bluuebunny/crossref_metadata_2025_split huggingface dataset:
An Essential Two-Component Signal Transduction System in
Mycobacterium tuberculosis
Authors: Zahrt Thomas C. , Deretic Vojo 
Published: July 2002

ABSTRACT

The bacterial two-component signal transduction systems regulate adaptation processes and are likely to play a role in
Mycobacterium tuberculosis
physiology and pathogenesis. The previous initial characterization of an
M. tuberculosis
response regulator from one of these systems,
mtrA-mtrB
, suggested its transcriptional activation during infection of phagocytic cells. In this work, we further characterized the
mtrA
response regulator from
M. tuberculosis
H37Rv. Inactivation of
mtrA
on the chromosome of
M. tuberculosis
H37Rv was possible only in the presence of plasmid-borne functional
mtrA
, suggesting that this response regulator is essential for
M. tuberculosis
viability. In keeping with these findings, expression of
mtrA
in
M. tuberculosis
H37Rv was detectable during in vitro growth, as determined by S1 nuclease protection and primer extension analyses of mRNA levels and mapping of transcript 5′ ends. The
mtrA
gene was expressed differently in virulent
M. tuberculosis
and the vaccine strain
M. tuberculosis
var.
bovis
BCG during infection of macrophages, as determined by monitoring of
mtrA-gfp
fusion activity. In
M. bovis
BCG,
mtrA
was induced upon entry into macrophages. In
M. tuberculosis
H37Rv, its expression was constitutive and unchanged upon infection of murine or human monocyte-derived macrophages. In conclusion, these results identify
mtrA
as an essential response regulator gene in
M. tuberculosis
which is differentially expressed in virulent and avirulent strains during growth in macrophages.
Metabolic Defects Caused by Mutations in the
isc
Gene Cluster in
Salmonella enterica
Serovar Typhimurium: Implications for Thiamine Synthesis
Authors: Skovran Elizabeth , Downs Diana M. 
Published: July 2002

ABSTRACT

The metabolic consequences of two insertions,
iscR1
::MudJ and
iscA2
::MudJ, in the
isc
gene cluster of
Salmonella enterica
serovar Typhimurium were studied. Each of these insertions had polar effects and caused a nutritional requirement for the thiazole moiety of thiamine. Data showed that IscS was required for the synthesis of nicotinic acid and the thiazole moiety of thiamine and that one or more additional
isc
gene products were required for a distinct step in the thiazole biosynthetic pathway. Strains with
isc
lesions had reduced succinate dehydrogenase and aconitase activities. Furthermore,
isc
mutants accumulated increased levels of pyruvate in the growth medium in response to exogenously added iron (FeCl
3
), and this response required a functional ferric uptake regulator, Fur.
Genome Structure of the GenusAzospirillum
Authors: Martin-Didonet Claudia C. G. , Chubatsu Leda S. , Souza Emanuel M. , Kleina Margareth , Rego Fabiane G. M. , Rigo Liu U. , Yates M. Geoffrey , Pedrosa Fabio O. 
Published: July 2002

ABSTRACTAzospirillumspecies are plant-associated diazotrophs of the alpha subclass ofProteobacteria. The genomes of five of the sixAzospirillumspecies were analyzed by pulsed-field gel electrophoresis. All strains possessed several megareplicons, some probably linear, and 16S ribosomal DNA hybridization indicated multiple chromosomes in genomes ranging in size from 4.8 to 9.7 Mbp. ThenifHDKoperon was identified in the largest replicon.
Positive Selection for Mutations Affecting Bioconversion of Aromatic Compounds inAgrobacterium tumefaciens: Analysis of Spontaneous Mutations in the Protocatechuate 3,4-Dioxygenase Gene
Authors: Parke Donna 
Published: July 2002

ABSTRACTA positive selection method for mutations affecting bioconversion of aromatic compounds was applied to a mutant strain ofAgrobacterium tumefaciensA348. The nucleotide sequence of the A348pcaHGBgenes, which encode protocatechuate 3,4-dioxygenase (PcaHG) and β-carboxy-cis,cis-muconate cycloisomerase (PcaB) for the first two steps in catabolism of the diphenolic protocatechuate, was determined. An omega element was introduced into thepcaBgene of A348, creating strain ADO2077. In the presence of phenolic compounds that can serve as carbon sources, growth of ADO2077 is inhibited due to accumulation of the tricarboxylate intermediate. The toxic effect, previously described forAcinetobactersp., affords a powerful selection for suppressor mutations in genes required for upstream catabolic steps. By monitoring loss of the marker inpcaB, it was possible to determine that the formation of deletions was minimal compared to results obtained withAcinetobactersp. Thus, the tricarboxylic acid trick in and of itself does not appear to select for large deletion mutations. The power of the selection was demonstrated by targeting thepcaHGgenes ofA. tumefaciensfor spontaneous mutation. Sixteen strains carrying putative second-site mutations inpcaHor -Gwere subjected to sequence analysis. All single-site events, their mutations revealed no particular bias toward multibase deletions or unusual patterns: five (−1) frameshifts, one (+1) frameshift, one tandem duplication of 88 bp, one deletion of 92 bp, one nonsense mutation, and seven missense mutations. PcaHG is considered to be the prototypical ferric intradiol dioxygenase. The missense mutations served to corroborate the significance of active site amino acid residues deduced from crystal structures of PcaHG fromPseudomonas putidaandAcinetobactersp. as well as of residues in other parts of the enzyme.
Escherichia coliTehB RequiresS-Adenosylmethionine as a Cofactor To Mediate Tellurite Resistance
Authors: Liu Mingfu , Turner Raymond J. , Winstone Tara L. , Saetre Andrea , Dyllick-Brenzinger Melanie , Jickling Glen , Tari Leslie W. , Weiner Joel H. , Taylor Diane E. 
Published: July 2002

ABSTRACTTheEscherichia colichromosomal determinant for tellurite resistance consists of two genes (tehAandtehB) which, when expressed on a multicopy plasmid, confer resistance to K2TeO3at 128 μg/ml, compared to the MIC of 2 μg/ml for the wild type. TehB is a cytoplasmic protein which possesses three conserved motifs (I, II, and III) found inS-adenosyl-l-methionine (SAM)-dependent non-nucleic acid methyltransferases. Replacement of the conserved aspartate residue in motif I by asparagine or alanine, or of the conserved phenylalanine in motif II by tyrosine or alanine, decreased resistance to background levels. Our results are consistent with motifs I and II in TehB being involved in SAM binding. Additionally, conformational changes in TehB are observed upon binding of both tellurite and SAM. The hydrodynamic radius of TehB measured by dynamic light scattering showed a ∼20% decrease upon binding of both tellurite and SAM. These data suggest that TehB utilizes a methyltransferase activity in the detoxification of tellurite.
A Set of Genes Encoding a Second Toluene Efflux System in
Pseudomonas putida
DOT-T1E Is Linked to the
tod
Genes for Toluene Metabolism
Authors: Mosqueda Gilberto , Ramos Juan-Luis 
Published: July 2002

ABSTRACT

Sequence analysis in
Pseudomonas putida
DOT-T1E revealed a second toluene efflux system for toluene metabolism encoded by the
ttgDEF
genes, which are adjacent to the
tod
genes. The
ttgDEF
genes were expressed in response to the presence of aromatic hydrocarbons such as toluene and styrene in the culture medium. To characterize the contribution of the TtgDEF system to toluene tolerance in
P. putida
, site-directed mutagenesis was used to knock out the gene in the wild-type DOT-T1E strain and in a mutant derivative, DOT-T1E-18. This mutant carried a Tn
5
insertion in the
ttgABC
gene cluster, which encodes a toluene efflux pump that is synthesized constitutively. For site-directed mutagenesis, a cassette to knock out the
ttgD
gene and encoding resistance to tellurite was constructed in vitro and transferred to the corresponding host chromosome via the suicide plasmid pKNG101. Successful replacement of the wild-type sequences with the mutant cassette was confirmed by Southern hybridization. A single
ttgD
mutant, DOT-T1E-1, and a double mutant with knock outs in the
ttgD
and
ttgA
genes, DOT-T1E-82, were obtained and characterized for toluene tolerance. This was assayed by the sudden addition of toluene (0.3% [vol/vol]) to the liquid culture medium of cells growing on Luria-Bertani (LB) medium (noninduced) or on LB medium with toluene supplied via the gas phase (induced). Induced cells of the single
ttgD
mutant were more sensitive to sudden toluene shock than were the wild-type cells; however, noninduced wild-type and
ttgD
mutant cells were equally tolerant to toluene shock. Noninduced cells of the double DOT-T1E-82 mutant did not survive upon sudden toluene shock; however, they still remained viable upon sudden toluene shock if they had been previously induced. These results are discussed in the context of the use of multiple efflux pumps involved in solvent tolerance in
P. putida
DOT-T1E.
Evidence for Na+Influx via the NtpJ Protein of the KtrII K+Uptake System inEnterococcus hirae
Authors: Kawano Miyuki , Abuki Ryoko , Igarashi Kazuei , Kakinuma Yoshimi 
Published: July 2002

ABSTRACTThentpJgene, a cistron located at the tail end of the vacuolar-type Na+-ATPase (ntp) operon ofEnterococcus hirae, encodes a transporter of the KtrII K+uptake system. We found that K+accumulation in thentpJ-disrupted mutant JEM2 was markedly enhanced by addition of valinomycin at pH 10. Studies of the membrane potential (ΔΨ; inside negative) by 3,3′-dihexyloxacarbocyanine iodide fluorescence revealed that the ΔΨ was hyperpolarized at pH 10 in JEM2; the ΔΨ values of the parent strain ATCC 9790 and JEM2, estimated by determining the equilibrium distribution of K+or Rb+in the presence of valinomycin, were −118 and −160 mV, respectively. ΔΨ generation at pH 10 was accomplished by an electrogenic Na+efflux via the Na+-ATPase, whose levels in the two strains were quite similar. Na+uptake driven by an artificially imposed ΔΨ (inside negative) was missing in JEM2, suggesting that NtpJ mediates Na+movement in addition to K+movement. Finally, the growth of JEM2 arrested in K+-limited high-Na+medium at pH 10 was restored by addition of valinomycin. These results suggest that NtpJ mediates electrogenic transport of K+as well as Na+, that it likely mediates K+and Na+cotransport, and that Na+movement via NtpJ is the major Na+reentry pathway at high pH values.
Improvement in K+-Limited Growth Rate Associated with Expression of the N-Terminal Fragment of One Subunit (KdpA) of the Multisubunit Kdp Transporter inEscherichia coli
Authors: Sardesai Abhijit A. , Gowrishankar J. 
Published: July 2002

ABSTRACTMutations in any one of three genes,kdpA, -B,or -C,inEscherichia coliabolish the activity of Kdp, a multisubunit K+-ATPase that belongs to the P-type ATPase family of cation transporters. We found in this study that expression in vivo of a 135-amino-acid-long N-terminal fragment (KdpA′), less than one-quarter the length of native KdpA, was able to mediate an improvement in K+-limited growth rates in two different contexts, even in the absence of both KdpC and the ATPase subunit KdpB. The first context was when KdpA′ was overexpressed in cells from a heterologous inducible promoter, and the second was when KdpA′ was provided with a C-terminally altered extension (following a spontaneous genetic rearrangement). Our results suggest that KdpA′ provides an incipient pathway for K+translocation which can serve to transport K+into the cells in response to the cytoplasmic membrane potential.
Characterization of theUstilago maydis sid2Gene, Encoding a Multidomain Peptide Synthetase in the Ferrichrome Biosynthetic Gene Cluster
Authors: Yuan Walter M. , Gentil Guillaume D. , Budde Allen D. , Leong Sally A. 
Published: July 2002

ABSTRACTUstilago maydis, the causal agent of corn smut disease, acquires and transports ferric ion by producing the extracellular, cyclic peptide, hydroxamate siderophores ferrichrome and ferrichrome A. Ferrichrome biosynthesis likely proceeds by hydroxylation and acetylation ofl-ornithine, and later steps likely involve covalently bound thioester intermediates on a multimodular, nonribosomal peptide synthetase.sid1encodesl-ornithineN5-oxygenase, which catalyzes hydroxylation ofl-ornithine, the first committed step of ferrichrome and ferrichrome A biosynthesis inU. maydis. In this report we characterizesid2, another biosynthetic gene in the pathway, by gene complementation, gene replacement, DNA sequence, and Northern hybridization analysis. Nucleotide sequencing has revealed thatsid2is located 3.7 kb upstream ofsid1and encodes an intronless polypeptide of 3,947 amino acids with three iterated modules of an approximate length of 1,000 amino acids each. Multiple motifs characteristic of the nonribosomal peptide synthetase protein family were identified in each module. A corresponding iron-regulatedsid2transcript of 11 kb was detected by Northern hybridization analysis. By contrast, constitutive accumulation of this large transcript was observed in a mutant carrying a disruption ofurbs1, a zinc finger, GATA family transcription factor previously shown to regulate siderophore biosynthesis inUstilago. Multiple GATA motifs are present in the intergenic region betweensid1andsid2, suggesting bidirectional transcription regulation byurbs1of this pathway. Indeed, mutation of two of these motifs, known to be important to regulation ofsid1, altered the differential regulation ofsid2by iron.
Juxtaposition of an Active Promoter to
vsp
Genes via Site-Specific DNA Inversions Generates Antigenic Variation in
Mycoplasma bovis
Authors: Lysnyansky Innesa , Ron Yael , Yogev David 
Published: July 2002

ABSTRACT

Mycoplasma bovis
, the most important etiological agent of bovine mycoplasmosis, undergoes extensive antigenic variation of major and highly immunogenic surface lipoprotein antigens (Vsps). A family of 13 related but divergent
vsp
genes, which occur as single chromosomal copies, was recently found in the chromosome of
M. bovis
. In the present study, the molecular mechanism mediating the high-frequency phase variation of two Vsps (VspA and VspC) as representatives of the Vsp family was investigated. Analysis of clonal isolates exhibiting phase transitions of VspA or of VspC (i.e., ON→OFF→ON) has shown that DNA inversions occur during Vsp phase variation. The upstream region of each
vsp
gene contains two sequence cassettes. The first (cassette no. 1), a 71-bp region upstream of the ATG initiation codon, exhibits 98% homology among all
vsp
genes, while the second (cassette no. 2), upstream of cassette no. 1, ranges in size from 50 to 180 bp and is more divergent. Examination of the ends of the inverted fragments during VspA or VspC phase variation revealed that in both cases, a change in the organization of
vsp
upstream cassettes involving three
vsp
genes had occurred. Primer extension and Northern blot analysis have shown that a specific cassette no. 2, designated A
2
, is an active promoter and that juxtaposition of this regulatory element to a silent
vsp
gene by DNA inversions allows transcription initiation of the recipient gene. Further genetic analysis revealed that phase variation of VspA or of VspC involves two site-specific DNA inversions occurring between inverted copies of a specific 35-bp sequence present within the conserved cassette no. 1. A model for the control of Vsp phase variation is proposed.
Pseudomonas aeruginosa
Exhibits Directed Twitching Motility Up Phosphatidylethanolamine Gradients
Authors: Kearns Daniel B. , Robinson Jayne , Shimkets Lawrence J. 
Published: July 2002

ABSTRACT

Pseudomonas aeruginosa
translocates over solid surfaces by a type IV pilus-dependent form of multicellular motility known as twitching. We wondered whether cells utilize endogenous factors to organize twitching, and we purified from wild-type cells a lipid that caused directed movement. Wild-type
P. aeruginosa
, but not a
pilJ
pilus-deficient mutant, showed biased movement up gradients of phosphatidylethanolamine (PE) established in agar. Activity was related to the fatty acid composition of the lipid, as two synthetic PE species, dilauroyl and dioleoyl PE, were capable of directing
P. aeruginosa
motility while many other species were inactive.
P. aeruginosa
PE did not contain either laurate or oleate, implying that the native attractant species contains different fatty acids. Uniform concentrations of PE increased cell velocity, suggesting that chemokinesis may be at least partly responsible for directed movement. We speculate that PE-directed twitching motility may be involved in biofilm formation and pathogenesis.
Functional Elements of the Strong
psbAI
Promoter of
Synechococcus elongatus
PCC 7942
Authors: Nair Usha , Thomas Colleen , Golden Susan S. 
Published: July 2002

ABSTRACT

The
psbAI
gene of the cyanobacterium
Synechococcus elongatus
PCC 7942 is one of three
psbA
genes that encode a critical photosystem II reaction center protein, D1. Regulation of the gene family in response to changes in the light environment is complex, occurs at transcriptional and posttranscriptional levels, and results in an interchange of two different forms of D1 in the membrane. Expression of
psbAI
is downregulated under high-intensity light (high light) in contrast to induction of the other two family members. We show that, in addition to a known accelerated degradation of the
psbAI
message, promoter activity decreases upon exposure to high light. Unlike the other
psbA
genes, additional sequences upstream of the
psbAI
−35 element are required for expression. Mutagenizing the atypical
psbAI
−10 element from TCTCCT to TATAAT increased the magnitude of expression from both
psbAI
::
lacZ
and
psbAI
::
luxAB
fusions but did not affect downregulation under high light. Inactivation of group 2 sigma factor genes
rpoD2
and
sigC
, in both wild-type and −10-element mutagenized backgrounds, resulted in elevated
psbAI
::
luxAB
expression but did not alter the response to high light. The results are consistent with redundancy of promoter recognition among cyanobacterial group 2 sigma factors. Electrophoretic mobility shift assays showed that the DNA sequence corresponding to the untranslated leader of the
psbAI
message binds one or more proteins from an
S. elongatus
extract. The corresponding region of
psbAII
efficiently competed for this binding activity, suggesting a shared regulatory factor among these disparately regulated genes.
Involvement of a CbbR Homolog in Low CO
2
-Induced Activation of the Bicarbonate Transporter Operon in Cyanobacteria
Authors: Omata Tatsuo , Gohta Satoshi , Takahashi Yukari , Harano Yoshimi , Maeda Shin-ichi 
Published: July 2002

ABSTRACT

The
cmpABCD
operon of
Synechococcus
sp. strain PCC 7942, encoding a high-affinity bicarbonate transporter, is transcribed only under CO
2
-limited conditions. In
Synechocystis
sp. strain PCC 6803, the
slr0040, slr0041, slr0043
, and
slr0044
genes, forming an operon with a putative porin gene (
slr0042
), were identified as the
cmpA, cmpB, cmpC
, and
cmpD
genes, respectively, on the basis of their strong similarities to the corresponding
Synechococcus cmp
genes and their induction under low CO
2
conditions. Immediately upstream of and transcribed divergently from the
Synechocystis cmp
operon is a gene (
sll0030
) encoding a homolog of CbbR, a LysR family transcriptional regulator of the CO
2
fixation operons of chemoautotrophic and purple photosynthetic bacteria. Inactivation of
sll0030
, but not of another closely related
cbbR
homolog (
sll1594
), abolished low CO
2
induction of
cmp
operon expression. Gel retardation assays showed specific binding of the Sll0030 protein to the
sll0030-cmpA
intergenic region, suggesting that the protein activates transcription of the
cmp
operon by interacting with its regulatory region. A
cbbR
homolog similar to
sll0030
and
sll1594
was cloned from
Synechococcus
sp. strain PCC 7942 and shown to be involved in the low CO
2
-induced activation of the
cmp
operon. We hence designated the
Synechocystis sll0030
gene and the
Synechococcus cbbR
homolog
cmpR
. In the mutants of the
cbbR
homologs, upregulation of ribulose-1,5-bisphosphate carboxylase/oxygenase operon expression by CO
2
limitation was either unaffected (strain PCC 6803) or enhanced (strain PCC 7942), suggesting existence of other low CO
2
-responsive transcriptional regulator(s) in cyanobacteria.
Involvement of Superoxide Dismutases in the Response ofEscherichia colito Selenium Oxides
Authors: Bébien Magali , Lagniel Gilles , Garin Jérôme , Touati Danièle , Verméglio André , Labarre Jean 
Published: July 2002

ABSTRACTSelenium can provoke contrasting effects on living organisms. It is an essential trace element, and low concentrations have beneficial effects, such as the reduction of the incidence of cancer. However, higher concentrations of selenium salts can be toxic and mutagenic. The bases for both toxicity and protection are not clearly understood. To provide insights into these mechanisms, we analyzed the proteomic response ofEscherichia colicells to selenate and selenite treatment under aerobic conditions. We identified 23 proteins induced by both oxides and ca. 20 proteins specifically induced by each oxide. A striking result was the selenite induction of 8 enzymes with antioxidant properties, particularly the manganese and iron superoxide dismutases (SodA and SodB). The selenium inductions ofsodAandsodBwere controlled by the transcriptional regulators SoxRS and Fur, respectively. Strains with decreased superoxide dismutase activities were severely impaired in selenium oxide tolerance. Pretreatment with a sublethal selenite concentration triggered an adaptive response dependent upon SoxRS, conferring increased selenite tolerance. Altogether, our data indicate that superoxide dismutase activity is essential for the cellular defense against selenium salts, suggesting that superoxide production is a major mechanism of selenium toxicity under aerobic conditions.
Molecular Analysis of the Gene Encoding a Novel Chitin-Binding Protease fromAlteromonassp. Strain O-7 and Its Role in the Chitinolytic System
Authors: Miyamoto Katsushiro , Nukui Eiji , Itoh Hiroyuki , Sato Takaji , Kobayashi Takeshi , Imada Chiaki , Watanabe Etsuo , Inamori Yoshihiko , Tsujibo Hiroshi 
Published: July 2002

ABSTRACTAlteromonassp. strain O-7 secretes several proteins in response to chitin induction. We have found that one of these proteins, designated AprIV, is a novel chitin-binding protease involved in chitinolytic activity. The gene encoding AprIV (aprIV) was cloned inEscherichia coli. DNA sequencing analysis revealed that the open reading frame ofaprIVencoded a protein of 547 amino acids with a calculated molecular mass of 57,104 Da. AprIV is a modular enzyme consisting of five domains: the signal sequence, the N-terminal proregion, the family A subtilase region, the polycystic kidney disease domain (PkdD), and the chitin-binding domain type 3 (ChtBD3). Expression plasmids coding for PkdD or both PkdD and ChtBD (PkdD-ChtBD) were constructed. The PkdD-ChtBD but not PkdD exhibited strong binding to α-chitin and β-chitin. Western and Northern analyses demonstrated thataprIVwas induced in the presence ofN-acetylglucosamine,N-acetylchitobiose, or chitin. Native AprIV was purified to homogeneity fromAlteromonassp. strain O-7 and characterized. The molecular mass of mature AprIV was estimated to be 44 kDa by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The optimum pH and temperature of AprIV were pH 11.5 and 35°C, respectively, and even at 10°C the enzyme showed 25% of the maximum activity. Pretreatment of native chitin with AprIV significantly promoted chitinase activity.