bluuebunny/crossref_metadata_2025_split huggingface dataset:
Natural Transformation of
Campylobacter jejuni
Requires Components of a Type II Secretion System
Authors: Wiesner Rebecca S. , Hendrixson David R. , DiRita Victor J. 
Published: August 2003

ABSTRACT

The human pathogen
Campylobacter jejuni
is one of more than 40 naturally competent bacterial species able to import macromolecular DNA from the environment and incorporate it into their genomes. However, in
C. jejuni
little is known about the genes involved in this process. We used random transposon mutagenesis to identify genes that are required for the transformation of this organism. We isolated mutants with insertions in 11 different genes; most of the mutants are affected in the DNA uptake stage of transformation, whereas two mutants are affected in steps subsequent to DNA uptake, such as recombination into the chromosome or in DNA transport across the inner membrane. Several of these genes encode proteins homologous to those involved in type II secretion systems, biogenesis of type IV pili, and competence for natural transformation in gram-positive and gram-negative species. Other genes identified in our screen encode proteins unique to
C. jejuni
or are homologous to proteins that have not been shown to play a role in the transformation in other bacteria.
Genetic Loci for Coaggregation Receptor Polysaccharide Biosynthesis inStreptococcus gordonii38
Authors: Xu De-Qi , Thompson John , Cisar John O. 
Published: August 2003

ABSTRACTThe cell wall polysaccharide ofStreptococcus gordonii38 functions as a coaggregation receptor for surface adhesins on other members of the oral biofilm community. The structure of this receptor polysaccharide (RPS) is defined by a heptasaccharide repeat that includes a GalNAcβ1→3Gal-containing recognition motif. The same RPS has now been identified fromS. gordoniiAT, a partially sequenced strain. PCR primers designed from sequences in the genomic database of strain AT were used to identify and partially characterize theS. gordonii38 RPS gene cluster. This cluster includes genes for seven putative glycosyltransferases, a polysaccharide polymerase (Wzy), an oligosaccharide repeating unit transporter (Wzx), and a galactofuranose mutase, the enzyme that promotes synthesis of UDP-Galf, one of five predicted RPS precursors. Genes outside this region were identified for the other four nucleotide-linked sugar precursors of RPS biosynthesis, namely, those for formation of UDP-Glc, UDP-Gal, UDP-GalNAc, and dTDP-Rha. Two genes for putative galactose 4-epimerases were identified. The first, designatedgalE1, was identified as a pseudogene in the galactose operon, and the second, designatedgalE2, was transcribed with three of the four genes for dTDP-Rha biosynthesis (i.e.,rmlA,rmlC, andrmlB). Insertional inactivation ofgalE2abolished (i) RPS production, (ii) growth on galactose, and (iii) both UDP-Gal and UDP-GalNAc 4-epimerase activities in cell extracts. Repair of thegalE1pseudogene in thisgalE2mutant restored growth on galactose but not RPS production. Cell extracts containing functional GalE1 but not GalE2 contained UDP-Gal 4-epimerase but not UDP-GalNAc 4-epimerase activity. Thus, provision of both UDP-Gal and UDP-GalNAc for RPS production byS. gordonii38 depends on the dual specificity of the epimerase encoded bygalE2.
Multiple Regulators Control Capsular Polysaccharide Production in
Vibrio parahaemolyticus
Authors: Güvener Zehra Tüzün , McCarter Linda L. 
Published: August 2003

ABSTRACT

Vibrio parahaemolyticus
, a biofouling marine bacterium and human pathogen, undergoes phase variation displaying translucent (TR) and opaque (OP) colony morphologies. Prior studies demonstrated that OP colonies produce more capsular polysaccharide (CPS) than TR colonies and that opacity is controlled by the
Vibrio harveyi
LuxR-type transcriptional activator OpaR. CPS has also been shown to be regulated by the
scrABC
signaling pathway, which involves a GGDEF-EAL motif-containing sensory protein. The present study identifies
cps
genes and examines their regulation. Transposon insertions in the
cps
locus, which contains 11 genes, abolished opacity. Such mutants failed to produce CPS and were defective in pellicle formation in microtiter wells and in a biofilm attachment assay. Reporter fusions to
cpsA
, the first gene in the locus, showed ∼10-fold-enhanced transcription in the OP (
opaR
+
) strain compared to a TR (Δ
opaR
) strain. Two additional transcriptional regulators were discovered. One potential activator, CpsR, participates in the
scrABC
GGDEF-EAL-signaling pathway; CpsR was required for the increased
cps
expression observed in
scrA
Δ
opaR
strains. CpsR, which contains a conserved module found in members of the AAA+ superfamily of ATP-interacting proteins, is homologous to
Vibrio cholerae
VpsR; however, unlike VpsR, CpsR was not essential for
cps
expression. CpsS, the second newly identified regulator, contains a CsgD-type DNA-binding domain and appears to act as a repressor. Mutants with
cpsS
defects have greatly elevated
cps
transcription; their high level of
cpsA
expression was CpsR dependent in ΤR strains and primarily OpaR dependent in OP strains. Thus, a network of positive and negative regulators modulates CPS production in
V. parahaemolyticus
.
Global Analyses of Transcriptomes and Proteomes of a Parent Strain and an
l
-Threonine-Overproducing Mutant Strain
Authors: Lee Jin-Ho , Lee Dong-Eun , Lee Bheong-Uk , Kim Hak-Sung 
Published: August 2003

ABSTRACT

We compared the transcriptome, proteome, and nucleotide sequences between the parent strain
Escherichia coli
W3110 and the
l
-threonine-overproducing mutant
E
.
coli
TF5015. DNA macroarrays were used to measure mRNA levels for all of the genes of
E
.
coli
, and two-dimensional gel electrophoresis was used to compare protein levels. It was observed that only 54 of 4,290 genes (1.3%) exhibited differential expression profiles. Typically, genes such as
aceA
,
aceB
,
icdA
,
gltA
,
glnA
,
leu
operon,
proA
,
thrA
,
thrC
, and
yigJ
, which are involved in the glyoxylate shunt, the tricarboxylic acid cycle, and amino acid biosynthesis (
l
-glutamine,
l
-leucine, proline, and
l
-threonine), were significantly upregulated, whereas the genes
dadAX
,
hdeA
,
hdeB
,
ompF
,
oppA
,
oppB
,
oppF
,
yfiD
, and many ribosomal protein genes were downregulated in TF5015 compared to W3110. The differential expression such as upregulation of
thr
operon and expression of
yigJ
would result in an accumulation of
l
-threonine in TF5015. Furthermore, two significant mutations,
thrA345
and
ilvA97
, which are essential for overproduction of
l
-threonine, were identified in TF5015 by the sequence analysis. In particular, expression of the mutated
thrABC
(pATF92) in W3110 resulted in a significant incremental effect on
l
-threonine production. Upregulation of
aceBA
and downregulation of b1795,
hdeAB
,
oppA
, and
yfiD
seem to be linked to a low accumulation of acetate in TF5015. Such comprehensive analyses provide information regarding the regulatory mechanism of
l
-threonine production and the physiological consequences in the mutant stain.
TodK, a Putative Histidine Protein Kinase, Regulates Timing of Fruiting Body Morphogenesis inMyxococcus xanthus
Authors: Rasmussen Anders A. , Søgaard-Andersen Lotte 
Published: August 2003

ABSTRACTIn response to starvation,Myxococcus xanthusinitiates a developmental program that results in the formation of spore-filled multicellular fruiting bodies. Fruiting body formation depends on the temporal and spatial coordination of aggregation and sporulation. These two processes are induced by the cell surface-associated C signal, with aggregation being induced after 6 h and sporulation being induced once cells have completed the aggregation process. We report the identification of TodK, a putative histidine protein kinase of two-component regulatory systems that is important for the correct timing of aggregation and sporulation. Loss of TodK function results in early aggregation and early, as well as increased levels of, sporulation. Transcription oftodKdecreases 10-fold in response to starvation independently of the stringent response. Loss of TodK function specifically results in increased expression of a subset of C-signal-dependent genes. Accelerated development in atodKmutant depends on the known components in the C-signal transduction pathway. TodK is not important for synthesis of the C signal. From these results we suggest that TodK is part of a signal transduction system which converges on the C-signal transduction pathway to negatively regulate aggregation, sporulation, and the expression of a subset of C-signal-dependent genes. TodK and the SdeK histidine protein kinase, which is part of a signal transduction system that converges on the C-signal transduction pathway to stimulate aggregation, sporulation, and C-signal-dependent gene expression, act in independent genetic pathways. We suggest that the signal transduction pathways defined by TodK and SdeK act in concert with the C-signal transduction pathway to control the timing of aggregation and sporulation.
Biochemical Characterization of a β-Galactosidase with a Low Temperature Optimum Obtained from an Antarctic
Arthrobacter
Isolate
Authors: Coker James A. , Sheridan Peter P. , Loveland-Curtze Jennifer , Gutshall Kevin R. , Auman Ann J. , Brenchley Jean E. 
Published: August 2003

ABSTRACT

A psychrophilic gram-positive isolate was obtained from Antarctic Dry Valley soil. It utilized lactose, had a rod-coccus cycle, and contained lysine as the diamino acid in its cell wall. Consistent with these physiological traits, the 16S ribosomal DNA sequence showed that it was phylogenetically related to other
Arthrobacter
species. A gene (
bgaS
) encoding a family 2 β-galactosidase was cloned from this organism into an
Escherichia coli
host. Preliminary results showed that the enzyme was cold active (optimal activity at 18°C and 50% activity remaining at 0°C) and heat labile (inactivated within 10 min at 37°C). To enable rapid purification, vectors were constructed adding histidine residues to the BgaS enzyme and its
E. coli
LacZ counterpart, which was purified for comparison. The His tag additions reduced the specific activities of both β-galactosidases but did not alter the other characteristics of the enzymes. Kinetic studies using
o
-nitrophenyl-β-
d
-galactopyranoside showed that BgaS with and without a His tag had greater catalytic activity at and below 20°C than the comparable LacZ β-galactosidases. The BgaS heat lability was investigated by ultracentrifugation, where the active enzyme was a homotetramer at 4°C but dissociated into inactive monomers at 25°C. Comparisons of family 2 β-galactosidase amino acid compositions and modeling studies with the LacZ structure did not mimic suggested trends for conferring enzyme flexibility at low temperatures, consistent with the changes affecting thermal adaptation being localized and subtle. Mutation studies of the BgaS enzyme should aid our understanding of such specific, localized changes affecting enzyme thermal properties.
Influence of Temperature on tRNA Modification in Archaea:
Methanococcoides burtonii
(Optimum Growth Temperature [
T
opt
], 23°C) and
Stetteria hydrogenophila
(
T
opt
, 95°C)
Authors: Noon Kathleen R. , Guymon Rebecca , Crain Pamela F. , McCloskey James A. , Thomm Michael , Lim Julianne , Cavicchioli Ricardo 
Published: August 2003

ABSTRACT

We report the first study of tRNA modification in psychrotolerant archaea, specifically in the archaeon
Methanococcoides burtonii
grown at 4 and 23°C. For comparison, unfractionated tRNA from the archaeal hyperthermophile
Stetteria hydrogenophila
cultured at 93°C was examined. Analysis of modified nucleosides using liquid chromatography-electrospray ionization mass spectrometry revealed striking differences in levels and identities of tRNA modifications between the two organisms. Although the modification levels in
M. burtonii
tRNA are the lowest in any organism of which we are aware, it contains more than one residue per tRNA molecule of dihydrouridine, a molecule associated with maintenance of polynucleotide flexibility at low temperatures. No differences in either identities or levels of modifications, including dihydrouridine, as a function of culture temperature were observed, in contrast to selected tRNA modifications previously reported for archaeal hyperthermophiles. By contrast,
S. hydrogenophila
tRNA was found to contain a remarkable structural diversity of 31 modified nucleosides, including nine methylated guanosines, with eight different nucleoside species methylated at O-2′ of ribose, known to be an effective stabilizing motif in RNA. These results show that some aspects of tRNA modification in archaea are strongly associated with environmental temperature and support the thesis that posttranscriptional modification is a universal natural mechanism for control of RNA molecular structure that operates across a wide temperature range in archaea as well as bacteria.
Characterization of Dominantly Negative Mutant ClyA Cytotoxin Proteins in
Escherichia coli
Authors: Wai Sun Nyunt , Westermark Marie , Oscarsson Jan , Jass Jana , Maier Elke , Benz Roland , Uhlin Bernt Eric 
Published: August 2003

ABSTRACT

We report studies of the subcellular localization of the ClyA cytotoxic protein and of mutations causing defective translocation to the periplasm in
Escherichia coli
. The ability of ClyA to translocate to the periplasm was abolished in deletion mutants lacking the last 23 or 11 amino acid residues of the C-terminal region. A naturally occurring ClyA variant lacking four residues (183 to 186) in a hydrophobic subdomain was retained mainly in the cytosolic fraction. These mutant proteins displayed an inhibiting effect on the expression of the hemolytic phenotype of wild-type ClyA. Studies in vitro with purified mutant ClyA proteins revealed that they were defective in formation of pore assemblies and that their activity in hemolysis assays and in single-channel conductance tests was at least 10-fold lower than that of the wild-type ClyA. Tests with combinations of the purified proteins indicated that mutant and wild-type ClyA interacted and that formation of heteromeric assemblies affected the pore-forming activity of the wild-type protein. The observed protein-protein interactions were consistent with, and provided a molecular explanation for, the dominant negative feature of the mutant ClyA variants.
In Vivo Reconstitution of the FhuA Transport Protein ofEscherichia coliK-12
Authors: Braun Michael , Endriss Franziska , Killmann Helmut , Braun Volkmar 
Published: August 2003

ABSTRACTThe FhuA protein in the outer membrane ofEscherichia coliactively transports ferrichrome and the antibiotics albomycin and rifamycin CGP 4832 and serves as a receptor for the phages T1, T5, and φ80 and for colicin M and microcin J25. The crystal structure reveals a β-barrel with a globular domain, the cork, which closes the channel formed by the barrel. Genetic deletion of the cork resulted in a β-barrel that displays no FhuA activity. A functional FhuA was obtained by cosynthesis of separately encoded cork and the β-barrel domain, each endowed with a signal sequence, which showed that complementation occurs after secretion of the fragments across the cytoplasmic membrane. Inactive complete mutant FhuA and an FhuA fragment containing 357 N-proximal amino acid residues complemented the separately synthesized wild-type β-barrel to form an active FhuA. Previous claims that the β-barrel is functional as transporter and receptor resulted from complementation by inactive complete FhuA and the 357-residue fragment. No complementation was observed between the wild-type cork and complete but inactive FhuA carrying cork mutations that excluded the exchange of cork domains. The data indicate that active FhuA is reconstituted extracytoplasmically by insertion of separately synthesized cork or cork from complete FhuA into the β-barrel, and they suggest that in wild-type FhuA the β-barrel is formed prior to the insertion of the cork.
Moritella
Cold-Active Dihydrofolate Reductase: Are There Natural Limits to Optimization of Catalytic Efficiency at Low Temperature?
Authors: Xu Ying , Feller Georges , Gerday Charles , Glansdorff Nicolas 
Published: August 2003

ABSTRACT

Adapting metabolic enzymes of microorganisms to low temperature environments may require a difficult compromise between velocity and affinity. We have investigated catalytic efficiency in a key metabolic enzyme (dihydrofolate reductase) of
Moritella profunda
sp. nov., a strictly psychrophilic bacterium with a maximal growth rate at 2°C or less. The enzyme is monomeric (
M
r
= 18,291), 55% identical to its
Escherichia coli
counterpart, and displays

T
m

and denaturation enthalpy changes much lower than
E. coli
and
Thermotoga maritima
homologues. Its stability curve indicates a maximum stability above the temperature range of the organism, and predicts cold denaturation below 0°C. At mesophilic temperatures the apparent

K
m

value for dihydrofolate is 50- to 80-fold higher than for
E. coli
,
Lactobacillus casei
, and
T. maritima
dihydrofolate reductases, whereas the apparent

K
m

value for NADPH, though higher, remains in the same order of magnitude. At 5°C these values are not significantly modified. The enzyme is also much less sensitive than its
E. coli
counterpart to the inhibitors methotrexate and trimethoprim. The catalytic efficiency (
k
cat

/K
m

) with respect to dihydrofolate is thus much lower than in the other three bacteria. The higher affinity for NADPH could have been maintained by selection since NADPH assists the release of the product tetrahydrofolate. Dihydrofolate reductase adaptation to low temperature thus appears to have entailed a pronounced trade-off between affinity and catalytic velocity. The kinetic features of this psychrophilic protein suggest that enzyme adaptation to low temperature may be constrained by natural limits to optimization of catalytic efficiency.
Clostridium pasteurianum
F
1
F
o
ATP Synthase: Operon, Composition, and Some Properties
Authors: Das Amaresh , Ljungdahl Lars G. 
Published: August 2003

ABSTRACT

The
atp
operon encoding F
1
F
o
ATP synthase in the fermentative obligate anaerobic bacterium
Clostridium pasteurianum
was sequenced. It consisted of nine genes arranged in the order
atpI
(i),
atpB
(a),
atpE
(c),
atpF
(b),
atpH
(δ),
atpA
(α),
atpG
(γ),
atpD
(β), and
atpC
(ε), which was identical to that found in many bacteria. Reverse transcription-PCR confirmed the presence of the transcripts of all nine genes. The amount of ATPase activity in the membranes of
C
.
pasteurianum
was low compared to what has been found in many other bacteria. The F
1
F
o
complexes solubilized from membranes of
C
.
pasteurianum
and
Escherichia coli
had similar masses, suggesting similar compositions for the F
1
F
o
complexes from the two bacteria. Western blotting experiments with antibodies raised against the purified subunits of F
1
F
o
detected the presence of eight subunits, α, β, γ, δ, ε, a, b, and c, in the F
1
F
o
complex from
C
.
pasteurianum
. The F
1
F
o
complex from
C
.
pasteurianum
was activated by thiocyanate, cyanate, or sulfhydryl compounds; inhibited by sulfite, bisulfite, or bicarbonate; and had tolerance to inhibition by dicyclohexylcarbodiimide. The target of thiol activation of the F
1
F
o
complex from
C
.
pasteurianum
was F
1
. Thiocyanate and sulfite were noncompetitive with respect to substrate Mg ATP but competitive with respect to each other. The F
1
and F
o
parts of the F
1
F
o
complexes from
C
.
pasteurianum
and
E
.
coli
bound to each other, but the hybrid F
1
F
o
complexes were not functionally active.
Interactions of FliJ with the
Salmonella
Type III Flagellar Export Apparatus
Authors: Fraser Gillian M. , González-Pedrajo Bertha , Tame Jeremy R. H. , Macnab Robert M. 
Published: August 2003

ABSTRACT

FliJ, a 17-kDa protein, is a soluble component of the
Salmonella
type III flagellar protein export system that has antiaggregation properties and several other characteristics that suggest it may have a chaperone-like function. We have now examined this protein in detail. Ten-amino-acid scanning deletions covering the entire 147-amino-acid sequence were tested for complementation of a
fliJ
null strain; only the first and last deletions complemented. A few of the deletions, especially towards the C terminus, exerted a dominant negative effect on wild-type cells, indicating that they were actively interfering with function. Two truncated versions of FliJ, representing its N- and C-terminal halves, failed to complement and were not dominant. We tested for FliJ self-association by several techniques. Size-exclusion chromatography (Superdex 200) indicated an apparent molecular mass of around 50 kDa, which could reflect either multimerization or an elongated shape or both. Multiangle light scattering gave a peak value of 20 kDa, close to the molecular mass of the monomer. Analytical ultracentrifugation gave evidence for weak self-association as a trimer or tetramer. It was known from previous studies that FliJ interacts with the N-terminal region of FliH, a negative regulator of the ATPase FliI. Using both truncation and deletion versions of FliJ, we now show that it is its C-terminal region that is responsible for this interaction. We also show that FliJ interacts with the soluble cytoplasmic domain of the largest membrane component of the export apparatus, FlhA; although small deletions in FliJ did not interfere with the association, both truncated versions failed to associate, indicating that a substantial amount of the central region of the FliJ sequence participates in the association. We present a model summarizing these multiple interactions.
Glutathione and Catalase Provide Overlapping Defenses for Protection against Respiration-Generated Hydrogen Peroxide in
Haemophilus influenzae
Authors: Vergauwen Bjorn , Pauwels Frederik , Van Beeumen Jozef J. 
Published: August 2003

ABSTRACT

Glutathione is an abundant and ubiquitous low-molecular-weight thiol that may play a role in many cellular processes, including protection against the deleterious effects of reactive oxygen species. We address here the role of glutathione in protection against hydrogen peroxide (H
2
O
2
) in
Haemophilus influenzae
and show that glutathione and catalase provide overlapping defense systems.
H. influenzae
is naturally glutathione deficient and imports glutathione from the growth medium. Mutant
H. influenzae
lacking catalase and cultured in glutathione-deficient minimal medium is completely devoid of H
2
O
2
scavenging activity and, accordingly, substantial amounts of H
2
O
2
accumulate in the growth medium.
H. influenzae
generates H
2
O
2
at rates similar to those reported for
Escherichia coli
, but the toxicity of this harmful metabolite is averted by glutathione-based H
2
O
2
removal, which appears to be the primary system for protection against H
2
O
2
endogenously generated during aerobic respiration. When H
2
O
2
concentrations exceed low micromolar levels, the
hktE
gene-encoded catalase becomes the predominant scavenger. The requirement for glutathione in protection against oxidative stress is analogous to that in higher and lower eukaryotes but is unlike the situation in other bacteria in which glutathione is dispensable for aerobic growth during both normal and oxidative stress conditions.