|
|
|
|
|
|
Surfaces of Spo0A and RNA Polymerase Sigma Factor A That Interact at the spoIIG Promoter in Bacillus subtilis. Amrita Kumar, 2004.In Bacillus subtilis, the DNA binding protein Spo0A activates transcription from two classes of promoters, those used by RNA polymerase containing the primary sigma factor,  A
(e.g., spoIIG), and those used by RNA polymerase containing
the secondary sigma factor,
H
(e.g., spoIIA) . Several single amino acid substitutions in
region 4 of
A
define positions in
A
that are specifically required for Spo0A-dependent promoter
activation . Similarly, several single amino acid substitutions in
Spo0A define positions in Spo0A that are required for
A-dependent
promoter activation but not for other functions of Spo0A . It is
unknown whether these amino acids in Spo0A interact directly with
those in region 4 of
A
or whether they interact with another subunit of RNA polymerase to
effect promoter activation . Here we report the identification of a
new amino acid in region 4 of
A,
arginine at position 355 (R355), that is involved in Spo0A-dependent
promoter activation . To further investigate the role of R355,
we used the coordinates of Spo0A and sigma region 4, each in complex
with DNA, to build a model for the interaction of
A
and Spo0A at the spoIIG promoter . We tested the model by examining
the effects of amino acid substitutions in the putative interacting
surfaces of these molecules . As predicted by the model, we found
genetic evidence for interaction of R355 of
A
with glutamine at position 221 of Spo0A . These results appear to
define the surfaces of Spo0A and
A
that directly interact during activation of the spoIIG
promoter .
Identification and Physical Characterization of the HbpR Binding Sites of the hbpC and hbpD Promoters. David Tropel, 2002.Pseudomonas azelaica HBP1 can use 2-hydroxybiphenyl (2-HBP) and 2,2'-dihydroxybiphenyl as sole carbon and energy sources by means of the hbp regulon . This regulon is composed of three genes, hbpCA and hbpD, coding for enzymes of a meta-cleavage pathway and the hbpR gene, which codes for a XylR/DmpR-type transcription regulator . It was previously shown that HbpR activates transcription from two  54-dependent promoters, PhbpC and PhbpD, in the presence of 2-HBP . In this study, by using gel mobility shift assays with a purified fusion protein containing calmodulin binding protein (CBP) and HbpR, we detected two binding regions for HbpR in PhbpC and one binding region in PhbpD . DNase I footprints of the proximal binding region of PhbpC and of the binding region in PhbpD showed that CBP-HbpR protected a region composed of two inverted repeat sequences which were homologous to the binding sites identified for XylR . Unlike the situation in the XylR/Pu system, we observed simultaneous binding of CBP-HbpR on the two upstream activating sequences (UASs) . Fragments with only one UAS did not show an interaction with HbpR, indicating that both pairs of UASs are needed for HbpR binding . The addition of both ATP and 2-HBP increased the DNA binding affinity of HbpR . These results showed for the first time that, for regulators of the XylR/DmpR type, the effector positively affects the recruitment of the regulatory protein on the enhancer DNA .
EvgA of the Two-Component Signal Transduction System Modulates Production of the YhiUV Multidrug Transporter in Escherichia coli. Kunihiko Nishino, 2002.Overexpression of the EvgA regulator of the two-component signal transduction system was previously found to modulate multidrug resistance of Escherichia coli by increasing efflux of drugs (K . Nishino and A . Yamaguchi, J . Bacteriol . 183:1455-1458, 2001) . Here we present data showing that EvgA contributes to multidrug resistance through increased expression of the multidrug transporter yhiUV gene . Effects of Carboxy-Terminal Modifications and pH on Binding of a Bacillus subtilis Small, Acid-Soluble Spore Protein to DNA. Jeffrey Kosman, 2003.Variants of the wild-type Bacillus subtilis  /ß-type small, acid-soluble spore protein (SASP) SspCwt were designed to evaluate the contribution of C-terminal residues to these proteins' affinity for DNA . SspC variants lacking one to three C-terminal residues were similar to SspCwt in DNA binding, but removal of six C-terminal residues greatly decreased DNA binding . In contrast, a C-terminal extension of three residues increased SspC's affinity for DNA 5- to 10-fold . C-terminal and N-terminal changes that independently caused large increases in SspC-DNA binding affinity were combined and produced an additive effect on DNA binding; the affinity of the resulting variant, SspC N11-D13K-C3, for DNA was increased
 20-fold over that of SspCwt . For most of the SspC variants tested, lowering the pH from 7 to 6 improved DNA binding two- to sixfold, although the opposite effect was observed with variants having additional C-terminal basic residues . In vitro, the binding of SspCN11-D13K-C3 to DNA suppressed the formation of cyclobutane-type thymine dimers and promoted the formation of the spore photoproduct upon UV irradiation to the same degree as the binding of SspCwt . However, B . subtilis spores lacking major
/ß-type SASP and overexpressing SspCN11-D13K-C3 had a 10-fold-lower viability and far less UV and heat resistance than spores overexpressing SspCwt . This apparent lack of DNA protection by SspCN11-D13K-C3 in vivo is likely due to the twofold-lower level of this protein in spores compared to the level of SspCwt, perhaps because of effects of SspCN11-D13K-C3 on gene expression in the forespore during sporulation . The latter results indicate that only moderately strong binding of
/ß-type SASP to DNA is important to balance the potentially conflicting requirements for these proteins in DNA transcription and DNA protection during spore formation, spore dormancy, and spore germination and outgrowth .
Use of In-Biofilm Expression Technology To Identify Genes Involved in Pseudomonas aeruginosa Biofilm Development. Antonio Finelli, 2003.Mature Pseudomonas aeruginosa biofilms form complex three-dimensional architecture and are tolerant of antibiotics and other antimicrobial compounds . In this work, an in vivo expression technology system, originally designed to study virulence-associated genes in complex mammalian environments, was used to identify genes up-regulated in P . aeruginosa grown to a mature (5-day) biofilm . Five unique cloned promoters unable to promote in vitro growth in the absence of purines after recovery from the biofilm environment were identified . The open reading frames downstream of the cloned promoter regions were identified, and knockout mutants were generated . Insertional mutation of PA5065, a homologue of Escherichia coli ubiB, was lethal, while inactivation of PA0240 (a porin homologue), PA3710 (a putative alcohol dehydrogenase), and PA3782 (a homologue of the Streptomyces griseus developmental regulator adpA) had no effect on planktonic growth but caused defects in biofilm formation in static and flowing systems . In competition experiments, mutants demonstrated reduced fitness compared with the parent strain, comprising less than 0.0001% of total biofilm cells after 5 days . Therefore, using in-biofilm expression technology, we have identified novel genes that do not affect planktonic growth but are important for biofilm formation, development, and fitness . Horizontal Transfer of phnAc Dioxygenase Genes within One of Two Phenotypically and Genotypically Distinctive Naphthalene-Degrading Guilds from Adjacent Soil Environments. Mark S. Wilson, 2003.Several distinct naphthalene dioxygenases have been characterized to date, which provides the opportunity to investigate the ecological significance, relative distribution, and transmission modes of the different analogs . In this study, we showed that a group of naphthalene-degrading isolates from a polycyclic aromatic hydrocarbon (PAH)-contaminated hillside soil were phenotypically and genotypically distinct from naphthalene-degrading organisms isolated from adjacent, more highly contaminated seep sediments . Mineralization of 14C-labeled naphthalene by soil slurries suggested that the in situ seep community was more acclimated to PAHs than was the in situ hillside community . phnAc-like genes were present in diverse naphthalene-degrading isolates cultured from the hillside soil, while nahAc-like genes were found only among isolates cultured from the seep sediments . The presence of a highly conserved nahAc allele among gram-negative isolates from the coal tar-contaminated seep area provided evidence for in situ horizontal gene transfer and was reported previously (J . B . Herrick, K . G . Stuart-Keil, W . C . Ghiorse, and E . L . Madsen, Appl . Environ . Microbiol . 63:2330-2337, 1997) . Natural horizontal transfer of the phnAc sequence was also suggested by a comparison of the phnAc and 16S ribosomal DNA sequences of the hillside isolates . Analysis of metabolites produced by cell suspensions and patterns of amplicons produced by PCR analysis suggested both genetic and metabolic diversity among the naphthalene-degrading isolates of the contaminated hillside . These results provide new insights into the distribution, diversity, and transfer of phnAc alleles and increase our understanding of the acclimation of microbial communities to pollutants . Development of a Sensitive Gene Expression Reporter System and an Inducible Promoter-Repressor System for Clostridium acetobutylicum. Laurence Girbal, 2003.A sensitive gene expression reporter system was developed for Clostridium acetobutylicum ATCC 824 by using a customized gusA expression cassette . In discontinuous cultures, time course profiles of ß-glucuronidase specific activity reflected adequately in vivo dynamic up- and down-regulation of acidogenesis- and/or solventogenesis-associated promoter expression in C . acetobutylicum . Furthermore, a new inducible gene expression system was developed in C . acetobutylicum, based on the Staphylococcus xylosus xylose operon promoter-repressor regulatory system .
|
© 2005
Transgalactic Ltd (manufacturer of Bioscreen C software) |
Privacy Statement | P.O. Box
1393, 00101 Helsinki, Finland,
Last modified: May 25, 2005
| ||||||
