bluuebunny/crossref_metadata_2025 huggingface dataset:
Contribution of NADH Oxidase to Aerobic Metabolism of
Streptococcus pyogenes
Authors: Gibson Carmela M. , Mallett T. Conn , Claiborne Al , Caparon Michael G.
Published: July 2002

An understanding of how the heme-deficient gram-positive bacterium
Streptococcus pyogenes
establishes infections in O
2
-rich environments requires careful analysis of the gene products important in aerobic metabolism. NADH oxidase (NOXase) is a unique flavoprotein of
S. pyogenes
and other lactic acid bacteria which directly catalyzes the four-electron reduction of O
2
to H
2
O. To elucidate a putative role for this enzyme in aerobic metabolism, NOXase-deficient mutants were constructed by insertional inactivation of the gene that encodes NOXase. Characterization of the resulting mutants revealed that growth in rich medium under low-O
2
conditions was indistinguishable from that of the wild type. However, the mutants were unable to grow under high-O
2
conditions and demonstrated enhanced sensitivity to the superoxide-generating agent paraquat. Mutants cultured in liquid medium under conditions of carbohydrate limitation and high O
2
tension were characterized by an extended lag phase, a reduction in growth, and a greater accumulation of H
2
O
2
in the growth medium compared to the wild-type strain. All of these mutant phenotypes could be overcome by the addition of glucose. Either the addition of catalase to the culture medium of the mutants or the introduction of a heterologous NADH peroxidase into the mutants eliminated the accumulation of H
2
O
2
and rescued the growth defect of the mutants under high-O
2
conditions in carbohydrate-limited liquid medium. Taken together, these data show that NOXase is important for aerobic metabolism and essential in environments high in O
2
with carbohydrate limitation.
Identification of RpoS (ς
S
)-Regulated Genes in
Salmonella enterica
Serovar Typhimurium
Authors: Ibanez-Ruiz Magdalena , Robbe-Saule Véronique , Hermant Daniel , Labrude Séverine , Norel Françoise
Published: July 2002

The
rpoS
gene encodes the alternative sigma factor ς
S
(RpoS) and is required for survival of bacteria under starvation and stress conditions. It is also essential for
Salmonella
virulence in mice. Most work on the RpoS regulon has been in the closely related enterobacterial species
Escherichia coli
. To characterize the RpoS regulon in
Salmonella
, we isolated 38 unique RpoS-activated
lacZ
gene fusions from a bank of
Salmonella enterica
serovar Typhimurium mutants harboring random Tn5B21 mutations. Dependence on RpoS varied from 3-fold to over 95-fold, and all gene fusions isolated were regulated by growth phase. The identities of 21 RpoS-dependent fusions were determined by DNA sequence analysis. Seven of the fusions mapped to DNA regions in
Salmonella
serovar Typhimurium that do not match any known
E. coli
sequence, suggesting that the composition of the RpoS regulon differs markedly in the two species. The other 14 fusions mapped to 13 DNA regions very similar to
E. coli
sequences. None of the insertion mutations in DNA regions common to both species appeared to affect
Salmonella
virulence in BALB/c mice. Of these, only three (
otsA
,
katE
, and
poxB
) are located in known members of the RpoS regulon. Ten insertions mapped in nine open reading frames of unknown function (
yciF
,
yehY
,
yhjY
,
yncC
,
yjgB
,
yahO
,
ygaU
,
ycgB
, and
yeaG
) appear to be novel members of the RpoS regulon. One insertion, that in mutant C52::H87, was in the noncoding region upstream from
ogt
, encoding a
O
6
-methylguanine DNA methyltransferase involved in repairing alkylation damage in DNA. The
ogt
coding sequence is very similar to the
E. coli
homolog, but the
ogt
5′ flanking regions were found to be markedly different in the two species, suggesting genetic rearrangements. Using primer extension assays, a specific
ogt
mRNA start site was detected in RNAs of the
Salmonella
serovar Typhimurium wild-type strains C52 and SL1344 but not in RNAs of the mutant strains C52K (
rpoS
), SL1344K (
rpoS
), and C52::H87. In mutant C52::H87, Tn
5
B21 is inserted at the
ogt
mRNA start site, with
lacZ
presumably transcribed from the identified RpoS-regulated promoter. These results indicate that
ogt
gene expression in
Salmonella
is regulated by RpoS in stationary phase of growth in rich medium, a finding that suggests a novel role for RpoS in DNA repair functions.
The Last RNA-Binding Repeat of the
Escherichia coli
Ribosomal Protein S1 Is Specifically Involved in Autogenous Control
Authors: Boni Irina V. , Artamonova Valentina S. , Dreyfus Marc
Published: July 2002

The
ssyF29
mutation, originally selected as an extragenic suppressor of a protein export defect, has been mapped within the
rpsA
gene encoding ribosomal protein S1. Here, we examine the nature of this mutation and its effect on translation. Sequencing of the
rpsA
gene from the
ssyF
mutant has revealed that, due to an IS
10
R insertion, its product lacks the last 92 residues of the wild-type S1 protein corresponding to one of the four homologous repeats of the RNA-binding domain. To investigate how this truncation affects translation, we have created two series of
Escherichia coli
strains (
rpsA
+
and
ssyF
) bearing various translation initiation regions (TIRs) fused to the chromosomal
lacZ
gene. Using a β-galactosidase assay, we show that none of these TIRs differ in activity between
ssyF
and
rpsA
+
cells, except for the
rpsA
TIR: the latter is stimulated threefold in
ssyF
cells, provided it retains at least ca. 90 nucleotides upstream of the start codon. Similarly, the activity of this TIR can be severely repressed in
trans
by excess S1, again provided it retains the same minimal upstream sequence. Thus, the
ssyF
stimulation requires the presence of the
rpsA
translational autogenous operator. As an interpretation, we propose that the
ssyF
mutation relieves the residual repression caused by normal supply of S1 (i.e., that it impairs autogenous control). Thus, the C-terminal repeat of the S1 RNA-binding domain appears to be required for autoregulation, but not for overall mRNA recognition.
Characterization of the Genes for Two Protocatechuate 3,4-Dioxygenases from the 4-Sulfocatechol-Degrading Bacterium
Agrobacterium radiobacter
Strain S2
Authors: Contzen Matthias , Stolz Andreas
Published: July 2002

The genes for two different protocatechuate 3,4-dioxygenases (P34Os) were cloned from the 4-sulfocatechol-degrading bacterium
Agrobacterium radiobacter
strain S2 (DSMZ 5681). The
pcaH1G1
genes encoded a P34O (P34O-I) which oxidized protocatechuate but not 4-sulfocatechol. These genes were part of a protocatechuate-degradative operon which strongly resembled the isofunctional operon from the protocatechuate-degrading strain
Agrobacterium tumefaciens
A348 described previously by D. Parke (FEMS Microbiol. Lett. 146:3–12, 1997). The second P34O (P34O-II), encoded by the
pcaH2G2
genes, was functionally expressed and shown to convert protocatechuate and 4-sulfocatechol. A comparison of the deduced amino acid sequences of PcaH-I and PcaH-II, and of PcaG-I and PcaG-II, with each other and with the corresponding sequences from the P34Os, from other bacterial genera suggested that the genes for the P34O-II were obtained by strain S2 by lateral gene transfer. The genes encoding the P34O-II were found in a putative operon together with two genes which, according to sequence alignments, encoded transport proteins. Further downstream from this putative operon, two open reading frames which code for a putative regulator protein of the IclR family and a putative 3-carboxymuconate cycloisomerase were identified.
Fate of the SpoIIAB*-ADP Liberated after SpoIIAB Phosphorylates SpoIIAA of
Bacillus subtilis
Authors: Lee Chung-Sheng , Lucet Isabelle , Yudkin Michael D.
Published: July 2002

Phosphorylation of SpoIIAA catalyzed by SpoIIAB helps to regulate the first sporulation-specific ς factor, ς
F
, of
Bacillus subtilis
. The steady-state rate of phosphorylation is known to be exceptionally slow and to be limited by the return of the protein kinase, SpoIIAB, to a catalytically active state. Previous work from this laboratory has suggested that, after catalyzing the phosphorylation, SpoIIAB is in a form (SpoIIAB*) that does not readily release ADP. We now show that the rate of release of ADP from the SpoIIAB*-ADP complex was much diminished by the presence of unreacted SpoIIAA, suggesting that SpoIIAA can form a long-lived ternary complex with SpoIIAB*-ADP in which the SpoIIAB* form is stabilized. In kinetic studies of the phosphorylation of SpoIIAA, the ternary complex SpoIIAA-SpoIIAB*-ADP could be distinguished from the short-lived complex SpoIIAA-SpoIIAB-ADP, which can be readily produced in the absence of an enzymatic reaction.
A Gene of
Synechocystis
sp. Strain PCC 6803 Encoding a Novel Iron Transporter
Authors: Katoh Hirokazu , Grossman Arthur R. , Hagino Natsu , Ogawa Teruo
Published: July 2002

A mutant of
Synechocystis
sp. strain PCC 6803 disrupted for
sll1878
exhibited greatly reduced Fe
3+
transport activity. The

K
m

value of
sll1878
-dependent Fe
3+
transport in cells grown in iron-replete medium was 0.5 μM. Both the maximal rate and

K
m

value were increased in iron-starved cells.
Posttranslational Processing ofMethanococcus voltaePreflagellin by Preflagellin Peptidases ofM. voltaeand Other Methanogens
Authors: Correia Jason D. , Jarrell Ken F.
Published: July 2002

Methanococcus voltaeis a mesophilic archaeon with flagella composed of flagellins that are initially made with 11- or 12-amino-acid leader peptides that are cleaved prior to incorporation of the flagellin into the growing filament. Preflagellin peptidase activity was demonstrated in immunoblotting experiments with flagellin antibody to detect unprocessed and processed flagellin subunits.Escherichia colimembranes containing the expressedM. voltaepreflagellin (as the substrate) were combined in vitro with methanogen membranes (as the enzyme source). Correct processing of the preflagellin to the mature flagellin was also shown directly by comparison of the N-terminal sequences of the two flagellin species.M. voltaepreflagellin peptidase activity was optimal at 37°C and pH 8.5 and in the presence of 0.4 M KCl with 0.25% (vol/vol) Triton X-100.
Crc Is Involved in Catabolite Repression Control of the
bkd
Operons of
Pseudomonas putida
and
Pseudomonas aeruginosa
Authors: Hester Kathryn L. , Lehman Jodi , Najar Fares , Song Lin , Roe Bruce A. , MacGregor Carolyn H. , Hager Paul W. , Phibbs Paul V. , Sokatch John R.
Published: July 2002

Crc (catabolite repression control) protein of
Pseudomonas aeruginosa
has shown to be involved in carbon regulation of several pathways. In this study, the role of Crc in catabolite repression control has been studied in
Pseudomonas putida
. The
bkd
operons of
P. putida
and
P. aeruginosa
encode the inducible multienzyme complex branched-chain keto acid dehydrogenase, which is regulated in both species by catabolite repression. We report here that this effect is mediated in both species by Crc. A 13-kb cloned DNA fragment containing the
P. putida crc
gene region was sequenced. Crc regulates the expression of branched-chain keto acid dehydrogenase, glucose-6-phosphate dehydrogenase, and amidase in both species but not urocanase, although the carbon sources responsible for catabolite repression in the two species differ. Transposon mutants affected in their expression of BkdR, the transcriptional activator of the
bkd
operon, were isolated and identified as
crc
and
vacB
(
rnr
) mutants. These mutants suggested that catabolite repression in pseudomonads might, in part, involve control of BkdR levels.
Feruloyl Esterase Activity of the
Clostridium thermocellum
Cellulosome Can Be Attributed to Previously Unknown Domains of XynY and XynZ
Authors: Blum David L. , Kataeva Irina A. , Li Xin-Liang , Ljungdahl Lars G.
Published: July 2002

The cellulosome of
Clostridium thermocellum
is a multiprotein complex with endo- and exocellulase, xylanase, β-glucanase, and acetyl xylan esterase activities. XynY and XynZ, components of the cellulosome, are composed of several domains including xylanase domains and domains of unknown function (UDs). Database searches revealed that the C- and N-terminal UDs of XynY and XynZ, respectively, have sequence homology with the sequence of a feruloyl esterase of strain PC-2 of the anaerobic fungus
Orpinomyces
. Purified cellulosomes from
C. thermocellum
were found to hydrolyze FAXX (
O
-{5-
O
-[(
E
)-feruloyl]-α-
l
-arabinofuranosyl}-(1→3)-
O
-β-
d
-xylopyranosyl-(1→4)-
d
-xylopyranose) and FAX
3
(5-
O
-[(
E
)-feruloyl]-[
O
-β-
d
-xylopyranosyl-(1→2)]-
O
-α-
l
-arabinofuranosyl-[1→3]}-
O
-β-
d
-xylopyranosyl-(1→4)-
d
-xylopyranose), yielding ferulic acid as a product, indicating that they have feruloyl esterase activity. Nucleotide sequences corresponding to the UDs of XynY and XynZ were cloned into
Escherichia coli
, and the expressed proteins hydrolyzed FAXX and FAX
3
. The recombinant feruloyl esterase domain of XynZ alone (FAE
XynZ
) and with the adjacent cellulose binding domain (FAE-CBD
XynZ
) were characterized. FAE-CBD
XynZ
had a molecular mass of 45 kDa that corresponded to the expected product of the 1,203-bp gene.

K
m

and
V
max
values for FAX
3
were 5 mM and 12.5 U/mg, respectively, at pH 6.0 and 60°C. PAX
3
, a substrate similar to FAX
3
but with a
p
-coumaroyl group instead of a feruloyl moiety was hydrolyzed at a rate 10 times slower. The recombinant enzyme was active between pH 3 to 10 with an optimum between pH 4 to 7 and at temperatures up to 70°C. Treatment of Coastal Bermuda grass with the enzyme released mainly ferulic acid and a lower amount of
p
-coumaric acid. FAE
XynZ
had similar properties. Removal of the 40 C-terminal amino acids, residues 247 to 286, of FAE
XynZ
resulted in protein without activity. Feruloyl esterases are believed to aid in a release of lignin from hemicellulose and may be involved in lignin solubilization. The presence of feruloyl esterase in the
C. thermocellum
cellulosome together with its other hydrolytic activities demonstrates a powerful enzymatic potential of this organelle in plant cell wall decomposition.
Expression of
ykdA
, Encoding a
Bacillus subtilis
Homologue of HtrA, Is Heat Shock Inducible and Negatively Autoregulated
Authors: Noone David , Howell Alistair , Devine Kevin M.
Published: July 2002

There are three members of the HtrA family of serine proteases, YkdA, YvtA, and YyxA, encoded in the chromosome of
Bacillus subtilis
. In this study, we report on the promoter structure and regulation of
ykdA
expression. The
ykdA
gene is heat inducible, exhibiting a biphasic pattern of expression during a 60-min interval after heat shock. Increased expression after heat shock occurs at the transcriptional level. The heat-shock-inducible promoter has a single mismatch with a SigA-type −10 motif, but does not exhibit similarity to a SigA −35 region. There are six octamer repeats with a consensus TTTTCACA positioned at, and upstream of, the normal position of a −35 region. While repeats V and VI appear dispensable, repeat IV is essential for normal thermoinducible expression. This promoter structure is also found in the control region of
yvtA
, encoding a second member of this family of proteases. Expression of
ykdA
is negatively autoregulated both during the growth cycle and during heat shock. Our evidence suggests that YkdA protease activity is not required for this form of regulation. Null mutants of
ykdA
display increased tolerance to heat and are 80-fold more resistant to 10 mM hydrogen peroxide than wild-type cells. However,
ykdA
expression is not induced by hydrogen peroxide. These results indicate that the regulon to which YkdA belongs is linked to the oxidative stress response in
B. subtilis
.
A Two-Component Multidrug Efflux Pump, EbrAB, in
Bacillus subtilis
Authors: Masaoka Yoko , Ueno Yasuhiro , Morita Yuji , Kuroda Teruo , Mizushima Tohru , Tsuchiya Tomofusa
Published: July 2002

Genes (
ebrAB
) responsible for ethidium resistance were cloned from chromosomal DNA of
Bacillus subtilis
ATCC 9372. The recombinant plasmid produced elevated resistance against ethidium bromide, acriflavine, pyronine Y, and safranin O not only in
Escherichia coli
but also in
B. subtilis
. It also caused an elevated energy-dependent efflux of ethidium in
E. coli
. EbrA and EbrB showed high sequence similarity with members of the small multidrug resistance (SMR) family of multidrug efflux pumps. Neither
ebrA
nor
ebrB
was sufficient for resistance, but introduction of the two genes carried on different plasmids conferred drug resistance. Thus, both EbrA and EbrB appear to be necessary for activity of the multidrug efflux pump. In known members of the SMR family, only one gene produces drug efflux. Thus, EbrAB is a novel SMR family multidrug efflux pump with two components.
High Spontaneous Mutation Rate in the Hyperthermophilic Archaeon
Sulfolobus solfataricus
Is Mediated by Transposable Elements
Authors: Martusewitsch Erika , Sensen Christoph W. , Schleper Christa
Published: July 2002

We have isolated uracil-auxotrophic mutants of the hyperthermophilic archaeon
Sulfolobus solfataricus
in order to explore the genomic stability and mutational frequencies of this organism and to identify complementable recipients for a selectable genetic transformation system. Positive selection of spontaneous mutants resistant to 5-fluoroorotate yielded uracil auxotrophs with frequencies of between 10
−4
and 10
−5
per sensitive, viable cell. Four different, nonhomologous insertion sequences (ISs) were identified at different positions within the chromosomal
pyrEF
locus of these mutants. They ranged in size from 1,058 to 1,439 bp and possessed properties typical of known transposable elements, i.e., terminal inverted repeats, flanking duplicated target sequences, and putative transposase genes encoding motifs that are indicative of the IS
4
-IS
5
IS element families. Between 12 and 25 copies of each IS element were found in chromosomal DNAs by Southern analyses. While characteristic fingerprint patterns created by IS element-specific probes were observed with genomic DNA of different
S. solfataricus
strains, no homologous sequences were identified in DNA of other well-characterized strains of the order
Sulfolobales
.
Escherichia coli
Strains Blocked in Tat-Dependent Protein Export Exhibit Pleiotropic Defects in the Cell Envelope
Authors: Stanley Nicola R. , Findlay Kim , Berks Ben C. , Palmer Tracy
Published: July 2002

The Tat system is a recently discovered protein export pathway that serves to translocate folded proteins, often containing redox cofactors, across the bacterial cytoplasmic membrane. Here we report that
tat
strains are associated with a mutant cell septation phenotype, where chains of up to 10 cells are evident. Mutant strains are also hypersensitive to hydrophobic drugs and to lysis by lysozyme in the absence of EDTA, and they leak periplasmic enzymes, characteristics that are consistent with an outer membrane defect. Both phenotypes are similar to those displayed by strains carrying point mutations in the
lpxC
(
envA
) gene. The phenotype was not replicated by mutations affecting synthesis and/or activity of all known or predicted Tat substrates.
Phylogeny of the Major Head and Tail Genes of the Wide-Ranging T4-Type Bacteriophages
Authors: Tétart Françoise , Desplats Carine , Kutateladze Mzia , Monod Caroline , Ackermann Hans-Wolfgang , Krisch H. M.
Published: July 2002

We examined a number of bacteriophages with T4-type morphology that propagate in different genera of enterobacteria,
Aeromonas
,
Burkholderia,
and
Vibrio
. Most of these phages had a prolate icosahedral head, a contractile tail, and a genome size that was similar to that of T4. A few of them had more elongated heads and larger genomes. All these phages are phylogenetically related, since they each had sequences homologous to the capsid gene (gene
23
), tail sheath gene (gene
18
), and tail tube gene (gene
19
) of T4. On the basis of the sequence comparison of their virion genes, the T4-type phages can be classified into three subgroups with increasing divergence from T4: the T-evens, pseudoT-evens, and schizoT-evens. In general, the phages that infect closely related host species have virion genes that are phylogenetically closer to each other than those of phages that infect distantly related hosts. However, some of the phages appear to be chimeras, indicating that, at least occasionally, some genetic shuffling has occurred between the different T4-type subgroups. The compilation of a number of gene
23
sequences reveals a pattern of conserved motifs separated by sequences that differ in the T4-type subgroups. Such variable patches in the gene
23
sequences may determine the size of the virion head and consequently the viral genome length. This sequence analysis provides molecular evidence that phages related to T4 are widespread in the biosphere and diverged from a common ancestor in acquiring the ability to infect different host bacteria and to occupy new ecological niches.
Some
Lactobacillus
l
-Lactate Dehydrogenases Exhibit Comparable Catalytic Activities for Pyruvate and Oxaloacetate
Authors: Arai Kazuhito , Kamata Takeo , Uchikoba Hiroyuki , Fushinobu Shinya , Matsuzawa Hiroshi , Taguchi Hayao
Published: July 2002

The nonallosteric and allosteric
l
-lactate dehydrogenases of
Lactobacillus pentosus
and
L. casei
, respectively, exhibited broad substrate specificities, giving virtually the same maximal reaction velocity and substrate

K
m

values for pyruvate and oxaloacetate. Replacement of Pro101 with Asn reduced the activity of the
L. pentosus
enzyme toward these alternative substrates to a greater extent than the activity toward pyruvate.
Regulation of Expression of the
vanD
Glycopeptide Resistance Gene Cluster from
Enterococcus faecium
BM4339
Authors: Casadewall Barbara , Reynolds Peter E. , Courvalin Patrice
Published: July 2002

A new open reading frame, encoding a putative integrase-like protein, was detected downstream from the six genes of the
vanD
glycopeptide resistance cluster in
Enterococcus faecium
BM4339 (B. Casadewall and P. Courvalin, J. Bacteriol. 181:3644–3648, 1999). In this cluster, genes coding for the VanR
D
-VanS
D
two-component regulatory system were cotranscribed from the
P

R
D

promoter, whereas transcription of the

vanY
D
, vanH
D
, vanD, vanX
D

, and
intD
genes was initiated from the
P

Y
D

promoter located between

vanS
D

and

vanY
D

(the D subscript indicates that the gene is part of the
vanD
operon). The VanR
D
-VanS
D
regulatory system is likely to activate transcription of the resistance genes from the promoter
P

Y
D

. Glycopeptide-susceptible derivatives of BM4339 were obtained by
trans
complementation of the frameshift mutation in the
ddl
gene, restoring functional
d
-alanine:
d
-alanine ligase activity in this strain. The glycopeptide-susceptible transformant BM4409, producing only
d
-alanyl-
d
-alanine-terminating peptidoglycan precursors, did not express the resistance genes encoding the VanY
D
d
,
d
-carboxypeptidase, the VanH
D
dehydrogenase, the VanD ligase, the VanX
D
d
,
d
-dipeptidase, and also the IntD integrase, although the regulatory region of the
vanD
cluster was still transcribed. In BM4409, the absence of VanR
D
-VanS
D
, apparently dependent, transcription from promoter
P

Y
D

correlated with the lack of
d
-alanyl-
d
-lactate-terminating precursors. The

vanX
D

gene was transcribed in BM4339, but detectable amounts of VanX
D
d
,
d
-dipeptidase were not synthesized. However, the gene directed synthesis of an active enzyme when cloned on a multicopy plasmid in
Escherichia coli
, suggesting that the enzyme was unstable in BM4339 or that it had very low activity that was detectable only under conditions of high gene dosage. This activity is not required for glycopeptide resistance in BM4339, since this strain cannot synthesize
d
-alanyl-
d
-alanine.
Requirement of Novel Competence Genes
pilT
and
pilU
of
Pseudomonas stutzeri
for Natural Transformation and Suppression of
pilT
Deficiency by a Hexahistidine Tag on the Type IV Pilus Protein PilAI
Authors: Graupner Stefan , Weger Nicole , Sohni Monika , Wackernagel Wilfried
Published: July 2002

The ubiquitous species
Pseudomonas stutzeri
has type IV pili, and these are essential for the natural transformation of the cells. An absolute transformation-deficient mutant obtained after transposon mutagenesis had an insertion in a gene which was termed
pilT
. The deduced amino acid sequence has identity with PilT of
Pseudomonas aeruginosa
(94%),
Neisseria gonorrhoeae
(67%), and other gram-negative species and it contains a nucleotide-binding motif. The mutant was hyperpiliated but defective for further pilus-associated properties, such as twitching motility and plating of pilus-specific phage PO4. [
3
H]thymidine-labeled DNA was bound by the mutant but not taken up. Downstream of
pilT
a gene, termed
pilU
, coding for a putative protein with 88% amino acid identity with PilU of
P. aeruginosa
was identified. Insertional inactivation did not affect piliation, twitching motility, or PO4 infection but reduced transformation to about 10%. The defect was fully complemented by PilU of nontransformable
P. aeruginosa
. When the
pilAI
gene (coding for the type IV pilus prepilin) was manipulated to code for a protein in which the six C-terminal amino acids were replaced by six histidine residues and then expressed from a plasmid, it gave a nonpiliated and twitching motility-defective phenotype in
pilAI
::Gm
r
cells but allowed transformability. Moreover, the mutant allele suppressed the absolute transformation deficiency caused by the
pilT
mutation. Considering the hypothesized role of
pilT
+
in pilus retraction and the presumed requirement of retraction for DNA uptake, it is proposed that the
pilT
-independent transformation is promoted by PilA mutant protein either as single molecules or as minimal pilin assembly structures in the periplasm which may resemble depolymerized pili and that these cause the outer membrane pores to open for DNA entry.
Monitoring Intracellular Levels of XylR inPseudomonas putidawith a Single-Chain Antibody Specific for Aromatic-Responsive Enhancer-Binding Proteins
Authors: Fraile Sofı́a , Roncal Fernando , Fernández Luis A. , de Lorenzo Vı́ctor
Published: July 2002

We have isolated a recombinant phage antibody (Phab) that binds a distinct epitope of the subclass of the ς54-dependent prokaryotic enhancer-binding proteins that respond directly to aromatic effectors, e.g., those that activate biodegradative operons ofPseudomonasspp. The DNA segments encoding the variable (V) domains of the immunoglobulins expressed by mice immunized with the C-terminal half of TouR (TouRΔA) ofPseudomonas stutzeriOX1 were amplified and rearranged in vitro as single-chain Fv (scFv) genes. An scFv library was thereby constructed, expressed in an M13 display system, and subjected to a panning procedure with TouR. One clone (named B7) was selected with high affinity for TouR and XylR (the regulator of the upper TOL operon of the pWW0 plasmid). The epitope recognized by this Phab was mapped to the peptide TPRAQATLLRVL, which seems to be characteristic of the group of enhancer-binding proteins to which TouR and XylR belong and which is located adjacent to the Walker B motif of the proteins. The Phab B7 was instrumental in measuring directly the intracellular levels of XylR expressed from its natural promoter in monocopy gene dosage inPseudomonas putidaunder various conditions. Growth stage, the physical form of the protein produced (XylR or XylRΔA), and the presence or absence of aromatic inducers in the medium influenced the intracellular pool of these molecules. XylR oscillated from a minimum of ∼30 molecules (monomers) per cell during exponential phase to ∼140 molecules per cell at stationary phase. Activation of XylR by aromatic inducers decreased the intracellular concentration of the regulator. The levels of the constitutively active variant of XylR named XylRΔA were higher, fluctuating between ∼90 and ∼570 molecules per cell, depending on the growth stage. These results are compatible with the present model of transcriptional autoregulation of XylR and suggest the existence of mechanisms controlling the stability of XylR protein in vivo.
Specificity of Signal Peptide Recognition in Tat-Dependent Bacterial Protein Translocation
Authors: Blaudeck Natascha , Sprenger Georg A. , Freudl Roland , Wiegert Thomas
Published: July 2002

The bacterial twin arginine translocation (Tat) pathway translocates across the cytoplasmic membrane folded proteins which, in most cases, contain a tightly bound cofactor. Specific amino-terminal signal peptides that exhibit a conserved amino acid consensus motif, S/T-R-R-X-F-L-K, direct these proteins to the Tat translocon. The glucose-fructose oxidoreductase (GFOR) of
Zymomonas mobilis
is a periplasmic enzyme with tightly bound NADP as a cofactor. It is synthesized as a cytoplasmic precursor with an amino-terminal signal peptide that shows all of the characteristics of a typical twin arginine signal peptide. However, GFOR is not exported to the periplasm when expressed in the heterologous host
Escherichia coli
, and enzymatically active pre-GFOR is found in the cytoplasm. A precise replacement of the pre-GFOR signal peptide by an authentic
E. coli
Tat signal peptide, which is derived from pre-trimethylamine
N
-oxide (TMAO) reductase (TorA), allowed export of GFOR, together with its bound cofactor, to the
E. coli
periplasm. This export was inhibited by carbonyl cyanide
m
-chlorophenylhydrazone, but not by sodium azide, and was blocked in
E. coli tatC
and
tatAE
mutant strains, showing that membrane translocation of the TorA-GFOR fusion protein occurred via the Tat pathway and not via the Sec pathway. Furthermore, tight cofactor binding (and therefore correct folding) was found to be a prerequisite for proper translocation of the fusion protein. These results strongly suggest that Tat signal peptides are not universally recognized by different Tat translocases, implying that the signal peptides of Tat-dependent precursor proteins are optimally adapted only to their cognate export apparatus. Such a situation is in marked contrast to the situation that is known to exist for Sec-dependent protein translocation.
Expression of the
Bacillus subtilis trpEDCFBA
Operon Is Influenced by Translational Coupling and Rho Termination Factor
Authors: Yakhnin Helen , Babiarz Joshua E. , Yakhnin Alexander V. , Babitzke Paul
Published: July 2002

The
trp
RNA-binding attenuation protein (TRAP) regulates expression of the
Bacillus subtilis trpEDCFBA
operon by transcription attenuation and translational control mechanisms. Both mechanisms require binding of tryptophan-activated TRAP to 11 (G/U)AG repeats in the
trp
leader transcript.
trpE
translational control involves formation of a TRAP-dependent RNA structure that sequesters the
trpE
Shine-Dalgarno (SD) sequence (the SD blocking hairpin). By comparing expression levels from
trpE
′
-
′
lacZ
translational fusions controlled by the wild-type leader or by a leader that cannot form the SD blocking hairpin, we found that translational control requires a tryptophan concentration higher than that required for transcription attenuation. We also found that inhibition of
trpE
translation by the SD blocking hairpin does not alter the stability of the downstream message. Since the coding sequences for
trpE
and
trpD
overlap by 29 nucleotides, we examined expression levels from
trpED
′
-
′
lacZ
translational fusions to determine if these two genes are translationally coupled. We found that introduction of a UAA stop codon in
trpE
resulted in a substantial reduction in expression. Since expression was partially restored in the presence of a tRNA suppressor, our results indicate that
trpE
and
trpD
are translationally coupled. We determined that the coupling mechanism is TRAP independent and that formation of the SD blocking hairpin regulates
trpD
translation via translational coupling. We also constructed a
rho
mutation to investigate the role of Rho-dependent termination in
trp
operon expression. We found that TRAP-dependent formation of the SD blocking hairpin allows Rho access to the nascent transcript, causing transcriptional polarity.
Bacterial Two-Hybrid Analysis of Interactions between Region 4 of the ς
70
Subunit of RNA Polymerase and the Transcriptional Regulators Rsd from
Escherichia coli
and AlgQ from
Pseudomonas aeruginosa
Authors: Dove Simon L. , Hochschild Ann
Published: July 2002

A number of transcriptional regulators mediate their effects through direct contact with the ς
70
subunit of
Escherichia coli
RNA polymerase (RNAP). In particular, several regulators have been shown to contact a C-terminal portion of ς
70
that harbors conserved region 4. This region of ς contains a putative helix-turn-helix DNA-binding motif that contacts the −35 element of ς
70
-dependent promoters directly. Here we report the use of a recently developed bacterial two-hybrid system to study the interaction between the putative anti-ς factor Rsd and the ς
70
subunit of
E. coli
RNAP. Using this system, we found that Rsd can interact with an 86-amino-acid C-terminal fragment of ς
70
and also that amino acid substitution R596H, within region 4 of ς
70
, weakens this interaction. We demonstrated the specificity of this effect by showing that substitution R596H does not weaken the interaction between ς and two other regulators shown previously to contact region 4 of ς
70
. We also demonstrated that AlgQ, a homolog of Rsd that positively regulates virulence gene expression in
Pseudomonas aeruginosa,
can contact the C-terminal region of the ς
70
subunit of RNAP from this organism. We found that amino acid substitution R600H in ς
70
from
P. aeruginosa
, corresponding to the R596H substitution in
E. coli
ς
70
, specifically weakens the interaction between AlgQ and ς
70
. Taken together, our findings suggest that Rsd and AlgQ contact similar surfaces of RNAP present in region 4 of ς
70
and probably regulate gene expression through this contact.
Interaction of MutS and Vsr: Some Dominant-Negative
mutS
Mutations That Disable Methyladenine-Directed Mismatch Repair Are Active in Very-Short-Patch Repair
Authors: Lieb Margaret , Rehmat Shehnaz , Bhagwat Ashok S.
Published: July 2002

In
Escherichia coli
and related bacteria, the very-short-patch (VSP) repair pathway uses an endonuclease, Vsr, to correct T · G mismatches that result from the deamination of 5-methylcytosines in DNA to C · G. The products of
mutS
and
mutL
, which are required for adenine methylation-directed mismatch repair (MMR), enhance VSP repair. Multicopy plasmids carrying
mutS
alleles that are dominant negative for MMR were tested for their effects on VSP repair. Some
mutS
mutations (class I) did not lower VSP repair in a
mutS
+
background, and most class I mutations increased VSP repair in
mutS
cells more than plasmids containing
mutS
+
. Other plasmid-borne
mutS
mutations (class II) and
mutS
+
decreased VSP repair in the
mutS
+
background. Thus, MutS protein lacking functions required for MMR can still participate in VSP repair, and our results are consistent with a model in which MutS binds transiently to the mispair and then translocates away from the mispair to create a specialized structure that enhances the binding of Vsr.
A Eukaryotic-Type Protein Kinase, SpkA, Is Required for Normal Motility of the Unicellular Cyanobacterium
Synechocystis
sp. Strain PCC 6803
Authors: Kamei Ayako , Yuasa Takashi , Orikawa Kumi , Geng Xiao Xing , Ikeuchi Masahiko
Published: July 2002

The genome of the unicellular cyanobacterium
Synechocystis
sp. strain PCC 6803 comprises many open reading frames (ORFs) which putatively encode eukaryotic-type protein kinase and protein phosphatase. Based on gene disruption analysis, a region of the hypothetical ORF
sll1575
, which retained a part of the protein kinase motif, was found to be required for normal motility in the original isolate of strain PCC 6803. Sequence determination revealed that in this strain
sll1575
was part of a gene (designated
spkA
) which harbored an entire eukaryotic-type Ser/Thr protein kinase motif. Strain ATCC 27184 and a glucose-tolerant strain derived from the same isolate as the PCC strain had a frameshift mutation dividing
spkA
into ORFs
sll1574
and
sll1575
. The structural integrity of
spkA
agreed well with the motility phenotype, determined by colony morphology on agar plates. The
spkA
gene was expressed in
Escherichia coli
as a His-tagged protein, which was purified by Ni
2+
affinity chromatography. With [γ-
32
P]ATP, SpkA was autophosphorylated and transferred the phosphate group to casein, myelin basic protein, and histone. SpkA also phosphorylated several proteins in the membrane fraction of
Synechocystis
cells. These results suggest that SpkA is a eukaryotic-type Ser/Thr protein kinase and regulates cellular motility via phosphorylation of the membrane proteins in
Synechocystis
.
Bacillus subtilis
NhaC, an Na
+
/H
+
Antiporter, Influences Expression of the
phoPR
Operon and Production of Alkaline Phosphatases
Authors: Prágai Zoltán , Eschevins Caroline , Bron Sierd , Harwood Colin R.
Published: July 2002

When
Bacillus subtilis
is subjected to phosphate starvation, genes of the Pho regulon are either induced or repressed. Among those induced are genes encoding alkaline phosphatases (APases). A set of isogenic mutants, with a β-galactosidase gene transcriptionally fused to the inactivated target gene, was used to identify genes that influence the operation of the Pho regulon. One such gene was
nhaC
(previously
yheL
). In the absence of NhaC, growth and APase production were enhanced, while the production of other non-Pho-regulon secretory proteins (proteases and α-amylase) did not change. The influence of NhaC on growth, APase synthesis, and its own expression was dependent on the external Na
+
concentration. Other monovalent cations such as Li
+
or K
+
had no effect. We propose a role for NhaC in the uptake of Na
+
.
nhaC
appears to be encoded by a monocistronic operon and, contrary to previous reports, is not in the same transcriptional unit as
yheK
, the gene immediately upstream. The increase in APase production was dependent on an active PhoR, the sensor kinase of the two-component system primarily responsible for controlling the Pho regulon. Transcriptional fusions showed that the
phoPR
operon and both
phoA
(encoding APaseA) and
phoB
(encoding APaseB) were hyperinduced in the absence of NhaC and repressed when this protein was overproduced. This suggests that NhaC effects APase production via
phoPR
.
Mutational Scanning and Affinity Cleavage Analysis of UhpA-Binding Sites in the
Escherichia coli uhpT
Promoter
Authors: Olekhnovich Igor N. , Kadner Robert J.
Published: July 2002

UhpA, a member of the NarL family of response regulators, activates transcription of the
Escherichia coli uhpT
gene for the sugar phosphate transporter UhpT in response to extracellular glucose-6-phosphate. UhpA binds with different affinities to adjacent regions in the
uhpT
promoter, termed the strong-binding (S) region from −80 to −50 and the weak-binding (W) region from −50 to −32. Transcription activation by UhpA is stimulated by the catabolite gene activator protein (CAP)-cyclic AMP complex and depends on the C-terminal domains of the RNA polymerase RpoA and RpoD subunits. Because single-base substitutions in the UhpA-binding region had little effect on promoter activity, nucleotide substitutions in successive 4-bp blocks throughout this region were examined for their effects on promoter activation and UhpA binding. Changes in three of four blocks within the W region substantially impaired the ability of UhpA to bind to this region, to drive expression of a
uhpT-lacZ
reporter, and to support UhpA-dependent in vitro transcription. These W region variant promoters were strongly stimulated by CAP. Changes in several parts of the S region impaired UhpA binding to both the S and W regions and decreased promoter activity in vivo and in vitro. Thus, binding of UhpA to the W region is crucial for UhpA-dependent activation and depends on occupancy of the S region. None of these substitutions eliminated promoter function. The orientation of UhpA-binding sites was assessed by the affinity cleavage method. The iron chelate FeBABE [iron (
S
)-1-(
p
-bromoacetamidobenzyl) EDTA] was covalently attached to engineered cysteine residues near the DNA-binding region in UhpA. Hydroxyl radicals generated by the iron chelate attached at position 187 resulted in DNA strand cleavages in two clusters of sites located in the middle of the S and W regions. These results are consistent with the binding of two dimers of UhpA. Each dimer binds to an inverted repeat of monomer-binding sites with the consensus sequence CCTGRR, where R is A or G, and each is separated by 6 bp. It is likely that members of the NarL family bind to dyad targets, in contrast to the binding of OmpR family response regulators to direct-repeat targets.
Activation of Prophage
eib
Genes for Immunoglobulin-Binding Proteins by Genes from the IbrAB Genetic Island of
Escherichia coli
ECOR-9
Authors: Sandt Carol H. , Hopper James E. , Hill Charles W.
Published: July 2002

Four distinct
Escherichia coli
immunoglobulin-binding (
eib
) genes, each of which encodes a surface-exposed protein that binds immunoglobulins in a nonimmune manner, are carried by separate prophages in
E. coli
reference (ECOR) strain ECOR-9. Each
eib
gene was transferred to test
E. coli
strains, both in the form of multicopy recombinant plasmids and as lysogenized prophage. The derived lysogens express little or no Eib protein, in sharp contrast to the parental lysogen, suggesting that ECOR-9 has an expression-enhancing activity that the derived lysogens lack. Supporting this hypothesis, we cloned from ECOR-9 overlapping genes,
ibrA
and
ibrB
(designation is derived from “immunoglobulin-binding regulator”), which together activated
eib
expression in the derived lysogens. The proteins encoded by
ibrA
and
ibrB
are very similar to uncharacterized proteins encoded by genes of
Salmonella enterica
serovar Typhi and
E. coli
O157:H7 (in a prophage-like element of the Sakai strain and in two O islands of strain EDL933). The genomic segment containing
ibrA
and
ibrB
has been designated the IbrAB island. It contains regions of homology to the Shiga toxin-converting prophage, Stx2, as well as genes homologous to phage antirepressor genes. The left boundary between the IbrAB island and the chromosomal framework is located near min 35.8 of the
E. coli
K-12 genome. Homology to IbrAB was found in certain other ECOR strains, including the other five
eib
-positive strains and most strains of the phylogenetic group B2. Sequencing of a 1.1-kb portion of
ibrAB
revealed that the other
eib
-positive strains diverge by ≤0.1% from ECOR-9, whereas
eib
-negative ECOR-47 diverges by 16%.
Gene Transfer in
Mycoplasma pulmonis
Authors: Teachman Amy M. , French C. Todd , Yu Huilan , Simmons Warren L. , Dybvig Kevin
Published: July 2002

Experiments were undertaken to examine gene transfer in
Mycoplasma pulmonis
. Parent strains containing transposon-based tetracycline and chloramphenicol resistance markers were combined to allow transfer of markers. Two mating protocols were developed. The first consisted of coincubating the strains in broth culture for extended periods of time. The second protocol consisted of a brief incubation of the combined strains in a 50% solution of polyethylene glycol. Using either protocol, progeny that had acquired antibiotic resistance markers from both parents were obtained. Analysis of the progeny indicated that only the transposon and not flanking genomic DNA was transferred to the recipient cell. Gene transfer was DNase resistant and probably the result of conjugation or cell fusion.
Analyses of the Roles of the Three
cheA
Homologs in Chemotaxis of
Vibrio cholerae
Authors: Gosink Khoosheh K. , Kobayashi Reiji , Kawagishi Ikuro , Häse Claudia C.
Published: July 2002

The
Vibrio cholerae
genome revealed the presence of multiple sets of chemotaxis genes, including three
cheA
gene homologs. We found that the
cheA
-2, but not
cheA
-1 or
cheA
-3, gene is essential for chemotaxis under standard conditions. Loss of chemotaxis had no effect on virulence factor expression in vitro.
Complete Genomic Sequence of SfV, a Serotype-Converting Temperate Bacteriophage of
Shigella
flexneri
Authors: Allison Gwen E. , Angeles Dario , Tran-Dinh Nai , Verma Naresh K.
Published: July 2002

Bacteriophage SfV is a temperate serotype-converting phage of
Shigella
flexneri.
SfV encodes the factors involved in type V O-antigen modification, and the serotype conversion and integration-excision modules of the phage have been isolated and characterized. We now report on the complete sequence of the SfV genome (37,074 bp). A total of 53 open reading frames were predicted from the nucleotide sequence, and analysis of the corresponding proteins was used to construct a functional map. The general organization of the genes in the SfV genome is similar to that of bacteriophage λ, and numerous features of the sequence are described. The superinfection immunity system of SfV includes a lambda-like repression system and a P4-like transcription termination mechanism. Sequence analysis also suggests that SfV encodes multiple DNA methylases, and experiments confirmed that
orf-41
encodes a Dam methylase. Studies conducted to determine if the phage-encoded methylase confers host DNA methylation showed that the two
S.
flexneri
strains analyzed encode their own Dam methylase. Restriction mapping and sequence analysis revealed that the phage genome has
cos
sites at the termini. The tail assembly and structural genes of SfV show homology to those of phage Mu and Mu-like prophages in the genome of
Escherichia
coli
O157:H7 and
Haemophilus
influenzae
. Significant homology (30% of the genome in total) between sections of the early, regulatory, and structural regions of the SfV genome and the e14 and KpLE1 prophages in the
E.
coli
K-12 genome were noted, suggesting that these three phages have common evolutionary origins.
Novel Carbohydrate-Binding Module of β-1,3-Xylanase from a Marine Bacterium,
Alcaligenes
sp. Strain XY-234
Authors: Okazaki Fumiyoshi , Tamaru Yutaka , Hashikawa Shinnosuke , Li Yu-Teh , Araki Toshiyoshi
Published: July 2002

A β-1,3-xylanase gene (
txyA
) from a marine bacterium,
Alcaligenes
sp. strain XY-234, has been cloned and sequenced.
txyA
consists of a 1,410-bp open reading frame that encodes 469 amino acid residues with a calculated molecular mass of 52,256 Da. The domain structure of the β-1,3-xylanase (TxyA) consists of a signal peptide of 22 amino acid residues, followed by a catalytic domain which belongs to family 26 of the glycosyl hydrolases, a linker region with one array of DGG and six repeats of DNGG, and a novel carbohydrate-binding module (CBM) at the C terminus. The recombinant TxyA hydrolyzed β-1,3-xylan but not other polysaccharides such as β-1,4-xylan, carboxymethylcellulose, curdlan, glucomannan, or β-1,4-mannan. TxyA was capable of binding specifically to β-1,3-xylan. The analysis using truncated TxyA lacking either the N- or C-terminal region indicated that the region encoding the CBM was located between residues 376 and 469. Binding studies on the CBM revealed that the

K
d

and the maximum amount of protein bound to β-1,3-xylan were 4.2 μM and 18.2 μmol/g of β-1,3-xylan, respectively. Furthermore, comparison of the enzymatic properties between proteins with and without the CBM strongly indicated that the CBM of TxyA plays an important role in the hydrolysis of β-1,3-xylan.