bluuebunny/crossref_metadata_2025 huggingface dataset:
Polyprenyl Phosphate Biosynthesis in
Mycobacterium tuberculosis
and
Mycobacterium smegmatis
Authors: Crick Dean C. , Schulbach Mark C. , Zink Erin E. , Macchia Marco , Barontini Silvia , Besra Gurdyal S. , Brennan Patrick J.
Published: July 2002

Mycobacterium smegmatis
has been shown to contain two forms of polyprenyl phosphate (Pol-P), while
Mycobacterium tuberculosis
contains only one. Utilizing subcellular fractions from
M. smegmatis
and
M. tuberculosis
, we show that Pol-P synthesis is different in these species. The specific activities of the prenyl diphosphate synthases in
M. tuberculosis
are 10- to 100-fold lower than those in
M. smegmatis
. In
M. smegmatis
decaprenyl diphosphate and heptaprenyl diphosphate were the main products synthesized in vitro, whereas in
M. tuberculosis
only decaprenyl diphosphate was synthesized. The data from both organisms suggest that geranyl diphosphate is the allylic substrate for two distinct prenyl diphosphate synthases, one located in the cell membrane that synthesizes ω,
E
,
Z
-farnesyl diphosphate and the other present in the cytosol that synthesizes ω,
E
,
E
,
E
-geranylgeranyl diphosphate. In
M. smegmatis
, the ω,
E
,
Z
-farnesyl diphosphate is utilized by a membrane-associated prenyl diphosphate synthase activity to generate decaprenyl diphosphate, and the ω,
E
,
E
,
E
-geranylgeranyl diphosphate is utilized by a membrane-associated activity for the synthesis of the heptaprenyl diphosphate. In
M. tuberculosis
, however, ω,
E
,
E
,
E
-geranylgeranyl diphosphate is not utilized for the synthesis of heptaprenyl diphosphate. Thus, the difference in the compositions of the Pol-P of
M. smegmatis
and
M. tuberculosis
can be attributed to distinct enzymatic differences between these two organisms.
Evidence of a Role for LytB in the Nonmevalonate Pathway of Isoprenoid Biosynthesis
Authors: Cunningham Francis X. , Lafond Toulouse P. , Gantt Elisabeth
Published: July 2002

It is proposed that the
lytB
gene encodes an enzyme of the deoxyxylulose-5-phosphate (DOXP) pathway that catalyzes a step at or subsequent to the point at which the pathway branches to form isopentenyl diphosphate (IPP) and dimethylallyl diphosphate (DMAPP). A mutant of the cyanobacterium
Synechocystis
strain PCC 6803 with an insertion in the promoter region of
lytB
grew slowly and produced greenish-yellow, easily bleached colonies. Insertions in the coding region of
lytB
were lethal. Supplementation of the culture medium with the alcohol analogues of IPP and DMAPP (3-methyl-3-buten-1-ol and 3-methyl-2-buten-1-ol) completely alleviated the growth impairment of the mutant. The
Synechocystis lytB
gene and a
lytB
cDNA from the flowering plant
Adonis aestivalis
were each found to significantly enhance accumulation of carotenoids in
Escherichia coli
engineered to produce these colored isoprenoid compounds. When combined with a cDNA encoding deoxyxylulose-5-phosphate synthase (
dxs
), the initial enzyme of the DOXP pathway, the individual salutary effects of
lytB
and
dxs
were multiplied. In contrast, the combination of
lytB
and a cDNA encoding IPP isomerase (
ipi
) was no more effective in enhancing carotenoid accumulation than
ipi
alone, indicating that the ratio of IPP and DMAPP produced via the DOXP pathway is influenced by LytB.
Inhibitory Effect of Heterologous Ribosome Recycling Factor on Growth of
Escherichia coli
Authors: Atarashi Kenji , Kaji Akira
Published: July 2002

Ribosome recycling factor (RRF) of
Thermotoga maritima
was expressed in
Escherichia coli
from the cloned
T. maritima
RRF gene and purified. Expression of
T. maritima
RRF inhibited growth of the
E. coli
host in a dose-dependent manner, an effect counteracted by the overexpression of
E. coli
RRF.
T. maritima
RRF also inhibited the
E. coli
RRF reaction in vitro. Genes encoding RRFs from
Streptococcus pneumoniae
and
Helicobacter pylori
have been cloned, and they also impair growth of
E. coli
, although the inhibitory effect of these RRFs was less pronounced than that of
T. maritima
RRF. The amino acid sequence at positions 57 to 62, 74 to 78, 118 to 122, 154 to 160, and 172 to 176 in
T. maritima
RRF differed totally from that of
E. coli
RRF. This suggests that these regions are important for the inhibitory effect of heterologous RRF. We further suggest that bending and stretching of the RRF molecule at the hinge between two domains may be critical for RRF activity and therefore responsible for
T. maritima
RRF inhibition of the
E. coli
RRF reaction.
Evidence for a Role of
rpoE
in Stressed and Unstressed Cells of Marine
Vibrio angustum
Strain S14
Authors: Hild Erika , Takayama Kathy , Olsson Rose-Marie , Kjelleberg Staffan
Published: July 2002

We report the cloning, sequencing, and characterization of the
rpoE
homolog in
Vibrio angustum
S14. The
rpoE
gene encodes a protein with a predicted molecular mass of 19.4 kDa and has been demonstrated to be present as a single-copy gene by Southern blot analysis. The deduced amino acid sequence of RpoE is most similar to that of the RpoE homolog of
Sphingomonas aromaticivorans
, ς
24
, displaying sequence similarity and identity of 63 and 43%, respectively. Northern blot analysis demonstrated the induction of
rpoE
6, 12, and 40 min after a temperature shift to 40°C. An
rpoE
mutant was constructed by gene disruption. There was no difference in viability during logarithmic growth, stationary phase, or carbon starvation between the wild type and the
rpoE
mutant strain. In contrast, survival of the mutant was impaired following heat shock during exponential growth, as well as after oxidative stress at 24 h of carbon starvation. The mutant exhibited microcolony formation during optimal growth temperatures (22 to 30°C), and cell area measurements revealed an increase in cell volume of the mutant during growth at 30°C, compared to the wild-type strain. Moreover, outer membrane and periplasmic space protein analysis demonstrated many alterations in the protein profiles for the mutant during growth and carbon starvation, as well as following oxidative stress, in comparison with the wild-type strain. It is thereby concluded that RpoE has an extracytoplasmic function and mediates a range of specific responses in stressed as well as unstressed cells of
V. angustum
S14.
Function of a Principal Na
+
/H
+
Antiporter, ShaA, Is Required for Initiation of Sporulation in
Bacillus subtilis
Authors: Kosono Saori , Ohashi Yoshiaki , Kawamura Fujio , Kitada Makio , Kudo Toshiaki
Published: July 2002

ShaA (sodium/hydrogen antiporter, previously termed YufT [or NtrA]), which is responsible for Na
+
/H
+
antiporter activity, is considered to be the major Na
+
excretion system in
Bacillus subtilis
. We found that a
shaA
-disrupted mutant of
B. subtilis
shows impaired sporulation but normal vegetative growth when the external Na
+
concentration was increased in a low range. In the
shaA
mutant, ς
H
-dependent expression of
spo0A
(P
S
) and
spoVG
at an early stage of sporulation was sensitive to external NaCl. The level of ς
H
protein was reduced by the addition of NaCl, while the expression of
spo0H
, which encodes ς
H
, was little affected, indicating that posttranscriptional control of ς
H
rather than
spo0H
transcription is affected by the addition of NaCl in the
shaA
mutant. Since this mutant is considered to have a diminished ability to maintain a low internal Na
+
concentration, an increased level of internal Na
+
may affect posttranscriptional control of ς
H
. Bypassing the phosphorelay by introducing the
sof-1
mutation into this mutant did not restore
spo0A
(P
S
) expression, suggesting that disruption of
shaA
affects ς
H
accumulation, but does not interfere with the phosphorylation and phosphotransfer reactions of the phosphorelay. These results suggest that ShaA plays a significant role at an early stage of sporulation and not only during vegetative growth. Our findings raise the possibility that fine control of cytoplasmic ion levels, including control of the internal Na
+
concentration, may be important for the progression of the sporulation process.
The TorR High-Affinity Binding Site Plays a Key Role in Both
torR
Autoregulation and
torCAD
Operon Expression in
Escherichia coli
Authors: Ansaldi Mireille , Simon Gwénola , Lepelletier Michèle , Méjean Vincent
Published: July 2002

In the presence of trimethylamine
N
-oxide (TMAO), the TorS-TorR two-component regulatory system induces the
torCAD
operon, which encodes the TMAO respiratory system of
Escherichia coli
. The sensor protein TorS detects TMAO and transphosphorylates the response regulator TorR which, in turn, activates transcription of
torCAD
. The
torR
gene and the
torCAD
operon are divergently transcribed, and the short
torR-torC
intergenic region contains four direct repeats (the
tor
boxes) which proved to be TorR binding sites. The
tor
box 1-box 2 region covers the
torR
transcription start site and constitutes a TorR high-affinity binding site, whereas box 3 and box 4 correspond to low-affinity binding sites. By using
torR-lacZ
operon fusions in different genetic backgrounds, we showed that the
torR
gene is negatively autoregulated. Surprisingly, TorR autoregulation is TMAO independent and still occurs in a
torS
mutant. In addition, this negative regulation involves only the TorR high-affinity binding site. Together, these data suggest that phosphorylated as well as unphosphorylated TorR binds the box 1-box 2 region in vivo, thus preventing RNA polymerase from binding to the
torR
promoter whatever the growth conditions. By changing the spacing between box 2 and box 3, we demonstrated that the DNA motifs of the high- and low-affinity binding sites must be close to each other and located on the same side of the DNA helix to allow induction of the
torCAD
operon. Thus, prior TorR binding to the box 1-box 2 region seems to allow cooperative binding of phosphorylated TorR to box 3 and box 4.
Gene Families Encoding Phase- and Size-Variable Surface Lipoproteins of
Mycoplasma hyorhinis
Authors: Citti Christine , Watson-McKown Robyn , Droesse Martina , Wise Kim S.
Published: July 2002

A prototype family of seven genes encoding the variable surface lipoproteins (Vlps) of
Mycoplasma hyorhinis
is characterized in the pathogenic SK76 strain, using long-range PCR to amplify and analyze the single chromosomal region containing expressed genes
vlpA
to -
G
, each of which is subject to phase and size variation. Smaller families of
vlp
genes in subclones of SK76 or in another strain of
M. hyorhinis
, GDL, can be attributed to deletions of specific
vlp
genes from the prototype array described here. Two genes,
vlpA
and the newly revealed
vlpG
, contain repeat motifs in their 3′ coding regions that differ from the short tandem repeats in other
vlp
genes yet retain structural features common to all
vlp
gene products. SK76 and GDL
vlp
gene families are similarly organized and show sequence similarity between corresponding individual
vlp
genes. In light of the extensive potential for diversity within the
vlp
gene system, such conservation provides a provisional basis to hypothesize that
vlp
genes may exist in specific arrays that endow selected functions while retaining common structural features required during phase-variable expression of this set of gene products.
Reinvestigation of the Proteolytic Activity of Neocarzinostatin
Authors: Heyd Bernadette , Lerat Guilhem , Adjadj Elisabeth , Minard Philippe , Desmadril Michel
Published: July 2002

Neocarzinostatin (NCS) is the most studied member of a family of chromoproteins secreted by a range of actinomycetes species. It has been proposed that in addition to their antitumoral activity related to the bound chromophores, this group of related proteins could be a secreted proteases superfamily. With the aim of dissecting the molecular basis of the proteolytic activity of NCS, an expression system allowing efficient expression of apo-NCS in
Escherichia coli
was constructed. The recombinant protein was properly folded and functional. Its histone-specific proteolytic activity was similar to the activity described for the natural protein. Further analyses unambiguously demonstrated that the proteolytic activity could be physically separated from NCS. This activity is therefore due not to NCS itself but to minor contaminating proteases, the nature of which differed in the recombinant and natural NCS preparations. The histone degradation test commonly used to monitor proteolytic activity is extremely sensitive and may easily generate false-positive results. These results strongly suggest that the possible proteolytic activity of the proteins of this family should be critically reconsidered.
Repair of DNA Lesions Induced by Hydrogen Peroxide in the Presence of Iron Chelators in
Escherichia coli
: Participation of Endonuclease IV and Fpg
Authors: Galhardo Rodrigo S. , Almeida Carlos E. B. , Leitão Alvaro C. , Cabral-Neto Januário B.
Published: July 2002

In
Escherichia coli
, the repair of lethal DNA damage induced by H
2
O
2
requires exonuclease III, the
xthA
gene product. Here, we report that both endonuclease IV (the
nfo
gene product) and exonuclease III can mediate the repair of lesions induced by H
2
O
2
under low-iron conditions. Neither the
xthA
nor the
nfo
mutants was sensitive to H
2
O
2
in the presence of iron chelators, while the
xthA nfo
double mutant was significantly sensitive to this treatment, suggesting that both exonuclease III and endonuclease IV can mediate the repair of DNA lesions formed under such conditions. Sedimentation studies in alkaline sucrose gradients also demonstrated that both
xthA
and
nfo
mutants, but not the
xthA nfo
double mutant, can carry out complete repair of DNA strand breaks and alkali-labile bonds generated by H
2
O
2
under low-iron conditions. We also found indications that the formation of substrates for exonuclease III and endonuclease IV is mediated by the Fpg DNA glycosylase, as suggested by experiments in which the
fpg
mutation increased the level of cell survival, as well as repair of DNA strand breaks, in an AP endonuclease-null background.
Contribution of Base Excision Repair, Nucleotide Excision Repair, and DNA Recombination to Alkylation Resistance of the Fission Yeast
Schizosaccharomyces pombe
Authors: Memisoglu Asli , Samson Leona
Published: July 2002

DNA damage is unavoidable, and organisms across the evolutionary spectrum possess DNA repair pathways that are critical for cell viability and genomic stability. To understand the role of base excision repair (BER) in protecting eukaryotic cells against alkylating agents, we generated
Schizosaccharomyces pombe
strains mutant for the
mag1
3-methyladenine DNA glycosylase gene. We report that
S. pombe mag1
mutants have only a slightly increased sensitivity to methylation damage, suggesting that Mag1-initiated BER plays a surprisingly minor role in alkylation resistance in this organism. We go on to show that other DNA repair pathways play a larger role than BER in alkylation resistance. Mutations in genes involved in nucleotide excision repair (
rad13
) and recombinational repair (
rhp51
) are much more alkylation sensitive than
mag1
mutants. In addition,
S. pombe
mutant for the flap endonuclease
rad2
gene, whose precise function in DNA repair is unclear, were also more alkylation sensitive than
mag1
mutants. Further,
mag1
and
rad13
interact synergistically for alkylation resistance, and
mag1
and
rhp51
display a surprisingly complex genetic interaction. A model for the role of BER in the generation of alkylation-induced DNA strand breaks in
S. pombe
is discussed.
Events during Initiation of Archaeal Transcription: Open Complex Formation and DNA-Protein Interactions
Authors: Hausner Winfried , Thomm Michael
Published: July 2002

Transcription in
Archaea
is initiated by association of a TATA box binding protein (TBP) with a TATA box. This interaction is stabilized by the binding of the transcription factor IIB (TFIIB) orthologue TFB. We show here that the RNA polymerase of the archaeon
Methanococcus
, in contrast to polymerase II, does not require hydrolysis of the β-γ bond of ATP for initiation of transcription and open complex formation on linearized DNA. Permanganate probing revealed that the archaeal open complex spanned at least the DNA region from −11 to −1 at a tRNA
Val
promoter. The
Methanococcus
TBP-TFB promoter complex protected the DNA region from −40 to −14 on the noncoding DNA strand and the DNA segment from −36 to −17 on the coding DNA strand from DNase I digestion. This DNase I footprint was extended only to the downstream end by the addition of the RNA polymerase to position +17 on the noncoding strand and to position +13 on the coding DNA strand.
The Type IV Fimbrial Subunit Gene (
fimA
) of
Dichelobacter nodosus
Is Essential for Virulence, Protease Secretion, and Natural Competence
Authors: Kennan Ruth M. , Dhungyel Om P. , Whittington Richard J. , Egerton John R. , Rood Julian I.
Published: July 2002

Dichelobacter nodosus
is the essential causative agent of footrot in sheep. The major
D. nodosus
-encoded virulence factors that have been implicated in the disease are type IV fimbriae and extracellular proteases. To examine the role of the fimbriae in virulence, allelic exchange was used to insertionally inactivate the
fimA
gene, which encodes the fimbrial subunit protein, from the virulent type G
D. nodosus
strain VCS1703A. Detailed analysis of two independently derived
fimA
mutants revealed that they no longer produced the fimbrial subunit protein or intact fimbriae and did not exhibit twitching motility. In addition, these mutants were no longer capable of undergoing natural transformation and did not secrete wild-type levels of extracellular proteases. These effects were not due to polar effects on the downstream
fimB
gene because insertionally inactivated
fimB
mutants were not defective in any of these phenotypic tests. Virulence testing of the mutants in a sheep pen trial conducted under controlled environmental conditions showed that the
fimA
mutants were avirulent, providing evidence that the
fimA
gene is an essential
D. nodosus
virulence gene. These studies represent the first time that molecular genetics has been used to determine the role of virulence genes in this slow growing anaerobic bacterium.
Genome Size Determination and Coding Capacity of
Sodalis glossinidius
, an Enteric Symbiont of Tsetse Flies, as Revealed by Hybridization to
Escherichia coli
Gene Arrays
Authors: Akman Leyla , Rio Rita V. M. , Beard Charles B. , Aksoy Serap
Published: July 2002

Recent molecular characterization of various microbial genomes has revealed differences in genome size and coding capacity between obligate symbionts and intracellular pathogens versus free-living organisms. Multiple symbiotic microorganisms have evolved with tsetse fly, the vector of African trypanosomes, over long evolutionary times. Although these symbionts are indispensable for tsetse fecundity, the biochemical and molecular basis of their functional significance is unknown. Here, we report on the genomic aspects of the secondary symbiont
Sodalis glossinidius
. The genome size of
Sodalis
is approximately 2 Mb. Its DNA is subject to extensive methylation and based on some of its conserved gene sequences has an A+T content of only 45%, compared to the typically AT-rich genomes of endosymbionts. Sodalis also harbors an extrachromosomal plasmid about 134 kb in size. We used a novel approach to gain insight into
Sodalis
genomic contents, i.e., hybridizing its DNA to macroarrays developed for
Escherichia coli
, a closely related enteric bacterium. In this analysis we detected 1,800 orthologous genes, corresponding to about 85% of the
Sodalis
genome. The
Sodalis
genome has apparently retained its genes for DNA replication, transcription, translation, transport, and the biosynthesis of amino acids, nucleic acids, vitamins, and cofactors. However, many genes involved in energy metabolism and carbon compound assimilation are apparently missing, which may indicate an adaptation to the energy sources available in the only nutrient of the tsetse host, blood. We present gene arrays as a rapid tool for comparative genomics in the absence of whole genome sequence to advance our understanding of closely related bacteria.
Membrane Topology of theStreptomyces lividansType I Signal Peptidases
Authors: Geukens Nick , Lammertyn Elke , Van Mellaert Lieve , Schacht Sabine , Schaerlaekens Kristien , Parro Victor , Bron Sierd , Engelborghs Yves , Mellado Rafael P. , Anné Jozef
Published: July 2002

Most bacterial membranes contain one or two type I signal peptidases (SPases) for the removal of signal peptides from export proteins. ForStreptomyces lividans, four different type I SPases (denoted SipW, SipX, SipY, and SipZ) were previously described. In this communication, we report the experimental determination of the membrane topology of these SPases. A protease protection assay of SPase tendamistat fusions confirmed the presence of the N- as well as the C-terminal transmembrane anchor for SipY. SipX and SipZ have a predicted topology similar to that of SipY. These threeS. lividansSPases are currently the only known prokaryotic-type type I SPases of gram-positive bacteria with a C-terminal transmembrane anchor, thereby establishing a new subclass of type I SPases. In contrast,S. lividansSipW contains only the N-terminal transmembrane segment, similar to most type I SPases of gram-positive bacteria. Functional analysis showed that the C-terminal transmembrane anchor of SipY is important to enhance the processing activity, both in vitro as well as in vivo. Moreover, for theS. lividansSPases, a relation seems to exist between the presence or absence of the C-terminal anchor and the relative contributions to the total SPase processing activity in the cell. SipY and SipZ, two SPases with a C-terminal anchor, were shown to be of major importance to the cell. Accordingly, for SipW, missing the C-terminal anchor, a minor role in preprotein processing was found.
Distribution of the
Ssu
DAT1I Restriction-Modification System among Different Serotypes of
Streptococcus suis
Authors: Sekizaki Tsutomu , Osaki Makoto , Takamatsu Daisuke , Shimoji Yoshihiro
Published: July 2002

The
Ssu
DAT1I restriction-modification (R-M) system, which contains two methyltransferases and two restriction endonucleases with recognition sequence 5′-GATC-3′, was first found in a field isolate of
Streptococcus suis
serotype 2. Isoschizomers of the R-M system were found in the same locus between
purH
and
purD
in a field isolate of serotype 1/2 and the reference strains of serotypes 3, 7, 23, and 26 among 29 strains of different serotypes examined in this study. The R-M gene sequences in serotypes 1/2, 3, 7, and 23 were very similar to those of
Ssu
DAT1I, whereas those in serotype 26 were less similar. These results indicate intraspecies recombination among them and genetic divergence through their evolution.
Autophosphorylation of Archaeal Cdc6 Homologues Is Regulated by DNA
Authors: Grabowski Beatrice , Kelman Zvi
Published: July 2002

The initiator protein Cdc6 (Cdc18 in fission yeast) plays an essential role in the initiation of eukaryotic DNA replication. In yeast the protein is expressed before initiation of DNA replication and is thought to be essential for loading of the helicase onto origin DNA. The biochemical properties of the protein, however, are largely unknown. Using three archaeal homologues of Cdc6, it was found that the proteins are autophosphorylated on Ser residues. The winged-helix domain at the C terminus of Cdc6 interacts with DNA, which apparently regulates the autophosphorylation reaction. Yeast Cdc18 was also found to autophosphorylate, suggesting that this function of Cdc6 may play a widely conserved and essential role in replication initiation.
Properties of a Thermostable Nitrate Reductase from the Hyperthermophilic Archaeon
Pyrobaculum aerophilum
Authors: Afshar Sepideh , Johnson Eric , de Vries Simon , Schröder Imke
Published: July 2002

The nitrate reductase of the hyperthermophilic archaeon
Pyrobaculum aerophilum
was purified 137-fold from the cytoplasmic membrane. Based on sodium dodecyl sulfate-polyacrylamide gel electrophoresis analysis, the enzyme complex consists of three subunits with apparent molecular weights of 130,000, 52,000, and 32,000. The enzyme contained molybdenum (0.8-mol/mol complex), iron (15.4-mol/mol complex) and cytochrome
b
(0.49-mol/mol complex) as cofactors. The
P. aerophilum
nitrate reductase distinguishes itself from nitrate reductases of mesophilic bacteria and archaea by its very high specific activity using reduced benzyl viologen as the electron donor (
V
max
with nitrate, 1,162 s
−1
(326 U/mg);
V
max
with chlorate, 1,348 s
−1
(378 U/mg) [assayed at 75°C]). The

K
m

values for nitrate and chlorate were 58 and 140 μM, respectively. Azide was a competitive inhibitor and cyanide was a noncompetitive inhibitor of the nitrate reductase activity. The temperature optimum for activity was >95°C. When incubated at 100°C, the purified nitrate reductase had a half-life of 1.5 h. This study constitutes the first description of a nitrate reductase from a hyperthermophilic archaeon.
Identification of the Partitioning Site within the
repABC-
Type Replicon of the Composite
Paracoccus versutus
Plasmid pTAV1
Authors: Bartosik Dariusz , Szymanik Michal , Wysocka Edyta
Published: July 2002

The replicator region of composite plasmid pTAV1 of
Paracoccus versutus
(included in mini-replicon pTAV320) belongs to the family of
repABC
replicons commonly found in plasmids harbored by
Agrobacterium
and
Rhizobium
spp. The
repABC
replicons encode three genes clustered in an operon, which are involved in partitioning (
repA
and
repB
) and replication (
repC
). In order to localize the partitioning site of pTAV320, the two identified incompatibility determinants of this mini-replicon (
inc1
, located in the intergenic sequence between
repB
and
repC
; and
inc2
, situated downstream of the
repC
gene) were PCR amplified and used together with purified RepB fusion protein (homologous to the type B partitioning proteins binding to the partitioning sites) in an electrophoretic mobility shift assay. The protein bound only
inc2
, forming two complexes in a protein concentration-dependent manner. The
inc2
region contains two long (14-bp) repeated sequences (R1 and R2). Disruption of these sequences completely eliminates RepB binding ability. R1 and R2 have sequence similarities with analogous repeats of another
repABC
replicon of plasmid pPAN1 of
Paracoccus pantotrophus
DSM 82.5 and with centromeric sequences of the
Bacillus subtilis
chromosome. Excess RepB protein resulted in destabilization of the
inc2
-containing plasmid in
Escherichia coli
. On the other hand, the
inc2
region could stabilize another unstable replicon in
P. versutus
when RepA and RepB were delivered in
trans
, proving that this region has centromere-like activity. Thus, it was demonstrated that
repA
,
repB
, and
inc2
constitute a functional system for active partitioning of pTAV320.
Contributions of UP Elements and the Transcription Factor FIS to Expression from the Seven
rrn
P1 Promoters in
Escherichia coli
Authors: Hirvonen Christine A. , Ross Wilma , Wozniak Christopher E. , Marasco Erin , Anthony Jennifer R. , Aiyar Sarah E. , Newburn Vanessa H. , Gourse Richard L.
Published: July 2002

The high activity of the
rrnB
P1 promoter in
Escherichia coli
results from a
cis
-acting DNA sequence, the UP element, and a
trans
-acting transcription factor, FIS. In this study, we examine the effects of FIS and the UP element at the other six
rrn
P1 promoters. We find that UP elements are present at all of the
rrn
P1 promoters, but they make different relative contributions to promoter activity. Similarly, FIS binds upstream of, and activates, all seven
rrn
P1 promoters but to different extents. The total number of FIS binding sites, as well as their positions relative to the transcription start site, differ at each
rrn
P1 promoter. Surprisingly, the FIS sites upstream of site I play a much larger role in transcription from most
rrn
P1 promoters compared to
rrnB
P1. Our studies indicate that the overall activities of the seven
rrn
P1 promoters are similar, and the same contributors are responsible for these high activities, but these inputs make different relative contributions and may act through slightly different mechanisms at each promoter. These studies have implications for the control of gene expression of unlinked multigene families.
TOR Modulates
GCN4
-Dependent Expression of Genes Turned on by Nitrogen Limitation
Authors: Valenzuela Lourdes , Aranda Cristina , González Alicia
Published: July 2002

In
Saccharomyces cerevisiae
, the rapamycin-sensitive TOR signaling pathway plays an essential role in up-regulating translation initiation and cell cycle progression in response to nutrient availability. One of the mechanisms by which TOR regulates cell proliferation is by excluding the
GLN3
transcriptional activator from the nucleus and, in consequence, preventing its transcriptional activation therein. We examined the possibility that the TOR cascade could also control the transcriptional activity of Gcn4p, which is known to respond to amino acid availability. The results presented in this paper indicate that
GCN4
plays a role in the rapamycin-sensitive signaling pathway, regulating the expression of genes involved in the utilization of poor nitrogen sources, a previously unrecognized role for Gcn4p, and that the TOR pathway controls
GCN4
activity by regulating the translation of
GCN4
mRNA. This constitutes an additional TOR-dependent mechanism which modulates the action of transcriptional activators.
Sinorhizobium meliloti
Plasmid pRm1132f Replicates by a Rolling-Circle Mechanism
Authors: Barran L. R. , Ritchot N. , Bromfield E. S. P.
Published: July 2002

pRm1132f isolated from
Sinorhizobium meliloti
is a group III rolling-circle-replicating (RCR) plasmid. At least seven of eight open reading frames in the nucleotide sequence represented coding regions. The minimal replicon contained a
rep
gene and single- and double-stranded origins of replication. Detection of single-stranded plasmid DNA confirmed that pRm1132f replicated via an RCR mechanism.
Regulation of thenfsAGene inEscherichia coliby SoxS
Authors: Paterson E. Suzanne , Boucher Sherri E. , Lambert I. B.
Published: July 2002

InEscherichia coli,the response to oxidative stress due to elevated levels of superoxide is mediated, in part, by thesoxRSregulon. One member of thesoxRSregulon,nfsA,encodes the major oxygen-insensitive nitroreductase inEscherichia coliwhich catalyzes the reduction of nitroaromatic and nitroheterocyclic compounds by NADPH. In this study we investigate the regulation ofnfsAin response to the superoxide generating compound paraquat. The transcription start site (TSS) ofnfsAwas located upstream of theybjCgene, a small open reading frame of unknown function located directly upstream ofnfsA,suggesting that these two genes form an operon. The activity of the promoter associated with this TSS was confirmed withlacZfusions and was shown to be inducible by paraquat. Footprinting and band shift analysis showed that purified His-tagged SoxS protein binds to a 20-base sequence 10 bases upstream of the −35 promoter sequence in the forward orientation, suggesting that theybjC-nfsApromoter is a class I SoxS-dependent promoter.
ArgR-Independent Induction and ArgR-Dependent Superinduction of theastCADBEOperon inEscherichia coli
Authors: Kiupakis Alexandros K. , Reitzer Larry
Published: July 2002

ForEscherichia coli, growth in the absence of ammonia is termed nitrogen limited and results in the induction of genes that assimilate other nitrogen sources, a response mediated by σ54and nitrogen regulator I (NRI, also called NtrC). TheastCADBEoperon, which is required for growth with arginine as the sole nitrogen source, is moderately expressed during general nitrogen limitation and maximally expressed in the presence of arginine. The operon is also induced in stationary phase. Primer extension analysis ofE. colirevealed the presence of a σ54-dependent promoter utilized in exponential phase during nitrogen limitation and a σS-dependent promoter active during stationary phase. We used anast-lacZfusion to show that arginine stimulates expression, that ArgR, the arginine repressor, enhances expression from both promoters but is not essential, and that transcription by the two forms of the RNA polymerase is competitive and mutually exclusive. We demonstrated the binding of RNA polymerase holoenzymes, NRI, and ArgR to the promoter region in vitro. We also reconstituted transcription from both promoters with purified components, which confirmed the accessory role of ArgR for the σ54-dependent promoter. Thus, theastoperon exhibits nitrogen source-specific induction that is unique for an NRI-dependent gene. The transcriptional regulation of theastoperon inE. colidiffers from that inSalmonella entericaserovar Typhimurium, in which ArgR is required forastoperon expression.
The N Terminus of the
Escherichia coli
Transcription Activator MalT Is the Domain of Interaction with MalY
Authors: Schlegel Anja , Danot Olivier , Richet Evelyne , Ferenci Thomas , Boos Winfried
Published: July 2002

The maltose system of
Escherichia coli
consists of a number of genes encoding proteins involved in the uptake and metabolism of maltose and maltodextrins. The system is positively regulated by MalT, its transcriptional activator. MalT activity is controlled by two regulatory circuits: a positive one with maltotriose as effector and a negative one involving several proteins. MalK, the ATP-hydrolyzing subunit of the cognate ABC transporter, MalY, an enzyme with the activity of a cystathionase, and Aes, an acetyl esterase, phenotypically act as repressors of MalT activity. By in vivo titration assays, we have shown that the N-terminal 250 amino acids of MalT contain the interaction site for MalY but not for MalK. This was confirmed by gel filtration analysis, where MalY was shown to coelute with the N-terminal MalT structural domain. Mutants in MalT causing elevated
mal
gene expression in the absence of exogenous maltodextrins were tested in their response to the three repressors. The different MalT mutations exhibited a various degree of sensitivity towards these repressors, but none was resistant to all of them. Some of them became nearly completely resistant to Aes while still being sensitive to MalY. These mutations are located at positions 38, 220, 243, and 359, most likely defining the interaction patch with Aes on the three-dimensional structure of MalT.
Respiration-Dependent Utilization of Sugars in Yeasts: a Determinant Role for Sugar Transporters
Authors: Goffrini Paola , Ferrero Iliana , Donnini Claudia
Published: July 2002

In many yeast species, including
Kluyveromyces lactis
, growth on certain sugars (such as galactose, raffinose, and maltose) occurs only under respiratory conditions. If respiration is blocked by inhibitors, mutation, or anaerobiosis, growth does not take place. This apparent dependence on respiration for the utilization of certain sugars has often been suspected to be associated with the mechanism of the sugar uptake step. We hypothesized that in many yeast species, the permease activities for these sugars are not sufficient to ensure the high substrate flow that is necessary for fermentative growth. By introducing additional sugar permease genes, we have obtained
K. lactis
strains that were capable of growing on galactose and raffinose in the absence of respiration. High dosages of both the permease and maltase genes were indeed necessary for
K. lactis
cells to grow on maltose in the absence of respiration. These results strongly suggest that the sugar uptake step is the major bottleneck in the fermentative assimilation of certain sugars in
K. lactis
and probably in many other yeasts.
Comparison of Antifungal Activities and 16S Ribosomal DNA Sequences of Clinical and Environmental Isolates of
Stenotrophomonas maltophilia
Authors: Minkwitz Arite , Berg Gabriele
Published: July 2002

In recent years, the gram-negative bacterium
Stenotrophomonas maltophilia
has become increasingly important in biotechnology and as a nosocomial pathogen, giving rise to a need for new information about its taxonomy and epidemiology. To determine intraspecies diversity and whether strains can be distinguished based on the sources of their isolation, 50
S. maltophilia
isolates from clinical and environmental sources, including strains of biotechnological interest, were investigated. The isolates were characterized by in vitro antagonism against pathogenic fungi and the production of antifungal metabolites and enzymes. Phenotypically the strains showed variability that did not correlate significantly with their sources of isolation. Clinical strains displayed remarkable activity against the human pathogenic fungus
Candida albicans
. Antifungal activity against plant pathogens was more common and generally more severe from the environmental isolates, although not exclusive to them. All isolates, clinical and environmental, produced a range of antifungal metabolites including antibiotics, siderophores, and the enzymes proteases and chitinases. From 16S ribosomal DNA sequencing analysis, the isolates could be separated into three clusters, two of which consisted of isolates originating from the environment, especially rhizosphere isolates, and one of which consisted of clinical and aquatic strains. In contrast to the results of other recent investigations, these strains could be grouped based on their sources of isolation, with the exception of three rhizosphere isolates. Because there was evidence of nucleotide signature positions within the sequences that are suitable for distinguishing among the clusters, the clusters could be defined as different genomovars of
S. maltophilia
. Key sequences on the 16S ribosomal DNA could be used to develop a diagnostic method that differentiates these genomovars.
Genetic Analysis for Virulence Factors in
Escherichia coli
O104:H21 That Was Implicated in an Outbreak of Hemorrhagic Colitis
Authors: Feng Peter , Weagant Stephen D. , Monday Steven R.
Published: July 2002

Isolates of enterohemorrhagic
Escherichia coli
(EHEC) of serotype O104:H21 implicated in a 1994 outbreak of hemorrhagic colitis in Montana were analyzed for the presence of trait EHEC virulence markers. By using a multiplex PCR that specifically amplifies several genes, the O104:H21 strains were found to carry only the Shiga toxin 2 gene (
stx2
) and to express Stx2. They did not have the
eaeA
gene for γ-intimin, which is typically found in O157:H7, or the α- or β-intimin derivatives, which are common in other EHEC and enteropathogenic
E. coli
serotypes. Results of the multiplex PCR also indicated that the
ehxA
gene for enterohemolysin was absent from O104:H21. This, however, was not consistent with the results of a phenotypic assay that showed them to be hemolytic or a PCR analysis with another set of
ehxA
-specific primers, which indicated the presence of
ehxA
. To resolve this discrepancy, the
ehxA
region in O104:H21 and O157:H7 strains, to which the multiplex PCR primers anneal, was cloned and sequenced. Comparison of the sequences showed that the upstream primer binding site in the
ehxA
gene of O104:H21 was not identical to that of O157:H7. Specifically, there were several base mutations, including an A-to-G substitution at the 3′ end of the primer binding site. These base mutations are presumably not unique to O104:H21, since other enterohemolytic serotypes were also not detected with the
ehxA
primers used in the multiplex PCR. Comparison of the
ehxA
sequences of O104:H21 strains with those of other Stx-producing
E. coli
strains showed that they more closely resembled those of O8:H19 strains, which have cluster II
ehxA
genes, than those of O157:H7 strains, which have cluster I
ehxA
sequences. By modifying the upstream
ehxA
primer, the multiplex PCR was able to detect
ehxA
genes in both O157:H7 and O104:H21 strains.
Extraction and Genotyping of
Cryptosporidium parvum
DNA from Fecal Smears on Glass Slides Stained Conventionally for Direct Microscope Examination
Authors: Amar C. , Pedraza-Dı́az S. , McLauchlin J.
Published: July 2002

A method was developed for extracting cryptosporidial DNA from stained fecal smears on glass microscope slides. The correct genotype of
Cryptosporidium parvum
was amplified by PCR from 89 (85%) of 105 smears following conventional staining but not from negative controls. This technique may have applications for analysis of other infectious agents.
Increased Prevalence of Class I Integrons in
Escherichia coli
,
Klebsiella
Species, and
Enterobacter
Species Isolates over a 7-Year Period in a German University Hospital
Authors: Schmitz Franz-Josef , Hafner Dieter , Geisel Roland , Follmann Patrick , Kirschke Christian , Verhoef Jan , Köhrer Karl , Fluit Ad C.
Published: July 2002

The prevalence of integrons in five enterobacterial species was analyzed in 900 blood culture isolates from 1993, 1996, and 1999. Remarkably, the prevalence increased from 4.7% in 1993 to 9.7% in 1996 and finally to 17.4% in 1999 (
P
< 0.01). Within 7 years the combined percentage of P1 strong promoters and P1 weak plus P2 active promoters with high transcription efficacies has increased from 23.1 to 33.3 and finally 60% (
P
< 0.05).
Enzyme-Linked Immunosorbent Assay for Serological Diagnosis of Chagas' Disease Employing a
Trypanosoma cruzi
Recombinant Antigen That Consists of Four Different Peptides
Authors: Ferreira A. W. , Belem Z. R. , Lemos E. A. , Reed S. G. , Campos-Neto A.
Published: July 2002

Serological tests to detect
Trypanosoma cruzi
antibodies have been used for screening blood donors, for epidemic studies, and for diagnosis of probably infected persons. Among different tests, the enzyme-linked immunosorbent assay (ELISA) with total, semipurified, or synthetic antigens has been widely used, mainly due to its easy automation. Aiming to improve serological studies concerning Chagas' disease, we have developed and evaluated a new test, the TcF-ELISA, using an artificially engineered recombinant antigen, which contains tandem sequences of different
T. cruzi
-specific peptides. The sensibility of the TcF-ELISA was determined with 101 serum samples from chagasic patients well-defined by clinical and epidemiological criteria. The specificity was determined with 39 serum samples from leishmaniasis or kala-azar patients and 150 serum samples from nonchagasic blood donors from Sao Paulo, Brazil. The TcF-ELISA showed 100% sensitivity and 98.94% of specificity. Compared with conventional ELISA (with semipurified
T. cruzi
epimastigote antigens), the TcF-ELISA showed advantages; for example, it distinguishes better between reagent and nonreagent serum and provides better precision and a lower occurrence of leishmaniasis cross-reactions. Our studies demonstrate high reproducibility between two different lots of the TcF ELISA and its applicability for the serological diagnosis of Chagas' disease.
Quantitative Real-Time PCR Detection of Rift Valley Fever Virus and Its Application to Evaluation of Antiviral Compounds
Authors: Garcia Stephan , Crance Jean Marc , Billecocq Agnes , Peinnequin Andre , Jouan Alain , Bouloy Michele , Garin Daniel
Published: July 2002

The Rift Valley fever virus (RVFV), a member of the genus
Phlebovirus
(family
Bunyaviridae)
is an enveloped negative-strand RNA virus with a tripartite genome. Until 2000, RVFV circulation was limited to the African continent, but the recent deadly outbreak in the Arabian Peninsula dramatically illustrated the need for rapid diagnostic methods, effective treatments, and prophylaxis. A method for quantifying the small RNA segment by a real-time detection reverse transcription (RT)-PCR using TaqMan technology and targeting the nonstructural protein-coding region was developed, and primers and a probe were designed. After optimization of the amplification reaction and establishment of a calibration curve with synthetic RNA transcribed in vitro from a plasmid containing the gene of interest, real-time RT-PCR was assessed with samples consisting of RVFV from infected Vero cells. The method was found to be specific for RVFV, and it was successfully applied to the detection of the RVFV genome in animal sera infected with RVFV as well as to the assessment of the efficiency of various drugs (ribavirin, alpha interferon, 6-azauridine, and glycyrrhizin) for antiviral activity. Altogether, the results indicated a strong correlation between the infectious virus titer and the amount of viral genome assayed by real time RT-PCR. This novel method could be of great interest for the rapid diagnosis and screening of new antiviral compounds, as it is sensitive and time saving and does not require manipulation of infectious material.
Differentiation of
Ehrlichia platys
and
E. equi
Infections in Dogs by Using 16S Ribosomal DNA-Based PCR
Authors: Hancock Susan I. , Breitschwerdt Edward B. , Pitulle Christian
Published: July 2002

We have encountered a previously unrecognized specificity problem when using the small-subunit ribosomal DNA (16S rDNA)-based PCR primers recommended for use in the identification of
Ehrlichia equi
in clinical samples. These PCR primers annealed to
E. platys
16S rDNA in blood samples containing high levels of
E. platys
organisms. Therefore, we designed and tested new PCR primers for the identification of
E. equi
.
Isolation of
Helicobacter cinaedi
from the Colon, Liver, and Mesenteric Lymph Node of a Rhesus Monkey with Chronic Colitis and Hepatitis
Authors: Fox J. G. , Handt L. , Sheppard B. J. , Xu S. , Dewhirst F. E. , Motzel S. , Klein H.
Published: July 2002

On the basis of biochemical, phenotypic, and 16S rRNA analyses,
Helicobacter cinaedi
was isolated from the colon, liver, and mesenteric lymph nodes of a 2-year-old rhesus monkey with chronic diarrhea. Histologically, the liver had mild to moderate biliary hyperplasia and hypertrophy with periportal inflammation and fibrosis. Colonic and cecal lesions consisted of diffuse chronic inflammation and glandular hyperplasia extending the length of the crypts. This is the first observation of
H. cinaedi
associated with active hepatitis and colitis in a nonhuman primate.
Vertebral Osteomyelitis Caused by
Enterococcus raffinosus
Authors: Sandoe Jonathan A. T. , Witherden Ian R. , Settle Christopher
Published: July 2002

Enterococcus raffinosus
is a rare isolate in clinical specimens. A case of vertebral osteomyelitis caused by
E. raffinosus
in an elderly patient is described and confirms this organism to be an opportunistic human pathogen.
Molecular Detection of Human Calicivirus in Young Children Hospitalized with Acute Gastroenteritis in Melbourne, Australia, during 1999
Authors: Kirkwood Carl D. , Bishop Ruth F.
Published: July 2002

Reverse transcription-PCR and sequence analysis identified calciviruses in 32 of 60 stool specimens (negative for other enteric pathogens) obtained from children admitted to our hospital with acute gastroenteritis. The overall annual incidence rate for calcivirus was 9% (32 of 354 children). Molecular analysis identified 30 “Norwalk-like virus” genogroup II (predominantly Lordsdale cluster) and 2 “Sapporo-like virus” strains.
Limited Level of Accuracy Provided by Available Rapid Diagnosis Tests for Malaria Enhances the Need for PCR-Based Reference Laboratories
Authors: Rubio J. M. , Buhigas I. , Subirats M. , Baquero M. , Puente S. , Benito A.
Published: July 2002

The rise of imported malaria cases and the high fatality rate in Europe make the search for new and easy diagnostic methods necessary. Rapid diagnosis tests (RDTs) are, in part, developed to cover the lack of diagnosis experience. Unfortunately, our data suggest that the accuracy of RDTs is insufficient and could increase the number of incorrect malaria diagnoses.