bluuebunny/crossref_metadata_2025_split huggingface dataset:
Two-Component System That Regulates Methanol and Formaldehyde Oxidation in
Paracoccus denitrificans
Authors: Harms N. , Reijnders W. N. M. , Koning S. , van Spanning R. J. M. 
Published: July 2002

ABSTRACT

A chromosomal region encoding a two-component regulatory system, FlhRS, has been isolated from
Paracoccus denitrificans
. FlhRS-deficient mutants were unable to grow on methanol, methylamine, or choline as the carbon and energy source. Expression of the gene encoding glutathione-dependent formaldehyde dehydrogenase (
fhlA
) was undetectable in the mutant, and expression of the
S
-formylglutathione hydrolase gene (
fghA
) was reduced in the mutant background. In addition, methanol dehydrogenase was immunologically undetectable in cell extracts of FhlRS mutants. These results indicate that the FlhRS sensor-regulator pair is involved in the regulation of formaldehyde, methanol, and methylamine oxidation. The effect that the FlhRS proteins exert on the regulation of C
1
metabolism might be essential to maintain the internal concentration of formaldehyde below toxic levels.
Urkinase: Structure of Acetate Kinase, a Member of the ASKHA Superfamily of Phosphotransferases
Authors: Buss Kathryn A. , Cooper David R. , Ingram-Smith Cheryl , Ferry James G. , Sanders David Avram , Hasson Miriam S. 
Published: July 2002

ABSTRACT

Acetate kinase, an enzyme widely distributed in the
Bacteria
and
Archaea
domains, catalyzes the phosphorylation of acetate. We have determined the three-dimensional structure of
Methanosarcina thermophila
acetate kinase bound to ADP through crystallography. As we previously predicted, acetate kinase contains a core fold that is topologically identical to that of the ADP-binding domains of glycerol kinase, hexokinase, the 70-kDa heat shock cognate (Hsc70), and actin. Numerous charged active-site residues are conserved within acetate kinases, but few are conserved within the phosphotransferase superfamily. The identity of the points of insertion of polypeptide segments into the core fold of the superfamily members indicates that the insertions existed in the common ancestor of the phosphotransferases. Another remarkable shared feature is the unusual, epsilon conformation of the residue that directly precedes a conserved glycine residue (Gly-331 in acetate kinase) that binds the α-phosphate of ADP. Structural, biochemical, and geochemical considerations indicate that an acetate kinase may be the ancestral enzyme of the ASKHA (acetate and sugar kinases/Hsc70/actin) superfamily of phosphotransferases.
In Vivo Expression from the RpoS-Dependent P1 Promoter of the Osmotically Regulated
proU
Operon in
Escherichia coli
and
Salmonella enterica
Serovar Typhimurium: Activation by
rho
and
hns
Mutations and by Cold Stress
Authors: Rajkumari K. , Gowrishankar J. 
Published: July 2002

ABSTRACT

Unlike the ς
70
-controlled P2 promoter for the osmotically regulated
proU
operon of
Escherichia coli
and
Salmonella enterica
serovar Typhimurium, the ς
s
-controlled P1 promoter situated further upstream appears not to contribute to expression of the
proU
structural genes under ordinary growth conditions. For
S
.
enterica proU
P1, there is evidence that promoter crypticity is the result of a transcription attenuation phenomenon which is relieved by the deletion of a 22-base C-rich segment in the transcript. In this study, we have sought to identify growth conditions and
trans
-acting mutations which activate in vivo expression from
proU
P1. The cryptic
S
.
enterica proU
P1 promoter was activated, individually and additively, in a
rho
mutant (which is defective in the transcription termination factor Rho) as well as by growth at 10°C. The
E
.
coli proU
P1 promoter was also cryptic in constructs that carried 1.2 kb of downstream
proU
sequence, and in these cases activation of in vivo expression was achieved either by a
rho
mutation during growth at 10°C or by an
hns
null mutation (affecting the nucleoid protein H-NS) at 30°C. The
rho
mutation had no effect at either 10 or 30°C on in vivo expression from two other ς
s
-controlled promoters tested, those for
osmY
and
csiD
. In cells lacking the RNA-binding regulator protein Hfq, induction of
E
.
coli proU
P1 at 10°C and by
hns
mutation at 30°C was still observed, although the
hfq
mutation was associated with a reduction in the absolute levels of P1 expression. Our results suggest that expression from
proU
P1 is modulated both by nucleoid structure and by Rho-mediated transcription attenuation and that this promoter may be physiologically important for
proU
operon expression during low-temperature growth.
Structural and Immunochemical Characterization of the Lipooligosaccharides Expressed byNeisseria subflava44
Authors: Tong Yanhong , Reinhold Vernon , Reinhold Bruce , Brandt Brenda , Stein Daniel C. 
Published: July 2002

ABSTRACTNeisserial lipooligosaccharides (LOSs) are a family of complex cell surface glycolipids. We used mass spectrometry techniques (electrospray ionization, collision-induced dissociation, and multiple step), combined with fluorophore-assisted carbohydrate electrophoresis monosaccharide composition analysis, to determine the structure of the two low-molecular-mass LOS molecules (LOSI and LOSII) expressed byNeisseria subflava44. We determined that LOSI contains one glucose on both the α and β chains. LOSII is structurally related to LOSI and differs from it by the addition of a hexose (either glucose or galactose) on the α chain. LOSI and LOSII were able to bind monoclonal antibody (MAb) 25-1-LC1 when analyzed by Western blotting experiments. We used a set of genetically definedNeisseria gonorrhoeaemutants that expressed single defined LOS epitopes and a group ofNeisseria meningitidisstrains that expresses chemically defined LOS components to determine the structures recognized by MAb 25-1-LC1. We found that extensions onto the β-chain glucose of LOSI block the recognition by this MAb, as does further elongation from the LOSII α chain. The LOSI structure was determined to be the minimum structure that is recognized by MAb 25-1-LC1.
Formation of Chromosomal Tandem Arrays of the SXT Element and R391, Two Conjugative Chromosomally Integrating Elements That Share an Attachment Site
Authors: Hochhut Bianca , Beaber John W. , Woodgate Roger , Waldor Matthew K. 
Published: July 2002

ABSTRACT

The SXT element, a conjugative, self-transmissible, integrating element (a constin) originally derived from a
Vibrio cholerae
O139 isolate from India, and IncJ element R391, originally derived from a South African
Providencia rettgeri
isolate, were found to be genetically and functionally related. Both of these constins integrate site specifically into the
Escherichia coli
chromosome at an identical attachment site within the 5′ end of
prfC
. They encode nearly identical integrases, which are required for chromosomal integration, excision, and extrachromosomal circularization of these elements, and they have similar
tra
genes. Therefore, these closely related constins have virtually identical mechanisms for chromosomal integration and dissemination. The presence of either element in a recipient cell did not significantly reduce its ability to acquire the other element, indicating that R391 and SXT do not encode surface exclusion determinants. In cells harboring both elements, SXT and R391 were integrated in tandem fashion on the chromosome, and homologous recombination appeared to play little or no role in the formation of these arrays. Interference between R391 and SXT was detected by measuring the frequency of loss of an unselected resident element upon introduction of a second selected element. In these assays, R391 was found to have a stronger effect on SXT stability than vice versa. The level of expression and/or activity of the donor and recipient integrases may play a role in the interference between these two related constins.
Regulation of Metalloprotease Gene Expression in
Vibrio vulnificus
by a
Vibrio harveyi
LuxR Homologue
Authors: Shao Chung-Ping , Hor Lien-I 
Published: July 2002

ABSTRACT

Expression of the
Vibrio vulnificus
metalloprotease gene,
vvp
, was turned up rapidly when bacterial growth reached the late log phase. A similar pattern of expression has been found in the metalloprotease gene of
Vibrio cholerae
, and this has been shown to be regulated by a
Vibrio harveyi
LuxR-like transcriptional activator. To find out whether a LuxR homologue exists in
V. vulnificus
, a gene library of this organism was screened by colony hybridization using a probe derived from a sequence that is conserved in various
luxR
-like genes of vibrios. A gene containing a 618-bp open reading frame was identified and found to be identical to the
smcR
gene of
V. vulnificus
reported previously. An isogenic SmcR-deficient (RD) mutant was further constructed by an in vivo allelic exchange technique. This mutant exhibited an extremely low level of
vvp
transcription compared with that of the parent strain. On the other hand, the cytolysin gene,
vvhA
, was expressed at a higher level in the RD mutant than in the parent strain during the log phase of growth. These data suggested that SmcR might not only be a positive regulator of the protease gene but might also be involved in negative regulation of the cytolysin gene. Virulence of the RD mutant in either normal or iron-overloaded mice challenged by intraperitoneal injection was comparable to that of the parent strain, indicating that SmcR is not required for
V. vulnificus
virulence in mice.
Physiological Basis for Conservation of the Signal Recognition Particle Targeting Pathway in
Escherichia coli
Authors: Bernstein Harris D. , Hyndman Janine B. 
Published: July 2002

ABSTRACT

The
Escherichia coli
signal recognition particle (SRP) is a ribonucleoprotein complex that targets nascent inner membrane proteins (IMPs) to transport sites in the inner membrane (IM). Since SRP depletion only partially inhibits IMP insertion under some growth conditions, however, it is not clear why the particle is absolutely essential for viability. Insights into this question emerged from experiments in which we analyzed the physiological consequences of reducing the intracellular concentration of SRP below the wild-type level. We found that even moderate SRP deficiencies that have little effect on cell growth led to the induction of a heat shock response. Genetic manipulations that suppress the heat shock response were lethal in SRP-deficient cells, indicating that the elevated synthesis of heat shock proteins plays an important role in maintaining cell viability. Although it is conceivable that the heat shock response serves to increase the capacity of cells to target IMPs via chaperone-based mechanisms, SRP-deficient cells did not show an increased dependence on either GroEL or DnaK. By contrast, the heat shock-regulated proteases Lon and ClpQ became essential for viability when SRP levels were reduced. These results suggest that the heat shock response protects SRP-deficient cells by increasing their capacity to degrade mislocalized IMPs. Consistent with this notion, a model IMP that was mislocalized in the cytoplasm as the result of SRP depletion appeared to be more stable in a
Δlon ΔclpQ
strain than in control cells. Taken together, the data provide direct evidence that SRP is essential in
E. coli
and possibly conserved throughout prokaryotic evolution as well partly because efficient IMP targeting prevents a toxic accumulation of aggregated proteins in the cytoplasm.
Interruption of thecydBLocus inBrucella abortusAttenuates Intracellular Survival and Virulence in the Mouse Model of Infection
Authors: Endley Seema , McMurray David , Ficht Thomas A. 
Published: July 2002

ABSTRACTBrucellosis is characterized by abortion in ruminants and a protracted undulant fever in humans, which often results in severe pathological manifestations. Scant information exists about the molecular mechanisms employed byBrucella abortusto combat host defenses or to persist and replicate within host cells. Transposon (Tn5) mutagenesis ofB. abortusand the subsequent screening of mutants for sensitivity to killing in murine macrophages and in the mouse model led to the identification of mutants which were severely attenuated for intracellular survival. One group of mutants was interrupted incydB, a gene that is part of thecydABoperon encoding cytochromebdoxidase, which catalyzes an alternate terminal electron transport step in bacterial respiration. The elevated affinity for molecular oxygen of this enzyme inEscherichia colihas suggested that it is involved in the protection of sensitive enzymatic activities such as those of hydrogenases and nitrogenases from damage.B. abortus cydB::Tn5strains exhibited heightened sensitivity to the respiratory inhibitors zinc and azide, highly reactive oxygen species such as hydrogen peroxide, low pH, and attenuated virulence in the mouse model of infection. Virulence was restored by an intact copy ofcydABor byB. abortusgenes encoding the oxidative radical-scavenging enzyme Cu/Zn superoxide dismutase or catalase. These results suggest a bifunctional role for the products of thecydABoperon, both in preventing the buildup of oxidative free radicals and in detoxifying the intracellular compartment, thus indicating the importance of these products in preventing intracellular destruction. Intracellular conditions that favor expression of thecydABoperon are under investigation and may be linked to the acid sensitivity also observed in this strain.
Characterization of the
Plesiomonas shigelloides
Genes Encoding the Heme Iron Utilization System
Authors: Henderson D. P. , Wyckoff E. E. , Rashidi C. E. , Verlei H. , Oldham A. L. 
Published: July 2002

ABSTRACT

Plesiomonas shigelloides
is a gram-negative pathogen which can utilize heme as an iron source. In previous work,
P. shigelloides
genes which permitted heme iron utilization in a laboratory strain of
Escherichia coli
were isolated. In the present study, the cloned
P. shigelloides
sequences were found to encode ten potential heme utilization proteins: HugA, the putative heme receptor; TonB and ExbBD; HugB, the putative periplasmic binding protein; HugCD, the putative inner membrane permease; and the proteins HugW, HugX, and HugZ. Three of the genes,
hugA, hugZ
, and
tonB
, contain a Fur box in their putative promoters, indicating that the genes may be iron regulated. When the
P. shigelloides
genes were tested in
E. coli
K-12 or in a heme iron utilization mutant of
P. shigelloides, hugA
, the TonB system genes, and
hugW, hugX
, or
hugZ
were required for heme iron utilization. When the genes were tested in a
hemA entB
mutant of
E. coli, hugWXZ
were not required for utilization of heme as a porphyrin source, but their absence resulted in heme toxicity when the strains were grown in media containing heme as an iron source.
hugA
could replace the
Vibrio cholerae hutA
in a heme iron utilization assay, and
V. cholerae hutA
could complement a
P. shigelloides
heme utilization mutant, suggesting that HugA is the heme receptor. Our analyses of the TonB system of
P. shigelloides
indicated that it could function in
tonB
mutants of both
E. coli
and
V. cholerae
and that it was similar to the
V. cholerae
TonB1 system in the amino acid sequence of the proteins and in the ability of the system to function in high-salt medium.
Regulation of theacuFGene, Encoding Phosphoenolpyruvate Carboxykinase in the Filamentous FungusAspergillus nidulans
Authors: Hynes Michael J. , Draht Oliver W. , Davis Meryl A. 
Published: July 2002

ABSTRACTPhosphoenolpyruvate carboxykinase (PEPCK) is a key enzyme required for gluconeogenesis when microorganisms grow on carbon sources metabolized via the tricarboxylic acid (TCA) cycle.Aspergillus nidulans acuFmutants isolated by their inability to use acetate as a carbon source specifically lack PEPCK. TheacuFgene has been cloned and shown to encode a protein with high similarity to PEPCK from bacteria, plants, and fungi. The regulation ofacuFexpression has been studied by Northern blotting and by the construction oflacZfusion reporters. Induction by acetate is abolished in mutants unable to metabolize acetate via the TCA cycle, and induction by amino acids metabolized via 2-oxoglutarate is lost in mutants unable to form 2-oxoglutarate. Induction by acetate and proline is not additive, consistent with a single mechanism of induction. Malate and succinate result in induction, and it is proposed that PEPCK is controlled by a novel mechanism of induction by a TCA cycle intermediate or derivative, thereby allowing gluconeogenesis to occur during growth on any carbon source metabolized via the TCA cycle. It has been shown that thefacBgene, which mediates acetate induction of enzymes specifically required for acetate utilization, is not directly involved in PEPCK induction. This is in contrast toSaccharomyces cerevisiae, where Cat8p and Sip4p, homologs of FacB, regulate PEPCK as well as the expression of other genes necessary for growth on nonfermentable carbon sources in response to the carbon source present. This difference in the control of gluconeogenesis reflects the ability ofA.nidulansand other filamentous fungi to use a wide variety of carbon sources in comparison withS.cerevisiae. TheacuFgene was also found to be subject to activation by the CCAAT binding protein AnCF, a protein homologous to theS.cerevisiaeHap complex and the mammalian NFY complex.
The High-Molecular-Weight Cytochrome
c
Cyc2 of
Acidithiobacillus ferrooxidans
Is an Outer Membrane Protein
Authors: Yarzábal Andrés , Brasseur Gaël , Ratouchniak Jeanine , Lund Karen , Lemesle-Meunier Danielle , DeMoss John A. , Bonnefoy Violaine 
Published: July 2002

ABSTRACT

A high-molecular-weight
c
-type cytochrome, Cyc2, and a putative 22-kDa
c
-type cytochrome were detected in the membrane fraction released during spheroplast formation from
Acidithiobacillus ferrooxidans
. This fraction was enriched in outer membrane components and devoid of cytoplasmic membrane markers. The genetics, as well as the subcellular localization of Cyc2 at the outer membrane level, therefore make it a prime candidate for the initial electron acceptor in the respiratory pathway between ferrous iron and oxygen.
Mutant Analysis and Cellular Localization of the AlgI, AlgJ, and AlgF Proteins Required for O Acetylation of Alginate in
Pseudomonas aeruginosa
Authors: Franklin Michael J. , Ohman Dennis E. 
Published: July 2002

ABSTRACT

Alginate is an extracellular polysaccharide produced by mucoid strains of
Pseudomonas aeruginosa
that are typically isolated from the pulmonary tracts of chronically infected cystic fibrosis patients. Alginate is a linear polymer of
d
-mannuronate and
l
-guluronate with
O
-acetyl ester linkages on the O-2 and/or O-3 position of the mannuronate residues. The presence of
O
-acetyl groups plays an important role in the ability of the polymer to act as a virulence factor, and the
algF
,
algJ
, and
algI
genes are known to be essential for the addition of
O
-acetyl groups to alginate. To better understand the mechanism of O acetylation of alginate, the cellular locations of the AlgI, AlgJ, and AlgF proteins were determined. For these studies, defined nonpolar
algI
,
algJ
, and
algF
deletion mutants of
P. aeruginosa
strain FRD1 were constructed, and each mutant produced alginate lacking
O
-acetyl groups. Expression of
algI
,
algJ
, or
algF
in
trans
in the corresponding mutant complemented each O acetylation defect. Random
phoA
(alkaline phosphatase [AP] gene) fusions to
algF
,
algJ
, and
algI
were constructed. All in-frame fusions to
algF
and
algJ
had AP activity, indicating that both AlgF and AlgJ were exported to the periplasm. Immunoblot analysis of spheroplasts and periplasmic fractions showed that AlgF was released with the periplasmic contents but that AlgJ remained with the spheroplast fraction. An N-terminal sequence analysis of AlgJ showed that its putative AlgJ signal peptide was not cleaved, suggesting that AlgJ is anchored to the cytoplasmic membrane by its uncleaved signal peptide. AP gene fusions were also used to map the membrane topology of AlgI, and the results suggest that it is an integral membrane protein with seven transmembrane domains. These results suggest that AlgI-AlgJ-AlgF may form a complex in the membrane that is the reaction center for O acetylation of alginate.
Spa32 Regulates a Switch in Substrate Specificity of the Type III Secreton of
Shigella flexneri
from Needle Components to Ipa Proteins
Authors: Magdalena Juana , Hachani Abderrahman , Chamekh Mustapha , Jouihri Noureddine , Gounon Pierre , Blocker Ariel , Allaoui Abdelmounaaïm 
Published: July 2002

ABSTRACT

Type III secretion systems (TTSS) are essential virulence determinants of many gram-negative bacteria and serve, upon physical contact with target cells, to translocate bacterial proteins directly across eukaryotic cell membranes. The
Shigella
TTSS is encoded by the
mxi/spa
loci located on its virulence plasmid. By electron microscopy secretons are visualized as tripartite with an external needle, a transmembrane domain, and a cytoplasmic bulb. In the present study, we generated a
Shigella spa32
mutant and studied its phenotype. The
spa32
gene shows low sequence homology to
Salmonella
TTSS1
invJ/spaN
and to flagellar
fliK
. The
spa32
mutant, like the wild-type strain, secreted the Ipas and IpgD, which are normally secreted via the TTSS, at low levels into the growth medium. However, unlike the wild-type strain, the
spa32
mutant could neither be induced to secrete the Ipas and IpgD instantaneously upon addition of Congo red nor penetrate HeLa cells in vitro. Additionally, the Spa32 protein is secreted in large amounts by the TTSS during exponential growth but not upon Congo red induction. Interestingly, electron microscopy analysis of the
spa32
mutant revealed that the needle of its secretons were up to 10 times longer than those of the wild type. In addition, in the absence of induction, the
spa32
mutant secreted normal levels of MxiI but a large excess of MxiH. Taken together, our data indicate that the
spa32
mutant presents a novel phenotype and that the primary defect of the mutant may be its inability to regulate or control secretion of MxiH.
Direct Quantitation of the Numbers of Individual Penicillin-Binding Proteins per Cell in
Staphylococcus aureus
Authors: Pucci Michael J. , Dougherty Thomas J. 
Published: July 2002

ABSTRACT

The penicillin-binding proteins (PBPs) are a set of enzymes that participate in bacterial peptidoglycan assembly. The absolute numbers of each PBP were determined by direct measurement and have been reported for two
Staphylococcus aureus
strains, RN4220 (methicillin-sensitive
S. aureus
) and RN450M (methicillin-resistant
S. aureus
). From the specific activity of the labeled penicillin and the absolute number of disintegrations per minute, and from the number of CFU per milliliter calculated from proteins and optical density, a determination of the number of PBPs per cell was made. These numbers ranged from approximately 150 to 825 PBPs/cell and represent the first direct determination of absolute numbers of PBPs in
S. aureus
.