bluuebunny/crossref_metadata_2025 huggingface dataset:
Protection of Mice against Brucellosis by Intranasal Immunization withBrucella melitensisLipopolysaccharide as a Noncovalent Complex withNeisseria meningitidisGroup B Outer Membrane Protein
Authors: Bhattacharjee Apurba K. , Van De Verg Lillian , Izadjoo Mina J. , Yuan Liang , Hadfield Ted L. , Zollinger Wendell D. , Hoover David L.
Published: July 2002

Intranasal immunization of mice with purifiedBrucella melitensislipopolysaccharide (LPS) as a noncovalent complex withNeisseria meningitidisgroup B outer membrane protein (GBOMP) elicited a high-titer anti-LPS systemic antibody response and a significant mucosal antibody response. The anti-LPS immunoglobulin G (IgG) antibody was predominantly of the IgG1 subtype, although there was some response of the IgG2a, IgG2b, and IgG3 subtypes. The antibody titer remained high for 16 weeks postimmunization. Immunized mice and sham-immunized control mice were challenged intranasally with 104CFU of virulentB. melitensisstrain 16 M 4 weeks after the second dose of vaccine. The numbers of bacteria in lungs, livers, and spleens at 3 days, 9 days, and 8 weeks postchallenge were determined. Bacteria were found in lungs of all mice on day 3, but there was no disseminated infection of liver or spleen. By day 9, 40% of the mice had infected spleens and livers. At 8 weeks postchallenge, spleens of 25 of 62 immunized mice were infected, compared to 61 of 62 control mice (P< 0.0001). The livers of 12 of 43 immunized mice were infected, compared to 22 of 36 control mice (P= 0.005). In contrast, the lungs of 26 of 46 immunized mice were still infected, compared to 27 of 44 control mice. The numbers of bacterial CFU in lungs of immunized and control animals were identical. These studies show that intranasal immunization withB. melitensisLPS-GBOMP subunit vaccine significantly protects mice against intranasal challenge with virulentB. melitensis.Vaccination reduces bacterial dissemination to spleen and liver but has no effect on the course of lung infection.
Human Monocytes KillShigella flexneribut Then Die by Apoptosis Associated with Suppression of Proinflammatory Cytokine Production
Authors: Hathaway Lucy J. , Griffin George E. , Sansonetti Philippe J. , Edgeworth Jonathan D.
Published: July 2002

Shigella flexneriinfection of human macrophages is followed by rapid bacterial escape into the cytosol and secretion of IpaB, which activates caspase-1 to mediate cell death and release of mature interleukin (IL)-1β. Here we report a different outcome following infection of human peripheral blood monocytes.S. flexneriinfects monocytes inefficiently in the absence of complement and, following complement-dependent uptake, cannot escape the endosomal compartment. Consequently, bacteria are killed within the first 60 min in the absence of monocyte cell death, as demonstrated by immunofluorescence and electron microscopy and enumeration of colonies in a gentamicin protection assay. Despite early bacterial death, wild-typeS. flexneriinfluenced the subsequent monocyte proinflammatory cytokine response and cell fate. Infection with wild-typeS. flexneriresulted in IpaB-dependent suppression of IL-1β, tumor necrosis factor alpha, and IL-6 compared with that of plasmid-cured avirulentS. flexneri-infected cells. Furthermore, over the following 6 to 8 h, virulentS. flexneri-infected monocytes died by apoptosis whereas avirulent infected monocytes died by necrosis. Together, these results imply that monocytes migrating into the inflammatory site during the early stages of shigellosis killS. flexneribut that during bacterial uptake, they receive virulence signals fromS. flexneriwhich induce delayed apoptosis associated with suppression of the proinflammatory cytokine response to bacterial phagocytosis. This delayed apoptosis may have important effects on the ordered initiation of the innate immune response, leading to the excessive inflammatory response characteristic of shigellosis.
Expression and Characterization of the Chitin-Binding Domain of Chitinase A1 from
Bacillus circulans
WL-12
Authors: Hashimoto Masayuki , Ikegami Takahisa , Seino Shizuka , Ohuchi Nobuhumi , Fukada Harumi , Sugiyama Junji , Shirakawa Masahiro , Watanabe Takeshi
Published: July 2002

Chitinase A1 from
Bacillus circulans
WL-12 comprises an N-terminal catalytic domain, two fibronectin type III-like domains, and a C-terminal chitin-binding domain (ChBD). In order to study the biochemical properties and structure of the ChBD, ChBD
ChiA1
was produced in
Escherichia coli
using a pET expression system and purified by chitin affinity column chromatography. Purified ChBD
ChiA1
specifically bound to various forms of insoluble chitin but not to other polysaccharides, including chitosan, cellulose, and starch. Interaction of soluble chitinous substrates with ChBD
ChiA1
was not detected by means of nuclear magnetic resonance and isothermal titration calorimetry. In addition, the presence of soluble substrates did not interfere with the binding of ChBD
ChiA1
to regenerated chitin. These observations suggest that ChBD
ChiA1
recognizes a structure which is present in insoluble or crystalline chitin but not in chito-oligosaccharides or in soluble derivatives of chitin. ChBD
ChiA1
exhibited binding activity over a wide range of pHs, and the binding activity was enhanced at pHs near its pI and by the presence of NaCl, suggesting that the binding of ChBD
ChiA1
is mediated mainly by hydrophobic interactions. Hydrolysis of β-chitin microcrystals by intact chitinase A1 and by a deletion derivative lacking the ChBD suggested that the ChBD is not absolutely required for hydrolysis of β-chitin microcrystals but greatly enhances the efficiency of degradation.
Distinctiveness of Genotypes ofHelicobacter pyloriin Calcutta, India
Authors: Mukhopadhyay Asish K. , Kersulyte Dangeruta , Jeong Jin-Yong , Datta Simanti , Ito Yoshiyuki , Chowdhury Abhijit , Chowdhury Sujit , Santra Amal , Bhattacharya Sujit K. , Azuma Takeshi , Nair G. Balakrish , Berg Douglas E.
Published: July 2002

The genotypes of 78 strains ofHelicobacter pylorifrom Calcutta, India (55 from ulcer patients and 23 from more-benign infections), were studied, with a focus on putative virulence genes and neutral DNA markers that were likely to be phylogenetically informative. PCR tests indicated that 80 to 90% of Calcutta strains carried thecagpathogenicity island (PAI) and potentially toxigenicvacAs1alleles of the vacuolating cytotoxin gene (vacA), independent of disease status. This was higher than in the West (wherecagPAI+vacAs1genotypes are disease associated) but lower than in east Asia. TheiceA2gene was weakly disease associated in Calcutta, whereas in the West the alternative but unrelatediceA1gene at the same locus is weakly disease associated. DNA sequence motifs ofvacAm1(middle region) alleles formed a cluster that was distinct from those of east Asia and the West, whereas thecagAsequences of Calcutta and Western strains were closely related. An internal deletion found in 20% of CalcuttaiceA1genes was not seen in any of ∼200 strains studied from other geographic regions and thus seemed to be unique to thisH. pyloripopulation. Two mobile DNAs that were rare in east Asian strains were also common in Calcutta. About 90% of Calcutta strains were metronidazole resistant. These findings support the idea thatH. pylorigene pools differ regionally and emphasize the potential importance of studies of Indian and other non-WesternH. pyloripopulations in developing a global understanding of this gastric pathogen and associated disease.
The Archaeon
Sulfolobus solfataricus
Contains a Membrane-Associated Protein Kinase Activity That Preferentially Phosphorylates Threonine Residues In Vitro
Authors: Lower Brian H. , Bischoff Kenneth M. , Kennelly Peter J.
Published: July 2002

The extreme acidothermophilic archaeon
Sulfolobus solfataricus
harbors a membrane-associated protein kinase activity. Its solubilization and stabilization required detergents, suggesting that this activity resides within an integral membrane protein. The archaeal protein kinase utilized purine nucleotides as phosphoryl donors in vitro. A noticeable preference for nucleotide triphosphates over nucleotide diphosphates and for adenyl nucleotides over the corresponding guanyl ones was observed. The molecular mass of the solubilized, partially purified enzyme was estimated to be ≈125 kDa by gel filtration chromatography. Catalytic activity resided in a polypeptide with an apparent molecular mass of ≈67 kDa by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Challenges with several exogenous substrates revealed the protein kinase to be relatively selective. Only casein, histone H4, reduced carboxyamidomethylated and maleylated lysozyme, and a peptide modeled after myosin light chains (KKRAARATSNVFA) were phosphorylated to appreciable levels in vitro. All of the aforementioned substrates were phosphorylated on threonine residues, while histone H4 was phosphorylated on serine as well. Substitution of serine for the phosphoacceptor threonine in the myosin light chain peptide produced a noticeably inferior substrate. The protein kinase underwent autophosphorylation on threonine and was relatively insensitive to a set of known inhibitors of “eukaryotic” protein kinases.
Influence of Deletions within Domain II of Exotoxin A on Its Extracellular Secretion from
Pseudomonas aeruginosa
Authors: Voulhoux Romé , Taupiac Marie-Pierre , Czjzek Mirjam , Beaumelle Bruno , Filloux Alain
Published: July 2002

Pseudomonas aeruginosa
is a gram-negative bacterium that secretes many proteins into the extracellular medium via the Xcp machinery. This pathway, conserved in gram-negative bacteria, is called the type II pathway. The exoproteins contain information in their amino acid sequence to allow targeting to their secretion machinery. This information may be present within a conformational motif. The nature of this signal has been examined for
P. aeruginosa
exotoxin A (PE). Previous studies failed to identify a common minimal motif required for Xcp-dependent recognition and secretion of PE. One study identified a motif at the N terminus of the protein, whereas another one found additional information at the C terminus. In this study, we assess the role of the central PE domain II composed of six α-helices (A to F). The secretion behavior of PE derivatives, individually deleted for each helix, was analyzed. Helix E deletion has a drastic effect on secretion of PE, which accumulates within the periplasm. The conformational rearrangement induced in this variant is predicted from the three-dimensional PE structure, and the molecular modification is confirmed by gel filtration experiments. Helix E is in the core of the molecule and creates close contact with other domains (I and III). Deletion of the surface-exposed helix F has no effect on secretion, indicating that no secretion information is contained in this helix. Finally, we concluded that disruption of a structured domain II yields an extended form of the molecule and prevents formation of the conformational secretion motif.
Metronidazole Activation Is Mutagenic and Causes DNA Fragmentation in
Helicobacter pylori
and in
Escherichia coli
Containing a Cloned
H. pylori rdxA
+
(Nitroreductase) Gene
Authors: Sisson Gary , Jeong Jin-Yong , Goodwin Avery , Bryden Louis , Rossler Norma , Lim-Morrison Sabrina , Raudonikiene Ausra , Berg Douglas E. , Hoffman Paul S.
Published: July 2002

Much of the normal high sensitivity of wild-type
Helicobacter pylori
to metronidazole (Mtz) depends on
rdxA
(HP0954), a gene encoding a novel nitroreductase that catalyzes the conversion of Mtz from a harmless prodrug to a bactericidal agent. Here we report that levels of Mtz that partially inhibit growth stimulate forward mutation to rifampin resistance in
rdxA
+
(Mtz
s
) and also in
rdxA
(Mtz
r
)
H. pylori
strains, and that expression of
rdxA
in
Escherichia coli
results in equivalent Mtz-induced mutation. A reversion test using defined
lac
tester strains of
E. coli
carrying
rdxA
+
indicated that CG-to-GC transversions and AT-to-GC transitions are induced more frequently than other base substitutions. Alkaline gel electrophoretic tests showed that Mtz concentrations near or higher than the MIC for growth also caused DNA breakage in
H. pylori
and in
E. coli
carrying
rdxA
+
, suggesting that this damage may account for most of the bactericidal action of Mtz. Coculture of Mtz
s
H. pylori
with
E. coli
(highly resistant to Mtz) in the presence of Mtz did not stimulate forward mutation in
E. coli
, indicating that the mutagenic and bactericidal products of Mtz metabolism do not diffuse significantly to neighboring (bystander) cells. Our results suggest that the widespread use of Mtz against other pathogens in people chronically infected with
H. pylori
may stimulate mutation and recombination in
H. pylori
, thereby speeding host-specific adaptation, the evolution of virulence, and the emergence of resistance against Mtz and other clinically useful antimicrobials.
Predicted Highly Expressed Genes of Diverse Prokaryotic Genomes
Authors: Karlin Samuel , Mrázek Jan
Published: July 2002

Our approach in predicting gene expression levels relates to codon usage differences among gene classes. In prokaryotic genomes, genes that deviate strongly in codon usage from the average gene but are sufficiently similar in codon usage to ribosomal protein genes, to translation and transcription processing factors, and to chaperone-degradation proteins are predicted highly expressed (PHX). By these criteria, PHX genes in most prokaryotic genomes include those encoding ribosomal proteins, translation and transcription processing factors, and chaperone proteins and genes of principal energy metabolism. In particular, for the fast-growing species
Escherichia coli
,
Vibrio cholerae
,
Bacillus subtilis
, and
Haemophilus influenzae
, major glycolysis and tricarboxylic acid cycle genes are PHX. In
Synechocystis
, prime genes of photosynthesis are PHX, and in methanogens, PHX genes include those essential for methanogenesis. Overall, the three protein families—ribosomal proteins, protein synthesis factors, and chaperone complexes—are needed at many stages of the life cycle, and apparently bacteria have evolved codon usage to maintain appropriate growth, stability, and plasticity. New interpretations of the capacity of
Deinococcus radiodurans
for resistance to high doses of ionizing radiation is based on an excess of PHX chaperone-degradation genes and detoxification genes. Expression levels of selected classes of genes, including those for flagella, electron transport, detoxification, histidine kinases, and others, are analyzed. Flagellar PHX genes are conspicuous among spirochete genomes. PHX genes are positively correlated with strong Shine-Dalgarno signal sequences. Specific regulatory proteins, e.g., two-component sensor proteins, are rarely PHX. Genes involved in pathways for the synthesis of vitamins record low predicted expression levels. Several distinctive PHX genes of the available complete prokaryotic genomes are highlighted. Relationships of PHX genes with stoichiometry, multifunctionality, and operon structures are discussed. Our methodology may be used complementary to experimental expression analysis.
Bacillus subtilis ccpA
Gene Mutants Specifically Defective in Activation of Acetoin Biosynthesis
Authors: Turinsky Andrew J. , Moir-Blais Tessa R. , Grundy Frank J. , Henkin Tina M.
Published: July 2002

A large number of carbon source utilization pathways are repressed in
Bacillus subtilis
by the global regulator CcpA, which also acts as an activator of carbon excretion pathways during growth in media containing glucose. In this study, CcpA mutants defective in transcriptional activation of the
alsSD
operon, which is involved in acetoin biosynthesis, were identified. These mutants retained normal glucose repression of
amyE
, encoding α-amylase, and
acsA
, encoding acetyl-coenzyme A synthetase, and normal activation of
ackA
, which is involved in acetate excretion; in these
ccpA
mutants the CcpA functions of activation of the acetate and acetoin excretion pathways appear to be separated.
Synergistic Hydrolysis of Carboxymethyl Cellulose and Acid-Swollen Cellulose by Two Endoglucanases (CelZ and CelY) from
Erwinia chrysanthemi
Authors: Zhou Shengde , Ingram Lonnie O.
Published: July 2002

Erwinia chrysanthemi
produces a battery of hydrolases and lyases which are very effective in the maceration of plant cell walls. Although two endoglucanases (CelZ and CelY; formerly EGZ and EGY) are produced, CelZ represents approximately 95% of the total carboxymethyl cellulase activity. In this study, we have examined the effectiveness of CelY and CelZ alone and of combinations of both enzymes using carboxymethyl cellulose (CMC) and amorphous cellulose (acid-swollen cellulose) as substrates. Synergy was observed with both substrates. Maximal synergy (1.8-fold) was observed for combinations containing primarily CelZ; the ratio of enzyme activities produced was similar to those produced by cultures of
E. chrysanthemi
. CelY and CelZ were quite different in substrate preference. CelY was unable to hydrolyze soluble cellooligosaccharides (cellotetraose and cellopentaose) but hydrolyzed CMC to fragments averaging 10.7 glucosyl units. In contrast, CelZ readily hydrolyzed cellotetraose, cellopentaose, and amorphous cellulose to produce cellobiose and cellotriose as dominant products. CelZ hydrolyzed CMC to fragments averaging 3.6 glucosyl units. In combination, CelZ and CelY hydrolyzed CMC to products averaging 2.3 glucosyl units. Synergy did not require the simultaneous presence of both enzymes. Enzymatic modification of the substrate by CelY increased the rate and extent of hydrolysis by CelZ. Full synergy was retained by the sequential hydrolysis of CMC, provided CelY was used as the first enzyme. A general mechanism is proposed to explain the synergy between these two enzymes based primarily on differences in substrate preference.
Defects in
d
-Alanyl-Lipoteichoic Acid Synthesis in
Streptococcus mutans
Results in Acid Sensitivity
Authors: Boyd David A. , Cvitkovitch Dennis G. , Bleiweis Arnold S. , Kiriukhin Michael Y. , Debabov Dmitri V. , Neuhaus Francis C. , Hamilton Ian R.
Published: July 2002

In the cariogenic organism,
Streptococcus mutans
, low pH induces an acid tolerance response (ATR). To identify acid-regulated proteins comprising the ATR, transposon mutagenesis with the thermosensitive plasmid pGh9:IS
S1
was used to produce clones that were able to grow at neutral pH, but not in medium at pH 5.0. Sequence analysis of one mutant (IS1A) indicated that transposition had created a 6.3-kb deletion, one end of which was in
dltB
of the
dlt
operon encoding four proteins (DltA-DltD) involved in the synthesis of
d
-alanyl-lipoteichoic acid. Inactivation of the
dltC
gene, encoding the
d
-alanyl carrier protein (Dcp), resulted in the generation of the acid-sensitive mutant, BH97LC. Compared to the wild-type strain, LT11, the mutant exhibited a threefold-longer doubling time and a 33% lower growth yield. In addition, it was unable to initiate growth below pH 6.5 and unadapted cells were unable to survive a 3-h exposure in medium buffered at pH 3.5, while a pH of 3.0 was required to kill the wild type in the same time period. Also, induction of the ATR in BH97LC, as measured by the number of survivors at a pH killing unadapted cells, was 3 to 4 orders of magnitude lower than that exhibited by the wild type. While the LTA of both strains contained a similar average number of glycerolphosphate residues, permeabilized cells of BH97LC did not incorporate
d
-[
14
C]alanine into this amphiphile. This defect was correlated with the deficiency of Dcp. Chemical analysis of the LTA purified from the mutant confirmed the absence of
d
-alanine-esters. Electron micrographs showed that BH97LC is characterized by unequal polar caps and is devoid of a fibrous extracellular matrix present on the surface of the wild-type cells. Proton permeability assays revealed that the mutant was more permeable to protons than the wild type. This observation suggests a mechanism for the loss of the characteristic acid tolerance response in
S. mutans.
Catalase-Peroxidases of
Legionella pneumophila
: Cloning of the
katA
Gene and Studies of KatA Function
Authors: Bandyopadhyay Purnima , Steinman Howard M.
Published: July 2002

Legionella pneumophila
, the causative organism of Legionnaires' pneumonia, contains two enzymes with catalatic and peroxidatic activity, KatA and KatB. To address the issue of redundant, overlapping, or discrete in vivo functions of highly homologous catalase-peroxidases, the gene for
katA
was cloned and its function was studied in
L. pneumophila
and
Escherichia coli
and compared with prior studies of
katB
in this laboratory.
katA
is induced during exponential growth and is the predominant peroxidase in stationary phase. When
katA
is inactivated,
L. pneumophila
is more sensitive to exogenous hydrogen peroxide and less virulent in the THP-1 macrophage cell line, similar to
katB
. Catalatic-peroxidatic activity with different peroxidatic cosubstrates is comparable for KatA and KatB, but KatA is five times more active towards dianisidine. In contrast with these examples of redundant or overlapping function, stationary-phase survival is decreased by 100- to 10,000-fold when
katA
is inactivated, while no change from wild type is seen for the
katB
null. The principal clue for understanding this discrete in vivo function was the demonstration that KatA is periplasmic and KatB is cytosolic. This stationary-phase phenotype suggests that targets sensitive to hydrogen peroxide are present outside the cytosol in stationary phase or that the peroxidatic activity of KatA is critical for stationary-phase redox reactions in the periplasm, perhaps disulfide bond formation. Since starvation-induced stationary phase is a prerequisite to acquisition of virulence by
L. pneumophila
, further studies on the function and regulation of
katA
in stationary phase may give insights on the mechanisms of infectivity of this pathogen.
Secretion of an Acid Phosphatase (SapM) by
Mycobacterium tuberculosis
That Is Similar to Eukaryotic Acid Phosphatases
Authors: Saleh Mazen T. , Belisle John T.
Published: July 2002

Mycobacterium tuberculosis
secretes a large number of polypeptides with broad biological and immunological functions. We describe here the characterization of a 28-kDa acid phosphatase of
M. tuberculosis
(SapM) localized to the culture filtrate. The mature protein demonstrated biochemical characteristics similar to those of the bacterial nonspecific acid phosphatases. However, SapM yielded significant sequence homology to fungal acid phosphatases and not those of bacteria. Thus, SapM may represent a new class of bacterial nonspecific acid phosphatases.
The 4-Oxalomesaconate Hydratase Gene, Involved in the Protocatechuate 4,5-Cleavage Pathway, Is Essential to Vanillate and Syringate Degradation in
Sphingomonas paucimobilis
SYK-6
Authors: Hara Hirofumi , Masai Eiji , Katayama Yoshihiro , Fukuda Masao
Published: July 2002

Sphingomonas paucimobilis
SYK-6 is able to grow on various dimeric lignin compounds, which are converted to vanillate and syringate by the actions of unique lignin degradation enzymes in this strain. Vanillate and syringate are degraded by the
O
-demethylase and converted into protocatechuate (PCA) and 3-
O
-methylgallate (3MGA), respectively. PCA is further degraded via the PCA 4,5-cleavage pathway, while the results suggested that 3MGA is degraded through another pathway in which PCA 4,5-dioxygenase is not involved. In a 10.5-kb
Eco
RI fragment carrying the genes for PCA 4,5-dioxygenase (
ligAB
), 2-pyrone-4,6-dicarboxylate hydrolase (
ligI
), and a portion of 4-carboxy-2-hydroxymuconate-6-semialdehyde dehydrogenase (
ligC
), we found the
ligJ
gene encoding 4-oxalomesaconate (OMA) hydratase, which catalyzes the conversion of OMA into 4-carboxy-4-hydroxy-2-oxoadipate. The
ligJ
gene is transcribed in the same direction as
ligABC
genes and consists of an 1,023-bp open reading frame encoding a polypeptide with a molecular mass of 38,008 Da, which is located 73-bp upstream from
ligA
. The
ligJ
gene product (LigJ), expressed in
Escherichia coli
, was purified to near homogeneity and was estimated to be a homodimer (69.5 kDa) by gel filtration chromatography. The isoelectric point was determined to be 4.9, and the optimal temperature is 30°C. The

K
m

for OMA and the
V
max
were determined to be 138 μM and 440 U/mg, respectively. LigJ activity was inhibited by the addition of thiol reagents, suggesting that some cysteine residue is part of the catalytic site. The
ligJ
gene disruption in SYK-6 caused the growth defect on and the accumulation of common metabolites from both vanillate and syringate, indicating that the
ligJ
gene is essential to the degradation of these two compounds. These results indicated that syringate is converted into OMA via 3MGA, and it enters the PCA 4,5-cleavage pathway.
Functional Analysis of the Small Component of the 4-Hydroxyphenylacetate 3-Monooxygenase ofEscherichia coliW: a Prototype of a New Flavin:NAD(P)H Reductase Subfamily
Authors: Galán Beatriz , Díaz Eduardo , Prieto María A. , García José L.
Published: July 2002

Escherichia coliW uses the aromatic compound 4-hydroxyphenylacetate (4-HPA) as a sole source of carbon and energy for growth. The monooxygenase which converts 4-HPA into 3,4-dihydroxyphenylacetate, the first intermediate of the pathway, consists of two components, HpaB (58.7 kDa) and HpaC (18.6 kDa), encoded by thehpaBandhpaCgenes, respectively, that form a single transcription unit. Overproduction of the small HpaC component inE. coliK-12 cells has facilitated the purification of the protein, which was revealed to be a homodimer that catalyzes the reduction of free flavins by NADH in preference to NADPH. Subsequently, the reduced flavins diffuse to the large HpaB component or to other electron acceptors such as cytochromecand ferric ion. Amino acid sequence comparisons revealed that the HpaC reductase could be considered the prototype of a new subfamily of flavin:NAD(P)H reductases. The construction of a fusion protein between the large HpaB oxygenase component and the choline-binding domain of the major autolysin ofStreptococcus pneumoniaeallowed us to develop a rapid method to efficiently purify this highly unstable enzyme as a chimeric CH-HpaB protein, which exhibited a 4-HPA hydroxylating activity only when it was supplemented with the HpaC reductase. These results suggest the 4-HPA 3-monooxygenase ofE. coliW as a representative member of a novel two-component flavin-diffusible monooxygenase (TC-FDM) family. Relevant features on the evolution and structure-function relationships of these TC-FDM proteins are discussed.
Native Plasmids of
Fusobacterium nucleatum
: Characterization and Use in Development of Genetic Systems
Authors: Haake Susan Kinder , Yoder Sean C. , Attarian Gwynne , Podkaminer Kara
Published: July 2002

Three native plasmids of
Fusobacterium nucleatum
were characterized, including DNA sequence analysis of one plasmid, pFN1. A shuttle plasmid, pHS17, capable of transforming
Escherichia coli
and
F. nucleatum
ATCC 10953 was constructed with pFN1. pHS17 was stably maintained in the
F. nucleatum
transformants, and differences in the transformation efficiencies suggested the presence of a restriction-modification system in
F. nucleatum
.
Components of the RP4 Conjugative Transfer Apparatus Form an Envelope Structure Bridging Inner and Outer Membranes of Donor Cells: Implications for Related Macromolecule Transport Systems
Authors: Grahn A. Marika , Haase Jana , Bamford Dennis H. , Lanka Erich
Published: July 2002

During bacterial conjugation, the single-stranded DNA molecule is transferred through the cell envelopes of the donor and the recipient cell. A membrane-spanning transfer apparatus encoded by conjugative plasmids has been proposed to facilitate protein and DNA transport. For the IncPα plasmid RP4, a thorough sequence analysis of the gene products of the transfer regions Tra1 and Tra2 revealed typical features of mainly inner membrane proteins. We localized essential RP4 transfer functions to
Escherichia coli
cell fractions by immunological detection with specific polyclonal antisera. Each of the gene products of the RP4 mating pair formation (Mpf) system, specified by the Tra2 core region and by
traF
of the Tra1 region, was found in the outer membrane fraction with one exception, the TrbB protein, which behaved like a soluble protein. The membrane preparation from Mpf-containing cells had an additional membrane fraction whose density was intermediate between those of the cytoplasmic and outer membranes, suggesting the presence of attachment zones between the two
E. coli
membranes. The Tra1 region is known to encode the components of the RP4 relaxosome. Several gene products of this transfer region, including the relaxase TraI, were detected in the soluble fraction, but also in the inner membrane fraction. This indicates that the nucleoprotein complex is associated with and/or assembled facing the cytoplasmic site of the
E. coli
cell envelope. The Tra1 protein TraG was predominantly localized to the cytoplasmic membrane, supporting its potential role as an interface between the RP4 Mpf system and the relaxosome.
Characterization and Evolution of Anthranilate 1,2-Dioxygenase from
Acinetobacter
sp. Strain ADP1
Authors: Eby D. Matthew , Beharry Zanna M. , Coulter Eric D. , Kurtz Donald M. , Neidle Ellen L.
Published: July 2002

The two-component anthranilate 1,2-dioxygenase of the bacterium
Acinetobacter
sp. strain ADP1 was expressed in
Escherichia coli
and purified to homogeneity. This enzyme converts anthranilate (2-aminobenzoate) to catechol with insertion of both atoms of O
2
and consumption of one NADH. The terminal oxygenase component formed an α
3
β
3
hexamer of 54- and 19-kDa subunits. Biochemical analyses demonstrated one Rieske-type [2Fe-2S] center and one mononuclear nonheme iron center in each large oxygenase subunit. The reductase component, which transfers electrons from NADH to the oxygenase component, was found to contain approximately one flavin adenine dinucleotide and one ferredoxin-type [2Fe-2S] center per 39-kDa monomer. Activities of the combined components were measured as rates and quantities of NADH oxidation, substrate disappearance, product appearance, and O
2
consumption. Anthranilate conversion to catechol was stoichiometrically coupled to NADH oxidation and O
2
consumption. The substrate analog benzoate was converted to a nonaromatic benzoate 1,2-diol with similarly tight coupling. This latter activity is identical to that of the related benzoate 1,2-dioxygenase. A variant anthranilate 1,2-dioxygenase, previously found to convey temperature sensitivity in vivo because of a methionine-to-lysine change in the large oxygenase subunit, was purified and characterized. The purified M43K variant, however, did not hydroxylate anthranilate or benzoate at either the permissive (23°C) or nonpermissive (39°C) growth temperatures. The wild-type anthranilate 1,2-dioxygenase did not efficiently hydroxylate methylated or halogenated benzoates, despite its sequence similarity to broad-substrate specific dioxygenases that do. Phylogenetic trees of the α and β subunits of these terminal dioxygenases that act on natural and xenobiotic substrates indicated that the subunits of each terminal oxygenase evolved from a common ancestral two-subunit component.
Characterization of Transmembrane Segments 3, 4, and 5 of MalF by Mutational Analysis
Authors: Steinke Angelika , Grau Sandra , Davidson Amy , Hofmann Eckhard , Ehrmann Michael
Published: July 2002

MalF and MalG are the cytoplasmic membrane components of the binding protein-dependent ATP binding cassette maltose transporter in
Escherichia coli
. They are thought to form the transport channel and are thus of critical importance for the mechanism of transport. To study the contributions of individual transmembrane segments of MalF, we isolated 27 point mutations in membrane-spanning segments 3, 4, and 5. These data complement a previous study, which described the mutagenesis of membrane-spanning segments 6, 7, and 8. While most of the isolated mutations appear to cause assembly defects, L
323
Q in helix 5 could interfere more directly with substrate specificity. The phenotypes and locations of the mutations are consistent with a previously postulated structural model of MalF.
Recombinant
Thermus aquaticus
RNA Polymerase, a New Tool for Structure-Based Analysis of Transcription
Authors: Minakhin Leonid , Nechaev Sergei , Campbell Elizabeth A. , Severinov Konstantin
Published: July 2002

The three-dimensional structure of DNA-dependent RNA polymerase (RNAP) from thermophilic
Thermus aquaticus
has recently been determined at 3.3 Å resolution. Currently, very little is known about
T. aquaticus
transcription and no genetic system to study
T. aquaticus
RNAP genes is available. To overcome these limitations, we cloned and overexpressed
T. aquaticus
RNAP genes in
Escherichia coli
. Overproduced
T. aquaticus
RNAP subunits assembled into functional RNAP in vitro and in vivo when coexpressed in
E. coli
. We used the recombinant
T. aquaticus
enzyme to demonstrate that transcription initiation, transcription termination, and transcription cleavage assays developed for
E. coli
RNAP can be adapted to study
T. aquaticus
transcription. However,
T. aquaticus
RNAP differs from the prototypical
E. coli
enzyme in several important ways: it terminates transcription less efficiently, has exceptionally high rate of intrinsic transcript cleavage, and is highly resistant to rifampin. Our results, together with the high-resolution structural information, should now allow a rational analysis of transcription mechanism by mutation.
Genetic Characterization of theKlebsiella pneumoniae waaGene Cluster, Involved in Core Lipopolysaccharide Biosynthesis
Authors: Regué Miguel , Climent Núria , Abitiu Nihal , Coderch Núria , Merino Susana , Izquierdo Luis , Altarriba Maria , Tomás Juan M.
Published: July 2002

A recombinant cosmid containing genes involved inKlebsiella pneumoniaeC3 core lipopolysaccharide biosynthesis was identified by its ability to confer bacteriocin 28b resistance toEscherichia coliK-12. The recombinant cosmid contains 12 genes, the wholewaagene cluster, flanked bykblandcoaDgenes, as was found inE. coliK-12. PCR amplification analysis showed that this cluster is conserved in representativeK. pneumoniaestrains. Partial nucleotide sequence determination showed that the same genes and gene order are found inK. pneumoniaesubsp.ozaenae, for which the core chemical structure is known. Complementation analysis of knownwaamutants fromE. coliK-12 and/orSalmonella entericaled to the identification of genes involved in biosynthesis of the inner core backbone that are shared by these three members of theEnterobacteriaceae. K. pneumoniae orf10mutants showed a two-log-fold reduction in a mice virulence assay and a strong decrease in capsule amount. Analysis of a constructedK. pneumoniae waaEdeletion mutant suggests that the WaaE protein is involved in the transfer of the branch β-d-Glc to the O-4 position ofl-glycero-d-manno-heptose I, a feature shared byK. pneumoniae, Proteus mirabilis, andYersinia enterocolitica.
PhcS Represses Gratuitous Expression of Phenol-Metabolizing Enzymes in
Comamonas testosteroni
R5
Authors: Teramoto Maki , Harayama Shigeaki , Watanabe Kazuya
Published: July 2002

We identified an open reading frame, designated
phcS
, downstream of the transcriptional activator gene (
phcR
) for the expression of multicomponent phenol hydroxylase (mPH) in
Comamonas testosteroni
R5. The deduced product of
phcS
was homologous to AphS of
C. testosteroni
TA441, which belongs to the GntR family of transcriptional regulators. The transformation of
Pseudomonas aeruginosa
PAO1c (phenol negative, catechol positive) with pROR502 containing
phcR
and the mPH genes conferred the ability to grow on phenol, while transformation with pROR504 containing
phcS
,
phcR
, and mPH genes did not confer this ability. The disruption of
phcS
in strain R5 had no effect on its phenol-oxygenating activity in a chemostat culture with phenol. The phenol-oxygenating activity was not expressed in strain R5 grown in a chemostat with acetate. In contrast, the phenol-oxygenating activity in the strain with a knockout
phcS
gene when grown in a chemostat with acetate as the limiting growth factor was 66% of that obtained in phenol-grown cells of the strain with a knockout in the
phcS
gene. The disruption of
phcS
and/or
phcR
and the complementation in
trans
of these defects confirm that PhcS is a
trans
-acting repressor and that the unfavorable expression of mPH in the
phcS
knockout cells grown on acetate requires PhcR. These results show that the PhcS protein repressed the gratuitous expression of phenol-metabolizing enzymes in the absence of the genuine substrate and that strain R5 acted by an unknown mechanism in which the PhcS-mediated repression was overcome in the presence of the pathway substrate.
Extracellular Synthesis, Specific Recognition, and Intracellular Degradation of Cyclomaltodextrins by the Hyperthermophilic Archaeon
Thermococcus
sp. Strain B1001
Authors: Hashimoto Yoshiteru , Yamamoto Tomoko , Fujiwara Shinsuke , Takagi Masahiro , Imanaka Tadayuki
Published: July 2002

A unique extracellular and thermostable cyclomaltodextrin glucanotransferase (CGTase) from the hyperthermophilic archaeon
Thermococcus
sp. strain B1001 produces predominantly (>85%) α-cyclomaltodextrin (α-CD) from starch (Y. Tachibana, et al., Appl. Environ. Microbiol. 65:1991–1997, 1999). Nucleotide sequencing of the CGTase gene (
cgtA
) and its flanking region was performed, and a cluster of five genes was found, including a gene homolog encoding a cyclomaltodextrinase (CDase) involved in the degradation of CDs (
cgtB
), the gene encoding CGTase (
cgtA
), a gene homolog for a CD-binding protein (CBP) (
cgtC
), and a putative CBP-dependent ABC transporter involved in uptake of CDs (
cgtDE
). The CDase was expressed in
Escherichia coli
and purified. The optimum pH and temperature for CD hydrolysis were 5.5 and 95°C, respectively. The molecular weight of the recombinant enzyme was estimated to be 79,000. The CDase hydrolyzed β-CD most efficiently among other CDs. Maltose and pullulan were not utilized as substrates. Linear maltodextrins with a small glucose unit were very slowly hydrolyzed, and starch was hydrolyzed more slowly. Analysis by thin-layer chromatography revealed that glucose and maltose were produced as end products. The purified recombinant CBP bound to maltose as well as to α-CD. However, the CBP exhibited higher thermostability in the presence of α-CD. These results suggested that strain B1001 possesses a unique metabolic pathway that includes extracellular synthesis, transmembrane uptake, and intracellular degradation of CDs in starch utilization. Potential advantages of this starch metabolic pathway via CDs are discussed.
Phosphoenolpyruvate Synthetase from the Hyperthermophilic Archaeon
Pyrococcus furiosus
Authors: Hutchins Andrea M. , Holden James F. , Adams Michael W. W.
Published: July 2002

Phosphoenolpyruvate synthetase (PpsA) was purified from the hyperthermophilic archaeon
Pyrococcus furiosus
. This enzyme catalyzes the conversion of pyruvate and ATP to phosphoenolpyruvate (PEP), AMP, and phosphate and is thought to function in gluconeogenesis. PpsA has a subunit molecular mass of 92 kDa and contains one calcium and one phosphorus atom per subunit. The active form has a molecular mass of 690 ± 20 kDa and is assumed to be octomeric, while approximately 30% of the protein is purified as a large (∼1.6 MDa) complex that is not active. The apparent

K
m

values and catalytic efficiencies for the substrates pyruvate and ATP (at 80°C, pH 8.4) were 0.11 mM and 1.43 × 10
4
mM
−1
· s
−1
and 0.39 mM and 3.40 × 10
3
mM
−1
· s
−1
, respectively. Maximal activity was measured at pH 9.0 (at 80°C) and at 90°C (at pH 8.4). The enzyme also catalyzed the reverse reaction, but the catalytic efficiency with PEP was very low [
k
cat
/

K
m

= 32 (mM · s)
−1
]. In contrast to several other nucleotide-dependent enzymes from
P. furiosus
, PpsA has an absolute specificity for ATP as the phosphate-donating substrate. This is the first PpsA from a nonmethanogenic archaeon to be biochemically characterized. Its kinetic properties are consistent with a role in gluconeogenesis, although its relatively high cellular concentration (∼5% of the cytoplasmic protein) suggests an additional function possibly related to energy spilling. It is not known whether interconversion between the smaller, active and larger, inactive forms of the enzyme has any functional role.
Definition of the
Bacillus subtilis
PurR Operator Using Genetic and Bioinformatic Tools and Expansion of the PurR Regulon with
glyA
,
guaC
,
pbuG
,
xpt-pbuX
,
yqhZ-folD
, and
pbuO
Authors: Saxild Hans H. , Brunstedt Katja , Nielsen Karin I. , Jarmer Hanne , Nygaard Per
Published: July 2002

The expression of the
pur
operon, which encodes enzymes of the purine biosynthetic pathway in
Bacillus subtilis
, is subject to control by the
purR
gene product (PurR) and phosphoribosylpyrophosphate. This control is also exerted on the
purA
and
purR
genes. A consensus sequence for the binding of PurR, named the PurBox, has been suggested (M. Kilstrup, S. G. Jessing, S. B. Wichmand-Jørgensen, M. Madsen, and D. Nilsson, J. Bacteriol. 180:3900–3906, 1998). To determine whether the expression of other genes might be regulated by PurR, we performed a search for PurBox sequences in the
B. subtilis
genome sequence and found several candidate PurBoxes. By the use of transcriptional
lacZ
fusions, five selected genes or operons (
glyA
,
yumD
,
yebB
,
xpt-pbuX
, and
yqhZ-folD
), all having a putative PurBox in their upstream regulatory regions, were found to be regulated by PurR. Using a machine-learning algorithm developed for sequence pattern finding, we found that all of the genes identified as being PurR regulated have two PurBoxes in their upstream control regions. The two boxes are divergently oriented, forming a palindromic sequence with the inverted repeats separated by 16 or 17 nucleotides. A computerized search revealed one additional PurR-regulated gene,
ytiP
. The significance of the tandem PurBox motifs was demonstrated in vivo by deletion analysis and site-directed mutagenesis of the two PurBox sequences located upstream of
glyA
. All six genes or operons encode enzymes or transporters playing a role in purine nucleotide metabolism. Functional analysis showed that
yebB
encodes the previously characterized hypoxanthine-guanine permease PbuG and that
ytiP
encodes another guanine-hypoxanthine permease and is now named
pbuO. yumD
encodes a GMP reductase and is now named
guaC
.
Conditional-Replication, Integration, Excision, and Retrieval Plasmid-Host Systems for Gene Structure-Function Studies of Bacteria
Authors: Haldimann Andreas , Wanner Barry L.
Published: July 2002

We have developed a series of powerful and versatile conditional-replication, integration, and modular (CRIM) plasmids. CRIM plasmids can be replicated at medium or high copy numbers in different hosts for making gene (or mutant) libraries. They can be integrated in single copies into the chromosomes of
Escherichia coli
and related bacteria to study gene function under normal physiological conditions. They can be excised from the chromosome, e.g., to verify that phenotypes are caused by their presence. Furthermore, they can be retrieved singly or en masse for subsequent molecular analyses. CRIM plasmids are integrated into the chromosome by site-specific recombination at one of five different phage attachment sites. Integrants are selected as antibiotic-resistant transformations. Since CRIM plasmids encode different forms of resistance, several can be used together in the same cell for stable expression of complex metabolic or regulatory pathways from diverse sources. Following integration, integrants are stably maintained in the absence of antibiotic selection. Each CRIM plasmid has a polylinker or one of several promoters for ectopic expression of the inserted DNA. Their modular design allows easy construction of new variants with different combinations of features. We also report a series of easily curable, low-copy-number helper plasmids encoding all the requisite Int proteins alone or with the respective Xis protein. These helper plasmids facilitate integration, excision (“curing”), or retrieval of the CRIM plasmids.
Homologues of Neisserial Heme Oxygenase in Gram-Negative Bacteria: Degradation of Heme by the Product of the
pigA
Gene of
Pseudomonas aeruginosa
Authors: Ratliff Melanie , Zhu Wenming , Deshmukh Rahul , Wilks Angela , Stojiljkovic Igor
Published: July 2002

The oxidative cleavage of heme to release iron is a mechanism by which some bacterial pathogens can utilize heme as an iron source. The
pigA
gene of
Pseudomonas aeruginosa
is shown to encode a heme oxygenase protein, which was identified in the genome sequence by its significant homology (37%) with HemO of
Neisseria meningitidis
. When the gene encoding the neisserial heme oxygenase,
hemO
, was replaced with
pigA
, we demonstrated that
pigA
could functionally replace
hemO
and allow for heme utilization by neisseriae. Furthermore, when
pigA
was disrupted by cassette mutagenesis in
P. aeruginosa
, heme utilization was defective in iron-poor media supplemented with heme. This defect could be restored both by the addition of exogenous FeSO
4
, indicating that the mutant did not have a defect in iron metabolism, and by in
trans
complementation with
pigA
from a plasmid with an inducible promoter. The PigA protein was purified by ion-exchange chromotography. The UV-visible spectrum of PigA reconstituted with heme showed characteristics previously reported for other bacterial and mammalian heme oxygenases. The heme-PigA complex could be converted to ferric biliverdin in the presence of ascorbate, demonstrating the need for an exogenous reductant. Acidification and high-performance liquid chromatography analysis of the ascorbate reduction products identified a major product of biliverdin IX-β. This differs from the previously characterized heme oxygenases in which biliverdin IX-α is the typical product. We conclude that PigA is a heme oxygenase and may represent a class of these enzymes with novel regiospecificity.
Cloning and Characterization of Benzoate Catabolic Genes in the Gram-Positive Polychlorinated Biphenyl Degrader
Rhodococcus
sp. Strain RHA1
Authors: Kitagawa Wataru , Miyauchi Keisuke , Masai Eiji , Fukuda Masao
Published: July 2002

Benzoate catabolism is thought to play a key role in aerobic bacterial degradation of biphenyl and polychlorinated biphenyls (PCBs). Benzoate catabolic genes were cloned from a PCB degrader,
Rhodococcus
sp. strain RHA1, by using PCR amplification and temporal temperature gradient electrophoresis separation. A nucleotide sequence determination revealed that the deduced amino acid sequences encoded by the RHA1 benzoate catabolic genes,
benABCDK
, exhibit 33 to 65% identity with those of
Acinetobacter
sp. strain ADP1. The gene organization of the RHA1
benABCDK
genes differs from that of ADP1. The RHA1
benABCDK
region was localized on the chromosome, in contrast to the biphenyl catabolic genes, which are located on linear plasmids.
Escherichia coli
cells containing RHA1
benABCD
transformed benzoate to catechol via 2-hydro-1,2-dihydroxybenzoate. They transformed neither 2- nor 4-chlorobenzoates but did transform 3-chlorobenzoate. The RHA1
benA
gene was inactivated by insertion of a thiostrepton resistance gene. The resultant mutant strain, RBD169, neither grew on benzoate nor transformed benzoate, and it did not transform 3-chlorobenzoate. It did, however, exhibit diminished growth on biphenyl and growth repression in the presence of a high concentration of biphenyl (13 mM). These results indicate that the cloned
benABCD
genes could play an essential role not only in benzoate catabolism but also in biphenyl catabolism in RHA1. Six rhodococcal benzoate degraders were found to have homologs of RHA1
benABC
. In contrast, two rhodococcal strains that cannot transform benzoate were found not to have RHA1
benABC
homologs, suggesting that many
Rhodococcus
strains contain benzoate catabolic genes similar to RHA1
benABC
.