bluuebunny/crossref_metadata_2025_split huggingface dataset:
Tricksy Business: Transcriptome Analysis Reveals the Involvement of Thioredoxin A in Redox Homeostasis, Oxidative Stress, Sulfur Metabolism, and Cellular Differentiation inBacillus subtilis
Authors: Smits Wiep Klaas , Dubois Jean-Yves F. , Bron Sierd , van Dijl Jan Maarten , Kuipers Oscar P. 
Published: June 2005

ABSTRACTThioredoxins are important thiol-reactive proteins. Most knowledge about this class of proteins is derived from proteome studies, and little is known about the global transcriptional response of cells to various thioredoxin levels. InBacillus subtilis, thioredoxin A is encoded bytrxAand is essential for viability. In this study, we report the effects of minimal induction of a strain carrying an IPTG (isopropyl-β-d-thiogalactopyranoside)-inducibletrxAgene (ItrxA) on transcription levels, as determined by DNA macroarrays. The effective depletion of thioredoxin A leads to the induction of genes involved in the oxidative stress response (but not those dependent on PerR), phage-related functions, and sulfur utilization. Also, several stationary-phase processes, such as sporulation and competence, are affected. The majority of these phenotypes are rescued by a higher induction level of ItrxA, leading to an approximately wild-type level of thioredoxin A protein. A comparison with other studies shows that the effects of thioredoxin depletion are distinct from, but show some similarity to, oxidative stress and disulfide stress. Some of the transcriptional effects may be linked to thioredoxin-interacting proteins. Finally, thioredoxin-linked processes appear to be conserved between prokaryotes and eukaryotes.
Mutations in Cytochrome Assembly and Periplasmic Redox Pathways inBordetella pertussis
Authors: Feissner Robert E. , Beckett Caroline S. , Loughman Jennifer A. , Kranz Robert G. 
Published: June 2005

ABSTRACTTransposon mutagenesis ofBordetella pertussiswas used to discover mutations in the cytochromecbiogenesis pathway called system II. Using a tetramethyl-p-phenylenediamine cytochromecoxidase screen, 27 oxidase-negative mutants were isolated and characterized. Nine mutants were still able to synthesizec-type cytochromes and possessed insertions in the genes for cytochromecoxidase subunits (ctaC, -D, and -E), hemeabiosynthesis (ctaB), assembly of cytochromecoxidase (sco2), or ferrochelatase (hemZ). Eighteen mutants were unable to synthesize allc-type cytochromes. Seven of these had transposons indipZ(dsbD), encoding the transmembrane thioreduction protein, and all seven mutants were corrected for cytochromecassembly by exogenous dithiothreitol, which was consistent with the cytochromeccysteinyl residues of the CXXCH motif requiring periplasmic reduction. The remaining 11 insertions were located in theccsBAoperon, suggesting that with the appropriate thiol-reducing environment, the CcsB and CcsA proteins comprise the entire system II biosynthetic pathway. Antiserum to CcsB was used to show that CcsB is absent inccsAmutants, providing evidence for a stable CcsA-CcsB complex. No mutations were found in the genes necessary for disulfide bond formation (dsbAordsbB). To examine whether the periplasmic disulfide bond pathway is required for cytochromecbiogenesis inB. pertussis, a targeted knockout was made indsbB. The DsbB−mutant makes holocytochromesclike the wild type does and secretes and assembles the active periplasmic alkaline phosphatase. AdipZmutant is not corrected by adsbBmutation. Alternative mechanisms to oxidize disulfides inB. pertussisare analyzed and discussed.
Peculiar Properties of DsbA in Its Export across the
Escherichia coli
Cytoplasmic Membrane
Authors: Shimohata Nobuyuki , Akiyama Yoshinori , Ito Koreaki 
Published: June 2005

ABSTRACT

Export of DsbA, a protein disulfide bond-introducing enzyme, across the
Escherichia coli
cytoplasmic membrane was studied with special reference to the effects of various mutations affecting translocation factors. It was noted that both the internalized precursor retaining the signal peptide and the periplasmic mature product fold rapidly into a protease-resistant structure and they exhibited anomalies in sodium dodecyl sulfate-polyacrylamide gel electrophoresis in that the former migrated faster than the latter. The precursor, once accumulated, was not exported posttranslationally. DsbA export depended on the SecY translocon, the SecA ATPase, and Ffh (signal recognition particle), but not on SecB. SecY mutations, such as
secY39
and
secY205
, that severely impair translocation of a number of secretory substrates by interfering with SecA actions only insignificantly impaired the DsbA export. In contrast,
secY125
, affecting a periplasmic domain and impairing a late step of translocation, exerted strong export inhibition of both classes of proteins. These results suggest that DsbA uses not only the signal recognition particle targeting pathway but also a special route of translocation through the translocon, which is hence suggested to actively discriminate preproteins.
The
Haloferax volcanii
FtsY Homolog Is Critical for Haloarchaeal Growth but Does Not Require the A Domain
Authors: Haddad Alex , Rose R. Wesley , Pohlschröder Mechthild 
Published: June 2005

ABSTRACT

The targeting of many Sec substrates to the membrane-associated translocation pore requires the cytoplasmic signal recognition particle (SRP). In
Eukarya
and
Bacteria
it has been shown that membrane docking of the SRP-substrate complex occurs via the universally conserved SRP receptor (Srα/β and FtsY, respectively). While much has been learned about the archaeal SRP in recent years, few studies have examined archaeal Srα/FtsY homologs. In the present study the FtsY homolog of
Haloferax volcanii
was characterized in its native host. Disruption of the sole chromosomal copy of
ftsY
in
H. volcanii
was possible only under conditions where either the full-length haloarchaeal FtsY or an amino-terminally truncated version of this protein lacking the A domain, was expressed in
trans
. Subcellular fractionation analysis of
H. volcanii ftsY
deletion strains expressing either one of the complementing proteins revealed that in addition to a cytoplasmic pool, both proteins cofractionate with the haloarchaeal cytoplasmic membrane. Moreover, membrane localization of the universally conserved SRP subunit SRP54, the key binding partner of FtsY, was detected in both
H. volcanii
strains. These analyses suggest that the
H. volcanii
FtsY homolog plays a crucial role but does not require its A domain for haloarchaeal growth.
Expression Levels Influence Ribosomal Frameshifting at the Tandem Rare Arginine Codons AGG_AGG and AGA_AGA in
Escherichia coli
Authors: Gurvich Olga L. , Baranov Pavel V. , Gesteland Raymond F. , Atkins John F. 
Published: June 2005

ABSTRACT

The rare codons AGG and AGA comprise 2% and 4%, respectively, of the arginine codons of
Escherichia coli
K-12, and their cognate tRNAs are sparse. At tandem occurrences of either rare codon, the paucity of cognate aminoacyl tRNAs for the second codon of the pair facilitates peptidyl-tRNA shifting to the +1 frame. However, AGG_AGG and AGA_AGA are not underrepresented and occur 4 and 42 times, respectively, in
E. coli
genes. Searches for corresponding occurrences in other bacteria provide no strong support for the functional utilization of frameshifting at these sequences. All sequences tested in their native context showed 1.5 to 11% frameshifting when expressed from multicopy plasmids. A cassette with one of these sequences singly integrated into the chromosome in stringent cells gave 0.9% frameshifting in contrast to two- to four-times-higher values obtained from multicopy plasmids in stringent cells and eight-times-higher values in relaxed cells. Thus, +1 frameshifting efficiency at AGG_AGG and AGA_AGA is influenced by the mRNA expression level. These tandem rare codons do not occur in highly expressed mRNAs.
Analysis of
Pseudomonas putida
KT2440 Gene Expression in the Maize Rhizosphere: In Vitro Expression Technology Capture and Identification of Root-Activated Promoters
Authors: Ramos-González María Isabel , Campos María Jesús , Ramos Juan L. 
Published: June 2005

ABSTRACT

Pseudomonas putida
KT2440, a paradigm organism in biodegradation and a good competitive colonizer of the maize rhizosphere, was the subject of studies undertaken to establish the genetic determinants important for its rhizospheric lifestyle. By using in vivo expression technology (IVET) to positively select single cell survival, we identified 28
rap
genes (
r
oot-
a
ctivated
p
romoters) preferentially expressed in the maize rhizosphere. The IVET system had two components: a mutant affected in aspartate-β-semialdehyde dehydrogenase (
asd
), which was unable to survive in the rhizosphere, and plasmid pOR1, which carries a promoterless
asd
gene. pOR1-borne transcriptional fusions of the
rap
promoters to the essential gene
asd
, which were integrated into the chromosome at the original position of the corresponding
rap
gene, were active and allowed growth of the
asd
strain in the rhizosphere. The fact that five of the
rap
genes identified in the course of this work had been formerly characterized as being related to root colonization reinforced the IVET approach. Up to nine
rap
genes encoded proteins either of unknown function or that had been assigned an unspecific role based on conservation of the protein family domains. Rhizosphere-induced fusions included genes with probable functions in the cell envelope, chemotaxis and motility, transport, secretion, DNA metabolism and defense mechanism, regulation, energy metabolism, stress, detoxification, and protein synthesis.
Sulfate-Dependent Repression of Genes That Function in Organosulfur Metabolism inBacillus subtilisRequires Spx
Authors: Erwin Kyle N. , Nakano Shunji , Zuber Peter 
Published: June 2005

ABSTRACTOxidative stress inBacillus subtilisresults in the accumulation of Spx protein, which exerts both positive and negative transcriptional control over a genome-wide scale through its interaction with the RNA polymerase α subunit. Previous microarray transcriptome studies uncovered a unique class of genes that are controlled by Spx-RNA polymerase interaction under normal growth conditions that do not promote Spx overproduction. These genes were repressed by Spx when sulfate was present as a sole sulfur source. The genes include those of theytmI,yxeI, andssuoperons, which encode products resembling proteins that function in the uptake and desulfurization of organic sulfur compounds. Primer extension and analysis of operon-lacZfusion expression revealed that the operons are repressed by sulfate and cysteine; however, Spx functioned only in sulfate-dependent repression. Both theytmIoperon and the divergently transcribedytlI, encoding a LysR-type regulator that positively controlsytmIoperon transcription, are repressed by Spx in sulfate-containing media. The CXXC motif of Spx, which is necessary for redox sensitive control of Spx activity in response to oxidative stress, is not required for sulfate-dependent repression. TheyxeL-lacZandssu-lacZfusions were also repressed in an Spx-dependent manner in media containing sulfate as the sole sulfur source. This work uncovers a new role for Spx in the control of sulfur metabolism in a gram-positive bacterium under nonstressful growth conditions.
Catabolism of Benzoate and Phthalate in
Rhodococcus
sp. Strain RHA1: Redundancies and Convergence
Authors: Patrauchan Marianna A. , Florizone Christine , Dosanjh Manisha , Mohn William W. , Davies Julian , Eltis Lindsay D. 
Published: June 2005

ABSTRACT

Genomic and proteomic approaches were used to investigate phthalate and benzoate catabolism in
Rhodococcus
sp. strain RHA1, a polychlorinated biphenyl-degrading actinomycete. Sequence analyses identified genes involved in the catabolism of benzoate (
ben
) and phthalate (
pad
), the uptake of phthalate (
pat
), and two branches of the β-ketoadipate pathway (
catRABC
and
pcaJIHGBLFR
). The regulatory and structural
ben
genes are separated by genes encoding a cytochrome P450. The
pad
and
pat
genes are contained on a catabolic island that is duplicated on plasmids pRHL1 and pRHL2 and includes predicted terephthalate catabolic genes (
tpa
). Proteomic analyses demonstrated that the β-ketoadipate pathway is functionally convergent. Specifically, the
pad
and
pat
gene products were only detected in phthalate-grown cells. Similarly, the
ben
and
cat
gene products were only detected in benzoate-grown cells. However,
pca
-encoded enzymes were present under both growth conditions. Activity assays for key enzymes confirmed these results. Disruption of
pcaL
, which encodes a fusion enzyme, abolished growth on phthalate. In contrast, after a lag phase, growth of the mutant on benzoate was similar to that of the wild type. Proteomic analyses revealed 20 proteins in the mutant that were not detected in wild-type cells during growth on benzoate, including a CatD homolog that apparently compensated for loss of PcaL. Analysis of completed bacterial genomes indicates that the convergent β-ketoadipate pathway and some aspects of its genetic organization are characteristic of rhodococci and related actinomycetes. In contrast, the high redundancy of catabolic pathways and enzymes appears to be unique to RHA1 and may increase its potential to adapt to new carbon sources.
Regulation and Function of Cytochrome
c
′ in
Rhodobacter sphaeroides
2.4.3
Authors: Choi Peter S. , Grigoryants Vladimir M. , Abruña Hector D. , Scholes Charles P. , Shapleigh James P. 
Published: June 2005

ABSTRACT

Cytochrome
c
′ (Cyt
c
′) is a
c
-type cytochrome with a pentacoordinate heme iron. The gene encoding this protein in
Rhodobacter sphaeroides
2.4.3, designated
cycP
, was isolated and sequenced. Northern blot analysis and β-galactosidase assays demonstrated that
cycP
transcription increased as oxygen levels decreased and was not repressed under denitrifying conditions as observed in another
Rhodobacter
species. CO difference spectra performed with extracts of cells grown under different conditions revealed that Cyt
c
′ levels were highest during photosynthetic denitrifying growth conditions. The increase in Cyt
c
′ under this condition was higher than would be predicted from transcriptional studies. Electron paramagnetic resonance analysis of whole cells demonstrated that Cyt
c
′ binds NO during denitrification. Mass spectrometric analysis of nitrogen oxides produced by cells and purified protein did not indicate that Cyt
c
′ has NO reductase activity. Taken together, these results suggest a model where Cyt
c
′ in
R. sphaeroides
2.4.3 may shuttle NO to the membrane, where it can be reduced.
Gene Conversion Tracts Associated with Crossovers in
Rhizobium etli
Authors: Santoyo Gustavo , Martínez-Salazar Jaime M. , Rodríguez César , Romero David 
Published: June 2005

ABSTRACT

Gene conversion has been defined as the nonreciprocal transfer of information between homologous sequences. Despite its broad interest for genome evolution, the occurrence of this mechanism in bacteria has been difficult to ascertain due to the possible occurrence of multiple crossover events that would mimic gene conversion. In this work, we employ a novel system, based on cointegrate formation, to isolate gene conversion events associated with crossovers in the nitrogen-fixing bacterium
Rhizobium etli
. In this system, selection is applied only for cointegrate formation, with gene conversions being detected as unselected events. This minimizes the likelihood of multiple crossovers. To track the extent and architecture of gene conversions, evenly spaced nucleotide changes were made in one of the nitrogenase structural genes (
nifH
), introducing unique sites for different restriction endonucleases. Our results show that (i) crossover events were almost invariably accompanied by a gene conversion event occurring nearby; (ii) gene conversion events ranged in size from 150 bp to 800 bp; (iii) gene conversion events displayed a strong bias, favoring the preservation of incoming sequences; (iv) even small amounts of sequence divergence had a strong effect on recombination frequency; and (v) the MutS mismatch repair system plays an important role in determining the length of gene conversion segments. A detailed analysis of the architecture of the conversion events suggests that multiple crossovers are an unlikely alternative for their generation. Our results are better explained as the product of true gene conversions occurring under the double-strand break repair model for recombination.
A Region ofBacillus subtilisCodY Protein Required for Interaction with DNA
Authors: Joseph Pascale , Ratnayake-Lecamwasam Manoja , Sonenshein Abraham L. 
Published: June 2005

ABSTRACTBacillus subtilisCodY protein is the best-studied member of a novel family of global transcriptional regulators found ubiquitously in low-G+C gram-positive bacteria. As for many DNA-binding proteins, CodY appears to have a helix-turn-helix (HTH) motif thought to be critical for interaction with DNA. This putative HTH motif was found to be highly conserved in the CodY homologs. Site-directed mutagenesis was used to identify amino acids within this motif that are important for DNA recognition and binding. The effects of each mutation on DNA binding in vitro and on the regulation of transcription in vivo from two target promoters were tested. Each of the mutations had similar effects on binding to the two promoters in vitro, but some mutations had differential effects in vivo.
Mutagenesis at Two Distinct Phosphate-Binding Sites Unravels Their Differential Roles in Regulation of Rubisco Activation and Catalysis
Authors: Marcus Yehouda , Altman-Gueta Hagit , Finkler Aliza , Gurevitz Michael 
Published: June 2005

ABSTRACT

Orthophosphate (P
i
) has two antagonistic effects on ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco), stimulation of activation and inhibition of catalysis by competition with the substrate RuBP. The enzyme binds P
i
at three distinct sites, two within the catalytic site (where 1P and 5P of ribulose 1,5-bisphosphate [RuBP] bind), and the third at the latch site (a positively charged pocket involved in active-site closure during catalysis). We examined the role of the latch and 5P sites in regulation of Rubisco activation and catalysis by introducing specific mutations in the enzyme of the cyanobacterium
Synechocystis
sp. strain PCC 6803. Whereas mutations at both sites abolished the P
i
-stimulated Rubisco activation, substitution of residues at the 5P site, but not at the latch site, affected the P
i
inhibition of Rubisco catalysis. Although some of these mutations substantially reduced the catalytic turnover of Rubisco and increased the

K
m

(RuBP), they had little to moderate effect on the rate of photosynthesis and no effect on photoautotrophic growth. These findings suggest that in cyanobacteria, Rubisco does not limit photosynthesis to the extent previously estimated. These results indicate that both the latch and 5P sites participate in regulation of Rubisco activation, whereas P
i
binding only at the 5P site inhibits catalysis in a competitive manner.
Crystal Structure of the “PhoU-Like” Phosphate Uptake Regulator from
Aquifex aeolicus
Authors: Oganesyan Vaheh , Oganesyan Natalia , Adams Paul D. , Jancarik Jaru , Yokota Hisao A. , Kim Rosalind , Kim Sung-Hou 
Published: June 2005

ABSTRACT

The
phoU
gene of
Aquifex aeolicus
encodes a protein called PHOU_AQUAE with sequence similarity to the PhoU protein of
Escherichia coli.
Despite the fact that there is a large number of family members (more than 300) attributed to almost all known bacteria and despite PHOU_AQUAE's association with the regulation of genes for phosphate metabolism, the nature of its regulatory function is not well understood. Nearly one-half of these PhoU-like proteins, including both PHOU_AQUAE and the one from
E. coli
, form a subfamily with an apparent dimer structure of two PhoU domains on the basis of their amino acid sequence. The crystal structure of PHOU_AQUAE (a 221-amino-acid protein) reveals two similar coiled-coil PhoU domains, each forming a three-helix bundle. The structures of PHOU_AQUAE proteins from both a soluble fraction and refolded inclusion bodies (at resolutions of 2.8 and 3.2Å, respectively) showed no significant differences. The folds of the PhoU domain and Bag domains (for a class of cofactors of the eukaryotic chaperone Hsp70 family) are similar. Accordingly, we propose that gene regulation by PhoU may occur by association of PHOU_AQUAE with the ATPase domain of the histidine kinase PhoR, promoting release of its substrate PhoB. Other proteins that share the PhoU domain fold include the coiled-coil domains of the STAT protein, the ribosome-recycling factor, and structural proteins like spectrin.
Pseudomonas syringae
Type III Chaperones ShcO1, ShcS1, and ShcS2 Facilitate Translocation of Their Cognate Effectors and Can Substitute for Each Other in the Secretion of HopO1-1
Authors: Guo Ming , Chancey Scott T. , Tian Fang , Ge Zhengxiang , Jamir Yashitola , Alfano James R. 
Published: June 2005

ABSTRACT

The
Pseudomonas syringae
type III secretion system (TTSS) translocates effector proteins into plant cells. Several
P. syringae
effectors require accessory proteins called type III chaperones (TTCs) to be secreted via the TTSS. We characterized the
hopO1-1
,
hopS1
, and
hopS2
operons in
P. syringae
pv. tomato DC3000; these operons encode three homologous TTCs, ShcO1, ShcS1, and ShcS2. ShcO1, ShcS1, and ShcS2 facilitated the type III secretion and/or translocation of their cognate effectors HopO1-1, HopS1, and HopS2, respectively. ShcO1 and HopO1-1 interacted with each other in yeast two-hybrid and coimmunoprecipitation assays. Interestingly, ShcS1 and ShcS2 were capable of substituting for ShcO1 in facilitating HopO1-1 secretion and translocation and each TTC was able to bind the other's cognate effectors in yeast two-hybrid assays. Moreover, ShcO1, ShcS1, and ShcS2 all bound to the middle-third region of HopO1-1. The HopS2 effector possessed atypical
P. syringae
TTSS N-terminal characteristics and was translocated in low amounts. A site-directed HopS2 mutation that introduced a common N-terminal characteristic from other
P. syringae
type III secreted substrates increased HopS2 translocation, supporting the idea that this characteristic functions as a secretion signal. Additionally,
hopO1-2
and
hopT1-2
were shown to encode effectors secreted via the DC3000 TTSS. Finally, a DC3000
hopO1-1
operon deletion mutant produced disease symptoms similar to those seen with wild-type DC3000 but was reduced in its ability to multiply in
Arabidopsis thaliana
. The existence of TTCs that can bind to dissimilar effectors and that can substitute for each other in effector secretion provides insights into the nature of how TTCs function.
The Transcription Termination Factor Rho Is Essential and Autoregulated in
Caulobacter crescentus
Authors: Italiani Valéria C. S. , Marques Marilis V. 
Published: June 2005

ABSTRACT

The impossibility of obtaining a
rho
null mutant and sensitivity to bicyclomycin have indicated that
rho
is essential for the viability of
Caulobacter crescentus
. Transcription gene fusions of sequences with serial deletions of the
rho
5′ untranslated region (5′-UTR) with a
lacZ
reporter gene indicated that
rho
is autoregulated at the level of attenuation of transcription in the 5′-UTR.