bluuebunny/crossref_metadata_2025 huggingface dataset:
The Clp Proteases ofBacillus subtilisAre Directly Involved in Degradation of Misfolded Proteins
Authors: Krüger Elke , Witt Elke , Ohlmeier Steffen , Hanschke Renate , Hecker Michael
Published: July 2002

The presence of the heat stress response-related ATPases ClpC and ClpX or the peptidase ClpP in the cell is crucial for tolerance of many forms of stress inBacillus subtilis. Assays for detection of defects in protein degradation suggest that ClpC, ClpP, and ClpX participate directly in overall proteolysis of misfolded proteins. Turnover rates for abnormal puromycyl peptides are significantly decreased inclpC,clpP, andclpXmutant cells. Electron-dense aggregates, most likely due to the accumulation of misfolded proteins, were noticed in studies of ultrathin cryosections inclpCandclpPmutant cells even under nonstress conditions. In contrast, in the wild type orclpXmutants such aggregates could only be observed after heat shock. This phenomenon supports the assumption thatclpCandclpPmutants are deficient in the ability to solubilize or degrade damaged and aggregated proteins, the accumulation of which is toxic for the cell. By using immunogold labeling with antibodies raised against ClpC, ClpP, and ClpX, the Clp proteins were localized in these aggregates, showing that the Clp proteins act at this level in vivo.
A Point Mutation in the
mma3
Gene Is Responsible for Impaired Methoxymycolic Acid Production in
Mycobacterium bovis
BCG Strains Obtained after 1927
Authors: Behr Marcel A. , Schroeder Benjamin G. , Brinkman Jacquelyn N. , Slayden Richard A. , Barry Clifton E.
Published: July 2002

BCG vaccines are substrains of
Mycobacterium bovis
derived by attenuation in vitro. After the original attenuation (1908 to 1921), BCG strains were maintained by serial propagation in different BCG laboratories (1921 to 1961). As a result, various BCG substrains developed which are now known to differ in a number of genetic and phenotypic properties. However, to date, none of these differences has permitted a direct phenotype-genotype link. Since BCG strains differ in their abilities to synthesize methoxymycolic acids and since recent work has shown that the
mma3
gene is responsible for O-methylation of hydroxymycolate precursors to form methoxymycolic acids, we analyzed methoxymycolate production and
mma3
gene sequences for a genetically defined collection of BCG strains. We found that BCG strains obtained from the Pasteur Institute in 1927 and earlier produced methoxymycolates in vitro but that those obtained from the Pasteur Institute in 1931 and later all failed to synthesize methoxymycolates, and furthermore, the
mma3
sequence of the latter strains differs from that of
Mycobacterium tuberculosis
H37Rv by a point mutation at bp 293. Site-specific introduction of this guanine-to-adenine mutation into wild-type
mma3
(resulting in the replacement of glycine 98 with aspartic acid) eliminated the ability of this enzyme to produce O-methylated mycolic acids when the mutant was cloned in tandem with
mma4
into
Mycobacterium smegmatis
. These findings indicate that a point mutation in
mma3
occurred between 1927 and 1931, and that this mutant population became the dominant clone of BCG at the Pasteur Institute.
An Abundant DNA Binding Protein from the Hyperthermophilic Archaeon
Sulfolobus shibatae
Affects DNA Supercoiling in a Temperature-Dependent Fashion
Authors: Xue Hong , Guo Rong , Wen Yunfei , Liu Danxu , Huang Li
Published: July 2002

The DNA binding protein Ssh10b, a member of the Sac10b family, has been purified from the hyperthermophilic archaeon
Sulfolobus shibatae
. Ssh10b constitutes about 4% of the cellular protein. Electrophoretic mobility shift assays showed that Ssh10b first bound a double-stranded DNA fragment with an estimated binding size of ∼∼12 bp, forming distinct shifts, until the DNA was coated with the protein. Binding of more Ssh10b resulted in the formation of smears of lower mobilities. The migration pattern of the smearing Ssh10b-DNA complexes was affected by temperature, whereas that of complexes associated with the distinct shifts was not. Interestingly, Ssh10b was capable of constraining negative DNA supercoils in a temperature-dependent fashion. While the ability of the protein to constrain supercoils was weak at 25°C, it was enhanced substantially at 45°C or higher temperatures (up to 80°C). Taken together, our data suggest that archaeal proteins of the Sac10b family may affect the topology of chromosomal DNA in thermophilic archaea at their growth temperatures.
Analysis of MinC Reveals Two Independent Domains Involved in Interaction with MinD and FtsZ
Authors: Hu Zonglin , Lutkenhaus Joe
Published: July 2002

In
Escherichia coli
FtsZ assembles into a Z ring at midcell while assembly at polar sites is prevented by the
min
system. MinC, a component of this system, is an inhibitor of FtsZ assembly that is positioned within the cell by interaction with MinDE. In this study we found that MinC consists of two functional domains connected by a short linker. When fused to MalE the N-terminal domain is able to inhibit cell division and prevent FtsZ assembly in vitro. The C-terminal domain interacts with MinD, and expression in wild-type cells as a MalE fusion disrupts
min
function, resulting in a minicell phenotype. We also find that MinC is an oligomer, probably a dimer. Although the C-terminal domain is clearly sufficient for oligomerization, the N-terminal domain also promotes oligomerization. These results demonstrate that MinC consists of two independently functioning domains: an N-terminal domain capable of inhibiting FtsZ assembly and a C-terminal domain responsible for localization of MinC through interaction with MinD. The fusion of these two independent domains is required to achieve topological regulation of Z ring assembly.
Identification of aMycobacterium tuberculosisGene That Enhances Mycobacterial Survival in Macrophages
Authors: Wei Jun , Dahl John L. , Moulder James W. , Roberts Esteban A. , O'Gaora Peadar , Young Douglas B. , Friedman Richard L.
Published: July 2002

Intracellular survival plays a central role in the pathogenesis ofMycobacterium tuberculosis. To identifyM. tuberculosisgenes required for intracellular survival within macrophages, anM. tuberculosisH37Rv plasmid library was constructed by using the shuttle vector pOLYG. This plasmid library was electroporated intoMycobacterium smegmatis1-2c, and the transformants were used to infect the human macrophage-like cell line U-937. BecauseM. smegmatisdoes not readily survive within macrophages, any increased intracellular survival is likely due to clonedM. tuberculosisH37Rv DNA. After six sequential passages ofM. smegmatistransformants through U-937 cells, one clone (p69) was enriched more than 70% as determined by both restriction enzyme and PCR analyses. p69 demonstrated significantly enhanced survival compared to that of the vector control, ranging from 2.4- to 5.3-fold at both 24 and 48 h after infection. DNA sequence analysis revealed three open reading frames (ORFs) in the insert of p69. ORF2 (1.2 kb) was the only one which contained a putative promoter region and a ribosome-binding site. Deletion analysis of the p69 insert DNA showed that disruption of ORF2 resulted in complete loss of the enhanced intracellular survival phenotype. This gene was named the enhanced intracellular survival (eis) gene. By using an internal region ofeisas a probe for Southern analysis,eiswas found in the genomic DNA of variousM. tuberculosisstrains and ofMycobacterium bovisBCG but not in that ofM. smegmatisor 10 other nonpathogenic mycobacterial species. Sodium dodecyl sulfate-polyacrylamide gel electrophoretic analysis showed that allM. smegmatis eis-containing constructs expressed a unique protein of 42 kDa, the predicted size of Eis. The expression of this 42-kDa protein directly correlated to the enhanced survival ofM. smegmatisp69 in U-937 cells. These results suggest a possible role foreisand its protein product in the intracellular survival ofM. tuberculosis.
Mobilization of Poly(3-Hydroxybutyrate) inRalstonia eutropha
Authors: Handrick René , Reinhardt Simone , Jendrossek Dieter
Published: July 2002

Ralstonia eutrophaH16 degraded (mobilized) previously accumulated poly(3-hydroxybutyrate) (PHB) in the absence of an exogenous carbon source and used the degradation products for growth and survival. Isolated native PHB granules of mobilizedR. eutrophacells released 3-hydroxybutyrate (3HB) at a threefold higher rate than did control granules of nonmobilized bacteria. No 3HB was released by native PHB granules of recombinantEscherichia coliexpressing the PHB biosynthetic genes. Native PHB granules isolated from chromosomal knockout mutants of an intracellular PHB (i-PHB) depolymerase gene ofR. eutrophaH16 and HF210 showed a reduced but not completely eliminated activity of 3HB release and indicated the presence of i-PHB depolymerase isoenzymes.
Cholic Acid Is Accumulated Spontaneously, Driven by Membrane ΔpH, in Many Lactobacilli
Authors: Kurdi Peter , van Veen Hendrik W. , Tanaka Hiroshi , Mierau Igor , Konings Wil N. , Tannock Gerald W. , Tomita Fusao , Yokota Atsushi
Published: July 2002

Many lactobacilli from various origins were found to apparently lack cholic acid extrusion activity. Cholic acid was accumulated spontaneously, driven by the transmembrane proton gradient. Accumulation is a newly identified kind of interaction between intestinal microbes and unconjugated bile acids and is different from extrusion and modification, which have been described previously.
Regulatable Arabinose-Inducible Gene Expression System with Consistent Control in All Cells of a Culture
Authors: Khlebnikov Artem , Risa Øystein , Skaug Tove , Carrier Trent A. , Keasling J. D.
Published: July 2002

The arabinose-inducible promoter

P
BAD

is subject to all-or-none induction, in which intermediate concentrations of arabinose give rise to subpopulations of cells that are fully induced and uninduced. To construct a host-vector expression system with regulatable control in a homogeneous population of cells, the
araE
gene of
Escherichia coli
was cloned into an RSF1010-derived plasmid under control of the isopropyl-β-
d
-thiogalactopyranoside-inducible

P
tac

and

P
taclac

promoters. This gene encodes the low-affinity, high-capacity arabinose transport protein and is controlled natively by an arabinose-inducible promoter. To detect the effect of arabinose-independent
araE
expression on population homogeneity and cell-specific expression, the
gfpuv
gene was placed under control of the arabinose-inducible
araBAD
promoter (

P
BAD

) on the pMB1-derived plasmid pBAD24. The transporter and reporter plasmids were transformed into
E. coli
strains with native arabinose transport systems and strains deficient in one or both of the arabinose transport systems (
araE
and/or
araFGH
). The effects of the arabinose concentration and arabinose-independent transport control on population homogeneity were investigated in these strains using flow cytometry. The
araE
, and
araE araFGH
mutant strains harboring the transporter and reporter plasmids were uniformly induced across the population at all inducer concentrations, and the level of gene expression in individual cells varied with arabinose concentration. In contrast, the parent strain, which expressed the native
araE
and
araFGH
genes and harbored the transporter and reporter plasmids, exhibited all-or-none behavior. This work demonstrates the importance of including a transport gene that is controlled independently of the inducer to achieve regulatable and consistent induction in all cells of the culture.
The Truncated Form of the Bacterial Heat Shock Protein ClpB/HSP100 Contributes to Development of Thermotolerance in the Cyanobacterium
Synechococcus
sp. Strain PCC 7942
Authors: Clarke Adrian K. , Eriksson Mats-Jerry
Published: July 2002

ClpB is a highly conserved heat shock protein that is essential for thermotolerance in bacteria and eukaryotes. One distinctive feature of all bacterial
clpB
genes is the dual translation of a truncated 79-kDa form (ClpB-79) in addition to the full-length 93-kDa protein (ClpB-93). To investigate the currently unknown function of ClpB-79, we have examined the ability of the two different-sized ClpB homologues from the cyanobacterium
Synechococcus
sp. strain PCC 7942 to confer thermotolerance. We show that the ClpB-79 form has the same capacity as ClpB-93 to confer thermotolerance and that the ClpB-79 protein contributes ca. one-third of the total thermotolerance developed in wild-type
Synechococcus
, the first in vivo demonstration of a functional role for ClpB-79 in bacteria.
The Amino Terminus of Bacteriophage λ Integrase Is Involved in Protein-Protein Interactions during Recombination
Authors: Jessop Lea , Bankhead Troy , Wong David , Segall Anca M.
Published: July 2002

Bacteriophage lambda integrase (Int) catalyzes at least four site-specific recombination pathways between pairs of attachment (
att
) sites. Protein-protein contacts between monomers of Int are presumed to be important for these site-specific recombination events for several reasons: Int binds to the
att
sites cooperatively, catalytic Int mutants can complement each other for strand cleavage, and crystal structures for two other recombinases in the Int family (Cre from phage P1 and Int from
Haemophilus influenzae
phage HP1) show extensive protein-protein contacts between monomers. We have begun to investigate interactions between Int monomers by three approaches. First, using a genetic assay, we show that regions of protein-protein interactions occur throughout Int, including in the amino-terminal domain. This domain was previously thought to be important only for high-affinity protein-DNA interactions. Second, we have found that an amino-terminal His tag reduces cooperative binding to DNA. This disruption in cooperativity decreases the stable interaction of Int with core sites, where catalysis occurs. Third, using protein-protein cross-linking to investigate the multimerization of Int during recombination, we show that Int predominantly forms dimers, trimers, and tetramers. Moreover, we show that the cysteine at position 25 is present at or near the interface between monomers that is involved in the formation of dimers and tetramers. Our evidence indicates that the amino-terminal domain of Int is involved in protein-protein interactions that are likely to be important for recombination.
Distribution of Twelve Linear Extrachromosomal DNAs in Natural Isolates of Lyme Disease Spirochetes
Authors: Palmer Nanette , Fraser Claire , Casjens Sherwood
Published: July 2002

We have analyzed a panel of independent North American isolates of the Lyme disease agent spirochete,
Borrelia burgdorferi
(sensu stricto), for the presence of linear plasmids with sequence similarities to the 12 linear plasmids present in the
B. burgdorferi
type strain, isolate B31. The frequency of similarities to probes from each of the 12 B31 plasmids varied from 13 to 100% in the strain panel examined, and these similarities usually reside on plasmids similar in size to the cognate B31 plasmid. Sequences similar to 5 of the 12 B31 plasmids were found in all of the isolates examined, and >66% of the panel members hybridized to probes from 4 other plasmids. Sequences similar to most of the
B. burgdorferi
B31 plasmid-derived DNA probes used were also found on linear plasmids in the related Eurasian Lyme agents
Borrelia garinii
and
Borrelia afzelii
; however, some of these plasmids had uniform but substantially different sizes from their
B. burgdorferi
counterparts.
Generation of Dominant Selectable Markers for Resistance to Pseudomonic Acid by Cloning and Mutagenesis of the
ileS
Gene from the Archaeon
Methanosarcina barkeri
Fusaro
Authors: Boccazzi Paolo , Zhang Jun Kai , Metcalf William W.
Published: July 2002

Currently, only one selectable marker is available for genetic studies in the archaeal genus
Methanosarcina
. Here we report the generation of selectable markers that encode resistance to pseudomonic acid (PA
r
) in
Methanosarcina
species by mutagenesis of the isoleucyl-tRNA synthetase gene (
ileS
) from
Methanosarcina barkeri
Fusaro. The
M. barkeri ileS
gene was obtained by screening of a genomic library for hybridization to a PCR fragment. The complete 3,787-bp DNA sequence surrounding and including the
ileS
gene was determined. As expected,
M. barkeri
IleS is phylogenetically related to other archaeal IleS proteins. The
ileS
gene was cloned into a
Methanosarcina-Escherichia coli
shuttle vector and mutagenized with hydroxylamine. Nine independent PA
r
clones were isolated after transformation of
Methanosarcina acetivorans
C2A with the mutagenized plasmids. Seven of these clones carry multiple changes from the wild-type sequence. Most mutations that confer PA
r
were shown to alter amino acid residues near the KMSKS consensus sequence of class I aminoacyl-tRNA synthetases. One particular mutation (G594E) was present in all but one of the PA
r
clones. The MIC of pseudomonic acid for
M. acetivorans
transformed with a plasmid carrying this single mutation is 70 μg/ml of medium (for the wild type, the MIC is 12 μg/ml). The highest MICs (560 μg/ml) were observed with two triple mutants, A440V/A482T/G594E and A440V/G593D/G594E. Plasmid shuttle vectors and insertion cassettes that encode PA
r
based on the mutant
ileS
alleles are described. Finally, the implications of the specific mutations we isolated with respect to binding of pseudomonic acid by IleS are discussed.
Twofold Reduction of Phosphofructokinase Activity in
Lactococcus lactis
Results in Strong Decreases in Growth Rate and in Glycolytic Flux
Authors: Andersen Heidi W. , Solem Christian , Hammer Karin , Jensen Peter R.
Published: July 2002

Two mutant strains of
Lactococcus lactis
in which the promoter of the
las
operon, harboring
pfk, pyk
, and
ldh
, were replaced by synthetic promoters were constructed. These
las
mutants had an approximately twofold decrease in the activity of phosphofructokinase, whereas the activities of pyruvate kinase and lactate dehydrogenase remained closer to the wild-type level. In defined medium supplemented with glucose, the growth rate of the mutants was reduced to 57 to 70% of wild-type levels and the glycolytic flux was reduced to 62 to 76% of wild-type levels. In complex medium growth was even further reduced. Surprisingly, the mutants still showed homolactic fermentation, which indicated that the limitation was different from standard glucose-limited conditions. One explanation could be that the reduced activity of phosphofructokinase resulted in the accumulation of sugar-phosphates. Indeed, when one of the mutants was starved for glucose in glucose-limited chemostat, the growth rate could gradually be increased to 195% of the growth rate observed in glucose-saturated batch culture, suggesting that phosphofructokinase does affect the concentration of upstream metabolites. The pools of glucose-6-phosphate and fructose-6-phosphate were subsequently found to be increased two- to fourfold in the
las
mutants, which indicates that phosphofructokinase exerts strong control over the concentration of these metabolites.
Analysis of the PilQ Secretin from
Neisseria meningitidis
by Transmission Electron Microscopy Reveals a Dodecameric Quaternary Structure
Authors: Collins Richard F. , Davidsen Linn , Derrick Jeremy P. , Ford Robert C. , Tønjum Tone
Published: July 2002

PilQ is a member of the secretin family of outer membrane proteins and is specifically involved in secretion of type IV pili in
Neisseria meningitidis, Neisseria gonorrhoeae
, and
Pseudomonas aeruginosa
. The quaternary structure of PilQ from
N. meningitidis
was analyzed by transmission electron microscopy by using a negative stain. Single particle averaging was carried out with a total data set of 650 individual particles, which produced a projection map generated from 296 particles at an estimated resolution of 2.6 nm. Oligomeric PilQ adopts a donut-like structure with an external ring that is 16.5 nm in diameter surrounding a central cavity that is 6.5 nm in diameter. Self-rotation and power spectrum analysis demonstrated the presence of 12-fold rotational symmetry, showing that PilQ is organized as a ring of 12 identical subunits. A model of the type IV meningococcal pilus fiber, based on the X-ray crystal structure of the
N. gonorrhoeae
pilin subunit, fitted neatly into the cavity, demonstrating how PilQ could serve as a channel for the growing pilus fiber.
Succinate Dehydrogenase and Other Respiratory Pathways in Thylakoid Membranes of
Synechocystis
sp. Strain PCC 6803: Capacity Comparisons and Physiological Function
Authors: Cooley Jason W. , Vermaas Wim F. J.
Published: July 2002

Respiration in cyanobacterial thylakoid membranes is interwoven with photosynthetic processes. We have constructed a range of mutants that are impaired in several combinations of respiratory and photosynthetic electron transport complexes and have examined the relative effects on the redox state of the plastoquinone (PQ) pool by using a quinone electrode. Succinate dehydrogenase has a major effect on the PQ redox poise, as mutants lacking this enzyme showed a much more oxidized PQ pool. Mutants lacking type I and II NAD(P)H dehydrogenases also had more oxidized PQ pools. However, in the mutant lacking type I NADPH dehydrogenase, succinate was essentially absent and effective respiratory electron donation to the PQ pool could be established after addition of 1 mM succinate. Therefore, lack of the type I NADPH dehydrogenase had an indirect effect on the PQ pool redox state. The electron donation capacity of succinate dehydrogenase was found to be an order of magnitude larger than that of type I and II NAD(P)H dehydrogenases. The reason for the oxidized PQ pool upon inactivation of type II NADH dehydrogenase may be related to the facts that the NAD pool in the cell is much smaller than that of NADP and that the NAD pool is fully reduced in the mutant without type II NADH dehydrogenase, thus causing regulatory inhibition. The results indicate that succinate dehydrogenase is the main respiratory electron transfer pathway into the PQ pool and that type I and II NAD(P)H dehydrogenases regulate the reduction level of NADP and NAD, which, in turn, affects respiratory electron flow through succinate dehydrogenase.
The
gdhB
Gene of
Pseudomonas aeruginosa
Encodes an Arginine-Inducible NAD
+
-Dependent Glutamate Dehydrogenase Which Is Subject to Allosteric Regulation
Authors: Lu Chung-Dar , Abdelal Ahmed T.
Published: July 2002

The NAD
+
-dependent glutamate dehydrogenase (NAD-GDH) from
Pseudomonas aeruginosa
PAO1 was purified, and its amino-terminal amino acid sequence was determined. This sequence information was used in identifying and cloning the encoding
gdhB
gene and its flanking regions. The molecular mass predicted from the derived sequence for the encoded NAD-GDH was 182.6 kDa, in close agreement with that determined from sodium dodecyl sulfate-polyacrylamide gel electrophoresis of the purified enzyme (180 kDa). Cross-linking studies established that the native NAD-GDH is a tetramer of equal subunits. Comparison of the derived amino acid sequence of NAD-GDH from
P. aeruginosa
with the GenBank database showed the highest homology with hypothetical polypeptides from
Pseudomonas putida
,
Mycobacterium tuberculosis
,
Rickettsia prowazakii
,
Legionella pneumophila
,
Vibrio cholerae
,
Shewanella putrefaciens
,
Sinorhizobium meliloti
, and
Caulobacter crescentus.
A moderate degree of homology, primarily in the central domain, was observed with the smaller tetrameric NAD-GDH (protomeric mass of 110 kDa) from
Saccharomyces cerevisiae
or
Neurospora crassa
. Comparison with the yet smaller hexameric GDH (protomeric mass of 48 to 55 kDa) of other prokaryotes yielded a low degree of homology that was limited to residues important for binding of substrates and for catalytic function. NAD-GDH was induced 27-fold by exogenous arginine and only 3-fold by exogenous glutamate. Primer extension experiments established that transcription of
gdhB
is initiated from an arginine-inducible promoter and that this induction is dependent on the arginine regulatory protein, ArgR, a member of the AraC/XyIS family of regulatory proteins. NAD-GDH was purified to homogeneity from a recombinant strain of
P. aeruginosa
and characterized. The glutamate saturation curve was sigmoid, indicating positive cooperativity in the binding of glutamate. NAD-GDH activity was subject to allosteric control by arginine and citrate, which function as positive and negative effectors, respectively. Both effectors act by influencing the affinity of the enzyme for glutamate. NAD-GDH from this organism differs from previously characterized enzymes with respect to structure, protomer mass, and allosteric properties indicate that this enzyme represents a novel class of microbial glutamate dehydrogenases.
Surface Display of Recombinant Proteins onBacillus subtilisSpores
Authors: Isticato Rachele , Cangiano Giuseppina , Tran Hoa T. , Ciabattini Annalisa , Medaglini Donata , Oggioni Marco R. , De Felice Maurilio , Pozzi Gianni , Ricca Ezio
Published: July 2002

We developed a novel surface display system based on the use of bacterial spores. A protein of theBacillus subtilisspore coat, CotB, was found to be located on the spore surface and used as fusion partner to express the 459-amino-acid C-terminal fragment of the tetanus toxin (TTFC). Western, dot blot and fluorescent-activated cell sorting analyses were used to monitor TTFC surface expression on purified spores. We estimated that more than 1.5 × 103TTFC molecules were exposed on the surface of each spore and recognized by TTFC-specific antibodies. The efficient surface presentation of the heterologous protein, together with the simple purification procedure and the high stability and safety record ofB. subtilisspores, makes this spore-based display system a potentially powerful approach for surface expression of bioactive molecules.
Identification of a Plasmid-Encoded Gene from
Haemophilus ducreyi
Which Confers NAD Independence
Authors: Martin Paul R. , Shea Robin J. , Mulks Martha H.
Published: July 2002

Members of the family
Pasteurellaceae
are classified in part by whether or not they require an NAD supplement for growth on laboratory media. In this study, we demonstrate that this phenotype can be determined by a single gene,
nadV
, whose presence allows NAD-independent growth of
Haemophilus influenzae
and
Actinobacillus pleuropneumoniae
. This gene was cloned from a 5.2-kb plasmid which was previously shown to be responsible for NAD independence in
Haemophilus ducreyi
. When transformed into
A. pleuropneumoniae
, this cloned gene allowed NAD-independent growth on complex media and allowed the utilization of nicotinamide in place of NAD on defined media. Sequence analysis revealed an open reading frame of 1,482 bp that is predicted to encode a protein with a molecular mass of 55,619 Da. Compared with the sequence databases, NadV was found to have significant sequence homology to the human pre-B-cell colony-enhancing factor PBEF and to predicted proteins of unknown function identified in the bacterial species
Mycoplasma genitalium, Mycoplasma pneumoniae, Shewanella putrefaciens, Synechocystis sp., Deinococcus radiodurans, Pasteurella multocida
, and
Actinobacillus actinomycetemcomitans. P. multocida
and
A. actinomycetemcomitans
are among the NAD-independent members of the
Pasteurellaceae
. Homologues of NadV were not found in the sequenced genome of
H. influenzae
, an NAD-dependent member of the
Pasteurellaceae
, or in species known to utilize a different pathway for synthesis of NAD, such as
Escherichia coli
. Sequence alignment of these nine homologues revealed regions and residues of complete conservation that may be directly involved in the enzymatic activity. Identification of a function for this gene in the
Pasteurellaceae
should help to elucidate the role of its homologues in other species.
Visualization of the Attachment Organelle and Cytadherence Proteins of
Mycoplasma pneumoniae
by Immunofluorescence Microscopy
Authors: Seto Shintaro , Layh-Schmitt Gerlinde , Kenri Tsuyoshi , Miyata Makoto
Published: July 2002

A method was developed for protein localization in
Mycoplasma pneumoniae
by immunofluorescence microscopy. The P1 adhesin protein was revealed to be located at least at one cell pole in all adhesive cells, as has been observed by immunoelectron microscopy. Cell images were classified according to P1 localization and assigned by DNA content. Cells with a single P1 focus at one cell pole had a lower DNA content than cells with two foci, at least one of which was positioned at a cell pole. Those with one focus at each cell pole had the highest DNA content, suggesting that the nascent attachment organelle is formed next to the old one and migrates to the opposite cell pole before cell division. Double staining revealed that the accessory proteins for cytadherence—HMW1, HMW3, P30, P90, P40, and P65—colocalized with the P1 adhesin in all cells. The localization of cytadherence proteins was also examined in cytadherence-deficient mutant cells with a branched morphology. In M5 mutant cells, which lack the P90 and P40 proteins, HMW1, HMW3, P1, and P30 were focused at the cell poles of short branches, and P65 showed no signal. In M7 mutant cells, which produce a truncated P30 protein, HMW1, HMW3, P1, P90, and P40 were focused, and P65 showed no signal. In M6 mutant cells, which express no HMW1 and a truncated P30 protein, the P1 adhesin was distributed throughout the entire cell body, and no signal was detected for the other proteins. These results suggest that the cytadherence proteins are sequentially assembled to the attachment organelle with HMW1 first, HMW3, P1, P30, P90, and P40 next, and P65 last.
Global Analysis of
Escherichia coli
Gene Expression during the Acetate-Induced Acid Tolerance Response
Authors: Arnold Carrie N. , McElhanon Justin , Lee Aaron , Leonhart Ryan , Siegele Deborah A.
Published: July 2002

The ability of
Escherichia coli
to survive at low pH is strongly affected by environmental factors, such as composition of the growth medium and growth phase. Exposure to short-chain fatty acids, such as acetate, proprionate, and butyrate, at neutral or nearly neutral pH has also been shown to increase acid survival of
E. coli
and
Salmonella enterica
serovar Typhimurium. To investigate the basis for acetate-induced acid tolerance in
E. coli
O157:H7, genes whose expression was altered by exposure to acetate were identified using gene arrays. The expression of 60 genes was reduced by at least twofold; of these, 48 encode components of the transcription-translation machinery. Expression of 26 genes increased twofold or greater following treatment with acetate. This included six genes whose products are known to be important for survival at low pH. Five of these genes, as well as six other acetate-induced genes, are members of the
E. coli
RpoS regulon. RpoS, the stress sigma factor, is known to be required for acid tolerance induced by growth at nonlethal low pH or by entry into stationary phase. Disruption of the
rpoS
gene by a transposon insertion mutation also prevented acetate-induced acid tolerance. However, induction of RpoS expression did not appear to be sufficient to activate the acid tolerance response. Treatment with either NaCl or sodium acetate (pH 7.0) increased expression of an
rpoS
::
lacZ
fusion protein, but only treatment with acetate increased acid survival.
Mutually Exclusive Distribution of IS
1548
and GBSi1, an Active Group II Intron Identified in Human Isolates of Group B Streptococci
Authors: Granlund Margareta , Michel François , Norgren Mari
Published: July 2002

The present study shows that active, self-splicing group II intron GBSi1 is located downstream of the C5a-peptidase gene,
scpB
, in some group B streptococcus (GBS) isolates that lack insertion sequence IS
1548
. IS
1548
was previously reported to be often present at the
scpB
locus in GBS isolated in association with endocarditis. Since none of 67 GBS isolates examined, 40 of which were of serotype III, harbored both IS
1548
and GBSi1, these two elements are suggested to be markers for different genetic lineages in GBS serotype III. The DNA region downstream of
scpB
in GBS isolates harboring either GBSi1, IS1548, or none of these mobile elements was found to encode the laminin binding protein, Lmb, which shows sequence similarities to a family of streptococcal adhesins. IS
1548
is inserted 9 bp upstream of the putative promoter for
lmb
, while the insertion site for GBSi1 is located 88 bp further upstream. Sequences highly similar to GBSi1 exist also in
Streptococcus pneumoniae
. An inverted repeat sequence, with features typical of transcription terminators, was identified immediately upstream of the insertion site for the group II intron both in the GBS and
S. pneumoniae
sequences. This motif is suggested to constitute a target for the GBS intron as well as for rather closely related introns in
Bacillus halodurans, Pseudomonas alcaligenes
, and
Pseudomonas putida
. When transcripts containing the GBSi1 intron were incubated at high concentrations of ammonium and magnesium, a major product with the expected length and sequence for the ligated exons was generated. Unlike, however, all members of group II investigated so far, the excised intron was in linear, rather than in a branched (lariat), form.
Biofilm Formation by
Staphylococcus epidermidis
Depends on Functional RsbU, an Activator of the
sigB
Operon: Differential Activation Mechanisms Due to Ethanol and Salt Stress
Authors: Knobloch Johannes K.-M. , Bartscht Katrin , Sabottke Axel , Rohde Holger , Feucht Heinz-Hubert , Mack Dietrich
Published: July 2002

Staphylococcus epidermidis
is a common pathogen in medical device-associated infections. Its major pathogenetic factor is the ability to form adherent biofilms. The polysaccharide intercellular adhesin (PIA), which is synthesized by the products of the
icaADBC
gene cluster, is essential for biofilm accumulation. In the present study, we characterized the gene locus inactivated by Tn
917
insertions of two isogenic,
icaADBC
-independent, biofilm-negative mutants, M15 and M19, of the biofilm-producing bacterium
S. epidermidis
1457. The insertion site was the same in both of the mutants and was located in the first gene,
rsbU
, of an operon highly homologous to the
sigB
operons of
Staphylococcus aureus
and
Bacillus subtilis
. Supplementation of Trypticase soy broth with NaCl (TSB
NaCl
) or ethanol (TSB
EtOH
), both of which are known activators of
sigB
, led to increased biofilm formation and PIA synthesis by
S. epidermidis
1457. Insertion of Tn
917
into
rsbU
, a positive regulator of alternative sigma factor ς
B
, led to a biofilm-negative phenotype and almost undetectable PIA production. Interestingly, in TSB
EtOH
, the mutants were enabled to form a biofilm again with phenotypes similar to those of the wild type. In TSB
NaCl
, the mutants still displayed a biofilm-negative phenotype. No difference in primary attachment between the mutants and the wild type was observed. Similar phenotypic changes were observed after transfer of the Tn
917
insertion of mutant M15 to the independent and biofilm-producing strain
S. epidermidis
8400. In 11 clinical
S. epidermidis
strains, a restriction fragment length polymorphism of the
sigB
operon was detected which was independent of the presence of the
icaADBC
locus and a biofilm-positive phenotype. Obviously, different mechanisms are operative in the regulation of PIA expression in stationary phase and under stress induced by salt or ethanol.
Interaction of α-Agglutinin and
a
-Agglutinin,
Saccharomyces cerevisiae
Sexual Cell Adhesion Molecules
Authors: Zhao Hui , Shen Zheng-Ming , Kahn Peter C. , Lipke Peter N.
Published: July 2002

α-Agglutinin and a-agglutinin are complementary cell adhesion glycoproteins active during mating in the yeast
Saccharomyces cerevisiae
. They bind with high affinity and high specificity: cells of opposite mating types are irreversibly bound by a few pairs of agglutinins. Equilibrium and surface plasmon resonance kinetic analyses showed that the purified binding region of α-agglutinin interacted similarly with purified a-agglutinin and with a-agglutinin expressed on cell surfaces. At 20°C, the

K
D

for the interaction was 2 × 10
−9
to 5 × 10
−9
M. This high affinity was a result of a very low dissociation rate (≈ 2.6 × 10
−4
s
−1
) coupled with a low association rate (= 5 × 10
4
M
−1
s
−1
). Circular-dichroism spectroscopy showed that binding of the proteins was accompanied by measurable changes in secondary structure. Furthermore, when binding was assessed at 10°C, the association kinetics were sigmoidal, with a very low initial rate. An induced-fit model of binding with substantial apposition of hydrophobic surfaces on the two ligands can explain the observed affinity, kinetics, and specificity and the conformational effects of the binding reaction.
Mapping Stress-Induced Changes in Autoinducer AI-2 Production in Chemostat-Cultivated
Escherichia coli
K-12
Authors: DeLisa Matthew P. , Valdes James J. , Bentley William E.
Published: July 2002

Numerous gram-negative bacteria employ a cell-to-cell signaling mechanism, termed quorum sensing, for controlling gene expression in response to population density. Recently, this phenomenon has been discovered in
Escherichia coli
, and while pathogenic
E. coli
utilize quorum sensing to regulate pathogenesis (i.e., expression of virulence genes), the role of quorum sensing in nonpathogenic
E. coli
is less clear, and in particular, there is no information regarding the role of quorum sensing during the overexpression of recombinant proteins. The production of autoinducer AI-2, a signaling molecule employed by
E. coli
for intercellular communication, was studied in
E. coli
W3110 chemostat cultures using a
Vibrio harveyi
AI-2 reporter assay (M. G. Surrette and B. L. Bassler, Proc. Natl. Acad. Sci. USA 95:7046–7050, 1998). Chemostat cultures enabled a study of AI-2 regulation through steady-state and transient responses to a variety of environmental stimuli. Results demonstrated that AI-2 levels increased with the steady-state culture growth rate. In addition, AI-2 increased following pulsed addition of glucose, Fe(III), NaCl, and dithiothreitol and decreased following aerobiosis, amino acid starvation, and isopropyl-β-
d
-thiogalactopyranoside-induced expression of human interleukin-2 (hIL-2). In general, the AI-2 responses to several perturbations were indicative of a shift in metabolic activity or state of the cells induced by the individual stress. Because of our interest in the expression of heterologous proteins in
E. coli
, the transcription of four quorum-regulated genes and 20 stress genes was mapped during the transient response to induced expression of hIL-2. Significant regulatory overlap was revealed among several stress and starvation genes and known quorum-sensing genes.
Cell-Surface-Anchoring Role of N-Terminal Surface Layer Homology Domains of
Clostridium cellulovorans
EngE
Authors: Kosugi Akihiko , Murashima Koichiro , Tamaru Yutaka , Doi Roy H.
Published: July 2002

engE
, coding for endoglucanase E, one of the three major subunits of the
Clostridium cellulovorans
cellulosome, has been cloned and sequenced (Y. Tamaru and R. H. Doi, J. Bacteriol. 181:3270-3276, 1999). The N-terminal-half region of EngE possesses three repeated surface layer homology (SLH) domains, which are homologous to those of some bacterial S-layer proteins. Also, the C-terminal-half region consists of a catalytic domain of glycosyl hydrolase family 5 and a duplicated sequence (dockerin) for binding EngE to scaffolding protein CbpA. Our hypothesis is that the SLH domains serve in the role of anchoring to the cell surface. This model was investigated by using recombinant EngEs (rEngE) with and without SLH domains that were synthesized in
Escherichia coli
and cell wall preparations from
C. cellulovorans
. When rEngE and SLH polypeptides of EngE were incubated with cell wall fragments prepared by sodium dodecyl sulfate treatment, these proteins bound strongly to the cell wall. However, rEngEs without SLH domains lost their ability to bind to cell walls. When rEngE was incubated with mini-CbpA, consisting of two cohesin domains, and cell wall fragments, the mini-CbpA was able to bind to the cell wall with rEngE. However, the binding of mini-CbpA was dramatically inhibited by addition of a chelating reagent, such as EDTA, which prevents cohesin-dockerin interactions. These results suggest not only that the SLH domains of EngE can bind to the cell surface but also that EngE plays an anchoring role for cellulosomes through the interaction of its dockerin domain with a CbpA cohesin.
Site-Directed Mutagenesis Studies of Selected Motif and Charged Residues and of Cysteines of the Multifunctional Tetracycline Efflux Protein Tet(L)
Authors: Jin Jie , Krulwich Terry A.
Published: July 2002

All of the transmembrane glutamates of Tet(L) are essential for tetracycline (TET) resistance, and E397 has been shown to be essential for all catalytic modes, i.e., TET-Me
2+
and Na
+
efflux and K
+
uptake. Loop residues D74 and G70 are essential for TET flux but not for Na
+
or K
+
flux. A cysteineless Tet(L) protein exhibits all activities.
Inferring Genome Trees by Using a Filter To Eliminate Phylogenetically Discordant Sequences and a Distance Matrix Based on Mean Normalized BLASTP Scores
Authors: Clarke G. D. Paul , Beiko Robert G. , Ragan Mark A. , Charlebois Robert L.
Published: July 2002

Darwin's paradigm holds that the diversity of present-day organisms has arisen via a process of genetic descent with modification, as on a bifurcating tree. Evidence is accumulating that genes are sometimes transferred not along lineages but rather across lineages. To the extent that this is so, Darwin's paradigm can apply only imperfectly to genomes, potentially complicating or perhaps undermining attempts to reconstruct historical relationships among genomes (i.e., a genome tree). Whether most genes in a genome have arisen via treelike (vertical) descent or by lateral transfer across lineages can be tested if enough complete genome sequences are used. We define a phylogenetically discordant sequence (PDS) as an open reading frame (ORF) that exhibits patterns of similarity relationships statistically distinguishable from those of most other ORFs in the same genome. PDSs represent between 6.0 and 16.8% (mean, 10.8%) of the analyzable ORFs in the genomes of 28 bacteria, eight archaea, and one eukaryote (Saccharomyces cerevisiae). In this study we developed and assessed a distance-based approach, based on mean pairwise sequence similarity, for generating genome trees. Exclusion of PDSs improved bootstrap support for basal nodes but altered few topological features, indicating that there is little systematic bias among PDSs. Many but not all features of the genome tree from which PDSs were excluded are consistent with the 16S rRNA tree.
Global Regulation by
gidA
in
Pseudomonas syringae
Authors: Kinscherf Thomas G. , Willis David K.
Published: July 2002

Analysis of two virulence mutants of
Pseudomonas syringae
B728a revealed that the Tn
5
sites of insertion were within the
gidA
open reading frame (ORF). These mutations were pleiotropic, affecting diverse phenotypic traits, such as lipodepsipeptide (syringomycin and syringopeptin) antibiotic production, swarming, presence of fluorescent pigment, and virulence. Site-specific recombination of a disrupted
gidA
gene into the chromosome resulted in the same phenotypic pattern as transposon insertion. Mutant phenotypes were restored by the
gidA
ORF on a plasmid. The
salA
gene, a copy number suppressor of the syringomycin-deficient phenotype in
gacS
and
gacA
mutants, was also found to suppress the antibiotic-negative phenotypes of
gidA
mutants, suggesting that
gidA
might play some role in
salA
regulation. Reporter studies with chromosomal
salA
-
lacZ
translational fusions confirmed that
salA
reporter expression decreased approximately fivefold in a
gidA
mutant background, with a concurrent decrease in the expression of the syringomycin biosynthetic reporter fusion
syrB
-
lacZ.
Wild-type levels of reporter expression were restored by supplying an intact
gidA
gene on a plasmid. Often described as being involved in cell division, more recent evidence suggests a role for
gidA
in moderating translational fidelity, suggesting a mechanism by which global regulation might occur. The
gidA
gene is essentially universal in the domains
Bacteria
and
Eucarya
but has no counterparts in
Archaea
, probably reflecting specific differences in the translational machinery between the former and latter domains.
Development of a Canadian Standardized Protocol for Subtyping Methicillin-Resistant
Staphylococcus aureus
Using Pulsed-Field Gel Electrophoresis
Authors: Mulvey M. R. , Chui L. , Ismail J. , Louie L. , Murphy C. , Chang N. , Alfa M. ,   the Canadian Committee for the Standardization of Molecular Methods
Published: July 2002

A panel of 24 methicillin-resistant
Staphylococcus aureus
strains was distributed to 15 laboratories in Canada to evaluate their in-house pulsed-field gel electrophoresis (PFGE) protocols and interpretation criteria. Attempts to compare fingerprint images using computer-aided analysis were not successful due to variability in individual laboratory PFGE protocols. In addition, individual site interpretation of the fingerprint patterns was inadequate, as 7 of 13 sites (54%) made at least one error in interpreting the fingerprints from the panel. A 2-day standardized PFGE protocol (culture to gel image) was developed and distributed to all of the sites. Each site was requested to use the standardized protocol on five strains from the original panel. Thirteen sites submitted gel images for comparisons. The protocol demonstrated excellent reproducibility and allowed interlaboratory comparisons with Molecular Analyst DST software (Bio-Rad) and 1.5% band tolerance.
Multiplex PCR Using Internal Transcribed Spacer 1 and 2 Regions for Rapid Detection and Identification of Yeast Strains
Authors: Fujita Shin-Ichi , Senda Yasuko , Nakaguchi Shigeki , Hashimoto Takuma
Published: July 2002

Multiplex PCR amplification followed by either agarose gel electrophoresis (PCR-AGE) or microchip electrophoresis (PCR-ME) was used to test a total of 120 fungal strains. The internal transcribed spacer 1 (ITS1) and ITS2 regions and the 5.8S ribosomal DNA (rDNA) region of the fungi were amplified by using universal primers ITS1 and ITS4. The ITS2 region was simultaneously amplified by using universal primers ITS3 and ITS4. Since
Trichosporon asahi
and
T. asteroides
showed similar lengths for two amplicons, 29 different gel patterns were demonstrated for 30 yeast species tested on the basis of differences in the lengths of one or two amplicons. Of 75 yeast isolates from clinical materials, 5 isolates (6.8%) which were incompletely identified or not identified by the phenotypic method were identified with our PCR-based method (2 isolates as
Candida guilliermondii
, 2 as
C. krusei
, and 1 as
C. zeylanoides
). No differences in discriminating power or sensitivity were observed between the PCR-AGE method and the PCR-ME method. These methods, prospectively applied to 24 yeast-positive blood culture bottles (16 patients), resulted in the correct detection of 24 yeast strains. In conclusion, multiplex PCR followed by electrophoresis seems to be a promising tool for the rapid identification of common and uncommon yeast strains from culture colonies and from yeast-positive blood culture bottles (5.5 h for the PCR-AGE method and 3 h for the PCR-ME method).