bluuebunny/crossref_metadata_2025 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

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

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

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

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

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

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

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

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

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

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

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

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

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

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
).
Characterization of Phenotypic Changes in
Pseudomonas putida
in Response to Surface-Associated Growth
Authors: Sauer Karin , Camper Anne K.
Published: July 2002

The formation of complex bacterial communities known as biofilms begins with the interaction of planktonic cells with a surface. A switch between planktonic and sessile growth is believed to result in a phenotypic change in bacteria. In this study, a global analysis of physiological changes of the plant saprophyte
Pseudomonas putida
following 6 h of attachment to a silicone surface was carried out by analysis of protein profiles and by mRNA expression patterns. Two-dimensional (2-D) gel electrophoresis revealed 15 proteins that were up-regulated following bacterial adhesion and 30 proteins that were down-regulated. N-terminal sequence analyses of 11 of the down-regulated proteins identified a protein with homology to the ABC transporter, PotF; an outer membrane lipoprotein, NlpD; and five proteins that were homologous to proteins involved in amino acid metabolism. cDNA subtractive hybridization revealed 40 genes that were differentially expressed following initial attachment of
P
.
putida
. Twenty-eight of these genes had known homologs. As with the 2-D gel analysis, NlpD and genes involved in amino acid metabolism were identified by subtractive hybridization and found to be down-regulated following surface-associated growth. The gene for PotB was up-regulated, suggesting differential expression of ABC transporters following attachment to this surface. Other genes that showed differential regulation were structural components of flagella and type IV pili, as well as genes involved in polysaccharide biosynthesis. Immunoblot analysis of PilA and FliC confirmed the presence of flagella in planktonic cultures but not in 12- or 24-h biofilms. In contrast, PilA was observed in 12-h biofilms but not in planktonic culture. Recent evidence suggests that quorum sensing by bacterial homoserine lactones (HSLs) may play a regulatory role in biofilm development. To determine if similar protein profiles occurred during quorum sensing and during early biofilm formation, HSLs extracted from
P
.
putida
and pure C
12
-HSL were added to 6-h planktonic cultures of
P
.
putida
, and cell extracts were analyzed by 2-D gel profiles. Differential expression of 16 proteins was observed following addition of HSLs. One protein, PotF, was found to be down-regulated by both surface-associated growth and by HSL addition. The other 15 proteins did not correspond to proteins differentially expressed by surface-associated growth. The results presented here demonstrate that
P
.
putida
undergoes a global change in gene expression following initial attachment to a surface. Quorum sensing may play a role in the initial attachment process, but other sensory processes must also be involved in these phenotypic changes.
Colicin A Immunity Protein Interacts with the Hydrophobic Helical Hairpin of the Colicin A Channel Domain in the
Escherichia coli
Inner Membrane
Authors: Nardi Angèle , Corda Yves , Baty Daniel , Duché Denis
Published: July 2002

The colicin A pore-forming domain (pfColA) was fused to a bacterial signal peptide (sp-pfColA). This was inserted into the
Escherichia coli
inner membrane in functional form and could be coimmunoprecipitated with epitope-tagged immunity protein (EpCai). We constructed a series of fusion proteins in which various numbers of sp-pfColA α-helices were fused to alkaline phosphatase (AP). We showed that a fusion protein made up of the hydrophobic α-helices 8 and 9 of sp-pfColA fused to AP was specifically coimmunoprecipitated with EpCai produced in the same cells. This is the first biochemical evidence that Cai recognizes and interacts with the colicin A hydrophobic helical hairpin.
Transcriptional Regulation of
furA
and
katG
upon Oxidative Stress in
Mycobacterium smegmatis
Authors: Milano Anna , Forti Francesca , Sala Claudia , Riccardi Giovanna , Ghisotti Daniela
Published: July 2002

The DNA region upstream of
katG
in
Mycobacterium smegmatis
was cloned and sequenced. The
furA
gene, highly homologous to
Mycobacterium tuberculosis furA
, mapped in this region. The
furA-katG
organization appears to be conserved among several mycobacteria. The transcription pattern of
furA
and
katG
in
M
.
smegmatis
upon oxidative stress was analyzed by Northern blotting and primer extension. Although transcription of both
furA
and
katG
was induced upon oxidative stress, transcripts covering both genes could not be identified either by Northern blotting or by reverse transcriptase PCR. Specific transcripts and 5′ ends were identified for
furA
and
katG
, respectively. By cloning
M
.
smegmatis
and
M
.
tuberculosis
DNA regions upstream of a reporter gene, we demonstrated the presence of two promoters,
pfurA
, located immediately upstream of the
furA
gene, and
pkatG
, located within the terminal part of the
furA
coding sequence. Transcription from
pfurA
was induced upon oxidative stress. A 23-bp sequence overlapping the
pfurA
−35 region is highly conserved among mycobacteria and streptomycetes and might be involved in controlling
pfurA
activity. Transcription from a cloned
pkatG
, lacking the upstream
pfurA
region, was not induced upon oxidative stress, suggesting a
cis
-acting regulatory role of this region.
Analysis of the
pmsCEAB
Gene Cluster Involved in Biosynthesis of Salicylic Acid and the Siderophore Pseudomonine in the Biocontrol Strain
Pseudomonas fluorescens
WCS374
Authors: Mercado-Blanco Jesús , van der Drift Koen M. G. M. , Olsson Per E. , Thomas-Oates Jane E. , van Loon Leendert C. , Bakker Peter A. H. M.
Published: July 2002

Mutants of
Pseudomonas fluorescens
WCS374 defective in biosynthesis of the fluorescent siderophore pseudobactin still display siderophore activity, indicating the production of a second siderophore. A recombinant cosmid clone (pMB374-07) of a WCS374 gene library harboring loci necessary for the biosynthesis of salicylic acid (SA) and this second siderophore pseudomonine was isolated. The salicylate biosynthesis region of WCS374 was localized in a 5-kb
Eco
RI fragment of pMB374-07. The SA and pseudomonine biosynthesis region was identified by transfer of cosmid pMB374-07 to a pseudobactin-deficient strain of
P. putida
. Sequence analysis of the 5-kb subclone revealed the presence of four open reading frames (ORFs). Products of two ORFs (
pmsC
and
pmsB
) showed homologies with chorismate-utilizing enzymes; a third ORF (
pmsE
) encoded a protein with strong similarity with enzymes involved in the biosynthesis of siderophores in other bacterial species. The region also contained a putative histidine decarboxylase gene (
pmsA
). A putative promoter region and two predicted iron boxes were localized upstream of
pmsC
. We determined by reverse transcriptase-mediated PCR that the
pmsCEAB
genes are cotranscribed and that expression is iron regulated. In vivo expression of SA genes was achieved in
P. putida
and
Escherichia coli
cells. In
E. coli
, deletions affecting the first ORF (
pmsC
) diminished SA production, whereas deletion of
pmsB
abolished it completely. The
pmsB
gene induced low levels of SA production in
E. coli
when expressed under control of the
lacZ
promoter. Several lines of evidence indicate that SA and pseudomonine biosynthesis are related. Moreover, we isolated a Tn
5
mutant (374-05) that is simultaneously impaired in SA and pseudomonine production.
d
-Alanylation of Lipoteichoic Acid: Role of the
d
-Alanyl Carrier Protein in Acylation
Authors: Kiriukhin Michael Y. , Neuhaus Francis C.
Published: July 2002

The
d
-alanylation of membrane-associated lipoteichoic acid (LTA) in gram-positive organisms requires the
d
-alanine–
d
-alanyl carrier protein ligase (AMP) (Dcl) and the
d
-alanyl carrier protein (Dcp). The
dlt
operon encoding these proteins (
dltA
and
dltC
) also includes
dltB
and
dltD. dltB
encodes a putative transport system, while
dltD
encodes a protein which facilitates the binding of Dcp and Dcl for ligation with
d
-alanine and has thioesterase activity for mischarged
d
-alanyl-acyl carrier proteins (ACPs). In previous results it was shown that
d
-alanyl-Dcp donates its ester residue to membrane-associated LTA (M. P. Heaton and F. C. Neuhaus, J. Bacteriol. 176: 681–690, 1994). However, all efforts to identify an enzyme which catalyzes this
d
-alanylation process were unsuccessful. It was discovered that incubation of
d
-alanyl-Dcp in the presence of LTA resulted in the time-dependent hydrolysis of this
d
-alanyl thioester.
d
-Alanyl-ACP in the presence of LTA was not hydrolyzed. When Dcp was incubated with membrane-associated
d
-alanyl LTA, a time and concentration-dependent formation of
d
-alanyl-Dcp was found. The addition of NaCl to this reaction inhibited the formation of
d
-alanyl-Dcp and stimulated the hydrolysis of
d
-alanyl-Dcp. Since these reactions are specific for the carrier protein (Dcp), it is suggested that Dcp has a unique binding site which interacts with the poly(Gro-P) moiety of LTA. It is this specific interaction that provides the functional specificity for the
d
-alanylation process. The reversibility of this process provides a mechanism for the transacylation of the
d
-alanyl ester residues between LTA and wall teichoic acid.
Melamine Deaminase and Atrazine Chlorohydrolase: 98 Percent Identical but Functionally Different
Authors: Seffernick Jennifer L. , de Souza Mervyn L. , Sadowsky Michael J. , Wackett Lawrence P.
Published: July 2002

The gene encoding melamine deaminase (TriA) from
Pseudomonas
sp. strain NRRL B-12227 was identified, cloned into
Escherichia coli
, sequenced, and expressed for in vitro study of enzyme activity. Melamine deaminase displaced two of the three amino groups from melamine, producing ammeline and ammelide as sequential products. The first deamination reaction occurred more than 10 times faster than the second. Ammelide did not inhibit the first or second deamination reaction, suggesting that the lower rate of ammeline hydrolysis was due to differential substrate turnover rather than product inhibition. Remarkably, melamine deaminase is 98% identical to the enzyme atrazine chlorohydrolase (AtzA) from
Pseudomonas
sp. strain ADP. Each enzyme consists of 475 amino acids and differs by only 9 amino acids. AtzA was shown to exclusively catalyze dehalogenation of halo-substituted triazine ring compounds and had no activity with melamine and ammeline. Similarly, melamine deaminase had no detectable activity with the halo-triazine substrates. Melamine deaminase was active in deamination of a substrate that was structurally identical to atrazine, except for the substitution of an amino group for the chlorine atom. Moreover, melamine deaminase and AtzA are found in bacteria that grow on melamine and atrazine compounds, respectively. These data strongly suggest that the 9 amino acid differences between melamine deaminase and AtzA represent a short evolutionary pathway connecting enzymes catalyzing physiologically relevant deamination and dehalogenation reactions, respectively.
Cytoplasmic RNA Polymerase in
Escherichia coli
Authors: Shepherd N. , Dennis P. , Bremer H.
Published: July 2002

To obtain an estimate for the concentration of free functional RNA polymerase in the bacterial cytoplasm, the content of RNA polymerase β and β′ subunits in DNA-free minicells from the minicell-producing
Escherichia coli
strain χ925 was determined. In bacteria grown in Luria-Bertani medium at 2.5 doublings/h, 1.0% of the total protein was RNA polymerase. The concentration of cytoplasmic RNA polymerase β and β′ subunits in minicells produced by this strain corresponded to about 17% (or 2.5 μM) of the value found in whole cells. Literature data suggest that a similar portion of cytoplasmic RNA polymerase subunits is in RNA polymerase assembly intermediates and imply that free functional RNA polymerase can form a small percentage of the total functional enzyme in the cell. On infection with bacteriophage T7, 20% of the minicells produced progeny phage, whereas infection in 80% of the cells was abortive. RNA polymerase subunits in lysozyme-freeze-thaw lysates of minicells were associated with minicell envelopes and were without detectable activity in an in vitro transcription assay. Together, these results suggest that most functional RNA polymerase is associated with the DNA and that little if any segregates into DNA-free minicells.
TraG-Like Proteins of DNA Transfer Systems and of theHelicobacter pyloriType IV Secretion System: Inner Membrane Gate for Exported Substrates?
Authors: Schröder Gunnar , Krause Sabine , Zechner Ellen L. , Traxler Beth , Yeo Hye-Jeong , Lurz Rudi , Waksman Gabriel , Lanka Erich
Published: July 2002

TraG-like proteins are potential NTP hydrolases (NTPases) that are essential for DNA transfer in bacterial conjugation. They are thought to mediate interactions between the DNA-processing (Dtr) and the mating pair formation (Mpf) systems. TraG-like proteins also function as essential components of type IV secretion systems of several bacterial pathogens such asHelicobacter pylori. Here we present the biochemical characterization of three members of the family of TraG-like proteins, TraG (RP4), TraD (F), and HP0524 (H. pylori). These proteins were found to have a pronounced tendency to form oligomers and were shown to bind DNA without sequence specificity. Standard NTPase assays indicated that these TraG-like proteins do not possess postulated NTP-hydrolyzing activity. Surface plasmon resonance was used to demonstrate an interaction between TraG and relaxase TraI of RP4. Topology analysis of TraG revealed that TraG is a transmembrane protein with cytosolic N and C termini and a short periplasmic domain close to the N terminus. We predict that multimeric inner membrane protein TraG forms a pore. A model suggesting that the relaxosome binds to the TraG pore via TraG-DNA and TraG-TraI interactions is presented.
Salt-Dependent Expression of Glucosylglycerol-Phosphate Synthase, Involved in Osmolyte Synthesis in the Cyanobacterium
Synechocystis
sp. Strain PCC 6803
Authors: Marin Kay , Huckauf Jana , Fulda Sabine , Hagemann Martin
Published: July 2002

The cyanobacterium
Synechocystis
sp. strain PCC 6803 is able to acclimate to levels of salinity ranging from freshwater to twice the seawater concentrations of salt by accumulating the compatible solute glucosylglycerol (GG). Expression of the
ggpS
gene coding for the key enzyme (glucosylglycerol-phosphate synthase) in GG synthesis was examined in detail. Under control conditions, the GgpS protein is stable, so that weak constitutive transcription of the
ggpS
gene resulted in a significant protein content. However, the enzyme activity was biochemically switched off, and no GG was detectable. After a salt shock, an immediate increase in mRNA content proportional to the salt content occurred, while the GgpS protein and GG contents rose in a linear manner. Furthermore, the stability of the
ggpS
mRNA increased transiently. In salt-acclimated cells expression of the
ggpS
gene, the GgpS protein content, and the amount of accumulated GG depended linearly on the external salt concentration. Mapping of the 5′ end of the
ggpS
transcript revealed a long nontranslated 5′ sequence and a putative typical cyanobacterial promoter, which did not show any obvious salt-regulatory element. The alternative σ factor σ
F
was found to be involved in salt-dependent regulation of
ggpS
, since in a σ
F
mutant induction of this gene was strongly reduced. The present study demonstrated that in addition to biochemical regulation of GgpS activity, alterations of
ggpS
expression are involved in regulation of GG synthesis in
Synechocystis
sp. strain PCC 6803. A model showing the interaction of the two regulatory levels is presented.
FpvA Receptor Involvement in Pyoverdine Biosynthesis in
Pseudomonas aeruginosa
Authors: Shen Jiangsheng , Meldrum Allison , Poole Keith
Published: July 2002

Alignment of the
Pseudomonas aeruginosa
ferric pyoverdine receptor, FpvA, with similar ferric-siderophore receptors revealed that the mature protein carries an extension of ca. 70 amino acids at its N terminus, an extension shared by the ferric pseudobactin receptors of
P. putida
. Deletion of
fpvA
from the chromosome of
P. aeruginosa
reduced pyoverdine production in this organism, as a result of a decline in expression of genes (e.g.,
pvdD
) associated with the biosynthesis of the pyoverdine peptide moiety. Wild-type
fpvA
restored
pvd
expression in the mutant, thereby complementing its pyoverdine deficiency, although a deletion derivative of
fpvA
encoding a receptor lacking the N terminus of the mature protein did not. The truncated receptor was, however, functional in pyoverdine-mediated iron uptake, as evidenced by its ability to promote pyoverdine-dependent growth in an iron-restricted medium. These data are consistent with the idea that the N-terminal extension plays a role in FpvA-mediated pyoverdine biosynthesis in
P. aeruginosa
.
Membrane Topology of the Multidrug Transporter MdfA: Complementary Gene Fusion Studies Reveal a Nonessential C-Terminal Domain
Authors: Adler Julia , Bibi Eitan
Published: July 2002

The hydrophobicity profile and sequence alignment of the
Escherichia coli
multidrug transporter MdfA indicate that it belongs to the 12-transmembrane-domain family of transporters. According to this prediction, MdfA contains a single membrane-embedded charged residue (Glu26), which was shown to play an important role in substrate recognition. To test the predicted secondary structure of MdfA, we analyzed complementary pairs of hybrids of MdfA-PhoA (alkaline phosphatase, functional in the periplasm) and MdfA-Cat (chloramphenicol acetyltransferase, functional in the cytoplasm), generated in all the putative cytoplasmic and periplasmic loops of MdfA. Our results support the 12-transmembrane topology model and the suggestion that except for Glu26, no other charged residues are present in the membrane domain of MdfA. Surprisingly, by testing the ability of the truncated MdfA-Cat and MdfA-PhoA hybrids to confer multidrug resistance, we demonstrate that the entire C-terminal transmembrane domain and the cytoplasmic C terminus are not essential for MdfA-mediated drug resistance and transport.
Genetic Variation at the O-Antigen Biosynthetic Locus in
Pseudomonas aeruginosa
Authors: Raymond Christopher K. , Sims Elizabeth H. , Kas Arnold , Spencer David H. , Kutyavin Tanya V. , Ivey Richard G. , Zhou Yang , Kaul Rajinder , Clendenning James B. , Olson Maynard V.
Published: July 2002

The outer carbohydrate layer, or O antigen, of
Pseudomonas aeruginosa
varies markedly in different isolates of these bacteria, and at least 20 distinct O-antigen serotypes have been described. Previous studies have indicated that the major enzymes responsible for O-antigen synthesis are encoded in a cluster of genes that occupy a common genetic locus. We used targeted yeast recombinational cloning to isolate this locus from the 20 internationally recognized serotype strains. DNA sequencing of these isolated segments revealed that at least 11 highly divergent gene clusters occupy this region. Homology searches of the encoded protein products indicated that these gene clusters are likely to direct O-antigen biosynthesis. The O15 serotype strains lack functional gene clusters in the region analyzed, suggesting that O-antigen biosynthesis genes for this serotype are harbored in a different portion of the genome. The overall pattern underscores the plasticity of the
P. aeruginosa
genome, in which a specific site in a well-conserved genomic region can be occupied by any of numerous islands of functionally related DNA with diverse sequences.
epr
Is Transcribed from a ς
D
Promoter and Is Involved in Swarming of
Bacillus subtilis
Authors: Dixit Madhulika , Murudkar Charuta S. , Rao K. Krishnamurthy
Published: July 2002

Epr is a minor extracellular protease secreted by
Bacillus subtilis
168. In this study, we show that
epr
is transcribed by Eς
D
, the RNA polymerase associated with transcription of genes involved in chemotaxis and motility. Disruption of
epr
abolished swarming of
Bacillus subtilis
, suggesting its involvement in motility.
Characterization of the
parB
-Like
yyaA
Gene of
Bacillus subtilis
Authors: Sievers Jörg , Raether Brian , Perego Marta , Errington Jeff
Published: July 2002

We have characterized the
yyaA
gene of
Bacillus subtilis
, located near the origin of chromosome replication (
oriC
). Its protein product is similar to the Spo0J protein, which belongs to the ParB family of chromosome- and plasmid-partitioning proteins. Insertional inactivation of the
yyaA
gene had no apparent effect on chromosome organization and partitioning during vegetative growth or sporulation. Subcellular localization of YyaA by immunofluorescence microscopy indicated that it colocalizes with the nucleoid, and gel retardation studies confirmed that YyaA binds relatively nonspecifically to DNA. Overexpression of
yyaA
caused a sporulation defect characterized by the formation of multiple septa within the cell. This phenotype indicates that YyaA may have a regulatory role at the onset of sporulation.
In Vitro Identification of Rns-Regulated Genes
Authors: Munson George P. , Holcomb Lisa G. , Alexander Heather L. , Scott June R.
Published: July 2002

To identify Rns-regulated genes, a maltose binding protein (MBP)-Rns fusion protein was used to isolate DNA fragments from enterotoxigenicEscherichia coligenomic DNA that carry Rns binding sites. In vivo transcription fusion analysis shows that Rns positively regulates the expression of the open reading frame ofyiiS, which lies immediately downstream of one MBP-Rns-bound fragment.
A Two-Component Regulator Mediates Population-Density-Dependent Expression of theBradyrhizobium japonicumNodulation Genes
Authors: Loh John , Lohar Dasharath P. , Andersen Brett , Stacey Gary
Published: July 2002

Bradyrhizobium japonicum nodgene expression was previously shown to be population density dependent. Induction of thenodgenes is highest at low culture density and repressed at high population densities. This repression involves both NolA and NodD2 and is mediated by an extracellular factor found inB. japonicumconditioned medium. NolA and NodD2 expression is maximal at high population densities. We demonstrate here that a response regulator, encoded bynwsB,is required for the full expression of theB. japonicum nodYABCoperon. In addition, NwsB is also required for the population-density-dependent expression of bothnolAandnodD2. Expression ofnolAandnodD2in thenwsBmutant remained at a basal level, even at high culture densities. ThenwsBdefect could be complemented by overexpression of a second response regulator, NodW. Consistent with the fact that NolA and NodD2 repressnodgene expression, the expression of anodY-lacZfusion in thenwsBmutant was unaffected by culture density. In plant assays withGUSfusions, nodules infected with the wild type showed nonodY-GUSexpression. In contrast,nodY-GUSexpression was not repressed in nodules infected with thenwsBmutant. Nodule competition assays between the wild type and thenwsBmutant revealed that the addition of conditioned medium resulted in a competitive advantage for thenwsBmutant.
tRNA Is the Source of Low-Level
trans-
Zeatin Production in
Methylobacterium
spp
Authors: Koenig Robbin L. , Morris Roy O. , Polacco Joe C.
Published: July 2002

Pink-pigmented facultatively methylotrophic bacteria (PPFMs), classified as
Methylobacterium
spp., are persistent colonizers of plant leaf surfaces. Reports of PPFM-plant dialogue led us to examine cytokinin production by PPFMs. Using immunoaffinity and high-performance liquid chromatography (HPLC) purification, we obtained 22 to 111 ng of
trans
-zeatin per liter from culture filtrates of four PPFM leaf isolates (from
Arabidopsis
, barley, maize, and soybean) and of a
Methylobacterium extorquens
type culture originally recovered as a soil isolate. We identified the zeatin isolated as the
trans
isomer by HPLC and by a radioimmunoassay in which monoclonal antibodies specific for
trans
-hydroxylated cytokinins were used. Smaller and variable amounts of
trans
-zeatin riboside were also recovered.
trans
-Zeatin was recovered from tRNA hydrolysates in addition to the culture filtrates, suggesting that secreted
trans
-zeatin resulted from tRNA turnover rather than from de novo synthesis. The product of the
miaA
gene is responsible for isopentenylation of a specific adenine in some tRNAs. To confirm that the secreted zeatin originated from tRNA, we mutated the
miaA
gene of
M. extorquens
by single exchange of an internal
miaA
fragment into the chromosomal gene. Mutant exconjugants, confirmed by PCR, did not contain zeatin in their tRNAs and did not secrete zeatin into the medium, findings which are consistent with the hypothesis that all zeatin is tRNA derived rather than synthesized de novo. In germination studies performed with heat-treated soybean seeds, cytokinin-null (
miaA
) mutants stimulated germination as well as wild-type bacteria. While cytokinin production may play a role in the plant-PPFM interaction, it is not responsible for stimulation of germination by PPFMs.