bluuebunny/crossref_metadata_2025_split huggingface dataset:
SarA Represses
agr
Operon Expression in a Purified In Vitro
Staphylococcus aureus
Transcription System
Authors: Chakrabarti Swarup K. , Misra Tapan K. 
Published: July 2002

ABSTRACT

Mutation and genetic complementation studies suggested that two chromosomal loci,
agr
and
sar
, are involved in the upregulation of several exotoxin genes and the downregulation of a number of surface protein genes in a growth phase-dependent manner in
Staphylococcus aureus
. We purified recombinant T7-tagged SarA from
Escherichia coli
and determined its effect on transcription from several
S. aureus
promoters by using purified RNA polymerase reconstituted with either ς
A
or ς
B
from
S. aureus
. Of the seven ς
A
-dependent promoters that we tested, SarA repressed transcription from
agr
P2,
agr
P3,
cna
,
sar
P1, and
sea
promoters and did not affect
sec
and
znt
promoters. Furthermore, SarA had no effect on transcription from the ς
B
-dependent
sar
P3 promoter. In vitro experimental data presented in this report suggest that SarA expression is autoregulated.
Aerobic Activity of
Escherichia coli
Alcohol Dehydrogenase Is Determined by a Single Amino Acid
Authors: Holland-Staley Carol A. , Lee KangSeok , Clark David P. , Cunningham Philip R. 
Published: July 2002

ABSTRACT

Expression of the alcohol dehydrogenase gene,
adhE
, in
Escherichia coli
is anaerobically regulated at both the transcriptional and the translational levels. To study the AdhE protein, the
adhE
+
structural gene was cloned into expression vectors under the control of the
lacZ
and
trp
c
promoters. Wild-type AdhE protein produced under aerobic conditions from these constructs was inactive. Constitutive mutants (
adhC
) that produced high levels of AdhE under both aerobic and anaerobic conditions were previously isolated. When only the
adhE
structural gene from one of the
adhC
mutants was cloned into expression vectors, highly functional AdhE protein was isolated under both aerobic and anaerobic conditions. Sequence analysis revealed that the
adhE
gene from the
adhC
mutant contained two mutations resulting in two amino acid substitutions, Ala267Thr and Glu568Lys. Thus,
adhC
strains contain a promoter mutation and two mutations in the structural gene. The mutant structural gene from
adhC
strains was designated
adhE
*. Fragment exchange experiments revealed that the substitution responsible for aerobic expression in the
adhE
* clones is Glu568Lys. Genetic selection and site-directed mutagenesis experiments showed that virtually any amino acid substitution for Glu568 produced AdhE that was active under both aerobic and anaerobic conditions. These findings suggest that
adhE
expression is also regulated posttranslationally and that strict regulation of alcohol dehydrogenase activity in
E. coli
is physiologically significant.
Second-Site Suppressor Mutations of Inactivating Substitutions at Gly247 of the Tetracycline Efflux Protein, Tet(B)
Authors: Saraceni-Richards Cynthia A. , Levy Stuart B. 
Published: July 2002

ABSTRACT
An Asp or Asn substitution for Gly247 in transmembrane helix 8 (TM-8) of Tet(B), the tetracycline efflux protein, eliminated tetracycline resistance. Second site suppressor mutations which partially restored resistance were located in TM-5, -8, -10, or -11 or in cytoplasmic loop 8-9 or loop 10-11. These results indicate physical proximity or functional relationships between TM-8 and these other regions of Tet(B).
Interspecies Complementation of
Escherichia coli ccm
Mutants: CcmE (CycJ) from
Bradyrhizobium japonicum
Acts as a Heme Chaperone during Cytochrome
c
Maturation
Authors: Schulz Henk , Thöny-Meyer Linda 
Published: July 2002

ABSTRACT

Biogenesis of
c
-type cytochromes in α- and γ-proteobacteria requires the function of a set of orthologous genes (
ccm
genes) that encode specific maturation factors. The
Escherichia coli
CcmE protein is a periplasmic heme chaperone. The membrane protein CcmC is required for loading CcmE with heme. By expressing CcmE (CycJ) from
Bradyrhizobium japonicum
in
E. coli
we demonstrated that heme is bound covalently to this protein at a strictly conserved histidine residue. The
B. japonicum
homologue can transfer heme to apocytochrome
c
in
E. coli
, suggesting that it functions as a heme chaperone. CcmC (CycZ) from
B. japonicum
expressed in
E. coli
was capable of inserting heme into CcmE.
TheSaccharomyces cerevisiae ICL2Gene Encodes a Mitochondrial 2-Methylisocitrate Lyase Involved in Propionyl-Coenzyme A Metabolism
Authors: Luttik Marijke A. H. , Kötter Peter , Salomons Florian A. , van der Klei Ida J. , van Dijken Johannes P. , Pronk Jack T. 
Published: July 2002

ABSTRACTTheSaccharomyces cerevisiae ICL1gene encodes isocitrate lyase, an essential enzyme for growth on ethanol and acetate. Previous studies have demonstrated that the highly homologousICL2gene (YPR006c) is transcribed during the growth of wild-type cells on ethanol. However, even when multiple copies are introduced,ICL2cannot complement the growth defect oficl1null mutants. It has therefore been suggested thatICL2encodes a nonsense mRNA or nonfunctional protein. In the methylcitrate cycle of propionyl-coenzyme A metabolism, 2-methylisocitrate is converted to succinate and pyruvate, a reaction similar to that catalyzed by isocitrate lyase. To investigate whetherICL2encodes a specific 2-methylisocitrate lyase, isocitrate lyase and 2-methylisocitrate lyase activities were assayed in cell extracts of wild-typeS. cerevisiaeand of isogenicicl1,icl2, andicl1 icl2null mutants. Isocitrate lyase activity was absent inicl1andicl1 icl2null mutants, whereas in contrast, 2-methylisocitrate lyase activity was detected in the wild type and singleiclmutants but not in theicl1 icl2mutant. This demonstrated thatICL2encodes a specific 2-methylisocitrate lyase and that theICL1-encoded isocitrate lyase exhibits a low but significant activity with 2-methylisocitrate. Subcellular fractionation studies and experiments with an ICL2-green fluorescent protein fusion demonstrated that theICL2-encoded 2-methylisocitrate lyase is located in the mitochondrial matrix. Similar to that ofICL1, transcription ofICL2is subject to glucose catabolite repression. In glucose-limited cultures, growth with threonine as a nitrogen source resulted in a ca. threefold induction ofICL2mRNA levels and of 2-methylisocitrate lyase activity in cell extracts relative to cultures grown with ammonia as the nitrogen source. This is consistent with an involvement of the 2-methylcitrate cycle in threonine catabolism.
ModE-Dependent Molybdate Regulation of the Molybdenum Cofactor OperonmoainEscherichia coli
Authors: Anderson Lisa A. , McNairn Elizabeth , Leubke Torben , Pau Richard N. , Boxer David H. 
Published: July 2002

ABSTRACTThe expression of themoalocus, which encodes enzymes required for molybdopterin biosynthesis, is enhanced under anaerobiosis but repressed when the bacterium is able to synthesize active molybdenum cofactor. In addition,moaexpression exhibits a strong requirement for molybdate. The molybdate enhancement ofmoatranscription is fully dependent upon the molybdate-binding protein, ModE, which also mediates molybdate repression of themodoperon encoding the high-affinity molybdate uptake system. Due to the repression ofmoain molybdenum cofactor-sufficient strains, the positive molybdate regulation ofmoais revealed only in strains unable to make the active cofactor. Transcription ofmoais controlled at two sigma-70-type promoters immediately upstream of themoaAgene. Deletion mutations covering the region upstream ofmoaAhave allowed each of the promoters to be studied in isolation. The distal promoter is the site of the anaerobic enhancement which is Fnr-dependent. The molybdate induction ofmoais exerted at the proximal promoter. Molybdate-ModE binds adjacent to the −35 region of this promoter, acting as a direct positive regulator ofmoa. The molybdenum cofactor repression also appears to act at the proximal transcriptional start site, but the mechanism remains to be established. Tungstate in the growth medium affectsmoaexpression in two ways. Firstly, it can act as a functional molybdate analogue for the ModE-mediated regulation. Secondly, tungstate brings about the loss of the molybdenum cofactor repression ofmoa. It is proposed that the tungsten derivative of the molybdenum cofactor, which is known to be formed under such conditions, is ineffective in bringing about repression ofmoa. The complex control ofmoais discussed in relation to the synthesis of molybdoenzymes in the bacterium.
Construction and Characterization of a
recA
Mutant of
Thiobacillus ferrooxidans
by Marker Exchange Mutagenesis
Authors: Liu Zhenying , Guiliani Nicolas , Appia-Ayme Corinne , Borne Françoise , Ratouchniak Jeanine , Bonnefoy Violaine 
Published: July 2002

ABSTRACT

To construct
Thiobacillus ferrooxidans
mutants by marker exchange mutagenesis, a genetic transfer system is required. The transfer of broad-host-range plasmids belonging to the incompatibility groups IncQ (pKT240 and pJRD215), IncP (pJB3Km1), and IncW (pUFR034) from
Escherichia coli
to two private
T. ferrooxidans
strains (BRGM1 and Tf-49) and to two collection strains (ATCC 33020 and ATCC 19859) by conjugation was analyzed. To knock out the
T. ferrooxidans recA
gene, a mobilizable suicide plasmid carrying the ATCC 33020
recA
gene disrupted by a kanamycin resistance gene was transferred from
E. coli
to
T. ferrooxidans
ATCC 33020 by conjugation under the best conditions determined. The two kanamycin-resistant clones, which have retained the kanamycin-resistant phenotype after growth for several generations in nonselective medium, were shown to have the kanamycin resistance gene inserted within the
recA
gene, indicating that the
rec
A::Ω-Km mutated allele was transferred from the suicide plasmid to the chromosome by homologous recombination. These mutants exhibited a slightly reduced growth rate and an increased sensitivity to UV and γ irradiation compared to the wild-type strain. However, the
T. ferrooxidans recA
mutants are less sensitive to these physical DNA-damaging agents than the
recA
mutants described in other bacterial species, suggesting that RecA plays a minor role in DNA repair in
T. ferrooxidans
.
Genetic Analysis of Functions Involved in Adhesion of
Pseudomonas putida
to Seeds
Authors: Espinosa-Urgel Manuel , Salido Amparo , Ramos Juan-Luis 
Published: July 2002

ABSTRACT

Many agricultural uses of bacteria require the establishment of efficient bacterial populations in the rhizosphere, for which colonization of plant seeds often constitutes a critical first step.
Pseudomonas putida
KT2440 is a strain that colonizes the rhizosphere of a number of agronomically important plants at high population densities. To identify the functions involved in initial seed colonization by
P. putida
KT2440, we subjected this strain to transposon mutagenesis and screened for mutants defective in attachment to corn seeds. Eight different mutants were isolated and characterized. While all of them showed reduced attachment to seeds, only two had strong defects in their adhesion to abiotic surfaces (glass and different plastics). Sequences of the loci affected in all eight mutants were obtained. None of the isolated genes had previously been described in
P. putida
, although four of them showed clear similarities with genes of known functions in other organisms. They corresponded to putative surface and membrane proteins, including a calcium-binding protein, a hemolysin, a peptide transporter, and a potential multidrug efflux pump. One other showed limited similarities with surface proteins, while the remaining three presented no obvious similarities with known genes, indicating that this study has disclosed novel functions.
The
chvH
Locus of
Agrobacterium
Encodes a Homologue of an Elongation Factor Involved in Protein Synthesis
Authors: Peng Wen-Tao , Banta Lois M. , Charles Trevor C. , Nester Eugene W. 
Published: July 2002

ABSTRACT

The virulence of
Agrobacterium tumefaciens
depends on both chromosome- and Ti plasmid-encoded gene products. In this study, we characterize a chromosomal locus,
chvH
, previously identified by Tn
phoA
mutagenesis and shown to be required for tumor formation. Through DNA sequencing and comparison of the sequence with identified sequences in the database, we show that this locus encodes a protein similar in sequence to elongation factor P, a protein thought to be involved in peptide bond synthesis in
Escherichia coli
. The analysis of
vir-lacZ
and
vir-phoA
translational fusions as well as Western immunoblotting revealed that the expression of Vir proteins such as VirE2 was significantly reduced in the
chvH
mutant compared with the wild-type strain. The
E. coli efp
gene complemented detergent sensitivity, virulence, and expression of VirE2 in the
chvH
mutant, suggesting that
chvH
and
efp
are functionally homologous. As expected, ChvH exerts its activity at the posttranscriptional level. Southern analysis suggests that the gene encoding this elongation factor is present as a single copy in
A. tumefaciens
. We constructed a
chvH
deletion mutant in which a 445-bp fragment within its coding sequence was deleted and replaced with an omega fragment. On complex medium, this mutant grew more slowly than the wild-type strain, indicating that elongation factor P is important but not essential for the growth of
Agrobacterium
.
Biosynthesis of the Glycolipid Anchor in Lipoteichoic Acid of
Staphylococcus aureus
RN4220: Role of YpfP, the Diglucosyldiacylglycerol Synthase
Authors: Kiriukhin Michael Y. , Debabov Dmitri V. , Shinabarger Dean L. , Neuhaus Francis C. 
Published: July 2002

ABSTRACT

In
Staphylococcus aureus
RN4220, lipoteichoic acid (LTA) is anchored in the membrane by a diglucosyldiacylglycerol moiety. The gene (
ypfP
) which encodes diglucosyldiacylglycerol synthase was recently cloned from
Bacillus subtilis
and expressed in
Escherichia coli
(P. Jorasch, F. P. Wolter, U. Zahringer, and E. Heinz, Mol. Microbiol. 29:419–430, 1998). To define the role of
ypfP
in this strain of
S. aureus
, a fragment of
ypfP
truncated from both ends was cloned into the thermosensitive replicon pVE6007 and used to inactivate
ypfP
. Chloramphenicol-resistant (
ypfP
::
cat
) clones did not synthesize the glycolipids monoglucosyldiacylglycerol and diglucosyldiacylglycerol. Thus, YpfP would appear to be the only diglucosyldiacylglycerol synthase in
S. aureus
providing glycolipid for LTA assembly. In LTA from the mutant, the glycolipid anchor is replaced by diacylglycerol. Although the doubling time of the mutant was identical to that of the wild type in Luria-Bertani (LB) medium, growth of the mutant in LB medium containing 1% glycine was not observed. This inhibition was antagonized by either
l
- or
d
-alanine. Moreover, viability of the mutant at 37°C in 0.05 M phosphate (pH 7.2)-saline for 12 h was reduced to <0.1%. Addition of 0.1%
d
-glucose to the phosphate-saline ensured viability under these conditions. The autolysis of the
ypfP
::
cat
mutant in the presence of 0.05% Triton X-100 was 1.8-fold faster than that of the parental strain. Electron microscopy of the mutant revealed not only a small increase in cell size but also the presence of pleomorphic cells. Each of these phenotypes may be correlated with either (or both) a deficiency of free glycolipid in the membrane or the replacement of the usual glycolipid anchor of LTA with diacylglycerol.
Ralstonia solanacearum
Needs Motility for Invasive Virulence on Tomato
Authors: Tans-Kersten Julie , Huang Huayu , Allen Caitilyn 
Published: July 2002

ABSTRACT

Ralstonia solanacearum
, a widely distributed and economically important plant pathogen, invades the roots of diverse plant hosts from the soil and aggressively colonizes the xylem vessels, causing a lethal wilting known as bacterial wilt disease. By examining bacteria from the xylem vessels of infected plants, we found that
R. solanacearum
is essentially nonmotile in planta, although it can be highly motile in culture. To determine the role of pathogen motility in this disease, we cloned, characterized, and mutated two genes in the
R. solanacearum
flagellar biosynthetic pathway. The genes for flagellin, the subunit of the flagellar filament (
fliC
), and for the flagellar motor switch protein (
fliM
) were isolated based on their resemblance to these proteins in other bacteria. As is typical for flagellins, the predicted FliC protein had well-conserved N- and C-terminal regions, separated by a divergent central domain. The predicted
R. solanacearum
FliM closely resembled motor switch proteins from other proteobacteria. Chromosomal mutants lacking
fliC
or
fliM
were created by replacing the genes with marked interrupted constructs. Since
fliM
is embedded in the
fliLMNOPQR
operon, the
aphA
cassette was used to make a nonpolar
fliM
mutation. Both mutants were completely nonmotile on soft agar plates, in minimal broth, and in tomato plants. The
fliC
mutant lacked flagella altogether; moreover, sheared-cell protein preparations from the
fliC
mutant lacked a 30-kDa band corresponding to flagellin. The
fliM
mutant was usually aflagellate, but about 10% of cells had abnormal truncated flagella. In a biologically representative soil-soak inoculation virulence assay, both nonmotile mutants were significantly reduced in the ability to cause disease on tomato plants. However, the
fliC
mutant had wild-type virulence when it was inoculated directly onto cut tomato petioles, an inoculation method that did not require bacteria to enter the intact host from the soil. These results suggest that swimming motility makes its most important contribution to bacterial wilt virulence in the early stages of host plant invasion and colonization.
Genome of the Bacterium
Streptococcus pneumoniae
Strain R6
Authors: Hoskins JoAnn , Alborn William E. , Arnold Jeffrey , Blaszczak Larry C. , Burgett Stanley , DeHoff Bradley S. , Estrem Shawn T. , Fritz Lori , Fu Dong-Jing , Fuller Wendy , Geringer Chad , Gilmour Raymond , Glass Jennifer S. , Khoja Hamid , Kraft Angelika R. , Lagace Robert E. , LeBlanc Donald J. , Lee Linda N. , Lefkowitz Elliot J. , Lu Jin , Matsushima Patti , McAhren Scott M. , McHenney Margaret , McLeaster Kevin , Mundy Christopher W. , Nicas Thalia I. , Norris Franklin H. , O'Gara MaryJeanne , Peery Robert B. , Robertson Gregory T. , Rockey Pamela , Sun Pei-Ming , Winkler Malcolm E. , Yang Yong , Young-Bellido Michelle , Zhao Genshi , Zook Christopher A. , Baltz Richard H. , Jaskunas S. Richard , Rosteck Paul R. , Skatrud Paul L. , Glass John I. 
Published: July 2002

ABSTRACT

Streptococcus pneumoniae
is among the most significant causes of bacterial disease in humans. Here we report the 2,038,615-bp genomic sequence of the gram-positive bacterium
S. pneumoniae
R6. Because the R6 strain is avirulent and, more importantly, because it is readily transformed with DNA from homologous species and many heterologous species, it is the principal platform for investigation of the biology of this important pathogen. It is also used as a primary vehicle for genomics-based development of antibiotics for gram-positive bacteria. In our analysis of the genome, we identified a large number of new uncharacterized genes predicted to encode proteins that either reside on the surface of the cell or are secreted. Among those proteins there may be new targets for vaccine and antibiotic development.
Improved Soybean Root Association of N-Starved
Bradyrhizobium japonicum
Authors: López-Garcı́a Silvina L. , Vázquez Tirso E. E. , Favelukes Gabriel , Lodeiro Anı́bal R. 
Published: July 2002

ABSTRACT

In this study, we addressed the effects of N limitation in
Bradyrhizobium japonicum
for its association with soybean roots. The wild-type strain LP 3001 grew for six generations with a growth rate of 1.2 day
−1
in a minimal medium with 28 mM mannitol as the carbon source and with the N source [(NH
4
)
2
SO
4
] limited to only 20 μM. Under these conditions, the glutamine synthetase (GS) activity was five to six times higher than in similar cultures grown with 1 or 0.1 mM (NH
4
)
2
SO
4
. The NtrBC-inducible GSII form of this enzyme accounted for 60% of the specific activity in N-starved rhizobia, being negligible in the other two cultures. The exopolysaccharide (EPS) and capsular polysaccharide (CPS) contents relative to cell protein were significantly higher in the N-starved cultures, but on the other hand, the poly-3-hydroxybutyrate level did not rise in comparison with N-sufficient cultures. In agreement with the accumulation of CPS in N-starved cultures, soybean lectin (SBL) binding as well as stimulation of rhizobial adsorption to soybean roots by SBL pretreatment were higher. The last effect was evident only in cultures that had not entered stationary phase. We also studied
nodC
gene induction in relation to N starvation. In the chromosomal
nodC
::
lacZ
fusion Bj110-573,
nodC
gene expression was induced by genistein 2.7-fold more in N-starved young cultures than in nonstarved ones. In stationary-phase cultures,
nodC
gene expression was similarly induced in N-limited cultures, but induction was negligible in cultures limited by another nutrient. Nodulation profiles obtained with strain LP 3001 grown under N starvation indicated that these cultures nodulated faster. In addition, as culture age increased, the nodulation efficiency decreased for two reasons: fewer nodules were formed, and nodulation was delayed. However, their relative importance was different according to the nutrient condition: in older cultures the overall decrease in the number of nodules was the main effect in N-starved cultures, whereas a delay in nodulation was more responsible for a loss in efficiency of N-sufficient cultures. Competition for nodulation was studied with young cultures of two wild-type strains differing only in their antibiotic resistance, the N-starved cultures being the most competitive.
Identification of Essential Charged Residues in Transmembrane Segments of the Multidrug Transporter MexB of
Pseudomonas aeruginosa
Authors: Guan Lan , Nakae Taiji 
Published: July 2002

ABSTRACT

The MexABM efflux pump exports structurally diverse xenobiotics, utilizing the proton electrochemical gradient to confer drug resistance on
Pseudomonas aeruginosa
. The MexB subunit traverses the inner membrane 12 times and has two, two, and one charged residues in putative transmembrane segments 2 (TMS-2), TMS-4, and TMS-10, respectively. All five residues were mutated, and MexB function was evaluated by determining the MICs of antibiotics and fluorescent dye efflux. Replacement of Lys342 with Ala, Arg, or Glu and Glu346 with Ala, Gln, or Asp in TMS-2 did not have a discernible effect. Ala, Asn, or Lys substitution for Asp407 in TMS-4, which is well conserved, led to loss of activity. Moreover, a mutant with Glu in place of Asp407 exhibited only marginal function, suggesting that the length of the side chain at this position is important. The only replacements for Asp408 in TMS-4 or Lys939 in TMS-10 that exhibited significant function were Glu and Arg, respectively, suggesting that the native charge at these positions is required. In addition, double neutral mutants or mutants in which the charged residues Asp407 and Lys939 or Asp408 and Lys939 were interchanged completely lost function. An Asp408→Glu/Lys939→Arg mutant retained significant activity, while an Asp407→Glu/Lys939→Arg mutant exhibited only marginal function. An Asp407→Glu/Asp408→Glu double mutant also lost activity, but significant function was restored by replacing Lys939 with Arg (Asp407→Glu/Asp408→Glu/Lys939→Arg). Taken as a whole, the findings indicate that Asp407, Asp408, and Lys939 are functionally important and raise the possibility that Asp407, Asp408, and Lys939 may form a charge network between TMS-4 and TMS-10 that is important for proton translocation and/or energy coupling.