bluuebunny/crossref_metadata_2025 huggingface dataset:
Yersinia enterocolitica
TyeA, an Intracellular Regulator of the Type III Machinery, Is Required for Specific Targeting of YopE, YopH, YopM, and YopN into the Cytosol of Eukaryotic Cells
Authors: Cheng Luisa W. , Schneewind Olaf
Published: July 2002

Pathogenic
Yersinia
species employ type III machines to target effector Yops into the cytosol of eukaryotic cells.
Yersinia tyeA
mutants are thought to be defective in the targeting of YopE and YopH without affecting the injection of YopM, YopN, YopO, YopP, and YopT into the cytosol of eukaryotic cells. One model suggests that TyeA may form a tether between YopN (LcrE) and YopD on the bacterial surface, a structure that may translocate YopE and YopH across the plasma membrane of eukaryotic cells (M. Iriarte, M. P. Sory, A. Boland, A. P. Boyd, S. D. Mills, I. Lambermont, and G. R. Cornelis, EMBO J. 17:1907–1918, 1998). We have examined the injection of Yop proteins by
tyeA
mutant yersiniae with the digitonin fractionation technique. We find that
tyeA
mutant yersiniae not only secreted YopE, YopH, YopM, and YopN into the extracellular medium but also targeted these polypeptides into the cytosol of HeLa cells. Protease protection, cell fractionation, and affinity purification experiments suggest that TyeA is located intracellularly and binds to YopN or YopD. We propose a model whereby TyeA functions as a negative regulator of the type III targeting pathway in the cytoplasm of yersiniae, presumably by preventing the export of YopN.
Evidence for a Signaling System in
Helicobacter pylori
: Detection of a
luxS
-Encoded Autoinducer
Authors: Joyce Elizabeth A. , Bassler Bonnie L. , Wright Andrew
Published: July 2002

Helicobacter pylori
possesses a homolog of the
luxS
gene, initially identified by its role in autoinducer production for the quorum-sensing system 2 in
Vibrio harveyi
. The genomes of several other species of bacteria, notably
Escherichia coli
,
Salmonella enterica
serovar Typhimurium, and
Vibrio cholerae
, also include
luxS
homologs. All of these bacteria have been shown to produce active autoinducers capable of stimulating the expression of the luciferase operon in
V. harveyi
. In this report, we demonstrate that
H. pylori
also synthesizes a functional autoinducer (AI-2) that can specifically activate signaling system 2 in
V. harveyi
. Maximal activity is produced during early log phase, and the activity is diminished when cells enter stationary phase. We show that AI-2 is not involved in modulating any of the known or putative virulence factors in
H. pylori
and that a
luxS
null mutant has a two-dimensional protein profile identical to that of its isogenic parent strain. We discuss the implications of having an AI-2-like quorum-sensing system in
H. pylori
and suggest possible roles that it may play in
H. pylori
infection.
Effects of Perturbations of the Nitrogenase Electron Transfer Chain on Reversible ADP-Ribosylation of Nitrogenase Fe Protein in
Klebsiella pneumoniae
Strains Bearing the
Rhodospirillum rubrum dra
Operon
Authors: Halbleib Cale M. , Zhang Yaoping , Roberts Gary P. , Ludden Paul W.
Published: July 2002

The redox state of nitrogenase Fe protein is shown to affect regulation of ADP-ribosylation in
Klebsiella pneumoniae
strains transformed by plasmids carrying
dra
genes from
Rhodospirillum rubrum
. The
dra
operon encodes dinitrogenase reductase ADP-ribosyltransferase and dinitrogenase reductase-activating glycohydrolase, enzymes responsible for the reversible inactivation, via ADP-ribosylation, of nitrogenase Fe protein in
R. rubrum
. In bacteria containing the
dra
operon in their chromosomes, inactivation occurs in response to energy limitation or nitrogen sufficiency. The
dra
gene products, expressed at a low level in
K. pneumoniae
, enable transformants to reversibly ADP-ribosylate nitrogenase Fe protein in response to the presence of fixed nitrogen. The activities of both regulatory enzymes are regulated in vivo as described in
R. rubrum
. Genetic perturbations of the nitrogenase electron transport chain were found to affect the rate of inactivation of Fe protein. Strains lacking the electron donors to Fe protein (NifF or NifJ) were found to inactivate Fe protein more quickly than a strain with wild-type background. Deletion of
nifD
, which encodes a subunit of nitrogenase MoFe protein, was found to result in a slower inactivation response. No variation was found in the reactivation responses of these strains. It is concluded that the redox state of the Fe protein contributes to the regulation of the ADP-ribosylation of Fe protein.
Characterization of a Second
tfd
Gene Cluster for Chlorophenol and Chlorocatechol Metabolism on Plasmid pJP4 in
Ralstonia eutropha
JMP134(pJP4)
Authors: Laemmli Caroline M. , Leveau Johan H. J. , Zehnder Alexander J. B. , van der Meer Jan Roelof
Published: July 2002

Within the 5.9-kb DNA region between the
tfdR
and
tfdK
genes on the 2,4-dichlorophenoxyacetic acid (2,4-D) catabolic plasmid pJP4 from
Ralstonia eutropha
JMP134, we identified five open reading frames (ORFs) with significant homology to the genes for chlorocatechol and chlorophenol metabolism (
tfdCDEF
and
tfdB
) already present elsewhere on pJP4. The five ORFs were organized and assigned as follows:
tfdD
II
C
II
E
II
F
II
and
tfdB
II
(in short, the
tfd
II
cluster), by analogy to
tfdCDEF
and
tfdB
(the
tfd
I
cluster). Primer extension analysis of mRNA isolated from 2,4-D-grown
R. eutropha
JMP134 identified a single transcription start site in front of the first gene of the cluster,
tfdD
II
, suggesting an operon-like organization for the
tfd
II
genes. By expressing each ORF in
Escherichia coli
, we confirmed that
tfdD
II
coded for a chloromuconate cycloisomerase,
tfdC
II
coded for a chlorocatechol 1,2-dioxygenase,
tfdE
II
coded for a dienelactone hydrolase,
tfdF
II
coded for a maleylacetate reductase, and
tfdB
II
coded for a chlorophenol hydroxylase. Dot blot hybridizations of mRNA isolated from
R. eutropha
JMP134 showed that both
tfd
I
and
tfd
II
genes are transcribed upon induction with 2,4-D. Thus, the functions encoded by the
tfd
II
genes seem to be redundant with respect to those of the
tfd
I
cluster. One reason why the
tfd
II
genes do not disappear from plasmid pJP4 might be the necessity for keeping the regulatory genes for the 2,4-D pathway expression
tfdR
and
tfdS
.
rpoS
Mutants in Archival Cultures of
Salmonella enterica
Serovar Typhimurium
Authors: Sutton Amy , Buencamino Raphael , Eisenstark Abraham
Published: July 2002

Long-term survival under limited growth conditions presents bacterial populations with unique environmental challenges. The existence of
Salmonella enterica
serovar Typhimurium cultures undisturbed in sealed nutrient agar stab vials for 34 to 45 years offered a unique opportunity to examine genetic variability under natural conditions. We have initiated a study of genetic changes in these archival cultures. We chose to start with examination of the
rpoS
gene since, among gram-negative bacteria, many genes needed for survival are regulated by RpoS, the stationary-phase sigma factor. In each of 27 vials examined, cells had the
rpoS
start codon UUG instead of the expected AUG of
Salmonella
and
Escherichia coli
strains recorded in GenBank. Ten of the 27 had additional mutations in the
rpoS
gene compared with the
X77752
wild-type strain currently recorded in GenBank. The
rpoS
mutations in the 10 strains included two deletions as well as point mutations that altered amino acid sequences substantially. Since these stored strains were derived from ancestral cells inoculated decades ago and remained undisturbed, it is assumed that the 10
rpoS
mutations occurred during storage. Since the remaining 17 sequences were wild type (other than in the start codon), it is obvious that
rpoS
remained relatively stable during decades of sealed storage.
Competition between the Yops of
Yersinia enterocolitica
for Delivery into Eukaryotic Cells: Role of the SycE Chaperone Binding Domain of YopE
Authors: Boyd Aoife P. , Lambermont Isabelle , Cornelis Guy R.
Published: July 2002

A type III secretion-translocation system allows
Yersinia
adhering at the surface of animal cells to deliver a cocktail of effector Yops (YopH, -O, -P, -E, -M, and -T) into the cytosol of these cells. Residues or codons 1 to 77 contain all the information required for the complete delivery of YopE into the target cell (release from the bacterium and translocation across the eukaryotic cell membrane). Residues or codons 1 to 15 are sufficient for release from the wild-type bacterium under Ca
2+
-chelating conditions but not for delivery into target cells. Residues 15 to 50 comprise the binding domain for SycE, a chaperone specific for YopE that is necessary for release and translocation of full-length YopE. To understand the role of this chaperone, we studied the delivery of YopE-Cya reporter proteins and YopE deletants by polymutant
Yersinia
devoid of most of the Yop effectors (ΔHOPEM and ΔTHE strains). We first tested YopE-Cya hybrid proteins and YopE proteins deleted of the SycE-binding site. In contrast to wild-type strains, these mutants delivered YopE
15
-Cya as efficiently as YopE
130
-Cya. They were also able to deliver YopE
Δ17–77
. SycE was dispensable for these deliveries. These results show that residues or codons 1 to 15 are sufficient for delivery into eukaryotic cells and that there is no specific translocation signal in Yops. However, the fact that the SycE-binding site and SycE were necessary for delivery of YopE by wild-type
Yersinia
suggests that they could introduce hierarchy among the effectors to be delivered. We then tested a YopE-Cya hybrid and YopE proteins deleted of amino acids 2 to 15 but containing the SycE-binding domain. These constructs were neither released in vitro upon Ca
2+
chelation nor delivered into cells by wild-type or polymutant bacteria, casting doubts on the hypothesis that SycE could be a secretion pilot. Finally, it appeared that residues 50 to 77 are inhibitory to YopE release and that binding of SycE overcomes this inhibitory effect. Removal of this domain allowed in vitro release and delivery in cells in the absence as well as in the presence of SycE.
Understanding the Growth Phenotype of the Yeast
gcr1
Mutant in Terms of Global Genomic Expression Patterns
Authors: López M. Cecilia , Baker Henry V.
Published: July 2002

The phenotype of an organism is the manifestation of its expressed genome. The
gcr1
mutant of yeast grows at near wild-type rates on nonfermentable carbon sources but exhibits a severe growth defect when grown in the presence of glucose, even when nonfermentable carbon sources are available. Using DNA microarrays, the genomic expression patterns of wild-type and
gcr1
mutant yeast growing on various media, with and without glucose, were compared. A total of 53 open reading frames (ORFs) were identified as
GCR1
dependent based on the criterion that their expression was reduced twofold or greater in mutant versus wild-type cultures grown in permissive medium consisting of YP supplemented with glycerol and lactate. The
GCR1
-dependent genes, so defined, fell into three classes: (i) glycolytic enzyme genes, (ii) ORFs carried by Ty elements, and (iii) genes not previously known to be
GCR1
dependent. In wild-type cultures,
GCR1
-dependent genes accounted for 27% of the total hybridization signal, whereas in mutant cultures, they accounted for 6% of the total. Glucose addition to the growth medium resulted in a reprogramming of gene expression in both wild-type and mutant yeasts. In both strains, glycolytic enzyme gene expression was induced by the addition of glucose, although the expression of these genes was still impaired in the mutant compared to the wild type. By contrast, glucose resulted in a strong induction of Ty-borne genes in the mutant background but did not greatly affect their already high expression in the wild-type background. Both strains responded to glucose by repressing the expression of genes involved in respiration and the metabolism of alternative carbon sources. Thus, the severe growth inhibition observed in
gcr1
mutants in the presence of glucose is the result of normal signal transduction pathways and glucose repression mechanisms operating without sufficient glycolytic enzyme gene expression to support growth via glycolysis alone.
A Single-Transformation Gene Function Test in Diploid
Candida albicans
Authors: Enloe Brian , Diamond Aviva , Mitchell Aaron P.
Published: July 2002

The fungal pathogen
Candida albicans
is naturally diploid, and current gene disruption strategies require two successive transformations. We describe here a genetic construct (
UAU1
) for which two copies may be selected. Insertion of
UAU1
into one genomic site, after a single transformation, allows selection for segregants with two copies of the insertion. Major classes of segregants are those carrying homozygous insertion mutations and allelic triplications, which have two insertion alleles and a wild-type allele. Thus nonessential and essential genes may be distinguished rapidly through PCR tests for homozygosis and triplication. We find that homozygous mutations may be isolated at three nonessential loci (
ADE2, RIM20
, and
YGR189
), while only allelic triplications were found at two essential loci (
SNF1
and
CDC28
). We have unexpectedly isolated homozygous mutants with mutations at
CDC25
; they are viable but defective in filamentation on serum-containing medium. The
UAU1
cassette is thus useful to assess rapidly the essentiality of
C. albicans
genes.
The
torYZ
(
yecK bisZ
) Operon Encodes a Third Respiratory Trimethylamine
N
-Oxide Reductase in
Escherichia coli
Authors: Gon Stéphanie , Patte Jean-Claude , Méjean Vincent , Iobbi-Nivol Chantal
Published: July 2002

The
bisZ
gene of
Escherichia coli
was previously described as encoding a minor biotin sulfoxide (BSO) reductase in addition to the main cytoplasmic BSO reductase, BisC. In this study,
bisZ
has been renamed
torZ
based on the findings that (i) the
torZ
gene product, TorZ, is able to reduce trimethylamine
N
-oxide (TMAO) more efficiently than BSO; (ii) although TorZ is more homologous to BisC than to the TMAO reductase TorA (63 and 42% identity, respectively), it is located mainly in the periplasm as is TorA; (iii)
torZ
belongs to the
torYZ
operon, and the first gene,
torY
(formerly
yecK
), encodes a pentahemic
c
-type cytochrome homologous to the TorC cytochrome of the TorCAD respiratory system. Furthermore, the
torYZ
operon encodes a third TMAO respiratory system, with catalytic properties that are clearly different from those of the TorCAD and the DmsABC systems. The
torYZ
and the
torCAD
operons may have diverged from a common ancestor, but, surprisingly, no
torD
homologue is found in the sequences around
torYZ
. Moreover, the
torYZ
operon is expressed at very low levels under the conditions tested, and, in contrast to
torCAD
, it is not induced by TMAO or dimethyl sulfoxide.
Acquisition of the
rfb-gnd
Cluster in Evolution of
Escherichia coli
O55 and O157
Authors: Tarr Phillip I. , Schoening Laura M. , Yea Yoo-Lee , Ward Teresa R. , Jelacic Srdjan , Whittam Thomas S.
Published: July 2002

The
rfb
region specifies the structure of lipopolysaccharide side chains that comprise the diverse gram-negative bacterial somatic (O) antigens. The
rfb
locus is adjacent to
gnd
, which is a polymorphic gene encoding 6-phosphogluconate dehydrogenase. To determine if
rfb
and
gnd
cotransfer, we sequenced
gnd
in five O55 and 13 O157 strains of
Escherichia coli. E. coli
O157:H7 has a
gnd
allele (allele
A
) that is only 82% identical to the
gnd
allele (allele
D
) of closely related
E. coli
O55:H7. In contrast,
gnd
alleles of
E. coli
O55 in distant lineages are >99.9% identical to
gnd
allele
D
. Though
gnd
alleles
B
and
C
in
E. coli
O157 that are distantly related to
E. coli
O157:H7 are more similar to allele
A
than to allele
D
, there are nucleotide differences at 4 to 6% of their sites. Alleles
B
and
C
can be found in
E. coli
O157 in different lineages, but we have found allele
A
only in
E. coli
O157 belonging to the DEC5 lineage. DNA 3′ to the O55
gnd
allele in diverse
E. coli
lineages has sequences homologous to
tnpA
of the
Salmonella enterica
serovar Typhimurium IS
200
element,
E. coli Rhs
elements (including an H-rpt gene), and portions of the O111 and O157
rfb
regions. We conclude that
rfb
and
gnd
cotransferred into
E. coli
O55 and O157 in widely separated lineages and that recombination was responsible for recent antigenic shifts in the emergence of pathogenic
E. coli
O55 and O157.
Bacteriophage T4 Self-Assembly: In Vitro Reconstitution of Recombinant gp2 into Infectious Phage
Authors: Wang G. R. , Vianelli A. , Goldberg E. B.
Published: July 2002

T4 gene 2 mutants have a pleiotropic phenotype: degradation of injected phage DNA by exonuclease V (ExoV) in therecBCD+host cell cytoplasm and a low burst size due, at least in part, to a decreased ability for head-to-tail (H-T) joining. The more N terminal the mutation, the more pronounced is the H-T joining defect. We have overexpressed and purified the recombinant gene 2 product (rgp2) to homogeneity in order to test its role in H-T joining, during in vitro reconstitution. When we mix extracts of heads from a gp2+phage infection (H+) with tails from a gp2+or gp2−phage infection (T+or T−), the H-T joining is fast and all of the reconstituted phage grow equally well on cells with or without ExoV activity. When heads from gene 2 amber mutants (H−) are used, addition of rgp2 is required for H-T joining. In this case, H-T joining is slow and only about 10% of the reconstituted phage can form plaques on ExoV+cells. When extracts of heads with different gene 2 amber mutations are mixed with extracts of tails (with a gene 2 amber mutation) in the presence of rgp2, we find that the size of the gp2 amber peptide of the head extract is inversely related to the fraction of reconstituted phage with a 2+phenotype. We conclude that free rgp2 is biologically active and has a direct role in H-T joining but that the process is different from H-T joining promoted by natural gp2 that is incorporated into the head in vivo. Furthermore, it seems that gp2 has a domain which binds it to the head. Thus, the presence of the longer gp2am mutants (with this domain) inhibits their replacement by full-length rgp2.
Autoinduction of 2,4-Diacetylphloroglucinol Biosynthesis in the Biocontrol Agent
Pseudomonas fluorescens
CHA0 and Repression by the Bacterial Metabolites Salicylate and Pyoluteorin
Authors: Schnider-Keel Ursula , Seematter Arnaud , Maurhofer Monika , Blumer Caroline , Duffy Brion , Gigot-Bonnefoy Cécile , Reimmann Cornelia , Notz Regina , Défago Geneviève , Haas Dieter , Keel Christoph
Published: July 2002

The antimicrobial metabolite 2,4-diacetylphloroglucinol (2,4-DAPG) contributes to the capacity of
Pseudomonas fluorescens
strain CHA0 to control plant diseases caused by soilborne pathogens. A 2,4-DAPG-negative Tn
5
insertion mutant of strain CHA0 was isolated, and the nucleotide sequence of the 4-kb genomic DNA region adjacent to the Tn
5
insertion site was determined. Four open reading frames were identified, two of which were homologous to
phlA
, the first gene of the 2,4-DAPG biosynthetic operon, and to the
phlF
gene encoding a pathway-specific transcriptional repressor. The Tn
5
insertion was located in an open reading frame, tentatively named
phlH
, which is not related to known
phl
genes. In wild-type CHA0, 2,4-DAPG production paralleled expression of a
phlA′-′lacZ
translational fusion, reaching a maximum in the late exponential growth phase. Thereafter, the compound appeared to be degraded to monoacetylphloroglucinol by the bacterium. 2,4-DAPG was identified as the active compound in extracts from culture supernatants of strain CHA0 specifically inducing
phlA′-′lacZ
expression about sixfold during exponential growth. Induction by exogenous 2,4-DAPG was most conspicuous in a
phlA
mutant, which was unable to produce 2,4-DAPG. In a
phlF
mutant, 2,4-DAPG production was enhanced severalfold and
phlA′-′lacZ
was expressed at a level corresponding to that in the wild type with 2,4-DAPG added. The
phlF
mutant was insensitive to 2,4-DAPG addition. A transcriptional
phlA-lacZ
fusion was used to demonstrate that the repressor PhlF acts at the level of transcription. Expression of
phlA′-′lacZ
and 2,4-DAPG synthesis in strain CHA0 was strongly repressed by the bacterial extracellular metabolites salicylate and pyoluteorin as well as by fusaric acid, a toxin produced by the pythopathogenic fungus
Fusarium
. In the
phlF
mutant, these compounds did not affect
phlA′-′lacZ
expression and 2,4-DAPG production. PhlF-mediated induction by 2,4-DAPG and repression by salicylate of
phlA′-′lacZ
expression was confirmed by using
Escherichia coli
as a heterologous host. In conclusion, our results show that autoinduction of 2,4-DAPG biosynthesis can be countered by certain bacterial (and fungal) metabolites. This mechanism, which depends on
phlF
function, may help
P. fluorescens
to produce homeostatically balanced amounts of extracellular metabolites.
Effect of Temperature on Stability and Activity of Elongation Factor 2 Proteins from Antarctic and Thermophilic Methanogens
Authors: Thomas Torsten , Cavicchioli Ricardo
Published: July 2002

Despite the presence and abundance of archaea in low-temperature environments, little information is available regarding their physiological and biochemical properties. In order to investigate the adaptation of archaeal proteins to low temperatures, we purified and characterized the elongation factor 2 (EF-2) protein from the Antarctic methanogen
Methanococcoides burtonii
, which was expressed in
Escherichia coli
, and compared it to the recombinant EF-2 protein from a phylogenetically related thermophile,
Methanosarcina thermophila
. Using differential scanning calorimetry to assess protein stability and enzyme assays for the intrinsic GTPase activity, we identified biochemical and biophysical properties that are characteristic of the cold-adapted protein. This includes a higher activity at low temperatures caused by a decrease of the activation energy necessary for GTP hydrolysis and a decreased activation energy for the irreversible denaturation of the protein, which indicates a less thermostable structure. Comparison of the in vitro properties of the proteins with the temperature-dependent characteristics of growth of the organisms indicates that additional cytoplasmic factors are likely to be important for the complete thermal adaptation of the proteins in vivo. This is the first study to address thermal adaptation of proteins from a free-living, cold-adapted archaeon, and our results indicate that the ability of the Antarctic methanogen to adapt to the cold is likely to involve protein structural changes.
Multiple Factors Independently RegulatehilAand Invasion Gene Expression inSalmonella entericaSerovar Typhimurium
Authors: Lucas Robin L. , Lostroh C. Phoebe , DiRusso Concetta C. , Spector Michael P. , Wanner Barry L. , Lee Catherine A.
Published: July 2002

HilA activates the expression ofSalmonella entericaserovar Typhimurium invasion genes. To learn more about regulation ofhilA, we isolated Tn5mutants exhibiting reducedhilAand/or invasion gene expression. In addition to expected mutations, we identified Tn5insertions inpstS,fadD,flhD,flhC, andfliA. Analysis of thepstSmutant indicates thathilAand invasion genes are repressed by the response regulator PhoB in the absence of the Pst high-affinity inorganic phosphate uptake system. This system is required for negative control of the PhoR-PhoB two-component regulatory system, suggesting thathilAexpression may be repressed by PhoR-PhoB under low extracellular inorganic phosphate conditions. FadD is required for uptake and degradation of long-chain fatty acids, and our analysis of thefadDmutant indicates thathilAis regulated by a FadD-dependent, FadR-independent mechanism. Thus, fatty acid derivatives may act as intracellular signals to regulatehilAexpression.flhDCandfliAencode transcription factors required for flagellum production, motility, and chemotaxis. Complementation studies withflhCandfliAmutants indicate that FliZ, which is encoded in an operon withfliA, activates expression ofhilA, linking regulation ofhilAwith motility. Finally, epistasis tests showed that PhoB, FadD, FliZ, SirA, and EnvZ act independently to regulatehilAexpression and invasion. In summary, our screen has identified several distinct pathways that can modulateS. entericaserovar Typhimurium's ability to expresshilAand invade host cells. Integration of signals from these different pathways may help restrict invasion gene expression during infection.
Characterization of the Promoter Elements for the Staphylococcal Enterotoxin D Gene
Authors: Zhang Shuping , Stewart George C.
Published: July 2002

Deletion analysis of the promoter for the
Staphylococcus aureus
enterotoxin D determinant indicated that a 52-bp sequence, from −34 to +18, was sufficient for
sed
promoter function and
agr
regulation. A consensus −10 Pribnow box sequence, a less conserved −35 sequence, and a TG dinucleotide motif were present. Transcribed sequences (+1 to +18) are essential for promoter activity.
Genetic Variation and Evolutionary Origin of the Direct Repeat Locus of
Mycobacterium tuberculosis
Complex Bacteria
Authors: van Embden J. D. A. , van Gorkom T. , Kremer K. , Jansen R. , van der Zeijst B. A. M. , Schouls L. M.
Published: July 2002

The direct repeat region in
Mycobacterium tuberculosis
complex strains is composed of multiple direct variant repeats (DVRs), each of which is composed of a 36-bp direct repeat (DR) plus a nonrepetitive spacer sequence of similar size. It has been shown previously that clinical isolates show extensive polymorphism in the DR region by the variable presence of DVRs, and this polymorphism has been used in the epidemiology of tuberculosis. In an attempt to better understand the evolutionary scenario leading to polymorphic DR loci and to improve strain differentiation by spoligotyping, we characterized and compared the DNA sequences of the complete DR region and its flanking DNA of
M. tuberculosis
complex strains. We identified 94 different spacer sequences among 26
M. tuberculosis
complex strains. No sequence homology was found between any of these spacers and
M. tuberculosis
DNA outside of the DR region or with any other known bacterial sequence. Although strains differed extensively in the presence or absence of DVRs, the order of the spacers in the DR locus was found to be well conserved. The data strongly suggest that the polymorphism in clinical isolates is the result of successive deletions of single discrete DVRs or of multiple contiguous DVRs from a primordial DR region containing many more DVRs than seen in present day isolates and that virtually no scrambling of DVRs took place during evolution. Because the majority of the novel spacer sequences identified in this study were confined to isolates of the rare
Mycobacterium canettii
taxon, the use of the novel spacers in spoligotyping led only to a slight improvement of strain differentiation by spoligotyping.
Probing the CD Lumenal Loop Region of the D2 Protein of Photosystem II in
Synechocystis
sp. Strain PCC 6803 by Combinatorial Mutagenesis
Authors: Keilty Anna T. , Ermakova-Gerdes Svetlana Y. , Vermaas Wim F. J.
Published: July 2002

The CD lumenal loop region of the photosystem II reaction center protein D2 contains residues involved in oxygen evolution. Since detailed structural information about this region is unavailable, an M13-based combinatorial mutagenesis approach was used to investigate structure-function relationships in this vital region of D2 in
Synechocystis
sp. strain PCC 6803. The CD loop coding region contains close to 100 nucleotides, and for effective mutagenesis, it was subdivided into four regions of seven to eight codons. A gain-of-function selection protocol was employed such that all mutants that were selected contained a functional D2 protein. In this way, conservation patterns of residues along with numbers and types of amino acid substitutions accommodated at each position for each set of mutants would indicate which residues in the CD loop may play important structural and functional roles. Results of this study have substantiated the importance of residues previously studied by site-directed mutagenesis such as Arg180 and His189 and have identified other previously unremarkable residues in the CD loop (such as Ser166, Phe169, and Ala170) that cannot be replaced by many other residues. In addition, the pliability of the CD loop was further tested using deletion and D1-D2 substitution constructs in M13. This showed that the length of the loop was important to its function, and in two cases, D2 could accommodate homologous sequences from D1, which forms a heterodimer with D2 in photosystem II, but not the other way around. This study of the CD loop in D2 provides valuable clues regarding the structural and functional requirements of the region.
Effects of
bfp
Mutations on Biogenesis of Functional Enteropathogenic
Escherichia coli
Type IV Pili
Authors: Anantha Ravi P. , Stone Kelly D. , Donnenberg Michael S.
Published: July 2002

Enteropathogenic
Escherichia coli
expresses a type IV fimbria known as the bundle-forming pilus (BFP) that is required for autoaggregation and localized adherence (LA) to host cells. A cluster of 14 genes is sufficient to reconstitute BFP biogenesis in a laboratory strain of
E. coli
. We have undertaken a systematic mutagenesis of the individual genes to determine the effect of each mutation on BFP biogenesis and LA. Here we report the construction and analysis of nonpolar mutations in six genes of the
bfp
cluster,
bfpG
,
bfpB
,
bfpC
,
bfpD
,
bfpP
, and
bfpH
, as well as the further analysis of a previously described
bfpA
mutant strain that is unable to express bundlin, the pilin protein. We found that mutations in
bfpB
, which encodes an outer membrane protein;
bfpD
, which encodes a putative nucleotide-binding protein; and
bfpG
and
bfpC
, which do not have sequence homologues in other type IV pilus systems, do not affect prebundlin expression or processing but block both BFP biogenesis and LA. The mutation in
bfpP
, the prepilin peptidase gene, does not affect prebundlin expression but blocks signal sequence cleavage of prebundlin, BFP biogenesis, and LA. The mutation in
bfpH
, which is predicted to encode a lytic transglycosylase, has no effect on prebundlin expression, prebundlin processing, BFP biogenesis, or LA. For each mutant for which altered phenotypes were detected, complementation with a plasmid containing the corresponding wild-type allele restored the wild-type phenotypes. We also found that association of prebundlin or bundlin with sucrose density flotation gradient fractions containing both inner and outer membrane proteins does not require any accessory proteins. These studies indicate that many
bfp
gene products are required for biogenesis of functional type IV pili but that mutations in the individual genes do not lead to the identification of new phases of pilus assembly.
Functional Analysis of the Galactosyltransferases Required for Biosynthesis of
d
-Galactan I, a Component of the Lipopolysaccharide O1 Antigen of
Klebsiella pneumoniae
Authors: Guan Shukui , Clarke Anthony J. , Whitfield Chris
Published: July 2002

d
-Galactan I is an O-antigenic polymer with the repeat unit structure [→3)-β-
d
-Gal
f
-(1→3)-α-
d
-Gal
p
-(1→], that is found in the lipopolysaccharide of
Klebsiella pneumoniae
O1 and other gram-negative bacteria. A genetic locus containing six genes is responsible for the synthesis and assembly of
d
-galactan I via an ATP-binding cassette (ABC) transporter-dependent pathway. The galactosyltransferase activities that are required for the processive polymerization of
d
-galactan I were identified by using in vitro reactions. The activities were determined with endogenous lipid acceptors in membrane preparations from
Escherichia coli
K-12 expressing individual enzymes (or combinations of enzymes) or in membranes reconstituted with specific lipid acceptors. The
d
-galactan I polymer is built on a lipid acceptor, undecaprenyl pyrophosphoryl-Glc
p
NAc, a product of the WecA enzyme that participates in the biosynthesis of enterobacterial common antigen and O-antigenic polysaccharide (O-PS) biosynthesis pathways. This intermediate is directed into
d
-galactan I biosynthesis by the bifunctional
wbbO
gene product, which sequentially adds one Gal
p
and one Gal
f
residue from the corresponding UDP-sugars to form a lipid-linked trisaccharide. The two galactosyltransferase activities of WbbO are separable by limiting the UDP-Gal
f
precursor. Galactosyltransferase activity in membranes reconstituted with exogenous lipid-linked trisaccharide acceptor and the known structure of
d
-galactan I indicate that WbbM catalyzes the subsequent transfer of a single Gal
p
residue to form a lipid-linked tetrasaccharide. Chain extension of the
d
-galactan I polymer requires WbbM for Gal
p
transferase, together with Gal
f
transferase activity provided by WbbO. Comparison of the biosynthetic pathways for
d
-galactan I and the polymannose
E. coli
O9a antigen reveals some interesting features that may reflect a common theme in ABC transporter-dependent O-PS assembly systems.
Combined, Functional Genomic-Biochemical Approach to Intermediary Metabolism: Interaction of Acivicin, a Glutamine Amidotransferase Inhibitor, with
Escherichia coli
K-12
Authors: Smulski Dana R. , Huang Lixuan L. , McCluskey Michael P. , Reeve Mary Jane Gladnick , Vollmer Amy C. , Van Dyk Tina K. , LaRossa Robert A.
Published: July 2002

Acivicin, a modified amino acid natural product, is a glutamine analog. Thus, it might interfere with metabolism by hindering glutamine transport, formation, or usage in processes such as transamidation and translation. This molecule prevented the growth of
Escherichia coli
in minimal medium unless the medium was supplemented with a purine or histidine, suggesting that the HisHF enzyme, a glutamine amidotransferase, was the target of acivicin action. This enzyme, purified from
E. coli
, was inhibited by low concentrations of acivicin. Acivicin inhibition was overcome by the presence of three distinct genetic regions when harbored on multicopy plasmids. Comprehensive transcript profiling using DNA microarrays indicated that histidine biosynthesis was the predominant process blocked by acivicin. The response to acivicin, however, was quite complex, suggesting that acivicin inhibition resonated through more than a single cellular process.
Multiple Mobile Promoter Regions for the Rare Carbapenem Resistance Gene of
Bacteroides fragilis
Authors: Podglajen I. , Breuil J. , Rohaut A. , Monsempes C. , Collatz E.
Published: July 2002

Two novel insertion sequences (IS), IS
1187
and IS
1188
, are described upstream from the carbapenem resistance gene
cfiA
in strains of
Bacteroides fragilis
. Mapping, with the RACE procedure, of transcription start sites of
cfiA
in these and two other previously reported IS showed that transcription of this rarely encountered gene is initiated close to a variety of
B. fragilis
consensus promoter sequences, as recently defined (D. P. Bayley, E. R. Rocha, and C. J. Smith, FEMS Microbiol. Lett. 193:149–154, 2000). In the cases of IS
1186
and IS
1188
, these sequences overlap with putative Eς
70
promoter sequences, while in IS
942
and IS
1187
such sequences can be observed either upstream or downstream of the
B. fragilis
promoters.
Efg1, a Morphogenetic Regulator in
Candida albicans
, Is a Sequence-Specific DNA Binding Protein
Authors: Leng Ping , Lee Philip R. , Wu Hong , Brown Alistair J. P.
Published: July 2002

Efg1 is essential for hyphal development in the human pathogen
Candida albicans
under most conditions. Efg1 is related to basic helix-loop-helix regulators, and therefore most workers presume that Efg1 is a transcription factor. Here we confirm that Efg1 is a DNA binding protein that can interact specifically with the E box.
Structural and Functional Characterization of IS
679
and IS
66
-Family Elements
Authors: Han Chang-Gyun , Shiga Yasuyuki , Tobe Toru , Sasakawa Chihiro , Ohtsubo Eiichi
Published: July 2002

A new insertion sequence (IS) element, IS
679
(2,704 bp in length), has been identified in plasmid pB171 of enteropathogenic
Escherichia coli
B171. IS
679
has imperfect 25-bp terminal inverted repeats (IRs) and three open reading frames (ORFs) (here called
tnpA, tnpB
, and
tnpC
). A plasmid carrying a composite transposon (Tn
679
) with the kanamycin resistance gene flanked by an intact IS
679
sequence and an IS
679
fragment with only IRR (IR on the right) was constructed to clarify the transposition activity of IS
679
. A transposition assay done with a mating system showed that Tn
679
could transpose at a high frequency to the F plasmid derivative used as the target. On transposition, Tn
679
duplicated an 8-bp sequence at the target site. Tn
679
derivatives with a deletion in each ORF of IS
679
did not transpose, finding indicative that all three IS
679
ORFs are essential for transposition. The
tnpA
and
tnpC
products appear to have the amino acid sequence motif characteristic of most transposases. A homology search of the databases found that a total of 25 elements homologous to IS
679
are present in
Agrobacterium, Escherichia, Rhizobium, Pseudomonas
, and
Vibrio
spp., providing evidence that the elements are widespread in gram-negative bacteria. We found that these elements belong to the IS
66
family, as do other elements, including nine not previously reported. Almost all of the elements have IRs similar to those in IS
679
and, like IS
679
, most appear to have duplicated an 8-bp sequence at the target site on transposition. These elements have three ORFs corresponding to those in IS
679
, but many have a mutation(s) in an ORF(s). In almost all of the elements,
tnpB
is located in the −1 frame relative to
tnpA
, such that the initiation codon of
tnpB
overlaps the TGA termination codon of
tnpA
. In contrast,
tnpC
, separated from
tnpB
by a space of ca. 20 bp, is located in any one of three frames relative to
tnpB
. No common structural features were found around the intergenic regions, indicating that the three ORFs are expressed by translational coupling but not by translational frameshifting.
Genome Sequence and Comparative Analysis of the Solvent-Producing BacteriumClostridium acetobutylicum
Authors: Nölling Jörk , Breton Gary , Omelchenko Marina V. , Makarova Kira S. , Zeng Qiandong , Gibson Rene , Lee Hong Mei , Dubois JoAnn , Qiu Dayong , Hitti Joseph ,   GTC Sequencing Center Production Finishing, and Bioinformatics Teams, Wolf Yuri I. , Tatusov Roman L. , Sabathe Fabrice , Doucette-Stamm Lynn , Soucaille Philippe , Daly Michael J. , Bennett George N. , Koonin Eugene V. , Smith Douglas R.
Published: July 2002

The genome sequence of the solvent-producing bacteriumClostridium acetobutylicumATCC 824 has been determined by the shotgun approach. The genome consists of a 3.94-Mb chromosome and a 192-kb megaplasmid that contains the majority of genes responsible for solvent production. Comparison ofC. acetobutylicumtoBacillus subtilisreveals significant local conservation of gene order, which has not been seen in comparisons of other genomes with similar, or, in some cases closer, phylogenetic proximity. This conservation allows the prediction of many previously undetected operons in both bacteria. However, theC. acetobutylicumgenome also contains a significant number of predicted operons that are shared with distantly related bacteria and archaea but not withB. subtilis. Phylogenetic analysis is compatible with the dissemination of such operons by horizontal transfer. The enzymes of the solventogenesis pathway and of the cellulosome ofC. acetobutylicumcomprise a new set of metabolic capacities not previously represented in the collection of complete genomes. These enzymes show a complex pattern of evolutionary affinities, emphasizing the role of lateral gene exchange in the evolution of the unique metabolic profile of the bacterium. Many of the sporulation genes identified inB. subtilisare missing inC. acetobutylicum, which suggests major differences in the sporulation process. Thus, comparative analysis reveals both significant conservation of the genome organization and pronounced differences in many systems that reflect unique adaptive strategies of the two gram-positive bacteria.
LcrG-LcrV Interaction Is Required for Control of Yops Secretion in
Yersinia pestis
Authors: Matson Jyl S. , Nilles Matthew L.
Published: July 2002

Yersinia pestis
expresses a set of plasmid-encoded virulence proteins called Yops and LcrV that are secreted and translocated into eukaryotic cells by a type III secretion system. LcrV is a multifunctional protein with antihost and positive regulatory effects on Yops secretion that forms a stable complex with a negative regulatory protein, LcrG. LcrG has been proposed to block the secretion apparatus (Ysc) from the cytoplasmic face of the inner membrane under nonpermissive conditions for Yops secretion, when levels of LcrV in the cell are low. A model has been proposed to describe secretion control based on the relative levels of LcrG and LcrV in the bacterial cytoplasm. This model proposes that under secretion-permissive conditions, levels of LcrV are increased relative to levels of LcrG, so that the excess LcrV titrates LcrG away from the Ysc, allowing secretion of Yops to occur. To further test this model, a mutant LcrG protein that could no longer interact with LcrV was created. Expression of this LcrG variant blocked secretion of Yops and LcrV under secretion permissive conditions in vitro and in a tissue culture model. These results agree with the previously described secretion-blocking activity of LcrG and demonstrate that the interaction of LcrV with LcrG is necessary for controlling Yops secretion.
DnaK Chaperone-Mediated Control of Activity of a ς
32
Homolog (RpoH) Plays a Major Role in the Heat Shock Response of
Agrobacterium tumefaciens
Authors: Nakahigashi Kenji , Yanagi Hideki , Yura Takashi
Published: July 2002

RpoH (
Escherichia coli
ς
32
and its homologs) is the central regulator of the heat shock response in gram-negative proteobacteria. Here we studied salient regulatory features of RpoH in
Agrobacterium tumefaciens
by examining its synthesis, stability, and activity while increasing the temperature from 25 to 37°C. Heat induction of RpoH synthesis occurred at the level of transcription from an RpoH-dependent promoter, coordinately with that of DnaK, and followed by an increase in the RpoH level. Essentially normal induction of heat shock proteins was observed even with a strain that was unable to increase the RpoH level upon heat shock. Moreover, heat-induced accumulation of
dnaK
mRNA occurred without protein synthesis, showing that preexisting RpoH was sufficient for induction of the heat shock response. These results suggested that controlling the activity, rather than the amount, of RpoH plays a major role in regulation of the heat shock response. In addition, increasing or decreasing the DnaK-DnaJ chaperones specifically reduced or enhanced the RpoH activity, respectively. On the other hand, the RpoH protein was normally stable and remained stable during the induction phase but was destabilized transiently during the adaptation phase. We propose that the DnaK-mediated control of RpoH activity plays a primary role in the induction of heat shock response in
A. tumefaciens
, in contrast to what has been found in
E. coli
.
CotA of
Bacillus subtilis
Is a Copper-Dependent Laccase
Authors: Hullo Marie-Françoise , Moszer Ivan , Danchin Antoine , Martin-Verstraete Isabelle
Published: July 2002

The spore coat protein CotA of
Bacillus subtilis
displays similarities with multicopper oxidases, including manganese oxidases and laccases.
B. subtilis
is able to oxidize manganese, but neither CotA nor other sporulation proteins are involved. We demonstrate that CotA is a laccase. Syringaldazine, a specific substrate of laccases, reacted with wild-type spores but not with Δ
cotA
spores. CotA may participate in the biosynthesis of the brown spore pigment, which appears to be a melanin-like product and to protect against UV light.
Promoter Specificity Elements in
Pseudomonas aeruginosa
Quorum-Sensing-Controlled Genes
Authors: Whiteley Marvin , Greenberg E. P.
Published: July 2002

The LasR-dependent and RhlR-dependent quorum-sensing systems are global regulators of gene expression in
Pseudomonas aeruginosa
. Previous studies have demonstrated that promoter elements of the quorum-sensing-controlled genes
lasB
and
hcnABC
are important in density-dependent regulation. We have identified LasR- and RhlR-dependent determinants in promoters of quorum-sensing-controlled genes qsc102, qsc117 (
acpP
), and qsc131 (
phzA
to -
G
) by in silico, deletion, point-mutational, and primer extension analyses. Each of these genes (in addition to
lasI
and
rsaL
) is activated by LasR, and qsc117 and qsc131 also respond to RhlR. Point mutations in the promoters of the LasR-specific gene, qsc102, relax specificity so that this promoter can respond to RhlR in addition to LasR. Our findings indicate that quorum-sensing-controlled promoters in
P. aeruginosa
are either specific for LasR or respond to both LasR and RhlR and that critical bases in the promoter elements determine specificity.
Peptide Methionine Sulfoxide Reductase (MsrA) Is a Virulence Determinant in
Mycoplasma genitalium
Authors: Dhandayuthapani S. , Blaylock M. W. , Bebear C. M. , Rasmussen W. G. , Baseman J. B.
Published: July 2002

Mycoplasma genitalium
is the smallest self-replicating microorganism and is implicated in human diseases, including urogenital and respiratory infections and arthritides.
M. genitalium
colonizes host cells primarily through adherence mechanisms mediated by a network of surface-associated membrane proteins, including adhesins and cytadherence-related proteins. In this paper, we show that cytadherence in
M. genitalium
is affected by an unrelated protein known as peptide methionine sulfoxide reductase (MsrA), an antioxidant repair enzyme that catalyzes the reduction of methionine sulfoxide [Met(O)] residues in proteins to methionine. An
msrA
disruption mutant of
M. genitalium
, constructed through homologous recombination, displayed markedly reduced adherence to sheep erythrocytes. In addition, the
msrA
mutant was incapable of growing in hamsters and exhibited hypersensitivity to hydrogen peroxide when compared to wild-type virulent
M. genitalium
. These results indicate that MsrA plays an important role in
M. genitalium
pathogenicity, possibly by protecting mycoplasma protein structures from oxidative damage or through alternate virulence-related pathways.
SdiA of
Salmonella enterica
Is a LuxR Homolog That Detects Mixed Microbial Communities
Authors: Michael Bindhu , Smith Jenee N. , Swift Simon , Heffron Fred , Ahmer Brian M. M.
Published: July 2002

Proteins of the LuxR family detect the presence of
N
-acylhomoserine lactones (AHLs) and regulate transcription accordingly. When AHLs are synthesized by the same species that detects them, the system allows a bacterium to measure the population density of its own species, a phenomenon known as quorum sensing. The
sdiA
genes of
Escherichia coli
and
Salmonella enterica
serovar Typhimurium are predicted to encode LuxR homologs. However, these species do not appear to synthesize AHLs or any other molecule detected by SdiA. It has previously been demonstrated that overexpression of
sdiA
results in the activation of the
ftsQAZ
locus in
E. coli
and four other loci in
Salmonella
serovar Typhimurium. Here we report that transcriptional fusions to these five loci fall into two classes. The first class requires overexpression of
sdiA
for activation. The second class responds to
sdiA
expressed from its natural position in the chromosome if the appropriate AHLs are added to the culture. The only member of the second class is a series of P
rck-luxCDABE
fusions in
Salmonella
serovar Typhimurium. SdiA responds with highest sensitivity to AHLs that have a keto modification at the third carbon and an acyl chain length of 6 or 8 (half-maximal response between 1 and 5 nM). Growth of
Salmonella
in proximity to species known to synthesize these AHLs results in
sdiA
-dependent activation of the P
rck-luxCDABE
fusions. SdiA appears to be the first AHL receptor discovered that detects signals emanating exclusively from other species.