bluuebunny/crossref_metadata_2025 huggingface dataset:
Barriers to Genetic Exchange between Bacterial Species:
Streptococcus pneumoniae
Transformation
Authors: Majewski Jacek , Zawadzki Piotr , Pickerill Paul , Cohan Frederick M. , Dowson Christopher G.
Published: July 2002

Interspecies genetic exchange is an important evolutionary mechanism in bacteria. It allows rapid acquisition of novel functions by transmission of adaptive genes between related species. However, the frequency of homologous recombination between bacterial species decreases sharply with the extent of DNA sequence divergence between the donor and the recipient. In
Bacillus
and
Escherichia
, this sexual isolation has been shown to be an exponential function of sequence divergence. Here we demonstrate that sexual isolation in transformation between
Streptococcus pneumoniae
recipient strains and donor DNA from related strains and species follows the described exponential relationship. We show that the Hex mismatch repair system poses a significant barrier to recombination over the entire range of sequence divergence (0.6 to 27%) investigated. Although mismatch repair becomes partially saturated, it is responsible for 34% of the observed sexual isolation. This is greater than the role of mismatch repair in
Bacillus
but less than that in
Escherichia
. The remaining non-Hex-mediated barrier to recombination can be provided by a variety of mechanisms. We discuss the possible additional mechanisms of sexual isolation, in view of earlier findings from
Bacillus
,
Escherichia
, and
Streptococcus
.
Transposon Insertions in the
Flavobacterium johnsoniae ftsX
Gene Disrupt Gliding Motility and Cell Division
Authors: Kempf Michael J. , McBride Mark J.
Published: July 2002

Flavobacterium johnsoniae
is a gram-negative bacterium that exhibits gliding motility. To determine the mechanism of flavobacterial gliding motility, we isolated 33 nongliding mutants by Tn
4351
mutagenesis. Seventeen of these mutants exhibited filamentous cell morphology. The region of DNA surrounding the transposon insertion in the filamentous mutant CJ101-207 was cloned and sequenced. The transposon was inserted in a gene that was similar to
Escherichia coli ftsX
. Two of the remaining 16 filamentous mutants also carried insertions in
ftsX
. Introduction of the wild-type
F. johnsoniae ftsX
gene restored motility and normal cell morphology to each of the three
ftsX
mutants. CJ101-207 appears to be blocked at a late stage of cell division, since the filaments produced cross walls but cells failed to separate. In
E. coli
, FtsX is thought to function with FtsE in translocating proteins involved in potassium transport, and perhaps proteins involved in cell division, into the cytoplasmic membrane. Mutations in
F. johnsoniae ftsX
may prevent translocation of proteins involved in cell division and proteins involved in gliding motility into the cytoplasmic membrane, thus resulting in defects in both processes. Alternatively, the loss of gliding motility may be an indirect result of the defect in cell division. The inability to complete cell division may alter the cell architecture and disrupt gliding motility by preventing the synthesis, assembly, or functioning of the motility apparatus.
Probable Identification of a Membrane-Associated Repressor of
Bacillus subtilis
DNA Replication as the E2 Subunit of the Pyruvate Dehydrogenase Complex
Authors: Stein Andrew , Firshein William
Published: July 2002

Two
Bacillus subtilis
lysogenic libraries were probed by an antibody specific for a previously described membrane-associated inhibitor of
B. subtilis
DNA replication (J. Laffan and W. Firshein, Proc. Natl. Acad. Sci. USA 85:7452–7456, 1988). Three clones that reacted strongly with the antibody contained an entire open reading frame. Sequencing identified one of the clones (R1-2) as containing the E2 subunit of the pyruvate dehydrogenase complex, dihydrolipoamide acetyltransferase. An AT-rich sequence in the origin region was identified initially as the site to which extracts from the R1-2 clone were bound. This sequence was almost identical to one detected in
Bacillus thuringiensis
that also bound the E2 subunit but which was involved in activating the Cry1 protoxin gene of the organism, not in inhibiting DNA replication (T. Walter and A. Aronson, J. Biol. Chem., 274:7901–7906, 1999). However, the exact sequence was not as important in
B. subtilis
as the AT-rich core region. Binding would occur as long as most of the AT character of the core remained. Purified E2 protein obtained by use of PCR and an expression vector reacted strongly with antibody prepared against the repressor protein and the protein in the R1-2 clone, but its specificity for the AT-rich region was altered. The purified E2 protein was capable of inhibiting membrane-associated DNA replication in vitro, but anti-E2 antibody was variable in its ability to rescue repression when added to the assay.
Roles of Horizontal Gene Transfer and Gene Integration in Evolution of 1,3-Dichloropropene- and 1,2-Dibromoethane-Degradative Pathways
Authors: Poelarends Gerrit J. , Kulakov Leonid A. , Larkin Michael J. , van Hylckama Vlieg Johan E. T. , Janssen Dick B.
Published: July 2002

The haloalkane-degrading bacteria
Rhodococcus rhodochrous
NCIMB13064,
Pseudomonas pavonaceae
170, and
Mycobacterium
sp. strain GP1 share a highly conserved haloalkane dehalogenase gene (
dhaA
). Here, we describe the extent of the conserved
dhaA
segments in these three phylogenetically distinct bacteria and an analysis of their flanking sequences. The
dhaA
gene of the 1-chlorobutane-degrading strain NCIMB13064 was found to reside within a 1-chlorobutane catabolic gene cluster, which also encodes a putative invertase (
invA
), a regulatory protein (
dhaR
), an alcohol dehydrogenase (
adhA
), and an aldehyde dehydrogenase (
aldA
). The latter two enzymes may catalyze the oxidative conversion of
n
-butanol, the hydrolytic product of 1-chlorobutane, to
n
-butyric acid, a growth substrate for many bacteria. The activity of the
dhaR
gene product was analyzed in
Pseudomonas
sp. strain GJ1, in which it appeared to function as a repressor of
dhaA
expression. The 1,2-dibromoethane-degrading strain GP1 contained a conserved DNA segment of 2.7 kb, which included
dhaR
,
dhaA
, and part of
invA
. A 12-nucleotide deletion in
dhaR
led to constitutive expression of
dhaA
in strain GP1, in contrast to the inducible expression of
dhaA
in strain NCIMB13064. The 1,3-dichloropropene-degrading strain 170 possessed a conserved DNA segment of 1.3 kb harboring little more than the coding region of the
dhaA
gene. In strains 170 and GP1, a putative integrase gene was found next to the conserved
dhaA
segment, which suggests that integration events were responsible for the acquisition of these DNA segments. The data indicate that horizontal gene transfer and integrase-dependent gene acquisition were the key mechanisms for the evolution of catabolic pathways for the man-made chemicals 1,3-dichloropropene and 1,2-dibromoethane.
Alternative Pathways for Siroheme Synthesis inKlebsiella aerogenes
Authors: Kolko M. Miriam , Kapetanovich Lori A. , Lawrence Jeffrey G.
Published: July 2002

Siroheme, the cofactor for sulfite and nitrite reductases, is formed by methylation, oxidation, and iron insertion into the tetrapyrrole uroporphyrinogen III (Uro-III). The CysG protein performs all three steps of siroheme biosynthesis in the enteric bacteriaEscherichia coliandSalmonella enterica. In either taxon,cysGmutants cannot reduce sulfite to sulfide and require a source of sulfide or cysteine for growth. In addition, CysG-mediated methylation of Uro-III is required for de novo synthesis of cobalamin (coenzyme B12) inS. enterica. We have determined thatcysGmutants of the related enteric bacteriumKlebsiella aerogeneshave no defect in the reduction of sulfite to sulfide. These data suggest that an alternative enzyme allows for siroheme biosynthesis in CysG-deficient strains ofKlebsiella. However,Klebsiella cysGmutants fail to synthesize coenzyme B12, suggesting that the alternative siroheme biosynthetic pathway proceeds by a different route. GenecysF, encoding an alternative siroheme synthase homologous to CysG, has been identified by genetic analysis and lies within thecysFDNCoperon; thecysFgene is absent from theE. coliandS. entericagenomes. While CysG is coregulated with the siroheme-dependent nitrite reductase, thecysFgene is regulated by sulfur starvation. Models for alternative regulation of the CysF and CysG siroheme synthases inKlebsiellaand for the loss of thecysFgene from the ancestor ofE. coliandS. entericaare presented.
Identification of Residues Involved in Catalytic Activity of the Inverting Glycosyl Transferase WbbE from
Salmonella enterica
Serovar Borreze
Authors: Keenleyside Wendy J. , Clarke Anthony J. , Whitfield Chris
Published: July 2002

Synthesis of the O:54 O antigen of
Salmonella enterica
is initiated by the nonprocessive glycosyl transferase WbbE, assigned to family 2 of the glycosyl transferase enzymes (GT2). GT2 enzymes possess a characteristic N-terminal domain, domain A. Based on structural data from the GT2 representative SpsA (S. J. Charnock and G. J. Davies, Biochemistry 38:6380–6385, 1999), this domain is responsible for nucleotide binding. It possesses two invariant Asp residues, the first forming a hydrogen bond to uracil and the second coordinating a Mn
2+
ion. Site-directed replacement of Asp41 (D41A) of WbbE, the analogue of the first Asp residue of SpsA, revealed that this is not required for activity. WbbE possesses three Asp residues near the position analogous to the second conserved residue. Whereas D95A reduced WbbE activity, activity in D93A and D96A mutants was abrogated, suggesting that either D93 or D96 may coordinate the Mn
2+
ion. Our studies also identified a C-terminal region of sequence conservation in 22 GT2 members, including WbbE. SpsA was not among these. This region is characterized by an ED(Y) motif. The Glu and Asp residues of this motif were individually replaced in WbbE. E180D in WbbE had greatly reduced activity, and an E180Q replacement completely abrogated activity; however, D181E had no effect. E180 is predicted to reside on a turn. Combined with the alignment of the motif with potential catalytic residues in the GT2 enzymes ExoM and SpsA, we speculate that E180 is the catalytic residue of WbbE. Sequence and predicted structural divergence in the catalytic region of GT2 members suggests that this is not a homogeneous family.
SpoVID Guides SafA to the Spore Coat in
Bacillus subtilis
Authors: Ozin Amanda J. , Samford Craig S. , Henriques Adriano O. , Moran Charles P.
Published: July 2002

Bacteria assemble complex structures by targeting proteins to specific subcellular locations. The protein coat that encases
Bacillus subtilis
spores is an example of a structure that requires coordinated targeting and assembly of more than 24 polypeptides. The earliest stages of coat assembly require the action of three morphogenetic proteins: SpoIVA, CotE, and SpoVID. In the first steps, a basement layer of SpoIVA forms around the surface of the forespore, guiding the subsequent positioning of a ring of CotE protein about 75 nm from the forespore surface. SpoVID localizes near the forespore membrane where it functions to maintain the integrity of the CotE ring and to anchor the nascent coat to the underlying spore structures. However, it is not known which spore coat proteins interact directly with SpoVID. In this study we examined the interaction between SpoVID and another spore coat protein, SafA, in vivo using the yeast two-hybrid system and in vitro. We found evidence that SpoVID and SafA directly interact and that SafA interacts with itself. Immunofluorescence microscopy showed that SafA localized around the forespore early during coat assembly and that this localization of SafA was dependent on SpoVID. Moreover, targeting of SafA to the forespore was also dependent on SpoIVA, as was targeting of SpoVID to the forespore. We suggest that the localization of SafA to the spore coat requires direct interaction with SpoVID.
ChvD, a Chromosomally Encoded ATP-Binding Cassette Transporter-Homologous Protein Involved in Regulation of Virulence Gene Expression in
Agrobacterium tumefaciens
Authors: Liu Zhenying , Jacobs Mark , Schaff Dennis A. , McCullen Colleen A. , Binns Andrew N.
Published: July 2002

A yeast two-hybrid screen searching for chromosomally encoded proteins that interact with the
Agrobacterium tumefaciens
VirB8 protein was carried out. This screen identified an interaction candidate homologous to the partial sequence of a gene that had previously been identified in a transposon screen as a potential regulator of
virG
expression,
chvD
. In this report, the cloning of the entire
chvD
gene is described and the gene is sequenced and characterized. Insertion of a promoterless
lacZ
gene into the
chvD
locus greatly attenuated virulence and
vir
gene expression. Compared to that of the wild-type strain, growth of the
chvD
mutant was reduced in rich, but not minimal, medium. Expression of
chvD
, as monitored by expression of β-galactosidase activity from the
chvD-lacZ
fusion, occurred in both rich and minimal media as well as under conditions that induce virulence gene expression. The ChvD protein is highly homologous to a family of ATP-binding cassette transporters involved in antibiotic export from bacteria and has two complete Walker box motifs. Molecular genetic analysis demonstrated that disruption of either Walker A box, singly, does not inactivate this protein's effect on virulence but that mutations in both Walker A boxes renders it incapable of complementing a
chvD
mutant strain. Constitutive expression of
virG
in the
chvD
mutant strain restored virulence, supporting the hypothesis that ChvD controls virulence through effects on
virG
expression.
Constitutive Septal Murein Synthesis in
Escherichia coli
with Impaired Activity of the Morphogenetic Proteins RodA and Penicillin-Binding Protein 2
Authors: de Pedro Miguel A. , Donachie William D. , Höltje Joachim-Volker , Schwarz Heinz
Published: July 2002

The pattern of peptidoglycan (murein) segregation in cells of
Escherichia coli
with impaired activity of the morphogenetic proteins penicillin-binding protein 2 and RodA has been investigated by the
d
-cysteine–biotin immunolabeling technique (M. A. de Pedro, J. C. Quintela, J.-V. Höltje, and H. Schwarz, J. Bacteriol. 179:2823–2834, 1997). Inactivation of these proteins either by amdinocillin treatment or by mutations in the corresponding genes,
pbpA
and
rodA
, respectively, leads to the generation of round, osmotically stable cells. In normal rod-shaped cells, new murein precursors are incorporated all over the lateral wall in a diffuse manner, being mixed up homogeneously with preexisting material, except during septation, when strictly localized murein synthesis occurs. In contrast, in rounded cells, incorporation of new precursors is apparently a zonal process, localized at positions at which division had previously taken place. Consequently, there is no mixing of new and old murein. Old murein is preserved for long periods of time in large, well-defined areas. We propose that the observed patterns are the result of a failure to switch off septal murein synthesis at the end of septation events. Furthermore, the segregation results confirm that round cells of
rodA
mutants do divide in alternate, perpendicular planes as previously proposed (K. J. Begg and W. D. Donachie, J. Bacteriol. 180:2564–2567, 1998).
Localization of c-di-GMP-Binding Protein with the Linear Terminal Complexes of
Acetobacter xylinum
Authors: Kimura Satoshi , Chen He Ping , Saxena Inder M. , Brown R. Malcolm , Itoh Takao
Published: July 2002

Specific labeling of a single row of cellulose-synthesizing complexes (terminal complexes, TC subunits, TCs, or TC arrays) in
Acetobacter xylinum
by antibodies raised against a 93-kDa protein (the cyclic dignanylic acid-binding protein) has been demonstrated by using the sodium dodecyl sulfate (SDS)–freeze-fracture labeling (FRL) technique. The antibodies to the 93-kDa protein specifically recognized the TC subunits on the protoplasmic fracture (PF) face of the outer membrane in
A. xylinum
; however, nonlabeled TCs were also observed. Two types of TC subunits (particles or pits) are observed on the PF face of the outer membrane: (i) immunogold-labeled TCs showing a line of depressions (pits) with an indistinct particle array and (ii) nonlabeled TC subunits with a distinct single row of particle arrays. The evidence indicates that the labeling patterns differ with respect to the presence or absence of certain TC subunits remaining attached to the replica after SDS treatment. This suggests the presence of at least two TC components, one in the outer membrane and the other in the cytoplasmic membrane. If the TC component in the outer membrane is preferentially fractured and remains attached to the ectoplasmic fracture face (or outer leaflet) of the outer membrane, subsequent replica formation reveals a pit or depression with positive antibody labeling on the PF face of the outer membrane. If the TC component in the outer membrane remains with the PF face (or inner leaflet) of the outer membrane, the innermost TC component is removed during SDS treatment and labeling does not occur. SDS-FRL of TCs in
A. xylinum
has enabled us to provide the first topological molecular analysis of component proteins in a cellulose-synthesizing TC structure in a prokaryotic organism.
Regulation of Potassium-Dependent Kdp-ATPase Expression in the Nitrogen-Fixing Cyanobacterium
Anabaena torulosa
Authors: Alahari Anuradha , Ballal Anand , Apte Shree Kumar
Published: July 2002

The KdpB polypeptides in the cyanobacterium
Anabaena torulosa
were shown to be two membrane-bound proteins of about 78 kDa, expressed strictly under K
+
deficiency and repressed or degraded upon readdition of K
+
. In both
Anabaena
and
Escherichia coli
strain MC4100, osmotic and ionic stresses caused no significant induction of steady-state KdpB levels during extreme potassium starvation.
Novel Posttranslational Activation of the
LYS2-
Encoded α-Aminoadipate Reductase for Biosynthesis of Lysine and Site-Directed Mutational Analysis of Conserved Amino Acid Residues in the Activation Domain of
Candida albicans
Authors: Guo Shujuan , Evans Sarah A. , Wilkes Mindy B. , Bhattacharjee J. K.
Published: July 2002

The α-aminoadipate pathway for lysine biosynthesis is present only in fungi. The α-aminoadipate reductase (AAR) of this pathway catalyzes the conversion of α-aminoadipic acid to α-aminoadipic-δ-semialdehyde by a complex mechanism involving two gene products, Lys2p and Lys5p. The
LYS2
and
LYS5
genes encode, respectively, a 155-kDa inactive AAR and a 30-kDa phosphopantetheinyl transferase (PPTase) which transfers a phosphopantetheinyl group from coenzyme A (CoA) to Lys2p for the activation of Lys2p and AAR activity. In the present investigation, we have confirmed the posttranslational activation of the 150-kDa Lys2p of
Candida albicans
, a pathogenic yeast, in the presence of CoA and
C. albicans lys2
mutant (CLD2) extract as a source of PPTase (Lys5p). The recombinant Lys2p or CLD2 mutant extract exhibited no AAR activity with or without CoA. However, the recombinant 150-kDa Lys2p, when incubated with CLD2 extract and CoA, exhibited significant AAR activity compared to that of wild-type
C. albicans
CAI4 extract. The PPTase in the CLD2 extract was required only for the activation of Lys2p and not for AAR reaction. Site-directed mutational analysis of G882 and S884 of the Lys2p activation domain (LGGHSI) revealed no AAR activity, indicating that these two amino acids are essential for the activation. Replacement of other amino acid residues in the domain resulted in partial or full AAR activity. These results demonstrate the posttranslational activation and the requirement of specific amino acid residues in the activation domain of the AAR of
C. albicans
.
The
lytB
Gene of
Escherichia coli
Is Essential and Specifies a Product Needed for Isoprenoid Biosynthesis
Authors: McAteer Sean , Coulson Andrew , McLennan Neil , Masters Millicent
Published: July 2002

LytB and GcpE, because they are codistributed with other pathway enzymes, have been predicted to catalyze unknown steps in the nonmevalonate pathway for isoprenoid biosynthesis. We constructed a conditional
Escherichia coli lytB
mutant and found that LytB is essential for survival and that depletion of LytB results in cell lysis, which is consistent with a role for this protein in isoprenoid biosynthesis. Alcohols which can be converted to pathway intermediates beyond the hypothesized LytB step(s) support limited growth of
E. coli lytB
mutants. An informatic analysis of protein structure suggested that GcpE is a globular protein of the TIM barrel class and that LytB is also a globular protein. Possible biochemical roles for LytB and GcpE are suggested.
Role for
draTG
and
rnf
Genes in Reduction of 2,4-Dinitrophenol by
Rhodobacter capsulatus
Authors: Sáez Lara P. , Garcı́a Patricia , Martı́nez-Luque Manuel , Klipp Werner , Blasco Rafael , Castillo Francisco
Published: July 2002

The phototrophic bacterium
Rhodobacter capsulatus
is able to reduce 2,4-dinitrophenol (DNP) to 2-amino-4-nitrophenol enzymatically and thus can grow in the presence of this uncoupler. DNP reduction was switched off by glutamine or ammonium, but this short-term regulation did not take place in a
draTG
deletion mutant. Nevertheless, the target of DraTG does not seem to be the nitrophenol reductase itself since the ammonium shock did not inactivate the enzyme. In addition to this short-term regulation, ammonium or glutamine repressed the DNP reduction system. Mutants of
R. capsulatus
affected in
ntrC
or
rpoN
exhibited a 10-fold decrease in nitroreductase activity in vitro but almost no DNP activity in vivo. In addition, mutants affected in
rnfA
or
rnfC
, which are also under NtrC control and encode components involved in electron transfer to nitrogenase, were unable to metabolize DNP. These results indicate that NtrC regulates dinitrophenol reduction in
R. capsulatus
, either directly or indirectly, by controlling expression of the Rnf proteins. Therefore, the Rnf complex seems to supply electrons for both nitrogen fixation and DNP reduction.
TFE, an Archaeal Transcription Factor in
Methanobacterium thermoautotrophicum
Related to Eucaryal Transcription Factor TFIIEα
Authors: Hanzelka Brian L. , Darcy Trevor J. , Reeve John N.
Published: July 2002

In the archaeon
Methanobacterium thermoautotrophicum
, MTH1669 encodes a protein with a sequence related to the N-terminal sequences of the α-subunits of eucaryal general transcription factor TFIIE. The recombinant MTH1669 gene product has been purified and shown to stimulate transcription in vitro from
M. thermoautotrophicum
promoters that were almost inactive or much less active in reaction mixtures that contained only
M. thermoautotrophicum
RNA polymerase, TATA-binding protein and transcription factor B. As all complete archaeal genome sequences contain an MTH1669 homolog, the protein encoded by this gene is apparently the first characterized example of a transcription activator, here designated TFE, that may be universally present in the
Archaea
.
Nitric Oxide Signaling and Transcriptional Control of Denitrification Genes inPseudomonas stutzeri
Authors: Vollack Kai-Uwe , Zumft Walter G.
Published: July 2002

The expression of denitrification by a facultatively anaerobic bacterium requires as exogenous signals a low oxygen tension concomitant with an N oxide. We have studied the role of nitric oxide (NO), nitrous oxide (N2O), and nitrite as signal molecules for the expression of the denitrification apparatus ofPseudomonas stutzeri. Transcriptional kinetics of structural genes were monitored by Northern blot analysis in a 60-min time frame after cells were exposed to an N oxide signal. To differentiate the inducer role of NO from that of nitrite, mRNA kinetics were monitored under anoxic conditions in anirFstrain, where NO generation from nitrite is prevented because of a defect in heme D1biosynthesis. NO-triggered responses were monitored from thenirSTBoperon (encoding cytochromecd1nitrite reductase), thenorCBoperon (encoding NO reductase),nosZ(encoding nitrous oxide reductase), andnosR(encoding a putative regulator). Transcription ofnirSTBandnorCBwas activated by 5 to 50 nM NO, whereas thenosZpromoter required about 250 nM. Nitrite at 5 to 50 nM elicited no response. At a threshold concentration of 650 nM N2O, we observed in the anoxic cell the transient appearance ofnosZandnosRtranscripts. Constant levels of transcripts of both genes were observed in an anoxic cell sparged with N2O. NO at 250 nM stimulated in this cell type the expression ofnosgenes severalfold. The transcription factor DnrD, a member of the FNR-CRP family, was found to be part of the NO-triggered signal transduction pathway. However, overexpression ofdnrDin an engineered strain did not result in NirS synthesis, indicating a need for activation of DnrD. NO modified the transcriptional pattern of thednrDoperon by inducing the transcription ofdnrNanddnrO, located upstream ofdnrD. Insertional mutagenesis ofdnrNaltered the kinetic response of thenirSTBoperon towards nitrite. Our data establish NO and DnrD as key elements in the regulatory network of denitrification inP. stutzeri. The NO response adds to the previously identified nitrate-nitrite response mediated by the NarXL two-component system for the expression of respiratory nitrate reductase encoded by thenarGHJIoperon.
Mechanisms Causing Rapid and Parallel Losses of Ribose Catabolism in Evolving Populations ofEscherichia coliB
Authors: Cooper Vaughn S. , Schneider Dominique , Blot Michel , Lenski Richard E.
Published: July 2002

Twelve populations ofEscherichia coliB all lostd-ribose catabolic function during 2,000 generations of evolution in glucose minimal medium. We sought to identify the population genetic processes and molecular genetic events that caused these rapid and parallel losses. Seven independent Rbs−mutants were isolated, and their competitive fitnesses were measured relative to that of their Rbs+progenitor. These Rbs−mutants were all about 1 to 2% more fit than the progenitor. A fluctuation test revealed an unusually high rate, about 5 × 10−5per cell generation, of mutation from Rbs+to Rbs−, which contributed to rapid fixation. At the molecular level, the loss of ribose catabolic function involved the deletion of part or all of the ribose operon (rbsgenes). The physical extent of the deletion varied between mutants, but each deletion was associated with an IS150element located immediately upstream of therbsoperon. The deletions apparently involved transposition into various locations within therbsoperon; recombination between the new IS150copy and the one upstream of therbsoperon then led to the deletion of the intervening sequence. To confirm that the beneficial fitness effect was caused by deletion of therbsoperon (and not some undetected mutation elsewhere), we used P1 transduction to restore the functionalrbsoperon to two Rbs−mutants, and we constructed another Rbs−strain by gene replacement with a deletion not involving IS150. All three of these new constructs confirmed that Rbs−mutants have a competitive advantage relative to their Rbs+counterparts in glucose minimal medium. The rapid and parallel evolutionary losses of ribose catabolic function thus involved both (i) an unusually high mutation rate, such that Rbs−mutants appeared repeatedly in all populations, and (ii) a selective advantage in glucose minimal medium that drove these mutants to fixation.
pfs-Dependent Regulation of Autoinducer 2 Production inSalmonella entericaSerovar Typhimurium
Authors: Beeston Anne L. , Surette Michael G.
Published: July 2002

Bacterial intercellular communication provides a mechanism for signal-dependent regulation of gene expression to promote coordinated population behavior.Salmonella entericaserovar Typhimurium produces a non-homoserine lactone autoinducer in exponential phase as detected by aVibrio harveyireporter assay for autoinducer 2 (AI-2) (M. G. Surette and B. L. Bassler, Proc. Natl. Acad. Sci. USA 95:7046-7050, 1998). TheluxSgene product mediates the production of AI-2 (M. G. Surette, M. B. Miller, and B. L. Bassler, Proc. Natl. Acad. Sci. USA 96:1639-1644, 1999). Environmental cues such as rapid growth, the presence of preferred carbon sources, low pH, and/or high osmolarity were found to influence the production of AI-2 (M. G. Surette and B. L. Bassler, Mol. Microbiol. 31:585-595, 1999). In addition to LuxS, thepfsgene product (Pfs) is required for AI-2 production, as well asS-adenosylhomocysteine (SAH) (S. Schauder, K. Shokat, M. G. Surette, and B. L. Bassler, Mol. Microbiol. 41:463-476, 2001). In bacterial cells, Pfs exhibits both 5′-methylthioadenosine (MTA) and SAH nucleosidase functions. Pfs is involved in methionine metabolism, regulating intracellular MTA and SAH levels (elevated levels of MTA and SAH are potent inhibitors of polyamine synthetases and S-adenosylmethionine dependent methyltransferase reactions, respectively). To further investigate regulation of AI-2 production inSalmonella, we constructedpfsandluxSpromoter fusions to aluxCDABEreporter in a low-copy-number vector, allowing an examination of transcription of the genes in the pathway for signal synthesis. Here we report thatluxSexpression is constitutive but that the transcription ofpfsis tightly correlated to AI-2 production inSalmonellaserovar Typhimurium 14028. NeitherluxSnorpfsexpression appears to be regulated by AI-2. These results suggest that AI-2 production is regulated at the level of LuxS substrate availability and not at the level ofluxSexpression. Our results indicate that AI-2-dependent signaling is a reflection of metabolic state of the cell and not cell density.
Chromosomal Locus That Affects Pathogenicity ofRhodococcus fascians
Authors: Vereecke Danny , Cornelis Karen , Temmerman Wim , Jaziri Mondher , Van Montagu Marc , Holsters Marcelle , Goethals Koen
Published: July 2002

The gram-positive plant pathogenRhodococcus fasciansprovokes leafy gall formation on a wide range of plants through secretion of signal molecules that interfere with the hormone balance of the host. Crucial virulence genes are located on a linear plasmid, and their expression is tightly controlled. A mutant with a mutation in a chromosomal locus that affected virulence was isolated. The mutation was located in genevicA, which encodes a malate synthase and is functional in the glyoxylate shunt of the Krebs cycle. VicA is required for efficient in planta growth in symptomatic, but not in normal, plant tissue, indicating that the metabolic requirement of the bacteria or the nutritional environment in plants or both change during the interaction. We propose that induced hyperplasia on plants represents specific niches for the causative organisms as a result of physiological alterations in the symptomatic tissue. Hence, such interaction could be referred to as metabolic habitat modification.
The Crystal Structure of Zn(II)-Free
Treponema pallidum
TroA, a Periplasmic Metal-Binding Protein, Reveals a Closed Conformation
Authors: Lee Yong-Hwan , Dorwart Michael R. , Hazlett Karsten R. O. , Deka Ranjit K. , Norgard Michael V. , Radolf Justin D. , Hasemann Charles A.
Published: July 2002

We previously demonstrated that
Treponema pallidum
TroA is a periplasmic metal-binding protein (MBP) with a distinctive alpha-helical backbone. To better understand the mechanisms of metal binding and release by TroA, we determined the crystal structure of the apoprotein at a resolution of 2.5 Å and compared it to that of the Zn(II)-bound form (Protein Data Bank accession code 1toa). apo-TroA shows a conformation even more closed than that of its Zn(II)-bound counterpart due to a 4° tilt of the C-terminal domain (residues 190 through 308) about an axis parallel to the poorly flexible backbone helix. This domain tilting pushes two loops (residues 248 through 253 and 277 through 286) towards the metal-binding site by more than 1 Å, resulting in an unfavorable interaction of I251 with D66. To avoid this contact, D66 shifts towards H68, one of the four Zn(II)-coordinating residues. The approach of this negative charge coincides with the flipping of the imidazole side chain of H68, resulting in the formation of a new hydrogen bond. The conformational change of H68, along with a slight rearrangement of D279, a C-terminal domain Zn(II)-coordinating residue, distorts the metal-binding site geometry, presumably causing the release of the bound metal ion. Ligand binding and release by TroA, and presumably by other members of the MBP cluster, differs from the “Venus flytrap” mechanism utilized by bacterial nonmetal solute-binding receptors.
Characterization of Plasmid pRT1 from
Pyrococcus
sp. Strain JT1
Authors: Ward Donald E. , Revet Ingrid M. , Nandakumar Renu , Tuttle Jon H. , de Vos Willem M. , van der Oost John , DiRuggiero Jocelyne
Published: July 2002

We discovered a 3,373-bp plasmid (pRT1) in the hyperthermophilic archaeon
Pyrococcus
sp. strain JT1. Two major open reading frames were identified, and analysis of the sequence revealed some resemblance to motifs typically found in plasmids that replicate via a rolling-circle mechanism. The presence of single-stranded DNA replication intermediates of pRT1 was detected, confirming this mode of replication.
Molecular Analysis of
Malassezia
Microflora on the Skin of Atopic Dermatitis Patients and Healthy Subjects
Authors: Sugita Takashi , Suto Hajime , Unno Tetsushi , Tsuboi Ryoji , Ogawa Hideoki , Shinoda Takako , Nishikawa Akemi
Published: July 2002

Members of the genus
Malassezia
, lipophilic yeasts, are considered to be one of the exacerbating factors in atopic dermatitis (AD). We examined variation in cutaneous colonization by
Malassezia
species in AD patients and compared it with variation in healthy subjects. Samples were collected by applying transparent dressings to the skin lesions of AD patients. DNA was extracted directly from the dressings and amplified in a specific nested PCR assay.
Malassezia
-specific DNA was detected in all samples obtained from 32 AD patients. In particular,
Malassezia globosa
and
M. restricta
were detected in approximately 90% of the AD patients and
M. furfur
and
M. sympodialis
were detected in approximately 40% of the cases. The detection rate was not dependent on the type of skin lesion. In healthy subjects,
Malassezia
DNA was detected in 78% of the samples, among which
M. globosa
,
M. restricta
, and
M. sympodialis
were detected at frequencies ranging from 44 to 61%, with
M. furfur
at 11%. The diversity of
Malassezia
species found in AD patients was greater (2.7 species detected in each individual) than that found in healthy subjects (1.8 species per individual). Our results suggest that
M. furfur
,
M. globosa
,
M. restricta
, and
M. sympodialis
are common inhabitants of the skin of both AD patients and healthy subjects, while the skin microflora of AD patients shows more diversity than that of healthy subjects. To our knowledge, this is the first report of the use of a nested PCR as an alternative to fungal culture for analysis of the distribution of cutaneous
Malassezia
spp.
Removal of PCR Inhibitors by Silica Membranes: Evaluating the Amplicor
Mycobacterium tuberculosis
Kit
Authors: Böddinghaus Boris , Wichelhaus Thomas A. , Brade Volker , Bittner Thomas
Published: July 2002

The effectiveness of PCR inhibitor removal by silica membranes in combination with the Amplicor
Mycobacterium tuberculosis
kit was analyzed for 655 respiratory and nonrespiratory specimens. The overall inhibition rate was reduced from 12.5%, when applying the Amplicor kit alone, to 1.1% with the addition of silica membrane DNA purification.
Phenotypic Identification of
Actinomyces
and Related Species Isolated from Human Sources
Authors: Sarkonen Nanna , Könönen Eija , Summanen Paula , Könönen Mauno , Jousimies-Somer Hannele
Published: July 2002

Recent advancements in chemotaxonomic and molecular biology-based identification methods have clarified the taxonomy of the genus
Actinomyces
and have led to the recognition of several new
Actinomyces
and related species.
Actinomyces
-like gram-positive rods have increasingly been isolated from various clinical specimens. Thus, an easily accessible scheme for reliable differentiation at the species level is needed in clinical and oral microbiology laboratories, where bacterial identification is mainly based on conventional biochemical methods. In the present study we designed a two-step protocol that consists of a flowchart that describes rapid, cost-efficient tests for preliminary identification of
Actinomyces
and closely related species and an updated more comprehensive scheme that also uses fermentation reactions for accurate differentiation of
Actinomyces
and closely related species.
Bacterial Genetic Fingerprint: a Reliable Factor in the Study of the Epidemiology of Human
Campylobacter
Enteritis?
Authors: Steinbrueckner B. , Ruberg F. , Kist M.
Published: July 2002

The rate of human intestinal infections with more than a single
Campylobacter
strain was determined and the genetic variabilities of
Campylobacter
strains throughout an infection episode were investigated by means of pulsed-field gel electrophoresis (PFGE) and enterobacterial repetitive intergenic consensus sequence PCR (ERIC-PCR). For 48 and 49 of 50 patients, all isolates from one sample showed identical patterns by PFGE and ERIC-PCR, respectively. Throughout an infection episode in 47 of 52 patients, the PFGE fingerprints of the isolates remained stable, while in 1 patient two different species were observed and in 4 patients different patterns were observed. Therefore, ERIC-PCR proved less discriminative than PFGE. These findings suggest that human infection with more than one
Campylobacter
strain is rare and should not significantly impair epidemiologic analyses. However, changes in the genetic fingerprint throughout an infection should be considered in the assessment of epidemiologic studies of
Campylobacter
spp.
Comparison of DNA Sequencing and a Line Probe Assay for Detection of Human Immunodeficiency Virus Type 1 Drug Resistance Mutations in Patients Failing Highly Active Antiretroviral Therapy
Authors: Servais Jean , Lambert Christine , Fontaine Elodie , Plesséria Jean-Marc , Robert Isabelle , Arendt Vic , Staub Thérèse , Schneider François , Hemmer Robert , Burtonboy Guy , Schmit Jean-Claude
Published: July 2002

The resistance of human immunodeficiency virus type 1 (HIV-1) to drugs is a major cause of antiretroviral treatment failure. We have compared direct sequencing to a line probe assay (LiPA) for the detection of drug resistance-related mutations in 197 clinical samples, and we have investigated the sequential appearance of mutations under drug pressure. For 26 patients with virological failure despite the use of two nucleoside analogues and one protease inhibitor (indinavir [
n
= 6], ritonavir [
n
= 10], and saquinavir [
n
= 10]), genotypic resistance assays were carried out retrospectively every 3 months for up to 2 years by using direct sequencing (TruGene; Visible Genetics) and a LiPA for detection of mutations in the reverse transcriptase (INNO-LiPA HIV-1 RT; Innogenetics) and the protease (INNO-LiPA HIV Protease, prototype version; Innogenetics) genes. Comparison of the results from both assays found rare major discrepancies (<1% of codons analyzed). INNO-LiPA detected more wild-type–mutant mixtures than sequencing but suffered from a high rate of codon hybridization failures for the reverse transcriptase. LiPA detected earlier and more frequently than sequencing the transient mixed virus population that contained I84V, which appears before V82A in the protease sequence. Mutations M461, G48V, and L90M were often transient and drug pressure related. In conclusion, direct sequencing and LiPAs give concordant results for most clinical isolates. LiPAs are more sensitive for the detection of mixed virus populations. Mutation I84V appears in minor populations in the early steps of the pathways of resistance to indinavir and ritonavir. The fact that some mutations can be found only transiently and in minor virus populations highlights the importance of a low detection limit for resistance assays.
Multicenter Comparison Trial of DNA Extraction Methods and PCR Assays for Detection of
Chlamydia pneumoniae
in Endarterectomy Specimens
Authors: Apfalter Petra , Blasi Francesco , Boman Jens , Gaydos Charlotte A. , Kundi Michael , Maass Matthias , Makristathis Athanasios , Meijer Adam , Nadrchal Reinhard , Persson Kenneth , Rotter Manfred L. , Tong C. Y. William , Stanek Gerold , Hirschl Alexander M.
Published: July 2002

The reported rate of detection of
Chlamydia pneumoniae
DNA within atherosclerotic lesions by PCR varies between 0 and 100%. In this study, identical sets of coded experimental atheroma samples (
n
= 15) and spiked controls (
n
= 5) were analyzed by 16 test methods in nine centers by means of PCR. The positive controls were correctly identified to levels of 1, 0.1, and 0.01 inclusion bodies of
C. pneumoniae
/ml of tissue homogenate by 16 (100%), 11 (69%), and 3 (19%) of the test methods, respectively. Three out of 16 negative controls (19%) were rated positive. Positivity rates for atheroma samples varied between 0 and 60% for the different test methods, with the maximum concordant result for positivity being only 25% for one carotid artery sample. There was no consistent pattern of positive results among the various laboratories, and there was no correlation between the detection rates and the sensitivity of the assay used.
Use of a LightCycler
gyrA
Mutation Assay for Rapid Identification of Mutations Conferring Decreased Susceptibility to Ciprofloxacin in Multiresistant
Salmonella enterica
Serotype Typhimurium DT104 Isolates
Authors: Walker R. A. , Saunders N. , Lawson A. J. , Lindsay E. A. , Dassama M. , Ward L. R. , Woodward M. J. , Davies R. H. , Liebana E. , Threlfall E. J.
Published: July 2002

A LightCycler-based PCR-hybridization
gyrA
mutation assay (GAMA) was developed to rapidly detect
gyrA
point mutations in multiresistant (MR)
Salmonella enterica
serotype Typhimurium DT104 with decreased susceptibility to ciprofloxacin (MIC, 0.25 to 1.0 mg/liter). Ninety-two isolates (49 human, 43 animal) were tested with three individual oligonucleotide probes directed against an Asp-87-to-Asn (GAC→AAC) mutation, an Asp-87-to-Gly (GAC→GGC) mutation, and a Ser-83-to-Phe (TCC→TTC) mutation. Strains homologous to the probes could be distinguished from strains that had different mutations by their probe-target melting temperatures. Thirty-seven human and 30 animal isolates had an Asp-87-to-Asn substitution, 6 human and 6 animal isolates had a Ser-83-to-Phe substitution, and 5 human and 2 animal isolates had an Asp-87-to-Gly substitution. The remaining six strains all had mismatches with the three probes and therefore different
gyrA
mutations. The sequencing of
gyrA
from these six isolates showed that one human strain and two animal strains had an Asp-87-to-Tyr (GAC→TAC) substitution and two animal strains had a Ser-83-to-Tyr (TCC→TAC) substitution. One animal strain had no
gyrA
mutation, suggesting that this isolate had a different mechanism of resistance. Fifty-eight of the strains tested were indistinguishable by several different typing methods including antibiograms, pulsed-field gel gel electrophoresis, and plasmid profiling, although they could be further subdivided according to
gyrA
mutation. This study confirmed that MR DT104 with decreased susceptibility to ciprofloxacin from humans and food animals in England and Wales may have arisen independently against a background of clonal spread of MR DT104.
Coexistence of SHV-4- and TEM-24-Producing
Enterobacter aerogenes
Strains before a Large Outbreak of TEM-24-Producing Strains in a French Hospital
Authors: Mammeri H. , Laurans G. , Eveillard M. , Castelain S. , Eb F.
Published: July 2002

In 1996, a monitoring program was initiated at the teaching hospital of Amiens, France, and carried out for 3 years. All extended-spectrum β-lactamase (ESBL)-producing
Enterobacter aerogenes
isolates recovered from clinical specimens were collected for investigation of their epidemiological relatedness by pulsed-field gel electrophoresis and enterobacterial repetitive intergenic consensus PCR (ERIC-PCR) and determination of the type of ESBL harbored by isoelectric focusing and DNA sequencing. Molecular typing revealed the endemic coexistence, during the first 2 years, of two clones expressing, respectively, SHV-4 and TEM-24 ESBLs, while an outbreak of the TEM-24-producing strain raged in the hospital during the third year, causing the infection or colonization of 165 patients. Furthermore, this strain was identified as the prevalent clone responsible for outbreaks in many French hospitals since 1996. This study shows that TEM-24-producing
E. aerogenes
is an epidemic clone that is well established in the hospital's ecology and able to spread throughout wards. The management of the outbreak at the teaching hospital of Amiens, which included the reinforcement of infection control measures, failed to obtain complete eradication of the clone, which has become an endemic pathogen.
Truncated Hantavirus Nucleocapsid Proteins for Serotyping Hantaan, Seoul, and Dobrava Hantavirus Infections
Authors: Araki Koichi , Yoshimatsu Kumiko , Ogino Michiko , Ebihara Hideki , Lundkvist Åke , Kariwa Hiroaki , Takashima Ikuo , Arikawa Jiro
Published: July 2002

Truncated recombinant nucleocapsid proteins (rNPs) of Hantaan virus (HTNV), Seoul virus (SEOV), and Dobrava virus (DOBV) were expressed by a baculovirus system. The truncated rNPs, which lacked 49 (rNP50) or 154 (rNP155) N-terminal amino acids of the NPs of HTNV, SEOV, and DOBV, were able to differentiate HTNV-, SEOV-, and DOBV-specific immune sera. Recombinant NP50s retained higher reactivities than rNP155s and were proven useful for enzyme-linked immunosorbent assay (ELISA). The ELISAs based on the rNP50s of HTNV, SEOV, and DOBV successfully differentiated three groups of patient sera, previously defined by neutralization tests: 17 with HTNV infection, 12 with SEOV infection, and 20 with DOBV infection. The entire rNP of Puumala virus (PUUV) distinguished PUUV infection from the other types of hantavirus infection. Serotyping with these rNP50s can be recommended as a rapid and efficient system for hantavirus diagnosis.
Waldenström's Disease Complicated by Recurrent Meningococcal Arthritis
Authors: Singwe-Ngandeu Madeleine , Buchs Nicolas , Rohner Peter , Gabay Cem
Published: July 2002

Meningococcal arthritis is rare. We report a patient in whom a first episode of meningococcal arthritis revealed Waldenström's disease and who experienced a second episode of meningococcal arthritis 8 years later. We suggest that an impaired immune response secondary to Waldenström's disease favored the recurrence of meningococcal arthritis.