bluuebunny/crossref_metadata_2025_split huggingface dataset:
The Net Charge of the First 18 Residues of the Mature Sequence Affects Protein Translocation across the Cytoplasmic Membrane of Gram-Negative Bacteria
Authors: Kajava Andrey V. , Zolov Sergey N. , Kalinin Andrey E. , Nesmeyanova Marina A. 
Published: July 2002

ABSTRACT

This statistical study shows that in proteins of gram-negative bacteria exported by the Sec-dependent pathway, the first 14 to 18 residues of the mature sequences have the highest deviation between the observed and expected net charge distributions. Moreover, almost all sequences have either neutral or negative net charge in this region. This rule is restricted to gram-negative bacteria, since neither eukaryotic nor gram-positive bacterial exported proteins have this charge bias. Subsequent experiments performed with a series of
Escherichia coli
alkaline phosphatase mutants confirmed that this charge bias is associated with protein translocation across the cytoplasmic membrane. Two consecutive basic residues inhibit translocation effectively when placed within the first 14 residues of the mature protein but not when placed in positions 19 and 20. The sensitivity to arginine partially reappeared again 30 residues away from the signal sequence. These data provide new insight into the mechanism of protein export in gram-negative bacteria and lead to practical recommendations for successful secretion of hybrid proteins.
The GacS Sensor Kinase Regulates Alginate and Poly-β-Hydroxybutyrate Production in
Azotobacter vinelandii
Authors: Castañeda Miguel , Guzmán Josefina , Moreno Soledad , Espín Guadalupe 
Published: July 2002

ABSTRACT

Azotobacter vinelandii
produces two polymers: the extracellular polysaccharide alginate and the intracellular polyester poly-β-hydroxybutyrate (PHB). A cosmid clone (pSMU588) from an
A. vinelandii
gene library diminished alginate production by
A. vinelandii
mucoid strain ATCC 9046. The nucleotide sequence and predicted amino acid sequence of the locus responsible for the mucoidy suppression revealed 65% identity to
Pseudomonas
GacS, a transmembrane sensor kinase of the two-component regulators, whose cognate response regulator, GacA, is a global activator regulating several products and virulence factors. Plasmid pMC15, harboring
gacS
, and a strain carrying a
gacS
nonpolar mutation were constructed. Either pMC15 or the
gacS
mutation significantly reduced alginate production and transcription of
algD
, the gene coding for the key enzyme GDP-mannose dehydrogenase of the alginate biosynthetic pathway. We found that the
gacS
mutation also reduced PHB accumulation and impaired encystment. Taken together, these data indicate that in
A. vinelandii
the
gacSA
global system regulates polymer synthesis.
Role of
Escherichia coli
Nitrogen Regulatory Genes in the Nitrogen Response of the
Azotobacter vinelandii
NifL-NifA Complex
Authors: Reyes-Ramirez Francisca , Little Richard , Dixon Ray 
Published: July 2002

ABSTRACT

The redox-sensing flavoprotein NifL inhibits the activity of the nitrogen fixation (
nif
)-specific transcriptional activator NifA in
Azotobacter vinelandii
in response to molecular oxygen and fixed nitrogen. Although the mechanism whereby the
A. vinelandii
NifL-NifA system responds to fixed nitrogen in vivo is unknown, the
glnK
gene, which encodes a PII-like signal transduction protein, has been implicated in nitrogen control. However, the precise function of
A. vinelandii glnK
in this response is difficult to establish because of the essential nature of this gene. We have shown previously that
A. vinelandii
NifL is able to respond to fixed nitrogen to control NifA activity when expressed in
Escherichia coli
. In this study, we investigated the role of the
E. coli
PII-like signal transduction proteins in nitrogen control of the
A. vinelandii
NifL-NifA regulatory system in vivo. In contrast to recent findings with
Klebsiella pneumoniae
NifL, our results indicate that neither the
E. coli
PII nor GlnK protein is required to relieve inhibition by
A. vinelandii
NifL under nitrogen-limiting conditions. Moreover, disruption of both the
E. coli glnB
and
ntrC
genes resulted in a complete loss of nitrogen regulation of NifA activity by NifL. We observe that
glnB ntrC
and
glnB glnK ntrC
mutant strains accumulate high levels of intracellular 2-oxoglutarate under conditions of nitrogen excess. These findings are in accord with our recent in vitro observations (R. Little, F. Reyes-Ramirez, Y. Zhang, W. Van Heeswijk, and R. Dixon, EMBO J. 19:6041–6050, 2000) and suggest a model in which nitrogen control of the
A. vinelandii
NifL-NifA system is achieved through the response to the level of 2-oxoglutarate and an interaction with PII-like proteins under conditions of nitrogen excess.
A Homolog of the CtrA Cell Cycle Regulator Is Present and Essential in
Sinorhizobium meliloti
Authors: Barnett Melanie J. , Hung Dean Y. , Reisenauer Ann , Shapiro Lucy , Long Sharon R. 
Published: July 2002

ABSTRACT

During development of the symbiotic soil bacterium
Sinorhizobium meliloti
into nitrogen-fixing bacteroids, DNA replication and cell division cease and the cells undergo profound metabolic and morphological changes. Regulatory genes controlling the early stages of this process have not been identified. As a first step in the search for regulators of these events, we report the isolation and characterization of a
ctrA
gene from
S. meliloti
. We show that the
S. meliloti
CtrA belongs to the CtrA-like family of response regulators found in several α-proteobacteria. In
Caulobacter crescentus
, CtrA is essential and is a global regulator of multiple cell cycle functions.
ctrA
is also an essential gene in
S. meliloti
, and it is expressed similarly to the autoregulated
C. crescentus ctrA
in that both genes have complex promoter regions which bind phosphorylated CtrA.
Rhizobial NodL
O
-Acetyl Transferase and NodS
N
-Methyl Transferase Functionally Interfere in Production of Modified Nod Factors
Authors: López-Lara Isabel M. , Kafetzopoulos Dimitris , Spaink Herman P. , Thomas-Oates Jane E. 
Published: July 2002

ABSTRACT

The products of the rhizobial nodulation genes are involved in the biosynthesis of lipochitin oligosaccharides (LCOs), which are host-specific signal molecules required for nodule formation. The presence of an
O
-acetyl group on C-6 of the nonreducing
N
-acetylglucosamine residue of LCOs is due to the enzymatic activity of NodL. Here we show that transfer of the
nodL
gene into four rhizobial species that all normally produce LCOs that are not modified on C-6 of the nonreducing terminal residue results in production of LCOs, the majority of which have an acetyl residue substituted on C-6. Surprisingly, in transconjugant strains of
Mesorhizobium loti, Rhizobium etli,
and
Rhizobium tropici
carrying
nodL,
such acetylation of LCOs prevents the endogenous
nodS
-dependent transfer of the
N
-methyl group that is found as a substituent of the acylated nitrogen atom. To study this interference between
nodL
and
nodS,
we have cloned the
nodS
gene of
M. loti
and used its product in in vitro experiments in combination with purified NodL protein. It has previously been shown that a chitooligosaccharide N deacetylated on the nonreducing terminus (the so-called NodBC metabolite) is the preferred substrate for NodS as well as for NodL. Here we show that the NodBC metabolite, acetylated by NodL, is not used by the NodS protein as a substrate while the NodL protein can acetylate the NodBC metabolite that has been methylated by NodS.
Activation from a Distance: Roles of Lrp and Integration Host Factor in Transcriptional Activation of
gltBDF
Authors: Paul Ligi , Blumenthal Robert M. , Matthews Rowena G. 
Published: July 2002

ABSTRACT

The leucine-responsive regulatory protein (Lrp) binds to three sites centered 252, 216, and 152 bp upstream of the transcription start site of the
Escherichia coli
glutamate synthase operon (
gltBDF
) and activates transcription. Activators of ς
70
-dependent promoters usually bind closer to the −35 hexamer of the core promoter sequence. To study the mechanism by which Lrp-dependent activation occurs over this relatively large distance, the
gltBDF
upstream region was sequentially replaced with corresponding portions from the well-characterized ς
70
-dependent promoter
lacZYAp
. The
glt-lac
promoter hybrids were placed upstream of
lacZ
, allowing transcriptional activity to be monitored via β-galactosidase assays. Even replacing all
gltBDF
sequences downstream of and including the −35 hexamer did not eliminate Lrp-dependent activation of transcription. When a 91-bp region between the −35 hexamer and the proximal Lrp binding site (−48 to −128) was replaced with heterologous DNA of the same length, transcription was reduced nearly 40-fold. Based on the presence of a consensus binding sequence, this region seemed likely to be a binding site for integration host factor (IHF). Experiments to study the effects of a
himD
mutant on expression of a
gltB
::
lacZ
transcriptional fusion, gel mobility shift analyses, and DNA footprinting assays were used to confirm the direct participation of IHF in
gltBDF
promoter regulation. Based on these results, we suggest that IHF plays a crucial architectural role, bringing the distant Lrp complex in close proximity to the promoter-bound RNA polymerase.
In Vivo Evidence for Two Active Nuclease Motifs in the Double-Strand Break Repair Enzyme RexAB of
Lactococcus lactis
Authors: Quiberoni Andréa , Biswas Indranil , El Karoui Meriem , Rezaı̈ki Lahcen , Tailliez Patrick , Gruss Alexandra 
Published: July 2002

ABSTRACT

In bacteria, double-strand DNA break (DSB) repair involves an exonuclease/helicase (exo/hel) and a short regulatory DNA sequence (Chi) that attenuates exonuclease activity and stimulates DNA repair. Despite their key role in cell survival, these DSB repair components show surprisingly little conservation. The best-studied exo/hel, RecBCD of
Escherichia coli
, is composed of three subunits. In contrast, RexAB of
Lactococcus lactis
and exo/hel enzymes of other low-guanine-plus-cytosine branch gram-positive bacteria contain two subunits. We report that RexAB functions via a novel mechanism compared to that of the RecBCD model. Two potential nuclease motifs are present in RexAB compared with a single nuclease in RecBCD. Site-specific mutagenesis of the RexA nuclease motif abolished all nuclease activity. In contrast, the RexB nuclease motif mutants displayed strongly reduced nuclease activity but maintained Chi recognition and had a Chi-stimulated hyperrecombination phenotype. The distinct phenotypes resulting from RexA or RexB nuclease inactivation lead us to suggest that each of the identified active nuclease sites in RexAB is involved in the degradation of one DNA strand. In RecBCD, the single RecB nuclease degrades both DNA strands and is presumably positioned by RecD. The presence of two nucleases would suggest that this RecD function is dispensable in RexAB.
Competence Repression under Oxygen Limitation through the Two-Component MicAB Signal-Transducing System in
Streptococcus pneumoniae
and Involvement of the PAS Domain of MicB
Authors: Echenique José R. , Trombe Marie-Claude 
Published: July 2002

ABSTRACT

In
Streptococcus pneumoniae
, a fermentative aerotolerant and catalase-deficient human pathogen, oxidases with molecular oxygen as substrate are important for virulence and for competence. The signal-transducing two-component systems CiaRH and ComDE mediate the response to oxygen, culminating in competence. In this work we show that the two-component MicAB system, whose MicB kinase carries a PAS domain, is also involved in competence repression under oxygen limitation. Autophosphorylation of recombinant MicB and phosphotransfer to recombinant MicA have been demonstrated. Mutational analysis and in vitro assays showed that the C-terminal part of the protein and residue L100 in the N-terminal cap of its PAS domain are both crucial for autokinase activity in vitro. Although no insertion mutation in
micA
was obtained, expression of the mutated allele
micA59DA
did not change bacterial growth and overcame competence repression under microaerobiosis. This was related to a strong instability of MicA59DA-PO
4
in vitro. Thus, mutations which either reduced the stability of MicA-PO
4
or abolished kinase activity in MicB were related to competence derepression under microaerobiosis, suggesting that MicA-PO
4
is involved in competence repression when oxygen becomes limiting. The
micAB
genes are flanked by
mutY
and
orfC
. MutY is an adenine glycosylase involved in the repair of oxidized pyrimidines. OrfC shows the features of a metal binding protein. We did not obtain insertion mutation in
orfC
, suggesting its requirement for growth. It is proposed that MicAB, with its PAS motif, may belong to a set of functions important in the protection of the cell against oxidative stress, including the control of competence.
ZitB (YbgR), a Member of the Cation Diffusion Facilitator Family, Is an Additional Zinc Transporter in
Escherichia coli
Authors: Grass Gregor , Fan Bin , Rosen Barry P. , Franke Sylvia , Nies Dietrich H. , Rensing Christopher 
Published: July 2002

ABSTRACT

The
Escherichia coli zitB
gene encodes a Zn(II) transporter belonging to the cation diffusion facilitator family. ZitB is specifically induced by zinc. ZitB expression on a plasmid rendered
zntA
-disrupted
E. coli
cells more resistant to zinc, and the cells exhibited reduced accumulation of
65
Zn, suggesting ZitB-mediated efflux of zinc.
Transcriptional Analysis and Regulation of Expression of the
Scr
FI Restriction-Modification System of
Lactococcus lactis
subsp.
cremoris
UC503
Authors: Butler Derek , Fitzgerald Gerald F. 
Published: July 2002

ABSTRACT

Scr
FI is a type II restriction-modification system from
Lactococcus
lactis
which recognizes the nucleotide sequence 5′-CC↓ NGG-3′, cleaving at the point indicated by the arrow, and it comprises an endonuclease gene that is flanked on either side by genes encoding two 5-methylcytosine methylases. An open reading frame (
orfX
) of unknown function is located immediately upstream of these genes. In this study Northern analysis was performed, and it revealed that
orfX, scrFIBM,
and
scrFIR
are cotranscribed as a single polygenic mRNA molecule, while
scrFIAM
is transcribed independently. 5′ extension analysis indicated that the start site for the
scrFIAM
promoter was a thymine located 4 bp downstream of the −10 motif. The transcriptional start site for the
orfX
promoter was also found to be a thymine which is more atypically located 24 bp downstream of the −10 motif proximal to the start codon. A helix-turn-helix motif was identified at the N-terminal end of one of the methylases (M.
Scr
FIA). In order to determine if this motif played a role in regulation of the
Scr
FI locus, M.
Scr
FIA was purified. It was then employed in gel retardation assays using fragments containing the two promoters found on the
Scr
FI operon, one located upstream of
orfX
and the other located just upstream of
scrFIAM
. M.
Scr
FIA was found to bind to the promoter region upstream of the gene encoding it, indicating that it may have a regulatory role. In further studies the two putative promoters were introduced into a vector (pAK80) upstream of a promoterless
lacZ
gene, and cloned fragments of the
Scr
FI locus were introduced in
trans
with each of these promoter constructs to investigate the effect on promoter activity. These results implicated M.
Scr
FIA in regulation of both promoters on the
Scr
FI locus.
matB
, a Common Fimbrillin Gene of
Escherichia coli
, Expressed in a Genetically Conserved, Virulent Clonal Group
Authors: Pouttu Riitta , Westerlund-Wikström Benita , Lång Hannu , Alsti Krista , Virkola Ritva , Saarela Ulla , Siitonen Anja , Kalkkinen Nisse , Korhonen Timo K. 
Published: July 2002

ABSTRACT

A novel fimbrial type in
Escherichia coli
was identified and characterized. The expression of the fimbria was associated with the O18acK1H7 clonal group of
E. coli
, which cause newborn meningitis and septicemia when grown at low temperature; hence, it was named the Mat (meningitis associated and temperature regulated) fimbria. The fimbriae were purified from a
fimA
::
cat sfaA
::Gm
fliC
::St derivative of the O18K1H7 isolate
E. coli
IHE 3034. The purified Mat fimbrillin had an apparent molecular mass of 18 kDa and did not serologically cross-react with the type 1 or S fimbria of the same strain. The
matB
gene encoding the major fimbrillin was cloned from the genomic DNA of the
fimA
::
cat sfaA
::Gm
fliC
::St derivative of IHE 3034. The predicted MatB sequence was of 195 amino acids, contained a signal sequence of 22 residues, and did not show significant homology to any of the previously characterized fimbrial proteins. The DNA sequence of
matB
was 97.8% identical to a region from nucleotides 17882 to 18469 in the 6- to 8-min region of the
E. coli
K-12 chromosome, reported to encode a hypothetical protein. The 7-kb DNA fragment containing
matB
of IHE 3034 was found by restriction mapping and partial DNA sequencing to be highly similar to the corresponding region in the K-12 chromosome. Trans complementation of the
matB
::
cat
mutation in the IHE 3034 chromosome showed that
matB
in combination with
matA
or
matC
restored surface expression of the Mat fimbria. A total of 27 isolates representing K-12 strains and the major pathogroups of
E. coli
were analyzed for the presence of a
matB
homolog as well as for expression of the Mat fimbria. A conserved
matB
homolog was found in 25 isolates; however, expression of the Mat fimbriae was detected only in the O18acK1H7 isolates. Expression of the Mat fimbria was temperature regulated, with no or a very small amount of fimbriae or intracellular MatB fimbrillin being detected in cells cultivated at 37
o
C. Reverse transcriptase PCR and complementation assays with
mat
genes controlled by the inducible
trc
promoter indicated that regulation of Mat fimbria expression involved both transcriptional and posttranscriptional events.
Influence of a Functional
sigB
Operon on the Global Regulators
sar
and
agr
in
Staphylococcus aureus
Authors: Bischoff M. , Entenza J. M. , Giachino P. 
Published: July 2002

ABSTRACT

The growth phase-dependent activity profile of the alternate transcription factor ς
B
and its effects on the expression of
sar
and
agr
were examined in three different
Staphylococcus aureus
strains by Northern blot analyses and by the use of reporter gene fusion experiments. Significant ς
B
activity was detectable only in the clinical isolates MSSA1112 and Newman, carrying the wild-type
rsbU
allele, but not in the NCTC8325 derivative BB255, which is defective in
rsbU
. ς
B
activity peaked in the late exponential phase and diminished towards the stationary phase when bacteria were grown in Luria-Bertani medium. Transcriptional analysis and a
sarP1-sarP2-sarP3
(
sarP1-P2-P3
)-driven firefly luciferase (
luc
+) reporter gene fusion demonstrated a strong ς
B
activity- and growth phase-dependent increase in
sar
expression that was totally absent in either
rsbU
or Δ
rsbUVWsigB
mutants. In contrast, expression of the
agr
locus, as measured by RNAIII levels and by an
hldp
::
luc
+ fusion, was found to be higher in the absence of ς
B
activity, such as in
rsbU
or Δ
rsbUVWsigB
mutants, than in wild-type strains. Overexpression of ς
B
in BB255 derivatives resulted in a clear increase in
sarP1-P2-P3
::
luc
+ expression as well as a strong decrease in
hldp
::
luc
+ expression. The data presented here suggest that ς
B
increases
sar
expression while simultaneously reducing the RNAIII level in a growth phase-dependent manner.
Cloning and Functional Analysis of the
pbr
Lead Resistance Determinant of
Ralstonia metallidurans
CH34
Authors: Borremans B. , Hobman J. L. , Provoost A. , Brown N. L. , van der Lelie D. 
Published: July 2002

ABSTRACT

The lead resistance operon,
pbr
, of
Ralstonia metallidurans
(formerly
Alcaligenes eutrophus
) strain CH34 is unique, as it combines functions involved in uptake, efflux, and accumulation of Pb(II). The
pbr
lead resistance locus contains the following structural resistance genes: (i)
pbrT
, which encodes a Pb(II) uptake protein; (ii)
pbrA
, which encodes a P-type Pb(II) efflux ATPase; (iii)
pbrB
, which encodes a predicted integral membrane protein of unknown function; and (iv)
pbrC
, which encodes a predicted prolipoprotein signal peptidase. Downstream of
pbrC
, the
pbrD
gene, encoding a Pb(II)-binding protein, was identified in a region of DNA, which was essential for functional lead sequestration. Pb(II)-dependent inducible transcription of
pbrABCD
from the
PpbrA
promoter is regulated by PbrR, which belongs to the MerR family of metal ion-sensing regulatory proteins. This is the first report of a mechanism for specific lead resistance in any bacterial genus.
Cloning and Characterization of a Gene Cluster for Cyclododecanone Oxidation in
Rhodococcus ruber
SC1
Authors: Kostichka Kristy , Thomas Stuart M. , Gibson Katharine J. , Nagarajan Vasantha , Cheng Qiong 
Published: July 2002

ABSTRACT

Biological oxidation of cyclic ketones normally results in formation of the corresponding dicarboxylic acids, which are further metabolized in the cell.
Rhodococcus ruber
strain SC1 was isolated from an industrial wastewater bioreactor that was able to utilize cyclododecanone as the sole carbon source. A reverse genetic approach was used to isolate a 10-kb gene cluster containing all genes required for oxidative conversion of cyclododecanone to 1,12-dodecanedioic acid (DDDA). The genes required for cyclododecanone oxidation were only marginally similar to the analogous genes for cyclohexanone oxidation. The biochemical function of the enzymes encoded on the 10-kb gene cluster, the flavin monooxygenase, the lactone hydrolase, the alcohol dehydrogenase, and the aldehyde dehydrogenase, was determined in
Escherichia coli
based on the ability to convert cyclododecanone. Recombinant
E. coli
strains grown in the presence of cyclododecanone accumulated lauryl lactone, 12-hydroxylauric acid, and/or DDDA depending on the genes cloned. The cyclododecanone monooxygenase is a type 1 Baeyer-Villiger flavin monooxygenase (FAD as cofactor) and exhibited substrate specificity towards long-chain cyclic ketones (C
11
to C
15
), which is different from the specificity of cyclohexanone monooxygenase favoring short-chain cyclic compounds (C
5
to C
7
).