bluuebunny/crossref_metadata_2025_split huggingface dataset:
Sites of positive and negative regulation in the Bacillus subtilis antiterminators LicT and SacY
Authors: Tortosa Pablo , Declerck Nathalie , Dutartre Hélène , Lindner Cordula , Deutscher Josef , Le Coq Dominique 
Published: March 2003

The Bacillus subtilis homologous transcriptional antiterminators LicT and SacY control the inducible expression of genes involved in aryl β‐glucoside and sucrose utilization respectively. Their RNA‐binding activity is carried by the N‐terminal domain (CAT), and is regulated by two similar C‐terminal domains (PRD1 and PRD2), which are the targets of phosphorylation reactions catalysed by the phosphoenolpyruvate: sugar phosphotransferase system (PTS). In the absence of the corresponding inducer, LicT is inactivated by BglP, the PTS permease (EII) specific for aryl β‐glucosides, and SacY by SacX, a negative regulator homologous to the EII specific for sucrose. LicT, but not SacY, is also subject to a positive control by the general PTS components EI and HPr, which are thought to phosphorylate LicT in the absence of carbon catabolite repression. Construction of SacY/LicT hybrids and mutational analysis enabled the location of the sites of this positive regulation at the two phosphorylatable His207 and His269 within LicT‐PRD2, and suggested that the presence of negative charges at these sites is sufficient for LicT activation in vivo. The BglP‐mediated inhibition process was found to essentially involve His100 of LicT‐PRD1, with His159 of the same domain playing a minor role in this regulation. In vitro experiments indicated that His100 could be phosphorylated directly by the general PTS proteins, this phosphorylation being stimulated by P∼BglP. We confirmed that, similarly, the corresponding conserved His99 residue in SacY is the major site of the negative control exerted by SacX on SacY activity. Thus, for both antiterminators, the EII‐mediated inhibition process seems to rely primarily on the presence of a negative charge at the first conserved histidine of the PRD1.
Population density‐dependent regulation of the Bradyrhizobium japonicum nodulation genes
Authors: Loh John T. , Yuen‐Tsai Joyce P.‐Y. , Stacey Minviluz G. , Lohar Dasharath , Welborn April , Stacey Gary 
Published: March 2003

The nodulation genes of Bradyrhizobium japonicum are essential for infection and establishment of a nitrogen‐fixing symbiosis. Here, we demonstrate that plant‐produced isoflavones induce nodulation gene expression in a population density‐dependent fashion. Nodulation gene induction is highest at a low population density and significantly reduced in more dense cultures. A quorum signal molecule in the conditioned medium of B. japonicum cultures mediates this repression. Repression in response to the quorum signal results from the induction of NolA which, in turn, induces NodD2 leading to inhibition of nod gene expression. Consistent with this, nolA–lacZ and nodD2–lacZ expression increased with increasing population density. Unlike the wild type, the ability to induce nodY–lacZ expression did not decline with population density in a NolA mutant. Normally, nod gene expression is repressed in planta (i.e. within nodules). However, expression of a nodY–GUS fusion was not repressed in a NolA mutant, suggesting that quorum‐sensing control may mediate in planta repression of the nod genes. Addition of conditioned medium to cultures significantly reduced nod gene expression. Treatment of inoculant cultures with conditioned medium also reduced the ability of B. japonicum to nodulate soybean plants.
InlA‐ but not InlB‐mediated internalization of Listeria monocytogenes by non‐phagocytic mammalian cells needs the support of other internalins
Authors: Bergmann Birgit , Raffelsbauer Diana , Kuhn Michael , Goetz Monika , Hom Silke , Goebel Werner 
Published: March 2003

SummaryTo determine the contribution of the previously identified internalins, InlA, InlB, InlC, InlE, InlG, and InlH, to internalization of Listeria monocytogenes by
non‐professional phagocytic mammalian cells, we constructed mutants with various
combinations of deletions in the respective inl genes. Internalization of
these mutants into the epithelial‐like Caco‐2 and the microvascular endothelial HBMEC
cell lines were studied. Deletion of the inlGHE gene cluster, or of the single
genes, led to a two to fourfold increased internalization by HBMEC and other non‐phagocytic
mammalian cells. Invasion into HBMEC was totally blocked in the absence of InlB,
and InlB‐dependent internalization did not require the presence of any of the other
internalins. Internalization by Caco‐2 cells was reduced to a level of about 1%
in the absence of InlA and InlB, and was most efficient in the presence of InlA,
InlB and InlC and in the absence of InlG, InlH and InlE. InlB and InlA, in each case
in the absence of the other internalins, led (compared with the wild‐type strain)
to reduced internalization of about 20% and less than 10% respectively.
InlA‐dependent internalization (in the absence of InlB) required the additional function
of InlC and InlGHE. The deletion of inlGHE enhanced the expression of InlA
and InlB. The increased amount of InlA led to an increase in early association of
L. monocytogenes with Caco‐2 cells without enhancing its uptake in the absence
of the other internalins, whereas the larger amount of InlB did not enhance early
association of L. monocytogenes with HBMEC but led to an increase in internalization
of L. monocytogenes. The results suggest that InlB is able to induce phagocytosis
in HBMEC and (at a lower efficiency) in Caco‐2 cells by itself, but InlA needs the
supportive functions of the other internalins to trigger phagocytosis. None of these
internalins seems to be required for cell‐to‐cell spread by L. monocytogenes,
as shown by microinjection of Caco‐2 cells with appropriate inl mutants.
A common drug‐responsive element mediates the upregulation of the Candida albicans ABC transporters CDR1 and CDR2, two genes involved in antifungal drug resistance
Authors: Micheli Michelle de , Bille Jacques , Schueller Christoph , Sanglard Dominique 
Published: March 2003

SummaryUpregulation of the ATP‐binding cassette (ABC) transporter genes CDR1 and
CDR2 (Candidadrug resistance 1 and 2) is a common
mechanism observed in Candida albicans clinical isolates developing resistance
to the class of azole antifungals. In this work, the regulatory elements of both
genes were delimited using a reporter system in an azole‐susceptible strain exposed
to oestradiol, which allows transient induction of these genes. We found two regulatory
elements in the CDR1 promoter: one responsible for basal expression (basal
expression element; BEE) and the other required for oestradiol responsiveness (drug‐responsive
element I; DREI). In the CDR2 promoter, a single regulatory element
responsible for oestradiol responsiveness (DREII) was detected. Both DREs
shared a consensus of 21 bp with the sequence 5′‐CGGA(A/T)ATCGGATATTTTTTTT‐3′
having no equivalent to known eukaryotic regulatory sequence. Consistent with this
finding, two other C. albicans genes identified by a search for the presence
of DRE in the C. albicans genome sequence database were responsive to oestradiol.
Finally, the regulatory elements found in CDR1 and CDR2 were also functional
in an azole‐resistant strain with constitutive high expression of both transporters.
These results suggest that, although CDR1 and CDR2 upregulation can
be obtained by transient drug‐induced and constitutive upregulation, these two processes
converge to the same regulatory elements and probably mobilize the same trans‐acting factors.
Immediate GTP hydrolysis upon FtsZ polymerization
Authors: Scheffers Dirk‐Jan , Driessen Arnold J. M. 
Published: March 2003

SummaryTo understand the polymerization dynamics of FtsZ, a bacterial cell division protein similar to tubulin, insight is required into the nature of the nucleotide bound to the polymerized protein. In a previous study, we showed that the FtsZ polymers contain mostly GDP. A recent study challenged this result, suggesting that the polymerized FtsZ is in a GTP‐bound state. Here, we show that, when radiolabelled [γ‐32P]‐GTP is used to polymerize FtsZ, GTP is hydrolysed instantaneously. The FtsZ polymer contains both GDP and the radiolabelled inorganic phosphate.
Expression of mutant alanine tRNAs increases spontaneous mutagenesis in Escherichia coli
Authors: Dorazi Robert , Lingutla Josephine J. , Humayun M. Zafri 
Published: March 2003

SummaryThe expression of mutA, an allele of the glycine tRNA gene glyV,
can confer a novel mutator phenotype that correlates with its ability to promote
Asp→Gly mistranslation. Both activities are mediated by a single base
change within the anticodon such that the mutant tRNA can decode aspartate codons
(GAC/U) instead of the normal glycine codons (GCC/U). Here, we investigate whether
specific Asp→Gly mistranslation is required for the unexpected mutator
phenotype. To address this question, we created and expressed 18 individual alleles
of alaV, the gene encoding an alanine tRNA, in which the alanine anticodon
was replaced with those specifying other amino acids such that the mutant (alaVX)
tRNAs are expected to potentiate X→Ala mistranslation, where X is one
of the other amino acids. Almost all alaVX alleles proved to be
mutators in an assay that measured the frequency of rifampicin‐resistant mutants,
with one allele (alaVGlu) being a stronger mutator than mutA.
The alaVGlu mutator phenotype resembles that of mutA in
mutational specificity (predominantly transversions), as well as SOS independence,
but in a puzzling twist differs from mutA in that it does not require a functional
recA gene. Our results suggest that general mistranslation (as opposed to
Asp→Gly alone) can induce a mutator phenotype. Furthermore, these findings predict that a large number of conditions that increase translational errors, such as genetic defects in the translational apparatus, as well as environmental and physiological stimuli (such as amino acid starvation or exposure to antibiotics) are likely to activate a mutator response. Thus, both genetic and epigenetic mechanisms can accelerate the acquisition of mutations.
Functional complexity of the twin‐arginine translocase TatC component revealed by site‐directed mutagenesis
Authors: Buchanan Grant , Leeuw Erik de , Stanley Nicola R. , Wexler Margaret , Berks Ben C. , Sargent Frank , Palmer Tracy 
Published: March 2003

SummaryThe Escherichia coli Tat apparatus is a membrane‐bound protein translocase that serves to export folded proteins synthesized with N‐terminal twin‐arginine signal peptides. The essential TatC component of the Tat translocase is an integral membrane protein probably containing six transmembrane helices. Sequence analysis identified conserved TatC amino acid residues, and the role of these side‐chains was assessed by single alanine substitution. This approach identified three classes of TatC mutants. Class I mutants included F94A, E103A and D211A, which were completely devoid of Tat‐dependent protein export activity and thus represented residues essential for TatC function. Cross‐complementation experiments with class I mutants showed that co‐expression of D211A with either F94A or E103A regenerated an active Tat apparatus. These data suggest that different class I mutants may be blocked at different steps in protein transport and point to the co‐existence of at least two TatC molecules within each Tat translocon. Class II mutations identified residues important, but not essential, for Tat activity, the most severely affected being L99A and Y126A. Class III mutants showed no significant defects in protein export. All but three of the essential and important residues are predicted to cluster around the cytoplasmic N‐tail and first cytoplasmic loop regions of the TatC protein.
The Xanthomonas oryzae pv.◊oryzae raxP and raxQ genes encode an ATP sulphurylase and adenosine‐5′‐phosphosulphate kinase that are required for AvrXa21 avirulence activity
Authors: Shen Yuwei , Sharma Parveen , Silva Francisco G. da , Ronald Pamela 
Published: March 2003

Summary Xanthomonas oryzae pv. oryzae (Xoo) Philippine race 6 (PR6) is unable to cause bacterial blight disease on rice lines containing the rice resistance gene Xa21 but is virulent on non‐Xa21 rice lines, indicating that PR6 carries avirulence (avrXa21) determinants required for recognition by XA21. Here we show that two Xoo genes, raxP and raxQ, are required for AvrXa21 activity. raxP and raxQ, which reside in a genomic cluster of sulphur assimilation genes, encode an ATP sulphurylase and APS (adenosine‐5′‐phosphosulphate) kinase. These enzymes function together to produce activated forms of sulphate, APS and PAPS (3′‐phosphoadenosine‐5′‐phosphosulphate). Xoo PR6 strains carrying disruptions in either gene, PR6ΔraxP or PR6ΔraxQ, are unable to produce APS and PAPS and are virulent on Xa21‐containing rice lines. RaxP and RaxQ are similar to the bacterial symbiont Sinorhizobium meliloti host specificity proteins, NodP and NodQ and the Escherichia coli cysteine synthesis proteins CysD, CysN and CysC. The APS and PAPS produced by RaxP and RaxQ are used for both cysteine synthesis and sulphation of other molecules. Mutation in Xoo xcysI, a homologue of Escherichia coli cysI that is required for cysteine synthesis, blocked APS‐ or PAPS‐dependent cysteine synthesis but did not affect AvrXa21 activity, suggesting that AvrXa21 activity is related to sulphation rather than cysteine synthesis. Taken together, these results demonstrate that APS and PAPS production plays a critical role in determining avirulence of a phytopathogen and reveal a commonality between symbiotic and phytopathogenic bacteria.
The transcription termination factor Rho is required for oxidative stress survival in Caulobacter crescentus
Authors: Italiani Valéria C. S. , Zuleta Luiz Fernando G. , Marques Marilis V. 
Published: March 2003

SummaryA transposon Tn5 mutagenesis library was generated from Caulobacter crescentus

strain NA1000, and clones with deficiency in survival in a high concentration of

NaCl were selected. One of these clones, 37G10, has the Tn5 integrated within

the coding region of the transcription termination factor Rho. Analysis of this mutant

phenotype showed that the cells are motile and present a normal cell cycle, but have

a longer generation time. This strain is sensitive to acidic pH, to the presence

of different salts and to heat shock, but it responds well to UV light and alkaline

pH. The most striking phenotype of the rho mutant is that it is extremely

sensitive to oxidative stress, in both exponential and stationary phases. Experiments

using a transcriptional fusion of the rho promoter region to the lacZ

gene showed that rho gene expression varies during the cell cycle, showing

very low expression levels at the swarmer cell stage and presenting maximum levels

in early predivisional cells. Transcription of the rho gene is increased in

the rho mutant strain, which is indicative of an autoregulatory circuit, and

there is a small variation in the cell cycle pattern of expression. Several peptides

have their synthesis altered in the mutant strain, as analysed by two‐dimensional

gel electrophoresis, most of which show a reduction in expression. These results

indicate that the Rho factor is essential for an efficient response to certain stresses

in Caulobacter.
A two‐component regulator induces the transmission phenotype of stationary‐phase Legionella pneumophila
Authors: Hammer Brian K. , Tateda Eiko S. , Swanson Michele S. 
Published: March 2003

SummaryPathogenic Legionella pneumophila evolved as a parasite of aquatic amoebae.
To persist in the environment, the microbe must be proficient at both replication
and transmission. In laboratory cultures, as nutrients become scarce a stringent
response‐like pathway coordinates exit from the exponential growth phase with induction
of traits correlated with virulence, including motility. A screen for mutants that
express the flagellin gene poorly identified five activators of virulence: LetA/LetS,
a two‐component regulator homologous to GacA/GacS of Pseudomonas and SirA/BarA
of Salmonella; the stationary‐phase sigma factor RpoS; the flagellar sigma
factor FliA; and a new locus, letE. Unlike wild type, post‐exponential‐phase
letA and letS mutants were not motile, cytotoxic, sodium sensitive
or proficient at infecting macrophages. L. pneumophila also required fliA
to become motile, cytotoxic and to infect macrophages efficiently and letE
to express sodium sensitivity and maximal motility and cytotoxicity. When induced
to express RelA, all of the strains exited the exponential phase, but only wild type
converted to the fully virulent form. In contrast, intracellular replication was
independent of letA, letS, letE or fliA. Together, the data indicate
that, as the nutrient supply wanes, ppGpp triggers a regulatory cascade mediated
by LetA/ LetS, RpoS, FliA and letE that coordinates differentiation of replicating
L. pneumophila to a transmissible form.
Genetics of the phage growth limitation (Pgl) system of Streptomyces coelicolor A3(2)
Authors: Sumby Paul , Smith Margaret C. M. 
Published: March 2003

SummaryThe phage growth limitation (Pgl) system, encoded by Streptomyces coelicolor A3(2), confers protection against the temperate bacteriophage φC31 and its homoimmune relatives. The Pgl phenotype is characterized by the ability of Pgl+ hosts to support a phage burst on initial infection but subsequent cycles are severely attenuated. Previously, two adjacent genes pglY and pglZ were shown to be required for Pgl. It had been shown by Southern blotting that Streptomyces lividans, a close relative of S. coelicolor and naturally Pgl−, does not contain homologues of pglYZ and that introduction of pglYZ into S. lividans is not sufficient to confer a Pgl+ phenotype. Moreover, the mechanism of the Pgl+↔ Pgl− phase variation associated with this phenotype is also not understood. Here we describe two novel genes, pglW and pglX, that were shown to be part of this system by complementation of Pgl− mutants and by insertional mutagenesis. pglW encodes a 169 kDa protein that includes putative motifs for both serine/threonine protein kinase activity and DNA binding. pglX encodes a 136 kDa protein with putative adenine‐specific DNA methyltransferase activity. pglW and pglX have overlapping stop‐start codons suggesting transcriptional and translational coupling. S1 mapping of transcripts initiating up‐stream of pglW indicated that, like pglYZ, pglWX is expressed in uninfected cultures. A homologue of pglX with 76% amino acid identity was identified in S. coelicolor, and insertional mutagenesis indicated that this gene was not required for the Pgl+ phenotype. Southern blots indicated that S. lividans does not contain homologues of pglW or pglX. A plasmid encoding pglWXYZ was able to confer the Pgl+ phenotype to S. lividans implying that these four genes constitute the whole system.
Complementary activities of SseJ and SifA regulate dynamics of the Salmonella typhimurium vacuolar membrane
Authors: Ruiz‐Albert Javier , Yu Xiu‐Jun , Beuzón Carmen R. , Blakey Abigail N. , Galyov Edouard E. , Holden David W. 
Published: March 2003

SummaryThe Salmonella pathogenicity island 2 (SPI‐2) type III secretion system
(TTSS) of Salmonella typhimurium is required for bacterial replication within
host cells. It acts by translocating effector proteins across the membrane of the
Salmonella‐containing vacuole (SCV). The SifA effector is required to maintain
the integrity of the SCV membrane, and for the formation in epithelial cells of Salmonella‐induced
filaments (Sifs), which are tubular extensions of SCVs. We have investigated the
role in S. typhimurium virulence of the putative SPI‐2 effector genes sifB,
srfJ, sseJ and sseI. An S. typhimurium strain carrying a
mutation in sseJ was mildly attenuated for systemic virulence in mice, but
strains carrying mutations in either srfJ, sseI or sifB had
very little or no detectable virulence defect after intraperitoneal inoculation.
Expression of SseJ in HeLa cells resulted in the formation of globular membranous
compartments (GMCs), the composition of which appears to be similar to that of SCV
membranes and Sifs. The formation of GMCs was dependent on the serine residue of
the predicted acyltransferase/lipase active site of SseJ. Transiently expressed SseJ
also inhibited Sif formation by wild‐type bacteria, and was found to associate with
Sifs, SCV membranes and simultaneously expressed SifA. Intracellular vacuoles containing
sseJ mutant bacteria appeared normal but, in contrast to a sifA mutant,
a sifA sseJ double mutant strain did not lose its vacuolar membrane, indicating
that loss of vacuolar membrane around sifA mutant bacteria requires the action of SseJ. Collectively, these results suggest that the combined action of SseJ and SifA regulate dynamics of the SCV membrane in infected cells.
The Pneumocystis carinii drug target S‐adenosyl‐L‐methionine:sterol C‐24 methyl transferase has a unique substrate preference
Authors: Kaneshiro Edna S. , Rosenfeld Jill A. , Basselin‐Eiweida Mireille , Stringer James R. , Keely Scott P. , Smulian A. George , Giner José‐Luis 
Published: March 2003

Summary Pneumocystis is an opportunistic pathogen that can cause pneumonitis in immunodeficient people such as AIDS patients. Pneumocystis remains difficult to study in the absence of culture methods for luxuriant growth. Recombinant protein technology now makes it possible to avoid some major obstacles. The P. carinii expressed sequence tag (EST) database contains 11 entries of a sequence encoding a protein homologous to S‐adenosyl‐L‐methionine (SAM):C‐24 sterol methyl transferase (SMT), suggesting high activity of this enzyme in the organism. We sequenced the erg6 cDNA, identified the putative peptide motifs for the sterol and SAM binding sites in the deduced amino acid sequence and expressed the protein in Escherichia coli. Unlike SAM:SMT from other organisms, the P. carinii enzyme had higher affinities for lanosterol and 24‐methylenelanosterol than for zymosterol, the preferred substrate in other fungi. Cycloartenol was not a productive substrate. With lanosterol and 24‐methylenelanosterol as substrates, the major reaction products were 24‐methylenelanosterol and pneumocysterol respectively. Thus, the P. carinii SAM:SMT catalysed the transfer of both the first and the second methyl groups to the sterol C‐24 position, and the substrate preference was found to be a unique property of the P. carinii SAM:SMT. These observations, together with the absence of SAM:SMT among mammals, further support the identification of sterol C‐24 alkylation reactions as excellent targets for the development of drugs specifically directed against this pathogen.
SecDFyajC forms a heterotetrameric complex with YidC
Authors: Nouwen Nico , Driessen Arnold J. M. 
Published: March 2003

SummaryThe Escherichia coli preprotein translocase is composed of a ‘preprotein conducting channel’ domain that consists of the peripherally bound translocation ATPase SecA and the heterotrimeric SecYEG membrane protein complex. SecD, SecF, and YajC form another heterotrimeric complex that can associate with the SecYEG complex. YidC is an essential membrane protein that plays a role in the integration of newly synthesized membrane proteins, and has been shown to co‐purify with SecYEG when all translocase components are overproduced. Here, we demonstrate that under conditions that YidC co‐purifies with overproduced SecDFyajC it does not co‐purify with overproduced SecYEG. Moreover, this interaction of YidC with the SecDFyajC complex is also found at chromosomal protein levels of SecD, SecF and YajC. Closer examination of the SecDFyajC–YidC complex showed that YidC binds to SecD and SecF, whereas YajC interacts only with SecF. As SecF and YajC have previously been shown to interact with SecY, we propose that these two proteins link the heterotetrameric SecDFyajC–YidC complex to the SecYEG complex.