bluuebunny/crossref_metadata_2025_split huggingface dataset:
The Completely Sequenced Plasmid pEST4011 Contains a Novel IncP1 Backbone and a Catabolic Transposon Harboring
tfd
Genes for 2,4-Dichlorophenoxyacetic Acid Degradation
Authors: Vedler Eve , Vahter Merle , Heinaru Ain 
Published: October 2004

ABSTRACT

The
herbicide 2,4-dichlorophenoxyacetic acid (2,4-D)-degrading bacterium
Achromobacter xylosoxidans
subsp.
denitrificans
strain EST4002 contains plasmid pEST4011. This plasmid ensures its host
a stable 2,4-D
+
phenotype. We determined the
complete 76,958-bp nucleotide sequence of pEST4011. This plasmid is a
deletion and duplication derivative of pD2M4, the 95-kb highly unstable
laboratory ancestor of pEST4011, and was self-generated during
different laboratory manipulations performed to increase the stability
of the 2,4-D
+
phenotype of the original strain,
strain D2M4(pD2M4). The 47,935-bp catabolic region of pEST4011 forms a
transposon-like structure with identical copies of the hybrid insertion
element IS
1071
::IS
1471
at the two
ends. The catabolic regions of pEST4011 and pJP4, the best-studied
2,4-D-degradative plasmid, both contain homologous,
tfd
-like
genes for complete 2,4-D degradation, but they have little sequence
similarity other than that. The backbone genes of pEST4011 are most
similar to the corresponding genes of broad-host-range
self-transmissible IncP1 plasmids. The backbones of the other three
IncP1 catabolic plasmids that have been sequenced (the
2,4-D-degradative plasmid pJP4, the haloacetate-catabolic plasmid pUO1,
and the atrazine-catabolic plasmid pADP-1) are nearly identical to the
backbone of R751, the archetype plasmid of the IncP1 β
subgroup. We show that despite the overall similarity in plasmid
organization, the pEST4011 backbone is sufficiently different (51 to
86% amino acid sequence identity between individual backbone
genes) from the backbones of members of the three IncP1 subgroups (theα
, β, and γ subgroups) that it belongs to a new
IncP1subgroup, the δ subgroup. This conclusion was also
supported by a phylogenetic analysis of the
trfA2
,
korA
, and
traG
gene products of different IncP1
plasmids.
Characterization of the
Escherichia coli
AaeAB Efflux Pump: a Metabolic Relief Valve?
Authors: Van Dyk Tina K. , Templeton Lori J. , Cantera Keith A. , Sharpe Pamela L. , Sariaslani F. Sima 
Published: October 2004

ABSTRACT

Treatment of
Escherichia coli
with
p
-hydroxybenzoic acid (pHBA) resulted in upregulation of
yhcP
, encoding a protein of the putative efflux protein family. Also upregulated were the adjacent genes
yhcQ
, encoding a protein of the membrane fusion protein family, and
yhcR
, encoding a small protein without a known or suggested function. The function of the upstream, divergently transcribed gene
yhcS
, encoding a regulatory protein of the LysR family, in regulating expression of
yhcRQP
was shown. Furthermore, it was demonstrated that several aromatic carboxylic acid compounds serve as inducers of
yhcRQP
expression. The efflux function encoded by
yhcP
was proven by the hypersensitivity to pHBA of a
yhcP
mutant strain. A
yhcS
mutant strain was also hypersensitive to pHBA. Expression of
yhcQ
and
yhcP
was necessary and sufficient for suppression of the pHBA hypersensitivity of the
yhcS
mutant. Only a few aromatic carboxylic acids of hundreds of diverse compounds tested were defined as substrates of the YhcQP efflux pump. Thus, we propose renaming
yhcS
,
yhcR
,
yhcQ
, and
yhcP
, to reflect their role in aromatic carboxylic acid efflux, to
aaeR
,
aaeX
,
aaeA
, and
aaeB
, respectively. The role of pHBA in normal
E. coli
metabolism and the highly regulated expression of the AaeAB efflux system suggests that the physiological role may be as a “metabolic relief valve” to alleviate toxic effects of imbalanced metabolism.
Membrane Topology Analysis of Cyclic Glucan Synthase, a Virulence Determinant of
Brucella abortus
Authors: Ciocchini Andrés E. , Roset Mara S. , Iñón de Iannino Nora , Ugalde Rodolfo A. 
Published: October 2004

ABSTRACT

Brucella abortus
cyclic glucan synthase (Cgs) is a 316-kDa (2,831-amino-acid) integral inner membrane protein that is responsible for the synthesis of cyclic β-1,2-glucan by a novel mechanism in which the enzyme itself acts as a protein intermediate.
B. abortus
Cgs uses UDP-glucose as a sugar donor and has the three enzymatic activities necessary for synthesis of the cyclic polysaccharide (i.e., initiation, elongation, and cyclization). Cyclic glucan is required in
B. abortus
for effective host interaction and complete expression of virulence. To gain further insight into the structure and mechanism of action of
B. abortus
Cgs, we studied the membrane topology of the protein using a combination of in silico predictions, a genetic approach involving the construction of fusions between the
cgs
gene and the genes encoding alkaline phosphatase (
phoA
) and β-galactosidase (
lacZ
), and site-directed chemical labeling of lysine residues. We found that
B. abortus
Cgs is a polytopic membrane protein with the amino and carboxyl termini located in the cytoplasm and with six transmembrane segments, transmembrane segments I (residues 419 to 441), II (residues 452 to 474), III (residues 819 to 841), IV (residues 847 to 869), V (residues 939 to 961), and VI (residues 968 to 990). The six transmembrane segments determine four large cytoplasmic domains and three very small periplasmic regions.
Ribose Utilization with an Excess of Mutarotase Causes Cell Death Due to Accumulation of Methylglyoxal
Authors: Kim Insook , Kim Eunjung , Yoo Seokho , Shin Daesung , Min Bumchan , Song Jeeyeon , Park Chankyu 
Published: October 2004

ABSTRACT

Methylglyoxal (MG) is a highly reactive metabolic intermediate, presumably accumulated during uncontrolled carbohydrate metabolism. The major source of MG is dihydroxyacetone phosphate, which is catalyzed by MG synthase (the
mgs
product) in bacteria. We observed
Escherichia coli
cell death when the ribose transport system, consisting of the RbsDACBK proteins, was overproduced on multicopy plasmids. Almost 100% of cell death occurs a few hours after ribose addition (>10 mM), due to an accumulation of extracellular MG as detected by
1
H-nuclear magnetic resonance (
1
H-NMR). Under lethal conditions, the concentration of MG produced in the medium reached approximately 1 mM after 4 h of ribose addition as measured by high-performance liquid chromatography. An excess of the protein RbsD, recently characterized as a mutarotase that catalyzes the conversion between the β-pyran and β-furan forms of ribose, was critical in accumulating the lethal level of MG, which was also shown to require ribokinase (RbsK). The intracellular level of ribose 5-phosphate increased with the presence of the protein RbsD, as determined by
31
P-NMR. As expected, a mutation in the methylglyoxal synthase gene (
mgs
) abolished the production of MG. These results indicate that the enhanced ribose uptake and incorporation lead to an accumulation of MG, perhaps occurring via the pentose-phosphate pathway and via glycolysis with the intermediates fructose 6-phosphate and glyceraldehyde 3-phosphate. It was also demonstrated that a small amount of MG is synthesized by monoamine oxidase.
The “Intracellular” Poly(3-Hydroxybutyrate) (PHB) Depolymerase of
Rhodospirillum rubrum
Is a Periplasm-Located Protein with Specificity for Native PHB and with Structural Similarity to Extracellular PHB Depolymerases
Authors: Handrick René , Reinhardt Simone , Kimmig Philipp , Jendrossek Dieter 
Published: October 2004

ABSTRACT

Rhodospirillum rubrum
possesses a putative intracellular poly(3-hydroxybutyrate) (PHB) depolymerase system consisting of a soluble PHB depolymerase, a heat-stable activator, and a 3-hydroxybutyrate dimer hydrolase (J. M. Merrick and M. Doudoroff, J. Bacteriol.
88:
60-71, 1964). In this study we reinvestigated the soluble
R. rubrum
PHB depolymerase (PhaZ1). It turned out that PhaZ1 is a novel type of PHB depolymerase with unique properties. Purified PhaZ1 was specific for amorphous short-chain-length polyhydroxyalkanoates (PHA) such as native PHB, artificial PHB, and oligomer esters of (
R
)-3-hydroxybutyrate with 3 or more 3-hydroxybutyrate units. Atactic PHB, (
S
)-3-hydroxybutyrate oligomers, medium-chain-length PHA, and lipase substrates (triolein, tributyrin) were not hydrolyzed. The PHB depolymerase structural gene (
phaZ1
) was cloned. Its deduced amino acid sequence (37,704 Da) had no significant similarity to those of intracellular PHB depolymerases of
Wautersia eutropha
or of other PHB-accumulating bacteria. PhaZ1 was found to have strong amino acid homology with type-II catalytic domains of extracellular PHB depolymerases, and Ser
42
, Asp
138
, and His
178
were identified as catalytic-triad amino acids, with Ser
42
as the putative active site. Surprisingly, the first 23 amino acids of the PHB depolymerase previously assumed to be intracellular revealed features of classical signal peptides, and Edman sequencing of purified PhaZ1 confirmed the functionality of the predicted cleavage site. Extracellular PHB depolymerase activity was absent, and analysis of cell fractions unequivocally showed that PhaZ1 is a periplasm-located enzyme. The previously assumed intracellular activator/depolymerase system is unlikely to have a physiological function in PHB mobilization in vivo. A second gene, encoding the putative true intracellular PHB depolymerase (PhaZ2), was identified in the genome sequence of
R. rubrum
.
Osa Protein Constitutes a Strong Oncogenic Suppression System That Can Block
vir
-Dependent Transfer of IncQ Plasmids between
Agrobacterium
Cells and the Establishment of IncQ Plasmids in Plant Cells
Authors: Lee Lan-Ying , Gelvin Stanton B. 
Published: October 2004

ABSTRACT

The
osa
(oncogenic suppressive activity) gene of the IncW group plasmid pSa is sufficient to suppress tumorigenesis by
Agrobacterium tumefaciens. osa
confers oncogenic suppression by inhibiting VirE2 protein export. This result is similar, but not identical, to that of oncogenic suppression by the IncQ plasmid RSF1010. We conducted a series of experiments to compare oncogenic suppression by these two systems.
Agrobacterium
strains harboring plasmids containing
osa
are more able to effect oncogenic suppression than are similar strains containing various RSF1010 derivatives. When
osa
is present within a donor
Agrobacterium
strain that also carries a derivative of RSF1010, the transfer of RSF1010 derivatives to recipient bacteria and their establishment in plants are blocked. Oncogenic suppression is still effected when the
osa
gene is integrated into the
Agrobacterium
chromosome, suggesting that it is the
osa
gene product that is active in suppression and that suppression does not require a protein-nucleic acid intermediate like that described for IncQ plasmids. Extracellular complementation experiments with tobacco leaf disks indicated that Osa blocks stable transfer of RSF1010 to plant cells by inhibiting transfer of VirE2, which is essential for the transfer of RSF1010 into plant cells, and not by inhibiting the actual transfer of RSF1010 itself. Our results suggest that Osa and RSF1010 cause oncogenic suppression by using different mechanisms.
Construction of anEnterococcus faecalisTn917-Mediated-Gene-Disruption Library Offers Insight into Tn917Insertion Patterns
Authors: Garsin Danielle A. , Urbach Jonathan , Huguet-Tapia Jose C. , Peters Joseph E. , Ausubel Frederick M. 
Published: October 2004

ABSTRACTSequencing the insertion sites of 8,865 Tn917insertions inEnterococcus faecalisstrain OG1RF identified a hot spot in the replication terminus region corresponding to 6% of the genome where 65% of the transposons had inserted. InE. faecalis, Tn917preferentially inserted at a 29-bp consensus sequence centered on TATAA, a 5-bp sequence that is duplicated during insertion. The regional insertion site preference at the chromosome terminus was not observed in another low-G+C gram-positive bacterium,Listeria monocytogenes, although the consensus insertion sequence was the same. The 8,865 Tn917insertion sites sequenced inE. faecaliscorresponded to only ∼610 different open reading frames, far fewer than the predicted number of 2,400, assuming random insertion. There was no significant preference in orientation of the Tn917insertions with either transcription or replication. Even though OG1RF has a smaller genome than strain V583 (2.8 Mb versus 3.2 Mb), the onlyE. faecalisstrain whose sequence is in the public domain, over 10% of the Tn917insertions appear to be in a OG1RF-specific sequence, suggesting that there are significant genomic differences amongE. faecalisstrains.
TouR-Mediated Effector-Independent Growth Phase-Dependent Activation of the σ54PtouPromoter ofPseudomonas stutzeriOX1
Authors: Solera Dafne , Arenghi Fabio L. G. , Woelk Tanja , Galli Enrica , Barbieri Paola 
Published: October 2004

ABSTRACTTranscription of the catabolictouABCDEFoperon, encoding the toluene-o-xylene monooxygenase ofPseudomonas stutzeriOX1, is driven by the σ54-dependent Ptoupromoter, whose activity is controlled by the phenol-responsive NtrC-like activator TouR. In this paper we describe for the first time a peculiar characteristic of this system, namely, that Ptoutranscription is activated in a growth phase-dependent manner in the absence of genuine effectors of the cognate TouR regulator. This phenomenon, which we named gratuitous activation, was observed in the native strainP. stutzeriOX1, as well as in aPseudomonas putidaPaW340 host harboring the reconstructedtouregulatory circuit. Regulator-promoter swapping experiments demonstrated that the presence of TouR is necessary and sufficient for imposing gratuitous activation on the Ptoupromoter, as well as on other σ54-dependent catabolic promoters, whereas the highly similar phenol-responsive activator DmpR is unable to activate the Ptoupromoter in the absence of effectors. We show that this phenomenon is specifically triggered by carbon source exhaustion but not by nitrogen starvation. An updated model of thetouregulatory circuit is presented.
Diversion of the Metabolic Flux from Pyruvate Dehydrogenase to Pyruvate Oxidase Decreases Oxidative Stress during Glucose Metabolism in Nongrowing
Escherichia coli
Cells Incubated under Aerobic, Phosphate Starvation Conditions
Authors: Moreau Patrice L. 
Published: October 2004

ABSTRACT

Ongoing aerobic metabolism in nongrowing cells may generate oxidative stress. It is shown here that the levels of thiobarbituric acid-reactive substances (TBARSs), which measure fragmentation products of oxidized molecules, increased strongly at the onset of starvation for phosphate (P
i
). This increase in TBARS levels required the activity of the histone-like nucleoid-structuring (H-NS) protein. TBARS levels weakly increased further in Δ
ahpCF
mutants deficient in alkyl hydroperoxide reductase (AHP) activity during prolonged metabolism of glucose to acetate. Inactivation of pyruvate oxidase (PoxB) activity decreased the production of acetate by half and significantly increased the production of TBARS. Overall, these data suggest that during incubation under aerobic, P
i
starvation conditions, metabolic flux is diverted from the pyruvate dehydrogenase (PDH) complex (NAD dependent) to PoxB (NAD independent). This shift may decrease the production of NADH and in turn the adventitious production of H
2
O
2
by NADH dehydrogenase in the respiratory chain. The residual low levels of H
2
O
2
produced during prolonged incubation can be scavenged efficiently by AHP. However, high levels of H
2
O
2
may be reached transiently at the onset of stationary phase, primarily because H-NS may delay the metabolic shift from PDH to PoxB.
Analysis of Promoter Elements Involved in the Transcriptional Initiation of RpoS-DependentBorrelia burgdorferiGenes
Authors: Eggers Christian H. , Caimano Melissa J. , Radolf Justin D. 
Published: October 2004

ABSTRACTBorrelia burgdorferi, the causative agent of Lyme disease, encodes an RpoS ortholog (RpoSBb) that controls the temperature-inducible differential expression of at least some of the spirochete's lipoprotein genes, includingospCanddbpBA. To begin to dissect the determinants of RpoSBbrecognition of, and selectivity for, its dependent promoters, we linked a green fluorescent protein reporter to the promoter regions of severalB. burgdorferigenes with well-characterized expression patterns. Consistent with the expression patterns of the native genes/proteins inB. burgdorferistrain 297, we found that expression of theospC,dbpBA, andospFreporters in the spirochete was RpoSBbdependent, while theospEandflaBreporters were RpoSBbindependent. To compare promoter recognition by RpoSBbwith that of the prototype RpoS (RpoSEc), we also introduced our panel of constructs intoEscherichia coli. In this surrogate, maximal expression from theospC,dbpBA, andospFpromoters clearly required RpoS, although in the absence of RpoSEctheospFpromoter was weakly recognized by anotherE. colisigma factor. Furthermore, RpoSBbunder the control of an inducible promoter was able to complement anE. coli rpoSmutant, although RpoSEcand RpoSBbeach initiated greater activity from their own dependent promoters than they did from those of the heterologous sigma factor. Genetic analysis of theospCpromoter demonstrated that (i) the T(−14) in the presumptive −10 region plays an important role in sigma factor recognition in both organisms but is not as critical for transcriptional initiation by RpoSBbas it is for RpoSEc; (ii) the nucleotide at the −15 position determines RpoS or σ70selectivity inE. colibut does not serve the same function inB. burgdorferi; and (iii) the 110-bp region upstream of the core promoter is not required for RpoSEc- or RpoSBb-dependent activity inE. colibut is required for maximal expression from this promoter inB. burgdorferi. Taken together, the results of our studies suggest that theB. burgdorferiandE. coliRpoS proteins are able to catalyze transcription from RpoS-dependent promoters of either organism, but at least some of the nucleotide elements involved in transcriptional initiation and sigma factor selection inB. burgdorferiplay a different role than has been described forE. coli.
RpoS Proteolysis Is Regulated by a Mechanism That Does Not Require the SprE (RssB) Response Regulator Phosphorylation Site
Authors: Peterson Celeste N. , Ruiz Natividad , Silhavy Thomas J. 
Published: October 2004

ABSTRACTInEscherichia colithe response regulator SprE (RssB) facilitates degradation of the sigma factor RpoS by delivering it to the ClpXP protease. This process is regulated: RpoS is degraded in logarithmic phase but becomes stable upon carbon starvation, resulting in its accumulation. Because SprE contains a CheY domain with a conserved phosphorylation site (D58), the prevailing model posits that this control is mediated by phosphorylation. To test this model, we mutated the conserved response regulator phosphorylation site (D58A) of the chromosomal allele ofsprEand monitored RpoS levels in response to carbon starvation. Though phosphorylation contributed to the SprE basal activity, we found that RpoS proteolysis was still regulated upon carbon starvation. Furthermore, our results indicate that phosphorylation of wild-type SprE occurs by a mechanism that is independent of acetyl phosphate.
AplA, a Member of a New Class of Phycobiliproteins Lacking a Traditional Role in Photosynthetic Light Harvesting
Authors: Montgomery Beronda L. , Casey Elena Silva , Grossman Arthur R. , Kehoe David M. 
Published: October 2004

ABSTRACT

All known phycobiliproteins have light-harvesting roles during photosynthesis and are found in water-soluble phycobilisomes, the light-harvesting complexes of cyanobacteria, cyanelles, and red algae. Phycobiliproteins are chromophore-bearing proteins that exist as heterodimers of α and β subunits, possess a number of highly conserved amino acid residues important for dimerization and chromophore binding, and are invariably 160 to 180 amino acids long. A new and unusual group of proteins that is most closely related to the allophycocyanin members of the phycobiliprotein superfamily has been identified. Each of these proteins, which have been named allophycocyanin-like (Apl) proteins, apparently contains a 28-amino-acid extension at its amino terminus relative to allophycocyanins. Apl family members possess the residues critical for chromophore interactions, but substitutions are present at positions implicated in maintaining the proper α-β subunit interactions and tertiary structure of phycobiliproteins, suggesting that Apl proteins are able to bind chromophores but fail to adopt typical allophycocyanin conformations. AplA isolated from the cyanobacterium
Fremyella diplosiphon
contained a covalently attached chromophore and, although present in the cell under a number of conditions, was not detected in phycobilisomes. Thus, Apl proteins are a new class of photoreceptors with a different cellular location and structure than any previously described members of the phycobiliprotein superfamily.
Biosynthesis of Cryptic Lipopolysaccharide Glycoforms in
Haemophilus influenzae
Involves a Mechanism Similar to That Required for O-Antigen Synthesis
Authors: Hood Derek W. , Randle Gaynor , Cox Andrew D. , Makepeace Katherine , Li Jianjun , Schweda Elke K. H. , Richards James C. , Moxon E. Richard 
Published: October 2004

ABSTRACT

It is generally thought that mucosal bacterial pathogens of the genera
Haemophilus
,
Neisseria
, and
Moraxella
elaborate lipopolysaccharide (LPS) that is fundamentally different from that of enteric organisms that express O-specific polysaccharide side chains.
Haemophilus influenzae
elaborates short-chain LPS that has a role in the pathogenesis of
H. influenzae
infections. We show that the synthesis of LPS in this organism can no longer be as clearly distinguished from that in other gram-negative bacteria that express an O antigen. We provide evidence that a region of the
H. influenzae
genome, the
hmg
locus, is involved in the synthesis of glycoforms in which tetrasaccharide units are added en bloc, not stepwise, to the normal core glycoforms, similar to the biosynthesis of an O-antigen.
Increased Stability ofbmr3mRNA Results in a Multidrug-Resistant Phenotype inBacillus subtilis
Authors: Ohki Reiko , Tateno Kozue 
Published: October 2004

ABSTRACTA spontaneous mutant isolated in the presence of a high concentration of puromycin acquired a multidrug-resistant phenotype. Expression of thebmr3gene was dramatically increased. A base substitution, T to A at the +4 position, detected in the mutant resulted in the stabilization ofbmr3mRNA.
The Aer Protein of
Escherichia coli
Forms a Homodimer Independent of the Signaling Domain and Flavin Adenine Dinucleotide Binding
Authors: Ma Qinhong , Roy Francis , Herrmann Sarah , Taylor Barry L. , Johnson Mark S. 
Published: October 2004

ABSTRACT

In vivo cross-linking between native cysteines in the Aer receptor of
Escherichia coli
showed dimer formation at the membrane anchor and in the putative HAMP domain. Dimers also formed in mutants that did not bind flavin adenine dinucleotide and in truncated peptides without a signaling domain and part of the HAMP domain.
Molecular Basis of the Indole-Negative Reaction in
Shigella
Strains: Extensive Damages to the
tna
Operon by Insertion Sequences
Authors: Rezwan Ferdousi , Lan Ruiting , Reeves Peter R. 
Published: October 2004

ABSTRACT

The molecular basis of the loss of tryptophan utilization (indole-negative phenotype) of
Shigella
strains, in effect clones of
Escherichia coli
, was investigated. Analysis of the
tna
operon of 23
Shigella
strains representing each of the indole-negative serotypes revealed that insertion sequence-mediated insertion and/or deletions damaged the
tna
operon, leading to inability to convert tryptophan to indole. These events differ for cluster 1, cluster 3, and the outlier
Shigella
strains, confirming our previous observation of independent origins of these lineages from within
E. coli
. Parallel loss of the trait and prevalence of indole-negative strains suggest that the trait is deleterious in
Shigella
strains and advantages those without it.