Genetic makeup of the Corynebacterium glutamicum LexA regulon deduced from comparative transcriptomics and in vitro DNA band shift assays

Microbiology ◽  
2009 ◽  
Vol 155 (5) ◽  
pp. 1459-1477 ◽  
Author(s):  
Nina Jochmann ◽  
Anna-Katharina Kurze ◽  
Lisa F. Czaja ◽  
Karina Brinkrolf ◽  
Iris Brune ◽  
...  
Keyword(s):  
Dna Damage ◽  
Corynebacterium Glutamicum ◽  
Intergenic Region ◽  
Binding Sites ◽  
Consensus Sequence ◽  
Differentially Expressed ◽  
Wild Type ◽  
Band Shift ◽  
Lexa Binding

The lexA gene of Corynebacterium glutamicum ATCC 13032 was deleted to create the mutant strain C. glutamicum NJ2114, which has an elongated cell morphology and an increased doubling time. To characterize the SOS regulon in C. glutamicum, the transcriptomes of NJ2114 and a DNA-damage-induced wild-type strain were compared with that of a wild-type control using DNA microarray hybridization. The expression data were combined with bioinformatic pattern searches for LexA binding sites, leading to the detection of 46 potential SOS boxes located upstream of differentially expressed transcription units. Binding of a hexahistidyl-tagged LexA protein to 40 double-stranded oligonucleotides containing the potential SOS boxes was demonstrated in vitro by DNA band shift assays. It turned out that LexA binds not only to SOS boxes in the promoter–operator region of upregulated genes, but also to SOS boxes detected upstream of downregulated genes. These results demonstrated that LexA controls directly the expression of at least 48 SOS genes organized in 36 transcription units. The deduced genes encode a variety of physiological functions, many of them involved in DNA repair and survival after DNA damage, but nearly half of them have hitherto unknown functions. Alignment of the LexA binding sites allowed the corynebacterial SOS box consensus sequence TcGAA(a/c)AnnTGTtCGA to be deduced. Furthermore, the common intergenic region of lexA and the differentially expressed divS-nrdR operon, encoding a cell division suppressor and a regulator of deoxyribonucleotide biosynthesis, was characterized in detail. Promoter mapping revealed differences in divS-nrdR expression during SOS response and normal growth conditions. One of the four LexA binding sites detected in the intergenic region is involved in regulating divS-nrdR transcription, whereas the other sites are apparently used for negative autoregulation of lexA expression.

scholarly journals Genetic Composition of the Bacillus subtilis SOS System

2005 ◽  
Vol 187 (22) ◽  
pp. 7655-7666 ◽  
Author(s):  
Nora Au ◽  
Elke Kuester-Schoeck ◽  
Veena Mandava ◽  
Laura E. Bothwell ◽  
Susan P. Canny ◽  
...  
Keyword(s):  
Dna Damage ◽  
Bacillus Subtilis ◽  
Binding Sites ◽  
Consensus Sequence ◽  
Transcriptional Response ◽  
Open Reading Frames ◽  
Mobility Shift ◽  
E Coli ◽  
Recombination Protein ◽  
Lexa Binding

ABSTRACT The SOS response in bacteria includes a global transcriptional response to DNA damage. DNA damage is sensed by the highly conserved recombination protein RecA, which facilitates inactivation of the transcriptional repressor LexA. Inactivation of LexA causes induction (derepression) of genes of the LexA regulon, many of which are involved in DNA repair and survival after DNA damage. To identify potential RecA-LexA-regulated genes in Bacillus subtilis, we searched the genome for putative LexA binding sites within 300 bp upstream of the start codons of all annotated open reading frames. We found 62 genes that could be regulated by putative LexA binding sites. Using mobility shift assays, we found that LexA binds specifically to DNA in the regulatory regions of 54 of these genes, which are organized in 34 putative operons. Using DNA microarray analyses, we found that 33 of the genes with LexA binding sites exhibit RecA-dependent induction by both mitomycin C and UV radiation. Among these 33 SOS genes, there are 22 distinct LexA binding sites preceding 18 putative operons. Alignment of the distinct LexA binding sites reveals an expanded consensus sequence for the B. subtilis operator: 5′-CGAACATATGTTCG-3′. Although the number of genes controlled by RecA and LexA in B. subtilis is similar to that of Escherichia coli, only eight B. subtilis RecA-dependent SOS genes have homologous counterparts in E. coli.

scholarly journals The abundance and in vitro DNA binding of three cellular proteins interacting with the adenovirus EIIa early promoter are not modified by the EIa gene products.

1987 ◽  
Vol 7 (10) ◽  
pp. 3806-3817 ◽  
Author(s):  
P Jalinot ◽  
B Devaux ◽  
C Kédinger
Keyword(s):  
Binding Sites ◽  
Adenovirus Type ◽  
Specific Protein ◽  
Gene Products ◽  
Wild Type ◽  
Band Shift ◽  
Transcription Complexes ◽  
Adenovirus Type 5 ◽  
Band Shift Assay

Specific protein binding on the EIa-inducible adenovirus EIIa early (EIIaE) promoter was analyzed by the sensitive electrophoretic band-shift assay and by protection against DNase I digestion. Three factors were identified, and precise mapping of the cognate-binding sites revealed their correspondence to promoter elements essential for constitutive EIIaE transcription. One binds to the major upstream element located between -82 and -64 (with respect to the major EIIaE cap site), another appears to interact with sequences on either side of this region, and the last one binds to an element located further upstream. Comparison of the binding activities of the factors present in extracts from cells infected with wild-type adenovirus (adenovirus type 5) or with the EIa deletion mutant dl312 did not reveal striking differences. Not only were the general binding patterns indistinguishable, but the concentration of each of the identified factors as well as their affinity for the cognate-binding sites were unchanged. Our results suggest that the EIa-mediated activation of the EIIaE transcription complexes involves appropriate interactions between transcription factors, rather than their increased binding to DNA.

The abundance and in vitro DNA binding of three cellular proteins interacting with the adenovirus EIIa early promoter are not modified by the EIa gene products

1987 ◽  
Vol 7 (10) ◽  
pp. 3806-3817
Author(s):  
P Jalinot ◽  
B Devaux ◽  
C Kédinger
Keyword(s):  
Binding Sites ◽  
Adenovirus Type ◽  
Specific Protein ◽  
Gene Products ◽  
Wild Type ◽  
Band Shift ◽  
Transcription Complexes ◽  
Adenovirus Type 5 ◽  
Band Shift Assay

Specific protein binding on the EIa-inducible adenovirus EIIa early (EIIaE) promoter was analyzed by the sensitive electrophoretic band-shift assay and by protection against DNase I digestion. Three factors were identified, and precise mapping of the cognate-binding sites revealed their correspondence to promoter elements essential for constitutive EIIaE transcription. One binds to the major upstream element located between -82 and -64 (with respect to the major EIIaE cap site), another appears to interact with sequences on either side of this region, and the last one binds to an element located further upstream. Comparison of the binding activities of the factors present in extracts from cells infected with wild-type adenovirus (adenovirus type 5) or with the EIa deletion mutant dl312 did not reveal striking differences. Not only were the general binding patterns indistinguishable, but the concentration of each of the identified factors as well as their affinity for the cognate-binding sites were unchanged. Our results suggest that the EIa-mediated activation of the EIIaE transcription complexes involves appropriate interactions between transcription factors, rather than their increased binding to DNA.

scholarly journals RuvAB and RecG Are Not Essential for the Recovery of DNA Synthesis Following UV-Induced DNA Damage in Escherichia coli

Genetics ◽  
2004 ◽  
Vol 166 (4) ◽  
pp. 1631-1640 ◽  
Author(s):  
Janet R Donaldson ◽  
Charmain T Courcelle ◽  
Justin Courcelle
Keyword(s):  
Dna Damage ◽  
Dna Synthesis ◽  
Dna Lesions ◽  
Replication Forks ◽  
Wild Type ◽  
Replication Machinery ◽  
Dna Junctions ◽  
Fork Regression

Abstract Ultraviolet light induces DNA lesions that block the progression of the replication machinery. Several models speculate that the resumption of replication following disruption by UV-induced DNA damage requires regression of the nascent DNA or migration of the replication machinery away from the blocking lesion to allow repair or bypass of the lesion to occur. Both RuvAB and RecG catalyze branch migration of three- and four-stranded DNA junctions in vitro and are proposed to catalyze fork regression in vivo. To examine this possibility, we characterized the recovery of DNA synthesis in ruvAB and recG mutants. We found that in the absence of either RecG or RuvAB, arrested replication forks are maintained and DNA synthesis is resumed with kinetics that are similar to those in wild-type cells. The data presented here indicate that RecG- or RuvAB-catalyzed fork regression is not essential for DNA synthesis to resume following arrest by UV-induced DNA damage in vivo.

DNA-binding and transcriptional regulatory properties of hepatic leukemia factor (HLF) and the t(17;19) acute lymphoblastic leukemia chimera E2A-HLF

1994 ◽  
Vol 14 (9) ◽  
pp. 5986-5996
Author(s):  
S P Hunger ◽  
R Brown ◽  
M L Cleary
Keyword(s):  
Dna Binding ◽  
Binding Site ◽  
Dna Sequences ◽  
Transcriptional Activation ◽  
Binding Sites ◽  
Lymphoblastic Leukemia ◽  
Consensus Sequence ◽  
Reporter Genes ◽  
Wild Type ◽  
Basic Leucine Zipper

The t(17;19) translocation in acute lymphoblastic leukemias results in creation of E2A-hepatic leukemia factor (HLF) chimeric proteins that contain the DNA-binding and protein dimerization domains of the basic leucine zipper (bZIP) protein HLF fused to a portion of E2A proteins with transcriptional activation properties. An in vitro binding site selection procedure was used to determine DNA sequences preferentially bound by wild-type HLF and chimeric E2A-HLF proteins isolated from various t(17;19)-bearing leukemias. All were found to selectively bind the consensus sequence 5'-GTTACGTAAT-3' with high affinity. Wild-type and chimeric HLF proteins also bound closely related sites identified previously for bZIP proteins of both the proline- and acidic amino acid-rich (PAR) and C/EBP subfamilies; however, E2A-HLF proteins were significantly less tolerant of certain deviations from the HLF consensus binding site. These differences were directly attributable to loss of an HLF ancillary DNA-binding domain in all E2A-HLF chimeras and were further exacerbated by a zipper mutation in one isolate. Both wild-type and chimeric HLF proteins displayed transcriptional activator properties in lymphoid and nonlymphoid cells on reporter genes containing HLF or C/EBP consensus binding sites. But on reporter genes with nonoptimal binding sites, their transcriptional properties diverged and E2A-HLF competitively inhibited activation by wild-type PAR proteins. These findings establish a spectrum of binding site-specific transcriptional properties for E2A-HLF which may preferentially activate expression of select subordinate genes as a homodimer and potentially antagonize expression of others through heteromeric interactions.

scholarly journals Phosphoinositides Regulate Membrane-dependent Actin Assembly by Latex Bead Phagosomes

2002 ◽  
Vol 13 (4) ◽  
pp. 1190-1202 ◽  
Author(s):  
Hélène Defacque ◽  
Evelyne Bos ◽  
Boyan Garvalov ◽  
Cécile Barret ◽  
Christian Roy ◽  
...  
Keyword(s):  
Binding Sites ◽  
Kinase Activity ◽  
Kinase Inhibitors ◽  
Membrane Surface ◽  
Latex Bead ◽  
Actin Assembly ◽  
Wild Type ◽  
Membrane Surfaces ◽  
Organelle Membrane

Actin assembly on membrane surfaces is an elusive process in which several phosphoinositides (PIPs) have been implicated. We have reconstituted actin assembly using a defined membrane surface, the latex bead phagosome (LBP), and shown that the PI(4,5)P2-binding proteins ezrin and/or moesin were essential for this process ( Defacque et al., 2000b ). Here, we provide several lines of evidence that both preexisting and newly synthesized PI(4,5)P2, and probably PI(4)P, are essential for phagosomal actin assembly; only these PIPs were routinely synthesized from ATP during in vitro actin assembly. Treatment of LBP with phospholipase C or with adenosine, an inhibitor of type II PI 4-kinase, as well as preincubation with anti-PI(4)P or anti-PI(4,5)P2 antibodies all inhibited this process. Incorporation of extra PI(4)P or PI(4,5)P2 into the LBP membrane led to a fivefold increase in the number of phagosomes that assemble actin. An ezrin mutant mutated in the PI(4,5)P2-binding sites was less efficient in binding to LBPs and in reconstituting actin assembly than wild-type ezrin. Our data show that PI 4- and PI 5-kinase, and under some conditions also PI 3-kinase, activities are present on LBPs and can be activated by ATP, even in the absence of GTP or cytosolic components. However, PI 3-kinase activity is not required for actin assembly, because the process was not affected by PI 3-kinase inhibitors. We suggest that the ezrin-dependent actin assembly on the LBP membrane may require active turnover of D4 and D5 PIPs on the organelle membrane.

scholarly journals Multiple Elements Controlling Adherence of Enterohemorrhagic Escherichia coli O157:H7 to HeLa Cells

2003 ◽  
Vol 71 (9) ◽  
pp. 4985-4995 ◽  
Author(s):  
Alfredo G. Torres ◽  
James B. Kaper
Keyword(s):  
Escherichia Coli ◽  
Hela Cells ◽  
Culture Conditions ◽  
Enterohemorrhagic Escherichia Coli ◽  
Differentially Expressed ◽  
Wild Type ◽  
Transposon Insertion ◽  
Reduced Adherence ◽  
Insertion Mutants

ABSTRACT Adherence of enterohemorrhagic Escherichia coli (EHEC) to the intestinal epithelium is essential for initiation of infection. Intimin is the only factor demonstrated to play a role in intestinal colonization by EHEC O157:H7. Other attempts to identify additional adhesion factors in vitro have been unsuccessful, suggesting that expression of these factors is under tight regulation. We sought to identify genes involved in the control of adherence of EHEC O157:H7 to cultured epithelial cells. A total of 5,000 independent transposon insertion mutants were screened for their ability to adhere to HeLa cells, and 7 mutants were isolated with a markedly enhanced adherence. The mutants adhered at levels 113 to 170% that of the wild-type strain, and analysis of the protein profiles of these mutants revealed several proteins differentially expressed under in vitro culture conditions. We determined the sequence of the differentially expressed proteins and further investigated the function of OmpA, whose expression was increased in a mutant with an insertionally inactivated tcdA gene. An isogenic ompA mutant showed reduced adherence compared to the parent strain. Disruption of the ompA gene in the tdcA mutant strain abolished the hyperadherent phenotype, and anti-OmpA serum inhibited adhesion of wild-type and tdcA mutant strains to HeLa cells. Enhanced adhesion mediated by OmpA was also observed with Caco-2 cells, and anti-OmpA serum blocked adherence to HeLa cells of other EHEC O157:H7 strains. Our results indicate that multiple elements control adherence and OmpA acts as an adhesin in EHEC O157:H7.

scholarly journals DNA-binding and transcriptional regulatory properties of hepatic leukemia factor (HLF) and the t(17;19) acute lymphoblastic leukemia chimera E2A-HLF.

1994 ◽  
Vol 14 (9) ◽  
pp. 5986-5996 ◽  
Author(s):  
S P Hunger ◽  
R Brown ◽  
M L Cleary
Keyword(s):  
Dna Binding ◽  
Binding Site ◽  
Dna Sequences ◽  
Transcriptional Activation ◽  
Binding Sites ◽  
Lymphoblastic Leukemia ◽  
Consensus Sequence ◽  
Reporter Genes ◽  
Wild Type ◽  
Basic Leucine Zipper

The t(17;19) translocation in acute lymphoblastic leukemias results in creation of E2A-hepatic leukemia factor (HLF) chimeric proteins that contain the DNA-binding and protein dimerization domains of the basic leucine zipper (bZIP) protein HLF fused to a portion of E2A proteins with transcriptional activation properties. An in vitro binding site selection procedure was used to determine DNA sequences preferentially bound by wild-type HLF and chimeric E2A-HLF proteins isolated from various t(17;19)-bearing leukemias. All were found to selectively bind the consensus sequence 5'-GTTACGTAAT-3' with high affinity. Wild-type and chimeric HLF proteins also bound closely related sites identified previously for bZIP proteins of both the proline- and acidic amino acid-rich (PAR) and C/EBP subfamilies; however, E2A-HLF proteins were significantly less tolerant of certain deviations from the HLF consensus binding site. These differences were directly attributable to loss of an HLF ancillary DNA-binding domain in all E2A-HLF chimeras and were further exacerbated by a zipper mutation in one isolate. Both wild-type and chimeric HLF proteins displayed transcriptional activator properties in lymphoid and nonlymphoid cells on reporter genes containing HLF or C/EBP consensus binding sites. But on reporter genes with nonoptimal binding sites, their transcriptional properties diverged and E2A-HLF competitively inhibited activation by wild-type PAR proteins. These findings establish a spectrum of binding site-specific transcriptional properties for E2A-HLF which may preferentially activate expression of select subordinate genes as a homodimer and potentially antagonize expression of others through heteromeric interactions.

Site-specific DNA binding of nuclear factor I: analyses of cellular binding sites

1985 ◽  
Vol 5 (5) ◽  
pp. 964-971
Author(s):  
R M Gronostajski ◽  
S Adhya ◽  
K Nagata ◽  
R A Guggenheimer ◽  
J Hurwitz
Keyword(s):  
Dna Binding ◽  
Hela Cell ◽  
Dna Sequences ◽  
Binding Sites ◽  
Consensus Sequence ◽  
Nuclear Factor ◽  
Site Specific ◽  
Factor I ◽  
Nuclear Factor I

Nuclear factor I is a cellular site-specific DNA-binding protein required for the efficient in vitro replication of adenovirus DNA. We have characterized human DNA sequences to which nuclear factor I binds. Three nuclear factor I binding sites (FIB sites), isolated from HeLa cell DNA, each contain the sequence TGG(N)6-7GCCAA. Comparison with other known and putative FIB sites suggests that this sequence is important for the binding of nuclear factor I. Nuclear factor I protects a 25- to 30-base-pair region surrounding this sequence from digestion by DNase I. Methylation protection studies suggest that nuclear factor I interacts with guanine residues within the TGG(N)6-7GCCAA consensus sequence. One binding site (FIB-2) contained a restriction endonuclease HaeIII cleavage site (GGCC) at the 5' end of the GCCAA motif. Digestion of FIB-2 with HaeIII abolished the binding of nuclear factor I. Southern blot analyses indicate that the cellular FIB sites described here are present within single-copy DNA in the HeLa cell genome.

Architecture of the maize mitochondrial atp1 promoter as determined by linker-scanning and point mutagenesis

1993 ◽  
Vol 13 (12) ◽  
pp. 7232-7238
Author(s):  
W D Rapp ◽  
D S Lupold ◽  
S Mack ◽  
D B Stern
Keyword(s):  
Promoter Region ◽  
Consensus Sequence ◽  
Point Mutations ◽  
In Vitro Transcription ◽  
Wild Type ◽  
Transcription Start ◽  
Transcription System ◽  
Mitochondrial Promoters ◽  
Point Mutagenesis

Plant mitochondrial promoters are poorly conserved but generally share a loose consensus sequence spanning approximately 17 nucleotides. Using a homologous in vitro transcription system, we have previously shown that an 11-nucleotide sequence within this region comprises at least part of the maize mitochondrial atp1 promoter (W. Rapp and D. Stern, EMBO J. 11:1065-1073, 1992). We have extended this finding by using a series of linker-scanning and point mutations to define the atp1 promoter in detail. Our results show that mutations at positions -12 to +5, relative to the major transcription start site, can decrease initiation rates to between < 10 and 40% of wild-type levels. Some mutations, scattered throughout this region, have lesser effects or no effect. Taken together, our data suggest a model in which the atp1 promoter consists of a central domain extending from -7 to +5 and an upstream domain of 1 to 3 bp that is centered around -11 to -12. Because many mutations within this promoter region are tolerated in vitro, the maize atp1 promoter is distinct from the highly conserved yeast mitochondrial promoters.

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