacterium and an important human pathogen. The production of several virulence factors by
P. aeruginosa
is controlled through two quorum-sensing systems,
las
and
rhl
. We have obtained evidence that both the
las
and
rhl
quorum-sensing systems are also required for type 4 pilus-dependent twitching motility and infection by the pilus-specific phage D3112
c
ts. Mutants which lack the ability to synthesize PAI-1, PAI-2, or both autoinducers were significantly or greatly impaired in twitching motility and in susceptibility to D3112
c
ts. Twitching motility and phage susceptibility in the autoinducer-deficient mutants were partially restored by exposure to exogenous PAI-1 and PAI-2. Both twitching motility and infection by pilus-specific phage are believed to be dependent on the extension and retraction of polar type 4 pili. Western blot analysis of whole-cell lysates and enzyme-linked immunosorbent assays of intact cells were used to measure the amounts of pilin on the cell surfaces of
las
and
rhl
mutants relative to that of the wild type. It appears that PAI-2 plays a crucial role in twitching motility and phage infection by affecting the export and assembly of surface type 4 pili. The ability of
P. aeruginosa
cells to adhere to human bronchial epithelial cells was also found to be dependent on the
rhl
quorum-sensing system. Microscopic analysis of twitching motility indicated that mutants which were unable to synthesize PAI-1 were defective in the maintenance of cellular monolayers and migrating packs of cells. Thus, PAI-1 appears to have an essential role in maintaining cell-cell spacing and associations required for effective twitching motility.
Molecular Survey of the
Salmonella
Phage Typing System of Anderson
Authors: Schmieger Horst 
Published: December 2019

ABSTRACT

Typing phages for
Salmonella
and the prophages of their typical propagation strains were analyzed at the DNA level. Most of them belong to the P22 branch of the lambdoid phages. Acquisition of new plating properties of the typing phages by propagation in particular strains can be due to different host specific modifications of the DNA or to recombination events with residing prophages which are reflected by changes in the respective DNA restriction patterns. It is concluded that the actually available set of typing phages is a historically unique combination of strains.
Functional Importance and Local Environments of the Cysteines in the Tetracycline Resistance Protein Encoded by Plasmid pBR322
Authors: Jewell Jean E. , Orwick Jill , Liu Jun , Miller Kurt W. 
Published: December 2019

ABSTRACT
The properties of the cysteines in the pBR322-encoded tetracycline resistance protein have been examined. Cysteines are important but not essential for tetracycline transport activity. None of the cysteines reacted with biotin maleimide, suggesting that they are shielded from the aqueous phase or reside in a negatively charged local environment.
The Polar Flagellar Motor of
Vibrio cholerae
Is Driven by an Na
+
Motive Force
Authors: Kojima Seiji , Yamamoto Koichiro , Kawagishi Ikuro , Homma Michio 
Published: December 2019

ABSTRACT

Vibrio cholerae
is a highly motile bacterium which possesses a single polar flagellum as a locomotion organelle. Motility is thought to be an important factor for the virulence of
V. cholerae
. The genome sequencing project of this organism is in progress, and the genes that are highly homologous to the essential genes of the Na
+
-driven polar flagellar motor of
Vibrio alginolyticus
were found in the genome database of
V. cholerae
. The energy source of its flagellar motor was investigated. We examined the Na
+
dependence and the sensitivity to the Na
+
motor-specific inhibitor of the motility of the
V. cholerae
strains and present the evidence that the polar flagellar motor of
V. cholerae
is driven by an Na
+
motive force.
Construction and Analysis of Hybrid
Escherichia coli-Bacillus subtilis dnaK
Genes
Authors: Mogk Axel , Bukau Bernd , Lutz Rolf , Schumann Wolfgang 
Published: December 2019

ABSTRACT

The highly conserved DnaK chaperones consist of an N-terminal ATPase domain, a central substrate-binding domain, and a C-terminal domain whose function is not known. Since
Bacillus subtilis dnaK
was not able to complement an
Escherichia coli dnaK
null mutant, we performed domain element swap experiments to identify the regions responsible for this finding. It turned out that the
B. subtilis
DnaK protein needed approximately normal amounts of the cochaperone DnaJ to be functional in
E. coli
. The ATPase domain and the substrate-binding domain form a species-specific functional unit, while the C-terminal domains, although less conserved, are exchangeable. Deletion of the C-terminal domain in
E. coli
DnaK affected neither complementation of growth at high temperatures nor propagation of phage λ but abolished degradation of ς
32
.
A Novel Membrane Protein Influencing Cell Shape and Multicellular Swarming of
Proteus mirabilis
Authors: Hay Nicole A. , Tipper Donald J. , Gygi Daniel , Hughes Colin 
Published: December 2019

ABSTRACT

Swarming in
Proteus mirabilis
is characterized by the coordinated surface migration of multicellular rafts of highly elongated, hyperflagellated swarm cells. We describe a transposon mutant, MNS185, that was unable to swarm even though vegetative cells retained normal motility and the ability to differentiate into swarm cells. However, these elongated cells were irregularly curved and had variable diameters, suggesting that the migration defect results from the inability of these deformed swarm cells to align into multicellular rafts. The transposon was inserted at codon 196 of a 228-codon gene that lacks recognizable homologs. Multiple copies of the wild-type gene, called
ccmA
, for curved cell morphology, restored swarming to the mutant. The 25-kDa CcmA protein is predicted to span the inner membrane twice, with its C-terminal major domain being present in the cytoplasm. Membrane localization was confirmed both by immunoblotting and by electron microscopy of immunogold-labelled sections. Two forms of CcmA were identified for wild-type
P. mirabilis
; they were full-length integral membrane CcmA1 and N-terminally truncated peripheral membrane CcmA2, both present at approximately 20-fold higher concentrations in swarm cells. Differentiated MNS185 mutant cells contained wild-type levels of the C-terminally truncated versions of both proteins. Elongated cells of a
ccmA
null mutant were less misshapen than those of MNS185 and were able to swarm, albeit more slowly than wild-type cells. The truncated CcmA proteins may therefore interfere with normal morphogenesis, while the wild-type proteins, which are not essential for swarming, may enhance migration by maintaining the linearity of highly elongated cells. Consistent with this view, overexpression of the
ccmA
gene caused cells of both
Escherichia coli
and
P. mirabilis
to become enlarged and ellipsoidal.
Isolation of RNase H Genes That Are Essential for Growth of
Bacillus subtilis
168
Authors: Itaya Mitsuhiro , Omori Akira , Kanaya Shigenori , Crouch Robert J. , Tanaka Teruo , Kondo Kanae 
Published: December 2019

ABSTRACT

Two genes encoding functional RNase H (EC
3.1.26.4
) were isolated from a gram-positive bacterium,
Bacillus subtilis
168. Two DNA clones exhibiting RNase H activities both in vivo and in vitro were obtained from a
B. subtilis
DNA library. One (28.2 kDa) revealed high similarity to
Escherichia coli
RNase HII, encoded by the
rnhB
gene. The other (33.9 kDa) was designated
rnhC
and encodes
B. subtilis
RNase HIII. The
B. subtilis
genome has an
rnhA
homologue, the product of which has not yet shown RNase H activity. Analyses of all three
B. subtilis
genes revealed that
rnhB
and
rnhC
cannot be simultaneously inactivated. This observation indicated that in
B. subtilis
both the
rnhB
and
rnhC
products are involved in certain essential cellular processes that are different from those suggested by
E. coli rnh
mutation studies. Sequence conservation between the
rnhB
and
rnhC
genes implies that both originated from a single ancestral RNase H gene. The roles of bacterial RNase H may be indicated by the single
rnhC
homologue in the small genome of
Mycoplasma
species.
Inducing Effect of Diamines on Transcription of the Cephamycin C Genes from the
lat
and
pcbAB
Promoters in
Nocardia lactamdurans
Authors: Leitão Ana Lucia , Enguita Francisco J. , De La Fuente Juan Luis , Liras Paloma , Martin Juan F. 
Published: December 2019

ABSTRACT

The diamines putrescine, cadaverine, and diaminopropane stimulate cephamycin biosynthesis in
Nocardia lactamdurans
, in shake flasks and fermentors, without altering cell growth. Intracellular levels of the P7 protein (a component of the methoxylation system involved in cephamycin biosynthesis) were increased by diaminopropane, as shown by immunoblotting studies. Lysine-6-aminotransferase and piperideine-6-carboxylate dehydrogenase activities involved in biosynthesis of the α-aminoadipic acid precursor were also greatly stimulated. The diamine stimulatory effect is exerted at the transcriptional level, as shown by low-resolution S1 protection studies. The transcript corresponding to the
pcbAB
gene and to a lesser extent also the
lat
transcript were significantly increased in diaminopropane-supplemented cultures, whereas transcription from the
cefD
promoter was not affected. Coupling of the
lat
and
pcbAB
promoters to the reporter
xylE
gene showed that expression from the
lat
and
pcbAB
promoters was increased by addition of diaminopropane in
Streptomyces lividans
. Intracellular accumulation of diamines in
Nocardia
may be a signal to trigger antibiotic production.
Utilization of Electrically Reduced Neutral Red by
Actinobacillus succinogenes
: Physiological Function of Neutral Red in Membrane-Driven Fumarate Reduction and Energy Conservation
Authors: Park D. H. , Zeikus J. G. 
Published: December 2019

ABSTRACT

Neutral red (NR) functioned as an electronophore or electron channel enabling either cells or membranes purified from
Actinobacillus succinogenes
to drive electron transfer and proton translocation by coupling fumarate reduction to succinate production. Electrically reduced NR, unlike methyl or benzyl viologen, bound to cell membranes, was not toxic, and chemically reduced NAD. The cell membrane of
A. succinogenes
contained high levels of benzyl viologen-linked hydrogenase (12.2 U), fumarate reductase (13.1 U), and diaphorase (109.7 U) activities. Fumarate reductase (24.5 U) displayed the highest activity with NR as the electron carrier, whereas hydrogenase (1.1 U) and diaphorase (0.8 U) did not. Proton translocation by whole cells was dependent on either electrically reduced NR or H
2
as the electron donor and on the fumarate concentration. During the growth of
Actinobacillus
on glucose plus electrically reduced NR in an electrochemical bioreactor system versus on glucose alone, electrically reduced NR enhanced glucose consumption, growth, and succinate production by about 20% while it decreased acetate production by about 50%. The rate of fumarate reduction to succinate by purified membranes was twofold higher with electrically reduced NR than with hydrogen as the electron donor. The addition of 2-(
n
-heptyl)-4-hydroxyquinoline
N
-oxide to whole cells or purified membranes inhibited succinate production from H
2
plus fumarate but not from electrically reduced NR plus fumarate. Thus, NR appears to replace the function of menaquinone in the fumarate reductase complex, and it enables
A. succinogenes
to utilize electricity as a significant source of metabolic reducing power.
Cloning of
fibA
, Encoding an Immunogenic Subunit of the Fibril-Like Surface Structure of
Peptostreptococcus micros
Authors: Kremer B. H. A. , Bijlsma J. J. E. , Kusters J. G. , de Graaff J. , van Steenbergen T. J. M. 
Published: December 2019

ABSTRACT

Although we are currently unaware of its biological function, the fibril-like surface structure is a prominent characteristic of the rough (Rg) genotype of the gram-positive periodontal pathogen
Peptostreptococcus micros
. The smooth (Sm) type of this species as well as the smooth variant of the Rg type (Rg
Sm
) lack these structures on their surface. A fibril-specific serum, as determined by immunogold electron microscopy, was obtained through adsorption of a rabbit anti-Rg type serum with excess bacteria of the Rg
Sm
type. This serum recognized a 42-kDa protein, which was subjected to N-terminal sequencing. Both clones of a λTriplEx expression library that were selected by immunoscreening with the fibril-specific serum contained an open reading frame, designated
fibA
, encoding a 393-amino-acid protein (FibA). The 15-residue N-terminal amino acid sequence of the 42-kDa antigen was present at positions 39 to 53 in FibA; from this we conclude that the mature FibA protein contains 355 amino acids, resulting in a predicted molecular mass of 41,368 Da. The putative 38-residue signal sequence of FibA strongly resembles other gram-positive secretion signal sequences. The C termini of FibA and two open reading frames directly upstream and downstream of
fibA
exhibited significant sequence homology to the C termini of a group of secreted and surface-located proteins of other gram-positive cocci that are all presumably involved in anchoring of the protein to carbohydrate structures. We conclude that FibA is a secreted and surface-located protein and as such is part of the fibril-like structures.